regulation of foxo stability and activity by mdm2 e3 ligase
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University of South FloridaScholar Commons
Graduate Theses and Dissertations Graduate School
2007
Regulation of FOXO stability and activity byMDM2 E3 ligaseWei FuUniversity of South Florida
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Scholar Commons CitationFu, Wei, "Regulation of FOXO stability and activity by MDM2 E3 ligase" (2007). Graduate Theses and Dissertations.http://scholarcommons.usf.edu/etd/2181
Regulation of FOXO Stability and Activity by MDM2 E3 Ligase
By
WEI FU
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy Department of Pathology and Cell Biology
College of Medicine University of South Florida
Major Professor: Wenlong Bai, Ph.D. George Blanck, Ph.D. Jiandong Chen, Ph.D. Santo V. Nicosia, M.D
John C.M. Tsibris, Ph.D.
Date of Approval: Nov., 2007
Key words: Forkhead Transcriptional Factors, MDM2, Ubiquitination, Protein Degradation, Apoptosis
©Copyright 2007, Wei Fu
Dedication
To my parents, my in-laws, my brother,
my husband, Zhigang and my little pumpkin, caroline.
Without their love, understanding and support, I could
not have done even one piece of it.
Acknowledgement
I would like to extend my sincere appreciation to Dr. Wenlong Bai for guiding me in the
development of this project and the invaluable support from him at each step throughout
my graduate studies.
My appreciation also goes to Dr. Santo V. Nicosia for being helped in every way.
I would like to thank my adviser and committee members, Drs Bai, Blanck, Chen,
Nicosia and Tsibris for their guidance, valuable suggestion and their word of
encouragement.
My appreciation also goes to Dr. Yanping Zhang for willing to be my outside chair.
Thanks to Dr. Jiandong Chen and the members in his lab, in particular to Lihong Chen,
for their generous supply of the cell lines, plasmids, antibodies and priceless technical
assistant.
Thanks to Dr. Xiaohong (Mary) Zhang for all her kindly help.
Thanks to all the colleagues in Dr. Bai’s and Dr. Zhang’s Lab: Qiuping Ma, Yonghua
Yang, Pengfei Li, Heehyoung Lee, Feng Jiang, Chunrong Li, Betty Bean, Xiaohui Zhang,
Junying Bao, Lucy Hou, Zheng Shen, Guoqing Liu, Latasha Lee, Ushiwa Jinwal, Tung
Hsieh, Yingtao Zhang, Sarah Yong, Huiqin Dong and Mu Zhang for sharing their
opinions and joy with me.
Thanks to Dr. Patricia Kruk and all the members in her lab.
Thanks to Dr. Jin Cheng and the members in his lab, especially to Mei Sun, Zengqiang
Yuan and Hua Yang for valuable suggestion.
I would like thank to all the members in the Department of Pathology and Cell Biology.
Addtionally, I would like to thank everyone in Graduate Affairs Office, especially to Dr.
Barber, Dr. Joseph Krzanowski, Ms Kathy Zahn, Ms Susan Chapman.
Thanks to Moffitt & College of Medicine core facility for some analysis.
Thanks to all my friends who guide me, encourage me and help me on the way.
This thesis is dedicated to my family. Word cannot express my grateful feeling. I want to
say thanks to my husband, Zhigang, for his loving and my daughter, Caroline, for her
shining smile that let me feel I am the happiest mother in the world. I would like to thank
my parents and my brother, for their life-long loving. I would also like to thank my in-laws
for their kindly help.
The research in this dissertation has been partially supported by Predoctoral Fellowship
from American Heart Association.
Thanks to Dr. Tsibris and Dr. Aslam for reading this thesis and their valuable
suggestions.
i
Table of Contents
List of Figures................................................................................................................... vi
List of Tables..................................................................................................................... x
Abstract ............................................................................................................................ xi
INTRODUCTION............................................................................................................... 1
1. FOXO Family...................................................................................................... 1
1.1. FOX Family................................................................................................ 1
1.2. FOXO Family ............................................................................................. 4
1.2.1. Classification of Human FOXO Factors ............................................ 5
1.2.2. Domain Structure of FOXO............................................................... 7
1.3. Function of FOXO Factors......................................................................... 9
1.3.1. FOXO and Cell Cycle Checkpoint and DNA Repair ....................... 10
1.3.1.1. FOXO and G1/S Checkpoint.................................................. 10
1.3.1.2. FOXO and G2/M Checkpoint and DNA Repair ...................... 11
1.3.2. FOXO and Apoptosis ...................................................................... 12
1.3.3. FOXO and Atrophy ......................................................................... 13
1.3.4. FOXO and ROS Detoxification in Stem Cells ................................. 14
1.3.5. FOXO and Tissue Differentiation.................................................... 15
1.3.6. FOXO and Glucose and Energy Metabolism.................................. 16
ii
1.3.7. FOXO and Longevity ...................................................................... 17
1.4. Posttranslational Modifications of FOXO Proteins................................... 18
1.4.1. Phosphorylation of FOXO............................................................... 18
1.4.1.1. PKB and FOXO...................................................................... 18
1.4.1.2. SGK and FOXO ..................................................................... 20
1.4.1.3. IκB Kinase and FOXO3A ....................................................... 21
1.4.1.4. CK1 and FOXO...................................................................... 21
1.4.1.5. CDK2 and FOXO ................................................................... 21
1.4.1.6. JNK and FOXO ...................................................................... 21
1.4.1.7. MST and FOXO3A................................................................. 22
1.4.2. Acetylation and Deacetylation of FOXO ......................................... 23
1.4.2.1. Acetylation and Deacetylation................................................ 23
1.4.2.2. Acetylation of FOXO .............................................................. 23
1.4.2.2.1. P300 and CBP .............................................................. 23
1.4.2.2.2. P300/CBP and FOXO Acetylation................................. 25
1.4.2.3. Deacetylation of FOXO by HDAC .......................................... 26
1.4.2.3.1. SIRT1 ............................................................................ 26
1.4.2.3.2. SIRT1 and FOXO Deacetylation ................................... 28
1.4.3. Ubiquitination and Deubiquitination of FOXO ................................. 30
1.4.3.1. Ubiquitination and Degradation of FOXO .............................. 30
1.4.3.2. Deubiquitination of FOXO ...................................................... 31
2. Ubiquitin, Proteasome and MDM2 as an E3 Ligase......................................... 32
2.1. Ubiquitin-proteasome System.................................................................. 32
2.1.1. Ubiquitin and Ubiqutination............................................................. 32
2.1.2. Deubiquitination .............................................................................. 33
iii
2.1.3. Ubiquitination Machinery and Proteasome Pathway ...................... 35
2.2. MDM2 as an Ubiquitin E3 Ligase ............................................................ 37
2.2.1. General Information about MDM2................................................... 37
2.2.2. Functions of MDM2......................................................................... 38
2.2.2.1. MDM2 and Cell Cycle ............................................................ 38
2.2.2.2. MDM2 and Differentiation ...................................................... 38
2.2.2.3. MDM2 and Ribosome Biogenesis.......................................... 39
2.2.2.4. MDM2 and Transcription........................................................ 40
2.2.2.5. MDM2 and Protein Ubiquitination and Degradation............... 40
2.2.3. Regulation of MDM2 E3 Activity ..................................................... 41
2.2.3.1. Sumoylation of MDM2............................................................ 41
2.2.3.1.1. SUMO and Sumoylation................................................ 41
2.2.3.1.2. Sumoylation and MDM2................................................ 43
2.2.3.2. Ubiquitination and Degradation of MDM2 .............................. 43
2.2.3.3. Phosphorylation of MDM2...................................................... 44
2.2.3.3.1. DNA-PK, ATM and MDM2 ............................................ 44
2.2.3.3.2. PKB and MDM2 ............................................................ 45
2.2.3.3.3. c-Abl and MDM2 ........................................................... 46
2.2.3.3.4. CK2 and MDM2............................................................. 46
3. Functional interaction among FOXO, p53, and MDM2..................................... 49
3.1. Interaction between MDM2 and p53........................................................ 49
3.2. Interaction between p53 and FOXO ........................................................ 50
HYPOTHESIS & OBJECTIVES ...................................................................................... 53
MATERIALS AND METHODS ........................................................................................ 54
1. Chemicals, Antibodies and Cell Lines .............................................................. 54
iv
2. Plasmids ........................................................................................................... 54
3. Transfections and Immunological Assays ........................................................ 55
4. Ni-NTA Pull-down Assay .................................................................................. 57
5. In vitro Transcription Coupled Translations and GST (Glutathione
S-transferase) Pull-down Assays .................................................................. 58
6. In Vitro Ubiquitination Assay............................................................................. 58
7. Apoptotic Analysis and Flow Cytometry ........................................................... 59
RESULTS........................................................................................................................60
1. MDM2 promotes the degradation of FOXO...................................................... 60
2. MDM2 interacts with FOXO in vivo and in vitro ................................................69
3. The fork head box of FOXO and the region of MDM2 controlling
nuclear-cytoplasmic shuttling mediate the interaction between
MDM2 and FOXO............................................................................................. 77
4. MDM2 promotes the ubiquitination of FOXO1 and FOXO3A ........................... 88
5. MDM2 suppresses the expression of FOXO target genes and
protects cells from FOXO1-induced cell death ................................................. 96
6. MDM2 transiently increases FOXO transcriptional activity............................. 103
7. p53 induces transient increase in the transcriptional activity of
FOXO factors, which is followed by FOXO degradation in an
MDM2-dependent manner.............................................................................. 108
8. Site-specific sumoylation of SIRT1 regulates FOXO1
transcriptional activity and stability ................................................................. 116
9. Genistein-induced FOXO1 expression is blocked by MDM2
expression in H1299 cells............................................................................... 121
10. ARF promotes the MDM2-induced FOXO ubiquitination................................ 123
v
DISCUSSION................................................................................................................ 125
1. MDM2 is the E3 ubiquitin ligase of FOXO ..................................................... 125
2. MDM2-promoted ubiquitination of FOXO depends on
phosphorylation on PKB sites......................................................................... 126
3. MDM2-promoted ubiquitination of FOXO happenes mainly in the
cytoplasm ....................................................................................................... 127
4. MDM2 affects both the transcriptional activity and ubiquitination of
FOXO ............................................................................................................. 128
5. Mammalian FOXO factors interact with p53 .................................................. 129
6. MDM2 regulates FOXO factors in a p53 independent way ............................ 130
7. Different domains of MDM2 play different roles in the regulation of
p53 and FOXO factors (see Table 2) ............................................................. 132
8. The sumoylation status of SIRT1 affects the stability and activity of
FOXO ............................................................................................................. 134
9. Genistein increases FOXO expression levels through down-
regulation of MDM2 ........................................................................................ 134
10. ARF differentially affects E3 function of MDM2 toward different
substrates....................................................................................................... 135
SUMMARY AND PERSPECTIVES............................................................................... 136
REFERENCES.............................................................................................................. 139
About the author.................................................................................................. End Page
vi
List of Figures
Figure 1 Structure of human FOXO............................................................................ 8
Figure 2 The regulation of FOXO ............................................................................. 29
Figure 3 Ubiquitin-proteasome degradation pathway ............................................... 36
Figure 4 Structure of MDM2 gene and protein ......................................................... 47
Figure 5 Stress-induced FOXO and p53 pathway .................................................... 52
Figure 6 Inverse correlation of MDM2 and FOXO3A protein expression in
different cancer cell lines ............................................................................ 62
Figure 7 Inverse correlation of MDM2 and FOXO protein expression in MEFs........ 63
Figure 8 Stably expressed MDM2 decreases the level of endogenous
FOXO3A expression................................................................................... 64
Figure 9 Knockdown of endogenous MDM2 by siRNA causes an increase in
endogenous FOXO3A protein .................................................................... 65
Figure 10 Knockdown of endogenous MDM2 by shRNA increases the level of
ectopic FOXO1 protein ............................................................................... 66
Figure 11 MDM2 overexpression causes a decrease in Flag-FOXO1 protein,
which is blocked by MG132........................................................................ 67
Figure 12 MDM2 overexpression results in decrease in the half-life of the
FOXO1 protein ........................................................................................... 68
Figure 13 Exogenous MDM2 and Flag-FOXO1 are colocalized in the nucleus
of MEF cells................................................................................................ 70
vii
Figure 14 Endogenous MDM2 and FOXO3A are colocalized in the nucleus of
H1299 cells................................................................................................. 71
Figure 15 Interaction of ectopic FOXO1 and MDM2 in H1299 cells ........................... 72
Figure 16 Interaction of ectopic FOXO3A and MDM2 in HEK293T cells.................... 73
Figure 17 Interaction of endogenous MDM2 and FOXO1 in H1299/V138 cells ......... 74
Figure 18 Interaction of endogenous MDM2 and FOXO3A in HEK293T cells ........... 75
Figure 19 The interaction between FOXO1 and GST-MDM2 in vitro ......................... 76
Figure 20 Diagram of different FOXO mutants used in this study .............................. 79
Figure 21 Mapping of the MDM2-interacting domain of FOXO1 by
immunoprecipitation ................................................................................... 80
Figure 22 Mapping of the MDM2-interacting domain of FOXO3A by GST pull-
down assay................................................................................................. 81
Figure 23 Alignment of FOXO members’ fork head box............................................. 82
Figure 24 Diagram of different MDM2 mutants used in this study.............................. 83
Figure 25 Mapping of the FOXO1-interacting domain of MDM2 by
coimmunoprecipitations in the absence of MG132..................................... 84
Figure 26 An N-terminal truncation mutant and an NLS-mutant of MDM2
interact with FOXO1 in the presence of MG132......................................... 85
Figure 27 Truncation of the central region of MDM2 abolishes the interaction
between MDM2 and FOXO1 ...................................................................... 86
Figure 28 Immunofluorescence images show the cellular localization of
various MDM2 mutants............................................................................... 87
Figure 29 MDM2 promotes the ubiquitination of FOXO1............................................ 90
Figure 30 MDM2 promotes the ubiquitination of FOXO3A ......................................... 91
viii
Figure 31 The MDM2 ring finger domain is critical for the ubiquitination of
FOXO1 ....................................................................................................... 92
Figure 32 Ubiquitination of FOXO1 requires MDM2 ubiquitin ligase function............. 93
Figure 33 The central region of MDM2 cannot promote the polyubiquitination
of FOXO1 ................................................................................................... 94
Figure 34 MDM2 promotes FOXO1 polyubiquitination in vitro....................................95
Figure 35 Stably expressed MDM2 in H1299 cells regulates the expression of
downstream targets of FOXO..................................................................... 97
Figure 36 MDM2 siRNA increases the expression of FOXO1 target gene
TRAIL ......................................................................................................... 98
Figure 37 MDM2 promotes the cell survival in the presence of FOXO1..................... 99
Figure 38 MDM2 protectes cells from FOXO1-induced cell death measured by
Flow Cytometry......................................................................................... 101
Figure 39 MDM2 increases the transcriptional activity of FOXO1 in a dose-
dependent manner in DU145 cells ........................................................... 104
Figure 40 MDM2 increases the transcriptional activity of FOXO1 in NIH3T3
cells .......................................................................................................... 105
Figure 41 MDM2 enhances the ability of FOXO1 to inhibit cyclin D1 ....................... 106
Figure 42 MDM2 increases the transcriptional activity of both wild type FOXO1
and FOXO1 (AAA) mutant in NIH3T3 and DU145 cells ........................... 107
Figure 43 P53 inhibits the transcriptional activity of FOXO, in a dose-
dependent manner, but not FOXO1-induced cell death ........................... 109
Figure 44 P53 inhibits the transcriptional activity of FOXO in a dose-
dependent manner in LNCaP cells........................................................... 110
Figure 45 MDM2 relieves the repression of FOXO1 activity by p53......................... 111
ix
Figure 46 MDM2 transiently increases FOXO transcriptional activity, which is
followed by FOXO degradation ................................................................ 112
Figure 47 MG132 relieves p53-induced decrease in the expression of
FOXO3A protein in H1299/V138 cells ...................................................... 113
Figure 48 p53 decreases the protein level of FOXO1 through MDM2...................... 114
Figure 49 Knockdown of MDM2 partially relieves p53-induced FOXO3A
downregulation ......................................................................................... 115
Figure 50 SIRT1 inhibits the transcriptional activity of FOXO................................... 117
Figure 51 Nicotinamide treatment increases the FOXO1 transcriptional activity...... 118
Figure 52 SIRT1 sumoylation at Lys 734 is required for FOXO1 deacetylation ....... 119
Figure 53 Mutation of Lys 734 relieves the inhibition of FOXO1 transcriptional
activity by SIRT1....................................................................................... 120
Figure 54 Genistein increases the expression of FOXO through MDM2
downregulation ......................................................................................... 122
Figure 55 ARF promotes the MDM2-induced FOXO1 ubiquitination........................ 124
Figure 56 A working model shows the functional interaction among FOXO,
p53 and MDM2 ......................................................................................... 131
x
List of Tables
Table 1 The FOXO family members in mammals ........................................................ 6
Table 2 Localization and function of different MDM2 mutants.................................. 133
xi
REGULATION OF FOXO STABILITY AND ACTIVITY BY MDM2 E3 LIGASE
WEI FU
ABSTRACT
Members of the forkhead class O (FOXO) transcription factors are tumor
suppressors and key molecules that control aging and lifespan. The stability of
mammalian FOXO proteins is controlled by proteasome-mediated degradation but
general ubiquitin E3 ligases for FOXO factors remain to be defined. The current studies
demonstrate that MDM2 bound to FOXO1 and FOXO3A and promoted their
ubiquitination and subsequent degradation, a process apparently dependent on FOXO
phopshorylation at PKB sites and on the E3 ligase activity of MDM2. The binding
occurred between endogenous proteins and was involved the forkhead box of FOXO1
and the region of MDM2 that controls its cellular localization. MDM2 promoted the
ubiquitination of FOXO1 in vitro in a cell free system. Knocking down MDM2 by siRNA
caused the accumulation of endogenous FOXO3A protein, and enhanced the
expression of FOXO target genes. In addition, MDM2 promoted the transcriptional
activity of FOXO in a transient transfection system. In cells stably expressing a
temperature sensitive mutant p53, activation of p53, by shifting to permissive
temperatures led to MDM2 induction and the degradation of endogenous FOXO3A.
These data suggested that MDM2 acts downstream of p53 as an E3 ubiquitin ligase to
promote the degradation of mammalian FOXO factors.
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INTRODUCTION
1. FOXO Family
1.1. FOX Family
Forkead proteins constitute a family of structurally related transcriptional factors
that have been identified in all eukaryotes ranging from yeast to human. Forkhead
transcription factors are characterized by the forkhead domain, which is a ~100 amino-
acid monomeric DNA binding domain. The three-dimensional structure of forkhead
domain consists of two W1 and W2 loops (or wings) and three α helices (Lehmann et al.,
2003). Due to its butterfly-like appearance, the domain is also known as “winged-helix”
domain.
The first member of the forkhead family was identified in 1989 as a nuclear
homeotic gene involved in the embryonic development of flies (Drosophila
melanogaster). To date, over 100 members of the forkhead family have been identified in
species ranging from Saccharomyces cerevisiae to humans (Kaestner et al., 2000). The
nomenclature of the forkhead transcription factors has recently been revised. These
genes, now termed Fox (after Forkhead box), are divided into 17 subclasses (A to Q),
according to the amino acid sequence of their conserved forkhead domains
(http://www.biology.pomona.edu/fox.html).
Comparative genome analyses have shown that the number of forkhead
transcription factors appears to have increased during evolution, with a greater number
identified in vertebrates than in invertebrates (Lehmann et al., 2003). The origin and
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expansion of forkhead genes is positively correlated with eukaryotic complexity because
among the organisms for which genome sequences are available, there is a correlation
between anatomical complexity and forkhead gene number: 4 FOX genes in S.
cerevisiae, 15 in Caenorhabditis elegans, 20 in Drosophila melanogaster and 43 in
humans (Mazet et al., 2003). The human FOX gene family consists of : FOXA1, FOXA2,
FOXA3, FOXB1, FOXC1, FOXC2, FOXD, FOXD2, FOXD3, FOXD4, FOXD5
(FOXD4L1), FOXD6 (FOXD4L3), FOXE1, FOXE2, FOXE3, FOXF1, FOXF2, FOXG1
(FOXG1B), FOXH1, FOXI1, FOXJ1, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2,
FOXM1, FOXN1, FOXN2 (HTLF), FOXN3 (CHES1), FOXN4, FOXN5 (FOXR1), FOXN6
(FOXR2), FOXO1 (FOXO1A, FKHR), FOXO2 (FOXO6), FOXO3 (FOXO3A), FOXO4
(MLLT7), FOXP1, FOXP2, FOXP3, FOXP4, and FOXQ1. Members of FOX subfamilies
A-G, I-L and Q were grouped into class-1 FOX proteins, while members of FOX
subfamilies H and M-P were grouped into class-2 FOX protein. The presence of a C-
terminal basic region within the FOX domain is the common feature of class-1 FOX
protein (Katoh and Katoh, 2004b).
FOX transcriptional regulators play various roles during development, from
organogenesis to language acquistion. Foxb1 is reported to control development of
mammary glands and regions of the central nervous system that regulate the milk
ejection reflex in mice (Kloetzli et al., 2001). FOXC1 and FOXC2 (Kume et al., 2000;
Kume et al., 1998; Wilm et al., 2004), FOXE1 (Aza-Blanc et al., 1993; Ortiz et al., 1997;
Zannini et al., 1997), FOXF1 (Kalinichenko et al., 2001; Kalinichenko et al., 2002),
FOXJ1 (Pelletier et al., 1998) and FOXJ2 (Granadino et al., 2000) play important roles in
the development of the organs, such as thyroid organogenesis, mesonephros, gonad,
liver, gallbladder, lung and intestinal tract. FOXD1 (Hatini et al., 1996), FOXE3 (Blixt et
al., 2007; Valleix et al., 2006) and FOXL2 (De Baere et al., 2001; De Baere et al., 2002)
- 3 -
play a critical role in the development of eyes. FOXD3 (Alkhateeb et al., 2005; Guo et
al., 2002), FOXD5 (Yu et al., 2002) and FOXJ3 (Landgren and Carlsson, 2004) are
involved in neural development. FOXD4 (Minoretti et al., 2007) and FOXP4 (Li et al.,
2004b) are related to heart development and differentiation. The FOXG1 (Pauley et al.,
2006) and FOXI1(Hulander et al., 2003) are required for the development of the
mammalian inner ear. FoxH1 (FAST) is a transcription factor that mediates signaling by
transforming growth factor-β, activin, and nodal. FoxH1 / embryos failed to orient the
anterior-posterior (A-P) axis correctly (Yamamoto et al., 2001). FOXJ1 is required for late
stages of ciliogenesis (You et al., 2004b). It is essential for nonrandom determination of
left-right asymmetry and development of ciliated cells. FOXN4 is necessary and
sufficient for commitment to the amacrine cell fate and is nonredundantly required for the
amacrine and horizontal cells (Li et al., 2004a). FOXP2 has been implicated in the
acquisition of grammatical skills (Hurst et al., 1990; Lai et al., 2001). FOXP3 is the
master regulator in the development and function of regulatory T cells and the selected
marker for them (Alvarado-Sanchez et al., 2006; Bennett et al., 2001; Brunkow et al.,
2001; Suri-Payer and Fritzsching, 2006). Human FOXN1 (Frank et al., 1999) and
FOXQ1 (Hong et al., 2001) function in hair differentiation. FOXN1 is also required for the
growth and differentiation of thymic epithelial cells (Balciunaite et al., 2002).
FOX factors are also very important for cell growth, differentiation and
tumorigenesis. FOXA1 binds to the promoters of more than 100 genes associated with
metabolic processes, the regulation of signaling and the cell cycle (Carlsson and
Mahlapuu, 2002; Kaestner, 2000; Tomaru et al., 2003). High expression of FOXA1 (Gao
et al., 2003; Lin et al., 2002b), FOXN6 (Katoh and Katoh, 2004d), FOXQ1 (Bieller et al.,
2001) and FOXM1(Nakamura et al., 2004) has been reported in various tumors,
including lung, esophageal, breast, prostate and pancreatic cancers. Several FOX
- 4 -
factors are thought of as the candidate tumor suppressor genes. This includes FOXC1
(Zhou et al., 2002), FOXK2 (Li et al., 1992), FOXN5 (Katoh and Katoh, 2004a, c) and
FOXO family (Medema et al., 2000). These factors are reported either to be inactive in
cancer or to suppress tumor cell growth when overexpressed. FOXM1 is expressed in
proliferating cells and becomes silenced in terminally differentiated cells (Korver et al.,
1997; Yao et al., 1997; Ye et al., 1997). FOXM1 might be beneficial to patients whose
lung endothelial-cell barrier has been damaged (Zhao et al., 2006).
FOX factors also regulate chromatin remodeling and transcription. The FOXA3
acts in chromatin remodeling (Kaestner et al., 1994). FOXK1 is essential for regulating
cell cycle progression in myogenic progenitor cells (Hawke et al., 2003). FOXL1 is
capable of remodeling chromatin higher-order structure and can cause stable and site-
specific changes of chromatin by either creating or removing DNase I hypersensitive
sites, resulting in changes the proliferation, differentiation, and positioning of epithelial
cells (Kaestner et al., 1997; Yan et al., 2006). FOXN3 binds to Ski-interacting protein
(SKIP, NCoA-62), which is a transcriptional co-regulator known to associate with a
repressor complex (Scott and Plon, 2005).
It is believed that FOX factors form a network. For example, FOXG1, a class-1
FOXO member, acts as a repressor of transcriptional activity of the FOXO family (Aoki et
al., 2004). FOXA and FOXO factors cooperate in complex regulatory networks of the
pancreas and liver (Czech, 2003).
1.2. FOXO Family
FOXO factors are members of the O-subclass of the class-2 FOX family. A
unique five amino acid (GDSNS) insertion immediatedly prior to helix H3 within the
forkhead domain is characteristic of this subfamily. This insertion is absent in all other
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FOX transcription factors (Arden, 2006). The first member of FOXO was identified in C.
elegans (Caenorhabditis elegans) and named DAF-16 and was shown to regulate dauer
formation in C. elegans (Thomas, 1993).
1.2.1. Classification of Human FOXO Factors
In invertebrates there is only one FOXO gene, termed daf-16 in the worm and
dFOXO in the fly. In mammals there are four functional FOXO genes, FOXO1, 3A, 4,
and 6 (Carter and Brunet, 2007) (Table 1).
The FOXO1 gene is located in chromosome 13q14.1. It is the most abundant
FOXO isoform in insulin-responsive tissues such as liver, adipose tissue, pancreatic
cells, kidney, spleen and brain (Armoni et al., 2006). Embryos of FOXO1 homozygous
mice are smaller and die at embryonic day 10.5 due to several embryonic defects
(Nakae et al., 2002), such as incomplete vascular development (Hosaka et al., 2004).
Analysis of heterozygote mutant mice indicates that FOXO1 is involved in the function of
pancreatic β-cells, hepatic glucose metabolism and adipocyte differentiation (Kitamura et
al., 2002; Nakae et al., 2002; Nakae et al., 2003).
The FOXO3A gene is located in chromosome 6q21. It is highly expressed in the
heart, spleen, lung, kidney, ovary, adipose tissue and brain (Zhu et al., 2004). FOXO3A
knockout mice reveal haematological abnormalities, decreased glucose uptake in
glucose tolerance tests (Castrillon et al., 2003) and a distinct ovarian phenotype due to
premature follicular activation that leads to oocyte death and subsequent depletion of
follicles. It has been suggested that FOXO3A acts as a suppressor of follicular activation
(Castrillon et al., 2003; Hosaka et al., 2004). In mature endothelial cells, FOXO1 and
FOXO3A are the most abundant FOXO isoforms. Overexpression of constitutively active
6
- 7 -
FOXO1 or FOXO3A significantly inhibits endothelial cell migration and tube formation in
vitro (Potente et al., 2005).
FOXO4 gene is located in chromosome Xq13.1. It is abundant in the lungs, brain,
heart and skeletal muscle and kidneys (Nakae et al., 2001). FOXO4 knockout mice show
no an obvious abnormalities (Hosaka et al., 2004).
FOXO6 gene is located in 1p34.1. Embryonic expression of FOXO6 is seen
predominantly in the developing brain where it is expressed in a specific temporal and
spatial pattern. In the adult animal, FOXO6 expression persists in the nucleus
accumbens, cingulated cortex, parts of the amygdale, and hippocampus. FOXO6 is also
expressed in kidney and thymus (Jacobs et al., 2003). The phenotype of FOXO6-
deficient animals is not reported, but its restricted expression pattern suggests that it
may play a role in embryologic development of the central nervous system (Hoekman et
al., 2006).
1.2.2. Domain Structure of FOXO
From the N-terminus to the C-terminus, the FOXO protein contains a proline-rich
domain, a forkhead domain, NLS (nuclear localization signal), NES (nuclear export
signal), a LxxLL motif and an activation domain (Figure 1). The proline rich motif binds to
the CH3 region of CBP/p300 and stabilizes the interaction between FOXO factors and
CBP/p300 (van der Heide and Smidt, 2005). The Forkhead domain is responsible for
binding to target gene promoters. The central region of FOXO includes the NLS and
NES and accounts for the cellular localization of FOXO. The LxxLL motif is reported to
bind to SIRT1 and has an important role in regulating its transcriptional activity (Nakae et
al., 2006). The C-terminus of FOXO contains the activation domain which stimulates the
promoter activity (Yang et al., 2005). In the nucleus, all FOXO proteins bind to DNA as
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FOX150-260
LxxLL461-466
AD596-655
NLS NESFOXO1 655aa
FOXO3 673aa
FOXO4 501aa
FOXO6 509aa
Nt Ct
FOX 148-258
FOX 88-198
FOX 78-188
368-377
303-327
300-308
PxPxP
Figure 1 Structure of human FOXO. Full-length FOXO and known motifs
are presented. PxPxP-proline rich motif; FOX-forkhead domain; LXXLL-L,
leucine; X, any amino acid; AD-activation domain; Nt-amino terminal; NLS-
nuclear localization signal; NES-nuclear export signal; Ct-carboxyl terminal.
The numbers above the drawings denote amino acid numbers.
- 9 -
monomers through the FOX domain. The core motif of the consensus recognition site for
FOXO on DNA is GTAAA (C/T) A and it is designated as the DBE for DAF-16 family
member-binding element.
1.3. Functions of FOXO Factors
Studies in C. elegans showed that direct activation of DAF-16, or mutation of the
insulin-PI3K (phosphotidylinositol 3 kinase)-PKB (protein kinase B) pathway results in
extension of lifespan, stress resistance and arrest at the dauer diapause stage (Ogg et
al., 1997). Besides cell-autonomous inputs, DAF-16 also responds to environmental
cues such as starvation, heat and oxidative stress. All these stress signals activate DAF-
16, whereas nutrient-rich conditions deactivate it. dFOXO, the unique FOXO homologue
in Drosophila, seems to play similar roles, which are negatively controlled by the insulin-
PI3K-PKB signaling cascade and nutrients (Junger et al., 2003; Kramer et al., 2003;
Puig et al., 2003), although dFOXO-knockout flies are viable and of normal size, they are
more vulnerable to the oxidative stress. These data suggest that dFOXO offers
protection against oxidative stress.
In humans, the pivotal role of FOXO on cell fate decisions depends on the
balance between growth factor stimulation versus cellular stress and damage. Both
circumstantial and direct evidences implicate a role of FOXO factors in cancer. The first
three members of FOXO were found at chromosomal translocations in tumors. The
FOXO1 gene is fused to PAX3 or PAX7 gene in rhabdomyosarcoma (Galili et al., 1993;
Sorensen et al., 2002). The FOXO3A gene is fused to MLL gene in secondary acute
myeloblastic leukemia (Hillion et al., 1997). The FOXO4 gene is fused to MLL gene in
acute lymphocytic leukemia (Parry et al., 1994). Nuclear exclusion of FOXO3A in
primary breast tumors correlates with PI3K activation and poor survival of the patients
(Hu et al., 2004). FOXO factors reduce tumorigenicity in nude mice (Hu et al., 2004;
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Ramaswamy et al., 2002). Furthermore, FOXO proteins interact with many oncogenes
such as β-catenin (Essers et al., 2005) or tumor suppressors such as p53 (Brunet et al.,
2004).
The physiological importance of FOXO transcription factors is diverse as
revealed by loss and gain of function experiments in transgenic and knockout mice. All
lines of evidence point to FOXO factors as central regulators of metabolism, aging,
proliferation and differentiation (Hosaka et al., 2004). Since a wide range of human
diseases, including cancer, have striking aging-dependent onset, FOXO factors are
believed to serve as molecular links between longevity and tumor suppression (Greer
and Brunet, 2005).
1.3.1. FOXO and Cell Cycle Checkpoint and DNA Repair
Mammalian cells have evolved an intricate defense network to maintain genomic
integrity by preventing the fixation of permanent damage from endogenous and
exogenous mutagens. Cell cycle checkpoints, a major genomic surveillance mechanism
acting at the G1/S and G2/M boundaries, are regulated in response to DNA damage
(Hartwell and Weinert, 1989). Defects in these steps may result in a mutated phenotype
that is associated with tumorigenesis.
1.3.1.1. FOXO and G1/S Checkpoint
p21 is a cyclin- dependent kinase inhibitor. Loss of p21 expression has been
described as a poor prognostic factor and as an independent predictor of bladder cancer
progression in muscle invasive cancer (Stein et al., 1998). p27 is a protein that binds to
cyclins and cdk to block the entry into S phase. Loss of p27 expression has been shown
to be associated with aggressive behavior in a variety of human epithelial tumors,
including prostate (Macri and Loda, 1998) and breast (Catzavelos et al., 1997; Gillett et
- 11 -
al., 1999; Tan et al., 1997; Wu et al., 1999) cancers. p130 belongs to the RB family. A
study in mice showed that p130 can induce cell cycle arrest (Classon et al., 2000).
Mammalian cyclins are classified into 12 different types, from cyclins A to I, based on
structural similarity, function period in the cell division cycle, and regulated protein
expression. cyclin D1 is a key regulator of the G1 phase of the cell division cycle where it
binds to and activates the cyclin-dependent kinases CDK4 and CDK6. In the cells that
have the capacity to divide, the main effect of the expression of active forms of FOXO
family members is to promote cell cycle arrest at the G1/S boundary. FOXO factors play
a major role in G1 arrest by upregulating cell cycle inhibitors such as p21 (Hauck et al.,
2007; Lawlor and Rotwein, 2000), p27 (Medema et al., 2000; Stahl et al., 2002) and
p130 (Chen et al., 2006) and by repressing cell cycles activators cyclin D1 and D2
(Schmidt et al., 2002).
1.3.1.2. FOXO and G2/M Checkpoint and DNA Repair
GADD45 is growth arrest and DNA damage-inducible protein 45. It was identified
on the basis of its rapid transcriptional induction after UV irradiation (Fornace et al.,
1989) and can induce G2/M arrest (Wang et al., 1999). Induction of GADD45 is also
observed after several types of pathological stimuli including various environmental
stresses: hypoxia, irradiation, genotoxic drug exposure and growth-factor withdrawal
(Papathanasiou et al., 1991).Three G-type cyclins have been identified: cyclins G1, G2,
and I. All three are expressed in terminally differentiated cells, act as cell cycle inhibitors
in certain cell types and may induce cell cycle arrest (Martinez-Gac et al., 2004).
Microarray analysis led to the identification of GADD45 and cyclin G2 as the
downstream target genes of FOXO3A for G2/M arrest and DNA repair (Tran et al.,
2002).
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In vivo experiment showed that FOXO3A and FOXO4 activate GADD45
promoter through direct interaction with the DBE. Oxidative stress activates the GADD45
promoter in a FOXO-dependent manner, resulting in increased level of GADD45 mRNA
and protein as well as G2 arrest (Furukawa-Hibi et al., 2002). In response to glucose
starvation, FOXO1 also affects GADD45 mRNA level in islets (Martinez et al., 2006).
Cyclin G2 is expressed in various amounts during the cell cycle in lymphocytes
(Horne et al., 1997; Horne et al., 1996). Ectopic expression of cyclin G2 inhibits cell
cycle progression (Bennin et al., 2002). FOXO3A interacts with cyclin G2 in mouse
fibroblasts. Active forms of FOXO3A increase cyclin G2 mRNA levels by activating the
cyclin G2 transactivation through interaction with the DBE in its promoter (Martinez-Gac
et al., 2004).
One mechanism by which cells protect themselves against stress is by repairing
the damage to their DNA and proteins that occurs upon exposure to environmental
stress. Furthermore, this capacity to repair DNA damage is closely correlated with an
increased longevity in mammals (Kirkwood and Austad, 2000). Gene array analysis
showed that FOXO3A is involved in nuclear excision DNA repair by modulating the
expression of GADD45 and DDB (damaged DNA binding) protein (Tran et al., 2002).
1.3.2. FOXO and Apoptosis
FOXO factors have been shown to mediate apoptosis by activating proapoptotic
genes in a variety of cells. FOXO promotes cell death through these downstream
targets: TRAIL (Tumor necrosis-related apoptosis-inducing ligand), FasL (Fas Ligand)
(Brunet et al., 1999), Bim (Stahl et al., 2002; Sunters et al., 2003; Urbich et al., 2005)
and PUMA (You et al., 2006a).
TRAIL is a member of the TNF family of cytokines. It induces apoptosis via death
receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells (Suliman
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et al., 2001). TRAIL is a direct target of FOXO3A. FOXO3A response element is located
in TRAIL promoter spanning nucleotides -138 to -121. Decreased activity of FOXO3A
and FOXO1 in prostate cancers resulting from loss of PTEN leads to a decrease in
TRAIL expression that can contribute to increased survival of the tumor cells (Modur et
al., 2002).
FasL is a type II transmembrane protein. It functions as a homotrimer, because it
trimerizes Fas receptor, which spans the membrane of the "target" cell. This
trimerization usually leads to apoptosis.There are three putative FOXO binding elements
in the FasL promoter. In the human leukemia T cell line Jurkat, FOXO3A induces
apoptosis by activating FasL expression and the Fas-FasL apoptotic pathway
(Yamamura et al., 2006).
Bim is a BH3-only Bcl2 family member. Bim (also known as Bcl2l11) provokes
apoptosis (O'Connor et al., 1998). There are two conserved FOXO binding sites in the
Bim promoter: one at position -204 relative to the transcription start site (bim1) and one
at the boundary between exon 1 and the first intro. FOXO3A directly activates the bim
promoter via the two conserved FOXO binding sites (Gilley et al., 2003).
PUMA is an essential mediator of p53-dependent and -independent apoptosis in
vivo. Promoter analysis identifies that there is a potential consensus FOXO-responsive
element in intron 1 of PUMA which is conserved in humans and mice. FOXO3A up-
regulates PUMA transcription in response to cytokine or growth factor deprivation (You
et al., 2006a).
1.3.3. FOXO and Atrophy
Atrophy is the partial or complete decompositon of a part of the body. Causes of
atrophy include poor nourishment, poor circulation, loss of hormonal support, loss of
nerve supply to the target organ, disuse or lack of exercise or disease intrinsic to the
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tissue itself. Hormone and nerve inputs that maintain an organ or body part are referred
to as trophic. Atrophy is a general physiological process of reabsorption and breakdown
of tissues, involving apoptosis on a cellular level. When it occurs as a result of disease
or loss of trophic support due to other disease, it is termed pathological atrophy,
although it can be a part of normal body development and homeostasis as well.
Skeletal muscle atrophy is a debilitating response to fasting, disuse, cancer, and
other systemic diseases. FOXO3A acts on the atrogin-1 promoter to cause atrogin-1
transcription and dramatic atrophy of myotubes and muscle fibers. When FOXO
activation is blocked by a dominant-negative construct in myotubes or by RNAi in mouse
muscles in vivo, atrogin-1 induction is prevented during starvation and atrophy of
myotubes induced by glucocorticoids (Sandri et al., 2004).
In fully differentiated skeletal and cardiac muscle cells, expression of a
constitutively active form of FOXO3A causes atrophy. The atrophy is not due to
apoptosis. Instead, it is due to a decrease in cell size.
1.3.4. FOXO and ROS Detoxification in Stem Cells
FOXO proteins have been reported to allow detoxification of ROS (reactivie
oxygen species) by upregulating free radical scavenging enzymes, including MnSOD
(manganese superoxide dismutase) and catalase (Storz, 2006).
FOXO3A protects quiescent cells from oxidative stress by directly increasing
MnSOD mRNA and protein levels. This increase in protection from reactive oxygen
species antagonizes the apoptosis caused by glucose deprivation. Increased resistance
to oxidative stress is also associated with longevity. The model of Forkhead involvement
in regulating longevity stems from genetic analysis in C. elegans, but the model also
can be extendable to mammalian systems (Kops et al., 2002).
- 15 -
Stem cells are primal cells found in all multi-cellular organisms. They have two
important characteristics that distinguish them from other types of cells. First, they are
unspecialized cells that renew themselves for long periods through cell division. The
second characteristic is that under certain physiologic or experimental conditions they
can be induced to become cells with special functions such as the beating cells of the
heart or the insulin-producing cells of the pancreas. Regulation of oxidative stress in the
HSC (hematopoietic stem cell) compartment is critical for the maintenance of HSC self-
renewal. FOXO-dependent signaling is required for the long-term regenerative potential
of the HSC compartment through regulation of HSC responses to physiologic oxidative
stress, quiescence and survival. Further analysis of ROS levels in FOXO knockout mice
showed that FOXOs affect HSC integrity by regulating ROS. Myeloid progenitor cells
isolated from FOXO1/3/4 conditional knockout animals show decreased ROS (Tothova
et al., 2007).
1.3.5. FOXO and Tissue Differentiation
Cell and tissue differentiation process systematically bases on a number of gene
expressions that commence successively along with the passage of time. In
differentiating cells, FOXO factors have been implicated in inhibiting and promoting
differentiation, depending on the cell types and different FOXO isoforms. In adipocytes,
FOXO1 is induced in the early stages of adipocyte differentiation but its activation is
delayed until the end of the clonal expansion phase. Constitutively active FOXO1
prevents the differentiation of preadipocytes, while dominant-negative FOXO1 restores
adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. FOXO1
directly inhibits the differentiation through upregulation of p21 (Nakae et al., 2003).
Hematopoiesis, or blood cell formation, is the process in which a limited set of
hematopoietic stem cells is able to give rise to all types of functional blood cells via
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commitment to specific hematopoietic lineages. FOXO3A can promote erythroid
differentiation through BTG1 (B-cell translocation gene 1). BTG1, in turn, modulates
arginine methylation necessary for erythroid differentiation (Bakker et al., 2004).
Furthermore, recent experiments demonstrated that FOXO3A directly binds and
represses the transcription of the ID1 (inhibitor of differentiation 1), a suppressor of
erythroid differentiation, through the recruitment of an HDAC1 (histone deacetylase 1)–
mSin3a complex (Lam et al., 2006) .
1.3.6. FOXO and Glucose and Energy Metabolism
FOXO factors play an important role in upregulating genes that control glucose
metabolism. They upregulate G6Pase (glucose-6-phosphatase) which is responsible for
converting glucose-6-phosphate to glucose (Nakae et al., 2001; Onuma et al., 2006) and
PEPCK (phosphoenolpyruvate carboxykinase) which converts oxaloacetate to
phosphoenolpyruvate (Samuel et al., 2006). In the absence of insulin, FOXO1 binds to
IRE (insulin response elements) in G6Pase and PEPCK promoters and stimulates their
promoter activity. However, in the presence of insulin, FOXO1 undergoes
phosphorylation and is exported out of the nucleus, ceasing its transcriptional activity
(Kitamura et al., 2002). In liver and muscle, FOXO proteins can stimulate PDK4 (
pyruvate dehydrogenase kinase-4) expression, which limits oxidative metabolism of
glucose and conserves glucose for utilization in other tissues (Furuyama et al., 2003).
FOXOs are also involved in lipid metabolism through the regulation of AdipoR1/2
(Tsuchida et al., 2004), LPL (lipoprotein lipase) (Kamei et al., 2003), HMGCS2
(mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase) (Nadal et al., 2002) and SCP
(sterol carrier protein) (Dansen et al., 2004) gene expression. Adiponectin/Acrp30 is a
hormone secreted by adipocytes, which acts as an antidiabetic and antiatherogenic
adipokine. AdipoR1 and -R2 serve as the receptors for adiponectin and mediate
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increased fatty acid oxidation and glucose uptake by adiponectin. Mouse study shows
that insulin reduces the expression of AdipoR1/R2 via the PI3K/FOXO1-dependent
pathway in vitro (Tothova et al., 2007). FOXO proteins also stimulate the expression of
both PDK4 (Furuyama et al., 2003; Kwon et al., 2004), which limits the flux of pyruvate
through the Krebs cycle, and the expression of LPL (Kamei et al., 2003). HMGCS2 is a
key enzyme controling ketogenesis. Deletion analysis showed that there is a FOXO3A-
responsive sequence AAAAATA located 211 bp upstream of the HMGCS2 gene
transcription start site. FOXO3A can stimulate transcription of HMGCS2 and this
stimulation is repressed by insulin (Nadal et al., 2002). The SCP (sterol carrier protein)
gene encodes two proteins, SCPx and SCP2, which are independently regulated by
separate promoters. SCPx has been shown to be the thiolase involved in the breakdown
of branched-chain fatty acids and in the biosynthesis of bile acids. Both SCPx and SCP2
are upregulated by FOXO3A (Dansen et al., 2004).
1.3.7. FOXO and Longevity
A number of diseases, including cancer, type 2 diabetes and neurodegenerative
disorders have a striking age-dependent onset. A number of studies now support the
hypothesis that longevity could be affected by simple changes in the environment.
Organismal longevity also has a genetic component. A pivotal breakthrough in
identifying genes involved in longevity came from studies in C. elegans. Some worm
mutants can live two to three times longer than wild type worms (Kenyon et al., 1993).
These long-living worms turned out to have mutations in the insulin receptor gene
(Kimura et al., 1997). Remarkably, flies and mice that have mutations in the insulin or
the IGF-1 (insulin like growth factor-1) receptor genes are also long-lived (Bluher et al.,
2003; Holzenberger et al., 2003; Tatar et al., 2001). These results indicate that insulin
and IGF-1 regulate longevity in a conserved manner throughout species.
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A link between FOXO factors and aging was also initially observed in
invertebrates. In C. elegans, DAF-16 regulates lifespan (Baumeister et al., 2006). In
Drosophila melanogaster, dFOXO regulates age-linked decline of cardiac function and
longevity (Giannakou et al., 2004; Giannakou and Partridge, 2004; Hwangbo et al.,
2004; Wessells et al., 2004).
But so far, there is no direct evidence to link FOXO factors and longevity in
mammals. Since FOXO factors are involved in cellular resistance to stress and promote
ROS detoxification, DNA repair, and cell cycle arrest to allow time for the repair process,
they are projected to reduce detrimental effects of aging in mammals (Glauser and
Schlegel, 2007). Thus, a role of FOXO factors in longevity of mammals is expected.
1.4. Posttranslational Modifications of FOXO Proteins
As described above, FOXO proteins have diverse cellular functions by acting as
transcription factors. The activity of FOXO proteins can be regulated by posttranslational
modifications including phosphorylation, acetylation and ubiquitination.
1.4.1. Phosporylation of FOXO
Phosphorylation is a process wherein phosphate groups are added to proteins by
protein kinases or removed from proteins by protein phosphatases. The addition or
removal of phosphate groups dramatically alters protein function and leads to a myriad
of biological responses. Phosphorylation is shown to play a major role in the regulation
of FOXO cellular localization, transcription activity and protein stability, and is mediated
mainly by PKB.
1.4.1.1. PKB and FOXO
PKB is a serine/threonine kinase. It plays a pivotal role in cell survival and
proliferation through a number of downstream effectors. All FOXO family members,
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except FOXO6, contain three PKB phosphorylation sites (RXRXX(S/T)X). DAF-16, the
C. elegans FOXO, contains four putative PKB recognition motifs. Two are located at end
of the N-terminal & C-terminal of DAF-16. The other two sites are located in the forkhead
domain and overlap. Disrupting PKB-consensus phosphorylation sites in DAF-16 causes
nuclear accumulation in wild-type animals but has little effect on lifespan (Lin et al.,
2001; Van Der Heide et al., 2004).
PKB is shown to phosphorylate human FOXO family members on the three PKB
motifs: an N-terminal threonine (Thr32), a forkhead box serine (Ser253), and a more C-
terminal serine (Ser315). Thr32 is preferentially phosphorylated by PKB. This
phosphorylation is shown to inhibit p300 binding to FOXO3A, resulting in the recruitment
of RanGTPase and CRM1 protein to transport FOXO3A to cytoplasm and to bind the 14-
3-3 chaperone (Mahmud et al., 2002). Mutation of Ser253 to a neutral residue, such as
alanine (S253A), inhibits further phosphorylation of FOXO3A in other regions of the
proteins by PKB and other kinases. This suggests that this site is necessary to prime
other sites for phosphorylation. The FOXO3A S253A mutant is constitutively nuclear
under different conditions including PKB activation. Therefore, the S253 is called the
“gatekeeper site”. This site is believed to inhibit DNA binding, to change the
conformation and to inhibit nuclear re-import (Brownawell et al., 2001; Brunet et al.,
1999; Brunet et al., 2001; Woods and Rena, 2002). Phosphorylation of Ser315 is shown
to enhance nuclear export (Brunet et al., 2001; Rena et al., 2002). All FOXO proteins
have been shown to require the consensus N-terminal PKB site and the PKB site
located in the forkhead domain in order to translocate from nucleus to cytosol
(Brownawell et al., 2001).
Interestingly, the regulation of FOXO6 is different from those of FOXO1,
FOXO3A and FOXO4. FOXO6 only contains two PKB/SGK regulatory phosphorylation
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sites (Thr26 and Ser184 in mouse FOXO6) (Jacobs et al., 2003). Unlike the other FOXO
isoforms, FOXO6 is mostly nuclear. However, FOXO6 phosphorylation at Thr26 and
Ser184 appears to decrease its transcriptional activity (van der Heide and Smidt, 2005),
which is similar to the effect of PKB on other FOXO factors transcriptional activity.
1.4.1.2. SGK and FOXO
SGKs (serum- and glucocorticoid-induced kinases) belong to a new family of
serine/threonine kinases that are regulated at both the transcriptional and
posttranslational levels by external stimulation. SGK mediates the biological effects of
PI3K in parallel with PKB. Although SGK is closely related to PKB, SGK and PKB
display unique features. First, SGK protein expression is induced by extracellular stimuli,
but PKB might not be. Second, SGK does not have a pleckstrin homology domain and
appears not to be recruited to the plasma membrane prior to its activation. Third, the
consensus sequence that is phosphorylated by SGK is not identical to the site
phosphorylated by PKB. Fourth, SGK, in contrast to PKB, is capable of phosphorylating
peptide substrates that do not have a bulky hydrophobic amino acid immediately C-
terminal to the phosphate-acceptor site (Kobayashi and Cohen, 1999).
Activated SGK promotes cell survival in part by phosphorylating and inactivating
FOXO3A. In the presence of DNA damage, caused by UV and irradiation treatment,
SGK is induced in a p53-dependent manner, leading to the phosphorylation of FOXO3A
(You et al., 2004a). SGK, like PKB, phosphorylates FOXO3A, thereby causing FOXO3A
translocation from the nucleus to the cytoplasm and inhibition of FOXO3A-dependent
transcription. However, SGK and PKB, when expressed at physiological levels, display
differences with respect to the efficacy with which they phosphorylate the regulatory
sites on FOXO3A. Specifically, Thr32 is phosphorylated by both protein kinases, but
SGK prefers Ser315 whereas PKB favors Ser253 (Brunet et al., 2001).
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1.4.1.3. IκB Kinase and FOXO3A
The IKK (IκB kinase) signaling pathway is a key survival and antiapoptotic
mechanism, aberrant expression of IKK has been implicated in constitutive activation of
NF-κB in human breast cancer cell lines and primary tumors. In breast cancer, IKK
physically interacts with, phosphorylates, and inhibits FOXO3A independently of PKB.
Cytoplasmic FOXO3A correlates with expression of IKKβ or phospho-PKB in many
tumors and associates with poor survival in breast cancer (Hu et al., 2004).
1.4.1.4. CK1 and FOXO
CK1 (Casein Kinase 1) is a serine/threonine protein kinase (Hathaway and
Traugh, 1979) that phosphorylates FOXO1 at Ser322 and Ser325 following the
phosphorylation of Ser319 by PKB or SGK. Multi-site phosphorylation of the region
containing Ser319, Ser322, Ser325 and Ser329 provides a signal for the nuclear
exclusion of FOXO1 (Rena et al., 2002).
1.4.1.5. CDK2 and FOXO
CDK2 phosphorylates FOXO1 at serine-249 (Ser249) in vitro and in vivo.
Phosphorylation of FOXO1 at Ser249 results in cytoplasmic localization of FOXO1 and
the inhibition of its transcriptional activity. This phosphorylation was abrogated upon DNA
damage through the cell cycle checkpoint pathway and was dependent on the protein
kinases Chk1 and Chk2 (Huang et al., 2006). Functional interaction between CDK2 and
FOXO1 provides a mechanism that regulates apoptotic cell death after double strand
DNA breakage.
1.4.1.6. JNK and FOXO
Besides IIS (insulin/IGF signaling) pathway, lifespan can also be increased by
activating the stress-responsive JNK (Jun-N-terminal kinase) pathway. In Drosophila,
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JNK requires FOXO to extend lifespan. JNK antagonizes IIS, causes nuclear localization
of FOXO and induces the expression of FOXO targets including the growth control and
stress defense genes. JNK and FOXO also restrict IIS activity systemically by repressing
IIS ligand expression in neuroendocrine cells (Wang et al., 2005).
In humans, an increase in ROS levels induces activation of the small GTPase
Ral, which in turn leads to the phosphorylation and activation of the stress kinase JNK.
Active JNK induces the phosphorylation of Thr447 and Thr451 on FOXO4.
Phosphorylation of these residues is essential for FOXO4 transcriptional activity.
Consistent with this, H2O2 treatment increases FOXO transcriptional activity and
translocation of FOXO4 from the cytoplasm to the nucleus and activation of the
transcription factor. Activation of FOXO4 through Thr447/451 phosphorylation can
induce transcription of MnSOD and catalase, leading to a decrease in ROS levels. Thus,
activation of FOXO4 by oxidative stress is part of a negative feedback loop to reduce the
levels of oxidative stress in a cell and to prevent damage to DNA, lipids and proteins
(Essers et al., 2004).
1.4.1.7. MST1 and FOXO3A
MST1 is a serine/threonine protein kinase that mediates cell death induced by
oxidative stress in primary mammalian neurons through direct activation of FOXO
transcription factors. MST1 phosphorylates FOXO proteins at a conserved site within the
FOX domain (Ser207) disrupting their interaction with 14-3-3 proteins, promoting FOXO
nuclear translocation, and thereby inducing cell death in neurons under stress conditions
such as hydrogen peroxide treatment. Knockdown of the C. elegans MST1 ortholog
CST-1 shortens lifespan and accelerates tissue aging, while overexpression of cst-1
promotes lifespan and delays aging. The cst-1-induced lifespan extension requires daf-
16 (Lehtinen et al., 2006).
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1.4.2. Acetylation and Deacetylation of FOXO
1.4.2.1. Acetylation and Deacetylation
Protein acetylation is a post-translational modification that transfers an acetyl
group to lysine residues. The best example is histone acetylation, which “opens”
chromatin’s structure and activates transcription. Deacetylation, on the other hand,
induces closed chromatin structure and repression of gene expression (Cohen and Yao,
2004). Protein acetylation is catalyzed by a group of acetyltranferases with different
specificities and target consensus sites. CBP/p300 (calcium response element-binding
(CREB)-binding protein), PCAF (p300/CBP-associated factor), members of p160 family,
Tip60, and TAFII250 are the main acetyltransferases involved in histone regulation and
transcription factor acetylation (http://www.chromatin.us/hatdesc.html). Proteins
acetylation is reversed by HDACs (histone deacetylases). There are three classes of
HDACs. Class I includes HDAC1, 2, 3, and 8. Class II includes HDAC4, 5, 6, 7, 9 and 10
which show high homology to HAD1 yeast deacetylase. There is one HDAC (HDAC11)
that shares homology with both class I and II HDACs. Class III HDACs (SIRTs) have an
absolute requirement for NAD in vivo and in vitro (de Ruijter et al., 2003; Imai et al.,
2000).
1.4.2.2. Acetylation of FOXO
1.4.2.2.1. P300 and CBP
p300 and CBP are large nuclear proteins encoded by two distinct genes.
p300/CBP have been implicated in numerous disease processes, including several
forms of cancer, cardiac hypertrophy and Huntington’s disease (Bandyopadhyay et al.,
2002; Borrow et al., 1996; Deguchi et al., 2003; Gayther et al., 2000; Gusterson et al.,
2003; Muraoka et al., 1996; Ross et al., 2002).
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The relative low abundance of p300 is rate-limiting in coactivation and
corepression of many transcription factors. Thus, p300 serves to integrate diverse
signaling pathways involved in metabolism, cellular differentiation and p53-mediated
apoptosis processes (Yao et al., 1998). Orchestration of these activities by p300
involves both a scaffolding function to tether transcription factors to target promoters and
its enzymatic activity as a HAT (histone acetyl transferase) (Bannister and Kouzarides,
1996; Ogryzko et al., 1996).
In addition to histones, several other non-histone proteins including transcription
factors are also acetylated by p300 (Goodman and Smolik, 2000), including p53,
estrogen receptor (Wang et al., 2001) and MyoD (Polesskaya et al., 2000). The activity
of p300 itself is also subjected to regulation via a number of post-translational
modification including phosphorylation, methylation, sumoylation and acetylation
(Banerjee et al., 1994; Chevillard-Briet et al., 2002; Girdwood et al., 2003; Thompson et
al., 2004; Yaciuk and Moran, 1991; Yadav et al., 2003).
In terms of differentiation, p300 and CBP appear to have numerous functions.
There is embryonic lethality of mice nullizygous for p300 (with defects in neurulation and
heart development) as well as in mice double heterozygous for p300 and CBP. Animals
which are nullizygous for p300 died between days 9 and 11.5 of gestation and showed
defects in neurulation, cell proliferation and heart development. Cells derived from p300-
deficient embryos displayed specific transcriptional defects and proliferated poorly.
Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality.
Moreover, double heterozygosity for p300 and CBP was invariably associated with
embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene
dosage of p300 and CBP (Yao et al., 1998). Further study in the mouse oocytes within
primordial follicles finds that p300 and CBP enter into the oocyte nucleus at different
- 25 -
stages of oocyte growth. From the two-cell stage to the blastocyst stage in the pre-
implantation mouse embryos, the localizations of p300 and CBP are different, which
provide the information that p300 and CBP have different functions in early mouse
embryogenesis (Kwok et al., 2006).
1.4.2.2.2. P300/CBP and FOXO Acetylation
Using immunoprecipitation, an interaction between FOXO1, FOXO4, and
FOXO3A and CBP as well as between FOXO3A and p300 was found in vivo and in vitro
(Brunet et al., 2004; Daitoku et al., 2004; Fukuoka et al., 2003; Motta et al., 2004; van
der Horst et al., 2004).
CBP and p300 play different roles in the regulation of FOXO1 and FOXO4.
Mutational analysis show that FOXO1 and FOXO4 interact with CBP via their C-terminal
domain, whereas the N-terminal CH1 domain of CBP is required to bind FOXO1 and
FOXO4. CBP binds and acetylates FOXO1 at the K (lysine) 242, K245, and K262
residues. Acetylation at these residues by CBP attenuates FOXO1’s ability to bind
cognate DNA sequence and promotes the PKB-dependent phosphorylation of FOXO1
(Matsuzaki et al., 2005). CBP interacts with FOXO4 and acetylates it at K186, K189, and
K408. Acetylation by CBP inhibited the transcriptional activity of FOXO4 (Daitoku et al.,
2004; Fukuoka et al., 2003; van der Horst et al., 2004). However, in transient
transfection experiments acetylation by p300 in the C-terminal region of FOXO1 showed
that it potently stimulates FOXO1-induced transcription of IGF-binding protein-1. The
intrinsic acetyltransferase activity of p300 is required for both activities (Perrot and
Rechler, 2005).
As for FOXO3A, p300 and CBP can bind and acetylate it both in vivo and in vitro,
but two different studies show some discrepancies. One study shows that FOXO3A
binds p300/CBP at the N-terminus masking the first 52 amino acids at the N-terminus
- 26 -
under serum starvation conditions (Mahmud et al., 2002). Another study shows that
CBP/p300 binds the C-terminus and forkhead domains of FOXO3A, leading to
acetylation at lysine residues K242, K259, K271, K290, and K569 (Brunet et al., 2004;
Mahmud et al., 2002). Overall, acetylation by p300 stimulates the transcriptional activity
of FOXO3A (Motta et al., 2004).
1.4.2.3. Deacetylation of FOXO by HDAC
1.4.2.3.1. SIRT1
The SIRT1 gene is located in chromosome 10q21.3. It contains 9 exons
(NC 000010). The 4,107 bp human Sirt1 mRNA has an open reading frame of 2,244 bp
and encodes a 747 aa protein with a predictive molecular weight of 81.7 kDa (kilodalton)
(Voelter-Mahlknecht and Mahlknecht, 2006). It belongs to the family of mammalian
sirtuins. The founding member of the sirtuin protein family was the silent information
regulator 2 (Sir2) of S. cervisiae (Saccharomyces cervisiae). Sirtuin proteins are highly
conserved from yeast to humans. In yeast, worms and flies, expression of Sir2 extends
longevity (Kaeberlein et al., 1999; Rogina and Helfand, 2004; Tissenbaum and
Guarente, 2001; Wood et al., 2004). In S. cervisiae, there are five sirtuin homologs,
Hst1-4 and Sir2. In mammals, there are seven sirtuins, SIRT1-7. SIRT1 is the closest
homolog of Sir2, plays important roles in diverse cellular processes including
transcriptional silencing, rDNA recombination, glucose metabolism and energy
homeostasis, DNA repair and cell survival. Due to its dependency on NAD+, the activity
of SIRT1 is regulated by NAD+/NADH ratio and thus sensitive to the redox status and
celluar metabolism. Similar to Sir2, SIRT1 is potentially a nutrient sensor that regulates
the lifespan of mammals in response to caloric restrication or nutrient starvation.
- 27 -
Several compounds have been shown to inhibit or activate the deacetylase
activity of the Sir2 family. One inhibitor, splitomicin, is a cell-permeable lactone derived
from β-naphthol; it inhibits the NAD+-dependent deacetylase activity of Sir2 in vitro
(Bedalov et al., 2001). Another inhibitor, nicotinamide, a product of the Sir2 deacetylation
reaction, is an inhibitor of Sir2 activity both in vivo and in vitro. Nicotinamide has been
shown to inhibit a Sir2 homolog, SIRT1, a negative p53 regulator, promoting p53-
dependent apoptosis in mammalian cells (Luo et al., 2001; Vaziri et al., 2001). The most
potent activator was resveratrol, a polyphenol found in red wine, which is implicated in a
number of health benefits. In human cells, treatment with a low concentration of
resveratrol increased cell survival following DNA damage. Moreover, low resveratrol
concentrations decreased the acetylation of p53 at lysine residue 382, a known SIRT1
substrate; however, high resveratrol concentrations caused the opposite (Howitz et al.,
2003).
By deacetylating and inactivating the substrates such as p53 (Langley et al.,
2002; Vaziri et al., 2001), NFκB (nuclear factor-κB), FOXO (Brunet et al., 2004; Daitoku
et al., 2004; Motta et al., 2004; van der Horst et al., 2004), Ku70 (Jeong et al., 2007),
PPARγ (Peroxisome proliferator-activated receptor γ) (Picard et al., 2004), PGC-1α
(Balaban et al., 2005), AceCS2 (acetyl-CoA synthetase 2), p300 (Bouras et al., 2005), α-
tubulin, and HES1 and HEY2 (basic helix–loop–helix transcriptional repressors) (Takata
and Ishikawa, 2003), SIRT1 plays important roles in regulating various cellular
processes, including stress response (Vaziri et al., 2001), embryogenesis (McBurney et
al., 2003), metabolism (Leibiger and Berggren, 2006), calorie restriction (Brunet et al.,
2004), neuronal cell survival (Anekonda and Reddy, 2006; Bordone et al., 2006), insulin
secretion (Bordone et al., 2006) and aging (Chua et al., 2005).
- 28 -
1.4.2.3.2. SIRT1 and FOXO Deacetylation
In mammalian cells, SIRT1 interacts with all the FOXO factors. Co-
immuoprecipitation experiments showed that SIRT1 interacts with FOXO1 in vivo.
Mutation analysis showed that the LxxLL motif in FOXO1 (amino acids 459-463) is
critical for the interaction with SIRT1. Mutagenesis of the LxxLL motif eliminates FOXO1
interaction with SIRT1, sustains the acetylated state of FOXO1 and makes FOXO1
insensitive to nicotinamide and resveratrol (Nakae et al., 2006). Yeast two hybridization
experiment showed that FHL2 (Four and a half LIM 2), which interacts with the N-
terminal of FOXO1 in vivo and in vitro, can enhance the interaction of FOXO1 and
SIRT1 (Yang et al., 2005).
Mammalian SIRT1 has dual effects on FOXO3A and FOXO4. SIRT1 deacetylates
FOXO3A, attenuating FOXO-induced apoptosis but potentiates FOXO-induced cell-cycle
arrest (Motta et al., 2004). SIRT1 and FOXO3A form a complex in cells in response to
oxidative stress, and SIRT1 deacetylates FOXO3A in vitro and in intact cells. The sites of
FOXO3A that appear to be primarily deacetylated by SIRT1 are K242, K245, and K262
(Daitoku et al., 2004). The SIRT1 and FOXO3A interaction requires PKB
phosphorylation of FOXO3A because constitutive FOXO3A, in which three PKB
phosphorylation sites were mutated to alanine, failed to bind SIRT1 under any stress
condition (Brunet et al., 2004; Motta et al., 2004). SIRT1 has a dual effect on FOXO3A
function: It increases FOXO3A's ability to induce cell cycle arrest and resistance to
oxidative stress, but inhibits FOXO3A's ability to induce cell death (Brunet et al., 2004).
In the case of FOXO4, acetylation by CBP inhibits its transcriptional activity.
SIRT1- mediated deacetylation precludes FOXO4 inhibition through acetylation
andthereby prolongs FOXO4-dependent transcription of stress-regulating genes. A
FOXO4 study revealed a molecular mechanism whereby SIRT1 can promote cellular
- 29 -
T24 S249 S256 S319 S322 S325 S329 T32 S253 S315 S318 S321 S325 S644 T28 S193 S258 S261 S264 S268 T447 T451T26 S184
FOXO1P
PKB/SGK CK1 DYRK JNK IKKβ
PP P P P P PP
FOXO1FOXO3FOXO4FOXO6
Figure 2 The regulation of FOXO. Full-length FOXO and known
phosphorylation sites and acetylation sites are presented. K-lysine; T-threonine;
S-serine; T-tyrosine; CDK2-cyclin-dependent kinase 2; PKB- protein Kinase B;
SGK- serum and glucocorticoid responsive kinase; CK1- casine kinase 1;DYRK-
dual-specificity YAK-1-related kinase; JNK- c-Jun N-terminal kinase; IKKβ- IκB
kinase β. The numbers above the drawings denote amino acid numbers. Full-
length Mdm2 and known motifs are also shown.
1 150 368 377 461 466 596 655
CDK2P
FOX
K245 K248 K259 K265 K274 K294K242 K245 K259 K262 K271 K290 K569 K182 K185 K199 K211 K233 K403 K173 K176 K190 K202 K229
FOXO1FOXO3FOXO4FOXO6
A A A A A A
P300/CBP P300/CBP1 150 208 655
SIRT1461-466
FOXG1150 260
SHP401 652
Crm1368-377
14-3-324 256
AR350 655∗ ∗
P53
SMAD3/4150 260
FHL21
- 30 -
survival and increase lifespan (van der Horst et al., 2004), a similar Sir2 function as
observed in worms.
1.4.3. Ubiquitination and Deubiquitination of FOXO
1.4.3.1. Ubiquitination and Degradation of FOXO
The degradation of FOXO transcription factors is mediated by the ubiqutin-
proteasome pathway. In the presence of insulin and other growth factors, FOXO proteins
are relocalized from the nucleus to the cytoplasm and to be degraded via the ubiquitin–
proteasome pathway. PKB activation is necessary for insulin promoted ubiquitin-
mediated degradation of FOXO1 and FOXO3A via the proteasome pathway (Aoki et al.,
2004; Plas and Thompson, 2003).
As for insulin induced proteasomal degradation of FOXO1, insulin treatment
decreases endogenous FOXO1 proteins in HepG2 cells and this decrease is suppressed
by proteasome inhibitors. FOXO1 is ubiquitinated in vivo and in vitro; insulin enhances its
ubiquitination in cells. In addition, the insulin signal to FOXO1 degradation is mediated
by the PI3K pathway. FOXO1 mutates at the serine or threonine residues that are
phosphorylated by PKB inhibit FOXO1 ubiquitination in vivo and in vitro. These data
suggest that efficient ubiquitination of FOXO1 requires both phosphorylation and
cytoplasmic retention in the cells (Matsuzaki et al., 2003).
Skp2 is an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex. It
inhibits FOXO1 during tumor suppression through ubiquitin-mediated degradation but it
only ubiquitinates and degrades FOXO1, not FOXO3A or FOXO4. The effect of Skp2 on
FOXO1 requires PKB-specific phosphorylation of FOXO1 at Ser-256. Decrease
ubiquitination is seen in the mutant of FOXO1 in which all three PKB sites are replaced
by alanine and FOXO1 is forced to remain in the cytoplasm, through a mutation in the
- 31 -
NLS. In addition, when phosphorylated FOXO1 is confined to the nucleus by a mutation
in the NES, FOXO1 ubiquitination is decreased (Huang et al., 2005). This study also
confirmed that only cytoplasmic FOXO1 is successfully ubiquitinated by an E3 ubiquitin
ligase and subsequently degraded.
IKKβ (I kappa B kinase β) also causes the proteasome-dependent degradation of
FOXO factors. IKKβ induces the phosphorylation of FOXO3A at Ser644, in the extreme
C-terminal portion of the molecule. This phosphorylation results in the ubiquitination and
subsequent degradation of FOXO3A. Since IKKβ-induced tumorigenesis can be
suppressed by overexpression of FOXO3A (Hu et al., 2004), the regulation of FOXO
protein degradation by IKKβ may play an important role in tumorigenesis. However,
Ser644 is not conserved in other FOXO isoforms and is not present in worms and flies. It
remains to be determined whether IKKβ phosphorylates and controls the other FOXO
isoforms. It is possible that the degradation of FOXO isoforms is regulated by different
protein kinases through other independent mechanisms.
Recently, it was showed that in the presence of oxidative stress FOXO4
becomes monoubiquitinated, resulting in its re-localization to the nucleus and increased
transcriptional activity (van der Horst et al., 2006). This study demonstrated that besides
FOXO degradation, the ubiqutination of FOXO may also have other biological functions.
1.4.3.2. Deubiquitination of FOXO
USP7/HAUSP is a herpes virus-associated ubiquitin-specific protease.
Deubiquitination of FOXO4 requires the enzyme USP7/HAUSP, which interacts with and
deubiquitinates FOXO4 in response to oxidative stress. Oxidative stress-induced
ubiquitination and deubiquitination by USP7 do not influence the half-life of FOXO4
- 32 -
protein. Moreover, USP7 does negatively affect FOXO transcriptional activity towards
endogenous promoters (van der Horst et al., 2006).
2. Ubiquitin, Proteasome and MDM2 as an E3 Ligase
Ubiquitination is a reversible post-translational modification of cellular proteins, in
which ubiquitin is attached to the target proteins. Aberrations in the ubiquitination system
are implicated in the pathogenesis of many diseases, including certain malignancies,
neurodegenerative disorders and pathologies of the inflammatory immune response.
Deubiqutination is a biological process in which one or more ubiqutin moieties are
removed from protein. Deubiquitination helps to maintain the pool of free ubiquitin.
2.1. Ubiquitin-Proteasome System
2.1.1. Ubiquitin and Ubiqutination
Ubiquitin (Ub) is a small protein composed of 76 amino acids. Ub is found only in
eukaryotic organisms and not in either eubacteria or archaebacteria. Among eukaryotes,
ubiquitin is highly conserved. Ub is a heat-stable protein folded into a compact globular
structure. Ub is found throughout the cell and can exist either in free form or as part of a
complex with other proteins. In the latter case, Ub is conjugated to proteins through a
covalent bond between glycine at the C-terminal end of Ub and the side chains of lysine
on the target proteins. A single Ub can be conjugated to the lysine of these proteins, or
more commonly, Ub-chains can be attached. Uiquitination depends on ATP.
Ub is encoded by a family of genes whose translation products are fusion
proteins. The Ub genes typically exist in two states: 1) The ubiquitin gene can be fused
to a ribosomal gene giving rise to a translation product that is an Ub-ribosomal fusion
protein; or 2) Ub genes can exist as a linear repeat, meaning that the translation product
consists of a linear chain of Ub-molecules fused together (a polyubiquitin molecule).
- 33 -
After the fusion proteins are synthesized, another protein called Ub-C-term hydrolase
cleaves the fusion proteins at the C-terminal end of Ub. This either liberates an individual
Ub and ribosomal protein or liberates a set of Ub monomers from the polyubiquitin.
Ubiquitination is a process in which ubiquitin is conjugated to the protein
substrates. The attachment of a single ubiquitin polypeptide, monoubiquitin, to a
substrate serves as an important regulatory modification (Hicke, 2001). Monoubiquitin
acts as a sorting signal throughout the endocytic pathway and regulates diverse proteins
including histones, endocytic machinery and transcription factors. Monoubiquitination
targets proteins to the lysosome, either by directing endocytosis of cell-surface receptors
or by sorting newly synthesized hydrolases from the Golgi to their resident lysosomal
compartment (Lee and Tyers, 2001). A polyubiquitin chain is formed when ubiquitin is
attached to a lysine within ubiquitin itself and this process is repeated. Polyubiquitin
chains linked through different lysine residues are involved in distinct cellular functions.
For instance, the signal necessary for degradation of substrates by the proteasome is a
polyubiquitin chain attached through Lys-48, whereas chains linked through Lys-63 are
crucial to the role of ubiquitin in DNA damage repair (Pickart, 2001). Polyubiquitination
typically targets proteins for rapid proteasomal degradation. Polyubiquitination is also
critical in protein quality control, where it helps to eliminate improperly processed or mis-
folded proteins from the ER (endoplasmic reticulum), in a process called ER-associated
degradation (Lee and Tyers, 2001).
2.1.2. Deubiquitination
Both poly- and monoubiquitination can be reversed by DUBs (deubiquitinating
enzymes) that specifically cleave the isopeptide bond at the C terminus of ubiquitin.
DUBs also generate the pool of free ubiquitin both by liberating ubiquitin from precursor
- 34 -
ubiquitin fusion proteins and by recycling ubiquitin from the branched polyubiquitin
chains of degraded proteins (D'Andrea and Pellman, 1998; Wilkinson, 1997).
The DUBs are comprised of two groups of enzymes, the UCHs (ubiquitin C-
terminal hydrolyases) and the USPs (ubiquitin-specific proteases; referred to as UBPs in
yeast). The UCHs are small (20-30 kDa), closely related proteases that are generally
involved in cleaving ubiquitin from small processed peptides. The USPs are more
numerous, much larger (60–300 kDa), and are thought to have specific protein targets.
USPs can be identified by conserved sequences within the active site, but sequences
outside of the catalytic domain are highly divergent, likely reflecting their role in mediating
interactions with different protein targets (Hu et al., 2002; Wilkinson, 1997; Wilkinson et
al., 1995).
There are 16 known UBPs in Saccharomyces cerevisiae, and 63 putative USP
genes in humans. Single and even multiple UBP deletions in yeast generally produce
minimal phenotypic abnormalities, suggestive of functional redundancies among the
yeast UBP family (Amerik et al., 2000). However, studies have shown that USPs can
play specific roles in various biological processes in higher eukaryotes, suggesting a
more specialized role as cellular regulators in multicellular organisms. Specific DUBs
have been shown to regulate eye development (Huang et al., 1995; Huang and Fischer-
Vize, 1996), cell growth in response to cytokines (Zhu et al., 1996), oncogenic
transformation (Gray et al., 1995; Jensen et al., 1998; Papa and Hochstrasser, 1993),
cell cycle regulation (Hu et al., 2002), chromatin structure (Robzyk et al., 2000), and
transcriptional regulation (Holowaty et al., 2003; Mimnaugh et al., 1997; Moazed and
Johnson, 1996).
- 35 -
2.1.3. Ubiquitination Machinery and Proteasome Pathway
Ubiquitination of a protein substrate requires the concerted action of three
classes of enzymes designated E1, E2, and E3. E1 (ubiquitin activating enzyme) initially
activates ubiquitin in an ATP-dependent reaction through the formation of a thiol-ester
bond between the carboxyl terminus of ubiquitin and the thiol group of a specific cysteine
residue of E1. Ubiquitin is then transferred to a specific cysteine residue on one of
several E2 (ubiquitin-conjugating enzymes, Ubcs). E2 enzymes in turn transfer the
ubiquitin either directly to a substrate or to the final class of enzymes known as E3
(ubiquitin protein ligases). E3 enzymes catalyze the formation of an isopeptide bond
between the carboxyl terminus of ubiquitin and the amino group of lysine residues on the
target protein. A substrate may then undergo multiply ubiquitinations through the
attachment of additional ubiquitin molecules to specific lysine residues of ubiquitin itself.
In many cases the E1, E2, and E3 enzymes form large, multi-protein complexes. This
increases the efficiency of the process by allowing the rapid thiol-ester transfer of
ubiquitin molecules between proteins.
This type of protein degradation plays a role in many cellular processes, such as
cell cycle regulation, antigen presentation, and the disposal of denatured, unfolded, or
oxidized proteins. Most intracellular protein degradation is through the ubiquitin-
proteasome pathway (Ciechanover and Schwartz, 2002; Hershko and Ciechanover,
1998).
In the ubiquitin-proteasome degradation pathway, the covalent attachment of
multiple ubiquitin molecules to lysine residues of a target protein serves to signal its
recognition and rapid degradation by the 26S proteasome. The proteasome is a large,
multisubunit complex that exists in cells in two forms: a 20S and a 26S species. The
- 36 -
Figure 3 Ubiquitin-proteasome degradation pathway. E1 (ubiquitin
activating enzyme) activates Ub in the presence of ATP. Activated Ub is then
transferred to E2 (ubiquitin conjugating enzyme). E2 in turn transfers Ub to E3
(ubiquitin protein ligase). E3 binds ubiquitin to the substrate protein.
Ubquitinated proteins are degraded by the proteasome. Ub – ubiquitin.
- 37 -
active protease sites sequestered in its central cavity, so that only proteins entering this
chamber are degraded. The openings to this cavity permit only denatured proteins to
enter, where they are progressively cleaved to small peptides. The addition of a 19S
regulator to either or both ends of the 20S proteasome creates the 26S proteasome. The
19S regulator contains ATPases and other proteins and serves numerous functions,
including the recognition of the substrate and its translocation to the catalytic core
(Voges et al., 1999).
2.2. MDM2 as an Ubiquitin E3 Ligase
2.2.1. General Information about MDM2
MDM2 is the product of the 'murine double minute 2' gene. The MDM2 gene was
originally identified as one of three genes (mdm1, 2, 3) which were overexpressed by
more than 50-fold through amplification in a spontaneously transformed mouse BALB/c
cell line (3T3-DM). The mdm2 genes are located on small, acentromeric
extrachromosomal nuclear bodies, called double minutes, which are retained in cells
only if they provide a growth advantage. The gene product of mdm2 was later shown to
be responsible for cell transformation when MDM2 was overexpressed (Cahilly-Snyder
et al., 1987; Fakharzadeh et al., 1991). In 1992, Oliner cloned the human MDM2 gene
and mapped it to chromosome 12q13-14 (Oliner et al., 1992). Both the mdm2 gene and
its human counterpart, MDM2, consist of 12 exons that can generate many different
MDM2 proteins, as shown in Figure 4. There are two different promoters, the second of
which is responsive to p53. These promoters generate two proteins, the full-length p90
and a shorter p76 protein that initiates at an internal ATG (Olson et al., 1993; Perry et
al., 1993; Saucedo et al., 1999). p76 is missing part of the p53-binding domain and it can
act as a dominant negative inhibitor of p90 and activate p53. Alternative splicing of
- 38 -
MDM2 to generate shorter proteins also occurs in many human and mouse tumors. More
than 40 different alternatively spliced transcript variants have been isolated from both
tumor and normal tissues. In humans, MDM2-a and MDM2-b are the major splice
variants that delete exons 4–9 and 4–11, respectively. Neither product contains the p53-
binding motif. MDM2-b, also named MDM2-ALT1, interacts with full-length MDM2 and
sequesters it in the cytoplasm (Bartel et al., 2002).
2.2.2. Functions of MDM2
2.2.2.1. MDM2 and Cell Cycle
Overexpression of MDM2 has been shown to correlate with the cyclin-dependent
kinase inhibitor p21. In breast cancer cells, overexpression of MDM2 correlates with lack
of p21 expression (Jiang et al., 1997). On the other hand, in squamous cell carcinoma,
overexpression of MDM2 is associated with high levels of p21 (Ng et al., 1999).
MDM2 reverses the growth inhibition at G1 imposed by p53 and RB (Chen et al.,
1996; Xiao et al., 1995). Overproduction of MDM2 can overcome the TGF-β-imposed
growth inhibition via the RB–E2F pathway (Sun et al., 1998).
Transgenic mice experiments showed that expression of a BLG (β-
lactoglobulin)/mdm2 transgene (BLGmdm2) in the epithelial cells of the mouse
mammary gland caused mammary epithelial cells to undergo multiple rounds of S phase
without cell division, and resulted in polyploidy and tumor formation. The effect of MDM2
on S phase is independent of the p53 status (Lundgren et al., 1997).
2.2.2.2. MDM2 and Differentiation
One phenotype of tumor cells is the lack of terminal differentiation. MDM2 plays a
very important role in epidermal differentiation (Dazard et al., 1997). MDM2
overexpression in the granular layer perturbs the differentiation program (Alkhalaf et al.,
- 39 -
1999). In rhabdomyosarcoma, forced expression of MDM2 inhibits MyoD function and
consequently inhibits muscle differentiation (Fiddler et al., 1996). A MDM2-conditional
mice experiment showed that MDM2 plays a very important role in bone organogenesis
and homeostasis through inhibition of p53 function, which is a prerequisite for master
osteoblast transcriptional regulator Runx2 activation, osteoblast differentiation, and
proper skeletal formation (Lengner et al., 2006). NUMb is a cell fate determinant protein.
MDM2 associates with NUMb and influences cell differentiation and survival through
translocation of NUMb to nucleus and degradation of NUMb (Juven-Gershon et al.,
1998).
However, microinjection of MDM2 mRNA in two-cell stage zebrafish embryos
caused inhibition of cellular convergence during gastrulation. Clones derived from MDM2
microinjected blastomeres were significantly smaller than those derived from control
microinjections. This indicates that MDM2 expression may be important during the
differentiation of neural and muscular tissues of zebrafish (Thisse et al., 2000).
2.2.2.3. MDM2 and Ribosome Biogenesis
Ribosome biogenesis is the process of making ribosomes. It takes place both in
the cell cytoplasm and in the nucleolus of eukaryotic cells. It involves the coordinated
action of over 200 proteins in the synthesis and processing of the four rRNAs, as well as
assembly of those rRNAs with the ribosomal proteins. Proper ribosome assembly is
essential for the health of a cell.
Ribosome proteins L5, L11 and L23 exist in the same complex with MDM2 in
response to ribosome stress, such as exposure to actinomycin D. They activate p53 by
inhibiting MDM2-mediated p53 suppression (Dai et al., 2004; Lohrum et al., 2003;
Marechal et al., 1994; Zhang et al., 2003).
- 40 -
2.2.2.4. MDM2 and Transcription
MDM2 affects the gene transcription by inhibiting p53-mediated transactivation
(Momand et al., 1992). MDM2 uses multiple mechanisms to inactivate p53 and to inhibit
its transcriptional activity. MDM2 targets p53 for ubiquitination and degradation by the
proteosome, shuttles p53 out of the nucleus, prevents its interaction with transcriptional
coactivators and recruits the known transcriptional corepressors, such as hCtBP2, to
p53.
2.2.2.5. MDM2 and Protein Ubiquitination and Degradation
E3 ubiquitin ligases are a large family of proteins engaged in the regulation of the
turnover and activity of many proteins. Together with ubiquitin-activating enzyme E1 and
ubiquitin-conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a
variety of biologically significant protein substrates leading to their degradation through
the 26S proteasome. Because they serve as the specific substrate-recognition element
of the system, E3 ligases play an important role in the ubiquitin-mediated proteolytic
cascade. There are approximately 1000 E3 ligases in the human genome.
MDM2 possesses the activity of an E3 ubiquitin ligase. MDM2 was initially found
to promote the proteasome–dependent degradation of p53 (Haupt et al., 1997; Honda et
al., 1997; Kubbutat et al., 1997). It functions as an E3 ubiquitin ligase for p53 and for
itself through its RING finger domain at the C-terminus (Fang et al., 2000; Honda et al.,
1997; Honda and Yasuda, 2000). It is now known that MDM2 also promotes the
degradation of several other proteins in intact cells, such as: Numb (Yogosawa et al.,
2003), RB (Miwa et al., 2006; Uchida et al., 2005) and MDMX (Pan and Chen, 2003).
- 41 -
2.2.3. Regulation of MDM2 E3 Activity
2.2.3.1. Sumoylation of MDM2
2.2.3.1.1. SUMO and Sumoylation
SUMOs (small ubiquitin-related modifiers) constitute a family of highly conserved
proteins found in all eukaryotes and are required for viability of most eukaryotic cells,
including budding yeast, nematodes, fruit flies, and vertebrate cells in culture
(Apionishev et al., 2001; Epps and Tanda, 1998; Fraser et al., 2000; Hayashi et al.,
2002; Jones et al., 2002). In multicellular organisms, SUMO conjugation takes place in
all tissues and at all developmental stages (Chen et al., 1998; Howe et al., 1998;
Joanisse et al., 1998; Kamitani et al., 1998; Kurepa et al., 2003; Lois et al., 2003;
Mannen et al., 1996; Shen et al., 1996). Since its discovery in 1996, SUMO has been
found covalently attached to more than 50 proteins, including the androgen receptor,
IκBα, c-Jun, HDACs and p53. Proteins that participate in transcription, DNA repair,
nuclear transport, signal transduction and the cell cycle have been found to be
sumoylated. In contrast to ubiquitination, however, sumoylation of a protein does not
appear to target it for rapid degradation, but rather affect the ability of the modified
protein to interact with cellular factors. Most SUMO-modified proteins that have been
characterized in mammalian systems are involved in transcription and they are often
repressed by SUMO conjugation. However, genetic studies in model organisms have
pointed to a role for SUMO in chromosome dynamics and higher order chromatin
structures, illustrating the diversity of SUMO function.
SUMO often has a positive effect on protein-protein interactions, and it promotes
assembly of several multi-protein complexes. However, the effects of SUMO on
interactions vary depending on the substrates. SUMO can also act by a completely
- 42 -
different mechanism, including the prevention of ubiquitination of a protein by blocking
lysine residue where Ub would normally be attached (Desterro et al., 1998; Hoege et al.,
2002; Lee et al., 2003; Lin et al., 2003a).
The linkage between SUMO and its substrates is an isopeptide bond between
the C-terminal carboxyl group of SUMO and the ε-amino group of a lysine residue in the
substrate. A three-step enzyme pathway attaches SUMO to specific substrates, and
other enzymes cleave SUMO off its targets. The enzymes of the SUMO pathway,
although analogous to those of the Ub pathway, are specific for SUMO and have no role
in conjugating Ub or any of the other ubiquitin-like modifiers. The SUMO pathway begins
with a SUMO-activating enzyme (also called E1). E1 catalyses an ATP-dependent
activation of the SUMO C-terminus and then transfers activated SUMO to a SUMO-
conjugating enzyme (E2), also known as Ubc9. SUMO is then transferred from Ubc9 to
the substrate with the assistance of one of several SUMO-protein ligases (E3s). In
contrast to the ubiquitin-conjugating system, where E3 ligase is responsible for target
recognition, the recognition of SUMO targets is mediated by both E2 and E3 enzymes.
Many of the Lys residues where SUMO becomes attached are in the short consensus
sequence ψΩKXE/D, where ψ denotes a bulky aliphatic residue, Ω denotes a large
hydrophobic amino acid, generally isoleucine, leucine, or valine; K is the lysine residue
being modified; X is any residue; and E/D is a glutamic or aspartic acid. This motif is
bound directly by Ubc9. E3 ligases probably enhance specificity by interacting with other
features of the substrate. Although most known SUMO targets contain this sequence,
other conjugation sites are now beginning to be known, such as TKET in S. cerevisiae
PCNA (Hoege et al., 2002) and VKYC in Smad4 (Lee et al., 2003; Lin et al., 2003b).
Sumoylation is a reversible modification, and removal of SUMO is carried out by
enzymes that specifically cleave at the C-terminus of SUMO (Johnson, 2004). All known
- 43 -
SUMO-cleaving enzymes belong to the family of ubiquitin like protease 1 (Ulp1) cysteine
proteases and contain a 200 amino acid C-terminal core domain (the Ulp domain). The
core domain has the SUMO cleaving activity and contains the catalytic triad Cys-His-Asn
(Mossessova and Lima, 2000). Mammals have seven members of the Ulp1 family:
SENP1-3 and SENP5-8. Only four of the SENP genes have been confirmed to encode
SUMO proteases, namely SENP1 (Bailey and O'Hare, 2002), SENP3 (SMT3IP1)
(Nishida et al., 2000), SENP6 (SUSP1) (Kim et al., 2000) and SENP2 (Axam,
SMT3IP2/Axam2, SuPr-1) (Best et al., 2002; Kadoya et al., 2002; Nishida et al., 2001).
2.2.3.1.2. Sumoylation and MDM2
SUMO-1 modification of MDM2 can differentially modulate the outcome of MDM2
E3 ligase activity in a manner that favors accmulation of p53. Upon Sumo-1 conjugation,
MDM2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide
ligase (E3) activity toward p53, thereby increasing its oncogenic potential. This switch in
modification status is stress-responsive, because UV irradiation leads to a decrease in
the interaction of MDM2 with Ubc9 and a corresponding loss of MDM2 sumoylation
(Buschmann et al., 2001). Further studies showed that Ubc9 can associate with MDM2
only if amino acids 40-59 within the N-terminus of MDM2 are present. Furthermore,
addition of a peptide corresponding to amino acids 40-59 of MDM2 efficiently inhibits
MDM2 sumoylation in vitro and in vivo (Buschmann et al., 2001)
2.2.3.2. Ubiqutination and Degradation of MDM2
As mentioned earlier, MDM2 mediates autoubiquitination as well as the
ubiquitination of other substrates. The balance between auto- and substrate-
ubiquitination of MDM2 is modulated physiologically by posttranslational modifications,
including sumoylation and phosphorylation. After SUMO conjugation to MDM2, MDM2
- 44 -
E3 ligase activity is shifted toward p53, while self-ubiquitination is minimized
(Buschmann et al., 2001).
P300 is an acetylase-possessing transcriptional co-activator that has been
shown to mediate transcription by numerous transcriptional activators. It binds to and
stabilizes MDM2. It stabilizes MDM2 by retaining it in a specific nuclear structure but
does not acetylate MDM2 in solution or in cells.
2.2.3.3. Phosphorylation of MDM2
The first demonstration of the complex nature of MDM2 phosphorylations was by
Henning who showed that the phosphorylation status of MDM2 was influenced by early
gene expression of SV40. MDM2 is stabilized in the presence of SV40. Moreover,
hyperphosphorylated MDM2 participates in a trimeric complex with p53 and T-Ag (T-
antigen, the transforming protein of SV40), which is thought to activate oncogenic
functions of MDM2 and enhance the transforming potential of T-Ag (Henning et al.,
1997).
Nearly 20% of the residues of MDM2 are either serine or threonine. MDM2
protein is phosphorylated at multiple sites in vivo. Two clusters of phosphorylation sites
are located at the N-terminal (amino acids 1–193) and central amino acids 194–293 of
murine Mdm2, respectively (Hay and Meek, 2000).
2.2.3.3.1. DNA-PK, ATM and MDM2
The PI3K family of enzymes generates lipid 'second messengers' that mediate
signal transduction. It includes four classes of proteins. Class IV of PI3K includes mTOR
(mammalian target of rapamycin), DNA-PK (DNA activated protein kinase), ATM (ataxia
telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases. MDM2 is
phosphorylated in vitro by both DNA-PK (Mayo et al., 1997) and ATM (Maya et al., 2001)
- 45 -
but phosphorylation by ATR has not yet been reported. Of eight potential DNA-PK
targets in MDM2, only Ser17 has been shown to be phosphorylated by this enzyme in
vitro (Mayo et al., 1997). Although physiological phosphorylation of Ser17 has been
confirmed, the phosphorylation site itself has been reported to have a significant impact
on the ability of MDM2 to regulate the response to p53. S17A mutant, mimics the
dephosphorylation form of MDM2, was significantly more effective in inhibiting p53-
dependent transactivation in cultured cells than wild-type MDM2. Nuclear magnetic
resonance studies also showed that MDM2 amino acids 16–24 can form a "flexible lid"
that folds over and stabilizes the MDM2 structure but competes only weakly with p53 for
binding to this cleft. The S17D mutant peptide which mimics the consititutive
phosphorylation form was found to have higher affinity for MDM2 than the wild-type
peptide (McCoy et al., 2003).
ATM is able to phosphorylate MDM2 at Ser395 in vitro. In response to ionizing
radiation and radiomimetic drugs, MDM2 undergoes rapid ATM-dependent
phosphorylation prior to p53 accumulation, which results in a decrease in its reactivity
with the 2A10 monoclonal antibody. MDM2 S395D is impaired in promoting p53
degradation and it is markedly less able to promote p53 cytoplasmic export (Maya et al.,
2001).
2.2.3.3.2. PKB and MDM2
Mitogen-induced activation of PKB results in phosphorylation of MDM2 on Ser166
and Ser186. Phosphorylation on these sites is necessary for translocation of MDM2 from
the cytoplasm into the nucleus (Mayo and Donner, 2001). Moreover, phosphorylation of
MDM2 not only enhances its nuclear localization but its interaction with p300, and
inhibits its interaction with p19ARF, resulting in increased p53 degradation (Zhou et al.,
2001).
- 46 -
PKB inhibits MDM2 self-ubiquitination via phosphorylation of MDM2 on Ser166
and Ser188. Stimulation of human embryonic kidney 293 cells with IGF-1 increased
MDM2 phosphorylation on Ser166 and Ser188 in a PI3K-dependent manner. Treatment
of both human embryonic kidney 293 and COS-1 cells with PI3K inhibitor LY-294002 led
to proteasome-mediated MDM2 degradation (Feng et al., 2004).
2.2.3.3.3. c-Abl and MDM2
c-Abl is an non-receptor tyrosine kinase that can shuttle between the cytoplasmic
and nuclear compartments. In response to stress, such as DNA damage, c-Abl promotes
cell growth arrest and apoptosis. The apoptotic activity of c-Abl is mediated partly via
p73 (Agami et al., 1999; Gong et al., 1999) and to a lesser extent through a p53-
dependent pathway (Yuan et al., 1997). One mechanism for the protection of p53 by c-
Abl is that c-Abl can neutralize the ability of MDM2 to ubiquitinate p53 and degrade it
(Sionov et al., 1999). C-Abl directly interacts with MDM2 at multiple sites in the nucleus,
enhances its accumulation in a p53-independent manner. c-Abl phosphorylates MDM2
at Tyr394. Substitution of Tyr394 by Phe394 enhances the ability of MDM2 to promote
p53 degradation and to inhibit the transcriptional and apoptotic activities of p53
(Goldberg et al., 2002).
2.2.3.3.4. CK2 and MDM2
Protein kinase CK2 is a ubiquitous Ser/Thr protein kinase required for cell cycle
progression and cell viability. Serine residue at position 269 of MDM2 was established
as the most important CK2 phosphorylation site by analyses with deletion mutants of
MDM2 and a peptide library. Phosphorylation of MDM2 by CK2 is stimulated in the
presence of the C-terminal sequences of p53, but binding studies revealed that the
- 47 -
I II III IV V VI VII VIII IX X XI XII
1 27 28 52 53 97 97 114 114 133 134 166166 220 221 272 273 298 299 491
P1 P2 ATG ATG
1 26 108 178 192 230 274 289 331 433 467 471 491
Zn-finger
NLS NES Ring-fingerAcidic domain
Zn-finger
NLS NES Ring-fingerAcidic domain
P P P PP PP NoLS
MDM2
P300
P5326 108
102 222 341 491
L5,L111 222 284 374
ARF210 224
P53 211 361
SP1,RB221 294
Numb1 134
PCAF50 384
AR
P
1 27 223 491
1 27 301 491MDM2-a
MDM2-b
MTBP 167 304
PML 50 166 202 304 340 488
PML
E2F, TFIIE
DNA polymerase ε
p731 150
1 52 223 491
1 27 389 491MDM2-c
MDM2-d1 75 483 491
MDM2-e
S17 S166 S186 S188 S269 S395DNA-PK PKB/AKT CK2 ATM
T216 Y394CyclinA/CDK2 c-Abl
Figure 4 Structure of MDM2 gene and protein
- 48 -
Figure 4 Structure and regulation of MDM2 gene and protein. MDM2 gene
consists of 12 exons. Two promoters are shown by arrows. Full-length MDM2 p90
is translated from the first start codon ATG in exon 3 and the short form, p76, is
translated from the second ATG in exon 4. Phosphorylation sites are indicated by
the letter P within an ellipse. Their locations relative to the functional domains of
MDM2 are shown. Protein kinases, where known, are indicated in boxes with the
target residue(s) shown above. Five major alternative splice variants MDM2-a,
MDM2-b, MDM2-c, MDM2-d, and MDM2-e are shown. Full-length Mdm2 and
known motifs are also represented. NLS-nuclear localization signal; NES-nuclear
export signal; Zn-finger-zinc finger domain; NoLS-nucleolar localization signal;
RING-finger-ring finger domain. The numbers above the drawings denote amino
acid numbers and roman numerals denote the exon numbers.
- 49 -
biological function of CK2 phosphorylation still needs to be established by further
studies because phosphorylation of MDM2 at Ser269 does not have any influence on
the binding of p53 to MDM2 (Gotz et al., 1999).
3. Functional interaction among FOXO, p53, and MDM2
3.1. Interaction between MDM2 and p53
P53 is a transcription factor that regulates the cell cycle and functions as a tumor
suppressor. p53 has been described as "the guardian of the genome" or the "master
watchman", referring to its role in conserving stability by preventing genome mutations.
In the absence of genetic damage, p53 is a very unstable protein with a half-life ranging
from 5-30 min and transcriptional activity is inert (Yuan et al., 1996). In the presence of
stress, such as DNA damage, hypoxia, telomere shortening, and oncogenic activation,
p53 becomes stable and activated by blocking its degradation (Caspari, 2000; Meek,
1994; Sakaguchi et al., 1998; Siliciano et al., 1997). p53 can kill cells via dual
transcription-dependent and -independent functions in the nucleus and mitochondria
(Mihara et al., 2003; Vousden and Lu, 2002).
The p53-MDM2 system forms a feedback loop, in which p53 upregulates MDM2
by activating MDM2 transcription (Barak et al., 1993). Experiments with knock-out mice
revealed that deletion of the mdm2 gene results in embryonic lethality, which can be
rescued by deletion of the p53 gene (Jones et al., 1995; Montes de Oca Luna et al.,
1995). Inhibition of cell growth and marked cell death is often seen in the absence of p53
regulation by MDM2, further emphasizing the importance of the p53–MDM2
autoregulatory loop in controling of cell growth and death.
MDM2 negatively regulates p53 in several ways:
- 50 -
1) MDM2 binds to p53 and this interaction is conformation based. Site-directed
experiments have demonstrated the importance of p53 residues Leu14, Phe19, Leu22,
and Trp23 (Lin et al., 1994). A minimal MDM2-binding site on p53 residues 18-23 was
mapped with p53-derived peptides (Picksley et al., 1994). On binding to the p53
transactivation domain, MDM2 inhibits its transcriptional activity. Crystallographic data
showed that the amino terminal domain of MDM2 forms a deep hydrophobic cleft into
which the transactivation domain of p53 binds, thereby concealing itself from interacting
with the transcriptional machinery (Kussie et al., 1996).
2) MDM2 functions as an E3 ubiqutin ligase for p53 (Honda and Yasuda, 1999;
Lohrum et al., 2000; Tao and Levine, 1999; Weber et al., 1999). The level of MDM2 is
very important for the ubiquitination level of p53. In vitro studies showed that low levels
of MDM2 activity induce monoubiquitination, whereas high levels promote
polyubiquitination and nuclear degradation of p53 (Li et al., 2003a).
3) MDM2 promotes the export of p53 from the nucleus. p53 shuttles between
nucleus and cytoplasm in the cells in response to stress. MDM2 contains an NES, but
the study showed that the MDM2 RING finger domain, not the MDM2 NES, is necessary
for the efficient export of p53 to the cytoplasm (Boyd et al., 2000; Geyer et al., 2000).
Another study also showed that low MDM2 levels induce cytoplasmic translocation of
p53 (Li et al., 2003a), whereas MDM2-mediated p53 monoubiquitylation promotes its
mitochondrial translocation (Marchenko et al., 2007).
3.2. Interaction between p53 and FOXO
p53 and FOXO factors share similar characteristics. Both are involved in stress
response and can be post-translationally modified by phosphorylation and acetylation.
They also have some common downstream targets, such as: Fas ligand (Greer and
- 51 -
Brunet, 2005), GADD45 (Greer and Brunet, 2005), PA26 (Greer and Brunet, 2005), p21
(Seoane et al., 2004) and PUMA (You et al., 2006a) (Figure 5).
Recently it was shown that p53 and FOXO3A interact with each other. In
response to oxidative stress, p53 binds to FOXO3A. In vivo, these two transcription
factors exhibit “crosstalk”. In response to DNA damage, p53 activation leads to FOXO3A
phosphorylation and subcellular localization change, which results in inhibition of
FOXO3A transcription activity. PKB is dispensable for p53-dependent suppression of
FOXO3A. By contrast, SGK1 was significantly induced in a p53-dependent manner after
DNA damage, and this induction is through extracellular signal-regulated kinase 1/2-
mediated posttranslational regulation. Furthermore, inhibition of SGK1 expression by a
small interfering RNA knockdown significantly decreased FOXO3A phosphorylation in
response to DNA damage.
Nuclear activated FOXO3A can impair p53 transcriptional activity. However,
activation of FOXO3A either by serum starvation or by expressing a constitutively active
form of FOXO3A can induce p53-dependent apoptosis, even in cells bearing a
transcriptionally inactive form of p53 (You et al., 2006b).
- 52 -
DNA damageOncogenactivation
OxidativeStress
FOXOs
P21
P27
GADD45
Fas Ligand
Bim PUMA
TRAIL
Cell cycle Arrest Apoptosis
DNA damageOncogenactivation
Hypoxia
p53
P21
14-3-3δ
GADD45
Fas Ligand
PTEN PUMA
BAX Noxa
Cell cycle Arrest Apoptosis
Figure 5 Stress-induced FOXO and p53 pathway.
- 53 -
HYPOTHESIS & OBJECTIVES
FOXO factors are known to be ubiquitinated, but so far no general E3 ubiquitin
ligases capable of ubiquitination of all FOXO factors have been identified. Skp2 is
reported to promote the ubiqutination and degradation of FOXO1, but this effect is
limited to FOXO1. A genetics study in C.elegans showed that skp expression is actually
needed for FOXO transcriptional activity.
Since MDM2 is known to be an E3 ubiquitin ligase for p53 and both p53 and
FOXO factors are important regulators in stress responses, aging and tumorigensis, we,
therefore, hypothesize that MDM2 interacts with and promotes the ubiquitination and
degradation of FOXO factors.
To substantiate the hypothesis, my thesis studies are to achieve the following
objectives:
1. Determine whether FOXO factors and MDM2 interact;
2. Determine whether MDM2 regulates the transcriptional activity of FOXO
factors;
3. Determine whether MDM2 decreases the stability of FOXO factors;
4. Determine whether MDM2 changes the biological function of FOXO;
5. Identify the signals that regulate effect of MDM2 on FOXO and investigate
the functional relationship among p53, FOXO and MDM2.
- 54 -
MATERIALS AND METHODS
Chemicals, Antibodies and Cell Lines:
Cycloheximide and nicotinamide were purchased from Sigma and MG132 from
Calbiochem. Antibodies against FOXO1 (H-128, Santa Cruz Biotechnology), FOXO3A
(H-144, Santa Cruz Biotechnology), MDM2 (SMP14, Santa Cruz Biotechnology; 2A10,
Calbiochem), c-Myc (A-14, Santa Cruz Biotechnology), Flag (M2-A-2220 and F-7452,
Sigma), HA (MMS-101P and PRB-101P, Covance), MnSOD (Upstate), TRAIL (BD
Pharmingen), p27 (N-20, Santa Cruz), acetyl-K (Upstate), α-Tubulin (Sigma), and β-actin
(AC-74, Sigma) were purchased from commercial sources.
H1299/V138 cells were cultured as described (Pochampally et al., 1999). HEK
293T, NIH 3T3, DU145, JCA1, PC3, HeLa, MCF-7, Saos-2, H1299, p53 null MEFs
(mouse embryonic fibrblasts )and p53 and MDM2 double null MEFs (Huang et al., 2005;
Peng et al., 2001) were maintained in DMEM (Dulbecco's modified Eagle's medium) with
10% FBS (fetal bovine serum). LNCaP cells were cultured in RPMI 1640 with 10% FBS
at 37°C. H1299 cells stably expressing human MDM2 were generated by cotransfecting
MDM2 with pcDNA3 and selecting in the presence of 750 μg/ml G418.
Plasmids:
pcDNA3-Flag-FOXO1, pcDNA3-Flag-FOXO1 (AAA), HA-FOXO3A, HA-FOXO3A
(AAA) (Li et al., 2003b), HA-SIRT1 (Yang et al., 2005), HA-SIRT1(734R) (Yang et al.,
2007), pcMDM2 and different MDM2 mutants were described previously (Armoni et al.,
- 55 -
2006; Chen et al., 1995; Chen et al., 1993; Freedman and Levine, 1999). To construct
pcDNA3-HA-FOXO1 (1-150), FOXO1 cDNA fragment coding the first 150 amino acids
were amplified by PCR with primers 5'-CGG GGG TCA CCG GAT CCA TGG CCG AGG
C-3' and 5' –GCG GCG GGA CGA TCT AGA CTA GCG CGG CTG C-3', which
generated BamHI and XbaI sites at 5' and 3' ends of the DNA fragment, respectively.
The amplified FOXO1 (1-150) fragment was cloned into the BamHI and XbaI sites of
pcDNA3.1 HA vector (Invitrogen). pcDNA3-HA-FOXO1 (1-270) and pcDNA3-HA-FOXO1
(256-655) were constructed similarly by generating a BamHI site at the 5' end and a XbaI
site at the 3' end of the corresponding FOXO1 cDNA fragments. The upstream primer of
FOXO1 (1-270) was same as the FOXO1 (1-150), and the downstream primer was 5'-
CTT GGC TCT AGA AGC TCG GCT TCG GCT CTT AG -3'. The upstream primer of
FOXO1 (256-655) was 5'-GGA GAA GAG CTG GAT CCA TGG ACA ACA AC-3' and the
downstream primers was 5'-CGG GCC CTC TAG ATC AGC CTG ACA CC -3'. MDM2
siRNAs were subcloned into pSilencer-Neo (Ambion). The corresponding
oligonucleotides for generating the MDM2 siRNA were 5'-GAT CCG CAG GTG TCA
CCT TGA AGG TTT CAA GAG AAC CTT CAA GGT GAC ACC TGT TTT TTG GAA A-
3'and 5'-GAT CCG TGG TTG CAT TGT CCA TGG CTT CAA GAG AGC CAT GGA CAA
TGC AAC CAT TTT TTG GAA A-3'. The oligonucleotides for GFP siRNA were from
Ambion.
Transfections and Immunological Assays:
For co-precipitation analysis, 106 cells were plated in 100 mm dishes in a
medium containing 10% fetal bovine serum. One day after plating, cells were transfected
with the indicated plasmids by Lipofectamine Plus following the protocol from Invitrogen.
Cellular extracts were prepared in a buffer containing 20 mM Tris-HCl (pH 7.5), 0.5% NP-
40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10
- 56 -
mM benzamidine and 1 mM phenylmethylsulfonyl fluoride. After pre-clearance by
incubating with protein A-agarose for 1 hour (h) followed by brief centrifugation, the
extracts were incubated sequentially with 1-3 µg antibody and protein G-agarose beads
for 4 h at 4°C. After four times washes with the lysis buffer, the immunoprecipitates were
eluted from the beads by boiling in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) loading buffer.
For immunoblotting, cellular extracts or immunoprecipitates were separated on
SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, probed with the
cognate antibody and visualized with enhanced chemiluminescence.
For immunofluorescence analyses, H1299 cells or MEFs transfected cells on
cover slips were cultured in DMEM for 16 h, washed once with PBS (phosphate buffered
saline) and fixed in 4% paraformaldehyde for 10 min at 37ºC. The cells were
permeabilized in buffer containing 1% Triton X-100 and 1% BSA at room temperature for
30 min and incubated for another 1 h in PBS containing 0.2% NP-40, 1% bovine serum
albumin and the primary antibody. After washing three times in PBS, the cells were
incubated for 45 min with goat anti-mouse IgG conjugated with Alexa Fluor 594 (red) or
fluorescein isothiocyanate (FITC; green) conjugated anti-rabbit IgG (Molecular Probes),
respectively and washed three times with PBS. The slides were dried and mounted with
Vectashield mounting medium with DAPI (4', 6'-diamidino-2-phenylindole). DAPI staining
was performed to visualize the nucleus. Regular fluorescent microscopic images were
obtained with a Nikon Diaphot microscope using a Photometrix PXL cooled CCD
camera. The microscope was equipped with the appropriate filters for three-color
imaging and a motorized stage for obtaining z-series images. Digital image files were
processed and deconvolved using the Oncor Image software (Oncor Inc.). High-
resolution images of the deconvolved and 3-D reconstructed image z-series stacks were
- 57 -
processed for presentation with Adobe Photoshop. For confocal analysis, samples were
viewed with a Leica DMI6000 inverted microscope, TCS SP5 confocal scanner, and a
100X/1.40NA Plan Apochromat oil immersion objective (Leica Microsystems, Germany).
405 Diode and HeNe 594 laser lines were applied to excite the samples and tunable
filters were used to minimize spectral overlap between fluorochromes. DIC imaging was
performed using an argon laser line. Scale bars were created with the LAS AF software
version 1.6.0 build 1016 (Leica Microsystems, Germany).
Ni-NTA Pull-down Assay:
H1299 cells or MEFs were plated in 100 mm dishes and transfected with 4 µg
His6-ubiquitin (His-Ub) plasmid, 4 µg FOXO vectors and 4 µg MDM2 vectors using
Lipofectamine Plus; 24 h post transfection, cells were harvested and separated into two
aliquots. One aliquot (10%) was subjected to immunoblotting analysis to detect the
expression of transfected proteins. The other aliquot of cells (90%) was used to purify
the proteins of interested using Ni2+-nitrilotriacetic acid (NTA) beads. Cell pellets were
lysed in a buffer containing 0.01 M Tris-Cl (pH 8.0), 6 M guanidinium-HCl, 0.1 M sodium
phsophate, 5 mM imidazole, 10 mM β-mercaptoethanol and incubated with Ni2+-NTA
beads (Qiagen) for overnight at room temperature. The beads were washed sequentially
with the lysis buffer, a buffer containing 0.01 M Tris-Cl (pH 8.0), 8 M urea, 0.1 M sodium
phsophate, 10 mM β-mercaptoethanol, and a buffer containing 0.01 M Tris-Cl (pH 6.3), 8
M urea, 0.1 M sodium phsophate, 10 mM β-mercaptoethanol. Proteins bound to the
beads were eluted with a buffer containing 0.15 M Tris-Cl (pH 6.7), 5% SDS, 200 mM
imidazole, 30% glycerol, 0.72 M β-mercaptoethanol and were subjected to
immunoblotting analysis for the presence of Ub-conjugated FOXO proteins.
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In vitro Transcription Coupled Translations and GST (Glutathione S-transferase)
Pull-down assays:
FOXO1 protein was produced with pcDNA3-Flag-FOXO1 as a template using T7
polymerase-based in vitro transcription coupled translations (Promega, Madison, Wis.).
GST-MDM2 plasmids were transformed into BL21 and cultured at 37°C until the optical
density at 600 nm reached 0.6. Then, 0.2 mM of isopropylthiogalactopyranoside (IPTG)
was added and the incubation continued for another 5 h at 30°C. Bacterial cultures were
lysed by sonication in a buffer containing 50 mM Tris (pH 8.0), 10 mM NaCl, 1 mM
EDTA, 6 mM MgCl2, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. GST
pull down analyses were performed using the MagneGST pull-down system (Promega
Madison, Wis) following the vendor’s protocols.
In Vitro Ubiquitination Assays:
Full length GST-MDM2 and GST-MDM2-NT (1-150) were expressed in E. coli
and bound to glutathione agarose beads. The substrate FOXO1 was produced by in vitro
transcription coupled translation in rabbit reticulocyte lysate using the TNT system
(Promega) in the presence of 35S- methionine. 4 µg GST-fusion proteins and 8 µl FOXO1
in vitro translation product were incubated to allow the enzyme-substrate interaction to
occur. After three times washes with PBS containing 0.2% NP-40, the bead-bound
enzyme-substrate complex was incubated at 37°C for 1 h with 250 ng GST-Ubc5Hb
(Boston Biochem), 250 ng purified rabbit E1 (Boston Biochem), 2 µg His6-Ub (Boston
Biochem) in 20 µl reaction buffer containing 50 mM Tris (pH 7.5), 2.5 mM MgCl2, 15 mM
KCl, 1 mM dithiothreitol, 0.01% Triton X-100, 1% glycerol and 8 mM ATP. The reactions
were terminated by boiling in SDS sample buffer and separated by SDS-polyacrylamide
gel electrophoresis. Gel was dried and the Ub-conjugated FOXO1 proteins were
detected by autoradiography.
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Apoptotic Analysis and Flow Cytometry:
The determination of the survival and apoptotic index of GFP (green fluorescent
protein)-transfected cells has been described (Li et al., 2001). In brief, transfected cells
were washed with PBS, fixed in 4% formaldehyde and stained with DAPI.
Representative micrographs were captured by a charge-coupled device camera with a
Smart Capture Program (Vysis, Downers Grove, Ill.) attached to a Leitz Orthoplan 2
fluorescence microscope. The viability of transfected cells in each well was determined
by counting the total number of green cells in each well. The apoptotic index of GFP-
positive cells was determined by scoring 300 GFP-positive cells for chromatin
condensation and apoptotic body formation.
To assay the apoptosis index induced by FOXO1, H1299 cells in 100 mm dishes
were transfected with GFP and FOXO1 with or without MDM2. Transfected cells were
collected in PBS containing 2.5 mM EDTA, washed twice with cold PBS, re-suspended
in 1× binding buffer containing 0.01 M Hepes (pH7.4), 0.14 M NaCl, 2.5 mM CaCl2 at a
concentration of 1 × 106 cells/ml and stained with Annexin-V APC and 7-AAD. Cell
sorting and flow cytometry analysis were performed on a FAC Scan (Becton Dickinson,
Mountain View, Calif.).
- 60 -
RESULTS
FOXO proteins are ubiquitinated and their expression level is regulated by
proteasome mediated degradation (Aoki et al., 2004; Plas and Thompson, 2003), but a
general ubiquitin E3 ligase for FOXO proteins remains to be identified.
Skp2 was reported to mediate the ubiquitination and degradation of FOXO1 but
such activity appears to be restricted to FOXO1 (Huang et al., 2005). So far, no
mutational analysis and in vitro enzyme assays have been performed to show the direct
involvement of Skp2 E3 activity in FOXO ubiquitination. It remains to be determined
whether Skp2 promotes FOXO1 ubiquitination directly, through its intrinsic E3 activity, or
indirectly through another E3. The present study showed that MDM2 is the E3 ubiquitin
ligase for FOXO factors. MDM2 interacts with FOXO, promotes the degradation and
affects downstream targets and the biological function of FOXO.
1. MDM2 promotes the degradation of FOXO factors.
In an effort to investigate the interaction between p53 and FOXO signaling
pathways, an inverse correlation was noticed between the expression of FOXO3A and
the p53 target gene MDM2 using a panel of human cancer cell lines including
osteosarcoma (Saos2), prostate (LNCaP, PC3, DU145, and JCA1), breast (MCF-7),
cervical (Hela) and lung (H1299, H1299/V138) cancer cells (Figure 6). To determine
whether the inverse correlation of these two proteins among cancer cells reflects a
negative effect of MDM2 on FOXO protein, the level of FOXO3A and FOXO1 protein
expression in p53 null and p53/MDM2 double null MEFs was measured by
- 61 -
immunoblotting. As shown in Figure 7, the knockout of MDM2 in MEFs resulted in an
increased expression of endogenous FOXO1 and FOXO3A proteins. Consistent with the
data from MEFs, the stable expression of MDM2 in H1299 cells reduced (Figure 8) and
the knockdown of endogenous MDM2 increased (Figure 9) the expression of
endogenous FOXO3A. Unlike MEFs, H1299/V138 cells express relatively low levels of
endogenous FOXO1, which is difficult to detect. Thus, we tested the effect of MDM2
silencing on the expression of ectopic FOXO1. As shown in Figure 10, the knockdown of
MDM2 by shRNA increased the level of ectopic Flag-FOXO1 expression.
Because MDM2 is an E3 ubiquitin ligase that promotes proteasome mediated
degradation of p53 (Honda et al., 1997) and other (Lin et al., 2002a) proteins, it is likely
that MDM2 decreases the expression of FOXO factors in a proteasome-dependent
manner. In transient transfection studies, co-expression of MDM2 reduced the
expression level of Flag-FOXO1 in DU145 prostate cancer cells, a process that was
prevented by treatment with two different concentrations of the proteasome inhibitor
MG132 (Figure 11).
To test whether the decreased FOXO protein expression by MDM2 is due to
protein degradation, the half-life of Flag-FOXO1 was measured in H1299 cells
cotransfected with control vector, full length MDM2 or an MDM2 (1-361) mutant which
lacks the C-terminal E3 ligase domain. As shown in Figure 12, the half life of FOXO1
was decreased by full length MDM2 from 6 h to 3 h, as compared to the control vector.
When FOXO1 was cotransfection of MDM2 (1-361), MDM2 (1-361) significantly
extended the half-life of Flag FOXO1; the exact meaning of the latter is unclear. Overall,
the data raise the possibility that MDM2 may function as an E3 ubiqitin ligase for
mammalian FOXO factors.
- 62 -
Figure 6 Inverse correlation of MDM2 and FOXO3A protein expression
in different cancer cell lines. DU145, JCA, H1299-V138, HeLa, LNCaP,
MCF7, PC3, Saos-2, H1299 cells were plated into 100 mm dishes. MDM2
and FOXO3A proteins were detected by Western blotting with anti-MDM2
(SMP-14 ) and anti-FOXO3A (H-144) antibodies, respectively.
MDM2
DU
145
JCA1
H12
99/V
-138
Hel
a
LNC
aP
MC
F7
PC3
Saos
2
H12
99
78
105
MW(kD)
FOXO3A
- 63 -
Figure 7 Inverse correlation of MDM2 and FOXO protein expression in MEFs.
p53-/- MEFs and p53-/-, MDM2-/- double null MEFs were plated in 100 mm dishes.
The cells were harvested the next day and cell lysates were subjected to
immunoblotting analyses with anti-MDM2 (SMP-14 ) antibody, anti-FOXO1 antibody,
and anti-FOXO3A (H-144) antibodies, respectively. The β-actin level showed the
equal loading.
P53-
/-
P53-
/-M
DM
2-/-
FOXO1
β-actin
150
100
75
50
150
100
75
50
IB:MDM2
IB:FOXO3A
MDM2
FOXO3A
IB:FOXO1
IB:β-actin
- 64 -
Figure 8 Stably expressed MDM2 decreases the level of endogenous
FOXO3A expression. H1299 cells stably expressing MDM2 (MDM2-1, MDM2-
2) or empty vector (Control) were established and cell extracts were subjected to
Western blot analyses with the indicated antibodies.
Con
trol
MD
M2-
1
MD
M2-
2
MDM2
H1299 Stable Clones
FOXO3A
β-actin
IB:MDM2
IB:FOXO3A
IB:β-actin
- 65 -
Figure 9 Knockdown of endogenous MDM2 by siRNA causes an
increase in endogenous FOXO3A protein. H1299 cells were transfected
with control GFP-siRNAs or siRNAs (MDM2-siRNA1, MDM2-siRNA2) for
MDM2, and cell extracts were subjected to Western blot analyses.
GFP
MD
M2-
1
MD
M2-
2
MDM2
FOXO3A
β -actin
siRNA
IB:MDM2
IB:FOXO3A
IB:β -actin
- 66 -
Figure 10 Knockdown of endogenous MDM2 by siRNA increases
the level of ectopic FOXO1 protein. A Flag-tagged FOXO1 expression
vector was cotransfected transiently with MDM2-siRNA or control siRNA
(GFP-siRNA) into H1299-V138 cells, and whole cell extracts were
prepared and subjected to Western blot analyses.
+
-+
MDM2-shRNA-+-
Control-shRNA+-+Flag-FOXO1+--
+
-+
MDM2-shRNA-+-
Control-shRNA+-+Flag-FOXO1+--
β-actin
MDM2
Flag-FOXO1
IB:MDM2
IB:M2
IB: β-actin
- 67 -
1010550 00MG132 (μM)+-+-+--MDM2++++++-Flag-FOXO1
1010550 00MG132 (μM)+-+-+--MDM2++++++-Flag-FOXO1
IB:M2
IB:β-Actin
IB:MDM2
FOXO1
MDM2
β-Actin
0
0.5
1
1.5
1 2 3 4 5 6 7
FOXO
1 Pr
otei
n D
ensi
ty
Figure 11 MDM2 overexpression causes a decrease in Flag-FOXO1 protein,
which is blocked by MG132. DU145 cells were transfected with Flag-FOXO1 and
pcMDM2. 24 h posttransfections, cells were incubated with or without different
concentrations of MG132 for 6 h. Cell lysates were separated in a 8% SDS-PAGE.
FOXO1 and MDM2 were detected by Western blotting with anti-Flag (M2) and anti-
MDM2 (SMP-14) antibodies, respectively. Equal amounts of protein were loaded.
- 68 -
Flag-FOXO1
β-actin
+++++-----------MDM2(1-361)241263024126302412630-CHX treatment (h)
+++++++++++++++-Flag-FOXO1-----+++++------MDM2+++++-----------MDM2(1-361)241263024126302412630-CHX treatment (h)
+++++++++++++++-Flag-FOXO1-----+++++------MDM2
Figure 12 MDM2 Overexpression results in a decrease in half-life of the
FOXO1 protein. H1299 cells were transfected with the plasmids as indicated. 24 h
after transfection, cells were treated with CHX for different times. The cell lysates
were separated by SDS−8% PAGE. Exogenous FOXO1 was detected by Western
blotting with anti-Flag M2 antibodies. Equal amounts of protein were loaded on each
sample.
0
20
40
60
80
100
120
0 3 6 12 24
CTLMDM2MDM2(1-361)
FOXO
1/ β
–act
in(%
)
(hrs)
- 69 -
2. MDM2 interacts with FOXO factors in vivo and in vitro.
Ubiquitin E3 ligases are known to make direct contact with their substrates.
Therefore, it was investigated whether or not MDM2 and FOXO factors form a complex
in cells. Deconvolution imaging analysis detected ectopic MDM2 and FOXO1 proteins in
both cytoplasmic and nuclear compartments of p53/MDM2 double null MEFs, but
colocalization was detected predominantly in the nucleus (Figure 13). Similarly, confocal
imaging gave the same result for endogenous MDM2 and FOXO3A in H1299 cells
(Figure 14).
To determine whether MDM2 and FOXO1 interact in mammalian cells, they were
ectopically expressed in H1299 cells and reciprocal coimmunoprecipitations performed.
MDM2 and FOXO1 were co-precipitated in cells expressing both proteins. In cells that
express either MDM2 or FOXO1, little or no coprecipitations were observed, suggesting
that co-precipitations were not due to cross reactivity of the antibodies (Figure 15).
Similar analysis showed that MDM2 and GFP-FOXO3A were specifically co-precipitated,
but not MDM2 and GFP (Figure 16).
To determine whether endogenous MDM2 and FOXO1 interacted in mammalian
cells, H1299/V138 cells expressing a temperature-sensitive p53 mutant (Pochampally et
al., 1999) were shifted to permissive temperature for 16 h to induce MDM2 expression
and cellular extracts were subjected to coimmunoprecipitation analysis. As shown in
Figure 17, FOXO1 was co-precipitated by the anti-MDM2 antibody, but not by the control
antibody, showing that the co-precipitations were not due to antibody cross reactivity.
To determine whether endogenous MDM2 and FOXO3A form a complex,
extracts of HEK293T cells were treated with DMSO or MG132 and co-
immunoprecipitations with an anti-MDM2 antibody. An anti-HA antibody was
- 70 -
Figure 13 Exogenous MDM2 and Flag-FOXO1 are colocalized in the nucleus
of MEF cells. p53-/-, MDM2-/- MEF in a medium containing 0.5% FBS were
transfected with 1 μg pcMDM2 and 1 μg Flag-FOXO1. Cells were stained with DAPI
and incubated with anti-MDM2 (SMP14) and anti-FOXO1 (H-128) antibodies.
Immunoreactivity was detected with IgG conjugated to Alexa Fluor 594 (red for
MDM2) or FITC (green for FOXO1). Colocalization was determined by high-
resolution imaging with deconvolution microscopy.
Flag-FOXO1MDM2
Merged
20 microns
- 71 -
Figure 14 Endogenous MDM2 and FOXO3A are colocalized in the nucleus of
H1299 cells. H1299 cells in a medium containing 1% FBS were stained with DAPI
and incubated with anti-MDM2 (SMP14) and anti-FOXO3A (H-144) antibodies.
Immunoreactivity was detected with IgG conjugated to Alexa Fluor 594 (red for
MDM2) or FITC (green for FOXO3A). Colocalization was visualized by high-
resolution imaging with confocal microscopy.
DAPI MDM2 FOXO3A
Merged
20 microns
- 72 -
Figure 15 Interaction of ectopic FOXO1 and MDM2 in H1299 cells.
H1299 cells were cotransfected with HA-tagged FOXO1 and pcMDM2. 24 h
posttransfection, cell extracts were immunoprecipitated with either anti-HA or
anti-MDM2 antibodies followed by immunoblotting with anti-MDM2 or anti-HA
antibody.
++
MDM2+-HA-FOXO1-+
++
MDM2+-HA-FOXO1-+
60
78
109
60
78
109
60
78
47
IP: HAIB: MDM2
IP:MDM2IB:HA
IB: HA
IB: MDM2
MW(kD)
MDM2
FOXO1
MDM2
FOXO1
IB: β-actin β-actin
- 73 -
Figure 16 Interaction of ectopic FOXO3A and MDM2 in HEK293T cells.
HEK293T cells were cotransfected with pcMDM2 and either GFP or GFP-
FOXO3A. 24 h posttransfection, cell extract were immunoprecipitated with either
an anti-MDM2 or an anti-GFP antibody followed by immunoblotting with an anti-
GFP or an anti-MDM2 antibody.
MDM2GFPGFP-FOXO3A
+-+
++
-
MDM2GFPGFP-FOXO3A
+-+
++
-
36
47
60
78
24
IP: MDM2IB: GFP
IP: GFP IB: MDM2
IB:GFP
IB:GFP
IB:MDM2
FOXO3A
MDM2
MDM2
FOXO3A
GFP
MW(kD)
- 74 -
Figure 17 Interaction of endogenous MDM2 and FOXO1 in H1299/V138
cells. Cells were shifted from 39°C and cultured in 33°C for overnight and
were treated with or without MG132 for 6 h. Cell extracts were
immunoprecipitated with an anti-FOXO1 antibody followed by
immunoblotting with an anti-MDM2 antibody.
IPMG132
M+
M+
IPMG132
M+
M-
HAMW(kD)
IB: MDM2
IB: FOXO1
Heavy Chain50
75
50
75 FOXO1
MDM2
- 75 -
Figure 18 Interaction of endogenous MDM2 and FOXO3A in HEK293T
cells. HEK 293T cells were treated with or without MG132 for 6 h before
harvest. Cell extracts were immunoprecipitated with either an anti-HA
antibody as control or an anti-MDM2 (SMP14) antibody followed by
immunoblotting with an anti-FOXO3A antibody.
-
HA
-
M
+
HA
+
M
MG132
IP
IB: FOXO3A
IB: MDM2
Heavy chain
78
60
60
78
109
MWkD
FOXO3A
MDM2
- 76 -
Figure 19 The interaction between FOXO1 and GST-MDM2 in vitro.
FOXO1 synthesized by in vitro transcription-translation reactions was incubated
with GST-MDM2 fusion proteins, precipitated with glutathione beads and
detected by immunoblotting with an anti-FOXO1 antibody. The amounts of GST
proteins used in the pull-down assays were visualized by Coomassie blue
staining after separation in a SDS-PAGE gel (bottom panel).
GSTFOXO1-AAAFOXO1-WT
+-+-
--+++
+--+---+--
++-- GST-MDM2GSTFOXO1-AAAFOXO1-WT
+-+-
--+++
+--+---+--
++---
GST Pull Down
IB: FOXO1
CB: GST-MDM2
CB: GST
IB: FOXO1
- 77 -
included as a control. As shown in Figure 18, FOXO3A proteins were co-precipitated
with MDM2 by an anti-MDM2 but not an anti-HA antibody. Treatment with MG132
increased the level of MDM2 and FOXO expression as well as the amount of proteins
co-precipitated. These studies showed that co-precipitations occur with both
endogenous and ectopically expressed proteins.
To determine whether MDM2 and FOXO1 interact in vitro, GST and GST-MDM2
fusion proteins were produced in bacteria, bound to glutathione beads and incubated
with FOXO1 proteins produced by in vitro transcription coupled translations. In these
GST pull down assays, wild type FOXO1 was precipitated with GST-MDM2, but not with
GST (Figure 19). An active form of FOXO1, FOXO1 (AAA) in which all three AKT
phosphorylation sites were mutated to alanine, was also precipitated with GST-MDM2
but not with GST (Figure 19). This analysis demonstrated that FOXO1 and MDM2 form a
complex in vitro and that complex formation occurs independently of the phosphorylation
of FOXO1 by AKT.
3. The fork head box of FOXO and the region of MDM2 controlling nuclear-
cytoplasmic shuttling mediate the interaction between MDM2 and FOXO.
To define the region in FOXO1 that mediates the interaction with MDM2, H1299
cells were transfected with full length MDM2 and FOXO1 deletion constructs fused to the
HA tag (Figure 20a). Cellular extracts were subjected to co-immunoprecipitation with an
anti-MDM2 antibody. As shown in Figure 21, the full length FOXO1 protein was co-
precipitated with MDM2. Deletion of the C-terminal region of FOXO1 did not alter the
interaction whereas further deletion into fork head box abolished it, suggesting that the
fork head box is required for the interaction.
Further define the region in FOXO3A that mediates the interaction with MDM2,
purified bacteria expressed GST or GST fused fragments of FOXO3A (peptide P1-P5)
- 78 -
encoding five nonoverlapping FOXO3A regions (Figure 20b) were incubated with
HEK293T cell extracts overexpressed MDM2. MDM2 coprecipitated only with peptide P2
(amio acid 154-259), which contains the forkhead domain (Figure 22). These results
suggest that MDM2 specifically interacts with the forkhead domain of FOXO3A. Further
alignment the fork head domain of mouse FOXO3A, human FOXO1, FOXO3A and
FOXO4 (Figure 23), we found that the fork head domain is highly conserved in these
four kinds of FOXO. All these data indicated MDM2 has a general effect on FOXO.
To define the region of MDM2 responsible for FOXO1 binding, different MDM2
constructs (Figure 24) were transfected into H1299 cells together with Flag-FOXO1.
Cellular extracts were immunoprecipitated with MDM2 antibody and the co-precipitated
FOXO1 was detected by anti-Flag antibody. As shown in Figure 25, all MDM2 mutants,
except MDM2 (50-491) (p53 binding deficient) and MDM2 NLS-mt (182R, nuclear
localization sequence defective), interacted with FOXO1. Immunoblot analysis showed
that all MDM2 mutants were expressed at significant levels and most MDM2 proteins
were detected in multiple forms, presumably due to cleavage by proteases (Chen et al.,
1997). In the presence of proteasome inhibitor MG132, however, both MDM2 (50-491)
and MDM2 NLS-mt were co-precipitated with Flag-FOXO1 by the anti-Flag antibody
(Figure 26). The exact reason for the lack of interaction with FOXO1 in the absence of
MG132 is unclear, presumably due to the degradation of the protein complex by the
proteasome.
Further analysis with additional MDM2 mutants in the presence of MG132,
showed that the deletion of 150-230 amino acids abrogated the FOXO1 binding (Figure
27). Consistent with the fact that this region contains the nuclear localization sequence,
it was also shown (Figure 28) that this mutant is mainly localized to cytoplasm. However,
cytoplasmic localization is clearly not the reason for the lack of interaction with MDM2
- 79 -
Figure 20 Diagram of different FOXO mutants used in this study.
a, The different mutants of human FOXO1 used in this study; b, The
different mutants of mouse FOXO3A used in this study.
FK150 2601 655
1-150
1-270
256-655
1-655
FL
FK148 2581 672
P1: 1-154
P2: 154-259
P3: 259-409
P4: 409-542
FLGST
P5: 542-672
GST
GST
GST
GST
GST
a
b
- 80 -
Figure 21 Mapping of the MDM2-interacting domain of FOXO1 by
immunoprecipitation. H1299 cells were transfected with 2 µg MDM2 and 2 µg
of different HA-tagged FOXO1 fragments. Anti-MDM2 immunoprecipitates were
subjected to immunoblotting with anti-HA antibody and anti-MDM2 as indicated.
Immunoblotting of total cell extracts with anti-HA antibody (lower panel) showed
the expression of different FOXO1 fragments.
IP: MDM2IB: HA
IB: HA
Vect
or
1-15
0
1-27
0
256-
655
HA-FOXO1
IP: MDM2IB: MDM2
1-65
5 (F
L)
1-655
1-270
1-655
256-6551-270
1-150
MDM2
- 81 -
++++++MDM2
P5P4P3P2P1GST
++++++MDM2
P5P4P3P2P1GST
GST pull down
IB:MDM2
MDM2
GST fusion protein
Figure 22 Mapping of the MDM2-interacting domain of FOXO3A by
GST pull-down assay. Recombinant GST or GST fused with fragments of
FOXO3A (P1-P5) were amplified by bacteria expression and purified by
GST beads. HEK 293T cells were transfected with 4 µg MDM2. Cell lysates
were subjected to GST pull down experiment. The amounts of GST proteins
used in the pull-down assays were visualized by Coomassie blue (CB)
staining after separation in a SDS-PAGE gel (bottom panel).
CB
- 82 -
Figure 23 Alignment of FOXO member’s fork head box. Grey box
– fork head box; Green box –unique FOXO sequence.
- 83 -
Figure 24 Diagram of different MDM2 mutants used in the study.
1-361
50-491
Δ150-230
E3-mt
NES-mt
NLS-mt
0 100 200 300 400 491
P53 binding NLS RingMDM2
Δ89-150
Δ50-89
Δ222-325
Δ222-437
361D-A
NES
- 84 -
IP: anti-MDM2IB: anti-Flag
IB: anti-MDM21-
361
50-4
91
361D
-A
NE
S m
utan
t
182R
Δ222
-325
Δ222
-437
MDM2
++++++Flag-FOXO1 + + + +++++++Flag-FOXO1
FOXO1
MDM2 mutants
Figure 25 Mapping of the FOXO1-interacting domain in MDM2 by
coimmunoprecipitations in the absence of MG132. H1299 cells were
transfected with 2 μg Flag-FOXO1 and 2.5 µg different MDM2 fragments. Anti-
MDM2 mmunoprecipitates were probed with anti-Flag M2 antibody (upper
panel). Immunoblotting of cellular extracts with anti-MDM2 antibody (lower
panel) showed the expression of different MDM2 fragments.
457S
MD
M2
- 85 -
Figure 26 An N-terminal truncation mutant and an NLS-mutant of MDM2
interact with FOXO1 in the presence of MG132. H1299 cells were
transfected with 2 μg Flag-FOXO1 together with 5 μg MDM2 or 5 μg mutant
MDM2. Cells were treated with MG132 for 6 h before harvest. Cells lysate were
immunoprecipitated with anti-Flag M2 antibody and detected with anti-MDM2
antibody. FL: full length MDM2, 50-491; MDM2(50-491); NLS: MDM2(NLS-mt).
FL 50-4
91
182R
75
50
IP:M2IB:MDM2
IB:MDM2
IB:M2
FL5% in
put
75
50
75
50
FOXO1
MDM2
MDM2/MDM2(182R)MDM2(50-491)
MW(kD)
- 86 -
Figure 27 Truncation of the central region of MDM2 abolishes the
interaction between MDM2 and FOXO1. H1299 cells were transfected with 2 μg
Flag-FOXO1 and 2.5 µg of different MDM2 fragments. Cells were treated with
MG132 for 6 h before harvesting. Anti-Flag immunoprecipitates were probed with
anti-MDM2 (2A10) antibody (upper panel). Immunoblottiong of cellular extracts
with anti-MDM2 antibody (lower panel) showed the expression of different MDM2
fragments.
FLVect
or
FL Δ50-
89
Δ89-
150
Δ150
-230
50-4
91
IP: M2IB: MDM2
IB: M2
IB: MDM2
FOXO160
78
110
170
47
60
78
110
170
++
MG132++++++Flag-FOXO1++++-+
++
MG132++++++Flag-FOXO1++++-+
MW(kD)
MDM2
MDM2
60
78
110
- 87 -
Figure 28 Immunofluorescence images show the cellular localization of
various MDM2 mutants. H1299 cells were transiently transfected with MDM2
or different MDM2 mutants. 24 h posttransfection, cells were fixed and
stained. The red signal showed the localization of MDM2 and the blue signal
showed the nucleus of the cells.
50-491
Δ222-325
Δ222-437
Δ50-89
Δ89-150
Δ150-230 E3-mt
1-361
NES-mt
NLS-mt
MDM2
MDM2 DAPI
361D-A
MDM2 DAPI
- 88 -
because co-precipitations were done with whole cell extracts and, under the same
conditions, nuclear localization sequence mutant, the MDM2 NLS-mt, interacted with
FOXO1 (Figure 26). Besides cellular localization sequences, the 150-230 region also
contains an inhibitory domain that suppresses cell cycle progression independently of
p53. This region is also involved in interactions with several proteins, including TBP and
p300.
4. MDM2 promotes the ubiquitination of FOXO1 and FOXO3A.
To test whether MDM2 promoted the ubiqutination of FOXO factors, H1299 cells
were transfected with Flag-tagged FOXO1, wild type or an AKT phosphorylation site
FOXO1 mutant. The effect of ectopic MDM2 on FOXO1 ubiquitination by cotransfected
Myc-tagged ubiquitin was measured in the anti-Flag precipitates. In the absence of
ectopic MDM2, limited FOXO1 ubiquitination was occured. This result is consistent with
the fact that H1299 cells are p53-deficient and contain low levels of endogenous MDM2.
Cotransfection of MDM2 with increased the level of ubiquitiation of the wild type FOXO1
but not of FOXO1 (AAA). The data argue that the positive effect of MDM2 on FOXO1
ubiquitination requires phosphorylation at the AKT sites (Figure 29).
To test whether the effect of MDM2 extends to other FOXO factors, FOXO3A
and His-tagged ubiquitin were transfected into H1299 cells and the effect of MDM2 on
FOXO3A ubiquitination was measured by immunblotting with anti-HA antibody following
nickel bead pull-down under denaturing conditions. As shown in Figure 30, FOXO3A
ubiquitination was increased by MDM2 in a manner dependent on the phosphorylation
on the AKT sites. The data suggest that the stimulation of ubiquitination by MDM2 is not
restricted to FOXO1 but among the FOXO factors.
In p53 and MDM2 double-null MEFs, wild type MDM2 stimulated the
ubiquitination of FOXO1 in a dose dependent manner, whereas the MDM2 mutant
- 89 -
lacking the C-terminal ring finger E3 region did not exert such an effect (Figure 31),
emphasizing the potential involvement of the E3 ligase activity. Nickel bead pull down
assays under denaturing conditions revealed that MDM2 containing a point mutation in
the E3 ligase domain, MDM2 E3 -mt (457S), did not stimulate FOXO1 ubiquitination
(Figure 32), confirming that FOXO1 ubiquitination by MDM2 requires its ubiquitin ligase
activity. Interestingly, on the one hand, a constitutively nuclear MDM2 in which the
nuclear export sequence was mutated, the NES-mt, did not promote FOXO1
ubiquitination (Figure 33), even though it contained an intact E3 ligase domain and
interacted with FOXO1 (Figure 25). On the other hand, two cytoplasmic MDM2 mutants,
MDM2 NLS-mt and MDM2 (∆89-150), stimulated FOXO1 ubiquitination (Figure 32 and
33), showing that the ubiquitiantion of FOXO1 by MDM2 is likely to occur in the
cytoplasm. MDM2 (∆150-230), which was located mainly in the cytoplasm (Figure 25)
and did not interact with FOXO1 (Figure 26), was unable to stimulate FOXO1
ubiquitination, suggesting that the ubiquitination requires FOXO1 interaction. Overall, the
data suggest that FOXO1 interacts with MDM2 in both the nucleus and the cytoplasm,
but the ubiquitination by MDM2 requires the interaction in the cytoplasm.
To fully establish MDM2 as an E3 ligase for FOXO1 ubiquitination, the ability of
recombinant MDM2 to catalyze the ubiquitination of FOXO1 was tested in vitro. In this
experiment, GST-MDM2 stimulated the ubiquitination of in vitro translated FOXO1 in the
presence but not in the absence of purified E1, E2 and ubiquitin (Figure 34, upper
panel). GST fused to an MDM2 N-terminal fragment had no effect on the ubiquitination of
FOXO1. In reactions performed with transcription-coupled translation product from a
control vector, the majority of the 35S-labeled ubiquitin conjugates disappeared, showing
that they were FOXO1 proteins (Figure 34, lower panel). In combination with the binding
- 90 -
Figure 29 MDM2 promotes the ubiquitination of FOXO1. H1299 cells were
transfected with the indicated plasmids and cell extracts were either
immunoprecipitated with M2 antibody, followed by immunoblotting with anti-Myc
antibody, or directly immunoblotted with antibodies to Flag or MDM2.
++-+ MDM2-+--
Myc-Ub+++-Flag-FOXO1 (AAA)+---Flag-FOXO1wt-+++
++-+ MDM2-+--
Myc-Ub+++-Flag-FOXO1 (AAA)+---Flag-FOXO1wt-+++
IP: M2IB: myc
IB: M2
IB: MDM2
170
110
78
60
MW(kD)
Ub-FOXO1
FOXO1
MDM2
- 91 -
Figure 30 MDM2 promotes the ubiquitination of FOXO3A. H1299 cells were
transfected with the indicated plasmids and treated with MG132 for 6 h before
harvest. Cell lysates were subjected to pull down analyses with Ni-NTA beads
followed by immunoblotting with anti-FOXO3A antibody. Cell extracts were also
subjected to direct immunoblotting by anti-MDM2 antibody.
MDM2+-+--His-Ub+++++
+-
FOXO3A (AAA)+---FOXO3Awt-++-
MDM2+-+--His-Ub+++++
+-
FOXO3A (AAA)+---FOXO3Awt-++-
MW(kD)
IB: FOXO3A
170110
7860
170
110
78
60
IB: MDM2
Ni-NTA pull down
Ub-FOXO3A
MDM2
- 92 -
Figure 31 The MDM2 ring finger domain is critical for the ubiquination of
FOXO1. p53-/-,MDM2-/-MEF cells were transfected with the indicated plasmids
including Myc-tagged ubiquitin and cell extracts were either immunoprecipitated
with M2 antibody followed by immunoblotting with an anti-Myc antibody or
immunoblotted directly with antibodies to Flag or MDM2.
4-++
MDM2 (μg)-42--MDM2 (1-361)2----
Myc-Ub+++++Flag-FOXO1++++-
4-++
MDM2 (μg)-42--MDM2 (1-361)2----
Myc-Ub+++++Flag-FOXO1++++-
IP: M2IB: Myc
IB:MDM2
IB:M2
170
110
78
MW(kD)
Ub-FOXO1
MDM2
MDM2(1-361)
FOXO1
- 93 -
-++++++++++FOXO1 -++++++++++FOXO1
CTL
MD
M2
MD
M2(
1-36
1)
MD
M2(
50-4
91)
MD
M2(
NES
mut
ant)
MD
M2(
NLS
mut
ant)
MD
M2(
457S
)
MD
M2(
Δ222
-325
)
MD
M2(
Δ222
-437
)
MD
M2(
361
mut
ant)
CTL
Ni-NTApull down
IB:FOXO1
IB:MDM2
IB:FOXO1
Figure 32 Ubiquitination of FOXO1 requires the MDM2 ubiquitin ligase
function. P53-/-, MDM2-/- MEFs were transfected with Flag-FOXO1, His-
Ubiquitin and MDM2 mutants. Cell extracts were subjected to pull down assays
with Ni-NTA beads followed by immunoblotting with anti-FOXO1 antibody.
170
110
78
63
47
M.W.(kD)
110
78
63
47
Ub-FOXO1
MDM2
FOXO1
- 94 -
Figure 33 The central region of MDM2 cannot promote the
polyubiquitination of FOXO1. H1299 cells were transfected with 2 μg Flag-
FOXO1, 4 μg MDM2, and 4 μg His-ubiquitin. Cell extracts were subjected to pull
down analyses with Ni-NTA beads followed by immunoblotting with anti-FOXO1
antibody.
Vect
or
FL Δ50-
89
Δ89-
150
Δ150
-230
Ni-NTAPull Down
IB: FOXO1
IB: MDM2
IB: FOXO1
75
100
150
MW(kD)
Ub-FOXO1
MDM2
FOXO1
- 95 -
Figure 34 MDM2 promotes FOXO1 polyubiquitination in vitro. GST-MDM2
and GST-N (containing MDM2 residues 1 to 150) were purified using glutathione
agarose beads. Loaded beads were incubated with in vitro-translated FOXO1 in
the presence or absence of E1 and E2 in an ubiquitination reaction as described
in Materials and Methods. Polyubiquitinated FOXO1 appears as a high
molecular weight smear above the unmodified FOXO1 band.
(35S labeled)
Ub-FOXO1
(35S labeled)
Ub-FOXO1
75
100
150
E1+E2+Ub+-+GST-MDM2++-GST-MDM2-NT--+FOXO1+++
E1+E2+Ub+-+GST-MDM2++-GST-MDM2-NT--+FOXO1+++
+++
E1+E2+Ub+GST-MDM2+FOXO1-
+++
E1+E2+Ub+GST-MDM2+FOXO1-
75
100
150
MW(kD)
MW(kD)
- 96 -
data and whole-cell ubiquitination analysis with MDM2 mutants, the in vitro data
established that MDM2 functions as an ubiquitin E3 ligase for FOXO proteins.
5. MDM2 suppresses the expression of FOXO target genes and protects cells
from FOXO1-induced cell death.
FOXO target genes tumor necrosis related apoptosis inducing ligand (TRAIL),
p27 CDK inhibitor and manganese superoxide dismutase (MnSOD) mediate the effect of
FOXO proteins on cell cycle arrest, apoptosis, and detoxification of reactive oxygen
species. Their transcription products are directly regulated by FOXO factors. Consistent
with the ubiquitination and degradation of FOXO factors by MDM2, stable expression of
MDM2 in H1299 cells decreased the level of TRAIL, p27 and MnSOD expression (Figure
35) whereas the knockdown of MDM2 increased the expression of TRAIL (Figure 36).
To test whether MDM2 protects cells from FOXO induced cell death, H1299 cells
were transiently transfected with GFP and either control vector, FOXO1 or FOXO1 in
combination with MDM2. The survival of the transfected cells was analyzed. The
expression of FOXO1 decreased the number of transfected (green) cells, an effect that
is relieved by MDM2 co-expression (Figure 37a). To confirm that the change in the
viability of transfected cells is the result of cell apoptosis, transfected cells were fixed
and stained with DAPI and their nuclear morphology was examined for features of
apoptosis under a fluorescence microscope that allows the simultaneous visualization of
blue and green fluorescence. Apoptotic index, as determined by scoring apoptotic cells
in 300 green cells per sample, was 5% for controls and 25% for cells transfected with
FOXO1. Co-expression of MDM2 suppressed FOXO1-induced cell death in a dose-
dependent manner (Figure 37b). The data were reproduced by independent
- 97 -
IB:MDM2
IB:FOXO3A
IB:MnSOD
IB:p27
IB:CyclinD
H129
9-VE
CH12
99-M
1
IB:FOXO1
Figure 35 Stable expressed MDM2 in H1299 cells regulates the
expression of downstream targets of FOXO. Extracts of control (H1299-VEC)
and MDM2 stable (H1299-M1) clones were subjected to immunoblotting with
different antibodies as indicated in the figure.
IB:β-actin
IB:TRAIL
MDM2
FOXO3A
FOXO1
MnSOD
p27
TRAIL
Cyclin D
β-actin
- 98 -
IB:MDM2
IB:β-actin
siRNA CTL MD
M2-
1
IB:FOXO3A
IB:TRAIL
Figure 36 MDM2 siRNA increases the expression of the FOXO1 target gene
TRAIL. H1299 cells were transiently transfected with either GFP-siRNA or MDM2-
siRNA. The cells were harvested at 48 h later. Cell extracts were subjected to
immunoblotting analyses with anti-MDM2, anti-FOXO3A and anti-TRAIL
antibodies.
MDM2
FOXO3A
TRAIL
β-actin
- 99 -
6h
FOXO1 FOXO1+MDM2
9h
20h
pcDNA3
0%
10%
20%
30%
0.40.200MDM2(ug)
+++-Flag-FOXO1
0.40.200MDM2(ug)
+++-Flag-FOXO1
Apop
tosi
s in
dex(
%)
a
b
Figure 37 MDM2 promotes the cell survival in the presence of FOXO1.
- 100 -
Figure 37 MDM2 promotes the cell survival in the presence of FOXO1.
a, H1299 cells were transfected with pLNCE and Flag-FOXO1 in the presence
or absence of MDM2. The viability of transfected cells in each well was scored
by counting the number of green cells. Representative micrographs were
captured by the fluorescence microscope that had a charge-coupled device
camera. b, H1299 cells were transfected with the same plasmids as in a,
Apoptotic index of GFP-positive cells was determined by scoring 300 GFP-
positive cells for chromatin condensation and nuclear fragmentation. Triplicate
samples were analyzed per data point, and the graph represents three
independent experiments.
- 101 -
0
10
20
30
40
Apop
tosi
s (%
)MDM2 (μg)
Flag-FOXO1
+
+
+
-
--
+-
MDM2 (μg)
Flag-FOXO1
+
+
+
-
--
+-
49.0%
32.4%
FOXO1
13.1%10.4%10.7%1
31.33%-16.7%2
FOXO1+MDM2MDM2ControlExp. No.
49.0%
32.4%
FOXO1
13.1%10.4%10.7%1
31.33%-16.7%2
FOXO1+MDM2MDM2ControlExp. No.
0
10
20
30
40
+
+
MDM2 (μg)--
Flag-FOXO1+-
+
+
MDM2 (μg)--
Flag-FOXO1+-
Apop
tosi
s (%
)
a
Figure 38 MDM2 protects cells from FOXO1-induced cell death measured
by Flow Cytometry.
- 102 -
Figure 38 MDM2 protects cells from FOXO1-induced cell death measured
by Flow Cytometry. H1299 cells were transfected with GFP-spectrin and Flag-
FOXO1 in the presence or absence of MDM2. The apoptotic GFP positive cells
were detected with Annexin-V APC and 7-AAD. a, bar graphs; b,representative
flow cytometry profiles.
0.6 1.1
10.7
1.72 2.12
10.4
1.52 4.13
32.4
1.18 2.84
13.1
7AA
D
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
101 102 103 104 101 102 103 104
101 102 103 104 101 102 103 104
Annexin-V APC
Control MDM2
87.6 85.7
62 82.9
FOXO1 FOXO1+MDM2
b
- 103 -
analysis of early apoptosis with the Annexin V method after FACS-based sorting. As
shown in Figure 36, two independent analyses of cells cotransfected with GFP-spectrin
showed that FOXO1 expression increased apoptosis in transfected cells by about 3-fold,
which is partially suppressed by the co-tranefection of MDM2. The degree of induction
by FOXO1 and the suppression by MDM2 varied between two experiments because the
basal line of the FACS machine varied from time to time.
6. MDM2 transiently increased FOXO transcriptional activity.
Since FOXO factors are transcriptional factors, an important question which must
be addressed is whether or not MDM2 affects the FOXO transcriptional activity. In order
to answer this question, the following reporter genes were used in our experiment.
Synthetic reporter 3 × IRSLuc which contained three conserved insulin response
sequence (IRS). FOXO1, like insulin, promotes the promoter activity through an IRS.
Cyclin D is the downstream target of FOXO factors in the cell cycle check point. FOXO
factors expression results in reduced levels of cyclin D protein expression. Cyclin D-Luc
is the reporter gene which can be used to specifically measure cyclin D activity in cells.
In order to measure the activity of the reporter gene, a transient transfection was
performed by transfecting the reporter gene, CMV-gal as an internal control, pcDNA3
control or Flag-FOXO1, and different doses of MDM2 into the DU145 cells and NIH3T3
cells. The data showed that MDM2 increased the 3 × IRSLuc activity, and this increase
occurred in a dose dependent manner (Figure 39 & Figure 40). FOXO1 decreased the
cyclin D-Luc activity and MDM2 decreased it further in a dose dependent manner
(Figure 41).
Our earlier data revealed that MDM2 interacted with both wild-type FOXO1 and
FOXO1 (AAA), however it only promoted the polyubiquitination of wild-type FOXO1.
- 104 -
05
1015202530354045
MDM2-0ugMDM2-0.1ugMDM2-0.3ugMDM2-0.5ug
3×IR
SLuc
act
ivity
(R
LU, ×
105 )
Endo-FOXO Flag-FOXO1wt
IB:M2
IB:MDM2
Figure 39 MDM2 increases the transcriptional activity of FOXO1 in a
dose-dependent manner in DU145 cells. DU145 cells were transfected with
0.5 μg 3×IRSLuc, 0.1 μg CMV-Gal, 0.1 μg control vector or Flag-FOXO1, and
different amounts of MDM2. FOXO activity was measured by the Promega
luciferase activity kit.
- 105 -
02468
10121416
3×IR
SLuc
act
ivity
(R
LU, ×
105 )
MDM2
NIH3T3
Figure 40 MDM2 increases the transcriptional activity of FOXO1 in
NIH3T3 cells. NIH3T3 cells were transfected with 0.5 μg 3×IRSLuc, 0.1 μg
CMV-Gal, 0.1 μg Flag-FOXO1, and different amounts of MDM2. FOXO
activity was measured by the Promega luciferase activity kit.
- 106 -
Cyc
linD
Luc
activ
ity (R
LU, ×
105 )
-MDM2
++++-FOXO1
-MDM2
++++-FOXO1
0
2
4
6
8
10H1299 cells
Figure 41 MDM2 enhances the ability of FOXO1 to inhibit cyclin D1.
H1299 cells were transfected with 0.5 μg cyclin D-Luc, 0.1 μg CMV-Gal, 0.1
μg Flag-FOXO1 and different amounts of MDM2. FOXO activity was
measured by the Promega luciferase activity kit.
- 107 -
Figure 42 MDM2 increases the transcriptional activity of both wild type
FOXO1 and the FOXO1(AAA) mutant in NIH3T3 and DU145 cells. Cells were
transfected with 0.5 μg 3×IRSLuc, 0.1 μg CMV-gal, 0.1 μg Flag-FOXO1 or
FOXO1 (AAA) and different amounts of MDM2. FOXO activity was measured by
the Promega luciferase activity kit. a, DU145; b, NIH3T3.
3×IR
SLuc
act
ivity
(R
LU, ×
105 )
3×IR
SLuc
act
ivity
(R
LU, ×
106 )
a
b
0
5
10
15
20
25
30
FOXO1wt FOXO1(AAA)
MDM2-0ugMDM2-0.3ugMDM2-0.5ug
0
5
10
15
20
25
30
endo-FOXO1 FOXO1wt FOXO1 (AAA)
MDM2-0ugMDM2-0.3ugMDM2-0.5ug
- 108 -
MDM2 also increased the transcriptional activity of FOXO1 (AAA) (Figure 42). The data
further indicated that MDM2 promotes FOXO transcriptional activity in an AKT-
independent fashion.
7. p53 induces transient increase in the transcriptional activity of FOXO factors,
which is followed by FOXO degradation in an MDM2-dependent manner.
To test whether p53 affects the transcriptional activity of FOXO1, reporter gene 3
× IRSLuc, CMV-gal, pcDNA3 control or Flag-FOXO1 or Flag-FOXO1 (AAA), and
different doses of p53 were transfected into DU145 cells (contain endogenous mutated
p53) and LNCaP cells (contain endogenous wild type p53) and the transcriptional activity
of FOXO1 was detemined. p53 decreased FOXO1 transcriptional activity in both cell
lines (Figure 43a & Figure 44), but did not inhibit FOXO1-induced decrease in cell
viability (Figure 43b). Introduction of MDM2 into the cells relieved the inhibition of FOXO
activity by p53 (Figure 45).
In order to better understand the interaction among FOXO factors, p53 and
MDM2, H1299/V138 stable cell line that expresses a temperature-sensitive p53 was
used. Shifting to a permissive temperature allows the activation of p53. At different time
points after p53 activation, the transcriptional activity of endogenous FOXO factors and
the level of FOXO3A and MDM2 protein expression were determined. Reporter analyses
showed that FOXO activity was transiently induced between 5 and 24 hours after p53
activation and subsequently decreased (Figure 46a). Immunoblotting analysis showed
that the MDM2 protein was induced to the highest level 5 hours after p53 activation
follwed by gradually decrease of FOXO3A and MDM2 level (Figure 46b). MG132
treatment prevented the time-dependent decrease in FOXO3A levels (Figure 47),
suggesting that the decrease is due to proteasome-mediated degradation. In order to
- 109 -
Figure 43 P53 inhibits the transcriptional activity of FOXO in a dose-
dependent manner in DU145 cells, but not FOXO1-induced cell death. a, DU145
cells were transfected with 0.5 μg 3×IRSLuc, 0.1 μg CMV-Gal, 0.1 μg Flag-FOXO1
and different amounts of p53. FOXO activity was measured by the Promega
luciferase activity kit. b, Cells were transfected with pLNCE and plasmids as
described in panel a. Representative micrographs were captured by the
fluorescence microscope that had a charge-coupled device camera.
0
20
40
60
80
100
120
pcDNA3 FOXO1wt FOXO1(AAA)
p53-0ugp53-0.02ugp53-0.1ugp53-0.3ug
3XIR
SLuc
act
ivity
(R
LU, ×
104 )
CTL FOXO1
FOXO1+p53 FOXO1+p53+MDM2
b
a
- 110 -
Figure 44 P53 inhibits the transcriptional activity of FOXO in a dose-
dependent manner in LNCaP cells. DU145 cells were transfected with 0.5
μg 3×IRSLuc, 0.1 μg CMV-Gal, 0.1 μg Flag-FOXO1 and different amounts
of p53. FOXO activity was measured by the Promega luciferase activity kit.
0
2
4
6
8
10
pcDNA3 FOXO1wt FOXO1(AAA)
p53-0ugp53-0.02ugp53-0.1ugp53-0.3ug
3XIR
SLuc
act
ivity
(R
LU, ×
104 )
- 111 -
3×IR
SLuc
act
ivity
(R
LU ×
106 )
Figure 45 MDM2 relieves the repression of FOXO1 activity by p53.
DU145 cells were transfected with 0.5 μg 3×IRSLuc, 0.1 μg CMV-gal, 0.1 μg
Flag-FOXO1, 0.1 μg HA-p53 and 0.5 μg of either control vector or MDM2.
FOXO activity was measured by luciferase activity kit (Promega).
0
1
2
3
4
5
6
FOXO1wt FOXO1(AAA)
mtp53wtp53wtp53+MDM2
- 112 -
FOXO3A
MDM2
p53
0 1 3 5 7 12 18 32 ºC (h)
Figure 46 MDM2 transiently increases FOXO transcriptional activity,
which is followed by FOXO degradation . H1299/V138 cells cultured at 39oC
were shifted to 32ºC for the indicated length of time. Cell extracts were
prepared and assayed by luciferase assay (panel a) or by immunoblotting
analyses (panel b).
0
0.5
11.5
2
2.5
3
0h 1h 3h 5h 9h 24h 48h 72h
3×IR
SLuc
act
ivity
(R
LU, ×
105 )
a
b
IB:FOXO3A
IB:MDM2
IB:p53
- 113 -
18950950
++++----
18950950
++++----
FOXO3A
β-actin
18 32ºC (h)
MG132
32ºC (h)
MG132
75
50
100
150
Figure 47 MG132 relieves p53-induced decrease in the expression of
FOXO3A protein in H1299/V138 cells. Cells cultured at 39°C were shifted to
32ºC for indicated length of time. Cells were treated with MG132 for 6 h before
cell extracts were prepared and subjected to immunoblot analysis.
IB: FOXO3A
IB: β-actin
- 114 -
HA-p53MDM2Flag-FOXO1
-++-+-++---+++++--
HA-p53MDM2Flag-FOXO1
-++-+-++---+++++--
IB:HA
IB:MDM2
IB:M2
IB: Tubulin
p53
MDM2
FOXO1
Tubulin
Figure 48 p53 decreases the protein level of FOXO1 through MDM2.
H1299 cells were transiently transfected as noted on the Figure. 24h after
transfection, cell lysates were subjected to immunoblotting with M2 antibody
(to detect FOXO1 expression), anti-HA antibody (to detect p53 expression),
and anti-MDM2 (SMP14). Tubulin blots was included to show even loading in
each sample.
- 115 -
FOXO3A
32-+
MDM2-siRNA-++Temperature (ºC)373237
GFP-siRNA+--
32-+
MDM2-siRNA-++Temperature (ºC)373237
GFP-siRNA+--
β-actin
MDM2
Figure 49 Knockdown of MDM2 partially relieves p53-induced FOXO3A
downregulation. H1299/V138 cells were transfected with scrambled or MDM2
siRNA. 24 h posttransfection, cells were shifted to 32ºC and cultured for
another 18 h. Then immunoblotting was performed with the indicated
antibodies.
IB: FOXO3A
IB:MDM2
IB:β-actin
- 116 -
determine which protein led to the degradation of FOXO factors, a transient transfection
experiment was performed in H1299 cells. Immunoblotting analyses showed that p53 did
not decrease the level of FOXO1 protein expression. MDM2, either alone or in
combination with p53, decreased the level of FOXO1 protein expression (Figure 48).
Knockdown of MDM2 by siRNA partially relieved FOXO3A down-regulation by active
p53, supporting the conclusion that the p53-induced decrease is MDM2-dependent
(Figure 49).
8. Site-specific sumoylation of SIRT1 regulates FOXO1 transcriptional activity
and stability.
Acetylation can affect protein degradation via ubiquitination (Ito et al., 2002;
Jeong et al., 2002; Li et al., 2002). The study in pancreatic β cells showed that FOXO1
acetylation inhibited the degradation of FOXO1 via the ubiquitin pathway (Kitamura et
al., 2005).
SIRT1 is a mammalian HDAC. As a potential nutrient sensor, it regulates the
lifespan of mammals in response to caloric restriction or nutrient starvation, and protects
cells from apoptosis induced by DNA damage.
Previously (Brunet et al., 2004) and our studies (Figure 50) showed that SIRT1
inhibited FOXO1 transcriptional activity. After treating the H1299/V138 cells with SIRT1
inhibitor nicotinamide, we found that nicotinamide caused further increase of FOXO1
activity from 24 hours to 48 hours post p53 activiation (Figure 51). Therefore SIRT1 has
a role in the action of p53-MDM2 on the activity and degradation of FOXO1.
Since SIRT1 affects the FOXO1 transcriptional activity and plays a potential role
in FOXO1 stability, two very interesting questions arise: How does SIRT1 affect the
transcriptional activity of FOXO1 and how is SIRT1 activity regulated. Our previous study
- 117 -
Figure 50 SIRT1 inhibits the transcriptional activity of FOXO. H1299 cells
were transfected with 0.5 μg 3×IRS Luc, 0.1 μg CMV-Gal, 0.1 μg Flag-FOXO1 and
0.5 μg HA-SIRT1. FOXO activity was measured by the Promega luciferase activity
kit.
3×IR
SLuc
act
ivity
(R
LU ×
105 )
0
5
10
15
20
25
1 2 3
+--SIRT1
++-FOXO1
+--SIRT1
++-FOXO1
- 118 -
3×IR
SLuc
act
ivity
(R
LU, ×
105 )
Figure 51 Nicotinamide treatment increases the FOXO transcriptional
activity. H1299 and H1299/V138 cells were transfected with 0.5 μg 3×IRS
Luc and 0.1 μg CMV-Gal. 16 h later, the cells were treated with nicotinamide
and transferred from 37°C to 32°C at indicated time points. FOXO activity
was measured by the Promega luciferase activity kit.
0
2
4
6
8
10
12
14
16
18
0h 1h 3h 5h 8h 24h 48h
H1299-V
H1299-V-NIC
H1299
H1299-NIC
- 119 -
Figure 52 SIRT1 sumoylation at Lys 734 is required for FOXO1
deacetylation. H1299 cells were co-transfected with Flag-FOXO1, HA-p300
and either wild-type HA-SIRT1 (WT) or HA-SIRT1 mutated at Lys 734
(734R). Anti-Flag M2 immunoprecipitates were immunoblotted with antibody
to acetylated FOXO1 or with an antibody to FOXO1. Cellular extracts were
also immunoblotted with antibody to HA.
IP:M2
IB:Acetyl -K
IB:M2
IB:HA
IB:HA
+++-HA-p300
734RWT--HA-SIRT1
++++Flag-FOXO1
+++-HA-p300
WT--HA-SIRT1
++++Flag-FOXO1
p300
SIRT1
- 120 -
CTL p300
Figure 53 Mutation of Lys 734 relieves the inhibition of FOXO1
transcriptional activity by SIRT1. The activity of transfected FOXO1 was
measured using an IGFBP1 promoter-based reporter in H1299 cells expressing
wild type SIRT1 or SIRT1 (734R) in the absence or presence of p300. CTL-
control.
0
5
10
15
20
25
30
35
1 2
pcDNA3FOXO1FOXO1+SIRT1FOXO1+SIRT1(734R)
3×IR
SLuc
act
ivity
(RLU
, ×10
5 )
- 121 -
showed that the sumoylation status of SIRT1 affects its deacetylase activity since a
sumoylation site specific mutation abolished the deacetylase activity of SIRT1.
FOXO1 is a known substrate of SIRT1. In order to determine whether this
sumoylation site affects the effect of SIRT1 on FOXO, Flag-tagged FOXO1 was
cotransfected with p300, wild-type SIRT1 or SIRT1 (734R) in which the sumoylation site
of SIRT1 was mutated, acetylated FOXO1 was detected by Western blotting of M2
immunoprecipitates with an antibody that recognizes acetylated FOXO1. As shown in
Figure 52, expression of wild type SIRT1 reduced amount of acetylated Flag-FOXO1
induced by p300, whereas expression of SIRT1 mutated at Lys 734 did not. When the
cells transfected either FOXO1 with SIRT1, SIRT1 decreased FOXO1 transcriptional
activity which is consistent with the previous study, whereas expression of SIRT1
mutated at Lys734 could not decrease the transcriptional activity of these two proteins
(Figure 53). These data showed that the sumoylation status of SIRT1 is critcal for its
effect on FOXO1.
9. Genistein-induced FOXO1 expression is blocked by MDM2 expression in
H1299 cells.
Genistein is considered the primary anticancer component of soybeans. Its in
vitro and/or in vivo activities include the antagonism of estrogen, inhibition of protein
tyrosine phosphorylation, suppression of angiogenesis, inhibition of hydrogen peroxide
formation induced by tumor promoters, inhibition of topoisomerases, induction of
apoptosis and cell differentiation, scavenging of free radicals, and inhibition of
carcinogenesis and tumor promotion. A previous paper showed that genistein down-
regulated the MDM2 oncogene, induced apoptosis and inhibited proliferation in a variety
of human cancer cell lines, regardless of p53 status(Li et al., 2005). This raised an
- 122 -
FOXO1
MDM2
Genistein (uM) 0 5 15 50 0 5 15 50Genistein (uM) 0 5 15 50 0 5 15 50
H1299-VEC H1299-M1
Figure 54 Genistein increases the expression of FOXO through MDM2
downregulation. H1299-VEC and H1299-M1 cells were plated in 100 mm
dishes and treated with indicated doses of genistein for 24 h. Cell lysates were
subjected to immunoblotting with anti-FOXO1 and anti-MDM2 antibodies.
IB:FOXO1
IB:MDM2
IB:β-actin β-actin
- 123 -
interesting question whether genistein’s anti-tumor activity is mediated through FOXO
factors.
To address this question, parental H1299 cells and H1299 cells that stably
express MDM2 (H1299-M) were plated in 100 mm dishes and treated with genistein at
different doses. The expression of MDM2 and FOXO1 was determined by Western blot
ananlysis. The analyses revealed that genistein decreased MDM2 and increased
FOXO1 level in a dose-dependent manner in H1299 parental cells. In H1299-M cells,
MDM2 expression was detected at a very high level. Genistein treatment had
little effect on the expression of MDM2 or FOXO1 proteins (Figure 54). The data
indicated that genistein increases FOXO1 level through MDM2 down regulation. It also
indicated that FOXO factors may be one of the nuclear targets of genistein in
suppressing tumorigenesis.
10. ARF promotes the MDM2-induced FOXO ubiquitination.
ARF is encoded by the INK4a-ARF locus and it is a known inhibitor of the MDM2
E3 ligase function on p53. To test whether ARF affects MDM2 E3 ligase function of
FOXO factors, H1299 cells were transfected with Flag-FOXO1, MDM2, myc-ARF and
His-ubiquitin as indicated in the figure 53. The cell lysates were purified by Ni-NTA
beads. The ubiquitinated-FOXO1 was detected by M2 anti-Flag antibody. As shown in
Figure 63, ARF increased FOXO1 ubiquitination level to a degree similar to that induced
by MDM2 (Figure 55). The combination of ARF and MDM2 increased the level of
FOXO1 ubiquitination to a degree comparable with that of MDM2 alone, suggesting that
AR may act through MDM2 to regulate FOXO ubiqutination (Figure 55). These data are
quite opposite to p53 ubiquitnation induced by MDM2 but are consistant with our
conclusion that FOXO ubiquitination occurs in the cytoplasm. It is known that ARF
inhibits the target of MDM2 to nucleoli to supprees the ubiquitination of p53.
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+++++His-Ubiquitin+-+--Myc-ARF++---MDM2++++-FOXO1
+++++His-Ubiquitin+-+--Myc-ARF++---MDM2++++-FOXO1
Ni-NTA pull down
IB:FOXO1
IB:myc
IB:M2
13
18
1107860
47
170
60
78
110
170
IB:MDM2
Ub-FOXO1
ARF
FOXO1
MDM2
Figure 55 ARF promotes the MDM2-induced FOXO1 ubiquitination. H1299
cells were transfected with 2μg Flag-FOXO1, 4 μg MDM2, 4 μg myc-ARF, 4 μg
His-ubiquitin as described on the Figure. Cell extracts were pull-down by Ni-NTA
beads and the expression levels were detected by an anti-FOXO1 antibody.
- 125 -
DISCUSSION
Mammlian FOXO factors play very important roles in development, glucose
metabolism, cancer, aging, and energy homeostatis. Several signaling pathways
regulate their activity, such as the insulin pathway, glucose, IGF1 and oxidative stress. In
this study, we found that MDM2 is the E3 ubiquitin ligase for FOXO factors. MDM2
interacts with FOXO factors and promotes their ubiquitination and degradation. It also
affects their biological function and downstream targets expression of FOXO.
1. MDM2 is the E3 ubiquitin ligase of FOXO.
FOXO proteins are known to be ubiquitinated and their level of expression is
regulated by proteasome mediated degradation (Aoki et al., 2004; Plas and Thompson,
2003), but a general ubiquitin E3 ligase for FOXO proteins remains to be identified.
Skp2 was reported to inhibit FOXO1 in tumor suppression through ubiquitin-
mediated degradation but such activity appears to be restricted to FOXO1 (Huang et al.,
2005).
The present study identified MDM2 as a general ubiquitin E3 ligase for
mammalian FOXO factors. We present multiple lines of evidences to support this
conclusion. First, the manipulation of MDM2 expression by genetic deletion, knock down
MDM2 with siRNA or overexpression of MDM2 using transient or stable transfection
leads to change of the level of FOXO1 and FOXO3A proteins in opposite directions.
Second, the down regulation of FOXO1 protein by MDM2 is relieved by treatment with a
proteasome inhibitor, MG132, and the half-life of FOXO1 protein is decreased by ectopic
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MDM2. Third, MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination.
The ubiquitination depends on both the ability of MDM2 to bind FOXO proteins as well
as its E3 ligase activity. This is supported by the evidence that mutant MDM2 (∆150-230)
that is missing in the FOXO binding region did not stimulate FOXO1 ubiquitination
although it contains an intact E3 ring finger domain. Moreover, MDM2 mutants with
either deletion MDM2 (1-361) or mutation MDM2 E3-mt (457S) at the C-terminus ring
finger region lost their ability to stimulate FOXO ubquitination even though they are still
able to interact with FOXO1. Finally, in test tube reaction, recombinant MDM2 catalyzed
the ubiquitination of FOXO1, supporting a direct substrate-enzyme relationship.
2. MDM2-promoted ubiquitnation of FOXO depends on phosphorylation on PKB
sites.
In response to stimulation by IL-3, insulin or PDGF (platelet derived growth
factor), the activated PKB oncogene triggered proteasome-dependent degradation of its
substrates including FOXO1 and FOXO3A (Aoki et al., 2004; Matsuzaki et al., 2003;
Plas and Thompson, 2003). In our study, ubiquitination was found to depend on
phosphorylation at sites that mediate the cytoplasmic localization of FOXO factors by
PKB. FOXO1 (AAA), in which all three PKB sites are mutated to non-phosphorylatable
alanine, did bind to MDM2 (Figure 19) but its level of ubiquitination was not affected by
MDM2 (Figure 29). Similarly, ubiquitination of FOXO3A (AAA) was unaffected by MDM2
(Figure 30). These data suggest that MDM2 acts as a conditional E3 ligase for FOXO
proteins, which is only functional after FOXO phosphorylation at AKT sites. Previous
studies (Feng et al., 2004; Zhou et al., 2001) showed that phosphorylation of MDM2 by
PKB inhibits its interaction with ARF, thereby increasing its stability and promoting its
ability to bind and degrade p53. It remains to be determined whether the MDM2
phosphorylation active PKB regulates its ability to stimulate FOXO ubiquitination.
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3. MDM2-promoted ubiqutination of FOXO happens mainly in the cytoplasm.
Protein ubiquitination and degradation occur in both the nucleus and cytoplasm
(Yu et al., 2000). In our analysis, the nuclear MDM2 in which the nuclear export
sequence is mutated contains an intact E3 region and is able to bind FOXO1 but unable
to increase FOXO1 ubiquitination, on the other hand, the cytoplasmic MDM2, both the
MDM2 NLS-mt and MDM2 (∆89-150), stimulated ubiquitination, suggesting that it is the
cytoplasmic MDM2 that functions as an E3 ligase for FOXO factors. In combination with
the data that showed that nuclear FOXO1 (AAA) is not ubiquitinated by MDM2, it is
reasonable to assume that FOXO ubiquitination occurs in the cytoplasm. However, such
a conclusion might seem incompatible with the immunofluorescence staining data
showing that the colocalization of MDM2 and FOXO occured mainly in the nucleus. It
should be pointed out that the colcoalization studies were performed in cells grown in a
medium containing low levels of serum, which minimizes PKB activity and inhibits FOXO
cytoplasmic localization and degradation. Consistent with the degradation in the
cytoplasm, the interaction between cytoplasmic MDM2 and FOXO1 was difficult to
detect unless proteasome activity was inhibited by MG132. The fact that MDM2 NLS-mt
that cannot enter the nucleus interacting with FOXO1 and stimulating its ubiquitination
suggests that a nuclear interaction between MDM2 and FOXO proteins is not required
for FOXO ubiquitination. Presently, the details of the nuclear interaction between MDM2
and FOXO proteins are unclear. It is possible that nuclear interaction between FOXO
factors and MDM2 may exert an effect on FOXO factors that is distinct from degradation.
In the case of p53 ubiquitination, it has been shown that monoubiquitination by MDM2
promotes the cytoplasmic localization of p53 whereas the polyubiquintination stimulates
the degradation of p53 in nucleus (Li et al., 2003a).
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4. MDM2 affects both the transcriptional activity and ubiquitination of FOXO.
FOXO factors are transcription factors. On one hand, FOXO1, FOXO3A and
FOXO4 directly bind to the promoters of TRAIL, Bim, p27, MnSOD, GADD45, G6Pase,
PEPCK, TAT and IGFBP-1 and of others. On the other hand, FOXO can also indirectly
regulate other genes, such as cyclin D1. In our studies, a positive effect of MDM2 on the
transcriptional activity of FOXO1 was detected in transient transfection reporter assays.
It also promoted the ubiquitination and degradation of FOXO factors, leading to FOXO
protein level decreases. It is not fully understood as to how MDM2 decreases FOXO
proteins expression level. There are three possible mechanisms that might be
responsible for MDM2’s positive effect on transcriptional activity of FOXO: 1) MDM2
promotes the transcriptional activity first, followed by its role in FOXO degradation
because transcriptional activity data in H1299/V138 cells showed that FOXO activity was
transiently induced between 5h and 48 h post p53 activation and subsequently FOXO
activity decreased (Figure 47a); 2) FOXO4 was recently shown to be monoubiquitinated,
which increases its transcriptional activity (van der Horst et al., 2006) and this raises the
second possibility. MDM2 plays two roles in the regulation of FOXO factor. It may
promote FOXO transcriptional activity through monoubiquitnation but degrade FOXO
protein through polyubiquitnation; 3) About 20 proteins are reported to interact with
MDM2, and p300 is reported to play a critical role in the MDM2-directed turnover of p53
(Grossman et al., 1998) and this raises the third possibility. Different cofactors are
involved in the regulation of FOXO factors by MDM2 leading to different outcomes.
In our p53 study, Mdm2-mediated monoubiquitylation of p53 greatly promoted its
mitochondrial translocation and thus its apoptosis in the mitochondria. Upon entrance in
the mitochondria, p53 undergoes rapid deubiquitylation by mitochondrial HAUSP via a
stress-induced mitochondrial p53-HAUSP complex (Marchenko et al., 2007). FOXO4
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was also reported to become monoubiquitinated in response to increased cellular
oxidative stress, resulting in its re-localization to the nucleus and an increase in its
transcriptional activity. Deubiquitination of FOXO is catalyzed by the deubiquitinating
enzyme HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts
with and deubiquitinates FOXO in response to oxidative stress. We found that MDM2
suppresses the expression of FOXO1 target genes and protects cells from FOXO1-
induced apoptosis. These observations are consistent with the effect of MDM2 on the
translocation change and degradation of FOXO facotors. Further study is required to
understand the biological effect of MDM2 promoted transcriptional activity of FOXO
factors and to estabolish whether FOXO factors have any mitochondrial function.
5. Mammalian FOXO factors interact with p53.
Mammalian FOXO factors and p53 are tumor suppressors and the regulators of
aging that act similarly in many ways. They induce apoptosis (Modur et al., 2002) and
cell cycle arrest (Medema et al., 2000; Nakamura et al., 2000; Schmidt et al., 2002) and
regulate cellular responses to DNA damages and stress (Essers et al., 2004; Kajihara et
al., 2006) through transcriptional induction of a similar set of target genes such as the
p21 CDK inhibitor, Fas ligand and GADD45. In our study, we found that ectopically
expressed p53 inhibited FOXO transcriptional activity in either DU145 cells (contain
inactive p53) or LNCaP (contain wild type p53) but did not decrease FOXO1 induced cell
death (Figure 44b). Two subsequent papers showed that p53 interacted with FOXO3A in
the presence of stress and active FOXO3A could induce p53-dependent apoptosis and
promote p53 cytoplasmic accumulation by increasing its association with the nuclear
exporting machinery. All these evidence indicate that the regulation of transcriptional
activity of p53 and FOXO factor is independent of the regulation of the proapoptotic
activity of p53 and of FOXO factors.
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p53 and MDM2 form a feedback loop, in which p53 induces MDM2 by activating
MDM2 transcription, and MDM2 in turn negatively regulates p53 by binding and
promoting p53 ubiquitination and degradation (Jin and Levine, 2001). This feedback loop
keeps the p53 activity in check under normal conditions. Upon DNA damages, the
interaction between MDM2 and p53 is suppressed, resulting in an increased p53 activity
that triggers apoptosis. Our data suggest that MDM2 might act as a general coordinator
to turn off multiple negative growth regulators when p53 senses that the DNA damage
has been repaired (Figure 56). During our investigations, several studies documented a
functional interaction between p53 and FOXO3A. In the presence of hydrogen peroxide,
p53 and FOXO3A form a complex (Brunet et al., 2004). DNA damage promotes
FOXO3A nuclear export through p53-dependent activation of serum and glucocorticoid
activated kinases (You et al., 2004a). In endothelial cells (Miyauchi et al., 2004) and
dermal fibroblasts (Kyoung Kim et al., 2005), inhibition of FOXO3A by PKB or siRNA
promoted senescence-like growth arrest in a p53- and p21-dependent manner. Overall,
p53 and FOXO factors appear to have a complex relationship, the outcome of which is
likely to depend on cellular status and environment.
6. MDM2 regulates FOXO factors in a p53 independent way.
In addition to the negative regulation of p53 activity, MDM2 is overexpressed in
tumor cells and functions as an oncogene to promote cancer cell growth independent of
p53 (Daujat et al., 2001; Ganguli and Wasylyk, 2003). Activated FOXO3A was also
reported to impair the transcriptional activity of p53, but enhanced its pro-apoptotic
function in mitochondria (You et al., 2006b). The present studies suggest that
suppression of FOXO factors may be one of the p53-independent mechanisms by which
MDM2 promotes cancer cell survival and cell growth. Decreased FOXO3A and FOXO1
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MDM2E3 Ligase
p53
FOXO
Proteasome
StimuliOxidative stress,DNA damages,& therapeutic treatments
Ub
UbUb
Ub
UbUb
Degradation
Tumor SuppressionApoptosis,
Senescence,cell cycle arrests,
& DNA repair
MDM2 Overexpressionin Tumor Cells
FOXO
p53
Figure 56 A working model shows the functional interaction among FOXO,
p53 and MDM2 .
- 132 -
activity from loss of PTEN led to a decrease in TRAIL expression and increased survival
of prostatic tumor cells (Modur et al., 2002). Loss of p27 expression is associated with
aggressive behavior in a variety of human epithelial tumors (Catzavelos et al., 1997;
Macri and Loda, 1998). Stable expression of MDM2 in H1299 cells decreased the
expression of FOXO3A together with TRAIL, p27 and MnSOD (Figure 35). Knockdown
of MDM2 by siRNA in H1299 cells increased the level of FOXO3A together with TRAIL
(Figure 36). These results suggest that MDM2 promotes tumorigenesis through down-
regulation of FOXO target genes and that the targeted interruption of FOXO
ubiquitination and degradation by MDM2 may represent an effective strategy for cancer
prevention and therapy.
7. Different domains of MDM2 play different roles in the regulation of p53 and
FOXO factors (see Table 2).
FOXO factor and p53 are the central regulators of cellular reponse to stress,
genotoxic insult and DNA damage. MDM2 interacts with and promotes the degradation
of p53. Our study shows that MDM2 also interacts with and promotes the ubiquitination
and degradation of FOXO. But it became quite clear that different domains of MDM2
play different roles in the regulation of these two proteins. MDM2 (50-491), MDM2 (Δ50-
89) and MDM2 (Δ 90-150) cannot bind with p53 and promotes its ubiquitination, but they
do bind with FOXO and can promote the ubquitination of FOXO factors. MDM2 (Δ 150-
230) binds with p53 and promotes the ubiquitinaion of p53 but it does not interact with
FOXO and promotes the ubiquitnation of FOXO. The nucleo-cytoplasmic shuttling is
critical for MDM2-promoted ubiquitination of p53 because even MDM2 (NES mutant)
and MDM2 (NLS mutant) are capable of binding to p53, but cannot promote its
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N+(Pochampally,1999)
+-+MDM2(361D-A)
N-(Pan,2003)
+-+MDM2(457S)
N-(Kubbutat,1998)
+++MDM2(Δ 222-437)
NND+-+MDM2(Δ 222-325)
C-(Tao, 1999)
+++MDM2(NLS mutant)
N-(Roth,1998)
+-+MDM2(NES mutant)
C+(Dai, 2004)
+--MDM2(Δ 150-230)
C/NND-+±MDM2(Δ 90-150)
NND-++MDM2(Δ50-89)
NND-++MDM2(50-491)
N-(Pochampally,1999)
+-+MDM2(1-361)
N+(Haupt,1997)
+++MDM2
Localization
Promotes the ubquitination of
p53
Binds to
p53
Increases ubiquitination of FOXO1
Binds to
FOXO1
N+(Pochampally,1999)
+-+MDM2(361D-A)
N-(Pan,2003)
+-+MDM2(457S)
N-(Kubbutat,1998)
+++MDM2(Δ 222-437)
NND+-+MDM2(Δ 222-325)
C-(Tao, 1999)
+++MDM2(NLS mutant)
N-(Roth,1998)
+-+MDM2(NES mutant)
C+(Dai, 2004)
+--MDM2(Δ 150-230)
C/NND-+±MDM2(Δ 90-150)
NND-++MDM2(Δ50-89)
NND-++MDM2(50-491)
N-(Pochampally,1999)
+-+MDM2(1-361)
N+(Haupt,1997)
+++MDM2
Localization
Promotes the ubquitination of
p53
Binds to
p53
Increases ubiquitination of FOXO1
Binds to
FOXO1
Table 2 Localization and function of different MDM2 mutants. ND- Not
done yet ; N-nucleus; C-cytoplasm.
- 134 -
ubiquination. MDM2 (Δ 222-437) is capable of binding to p53 and FOXO; as for
ubiquitnaiton, this mutant can only promote the ubiquitnation of FOXO but not p53. One
possible reason for this effect is the nucleolar localization of MDM2 (Δ 222-437). MDM2
(1-361) is a mutant which is deleted of the two highly conserved RING finger motifs in
the C-terminus. MDM2 (457S) carries a mutation critical for E3 function of MDM. It
prevents the polyubiquitination of p53, significantly lowers the efficiency of MDM2
interaction with MDMX and promotes MDMX polyubiquitination. Both of these mutants
can bind to p53 and FOXO factors but cannot promote their ubiquitination. This indicates
the essential role of the E3 region of MDM2 in the ubiquitination of both p53 and FOXO.
8. The sumoylation status of SIRT1 affects the stability and activity of FOXO.
Acetylation affects protein degradation via ubiquitination pathway (Ito et al., 2002;
Jeong et al., 2007; Li et al., 2002). In β-cells, acetylation of FOXO1 decreased its
degradation via the ubiquitin pathway (Kitamura et al., 2005). The current study showed
that sumoylation of wild type SIRT1 increased its deacetylase activity,wild type SIRT1,
but not SIRT1 (734R), decreased the acetylation level and transcriptional activity of
FOXO1. This study suggests that sumoylation of SIRT1 affects its ability to regulate the
transcriptional activity of FOXO1. The study also raised the possibility that SIRT1
sumoylation may regulate the stability of FOXO1.
9. Genistein increases FOXO expression level through down-regulation of MDM2.
Genistein (5,7,4'-trihydroxyisoflavone), a kind of isoflavone, is now considered to
be the primary anticancer component of soybeans. Experimental data showed that in
H1299 lung cancer cells, genistein increases the FOXO1 protein expression level in a
dose dependent manner, overexpression of MDM2 abolishes the effect of genestein on
FOXO1 (Figure 54). The data indicates that genstein increases FOXO1 protein
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expression level and this effect is MDM2-dependent. Previous data showed that
genistein down-regulates MDM2 expression at both the transcriptional and
posttranslational levels, independently of p53, in both human cancer cell lines and
primary cells. Up-regulateion of the tumor suppressor p21Waf1/CIP1 by genistein can be
regulated by both p53 and FOXO factors. Thus, the inhibition of MDM2 expression by
genistein may be essential for its antitumor activities.
10. ARF differentially affects E3 function of MDM2 toward different substrates.
p19ARF is the product of an alternative open reading frame of the mouse INK4a-
ARF locus. It is a known fact that ARF is an inhibitor of the MDM2 E3 ligase function on
p53 (Zhang and Xiong, 2001). In our study, also it was found that ARF stimulated
FOXO1 ubiqutination as well (Figure 55). The data indicate that ARF interaction with
MDM2 differentially affects its E3 function toward different substrates rather than
inactivating its E3 function in general. This effect is plausible because ARF does not
directly interact with the RING domain of MDM2, which may be involved in recruiting E2.
Binding of ARF to the acidic domain of MDM2 may simply alter its ability to properly
orient E2 for the transfer of ubiquitin to certain substrates. In the case of p53, ubiquitin
conjugation is blocked. In the case of FOXO1, ubiquitination is stimulated. There are two
possible explanations for this. The first one is that ARF may stablize MDM2 interaction
with FOXO, which may account for increased FOXO1 ubiqutination. The second one is
that ARF may also qualitatively stimulate the E3 activity of MDM2 toward FOXO. Further
experiments using the in vitro ubiquitination system will be required to address this
matter.
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SUMMARY AND PERSPECTIVES
Cancer is a major health problem in the USA and worldwide. One third of the
people suffer from some form of cancer and 20% of all deaths are cancer related. In
developed countries, cancer care represents about 10% of total health costs. Since the
incidence of cancer increases with age and people live longer, some important questions
need to be addressed: Why is cancer more prominent in older people and should cancer
treatment be different for the various age groups. Our studies focus on the regulation of
FOXO factors which are key elements of tumor initiation and of the mechanisms that
regulate an organism’s lifespan. They are potential candidates to serve as molecular
linker between longevity and cancer. FOXO factors also regulate numerous cellular
processes, such as stress resistance, the cell cycle, apoptosis, DNA repair/metabolism
and tumorigenesis
Ubiqitination controls the stability/degradation of FOXO factors, although the
mechanism is not clearly defined. Our data allow us to draw the following conclusions:
(1) MDM2 binds directly to FOXO1. Specifically, the binding between MDM2 and FOXO1
is through the central region of MDM2 and the N-terminal region of FOXO1.
Interestingly, FOXO protein phosphorylated by AKT is not required for this binding; (2)
MDM2 binding promotes the ubiquitin-dependent degradation of FOXO1 and FOXO3A.
Knockdown of MDM2 by siRNA caused accumulation of both FOXO1 and FOXO3A.
MDM2 mediated polyubiquitination of FOXO appears to occur in the cytoplasm and is an
AKT phosphorylation dependent; (3) Recombinant MDM2 catalyzes the ubiquitination of
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FOXO1 in test tube reactions, demonstrating a direct substrate-enzyme relationship; (4)
MDM2 affects the expression of downstream FOXO targets and protects cells from
FOXO1 induced cell death; (5) p53 transiently induced the transcription activity of
FOXO3A, followed by degradation of FOXO3A protein through MDM2.
In addition to the above conclusions, we also made several preliminary but
potentially important observations that need to be investigated in further studies.
1) In the transient transfection experiments, MDM2 increased the transcriptional
activity of both wild type FOXO factors and FOXO (AAA), which is no longer
phosphorylated by AKT anymore. The molecular mechanism underlying this effect
needs further clarification and monoubiquitination could play a decisive role. Other
important questions to be addressed are: whether monoubiquiyination of FOXO factors
is induced by MDM2 and is yes, whether monoubiquitination and polyubiquitination play
different roles in FOXO function; whether monoubiquitination increase the transcriptional
activity of FOXO without an effect on protein stability; whether MDM2 levels has a
differential effect on the ubiqutination status of FOXO; and whether cofactors, such as
p300 and SIRT1, have any effect on the ubiqutination status of FOXO1.
2) Genistein increases the expression of FOXO1 protein in a dose dependent
manner in H1299 lung cancer cells. This effect is abolished in the H1299 cells that stably
express MDM2. Our data suggested that the anti-tumor effect of genistein may be
mediated, at least in part, through the down-regulation of MDM2, causing the up-
regulation of FOXO factors (our present data) and p53 (earlier data of others). It remains
to be seen whether this effect of genistein on FOXO1 can be extended to other FOXO
factors and other cells lines and if so, whether it only affects FOXO protein stability or
through other mechanisms. It is also important to determine how genistein may affect
downstream targets of FOXO factors.
- 138 -
3) It is an estabolished fact that PKB and insulin promote FOXO ubiquitination
and degradation. It remains to be elucidated whether MDM2 is required for the PKB and
insulin promoted degradation of FOXO factors and whether the growth factors control
the levels of mono- vs. poly-ubiquitination.
4) SIRT1 acts as a “double-edged sword” promoting survival of aging cells, but
also increasing the cancer risk. Several studies had shown that acetylation inhibits
FOXO protein degradation. One key question is whether SIRT1 affects MDM2 promoted
ubiquitination and degradation, and if so, whether sumoylation mutant of SIRT1 will
affect stability of FOXO since the mutant is associated with a decrease in its deacetylase
activity and with reduced ability to inhibit the transcriptional activity of FOXO1.
5) ARF is known to inhibit MDM2-induced p53 ubiquitination, but enhance
MDM2-induced MDMX ubiquitination. In our study, ARF promoted FOXO ubiquitination.
It is very important to define the molecular mechanism behind the differential effect of
ARF on the MDM2-induced ubiquitination of p53 and FOXO factors.
In summary, my thesis work is the first to identify MDM2 as an E3 ubiquitin ligase
for FOXO factors and suggests that the targeted inhibition of MDM2 E3 activity may be a
more effective strategy for tumor supprerssion than the current strategy that targets
solely the interaction with p53.
- 139 -
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About the author
Wei Fu received her Bachelor’s degree in Biology from Shaanxi Normal
University in China in 1994 and her Master’s degree in Medical Microbiology and
Immunology from Xi’an Medical University in 1997. During her graduate year she worked
in the laboratory of Dr. Yonglie Chu and completed her thesis was on the molecular
biology of the human papillomavirus in cervical cancer. Wei worked also as a research
assistant in the Molecular Center of the First Clinical Hospital of Xi’an Jiaotong
Univeristy where she carried out both basic and clinical study. Wei started her Graduate
School studies in the fall of 2001 at University of South Florida, Tampa, Florida, and
persued her Ph.D thesis work under the tutelage of Dr. Wenlong Bai studying the
regulation of FOXO in stress response and tumorigenesis. She successfully defended
her doctoral dissertation in November 2007 at the University of South Florida. Wei is a
member of the Amercian Cancer Association and the American Heart Association.