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TRANSCRIPT
ROLE OF THE PTEN PHOSPHATASE IN REGULATING T CELL FUNCTION
Karen Andrea Berg
A thesis submitted in conformity with the requirements for the degree of Mater of Science
Graduate Department of Immunology University of Toronto
O Copyright by Karen Andrea Berg (2001)
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Title: Role of the PTEN phosphatase in regulriting T ce11 function DegredYear: M.Sc/ 200 1 Karen Andrea Berg, Department of Imrnunology, University of Toronto
ABSTRACT
While an important role for the PTEN phosphatase in the modulation of TCR
signriling is suggested by many lines of evidence. little is known about the biochemical
mechanisms whereby PTEN impacts on TCR function. To address this issue, affinity
chromatography and mass spectrometry were used to isolate and identify T ce11 protrins
representing binding-panners for PTEN. This approach led to the identification of seven
potential ETEN ligands including the SET. PHAPI2b. PHAPI. tubulin beta-1. eta and therü
subunits of chaperonin containing T-complex protein 1, nucleolin and rnoesin proteins. with
apparent rnolecular weights of 33. 25. 30.50.55. 67 and 67 kDa, respectively.
An analysis of PTEN roles in T ce11 activation was also undertaken and the results
revealed PTEN to be inducibly tyrosine phosphorylated consequrnt to cross-linking of the T
ce11 antigen and CD28 receptors. AKT phosphorylation was also observed following
TCRICD28 costimulation and was found to be relatively increased in thymocytes lacking the
SHP- 1 tyrosine phosphatase. These results suggest that the Pi3 WAKT signaling cascade
plays a role in TCR-elicited T ceIl activation and that this pathway is regulated by SHP-1.
As PTEN plays a critical role in downregulation the coupling of TCR engagement to AKT
phosphorylation/activation, these results raise the possibility that dephosphorylation of PTEN
represents a mechanism whereby SHP-L inhibits TCR signaling.
ACKNOWLEDGEMENTS
1 am grateful to my supervisor, Dr. Katherine Siminovitch, and to the members of my
supervisory cornmittee. Dr. Michael Julius and Dr. Robert Rottapel. for their valuable advice
and construciive criticism through these studies. 1 would also like to thank Dr. Jinyi Zhang
for al1 the help and encouragement and for his friendship. I am also grateful to several
individuals who contri buted with reagents for the experiments descn bed herein and provided
me with trernendous help and invaluable technical advice and who are acknowledged in the
relevant chapters.
Finally, 1 would like to thank al1 my friends and family for their continuous
encouragement and support. In particular, 1 would like to thank my parents and my brother
for their unconditional love and immeasunble patience and encouragement. which made this
endeavor possible.
TABLE OF CONTENTS
Abstrm ................................................................................................................................... ... 11
Acknowledgements .............................................................................................................. 111
Table of Contents............ ..................................................................................................... i v List of Tables ....................................................................................................................... v i List of Figures ..................................................................................................................... vii ... List of Abbreviations .......................................................................................................... V l l l
CHM"rER 1: INTRODUCTION .............................................................................................. 1 btroductory ovel-view and rationi-lle ...................................................................................... 1 T ce11 signüling ..................................................................................................................... 2
Role of phosphatases in regulating T ce11 function ..................................................... 4 n'EN is a phosphatase and a umor suppressor ...................................................................... 7
Initial charactuktion of m'EN ................................................................................ 7 Gennline PTEN mutations in cancer predisposition syndromes ................................. 8 EyrJ2N is inactivated in multiple h.lrmn hmlors .......................................................... 3 F"n3 is inactivated by multiple mechanisms ............................................................ 9
d e f i c h mice ................................................................................................. 9 SmJcture and catalytic properties of l'T'EN .......................................................................... 11
Overd1 !n-ucture of F'mN ....................................................................................... 11 Catalytic properties of P'I'EN .................................................................................. 13 Structure of the PTEN phosphatase domriin... implications ...................................... 14 s tmmre of the PTEN C:! dormin ........................................................................... 17 The pm-d~main binding site ................................................................................. 17 Effect of I-llutations on P n N tumor suppressor f'nction ......................................... 18
The PI3K pathw3y ............................................................................................................... 19 The PI3K family of ~roteins.~ .................................................................................. 19 Activity and regulation of Class IA PI3Ks ................................................................ 20 A m is 3 ~ownsueam target of PI3K... .................................................................... 31
' 5 bas multiple physiological fmxions ......................................................................... ..- Inhibition of apoptosis ............................................................................................ 25 Gene expression ...................................................................................................... 26
P'EN down-regulates the FWUAKT pathway .................................................................... 30 ........................ ETEN suppresses ceIl growth by inhibiting the PI3WAKT pathway 30
I'-'rEN and ~aenorhabditis elesans .......................................................................... 33 A rote for P7-m in T ceIl signal@ ..................................................................................... 34
A mXhanism for regulation ........................................................................ 36 Role of PTEN protein phosphatase rictivity in tumor suppression ......................................... 37 s u m w ........................................................................................................................... 39
Idencification of novel ligands/substrates for PTEN ............................................................. 51 p~rification of l'''EN for affinity c h o ~ t ~ g w h y ................................................... 51 Affinity chromtography ......................................................................................... 52 Identification of potential PTEN ligands by mass spectrornetry ............................... 55
De finition of E"TEN effects on T ce11 function ...................................................................... 59 PTEN is tyrosine-phosphorylated in thymocytes upon TCR engagement ................. 59 TCR-induced activation of AKT is increased in SHP-l-deficient T cells ................. 60
Menti fication of novel l'.EN liganddsubstrates ................................................................... 63 analy sis of the potential PTEN ligands .............................................................. 64
Affinity chromatography/mass spectrometry is a powerful approach ....................... 67 A d e for in T cd1 sipaling ..................................................................................... 71
LIST OF FIGURES
Figure 1-6.. Promotes ceIl survivd though multiple substrates -...---...---.---------.--.-. .-- ..-........-. - ------.- 29
Figure 1-7, antagonizes the P I 3 K A m pathway in C- e l e w w --------- * .---.-.--+----.------**.-*.***...* * 33
Figure 3- 1. GST-k'-rEN fusion protein*.**-** --*-*-*.*..*.-.-**.-..------ * * -.---...-..--. * ..--.---.-------.-. ...*- *.**** ...*.----------- -U
Figure 2-3- Sequence of PTEN c w k i ----.-*-*....*+.*.* * * .-.**..... * ...- **-+** - - * * m---------...----....----.-.*..-...** * -.*...... ..---- 45
Figure 1-3. Affinity chromWF~phy... ..----------.-.....-....-.----- - - - - - - . -....a - ....... - ............................................. 46
Figure 3- 1. Expression md purification of cN=pT'EN ..*.-..---------.----.-.---.--* * - - - - - - - - * -**.....--... * -.... * *... 53
Figure 3-3. Potential binding-partners for F"rEN ..*.*...*.-* * * *..- *.* .***.*....- - -------------..------.---- *-..*.* -..*-..... .. .. - - - 54
Figure 3-3- Y E N is tyrosine-phosphor~lated v o n TCR engagement - -.-...---.....---- - .--... -..-.*- .......-.------ 6 1
Figure 3 4 . TCR-induced activation of AKT is increased in SHP- 1 deficient T cells -_------------....--..*+..... 62
Figure 4- 1 - Schematic of a ~ALDI-ToF mass spectrometer -------.-------.-...........+-........ .. .-..---------------------- 70
Figure 4-2. A rnechanisrn for PTEN regulation: a role for SHP- 1 in the PI3K,AKT/PTEN pathway,-----.74
vii
LIST OF ABBREVIATIONS
aa ACB AICD AP- 1 Apaf- 1 MC ATP BCR B rdU b.v. BZS CBP CD cDNA c-LAP CREB C-terminus DMEM DNA DSP DTT EDTA rlF2B ES FAK FasL FBS FH FPLC Grb2 GSK-3 GST GTP HEPES HRP 12PP2A IGFB P 1 IgG IKK IL-3 IL-2R L K IPTG IRS ITAM It k D a
Arnino acids Affinity chromatognphy buffer Activation induced ce11 death Activating protein 1 Apoptosis protease-activating factor 1 Antigen presenting ce11 Adenosine triphosphate B ce11 receptor 5-bromo-2' deoxyuridine Bed volume Bannayan-Zonana syndrome CREB binding protein Cowden's disease CompIementary DNA Cellular inhibitor of apoptosis CAMP response elrment binding protein Carboxy-terminal Dulbecco's Modified Eagle Medium Deoxyribonucleic acid Dual-speci ficity phosphatase Dithiothreitol Ethy lenediamine trtraacetic acid Eukaryotic initiation factor 2B Embryonic stem Focal adhesion kinase Fris ligand Fetal bovine serum Forkhead homoIogy Fast Performance Liquid Chromatography Growth-factor receptor bound 2 GI ycogrn synthase kinase-3 Glutathione S-transferase Guanidy l triphosphate N[Z-hydroxyethyllpiperazine-N'-(2-ethanesulfonic acid] Horseradish peroxide Protein phosphatase 2A inhibitor lnsulin-like growth factor binding protein 1 Imrnunoglobulin G 1 kappa B kinase tnterleukin-2 Interieukin-2 receptor Integrin-linked kinase Isopropyl fi-D-thiogalactoside Insulin-responsive sequence [mmunoreceptor tyrosine-based activation motif Inducible T ceil tyrosine kinase Kilo Dalton
LAT LB LOH rnAb MAGI MAGUK MALDI-ToF MAPK Me Mev MHC MMAC 1 mRNA MS MW NCBI NF-KB NF-AT N-terminus ORF p l30Cas PA^ PAGE PBS PCR PDGF PDK 1 PDZ PH P W Pf3K PIF PKA PKB PKC PLCy PMSF PP2A PRK2 PtdIns PtdIns(3)P Ptdhs(3.4)P2 PtdIns(3.4,5)P3 PtdIns(3 S)PZ Ptdhs (4) P PTEN PTK m P-Tyr RAC RNA
Linker for activation of T cells Luria broth Loss of heterozygosity Monoclonal antibod y Membrane-associated guan ylate kinase wi t h inverted orientation Membrane-associated guanylate kinases Matrix-assisted laser desorption ionization-time of flight Mitogen-activated protein kinase Motheriten Motheaten viable Major histocompatibility complex Mutated in multiple advance cancers I Messenger RNA blass spectrometry iMolecular weight National center for biotechnology information Nuclear factor kappa B Nuclear factor of activated T ceIls Amino-terminal Open reading frrirne p 130 Crk-associated substrrite Pol yclonal rintibody Polyricrylamide gei electrophoresis Phosphate-buffered saline Polyrnerast: chah reaction Piatelet derived growth factor 3 '-phosp hoinositidedependent kinase- 1 PSD95 DiscsLarge ZO 1 Pleckstrin homology Putative HLA-DR associated protein Phosphatidyl inositol 3-kinase PDK 1 -interacting fragment Protein kinase A Protein kinase B Protein kinase C Phospholipase C g a m Pheny lmethy Isul fonyl fluoride Protein phosphatase 2A Protein kinase C-related protein kinase 3 Phosphatidylinositol Phosphatid y linositol 3-monophosphate Phosphatidylinositol 3.4-biphosphate f hosphatidylinositol 3.4.5-triphosphate Phosphatidylinositol 3.5-biphosphate Phosphatidylinositol4-monnphosphate Phosphatase and tensin homologue deleted on chromosome ten Protein tyrosine kinase Protein tyrosine phosphatase Phosphotyrosine ReIated to A and C protein kinases Ribonucleic acid
rpm SDS Sm SH3 Sm- 1 SLP-76 TBB 1 TCP 1 TCR TEP1 TGF-B TRIM w ZAP-70
Revolutions per minute Sodium dodecyl sulphate Src homology 2 Src homoIogy 3 SH2 domain-containing ETP 1 Src-hornology domin SHî-containing leukocyte protein 76 Tubulin beta- 1 chah T-complex protein 1 T ce 11 receptor TGF-p-regulated and epithelial crll-rrnrichrd phosphatase Transforming growth factor beta T ce11 receptor interacting rnokcule Ultraviolet 6-associated protein of 70 D a
(The one- and the-letter abbreviation system is used for amino acids. X refen to any amino acid)
Chapter 1: Introduction
INTRODUCTORY OVERVIEW AND RATIONALE
The recently discovered PTEN tumor suppressor gene is one of the most cornmon
sites of mutation in human cancers. Gemline mutations of PTEN give rise to a related set of
disorders, including Cowden's disease and Bannayan-Zonana syndrome, which are
characterized by the development of numerous benign tumors and an increased incidence for
developing cancer. The PTEN phosphatase has been implicated as an important modulator
of ce11 survival and apoptosis. rnainly by regulating the PI3WAKT pathway. but it is now
becoming clear that PTEN also plays a significant role in other aspects of ce11 physiology,
including the regulation of ce11 adhesion, migration and differentiation. At present many
lines of evidence indicate a key role for PTEN in regulating T cell function and TCR
signaling. including the finding that heteroz ygous PTEN mice exhibi t an increased incidence
of T-cell lymphomas. Similarly. TCR engagement has been shown to evoke AKT activation.
the activity of which is down-regulated by PTEN. Since its discovery, PTEN has been
extensively studied and despite several important advances many aspects of PTEN remain to
be clarified.
In this context, my research has focused on elucidating the effects of PTEN on T ce11
signaling, characterizhg biochemical mechanisms whereby PTEN might exert its function,
and finding whether additional PTEN substrates exist. Addressing these questions would not
only provide new insights into the function of PTEN and clarify the extent to which the
PTEN and PI3 WAKT pathways intersect. but could ultimately provide dues relevant to
developing novel strategies for cancer therapy.
T CELL SIGNALING
To participate in an adaptive immune response, niiive T cells must be activated in an
antigen-specific manner in order to differentiate into effector T cells capable of recognizing
and responding to foreign antigenic peptides. T ce11 activation requires a minimum of two
distinct signals. The first signal is delivered when the T cell antigen receptor (TCR)ICD3
complex recognizes specific antigen peptide within the context of the Major
Histocompatibility Cornplex (MHC) on antigen presenting cells (APCs) (1). Costirnulatory
molecules present on the surface of T cells provide the second signal. referred to as the
costimulatory signal. The best characterized costimulatory signal is delivered when the T
ceil surface rnolecule CD28 binds to B7 ligands on @Cs. This signal plays a critical role in
augmenting and sustaining ü T cell response initiated through TCR engagement. leading to T
ceIl clonal expansion and initiation of effector functions such as IL-2 production. Activated
T cells express a second receptor for B7 molecules on their surface. CTLAJ, which plays a
critical role as a negative regulator of lymphocyte homeostasis (2).
The ability of the TCR to transduce signais ücross the membrane is mediated by the
cytoplasmic domains of the CD3 subunits and TCRC chahs (3), which contain conserved
signaling motifs known as the ITAMs ammunoreceptor tyrosine-&ed gtivation motifs) (4,
5). These motifs are crucial for coupling to intracellulu tyrosine kinases and therefore
absolutely required for al1 subsequent TCR sipaling responses (6,7).
The earliest biochemical response elicited upon TCR engagement is the activation of
protein tyrosine kinases (FilCs), which regulate the function and interaction of a divergent
array of signaling molecules (8- 10). The Src-family tymsine kinases Lck and Fyn are the
fint PT& to be activated (3. 9). Once activated, these enzymes phosphorylate the ïïAMs of
the CD3 and chains allowing them to associate with ZAP-701Syk protein tyrosine kinase
(11-13). ITAM-bound ZAP-70 is tyrosine phosphorylated and activated by Lck, which in
tum associates with phosphorylated ZAP-70 by means of its S R domain (14). The Tec-
family tyrosine kinase Itk is also tyrosine-phosphorylated and activated by TCR signaling
(LS), and is required for efficient signaling through the TCR (16). Activation of PTKs
following TCR engagement initiates a cascade of sipnaling pathways; the key events are the
activation of PLCy, pZlras and PI3K (6). The signaling molecules implicated in TCR
signaling and that are relevant to the work presented herein are presented in Figure 1-1.
PLCy @hospho!ipase C gamma) controls the production of inositol polyphosphates and
diacylgl ycerols. which induce intracellular calcium mobi lization and acii vûte the
serinelthreonine protein kinase C (PKC) family. respectively. Active GTP-bound Ras
couples the TCR to multiple biochemical effector signaling pathways includin; the MAPK
(mitogen-gctivated protein kinase) pathway. TCR-engagement also controls inositol lipid
metabolism through the activity of phosphatidyl inositol 3-kinase (PI3K). The activation of
P13K is necessary for the synthesis of PtdIns(3,4,S)P3 and PtdIns(3,4)Pr required for the
recruitment of pleckstrin homology (PH) domain-containing proteins to the plasma
membrane and for the activation of proteins such as the serinelthreonine kinase AKT (11).
As it will be discussed later. the PI3WAKT signaling pathway has been implicated in
numerous cellular functions such as the regulation of ce11 sumival, apoptosis and cell-cycle.
to name a few.
In summary. T ceIl sipaling is initiated by the action of tyrosine kinases and
propagated by the combined action of multiple signaling pathways involving numerous
downstream adapter and effector molecules (17). These signaling events. summarized in
Figure 1-2, culminate in the reorganization of the cytoskeleton (18) as well as the
transcriptional activation of multiple genes leading to T ce11 proliferation, differentiation
andor effector function (19). The induction of the U--2 gene, for example. requires the
coordinate action of several signaling pathways that integrate at the level of multiple
transcription factors that include NFAT, AP- 1, NF-KB. and Oct-1 (19). Among the proteins
implicated in T cell signaling, PI3K is most relevant with respect to the role of F E N in
regulating T cell function. as i t is the PI3K signaling pathway that is negatively regulated by
PTEN.
Role of phosphatases in regulating T cell function
The activation of PTKs and subsequent tyrosine phosphorylation of downstream
signaling components is crucial to normal T cell function. and is therefore, tightly regulated
by the opposing action of protein tyrosine phosphatases (PTPs) (9). The disruption of this
balance has been shown to be an important hallmark of cellular transformation (70.21).
Figure 1-1. Signaling molecules involved in TCR signaling
Lc k/ fyn
ZAP-7OISy k
f LCy
PI3K(p85)
S hc
Grb2
[t k
S T
SHP- L
- SH3 - - S H 2 - Kinase - - S H 2 - SH2 - Kinase - - PH - PLC - SH2 - SH2 - SH3 - PLC -
. -- L-
- SHZ - Si42 - Phosphatase -
SH2- Src homology 2 SH3- Src hornology 3 PH- Piecksuin homology TH- Tec homology Pro- Proline-rich
Figure 1-2. Signaling pathways involved in T ce11 activation
TCR CD4/CD8
CD38
Protein Tyrosine Kinases
Protein Tyrosine Phosp hatases
Ras PI3K PLCy + J k MAPK Ip3 DAG
I I [ca2+] PKC
Cytoskeletal change Transcriptional activation
Tyrosine phosphorylation is crucial for the initiation of TCR-mediated signaling and is regulated by the opposing actions of PTKs and PT'Ps. At les t three signalhg pathways are involved in TCR-rnediated sipaling; those mediated by pZlRas, PLCy and PI3K. The nuclear iarget of each signahg pathway is a transcription factor. The coordinate action of multiple transcription factors is required for the transcriptional activation of cytokine genes such as IL-2.
PTEN IS A PHOSPHATASE AND A TUMOR SUPPRESSOR
While sevenl protein kinases have been implicated as oncogenes. i t has long been
postulated that some phosphatases may act as tumor suppressors. The identification of the
PTEN phosphatase as the tumor suppressor residing on 10q73 generated a great deal of
interest. While initial reports suggested that PTEN might act as a protein phosphatase. more
recent evidence by Maehama et al. demonstrated that PTEN can also function as a lipid
phosphatase that dephosphorylates by-products of PDK and thereby functionally antagonizes
signaling pathways that rely on PI3K activity (22). The catalytic properties of PTEN will be
discussed in more detail in another section.
Four lines of evidence (discussed below) have established PTEN as 3 turnor
suppressor gene. First. germline mutations in PTEN are associated wi th autosomal dominant
cancer predisposition syndromes. Second. the PTEN gene is homozygously inactivated in a
variety of sporadic human cancers. Third, the introduction of PTEN into cancer cell lines
leads to turnor suppression. and finally, germline heterozygous mutations in ETEN
predisposes mice to a variety of different tumors.
Initial characterization of PTEN
Loss of heterozygosity (LOH) at chromosome lOq23 occurs at a high frequency in a
variety of human cancers, reaching 60-809 in the case of prostate cancer, endometrial
carcinoma, and advanced glial tumon. By means of representational difference analysis and
positional cloning, PTEN (zhosphatase and m s i n homologue deleted on chromosome
m/MMACl (mutated in multiple gcivanced Gncers 1) was identified by two groups almost
simuItaneously as a candidate tumor suppressor gene located at lOq23.3 (33, 24). Another
group (25) identified the same gene in a search for new dual-specificity phosphatases (DSPs)
7
and named it TEP1 (TGF-P-regulated and gpithelial cell-enriched phosphatase) because its
transcription was modulated by TGF-P.
Germline PTEN mutations in cancer predisposition syndromes
Germline mutations of the PTEN gene are responsible for at least two rare autosornal
dominant inherited cancer predisposition syndromes: Cowden's disease (CD) (26-28) and
Bannayan-Zonwa syndrome (BZS) (58,79). These findings verified that PTEN functions as
a tumor suppressor. These disorders share similar pathological features. such as the
formation of multiple hamartomas (non-malignant growths) in multiple organs and a higher
incidence of developing malignant tumors. The predominant phenotype of CD is
hamartomas of the skin. Other organ si tes developing hamanomas include breast, th yroid,
endornetnum. gastrointestinal tract. and central nervous system, with breast cancers
developing in 25%-50% of affected women and thyroid cancer in 38-106 of affected
individuals (27. 30). BZS is characterized by mental retardation, macrocephaly. benign
neoplasms (lipomas, intestinal polyps), speckled penis and vascular malformations (29, 3 1).
The non-neoplastic features of these syndromes suggest that PTEN plays a role in the normal
development and formation of certain tissues.
PTEN is inactivated in multiple human tumors
In addition to germline mutations, sornatic mutations in the PTEN gene are frequently
found in a variety of sporadic human tumors. For example. in glioblastomas (the most
malignant form of glioma) the F I E N gene is inactivated with approxirnately 24% frequency
(23. 24). with mutations detected at a much lower frequency in many other types of bnin
tumon (32, 33). Several reports show that PTEN is the most frequently mutated gene
identified yet in endometrial cancers (30-504) and in endometrioid ovarian tumors PTEN
mutations have been found with a 26% frequency (34-36). An important observation is that
endometrial as well as ovarian tumon of endometrioid histology that contain PTEN gene
mutations are well or moderately differentiated, suggesting the involvement of the tumor
suppressor in the initiation of disease (37). Other tumors where ETEN mutations are
commonly found include advanced prostate cancers (3840). melanomas (>XI%) (4 I . 12). and lymphomas (4345). A detailed analysis of the nature and frequency of PTEN gene
mutations in different cancers may provide insights into the underlying genetic mechanisms
of tumongenesis in specific tissue types.
PTEN is inactivated by multiple mechanisms
The PTEN g n e is inactivated by multiple mechanisms, including homozygous
genomic deletions. In tumors with hemizygous deletions at chromosome 10q23. frameshift
or nonsense mutations, or mutations resulting in splice variants. prernaturely terminate the
ORF of the remaining copy of the gene. thus producing a truncated and nonfunctional
protein. Altematively. the remaining copy of the gene is altered by missense mutations that
are predicted to severely impair the function of the protein (37). Inactivation of the PTEN
gene may also occur by mechanisms other than deletions and mutations. Loss of expression
or reduced expression of PTEN. both at the mRNA and pmtein levels, was recently
demonstrated in xenografred prostate tumors (38). Although this mechanism requires further
characterization, restoration of PTEN expression by demethylation in cells derived from the
xenogafts suggested loss of expression by promoter methylation (38).
PTEN deficient mice
Further evidence that strongly implicated PTEN as a tumor suppressor carne from
several studies using knockout mice. A number of PTEN mutant mice have been descnbed
(46-48). Although the spectrum of disorders affecting these mice differed drarnatically
arnong the different strains, al1 the homozygous PTEN mutants manifested early embryonic
lethality (E7.5-E9.5) whereas the heterozygous mice exhi bited a predisposi tion to developing
tumon. These results supponed the notion that PTEN is a tumor suppressor essential for
embryonic development. The YIEN+'- mice and chimenc mice genented by eliminating
exons 4-6 (48) exhibited a high incidence of tumor formation involving the prostate, skin and
colon. which are characteristic of CD and BZS. They also spontaneously developed germ
cell, gonadostromal. rhyroid and colon tumors. The PTEN heterozygotes generated through
deletion of exons 4 and 5 displayed a high incidence of lymph node hyperplasia with
consequent disrupiion of lymphoid (B and T cells) organization (46). A high incidence of T
cell lymphomûs was observed in the heterozygous mice generated by eliminating exons 3-5
( 7 ) The appeannce of these turnors wlis markedly enhanced by irradiation and exhibited
elevated levels of phosphorylated AKT, a crucial reguiator of cell survival (see section on
PI3K). Moreover. Fas-mediated apoptosis was impaired in the PTEN"' mice lacking exons
4-6 and the responsiveness to Fas could be restored with PI3K inhibitors. Similarly, T
lymphocytes from these mice showed reduced activation-induced ce11 death (AICD) and
increased proliferation upon activation (49). These results not only indicated that PTEN is an
essential mediator of the Fas response but also implicated the PDWAKT pathway in Fas-
mediated apoptosis.
STRUCTURE AND CATALYTIC PROPERTIES OF PTEN
Overall stmcture of PTEN
The PTEN gene has 9 exons and its product is a 403-mino acid protein (55 kDa) that
is ubiquitously expressed. The structure of PTEN is illustrated in Figure 1-3. PTEN has
been described mostly as a cytosolic protein although one study daims that PTEN is
expressed in both the nucleus and the cytoplasm dunng neuronal differentiation (50). PTEN
contains the signature motif HCXXGXXRSrï present in the active sites of protein tyrosine
phosphatases (PTPs) and duai-specifici ty phosphatases (DSPs). but i t has little sequence
similarity to these protein fimilies outside this motif. Rather. homology searches reveal that
the amino-terminal 190-ürnino acid region of FTEN encompassing the signature motif has
sequence similarity to chicken tensin and bovine auxillin (33. 24). Tensin is an actin-binding
protein locaiized to focal adhesions complexes and auxillin is involved in the uncoating of
clathnn-coated vesicles. Recent crystallographic studies revealed that the carboxy-terminal
region of PTEN contains a C 2 domain. a structure that interacts with phospholipid
membranes in vitro (51). The C3 and phosphatase domains associate across an extensive
interface that is adjacent to the phosphatase active site. The last three amino acids of PTEN.
TKV. fom the consensus sequence (T-X-V-COOH) for binding PDZ domains. PDZ
domains are protein-protein interaction domains that bind to consensus motifs (SîTXV) in the
C terminus of partner proteins or. altematively, to other PDZ domains (52). In addition to
these features, ETEN has two putative consensus binding sites for the SHP-1 tyrosine
phosphatase SH2 domains (MYFEF at residues 239-243 and RYFSP at residues 335-339),
one for the Crk S H 2 domain (RYFSP at residues 335-339) and one for Shc (EYLVL at
residues 3 14-3 1 8).
Figure 1-3. Structure of PTEN
PDZ-BD
HCKAGKGRT
t t TKV EYLVL
Shc binding site MY FEF
SW-1 SH2 domain binding site
f RYFSP
SHP- 1 , Crk SH2 binding site
1 Potencial Tyrosine 1 Phos~horvlrition sites
The PTEN phosphatase is a 403 aa protein and contains the signature motif that defines PTPs and DSPs. Overlapping the phosphatase domain there is a region of sequence homology to the cytoskeletal proteins tensin and auxillin. The carboxy-terminal region contains a C3 domain and a consensus sequence (TKV) for binding PDZ domains. In addition, there are putative consensus sites, which if phosphorylated can potentially bind the SH2 domains of SHP- 1 , S hc and Crk.
Catalytic properties of PTEN
In early reports by Myen et al., PTEN was originally described as a dual-specificity
phosphatase (DSP) capable of dephosphorylating tyrosine. serine and threonine residues in
vitro. However, unlike other DSPs ETEN would not dephosphorylate members of the
MAPK famil y and showed preference towards extremel y acidic substrates (53).
Interestingly, a Cowden's disease-denved PTEN mutation (Glyl29Glu) was observed to
retain phosphatase acti vi ty against the substrates used in these studies. Because PTEN
mutations derived from CD samples rnust be. by definition. biologically inactive. this
observation implied that the G179E mutation might interfere with the ability of PTEN to
dephosphorylate its physiologie targets. Consequently. a later report by Maehama et al.
demonstrated that PTEN dephosphorylates the position D3 of phosphatidylinositol (PtdIns)
phosphates both in vivo and Ni vitro (22). Analysis of the various mutations found in tumors
and patients with CD. including the G119E mutation, revealed that they specifically ablate
the abili ty of PTEN to dephosphory late 3'-phosphorylated PtdIns (54. 55 ) . These results
provided strong evidence that it is the lipid phosphatase activity of PTEN that is required for
its tumor suppressor activity. In agreement with this idea. PTEN-deficient cell lines (56) and
immonalized fïbroblasts from ETEN'" mice (57) have elevated levels of intracellular
PtdIns(3,4,5)P3.
Although the protein phosphatase activity of PTEN seems not to be critical for its role
as a tumor suppressor, it may still be of physiological importance. At present. only two
proteins. the focal adhesion kinase (FAK) and Shc have been identified as targets for ETEN
tyrosine phosphatase activity. PTEN has been shown to directly associate with and
dephosphorylate both proteins thereb y inhi biting integrin-mediated ce11 spreading, migration.
invasion and cytoskeletal organization (58-60). The role of PTEN in the signaling pathways
mediated by the signaiing molecules aforementioned is discussed in more detail in another
section.
Structure of the PTEN phosphatase domain---implications
The phosphatase domain of PTEN consists of a central five-stranded sheet that
packs with two u helices on one side and four on the other (51). The signature motif
HCXXGXXR at residues 113-130, forms a loop (P loop) located at the bottom of the active
site pocket. in comrnon with DSPs and PTPs. In this motif (see Figure 1-4)' the Cys-174 and
Arg- 130 residues are essential for catalysis whereas the His-123 and Gly- 127 residues are
imponant for the conformation of the P loop (6 1.62). The C ys- 1% is absolutel y required for
catalysis. as it executes a nucleophilic attack upon the phosphate moiety of the substrate.
forming a thiol-phosphate intemediate (63). Mutation of this residue to senne or alanine
results in the complete loss of phosphatase activity (64). Another catalytically important and
conserved residue of PTEN is the aspartic acid at residue 97 (Asp-92). It is located on a
mobile loop and it acts as a general acid to facilitate protonation of the phenolic oxygen atom
of the tyrosyl leaving group (65.66). Mutation of this residue to alanine in PTEN results in a
700-fold reduction in catal ytic activit y toward PtdIns(3.4,5)P3 (5 1 ).
The P loop also has two basic residues in its center. This feature gives the PTEN
pocket a unique highly basic character. which is consistent with PTEN's preference for
highly acidic polypeptide substrates as well for the negative charge of PtdIns(3.4,5)P3 (53).
Furthemore, the active pocket is both deep and wide enough to accommodate the bulky and
polyphosphorylated sugar moiety of PtdIns(3.4.5)P3 (5 1). The crystal structure of PTEN
reveded that Gly-129 is at the bottom of the pocket and suggested that the mutation of this
residue to glutamic acid would reduce the size of the ETEN pocket, preventing
PtdIns(3,4,5)Pj from binding but allowing the smaller phosphorylated protein substrates to
bind (51). This provided the structural basis for an explanation of the lack of lipid
phosphatase activit y of the tumor-derived G L29E mutation w hich retained protein
phosphatase activity. However. further characterization is required since the mutation of the
sarne residue to arginine (found in glioblastoma) abolishes both the lipid and the protein
phosphatase activity (53).
The homology of PTEN to tensin and auxillin maps primarily to the hydrophobic core
and to residues that pack with the C2 domain. These residues are highly conserved
suggesting that tensin and auxillin may also contain a C2 domain. an idea thüt is supponed
by the fact that rnost of the PTEN C2 residues that interact with the phosphatase domain are
similar in both proteins (5 1 ).
Figure 1-4. Prirnary structure of the signature motif of PTEN phosphatase domain
1 . 1 - 1 - 1 Lipid Phosphatase
r 1 - 1 + 1 Protein Phosphatase
PTP signature motif
PTEN signature motif
The active-site sequence motif that characterizes the catalytic domains of PTPs and DSPs is illustrated dong with that of PTEN. Cys-124 and Arg-130 are essential for catalysis (in bold) whereas His-123 and Gly-117 are required for the conformation of the P loop (underlined). Asp-93 (double line), dthough not located within the signature motif, is important for catalytic activity (see text). The catalytic consequences of important mutations within the signature motif are also indicated.
Structure of the PTEN C2 domain
The C2 domain (170 amino acids) folds into a sandwich structure consisting of two
antiparallel sheets with two shon u helices intervening between the strands. Its structure is
similar to the C2 domains of PLCy, PKC6 and phospholipaseA7, but i t differs to these in that
it does not require calcium to bind the cell membrane as i t lacks al1 but one of the ca2'
ligands (5 1). The C? domain of PTEN was shown to have affinity to phospholipid
membranes in vitro. Moreover, the C2 domain is tightly associated with the ETEN
phosphatase domain. suggesting that i t may also serve to position the catal ytic site correctl y
with respect to its substrate. Consistent with this hypothesis, mutagenesis of residues in the
C? dornain reduced the tumor-suppressive iictivity of PTEN without interfering with its
enzymatic activity in vitro ( 5 1 ).
The PDZ domain-binding site
PDZ domains mediate protein-protein interactions that can result in the formation of
localized rnulti-protein complexes (67. 68). One study found that deletion of the PDZ-
binding motif of PTEN did not have a detectable effect on its ability to reduce the P13K-
dependent activation of AKT when overexpressed in U87-MG cells (69). On the other hand,
the effects of PTEN on membrane ruffling. in response to stimulation of Swiss 3T3 cells with
PDGF, were completel y blocked by the deletion. Membrane ruffiing due to PDGF is thought
to involve the PDK-dependent but AKT-independent activation of Rac. This effect reflects
the possibility that a PDZ-domain based interaction, although not required for AKT down-
regulation. may facilitate the regulation of other lipid signaiing responses. In a recent report,
PTEN was found to
MAGI3 (70, 7 1).
interact with a specific PDZ domain of MAGE and of the novel protein
These proteins belong to a family of proteins known as MAGUKs
17
(membrane-gsociated guanylate kinases), which appear to function as scaffold proteins to
assemble multiprotein signaling complexes and enhance their stability. Both proteins.
through a PDZ domain-mediated interaction with PTEN, were shown to enhance the
efficiency of PTEN to inhibit AKT. Furthetmore. the PTEN PDZ domain-binding motif was
shown to be important for contriburing to its association to the membrane and for
rnaintaining PTEN protein stability (70). The latter observation is in agreement to other
studies (69. 72) assigning a role for the C-terminus of PTEN in maintaining its protein
stability. More studies need to focus on determining whether PTEN-PD2 domain
interactions may be pathologically significant.
Effect of mutations on PTEN tumor suppressor function
Germline mutations in the PTEN gene detected in CD and BZS as well as somatic
mutations in various tumon are distributed over the entire gene with a clustering in three
regions. the phosphatase signature motif in exon 5 and phosphorylation sites in exons 7 and
8. A great majority of these mutations. 67-838. result in premature temination of the ORF
producing a truncated protein (53) (73). Moreover, the C-terminal region of PTEN contains
predicted secondary structure elements that are ais0 essential for the tumor suppressor
function of PTEN. Furthemore, the majority of residues that make interdomain hydrogen
bonds are found mutated in cancer. Two of them are among the eight most frequently
mutated residues in PTEN. indicatîng that the integity of the interface is also important to
the function of PTEN (51). In support of this contention, a tumor-derived mutation
(Asp252Tyr) which disrupts an interdomain hydrogen bond causes an 85% reduction in
PtdIns(3,4.5)P3 phosphatase activity ( 5 1 ).
THE PI3K PATHWAY
The finding that PTEN dephosphorylates 3'-phosphorylated PtdIns led to a mode1 for
how PTEN acts as a tumor suppressor, linking FTEN to the control of at least two known
cellular protooncogenes, P13K and AKT. PTEN inhibits PDK-dependent activation of AKT.
and deletion or inactivation of PTEN results in constitutive activation of AKT. An overview
of the PDK and AKT family of kinases is provided in the following sections.
The PI3K family of proteins
PI3Ks are enzymes that phosphorylate the position D3 of the inositol ring of PtdIns
and thus give rise to lipid molecules that function as second rnessengers. including
PtdIns(3)P. PtdIns(3.4)P2, PtdIns(3.5)P2, and PtdIns(3,J.5)P3. Three classes of PI3Ks (types
1. II and III) have been identified based upon substrate preference (74, 75,75). Class 1 PI3Ks
consist of heterodimeric enzymes composed of an 85kDa SH2 domain-containing regulatory
subuni t and a 1 lOkDa catalytic subunit (76). There are two subclasses: IA and tB. Subclass
IA (pl LOU, pl IOP. and pl 106) enzymes are regulated by tyrosine-phosphorylated proteins or
by proteins containing proline-rich domains. There are three known regulatory subunits
(p85a, p85P and p55y) (75. 77), as well as sevenl additional isofonns of p85a generated
through differential splicing of the gene (78.79). Both p85u and p8@ contain two carboxy-
terminal S E domains that are separated by an inter-SHZ region that serves as the docking
site for the catalytic subunit. Class IB (pl lOy) transduces signals from G protein-coupled
receptors by binding the fly subunit and does not interact with p85 proteins (80). Class II
PUKs are defined by a carboxy-terminal C2 domain which may confer phospholipid-binding
ability. They seem to preferentially phosphorylate PtdIns and Ptdns(4)P (75). The class Kt
PI3Ks phosphorylate only PtdIns. The yeast homologue VPS34 for this class seems to be
essential for accurate transport of newly synthesized proteins from the Golgi to the vacuole
(75).
For the purposes of this thesis, 1 will focus on class 1 PI3Ks. which regulate a
multitude of cellular functions including apoptosis, cellular proliferation, vesicular
trafficking, cytoskeletal structure and cellular morphology, glucose utilization. protein
biosynthesis. and lipid metabolism. Although this class of PI3Ks has been the focus of the
majonty of studies in signal transduction, the possible effects of the other family members
cannot be disregarded.
Activity and regulation of Class IA PI3K.s
The activation of PI3K clin be accomplished through receptor tyrosine kinase
activation. the activation of non-receptor tyrosine kinases and through interaction with the
activared form of Ras. Receptor activation followed b y dimerization. triggers receptor
autophosphorylation on tyrosine residues creating docking sites for the S H 2 domains of p85
8 8 ) . Non-receptor tyrosine kinases have been implicated in the activation of PI3Ks by
B- and T-cell antigen receptors. many cytokine receptors and CO-stimulatory molecules (such
as CD28). as well as by cell-ce11 and cell-rnatrix adhesion (74). The non-receptor tyrosine
kinase Lck, for example, may recruit p85 via an SH3-mediated interaction (83-85). p85 thus
brings the PDK into proximity with the ce11 membrane where its lipid substrates are located.
GTP-bound Ras also leads to the recruitment and activation of the catalytic subunit (pl 10) of
PDK (86). Because Ras is also a membrane-associated protein, its interaction with PDK
also helps recruit PUK to the membrane where it c m it interact with its substrates. Although
the activation of PDK has been shown to be necessary for TCR signaling, the biochemical
events linking its activation to TCR engagement remain elusive. Recent data has implicated
the adapter molecules TRiM (T ce11 receptor interacting ~olecule) and LAT Uinker for
activation of 1 cells) in the recruitment of PI3K to the ce11 membrane following TCR -
engagement (17. 87, 88). These proteins become phosphorylatrd in multiple tyrosine
residues after T ce11 activation allowing their association with many important signaling
molecules such as p85-PI3K in an SHZdomain dependent manner. Once activated and
brought to the membrane. PI3K specifically phosphorylates the D3 position of the inositol
ring of PtdIns to generate PtdIns(3,4)P2 and PtdIns(3,4.5)P3 (80. 8 1). These localized second
messengen recruit certain cytosolic proteins to the membrane. an event mediated by a lipid-
protein interaction domain known as the Pleckstrin homology (PH) domain. The class IA
PI3Ks. hereafter referred to as PI3Ks. possesses an intrinsic protein kinase activity in virro
directed towards senne residues within the catalytic subunit itself andfor its associated
regulatory subunit. The phosphorylntion of a senne in p85 by the pl LO catalytic subunit has
been documented to result in the down-regulation of PI3K lipid kinase activity (89).
AKT is a downstream target of PI3K
Several molecular targets for Ptd1ns(3.4.5)Pj and PtdIns(3.4)P2 have been identified.
which are tnnslocated and activated upon interaction with PtdIns. A well documented
example of a PDK downstrearn target is the AKT family of serinekhreonine kinases. AKT
(also known as PKB or RAC-PK) is the cellular homologue of the transfoming v-akt
oncogene and has been shown to deliver cell survival sipals in many systems involving a
variety of stimuli including growth factor withdnwal, loss of ce11 adhesion and DNA damage
(90-93).
AKT proteins are distinguished by the presence of an arnino-terminal PH domain that
preferentially binds PtdIns(3,4,5)P3 and PtdIns(3.4)P2 (94). PH domains are conserved lipid-
protein interaction domains that are found in a large variety of proteins involved in cellular
signaiing or cytoskeletal functions (95, 96). Different PH domains exhibit different
phosphoinositide-binding specificity.
There are two major PI3K-dependent events that are required for the full activation of
AKT: membrane localization (97) and senne/threonine phosphorylation (98. 99). The
translocation of AKT from the cytosol to the plasma membrane is accomplished by the
binding of the PH domain of AKT to PtdIns(3,4)P2 (100, 101), which in tum causes a
conformational change in the activation loop of AKT. In addition. two specific sites in AKT
need to be phosphorylated for full activation, one in the kinase domain (Thr-308) and the
other in the C-terminal reguiatory region (Ser-473). The enzyme thiit phosphorylates Thr-
308 was shown to be absolutely dependent on the presence of synthetic PtdIns(3.4.5)P3 in
vitro and therefore it was termed 3'-phosphoinositide-dependent kinase- 1 (PDK1) ( 102- 104).
Like AKT. PDKl also contains a PH domain that binds PDK-phosphorylated lipids. The
identity of the kinase that phosphorylates Ser-473. tentatively terrned PDKî. has remained
elusive. It has been claimed that integrin-linked kinase (LK) is capable of phosphorylating
Ser-473 in vitro and when overexpressed in cells ( 105). A recent study demonstrates that the
activity of ILK c m be regulated by PTEN (106). L K is constitutively activated in human
prostate carcinoma cells lacking PTEN expression presumably due to increased
PtdIns(3,4.5)P3 levels. The activity of L K in these cells was inhibited upon restoration of
PTEN expression by tnnsfection. In addition. selective inhibitors of ILK activity resulted in
inhibition of AKT-Ser-473 phosphorylation (106). These data not only places PTEN
upstream of ILK, but also demonstrates that inhibition of ILK affects the phosphorylation of
Ser-473. Moreover, recent findings have shown that PDKl can interact with a frqgnent of
the C-terminus of PRK2 (107), which w u termed PDK1-interacting fragment (Pm. Remarkably. this interaction converts PDKl to an enzyme that can phosphorylate both Thr-
308 and Ser-473 residues of AKT. Therefore, the activity of PD= might. in fact, be PDKl
itself or, alternative1 y. L K . Important1 y, the phosphorylation of both Thr-308 and Ser-473
and subsequent activation of AKT are abolished if the cells are incubated with PI3K
inhibiton pnor to stimulation with agonists (98). A schematic representation of growth
factor-induced activation of AKT is presented in Figure 1-5.
Figure 1-5. Growth factor-induced activation of the PDKfAKT signaling pathway
'1'
Thr-308 ' Inactive
Ser- 473 AKT
The growth factor-mediated activation of PUK results in increases in 3'-phosphorylated Ptduis. which allows for the translocation of AKT to the plasma membrane followed by a conformational change that renden the phosphorylation sites Thr-308 and Ser-473 available to PDKs. Once activated, AKT is released from the membrane to phosphorylate specific protein targets. PTEN negatively regulates AKT-mediated signaling by its ability to specificaily dephosphorylate PtdIns(3,4.5)P3.
AKT HAS MULTIPLE PHYSIOLOGICAL FUNCTIONS
When activated. AKT transduces signals that regulate multiple biological processes
including apoptosis, gene expression. and cellular proliferation by phosphorylating proteins
on senne and threonine residues. The wide range of biological functions of activated AKT
suggests that it may phosphorylate several targets. The definition of the AKT consensus
phosphorylation sequence (RXRXXSn) has aided the identification of a large number of
proteins that müy represent targets of AKT. many of which are components of the apoptotic
rnachinery. Below is a description of some of the proteins that have been reported to be
targets of AKT and a summary of sorne of rhe sipaling events that are regulated by these
proteins that are likely to be relevant to T ce11 signaling. The role of PTEN as an antagonist
to some of these signais is discussed in a later section.
Inhibition of apoptosis
The fint component of the apoptotic machinery found to be phosphorylated by AKT
was the Bcl-2 fmily member B A D (for a review on Bcl-2 family see 108). This protein
forms a heterodimer with the anti-apoptotic proteins Bcl-2 or Bcl-XL and thereby prevents
(hem from exening their anti-apoptotic function. When BAD is phosphorylated on Ser-112
or Ser- 136 ( log), it associates with cytoplasmic 14-3-3 proteins ( 1 10) and thus can no longer
interaci with Bcl-2 or Bcl-XL. AKT can phosphorylate BAD on Ser- 136 (1 1 1. 1 12) which
inactivates its ability to cause ceIl death and thereby promotes ce11 survival. However. the
fact that BAD is not ubiquitously expressed and that cell survival has been shown to be
regulated independently of both AKT activation and BAD phosphorylation (113, 114).
suggests that BAD is unlikely to be the major mechanism by which AKT blocks apoptosis.
In addition, BAD cm be phosphorylated by other kinases including PKA and kinases
activated through the MAPK pathway (1 15- 1 17).
Another mechanism whereby AKT can promote ce11 survival is by phosphorylating
the ceIl death protease caspase-9. The phosphorylation of human caspase-9 at Ser-196 by
AKT inhibits its proteolytic activation, although the exact basis for this inhibition is not clear
yet (1 18). Dunng the course of apoptosis, mitochondna release cytochrome c from the
intermembrane space into the c ytosol. Cytochrome c binds to Apaf- 1 (apoptosis grotease-
activating factor iJ in a dATP-dependent fashion, recruiting caspase-9 into a complex known -
as the apoptosome. These events activare caspase-9 (1 19, 170) which in turn cleaves and
activates other caspases including caspase-3. thereby initiating an apoptotic protease cascade
that ultimately leads to the cleavage of various key substrates and subsequently to the death
of the cell. Interestingly. the phosphorylation of caspase-9 by AKT does not appear to be a
generalized mechanism for its inactivation ricross species. Not only are AKT
phosphorylation sites and motifs found in humm caspase-9 absent in the mouse, rat and
monkey homologues. but mouse caspase-9 is not phosphorylated by AKT in vitro (121).
Gene expression
The observation that activated AKT translocates to the nucleus (97) led to examining
the possibility that AKT might regulate the activiiy of transcription factors that control genes
involved in cell death. Genetic studies done on C. elegans (discussed in another section)
implicated the PI3WAKT signaling pathway in the suppression of the activity of the
transcription factor DAF16, a member of the Forkhead (FH) family ( 122). Three memben of
the FH family of transcriptional activaton have been identified in mammalian cells, FKHR,
FKHRLl and AFX. XKT modulates the FH farnily members pnmarily though regulation of
their subcellular localization (133). Upon phosphorylation by AKT, the FH family is
exponed from the nucleus to the cytoplasm where they are retained by means of their
association with 11-3-3 proteins (123, 124). When not phosphorylated, members of the Ri
family are localized to the nucleus where they bind specific DNA elements such as IRS
within the IGFBPl promoter (125- 128). FKHRLl has also been found to bind to DNA
sequences found within the regulatory region of the Fas ligand (FüsL) sene (1 23). Thus,
when FKHRLl is phosphorylated. FasL is not expressed allowing the cells to survive.
AKT can also phosphorylate the transcriptional activator CREB. which increases the
binding of CREB to the coxtivator CBP and thus enhances CREB-mediated transcription
( 129). A study using dominant-negative CREB transgenic thymocytes, shows that CREB
might be lictivated upon T cell activation and required for the normal induction of the
transcription factor AP- 1 and subsequent IL-2 production ( 130). Furthemore. recent data
has irnplicated AKT in the modulation of NF-KB-dependen t expression of survi val genes
including the anti-apoptotic Bcl-2 family member Bfl-1/Al and the caspase inhibiton c-iAP I
and c-IAP2 (13 1). The induction of NF-KB by AKT has also been implicated in transcription
from the IL-2 promoter thereby suggesting an important role for the control of T ceII growth
and survival (132). NF-KB is sequestered in the cytoplasm by its cytosolic inhibitor IKB.
Phosphorylation of IKB by MKs (IKB kinases) targets i t for degradation, which allows NF-
KB to move to the nucleus and activate transcription. AKT activates NF-icB by enhancing
the degndation of IKB through phosphorylation and activation of IKKs (133).
Finally, another major downstream target of AKT is glycogen synthase kinase-3
(GSK-3), a serinekhreonine kinase whose activity is inhibited by AKT in response to growth
factor stimulation. GSK-3 negatively regulates a broad range of substrates, including several
transcription factors and the translation initiation factor eIF2B (80, 134). and is involved in
multiple cellular processes, such as metabolism (135, 136) and the regulation of apoptosis
(137). GSK-3 has also been shown to phosphorylate cyclin D l resulting in its degradation
(80). suggesting a role for AKT in the regulation of the cell-cycle partly through its ability to
inactivate GSK-3. In addition. GSK-3 has been identified as the kinase that phosphorylates
NF-AT (nuclear u t o r of ~ t i v a t e d cells) resulting in its expon from the nucleus (138). In
B cells, the engagement of the BCR has been shown to induce the PI3K-dependent
phosphorylation and inactivation of GSK-3 by AKT causing NF-AT to accumulate and
translocate to the nucleus where i t can promote the transcription of MAT-dependent genes
(139). Figure 1-6 provides a sumrnary of some of the biological functions associated with
the activity of AKT and that are discussed in this section.
Figure 1-6. AKT promotes ce11 survival through multiple substrates
Transcriptional activation Metabolisrn
.-.A....CI.- . .. Cell-cycle
death g e n s 1 -"uu
I
APOPTOS lS
I I survival genes I I I 1 I I 1 1 1 1 1 1 1 1 1 I I I
SURVIVAL
M T regulates ceIl survival through the phosphorylation of multiple substntes. AKT can block apoptosis by regulating the activity of transcription factors for death-inducing or survival-promoting genes. AKT c m also prevent apoptosis by phosphorylating and inactivating members of the apoptotic machinery. The phosphorylation and inactivation of GSK-3 by AKT can regulate other cellular functions neccesary for cell survival such as metabolism and ce11 cycle.
PTEN DOWN-REGULATES THE P13WAKT PATHWAY
As previously discussed, PTEN c m specifically dephosphorylate PI(3,1,5)P3 (22.57).
an important second messenger generated by PI3K. which in turn activates AKT-mediated
signals. Overexpression of PTEN or the catalytically inactive mutant (C124S) of PTEN in
393 cells reduces the Ievels of insulin-induced PI(3,4.5)Pi in a dose-dependent manner (22).
Conversely, F'TEN-deficient mouse embryonic fibroblasts display elevated levels of
PI(3.4,5)P3 when compared to their heterozygous counterparts. accompanied by increased
activity of AKT CO a peptide substrate (57). In addition. the CD-derived G129E PTEN
mutation retains protein phosphatase activi ty but lacks PTEN lipid phosphatase activity (54).
This indicates that the ability of F I E N to function as a tumor suppressor is due, in part, to its
ability to dephosphorylate PI(3.4.5)P; and thus affect the downstream target of PI3K. AKT.
PTEN suppresses ce11 growth by inhibiting the PI3WAKT pathway
Interestingly. PTEN appears to suppress cell growth by distinct mechanisms. For
example, while PTEN rnediates a GI cell-cycle arrest in glioblastoma cells ( 5 3 , i t induces
apoptosis/anoikis in other ce11 types (57. 140. 141). PTEN has been demonstrated to induce a
G l block when reconstituted in the PTEN-deficient U87 MG and U178 glioma ceIl lines and
the 786-0 rend carcinoma ce11 line ( 55 . 142-144). The ability of PTEN to induce a G1 block
was correlated with its lipid phosphatase activity since the G l29E PTEN mutant failed to
alter growth suppression. PTEN efficiently down-regulated the activity of AKT, whereas the
G129E mutant did not. Moreover, the coexpression of a mynstoylated form of AKT, but not
wild-type AKT, was able to ovemide the ETEN-mediated cell-cycle block (142). These
resulrs suggest that PTEN-mediated cell-cycle inhibition depends upon the inhibition of the
PUWAKT signaling pathway. In addition. the ability of PIW to block cell-cycle
30
progression correlated with a significant increase of the cell-cycle kinase inhibitor p27KIPl
and a concomitant decrease in the activities of the Gl cyclin-dependent kinases after
restoring PTEN expression (143). The levels of p27KIPl are also found to be reduced in
PTEN-deficient embryonic stem (ES) cells. which correlated with increased phosphorylation
of AKT (145). Consistent with a role of ectopic ETEN in suppressing ce11 growth is the
observation that ETEN-deficient mouse embryos display regions of increased proliferation.
confirmed by high rates of BrdU incorporation (57).
Numerous studies have implicated PTEN as an important regulator of ceil survival by
inducing apoptosis through the inhibition of the PI3WAKT pathway (57. 146). PTEN-
deficient immonalized mouse embryonic fibroblasts display decreased sensitivity to
apoptosis in response to apoptotic stimuli such as UV irradiation. a defect that is restored
upon expression of exognous PTEN (57). Similürly, expression of ETEN in a PTEN-
deficient prostate carcinoma ceIl line induced apoptosis ( 1-16). Interestingl y. enhanced
apoptosis was also observed in mN-deficient prostate carcinoma cells upon transfection of
a kinase-inactive. dominant-negative LK. which inhibited AKT activity (106). As explained
earlier. ILK has been shown to phosphorylate AKT on Ser-473 on a PDK-dependent manner
and its activity was demonstrated to be regulated by PTEN. The dominant-negative ILK also
induced a G1 phase cell-cycle arrest in ihese cells (106). The implication of these findings is
that another mechanism whereby PTEN might suppress ce11 growth is by negatively
regulating the activity of ILK. It is important to mention. however. that ILK may also
regulate cell-cycle arrest via an AKT-independent mechanism involving GSK-3 (105).
Finally, Ci25 1 glioma cells with infected PTEN were induced to undergo anoikis (140).
which is a form of apoptosis that occurs when cells dissociate from the extracellular matrix
and thus plays an important role in preventing transformed cells from becoming metastatic or
invasive. This data in addition to the implication of the PUWAKT pathway in the inhibition
of anoikis (92) strongly suggests an important role for PTEN in mediating this survival
pathway. In conclusion. PTEN-mediated growth suppression. either through the induction of
cell-cycle arrest or apoptosis. requires the inhibition of the PI3 WAKT pathway.
PTEN and Caenorhabditis elegans
Genetic evidence that PTEN acts as a negative regulator of PDK-regulated pathways
has been recently obtained through genetic analysis of a PI3K pathway in the nematode C.
elegans that controls dauer formation, a developmental stage which ensures survival under
stress conditions by suppressing feeding and metabolism. In this state. the animal displays a
prolonged life-span (147). The charxterization of mutants defective in dauer formation led
to the identification of the genes involved. called daf genes. Mutations in the genes daf-2
(insulin-receptor homologue). age-1 (PI3K homologue). pdk-1 (PDKl homologue) and
AKT-1 and AKT-2 (AKT homologues) result in a dramatic increase in longevity which can
be suppressed by mutations in daf-16 (homologue of the marnmalian transcription factors
FKHR, AFX and FKHRLl). Under normal reproductive growth conditions. the DAF-16
protein is phosphorylated and inactivated to allow the expression of metabolic genes
necessary for reproductive development and short lifespan ( 148). D M - 18, the C. elegans
homologue of PTEN. functions as a negative regulator of the DAF-2 and AGE-1 signaiing
pathways. Loss of DAF-18 rescues the extended longevity associated with loss of AGE-1
(149-152). Figure 1-7 illustrates the insulin receptor-like metabolic signaling pathway in C.
Figure 1-7. PTEN antagonizes the P13WAKT pathway in C. elegnns
AGE- I A Insulin Receptor
PTEN
PDK 1
important genetic evidence implicating PTEN as a negative regulator of the PDWAKT pathway has been derived from studies in C. eiegans. This pathway is important for the regulation of dauer formation (explained in text). The PTEN homologue in the nematode, DAF- 18, negativel y regulates the signals mediated by the PI3K homologue AGE- 1 .
A ROLE FOR PTEN IN T CELL SIGNALING
Many lines of evidence indicate a B y role for PTEN in regulating TCR signaling and
T cell function. For example, PTEN knockout mice can-ying one copy of the nul1 allele
develop a high frequency of T cell lymphomas (47). T cells from heterozygous PTEN mice
generated by another group (49) display reduced activation-induced ce11 death (AICD) and
increased proliferation upon activation. indicating that PTEN may act to suppress the
growthlproliferation of T lymphocytes. P13K. whose activity is antagonized by the action of
F E N , has been implicated in the mediation of signals transduced through sevenl surface
molecules involved in T ceIl activation and proliferation such as the TCR. CD28, and the IL-
2R (153).
Insight into the involvement of P13K in T ceIl function in vivo and its role in
tumorigenesis has been recently provided through transgenic mice expressing an active form
of P13K (p65PI3K) in T cells (154). These mice displayed an increased AKT kinase activity
and developed an infiltrating lymphoprolifentive disorder as well as autoimmune rend
disease. Interestingly, a similar phenotype was described for heterozygous PTEN mice (49).
suggesting that it is the lipid substrates of ETEN that are responsible for the
l yrnphoproli ferative and autoimmune disorders seen in these mice. PI3K has also been
shown to play important roles in BCR signaling. Both PI3K activity and the products of this
activity have been shown to increase following BCR engagement (155). Genetic evidence
demonstrating the importance of PDK for B cells has been provided by examination of the
phenotype of mice lacking p85-PI3K expression. which showed defects in B ceIl function
and development ( 156).
While work for this thesis was carried out, a report demonstrated that PTEN
antagonizes the PDK pathway in Jurkat T cells. such that cells transfected with PTEN
displayed an increased rate of apoptosis which was reversed when a constitutively active
membrane-bound form of AKT was coexpressed (157). Similady. the activation of AKT has
been assessed in T cells, revealing that AKT is activated upon TCR engagement or upon
expression of an active form of PI3K in T cells. The TCR-mediated activation of AKT could
in tum be inhibited by exposure to PI3K inhibiton (158). The function of AKT in T ceil
survival was elucidated further by generating mice expressing a constitutively active fom of
AKT in the T cell lineage (159). The active AKT increased resistance to apoptosis to various
stimuli. a property that was correlated to the increased levels of the anti-apoptotic molecule
Bcl-XL. In contrast to other studies (160). AKT did not have an effect on the
phosphorylation of BAD, which in tum was found to be poorly expressed in T cells. Thus
BAD may not be a relevant substnte for AKT in T cells. However, nuclear NF-KB was
elevated in the active AKT transgenic T cells upon TCR engagement, which correlated with
the accelerated degradation of the NF-KB inhibitory protein IKB. This finding suggested that
AKT might enhance the nuclear translocation of NF-KB upon TCR cross-linking.
AKT is constitutively phosphorylated in resting ETEN-deficient Jurkat T cells. a
reflection of the increased levels of PUK products in the cell membrane (161). The
dysregulation of PtdIns in this ce11 line results in the constitutive membrane association of the
PH domain-containing kinase Itk (161). The expression of PTEN or the use of PI3K
inhibiton was shown to not only block the constitutive activation of AKT, but also io induce
the redistribution of Itk to the cytosol.
In summary, these observations pertaining PTEN provide evidence that PTEN plays
an important role in the modulation of TCR signaling and the growth/proliferation of T cells,
primarily through its ability to regulate the PDKIAKT pathway.
A mechanism for PTEN regulation
Very little is known about the regulation of PTEN expression, an event that could
have a dramatic impact on ceIl survival and tumorigenesis. Expression of PTEN mRNA is
rapidly down-regulated upon TGF-8 treatment of a human keratinocyte tumor ce11 Iine (25),
but the biological signi ficance of this regulntory pathway is still unclear. In collaboration
with Our group, Dr. G. Mills found that PTEN is inducibly tyrosine-phosphorylated in BCR-
stimulated CH 12 (B Iymphoma) cells (G. Mills, persona1 communication). Moreover,
phosphorylated PTEN is capable of associating with a catal ytically inactive form of the SHP-
1 tyrosine phosphatase (C435S) when both proteins are expressed in COS7 cells with a
constitutively active form of Lck, capable of phosphorylating PTEN as well as SHP- 1. SHP-
1 (SH2 domain-containing phosphütase I) is a cytosolic protein tyrosine phosphatase highiy
expressed in T cells. SHP-1 has been shown to exert a predorninantly negative effect on the
sigrialing events linking TCR engagement to prolifention (162). This inhibitory effect is
evidenced b y the increased TCR-induced proli ferative response of th ymoc ytes and T cells
from SHP-l-deficient motheaten (me) and viable motheaten (me') mice upon TCR
stimulation (163). The association between PTEN and SHP-1, however, was not observed in
cells expressing wild-type SHP- I instead of the catal ytically inactive SHP- 1. This
observation suggested that &N rnight represent a substrate for SHP- 1. In agreement. the
lipid phosphatase activity of PTEN was found to be down-regulated when PTEN is
phosphorylated (G. Mills, penonal communication).
Although none of these findings have been investigated in T cells yet, the above
observations suggest that the ability of SHP-1 to interact with phosphorylated PTEN may
provide for a mechanism to modulnte the enzymatic activity of PTEN as well as its capacity
to interact with other targets.
ROLE OF K E N PROTEIN PHOSPHATASE ACTIVITY IN TUMOR
SUPPRESSION
As stated earlier, PTEN can dephosphoryiate protein substrates. In particular
overexpression of PTEN leads to the dephosphorylation of focal adhesion kinase (FAK)
resulting in a decrease in ceIl spreading. cell motility and the formation of focal ûdhesions
(58. 164). The dephosphoryliition of FAK ût tyrosine 397 by PTEN also seems to decrease
the tyrosine phosphorylation of its potential downstream effector. pl30Cas (58). FAK is a
non-receptor tyrosine kinase that colocalizes to cellular adhesions with integrins and is an
important element in some integrin-regulated pathways (165). FAK is expressed in most
tissues. and becomes tyrosine phosphorylated and activated after the stimulation of various
receptors and it has been linked to signaling pathways that regulate MAPKs (166). There
exists evidence irnplicating FAK in receptor-initiated activation of T cells showing that FAK
becomes phosphorylated after TCR ligation (166-168). Moreover. the expression of FAK is
increased in invasive and metastatic turnors (166). FAIS Iacks SH3 or SH3 binding domains
so its phosphorylation on tyrosine residues is critical for its interaction with the S H 2 domains
of other signaling molecuIes such as the adapter molecule Grb3 and p85-PI3K (164. 166,
169). The association of PUK and FAK occurs upon attachment of cells to the matrix and
results in a rapid increase of PI3K lipid products and AKT and protection from apoptosis
( 164. 166). Thus PTEN seems to regulate the extracellular rnatrix-dependent PI3 WAKT ce11
survival pathway by a mechanism that may involve FAK. An anti-apoptotic role for F M
has also been recently demonstrated against oxidative stress-induced apoptosis in HL-60
cells and a glioblastoma ce11 line (170). In addition. ETEN has been observed to inhibit
integrin- and growth factor-mediated MAPK signaling pathways. This inhibition was
associated with the ability of PTEN to dephosphorylate Shc. which in tum inhibits the
interaction of Shc with the adapter protein Grb2 and the subsequent activation of the
RaslMAPK pathway (60, 171). These results indicate that PTEN can suppress ceIl motility
by modulating two independent pathways involving Shc and FAK (60).
Importantly, ü PTEN mutation (G119E) that lacks lipid phosphatase activity but
retains protein phosphatase activity (51) is still capable of inhibiting ceIl spreading and
motility (58). This finding indicates that the inhibitory activity of PTEN in these cellular
processes is independent of PI3K regulation. Although the tumor-suppressive functions of
PTEN appear to depend only on its lipid phosphatase activity. the tyrosine phosphatase
activity of ETEN might still be necessary for other aspects of tumorigenesis. However. it is
important to keep in mind that one group only has reponed these findings. and that the direct
interaction between PTEN and FAK. and FAK dephosphorylation upon PTEN transfection.
have not been reproduced by other groups. Therefore. this issue remains rather controversial
and requires a more thorough analysis. Cenainly, the availability of a mutant that retains
only lipid phosphatase activity would allow a better understanding as to whether the lipid
phosphatase activity of PTEN is sufficient for tumor suppression and whether the protein
phosphatase activity is a necessary function.
SUMMARY
One of the challenging questions that remain to be addressed in the future is whether
additional PTEN substrates exist. The evidence indicating that PTEN down-regulates PI3K-
dependent pathways does not rule out that PTEN might control other pathways irnplicated in
functions other than the control of cell growth and survival. Given the importance of PTEN
in regulating cell survival pathways. it is critical to target future experiments into
undentanding the mechanism or mechanisms involved in the regulation of PTEN expression
and function.
In this context, my research has focused on elucidating mechanisms whereby PTEN
influences some of the signaling pathways involved in the activation and proliferation of T
cells. Part of my work focused on finding new substriites for PTEN. by coupling the
technologies of affini ty chrornatograph y wi th the sub-picomolar protein identification
capabilities of rnass spectrometry. These powerful techniques are discussed in more detail in
Chapter 4. In addition. based on preliminary data 1 proposed a novel mechanism for the
regulation of PTEN activity in T lymphocytes.
Chapter 2: Materials and methods
CELL LINES
The human T lymphoblastic leukemia cell line Sup-Tl was kindly provided by Dr. D.
Branch (Canadian Blood Services, Toronto, ON). Cells were cultivated in Dulbecco's
Modi fied Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine
semm (FBS) (Sigma), 1mM L-glutamine (GibcoBRL), 5x lo-' M B-mercaptoethanol
(GibcoBRL) and 100 UImL ampicillin, at 37°C in a humidified atrnosphere containing 5%
CO..
GENERATION AND PURIFICATION OF GST-PTEN FUSION PROTEINS
The cDNA encoding human PTEN (C124S) was PCR amplitïed with the forward
primer Y-TAGGGATCCATGACAGCCATCATC-3' and the reverse primer 5'-
GGCGAAM'CTCAGACTMTGTAAT-3' from the plasmid MMAClPGEM3 (C124S). a
generous gift from Dr. G. Mills (Houston. Texas). The fonvard and reverse primers contain
an added BamHl site and an EcoR 1 site respectively (underlined). The PCR reaction was
cmied out for 25 cycles (denaturing for 1 min at 94°C: annealing for 1 min at 55°C;
extension For 2.5 min at 72OC) using a Perkin Elmer Cetus DNA thermal cycler. The DNA
polymerase pfu (Stratagene) was used to minimize the introduction of mutations. The 1230
base pair PCR product was isolated from a 1% agarose gel using a QIAquick Gel Extraction
Kit (Qiagen), digested with BarnHl and EcoRl (Amersharn Pharmacia Biotech) and cioned
into the pGEX-2T vector (Amersham Pharmacia Biotech), which had been linearized using
the same enzymes (Figure 2-1). The sequence of PTEN (C124S) once in the pGEX-3T
plasmid was verified by using the PGEX forward and reverse pnmers (Amersham Pharmacia
Biotech) and sequencing facilities at Samuel Lunenfeld Research Institute (S W). Mount
Sinai Hospital. The sequence of PTEN (C 124s) cDNA is provided in Figure 2-2.
Ovemight cultures of E. coli strain BL21 cells (Stratagene) transfomed with the
pGEX plasmid or the recombinant PTEN (C124S)-pGEX plasmid, were diluted 1 5 in 2L of
fresh LB medium + ampicillin (50 pg/rnL) and grown at 37°C to an between 1 and 1.5.
To induce the expression of the GST protein or the GST-ETEN fusion protein. 0.2mM IPTG
was added to the bacterial cultures for an additional 2-3 hours. Cells were harvested by
centrifugation for 20 min at 12.000 rpm in a Sorvall SLA-3000 rotor at 4OC. The cells were
resuspended in 20mL of ice-cold sonication buffer (1% PBS. 150rnM NaCI. lOOrnM EDTA.
2mM PMSF, Zpg/rnL aprotinin. IpglmL leupeptin. Zpg/mL pepstatin. 5mM DTT) and
sonicated. After adding Triton X-LOO (Sigma) to a final concentration of 1%. the lysates
were centrifuged for an additional 10 min at 15.000 rpm and 4"C in a Sorvall SS-34 rotor.
To funher clarify the supernatant from cellular debris. the lysates were centrifuged a second
time under the same conditions. Al1 subsequent steps were performed at 4°C. The lysates
were mixed with 3mL of 75% slurry of Glutathione Sepharose 4B beads (Amenham
Pharmacia Biotech) in a 50mL polypropylene tube on a rotating platform for lh. The beads
were packed into a disposable 5mL PD- IO column (Amersham Pharmacia Biotech) and
washed three times with 4OmL of ice-cold sonication buffer with 1% Triton X-100 and two
times with ice-cold 1X PBS. The GST or GST-&N fusion proteins were eluted off the
beads by incubating with 1OrnM reduced glutathione (Amersharn Pharmacia Biotech). 25mM
Tris-HCI (pH8.0) and 1rnM DTT, as recommended by the manufacturer. Cleavage of the
GST affinity tag was accomplished by incubating the colurnn with the site-specific thrombin
protease instead of reduced glutathione (according to manufacturer's instructions). The flow-
through containing purified PTEN or GST-PTEN were loaded ont0 a lm1 HI Tnp Q
Sepharose column (Arnersham Pharmacia Biotech) equilibrated wi th Buffer A (20mM Tris-
HCI (pH 8.0) and ImM D'TT) and at an approximate rate of 1 mumin. The proteins were
eluted with a linem gradient of Buffer B (500mM KCI, 25mM Tris-HCI pH 8.0 and 1mM
DTT) at a rate of 0.5 Wrnin. Ten fractions of 1mL each were collected. Al1 this was done
using an FPLC system (Amersham Phmacia Biotech). The punty and relative amount of
purified PTEN or YTEN fusion proteins in each fraction were analyzed by coomasie blue-
staining of an 10% SDS-PAGE gel. The fractions containing the most amount of
PTEN/GST-PTEN were pooled and immediately dialyzed against ACB (20rnM HEPES pH
7.5. 10% glycerol. 1mM DTT. 1mM EDTA. 1miM PMSF and 2 pM of each leupeptin.
aprotinin. pepstatin) containing lOOmM NaCl. The amount of K E N present after dialysis
was estimated by the BCA Protein Assay system (Pierce).
AFFTMTY CHROMATOGRAPHY
Sup-Tl ce11 entract was prepared as follows. The cells were grown and collected by
centrifugation at 500 x g at 4'C in a Sorvall SLA-3000 rotor. A cell pellet of approximately
3g was lysed in ice-cold ACB containing J O O m M NaCl and subjected to mild sonication.
Cellular debris was removed by centrifugation for 3h at 4OC and 37.000 rpm in a Beckman
70Ti rotor. The supemantant was dialysed against ACB containing lOOrnM NaCl and
centnfuged at 4°C at 14,000 in a table-top microcentrifuge for 15 min. The foiiowing steps
were performed at the laboratory of Dr. J. Greenblatt (The Banting institute. University of
Toronto) and under the supervision of Dr. D. Awrey. A series of 4 columns were prepared
by coupling each with an increasing arnount of GST, GST-PTEN. or PTEN (0.1.0.5, 1,7 pg)
to the column matrix (Affigel 10, Bio Rad). The matrix was then blocked at both specific
and non-specific binding sites by incubating with ethanolamine and BSA, respectively. A
column containing no protein was used as a control column for identifying background
proteins that would interact with the column matnx under these conditions. The ce11 extract
from Sup-T 1 cells was loaded onto the columns, washed. and eluted sequentially with ACB
containing 1% Triton X- 100. LM NaCl or 1% SDS. Another control column containing 2 pg
of cross-linked PTEN. but ro which no ce11 extract was added. was used to identify
components of the cross-linked protein that may dissociate from the rnatrix during washing
and elution. The eluates (90pL) from the column washings were collected and the proteins
separated by an 12.5% SDS-PAGE gel ( U m m thick) at 30-35 mArnps for 4.5 hrs. The
proteins were visuaiized by silver staining safe for MS analysis. as follows.
Silver staining
The gel was fixed in 50% methanol and 10% acetic acid for 20 min. This step was
repeated once. after which the gel was rinsed in 10% ethanool for 10 min followed by a rinse
in water for 10 min. The gel was reduced with sodium thiosulfate (O.?@) for 1 min and
rinsed twice with water for 20 sec. The ge1 was then incubated in silver nitrate (2@) for 30
min and washed once with water for 20 sec. To develop the staining. the gel was incubated
with developing solution (30zA sodium carbonate, formaldehyde ( 1 A d of 37% solution/L)
and LOmgL sodium thiosulfate) until the desired intensity was attained. The reaction was
immediately stopped by replacing the developing solution with 1% acetic acid and incubated
for a minimum of 20 min. The stained gel was photogaphed using a digital canera. Figure
2-3 summarizes the various steps involved in the affi ni ty chromatograph y expenmen ts.
Proteins that specifically interacted with cross-Iinked ETEN were seen to increase in quantity
with increasing amounts of bound F E N , whereas background proteins remained constant
regardless of the arnount of cross-linked PTEN.
MASS SPECTROMETRY AND IDENTIFICATION OF PROTEINS
Candidate protein bands were excised from the ge1 and digested ovemight with
trypsin. This step and the preparation of the sample were done by B. Cox of Dr. J.
Greenblatt's Iaboratory. The analysis of the samples were performed using a PerSeptives
Biosystems Voyager STR MALDI-ToF in reflector positive ion mode, using the following
settings; ücceierating voltage: 10000. Grid voltage: 72.000%. Guide wire voltage: 0.050%
and Delay: 200 ON. The protein identification by peptide mapping was done using
ProFound search engine from ProteoMetrics (at www.proteometrics.com).
Figure 2-1. GST-PTEN fusion protein
Thrombin cleavage-site
H3N
The GST-PTEN fusion protein was generated by subcloning the cDNA of human PTEN into the pGEX-2T vector containing the GST affinity tag. The BamHl and EcoR 1 sites were introduced into PTEN by PCR. The pGEX-7T protein expression vectcr contains an accessible thrombin recognition sequence, which allowed the site-specific cleavage of the GST affinity tag from the PTEN protein once expressed and purified.
GST COOH
t- F t- w w W t - W O @ln 4 t J U l m LIVU & n u m w
" r 2 " " C "Z 1') C l x
E; 3 y
O 'jiï
m $
W C > U C J
1: K " E " f
< L i X 3 ' r! " ; "' :l xl: 2 ' i
*4 Pl
L-l L I R " il z*
" 5 Y
I d la tnrn u o cnw u i w
Figure 2-3. Affinity chromatography
Ce11 extract ACB
O O. 1 0.5 1 2 - 3 (pg of cross-linked PTEN) y-.. ,-. , - - y - ',-. .-. 5 4 :-- 1, \ --q +----j \
+ protein elution
Increasing amounts of purified PTEN (CIZJS) protein were cross-linked to a series of columns. Proteins from ce11 extract that associated with PTEN were eluted from the columns, resolved by SDS-PAGE and silver-stained. Proteins that were found to interact with FIEN were recognized as bands that increased in quantity with increasing amounts of cross-linked PTEN. These bands were excised from the gel and digested with trypsin. The resulting peptides were analysed by mass spectrometry.
MlCE
Motheaten mice for these studies were obtained by mliting heterozygous motheaten
mice (mec'-) in C57BU6 background breeding pairs from stock maintained at SLRI and have
been previously described (172). Fernale mice 3 4 weeks old were used in the experiments.
~ e " ' mice used in these experiments were littermates of the me"' mice.
ANTIBODIES
The monoclonal CD3 and CD28 mouse antibodies were purchased from Pharmingen
and anti-hamster IgG from Jackson Laboratones. Goat anti-mouse PTEN (N-19) pAb and
rnouse PTEN (AIB 1) mAb and IgG mAb were purchased from Santa Cruz Biotechnology.
Mouse phosphotyrosine (G410) mAb was purchased from Upstate Biotechnology and
phospho-AKT (Ser.173) nbbit pAb and AKT rabbit pAb were purchased from New England
BioLabs. The secondüry antibodies used were honeradish peroxide (HRP)-conjugated rabbit
ünti-goat IgG (Santa Cruz Biotechnology), HRP-conjugated goat anti-rnouse IgG (BioRad)
and HRP-conjugated Protein A ( Bio-Rad).
ACTIVATION OF THYMOCYTES
Freshly isolated thymocytes (3 x 10') from rnotheaten (meime) and their littermate
(+/+) rnice were resuspended in cold serum-free RPMI-1640 media. After 2 hours on ice, the
cells were spun down briefly and resuspended in 0.2mL serum-free RPMI-1640 media in
1.5rnL Eppendorf tubes. Anti-CD3 and anti-CD28 were added to each sample alone or
together to a final concentration of 25pdmL and 15pdrnL respectively. The cells were
incubated on ice for 20 minutes followed by the rernoval of the antibody-containing media
and resuspension in fresh media. Anti-hamster IgG was added to the sarnples to a final
47
concentration of 25pg/pl in order to crosslink the bound antibodies. The sarnples were
incubated at 37OC water bath for 2 , 5 and 10 min. time after which the cells were quickly
spun down and resuspended in 0.2m.L cold lysis buffer (1% Nonidet P-40, 50mM HEPES
(pH 7.4), l5OrnM NaCI2, 50mM NaF. 50mM sodium phosphate. 3mM EDTA, 3mM sodium
onhovanadate, 2mM PMSF and 2 pM of each leupeptin. aprotinin. pepstatin). The O min
group was lysed immediately after the 20 min incubation on ice. Following lysis, the
samples were transferred to ice for an additional 10 min and centrifuged at 14,000 rpm at 4°C
for 15 min to remove cellular debns. The total protein concentration of the supernatant was
determined by using BCA protein detemination system (Pierce). In order to check whether
thymocyte activation was successful, 50pg of activated and non-activated lysate were loaded
onto a 12% SDS-PAGE gel. transferred ont0 a nitrocellulose membrane and blotted using an
antibody against phosphorylated tyrosine residues. The membrane was re-blotted with p-
actin antibody to check for equal loadin;. Details on western blotting are explained in the
next page.
IMMUNOPRECIPITATION OF PTEN A M ) ELECTROPHORESIS
The lysate frorn activated or non-activated thymocytes (500pg total protein) was pre-
cleared with 15pL of Protein G sepharose 1FF beads (Arnersharn Pharmacia Biotech) for 30
min at 4OC. PTEN was immunoprecipitated by incubating the pre-cleared lysates with anti-
PTEN (A2B1) conjugated to Protein G sepharose beads for 2 4 hours at 4OC. The beads
were washed three tirnes with cold lysis buffer and three times with PBS. and then boiled for
5 minutes in Laemmli buffer (2%SDS, l O O m M Tris-HCI (pH 6.8). 20% fhnercaptoethanol,
0.0 1 % bromophenol blue).
The proteins were resolved by SDS-PAGE (10%) at LOO V and
electroblotted ont0 a nitrocellulose membrane (Xyrnotech Biosysterns) for 2
Amps. Purified PTEN obtained frorn the purification procedures described ear
i mmedi atel y
hours at 0.5
*lier was used
as a positive control for the PTEN blots. The anti-PTEN (A2B1) antibody was covaalently
coupled to Protein G sepharose beads as follows. Protein G sepharose 4FF (0.2rnL of slurry)
was washed twice with 3 bed volumes (b-v.) of 20mM sodium phosphate (pH 8.2). The
beads were bnetly spun down and the buffer removed carefully by suction. The beads were
then incubated with O.5rnL of sodium phosphate buffer and anti-PTEN antibody (O.lmg/rnL)
with gentle rotation for 2 h at room temperature, time after which the beads were spun down
and the buffer removed. This was followed by washing the beads with 6 b.v. of 30mM of
sodium phosphate buffer. The antibody was then crosslinked by adding 1mL of the
crosslinking solution (8mg of Dimethyl pimelirnidate. Sigma. dissolved in Z m L of O.2M
triethanolamine pH 8.1) and rotated for 1 h at room temperature. After removing the
crosslinking buffer. the beads were wüshed sequentially with 6 b.v. of 0.2M triethanolamine
(pH 8.2), 2 b.v. of 0. iM ethanolamine (pH 8.2) for 15 min. 3 b.v of O.iM glycine (pH 2.8)
and 6 b.v. of 20mM sodium phosphate (pH 8.2).
WESTERN BLOTTING
For detection of phosphorylated PTEN or phosphoryiated proteins following
thyrnocyte activation. the membranes were blocked for 1 h at room temperature with 3%
(w/v) of gelatin in TBS-T (20mM Tris-HCi (pH 7.6). 150mM NaCl. 0.05% Tween-20). The
membranes were rinsed three times with TBS-T before ovemight incubation at 4OC with the
phosphotyrosine antibody ( 1:5000). The membranes were washed three times for 5 min with
TBS-T and then incubated for 1 h at room temperature with HRP-conjugated goat anti-mouse
IgG (1:10,000) in TBS-T containing 5% (wlv) nonfat dry milk powder. The membranes
were washed three times for 10 min with TBS-Tl and immunoreactive bands were visualized
by enhanced cherniluminescence detection (Amersham Phmacia Biotech). To reprobe these
blots, bound Abs were eluted by incubating the blots for 30 min at 55 OC with stripping buffer
(62.5rnM Tris-HCI (pH 6.7). 2% SDS, lOOmM P-mercaptoethanol). The membranes were
blocked with 5% (wlv) nonfat dry milk for 1 h at room temperature followed by overnight
incubation with with anti-PTEN (N-19) Ab (diluted 1:200 in TBS-T) at 4*C. The KRP-
conjugated rabbi t anti-goat was used at a dilution 1 :3000 in TBS-T.
For the detection of phosphorylated AKT. 50yg of total protein lysate from activated
or non-activated lysates were sepanted on a 12% SDS-PAGE gel and transferred ont0 a
nitrocellulose membrane. The membranes were blotted using an antibody specific to Ser-473
and in ;i similar manner as described above with the following three exceptions. Fint, the
membranes were blocked with 5 '70 nonfat dry milk in TBS-T. the pnmary Ab was diluted
1:1000 in TBS-T plus 5% BSA, and visualized using HRP-conjugated Protein A (1:3000).
The membranes were reprobed with an antibody to AKT (1: 1000) to check for equal Ioading.
Chapter 3: Results
IDENTIFICATION OF NOVEL LIGANDSISUBSTRATES FOR PTEN
As mentioned in Chapter 1. the spectmm of proteins which physically associate with
PTEN remain elusive. In order to identify proteins that may represent substrates or
interacting proteins of F I E N in vivo, the technique of affinity chromatography was used to
Rnd potential protein ligands/substrates for PTEN followed by the identification of such
proteins by mass spectrometry. In an attempt to maximize the likelihood of obtaining
potential ETEN-interacting proteins, the catalytically inactive fom of PTEN containing the
C 124s mutation was used in these expenments. The alteration of the nucleophilic Cys to Ser
has been shown to allow some PTPs to be isolated in a complex with their target substntes
(64).
Purification of PTEN for affinity chrornatography
The purification of GST-PTEN was initially performed using the E. coZi competent
cells of the strain DH5u instead of BLIl to express the fusion proteins. However. numerous
contaminating bands were obtained in the final product. To resolve this problem and thus
minimize the chance of acquiring unwanted and irrelevant proteins. the PTEN fusion proteins
were expressed in B L l l E. coli strain instead (Figure 3-1). BL21 cells are deficient in
certain bacterial proteases that may potentially cleave the protein of interest thus generating
multiple bands. To funher clean the purified fusion PTEN to be used in subsequent steps,
FPLC (Mt performance liquid bromatography) was carried out using an anion-exchange
column.
Affinity Chromatography
Affinity chromatography was tint canied out by coupling purified GST-PTEN
protein to a column matrix. As a control, this experirnent was done in parallel to another
where GST protein was coupled to the column matnx. The protein elution steps revealed
that no proteins from the ce11 extract bound to the cross-linked GST-PTEN. In view of these
results. the GST portion of the GST-PTEN was cleaved off (Figures 3-LB). as explained in
Chapter 2. This was done under the assumption that the GST affinity tag might be
responsible for this negative result. After cleavage of the GST moiety. the experirnent was
repeated by cross-linking PTEN to the column rnütrix. The cell extract was loaded ont0 the
columns and the proteins eluted as explained in Materials and Methods. The eluted proteins
were separated on an SDS-PAGE gel and subjected to silver-staining, which revealed 8
bands. These bands were seen to increase in quantity as the amount of cross-linked PTEN
increased, and were absent in the control colurnns. These bands were then selected 3s
potential ligands for PTEN. Figure 3-2 shows a picture of the silver-stained gel containing
the proteins eluted after the LM salt and 1% SDS wash. The bands identified were of
approximate sizes 23. 28. 30. 45, 50. 55. 67 and 110 kDa. and were eluted off the columns
after washing with 1M salt. The purified PTEN that was cross-linked to the columns was
seen to corne off with the l%SDS wash. The 8 bands identified were then excised from the
silver-stained gel and trypsinized. as explained in Chapter 2. The choice of Sup-Tl cells as
the source of cell extract from which PTEN ligands were to be found was subjective. This
ce11 was shown to express PTEN, and thus favored, in a screen of various T ce11 lines (cell
Iysates were provided by Dr. D. Branch). as seen in a western blot using monoclonal PTEN
antibodies (Figure 3-IC). The implications of using this ce11 line in these experiments are
discussed in Chapter 4.
Figure 3-1. Expression and purification of GST-PTEN
B GST kDri kI GST PTEN PTEN -
Jurkrit SiT Sup-Tl Molt-l
(A) Expression of GST-PTEN in BL21 E. d i cells after 2 hr induction with IPTG visualized by coornasie blue staining after separation of cell lysate by SDS-PAGE. (B) After induction of protein expression, GST. GST-FIEN and PTEN were purified from bacterial ce11 lysate as explained in Materials and Methods. The purified proteins were resolved by SDS-PAGE and stained with coomasie blue to check for purification quality. (C) 50pg of protein lysate from the human T ce11 lines indicated were immunoblotted using PïEN-specific antibodies.
Figure 3-2. Potential binding-partnen for PTEN
Salt SDS
kDa O O.! 0.5 1 2 ACB O 0.1 0.5 1 7 ACB - (pg of cross-linked PTEN)
+ PTEN
A series of 5 columns were prepared by cross-linking 0, 0.1. 0.5. 1 and 2 pg of puified PTEN to column matrix. Cell extract derived from a human T ce11 line was loaded onto the columns. A sixth colurnn with I pg of FTEN was loaded with buffer instead of cell extnct. This colurnn was used as a control for determining components of the cross-Iinked PTEN that rnight corne off with the various protein elution steps. Proteins that associated with the bound F E N were eluted sequeniially with 1M NaCI, 1% Triton X-100 and 1% SDS. The protein eluates collected were separated by SDS-PAGE and visualized by silver staining. Because the 1% Triton X-100 wash did not provide any novel bands, only the proteins eluted with 1M NaCl and 1% SDS are shown in this figure. The position of the proteins that were identified as potential PTEN ligands are indicated by short arrows. These proteins were seen to increase in levels with increasing amount of cross-linked PTEN and were absent from the column to which buffer was added instead of ce11 extract, whereas Ievels of irrelevant proteins remained constant throughout the various columns. Cross-linked PTEN carne of the columns after washing with 1% SDS, shown with a long m o w .
Identification of potential PTEN ligands by mass spectrometry
Protein digestion of the 8 bands generated numerous specific peptide fragments,
whic h were subjected for anal ysis b y MALDI-ToF (Matrix-bsisted @er Bsorption
bnization-Iime of Elight) mass spectrometry. This technique is explained in more detail in
Chapter 4. In brief, MALDI-ToF determines the mass of each digest fragment, generating
unique spectra for each protein. These data are compiued, by means of a cornputer program.
to theoretical data that would have been generated if the protein had been digested by trypsin.
The proteins are thus identified by the masses of their proteolytic peptides. From the 8
protein bands initially isolated in this experiment. only 6 provided spectra with the masses of
their peptides. which allowed their identification. The names of these proteins and their
approximate molecular size. as derived frorn the silver-stained gel. are summarized in Table
1. The matching of pruteins was perfonned with +/- 0.1 Da error. The measured mass and
the calculated mass of each peptide measured by mass spectrometry are indicated in Table II
dong with their sequence and their position in the stipulated protein. It remains to be
mentioned that these results do not. by any meüns. assert that the proteins found to interact
with PTEN represent physiologie ligands for PTEN. Whether these proteins may be relevant
to the role of PTEN in T ceIl function. is discussed in Chapter 4. Given the complexity of
these experiments and time limitations for my thesis work. this part of my research was
discontinued at this stage.
Table 1. Summary of proteins identified by MALDI-ToF mass spectrometry* after
performing affinity chromatogruphy using purified FFEN
Protein name (as identified by MAI,I>I-ToF MS) NCBI protein accession number
Hurnün SET protein (Protein phosphütüse 2A inhibi tor-I2PPîA) (Putative HLA-DR Associüted Protein ILPHAPII)
Humun PHAPI2b protein
PHAPI (putative humün HLA clüss I I associated proicin 1 ) (humiin potent heitt- stable prolein
Humün chüperonin contüining T-complex protein 1 (TCPI ), eta subunit Hurnün T-complcx pmtein 1 (TCP 1 ), thetü subunir
Humün tubulin bela- 1 chuin (TBB I )
Humün nucleolin Human moesin (membrine-orgünizing extension spike protein)
135448
* The protein mutching wiis donc wi th ü +/- 0.1 Dn error
Table II . Information data on the proteolytic peptides by Mass Spect rometry (Continued)
Name of Protein (MW)
'I'CP 1 (55 )
Nuclcolin (67)
Mocsin
- Nurnbcr of rneasurcd peptides
35
Number of niatchcd peptides I
1 O
Covcrrrgc of protcin sequencc
2O'Xl
M casurcd Mnss (M)
Coniputed M a s (M)
- Hcsidues
44 1-447 309-375 44-53
43 1-440 358-366 388-397 370-387 3 19-230 345-357 107- 133
Peptide Scquence
AIXIIPK r ~ s c r ~ ~ ~ LU GMDK1,IVI)GR QQLLIGAY AK Y NI;I;RGC13K SLI IIIAIMIVK GGAEQFMEI'I'EK TFSY AGFEMQPK CQVIWITQIGGER QVKI'Y VEEGLI-1PQIIIK
1,lIL.QGPK GGFGGKGGGK GGCiRGGIGGK NDLAVVDVK Ai RL1il.QGPK 'I'I,I,AKNI,PY K SISLY YTGEK HVFEDAAEIR VIGNEIKLEKPK G1:GFVDGNSIIIIDAK GY AFIEFASFEDAK FGY VDGESAEDLEK KFGY VDESAEDLEK SlSLY YTGEKGQNQDYR G1,SED'I"I'EETLKESl:IlGSVR
IGFPWSEIR APDFVFY APR LNKDQWEER IQVWHEEHR ESEAVEWQQK SGY LAGDKLLPQR
DEFINITION OF PTEN EFFECTS ON T CELL FUNCTION
PTEN is tyrosine-phosphorylated in thymocytes upon TCR engagement
As mentioned earlier, Dr. G. Mills showed. in collaboration with our group. that
PTEN is inducibly phosphorylated upon BCR engagement in a B ce11 line and that its
phosphorylation. in vitro, resulted in a reduction of its lipid phosphatase activity (G. Mills,
persona1 communication). Consequently. we wanted to examine these findings in T cells and
thus specifically determine if the phosphorylation of PTEN could be induced in normal
thymocytes upon TCR ligation.
In order to examine this possibility. thymocytes from normal mice were stimulated
with monoclonal anti-CD3- and -CD28 antibodies followed by crosslinking with anti-
hamster IgG for 2 minutes and lysed. Lysates were resolved by SDS-PAGE and
immunoblotted wi th antibodies speci fic to phosphorylated-tyrosine residues (p-Tyr). to
ascenain that the stimulation procedure resulted in an increase of phosphorylated proteins
compared to lysate from non-stimulated cells, a characteristic result of the ligation of the
TCR. PTEN was then immunoprecipitated from the remaining activated lysate with a mouse
monoclonal anti body that had been previousl y chemicall y cross-linked to beads. which was
required to prevent phosphorylated IgG heav y-chains. w tiose size approxirnate that of PTEN.
from masking the PTEN band in the p-Tyr biots. Similady. the immunoprecipitated proteins
were resolved by SDS-PAGE for an extended period of time to maximize the sepantion of
proteins and facilitate the identification of PTEN. Figure 3-3A shows that TCRKD28
engagement results in the tyrosine phosphorylation of PTEN. The identity of the
phosphorylated band was confirmed to be PTEN by re-blotting the membrane with
antibodies specific to PTEN (Figure 3-3A. bottom panel). In addition. the
59
irnmunoprecipitation of ETEN with the aforementioned antibodies is specific, since no bands
are detected when mouse IgG is used instead (Figure 3-3B). The conditions for the
activation of thymocytes were worked out pnor to PTEN immunoprecipitation. Thymocytes
were stimulated for various induction times (2. 5, 10. 30, 60 min). Stimulation using
CD3+CD28 anti bodies for 1 minutes, resulted in the most protein phosphory lation, and thus
was chosen for immunoprecipitating PTEN (Figure 3-43).
TCR-induced activation of AKT is increased in SHP-1 deficient T ceIls
As explained earlier the phosphorylation of AKT on Ser-473 is essential for its
activation. To determine whether TCR engagement might activate AKT. the thymocytes
from both motheaten and wild type mice were stimulated using CD3+CD28 antibodies for 2.
5 and 10 min and then anti-phospho-AKT immunoblotting was performed on the lysates
using an antibody specific for AKT phosphorylated at Ser-473. Fig 3 4 A shows that AKT
was not phosphorylated to a significant extent in unstimulated cells, but that TCR ligation
caused a substantial increase in the phosphorylation of AKT on Ser-473. The
phosphorylation of AKT peaked at 2 min and progressively declined to lower levels at 5-10
min.
In order to examine the possibility that SHP-1 does indeed play a role in modulating
the activity of PTEN in T cells in vivo. we took advantage of the availability of naturally
occumng SHP- 1-deficient motheaten (Me) mice and examined the phosphorylation state of
AKT. Figure 3 4 A also shows that the lack of SHP-1 in Me thymocytes clearly has an effect
on the phosphorylation of AKT on Ser-473. The level of phosphorylated AKT is elevated in
SKP-1-deficient cells compared to control ceils throughout the various stimulation time
points. Tu confirm that the protein being recognized by the anti-phospho-AKT antibody was
in fact AKT. the membrane was re-blotted with an antibody specific to AKT. shown in
Figure 3-4 (lower panel). This figure also demonstrates that the totai levels of AKT in the
cells are not affected by TCR ligation or SHP- 1 expression.
Figure 3-3. PTEN is tyrosine-phosphorylated upon TCR engagement
(min)
Blot: mi-pTyr
B Iot: rtnti-REN
Thymocytes from wild-type mice were isolated and either stimulated with CD3- and CD28- specific antibodies followed by cross-linking with anti-hamster IgG for 2 min or left unstimulated and lysed. Purified PTEN was used as a positive control for the PTEN antibody. (A) PTEN-specific antibodies covalently linked to Protein G Sepharose beads were used to immunoprecipitate P E N , the resulting protein complexes were analysed by Western blotting using phosphotyrosine-specific antibodies. The bottorn panel was immunoblotted with anti-ETEN. (B) The experiment in A was repeated. including a non-specific Ig conuol.
Figure 3-4. TCR-induced activation of AKT is increased in SHP-1 deficient T cells
W ild-type Me
O 2 5 10 O 2 5 10 Stimulation (min)
O 2 5 LO Stimulation (min) -
Thymocytes from Me and wild-type mice were isolated and stimulated with ûnti-CD3 and anti-CD28 antibodies followed by cross-linking with anti-hamster IgG for 0, 2. 5, and 10 min and lysed. The cells were lysed and 50pg of lysate (total protein) was sepanted by SDS- PAGE, (A) Western blot analysis using an antibody specific for the phosphorylated Ser-473 residue of AKT is shown. The bottom panel was immunoblotted with anti-AKT to check for equal loading. These data represent one of two sirnilar experiments. (B) The same as above, except an antibody against phosphotyrosine residues was used.
Chapter 4: Discussion Signais delivered through the TCR and CD28 play an integral role in T ceIl functions
such as proliferation. survival and apoptosis. Given the importance of these signals for T cell
homeostasis. the elucidation of how these signals are regulated seems critical. In this context
the recent discovery of the tumor suppressor PTEN, a lipid/protein phosphatase. has
stimulated new ideas and challenged others. Many lines of evidence implicate PTEN as an
important regulator of some of the sipals originating frorn the TCR. in particular those
mediated by the PUWAKT pathway. This pathway affects many different cellular functions
uitimately responsible for the survival of the cell and thus represents an imponant
mechanism for the regulation of T cell homeostasis during immune responses. However.
many aspects of PTEN function remain to be elucidated. Although it is clear that the
function of PTEN as a tumor suppressor is due in part ro its ability to down-regulate the
PDWAKT pathway, the possibility that it may play a role in other signaling pathways cannot
be excluded. In this context. I set out to unravel some questions. Does PTEN have other
protein substrates aside from FAK and Shc? Does PTEN interact with other signahg
molecules? Does PTEN function downstream of the TCR? How is PTEN activity regulated?
IDENTIFICATION OF NOVEL PTEN LIGANDS/SUBSTRATES
One of the goals of the work presented in this thesis was to identify additional
proteins that may bind PTEN and might represent substrates of its protein phosphatase
activity. This objective is derived from the observation that the GL29E mutation. which is
lipid phosphatase inactive. behaves like ETEN in some biological assays (57). This finding
immediately suggested that the protein phosphatase activity of PTEN might be relevant to
sorne of its physiologie functions. The approach 1 used to identify novel substrates for PTEN
63
involved the use of two techniques, affinity chromatography and mass spectrometry. These
techniques constitute a powerful combination for chancterizing protein biochemical
pathways through the definition of protein-protein interactions. The results presented in
Table I demonstrate that these techniques can be used successfully to identify potential
binding panners for PTEN. However, to establish w hether these proteins represent PTEN
binding partners in vivo requires additional experiments. Therefore. whether these findings
may be of physiologic relevance will be eaamined in future experirnents. Figure 4-1 shows a
schematic of a MALDI-ToF mus spectrometer and brie fl y summarizes how this technology
operates.
An analysis of the potential PTEN ligands
Table 1 shows a summary of the proteins that were identified. In the following
section, I will introduce these proteins and their functions and attempt to propose when
appropriate whether they might represent relevant PTEN substrates.
One of the proteins positively identified is the beta-subunit of tubulin. Tubulin is a
major cytosolic protein whose assembly into microtubules is criticai to many cellular
processes, such as ceIl mobiiity. intracellular transport and T ceil polarization (173. 174).
Microtubules are heterodimers of two soluble proteins, alpha- and beta-tubulin. Tubulins or
microtubules might function in pan to promote the formation of signaling complexes or
perhaps aid in the intracellula. localization of these signaling molecules. In support of this
idea. tubulin has been found to form protein-protein interactions with a wide variety of
signaiing components, including Zap-70, Fyn. CD?. Vav, and P13K (173, 175. 176). In some
cases, the stimulation of T cells affects these interactions suggesting that they may play a role
in T ce11 activation ( 175). Through the experiments 1 performed, 1 was only able to detect the
beta subunit. It has been proposed that polymerization of tubulin heterodimers rnay be
negatively regulated by tyrosine phosphorylation of the alpha-subunit during T cell activation
(177). Cell lines by definition are not normal and thus expected to have an increased level of
activation. This might explain why the alpha-subunit was not copurified with the beta-
subunit. In the event thüt the interaction between PTEN and tubulin is physiologically
relevant. it can be speculated that tubulin rnight be acting to place PTEN properly in the
context of other sipnaling molecules or complexes nearby the cytoskeleton machinery.
Altematively, PTEN might be acting directly to regulate the polymerization of tubulin by
affecting its phosphorylation state or indirectly by affecting the phosphorylation state of a
protein required for tubulin stabilization.
In addition to tubulin. two subunits (eta and theta) of a chaperonin complex required
for the proper folding of tubulins were also copurified. These subunits form part of the T-
cornplex polypeptide (TCPI), the only known chaperonin in the cytosol of eukaryotes. It is
usuall y composed of eight di fferent but homologous subuni ts assembled into a hexadecamer
of two double rings, but recent studies show that they can also exist in the cell as individual
subunits or smaller oligomers (173), explaining in part why only two subunits were identified
herein. Subunits of TCPl do frequently copunfy with microtubules, providing a likely
explanation for their presence in these experiments (178).
Affinity chmmatography also provided a band of approximately 67 kDa. Based on
the information collected by mass spectrornetry two proteins with different functions were
identified. nucleolin and moesin. Nucleolin is an acidic phosphoprotein abundantly
expressed in exponentially growing cells and is located mainly in dense fibrillar regions of
the nucleolus. It is a multifaceted protein involved either directly or indirectly in modulating
transcriptional processes. cytokinesis. nucleogenesis, signal transduction. apoptosis.
induction of chromatin decondensation. and replication. In addition to several functions in
the nucleolus, nucleolin functions cytoplasrnically and on the ce11 surface to provide a
shuttling mechanism for cytoplasmic and extracellular regulation of nuclear activities (179.
180). For these reasons nucleolin is fundamental to the survival and proliferation of cells, yet
many of its functions rernain to be elucidated.
However. nucleolin is a 110 kDa protein, and the protein derived by affinity
chromatography in these expenments identified a protein of approximately 67 kDa. By
analyzing the position of the peptides measured by the mass spectrometer with respect to the
707 aa nucleolin protein. it can be seen that the peptides cover a stretch of 333 an situated
towards the C-terminus of the protein. This stretch corresponds to approximately 47% of the
complete nucleolin sequence. Given these observations, it is likely that the protein found
might represent a fragment of nucleolin derived from its own degradation or proteolysis. It
would be of interest to determine if PTEN binds to the cornpiete nucleolin, and subsequently
determine if this interaction might be physiologically relevant. However. given that this
protein is involved in so many functions and that it seems to act more as a housekeeping
protein, it is most likely that that the interaction found herein is not physiologically relevant.
On the other hand, moesin (for membrane-grganizing -tension spike pmten) is
widely expressed in different tissues in cells, where it is localized to filopodia and other
membranous protrusions. It is a critical regulator of cytoskeletal-plasma membrane
interactions, especially in polarized cells. as well as important components of signal
transduction pathways. During activation, T lymphocytes become motile cells, switching
from a spherical to a polarized shape with the formation of a uropod in the rear pole. Moesin
seems to be important for redistribution of adhesion molecules to this cellular structure (18 1.
182). As in the case of nucleolin, moesin is highly expressed in malignant ce11 types. which
might have increased the chances for copurification with PTEN. More detailed analysis of
these interactions needs to be performed.
Finally, the low-molecular weight proteins identified seem to be the same protein.
The putative HLA class II-associated proteins PHAP-1 and PHAP-II were purified and
cloned on the basis of their ability to bind to the cytoplasmic domain of the HLA DR-alpha
chah. and may be components of the transmembrane sipaling pathway activated üfter
extracellular binding of ligands durin; the immune response. Sequence identity
demonstrates that PHAP-U is identicai to the protein named SET (1 83). In addition, PHAP-1
has been shown to be equivalent to I2PP2A (potent heat-stable protein inhibitor of protein
phosphatase 2A) ( 184) and crin inhibit PP3-A. a major mammalian serinefthreonine
phosphatase involved in the regulation of most major metaboiic pathways. PP2A may
function as either a tumor promoter or tumor suppressor. depending on the cell type or the
tnnsforming agent (184). It would be interesting to examine whether these proteins rnight
function in regulating the function of PTEN.
Affinity chromatography/Mass spectrometry is a powerful approach
Much of what is known about protein-protein interactions and protei n biochemical
pathways has been denved through the use of protein affinity chromatography and
immunoprecipitation techniques. 1 have used affinity chromatography as a rneans to find
proteins that may interacr with PTEN in vivo. followed by mass spectrometry (MS) for the
identification of these proteins. However, together with these techniques are a number of
factors and variables that need to be taken into consideration for their success. MALDI-ToF
MS c m detect as little as -100 fmol of protein (the limit of silver stain sensitivity), which
means that most procedures have to be performed carefully in order to minimize the presence
of contaminating proteins. However. aside from these obvious considentions. other
decisions need to be made such as the source of ce11 extract to be used. In my expenrnents 1
used a human T cell line because we were interested in defining biochemical pathways for
PTEN in T cells. The cell line 1 used expresses PTEN, which rnight have implications in
itself. Given that PTEN is commonly mutated in ce11 lines. it is not implausible that the
protein expressed might be a truncated or a substr~te-trap mutant (64) which might "trap"
PTEN substrates thereby preventing their association with PTEN cross-linked to the affinity
chromatopph y column. Altemati vel y. wild-type PTEN may be expressed in these cells
thereby impeding the isolation of phosphorylated target substrates.
The purified PTEN to be cross-linked to the column needs to be preferably void of
contarninating proteins which may be minimized by choosing an appropriate expression
system. The stringency and pH of lysis buffen and the use of protease inhibitors are just a
few examples of parameters that can be examined when trying to minimize degradation of
proteins and contamination with unwanted proteins. It is also important to consider which
affinity tag to use for protein purification purposes and whether it should be cleaved or not. I
found that when GST-PTEN was used. no detectable F IEN binding proteins were revealed.
GST is a relatively big protein. -27 kDa and as such it might have interfered with the binding
of proteins perhaps by blocking a binding area or by interfering with the proper folding of
ETEN. Another factor affecting the folding of the protein in the column is the nature of the
matrix used to cross-link it. The matnx used in my expenments cross-links FTEN at pnmary
amino acids. As mentioned earlier. the crystal structure of PTW reveals that the
phosphatase domain and the C2 domain are tightly associated and mutations that disrupt this
structure are tumorigenic (5 1). These observations suggest that the structural state of PTEN
in the column influences the way it binds to candidate ligands or, altematively, rnight
promote the binding of proteins that are not physiologically relevant. As a consequence, in
future experiments we will experiment with other affinity tags such as 6XHIS located either
at the C- or N-terminus as well as with other column matrix. The purpose of the latter is to
avoid disturbing the conformation of PTEN and as a consequence promote the binding of
physiologically relevant substrates. In addition. a more efficient PTEN substrate-tnpping
mutant, D92A, will be used to rnaximize the likelihood of finding PTEN protein substraies.
The wild-type PTEN will be used in parallel as a control for detemining whether these
interactions are mediated through the phosphatase domain.
Figure 4-1. Schematic of a MALDI-ToF mass spectrometer
Accelenting field
Detectors
Proteins to be identified are isolated from the silver-stained gel and trypsinized. The generated peptides are then mixed with a suitable matrix. applied to the sample plate and dned. The plate is inserted into the source where the laser strikes the sample. When the laser hits the plate. some of the sample and matrix are ejected into a chamber where ionization takes place. This means that protons and other cations attach to the sample molecules. When the ions are formed. a voltage pulse is applied sending the ions on their flight. They continue to travel until they hit the plate detector producing an electncal signal. The ions disperse as a function of time. with lighter ions traveling faster than heavier ones. Al1 ions of the same m a s hit the detector at nearly the same kinetic energy. The collection of al1 the signais generates a spectmm with the mass of each digest fragment ion.
To identify the protein, the empirical data from the MALDI analysis is compared to data calculated from a virtual digestion. In the virtual digestion. a cornputer program analyses the sequence of a known protein and calculates the mass values of the fragments that would be genented by trypsin digestion. These calculated results are compared with experimental results. During the database search, any protein containing a specific number of identical fragments is selected as a potential match.
A ROLE FOR PTEN IN T CELL SIGNALING
Our data (Figure 3-3) demonstrated that signals transduced through the TCR in
combination with CD28 c m induce the tyrosine-phosphoryiation of PTEN. These results are
consistent with those by another group. (G. Mills, personal communication) which showed
that BCR engagement could also induce the tyrosine-phosphorylation of PTEN and
suggested that this biochemical modification obsewed following TCWCD28 ligation might
represent an important event relevant to the function of PTEN. The tyrosine phosphorylation
of PTEN might allow its association with SH2-dornain containing molecules resulting in
their dephosphorylation by PTEN and thereby affecting their association with other signaling
molecules. Alternative1 y. the tyrosine phosphorylation of PTEN might be necessary for its
own activity. This possibility 1s very attractive since there is no current understanding of
how PTEN is regulated.
In collaboration with Our lab. Dr. G. Mills recently overexpressed a catalytically
inactive form of PTEN (C121S) in COS7 cells and examined the capacity of tyrosine
phosphorylated PTEN to interact with other proteins (G. Mills. unpublished data). In view of
the presence in PTEN of a consensus motif for binding to the SH2 domains of the SHP-1
cytosolic tyrosine phosphatase, the fint experirnent cmied out in this system involved
investigation as to whether SHP- L interacts with phosphorylated K E N . To address this
issue. a consti tutivel y active form of Lck (Lck Y505F). previousl y shown to phosphorylate
both ETEN and SHP-1. were also expressed in these cells. The results of this study revealed
that PTEN is capable of binding the inert form of SHP-1 (C435S) if PTEN was tyrosine-
phosphorylated. This association was also observed in cells expressing wild-type PTEN. but
not in cells expressing wild-type rather than catalytically inert SHP-1. suggesting that PTEN
might be a substrate of SHP-1. In addition. the lipid phosphatase activity of PTEN was
diminished in vitro when PTEN was tyrosine-phosphorylated (G. Mills. unpublished data).
These findings led us to investigate funher whether the association of PTEN and
SHP-1 was relevant and necessary for TCR signding. Specifically, we set out to explore
whether SHP-1 may modulate PTEN activity and/or its capacity to interact with downstream
targets relevant to TCR signaling. To explore this idea, 1 took advantage of our availability
of motheaten mice that lack SHP-1 protein and examined the levels of phosphorylated AKT
in freshly isolated thymocytes following TCR engagement. The data 1 obtained from these
expenments (Figure 3-4A) demonstrate that the levels of phosphorylated AKT. detected by
using an antibody specific to phosphorylated Ser-473 residue. were elevated in rnotheaten
mice following TCR/CD?8 ligation when compared to thymocytes from wild-type mice
activated in the same fashion. The overall expression of AKT was not affected by
TCWCD28 ligation or by SHP-1 deficiency (Figure 3-4. lower panel), an indication that the
lack of SKP-1 affects the phosphorylation of AKT only. These findings are therefore
consistent with a role for SHP-I in the regulation of PTEN lipid phosphatase activity.
A recent study by Cuevas ri al. that is relevant to the data presented in this thesis has
shown that SHP-1 associates with P13K in TCR-stimulated Jurkat T cells (185). This
association is mediated through binding of a p85 S E domain to phosphotyrosine residue(s)
within the SHP-1 carboxy terminus and appean to reduce PI3K activation. This study
suggested that SHP-1 might work in vivo to down-regulate the activity of the PI3WAKT
pathway.
The data that 1 present herein provide an alternative mechanism by which SHP-1
might regulate the PI3WAKT pathway and in tum inhibit TCR signaling. 1 propose that
TCR signaling induces the tyrosine-phosphorylation of PTEN as a means of enhancing the
activation of AKT. an event required for many cellular functions necessary for the ultimaie
activation of T cells. SHP-1 is an important protein tyrosine-phosphatase in T cells required
for the down-regulation of T ceIl signal transduction. Consistent with its role as a down-
regulator of T ce11 signaling. 1 propose that SHP-1 dephosphorylates PTEN resulting in its
activation or enhanced catalytic activity, which in tum leads to the down-regulation of the
PDWAKT pathway. A schematic representation of this model is presented in Figure 4-2.
Although many aspects of this model remain to be clarified, these preliminary data
offer an exciting direction of study. In future experiments. we will attempt to elucidate this
mechanism further. We will confirrn the association of PTEN with SHP-1 and determine
whether it is dependent on TCR-induced PTEN tyrosine phosphorylation. The
phosphorylation stüte of F E N will be analyzed following longer stimulation times to
detemine the extent and duration of PTEN tyrosine phosphorylation. It is also important to
detemine whether the phosphorylation of PTEN is indeed dependent on PI3K activity. This
can easily shown by pre-treating thymocytes with PDK inhibitors prior to lysis and
immunoprecipi tation.
We will also examine SHP-1-deficient thymocytes in more detail. If the model
presented here is correct, we should be able to detect higher levels of phosphorylated PTEN
in these cells upon TCR engagement. It is also of interest to determine which kinase(s)
phosphorylates PTEN. We will also transfect T ce11 lines with various catalytic foms of
PTEN to further chmcterize some of these findings Ni vitro. For example. tnnsfection of
PTEN protein with a mutated SKP-1 binding site will also help confirm these findings.
Figure 4-2. A mechanism for PTEN regulation: a role For SHP-1 in the
PIJWAKTlPTEN pathway
i INACTIVE)
1 AKT 1
FIEN plays an important role in T ce11 signaling, that of down-regulating the PI3WAKT pathway. T ceIl Iigation leads to a series of signaling events including the activation of PDWAKT. which is required along other signaling molecules for the activation of T cells. The mode1 presented in this thesis suggests that the engagement of the TCR leads to tyrosine- phosphorylation of PTEN resulting in the suppression of its lipid phosphatase activity. This inhibition relieves the inhibition of AKT by PTEN. Similarly, the tyrosine phosphatase SHP- 1, an important down-modulator of TCR signaling, helps down-regulate the augmentation of AKT activity following TCR ligation by dephosphorylating PTEN and PDK. In this manner. the inhibitory effects of SHP- I on TCR signaihg are mediated via different mechanisms, by restonng the negative and positive influence of PTEN and PI3K on the activity of AKT, respective1 y.
The work presented in this thesis has focused on answering some important questions
regarding the function of PTEN. I attempted to identify novel PTEN binding partners
through the combination of affinity chromatography and mass spectrometry. 1 showed that
through this approach it is possible to identify potential PTEN ligands. Future experiments
will attempt to find if these proteins are relevant to PTEN function in T cells. In addition. the
results obtained through this work provide a framework for the characterization of a
mechanism for PTEN regulation. The elucidation of this mode1 will allow for better
understanding of the involvement of PTEN in TCR signaling.
References Davis. M. M. and P. J. Bjorkmn. 1988. T-ce11 antigen receptor genes and T-ce11 recognition [published emtum appeiirs in Nature 1988 Oct 2O;335(6 l92):7#]. Nature 334:395402.
Walunas, T. L., C. Y. Bakker. and J. A. Bluestone. 1996. CTLA4 ligation blocks CD28- dependent T ce11 activation [published erratum appears in J Exp Med 1996 h l 1; 184(1):3Ol]. J.Erp. Med. 183:254 1-2550.
Weiss, A. and D. R. Littman. 1993. Sipal transduction by lymphocyte antigen receptors. Ce11 76: 263-274.
Samelson, L. E.. A. F. Phillips, E. T. Luong, and R. D. Klausner. 1990. Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc.Narl.Acad.Sci.U.S.A 87:4358- 4362.
Veillette, A.. M. A. Bookman, E. M. Horak, and J. B. Bolen. 1988. The CD4 and CD8 T ce11 surface antigens are associated with the interna1 membrane tyrosine-protein kinase p561ck. Ce11 55:30 1-308.
Cantrell. D. 1996. T ce11 antigen receptor signal transduction pathways. Annu.Rev.Irnmiinol. 14:259-74:259-274.
Qian. D. and A. Weiss. 1997. T cell rintigrn receptor signal transduction. Crrrr.Opin.Cell Biol. 9:2O5-2 11.
June. C. H.. M. C. Fletcher, J. A. Ledbetter. and L. E. Samelson. 1990. incterises in tyrosine phosphorylation Lire detectable before phospholipase C activation after T ce11 receptor stimulation. J. Irnniunol. 144: 159 1 - 1599.
Zcnner. G.. H. J. Dirk zur. P. Burn. and T. Mustelin. 1995. Towards unnveling the complexity of T ce11 signal transduction. Bioessczys 17:967-975.
Wange. R. L. and L. E. Samelson. 1996. Complex complexes: signaling at the TCR. Immuni~. 5 : 197-205.
van Oers. N. S. and A. Weiss. 1995. The S y W - 7 0 protein tyrosine kinase connection to iintigen receptor signalling processes. Semin. Immwzol. 7237-736.
Chan, A. C.. N. S. van Oers. A. Tran. L. Turkri. C. L. Law. I. C. Ryan, E. A. Clark, and A. Weiss. 1994. Differential expression of ZAP-70 and Syk protein tyrosine kinases. and the roIe of this family of protein tyrosine kinases in TCR signaling. J. Immnol. 152:4758-4766.
Howe. L. R. and A. Weiss. 1995. Multiple kinases mediate Tcell-receptor signaling. Trends Biochem. Sci 3059-64.
Acuto. 0. and D. Cantrell. 2000. T ceIl activation and the cytoskeleton. Annu.Rer.Immuno1. 18: 165-83: 165- 184.
Gibson. S.. A. August. Y. Kawakami. T. Kawakami, B. Dupont. and G. B. Milis. 1996. The EMTIITWSK (EMT) tyrosine kinase is activated during TCR signaling: LCK is required for optimal activation of EMT. J. Intmunol. 1%: 27 16-2722.
Liao, X. C. and D. R. Littman. 1995. Altered T ceil receptor signaling and disrupted T ce11 development in mice Iacking Itk. Imlrnity. 3:757-769.
Zhang. W . and L. E. Samelson. 2000. The role of membrane-associated adaptors in T ce11 receptor signalling. Semin. Immunol. 1 Xj4t 1.
18. Lowin-Kropf, B., V. S. Shapiro, and A. Weiss. 1998. Cytoskeletal polarkation of T cells is regulated by an immunoreceptor tyrosine-based activation motifdependent mechanism. J-Cell Biol. l4O:86 1-87 1.
19. Crabtree, G. R. and N. A. Clipstone. 1994. Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu. Rev.Biochem. 63: 1045-83: 1W5- 1083.
20. Smith. A. and A. Ashworth. 1998. Cancer predisposition: w here's the phosphate? Curr-Biol. 8:R.X 1-R343.
2 1. Parsons. R. 1998. Phosphatases and tumorigenesis. Curr. Opin. Oncol. 10188-9 1.
22. Maehama, T. and J. E. Dixon. 1998. The tumor suppressor, PTEN/MMACl. dephosphorylates the lipid second messenger. phosphatidylinositol 3.4.5-trisphosphate. J.BioL.Clrertz. 273: 13375- 13378.
33. Steck, P. A., M. A. Pershouse. S. A. Jasser, W. K. Yung, H. Lin, A. H. Ligon. L. A. Langford. M. L. Baumgard, T. Hattier. T. Davis. C. Frye. R. Hu. B. Swedlund. D. H. Teng, and S. V. Tavtigian. 1997. Identification of a candidate tumour suppressor gene, MMAC1. at chromosome lOq33.3 that is mutated in multiple advanced cancers. Nat. Genet. 15:356-362.
24. Li. J., C. Yen. D. Liaw. K. Podsypanina. S. Bose. S. 1. Wang. J. Puc. C. Miliaresis. L. Rodgers, R. McCombie. S. H. Bigner. B. C. Giovanella. M. Ittmann. B. Tycko. H. Hibshoosh. M. H. Wigler. and R. Parsons. 1997. PTEN, a putative protein tyrosine phosphatase gene mutated in humn brain, breast. and prostate cancer [see commentsj. Science 275: 1943-1947.
35. Li, D. M. and H. Sun. 1997. TEPl. encoded by ri candidate tumor suppressor locus. is a novel protein tyrosine phosphatasc regulated by transforming growth factor beta. Cancer Rcs. 57:2124-2129.
26. Liaw. D., D. J. Marsh. J. Li. P. L. Dahia, S. 1. Wang. Z. Zheng, S. Bose. K. M. CriIl. H. C. Tsou. M. Peacocke, C. Eng, and R. Parsons. 1997. Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat. Genet. 16:W-67.
27. Nelen, M. R.. W. C. van Staveren. E. A. Peeters. M. B. Hassel, R. J. Gorlin, H. Hamm, C. F. Lindbw, J. P. Fryns. R. H. Sijmons. D. G. Woods, E. C. bIarîman, G. W. Padberg. and H. Kremer. 1997. Gemline mutations in the PTEN/MMACl gene in patients with Cowden disease. Htinr. Mol. Genet. 6: 1383- 1387.
28. Marsh. D. J.. V. Coulon, K. L. Lunetta. P. Rocca-Serra. P. L. Dahia, Z. Zheng, D. Liaw, S. Caron, B. Duboue, A. Y. Lin. A. L. Richardson, J. M. Bonnetblanc. J. M. Bressieux, A. Cabarrot-Moreau. A. Chompret. L. Demange. R. A. Eeles. A. M. Yahanda. E. R. Fearon, J. P. Fricker. R. J. Gorlin. S. V. Hodgson. S. Huson, D. Lacombe, and C. Eng. 1998. Mutation spectrum and genotype-phenotype andyses in Cowden disease and Bannayan-Sonana syndrome. two hamartoma syndromes with germIine PTEN mutation. Httm.Mol.Genet. 7507- 5 15.
29. Marsh, D. J., P. L. Dahia. Z. Zheng, D. Liaw, R. Parsons. R. J. Gorlin, and C. Eng. 1997. Germiine mutations in ITEN are present in Bannayan-Zonana syndrome [letter]. NatGenet. 16:333-334.
30. Eng. C. 1998. Genetics of Cowden syndrome: through the looking glass of oncology. Int. J. Oncol. 12: 70 1-7 1 O.
31. Longy. M., V. Coulon. B. Duboue, A. David. M. Larregue. C. Eng, P. Amati. J. L. Knimps, A. Bottani, D. Lacombe. and D. Bonneau. 1998. Mutations of PTEN in patients with Bannayan- Riley-Ruvalcaba phenotype. J.Med,Genet. 35:886-889.
32. Rasheed, B. K.. T. T. Stenzel, R. E. McLendon, R. Parsons, A. H. Friedman. H. S. Friedman, D. D. Bigner, and S. H. Bigner. 1997. PTEN gene mutations are seen in high-grade but not in low-grade gliomas. Cancer Res. 57:4 187-4 190.
Duerr, E. M.. B. Rollbrocker, Y. Hayashi, N. Peters, B. Meyer-Puttlitz, D. N. Louis. J. Schramm, O. D. Wiestler, R. Parsons, C. Eng, and A. von Deimling. 1998. PTEN mutations in gliomas and gfioneuronal tumors. Oncogene 16:2359-2264.
Tashiro, H., M. S. Blazes, R. Wu, K. R. Cho, S. Bose, S. 1. Wang, J. Li, R. Parsons, and L. H. Ellenson. 1997. Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological rnalignancies. Cancer Res. 57:3935-3940.
Risinger. J. 1.. A. K. Hayes, A. Berchuck, and J. C. Bmett. 1997. PTEN/MMACl mutations in endometrial cancers. Cancer Res. 57:47364738.
Mutter, G. L.. M. C. Lin, J. T. Fitzgerald. J. B. Kum. J. P. Baak, J. A. Lees, L. P. Weng. and C. Eng. 2000. Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers [see comments 1. J. Nntl. Cancer Insr. 92:924-930.
Ali. 1. U.. L. M. Schriml. and M. Dean. 1999. Mutational spectn of WEN/MMACl gene: a tumor suppressor with Iipid phosphatase activity. J. N d C m c e r Inst. 9 1: 1922- 1933.
Whang, Y. E.. X. Wu. H. Suzuki. R. E. Reiter, C. Tran, R. L. Vesselia. J. W. Said, W. B. Isaacs, and C. L. Sriwyers. 1998. Inactivation of the tumor suppressor PTENIMMACI in advanced human prostate cancer through loss of expression. Proc.Natl.Acad.Sci. U.S..4 95:5246-5250.
Feilotter. H. E.. M. A. Nagai. A. H. Boag, C. Eng, and L. M. Mulligan. 1998. Analysis of PTEN and the 10q23 region in primary prostate carcinomas. Oncogene 16: 1743-1748.
Cairns. P.. K. Okami, S. Hafachmi, N. Halachmi, M. Esreller. J. G. Hennan, J. len. W. B. Isaacs, G. S. Bova. and D. Sidransky. 1997. Frequent inactivation of FTEN/MMACl in primary prostate cancer. Cancer Res. 57:4997-5000.
Robertson, G. P.. F. B. Furnari. M. E. Miele. M. J. Glendening, D. R. Welch. J. W. Fountain. T. G. Lugo. H. J. Huang. and W. K. Cavence. 1998. In vitro loss of heterozygosity targets the PTEN/MMAC 1 gene in melanoma. froc. Natl.Acd.Sci. U.S.A 95:94 18-9423.
Guldberg, P.. S. P. thor. A. Birck, V. Ahrenkiel. A. F. Kirkin. and J. Zeuthen. 1997. Disruption of the MMAC IPTEN gene by deletion or mutation is ri frequent event in maliflant melanoma. Cancer Res. 57:3660-3663.
Nakahara. Y., H. Nagai. T. Kinoshita. T. Uchida, S. Hatano, T. Murate, and H. Saito. 1998. Mutational analysis of the ITEN/MMACl gene in non-Hodgkin's lymphoma. Leukemia 12: 1277- 1280.
Sakai, A.. C. Thieblemont. A. Wellmann. E. S. Jaffe. and M. Raffeld. 1998. PTEN gene alterations in lymphoid neoplasm. Blood 9234 10-34 15.
Gronbaek. K.. J. Zeuthen. P. Guldberg, E. Ralfiüaer. and K. Hou-Jensen. 1998. Altentions of the MMACi/PTEN gene in lymphoid malipancies [letterl. BZood 9 l:J388439O.
Podsypanina. K.. L. H. Ellenson. A. Nemes. J. Gu. M. Tamura, K. M. Yamada, C. Cordon- Cardo, G. Catoretti. P. E. Fisher, and R. Parsons. 1999. Mutation of Pten/Mmacl in mice causes neoplasia in multiple organ systerns. Proc.Natl.Acad.Sci. U.S.A 96: 1563-1568.
Suzuki, A.. J. L. de la Pompa, V. Stambolic. A. J. Elia, T. Sasaki. B. B. del, 1, A. Ho. A. Wakeham. A. Itie, W. Khoo, M. Fukumoto, and T. W. Mak. 1998. High cancer susceptibility and embryonic Iethality associated with mutation of the F E N tumor suppressor gene in mice. Curr.Bio1. 8: L 169- 1 178.
48. Di Cristofano, A., B. Pesce, C. Cordon-Cardo, and P. P. Pandolfi. 1998. Pten is essential for embryonic development and tumour suppression. Nat.Grnet. 19:343-355.
Di Cristofano, A., P. Kotsi, Y. F. Peng. C. Cordon-Cardo, K. B. Elkon, and P. P. Pandolfi. 1999. Impaired Fas response and autoirnmunity in Pten+/- mice. Science 122-2 125.
Lachyankar. M. B.. N. Sultana, C. M. Schonhoff, P. Mitra, W. Poluha. S. Lambert, P. J. Quesenkrry, N. S. Litofsky. L. D. Recht, R. Nabi, S. J . MilIer, S. Ohta, B. G. Neel, and A. H. Ross. 2000. A role for nuclçar PTEN in neuronal differentiation. J. Neilrusci. 20: 1404- 14 13.
Lee, J. O., H. Ymg. M. M. Gcorgescu, A. Di Cristofano. T. Maehama, Y. Shi, J. E. Dixon, P. Pandolfi. and N. P. PavIetich. 1999. Crystal structure of the K E N tumor suppressor: imptications for its phosphoinositide phosphatase activity and membrane association. CeII 99~323-334.
Songyang. Z., A. S. Fünning, C. Fu. J. Xu. S. M. hiarfatia. A. H. Chishti. A. Crompton, A. C. Chan. J. M. Anderson. and L. C. Cantley. 1997. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 37573-77.
Myers, M. P.. I. P. Stolarov. C. Eng. J. Li. S. 1. Wang. M. H. Wigler. R. Parsons, and N. K. Tonks. 1997. P-TEN. the tumor suppressor from hurnan chromosome lOq23. is a dual- specificity phosphatase. Proc.Natl.Acnd.Sci. U.S.A % 19;94:9052-9057.
Myers. M. P.. 1. Pass, 1. H. Batty. K. J. Van der. J. P. Stoliirov, B. A. Hemmings, M. H. Wigler. C. P. Downes. and N. K. Tonks. 1998. The lipid phosphatase activity of PTEN is critical for its tumor supressor Function. Proc.~Vatl.Acatl.Sci. U.S.A 95: t 35 13- 135 18.
Furnari. F. B., H. J. Huang, and W. K. Cavenee. 1998. The phosphoinositol phosphatase activity of PTEN mediates a serurn-sensitive G1 growth arrest in glioma celIs. Cancer Res. 58:5003-5008.
Haas-Kogan, D., N. Shalev. M. Wong, G. Mills, G. Yount, and D. Stokoe. 1998. Protein kinase B (PKBIAkt) activity is elevated in glioblastomii ceIls due to mutation of the tumor suppressor PTENIMMAC. Crtrr.Bio1. 8: 1 195-1 198.
Stambolic. V.. A. Suzuki. J. L. de la Pompa. G. M. Brothers. C. Mirtsos. T. Sasaki, J. Ruland. J. M. Penninger. D. P. Siderovski. and T. W. Mak. 1998. Negative regulation of PKBtAkt- dependent cell survival by the tumor suppressor PTEN. Ce11 9529-39.
Tamura, M.. J. Gu. K. Matsumoto, S. Aota. R. Parsons. and K. M. Yamada. 1998. Inhibition of ce11 migration. spreading, and focal adhesions by tumor suppressor PTEN. Science 380: 16 13- 16 17.
Tamura, M., J. Gu. T. Takino. and K. M. Yamada. 1999. Tumor suppressor ETEN inhibition of cell invasion. migration. and growth: differential involvement of focal adhesion kinase and p l30Cas. Cancer Res. 59~44249 .
Gu, J., M. Tarnun. R. Pankov, E. H- Danen. T. Takino, K. Matsumoto, and K. M. Yamada. 1999. Shc and FAK differentially regulate ce11 motility and directionality modulated by PTEN. J. CelZ Biol. 146:389403.
Bzirford. D.. A. J. Flint. and N. K. Tonks. 1994. Crystal structure of humn protein tyrosine phosphatase 1 B [see comments]. Science 263: 1397- 1404.
Denu, 3. M.. 5. A. Stuckey, M. A. Saper. and J. E. Dixon. 1996. Fom and function in protein dephosphorylation [comment]. CeZl87:36 1-364.
Hopkin. K. 1998. A surprising function for the PTEN tumor suppressor [news]. Science 282: lO27.lO29-lO?7.lO3O.
64. Flint, A. J., T. Tiganis, D. Barford, and N. K. Tonks. 1997. Development of "substrate- trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. Proc.Natl.Acad.Sci. U3.A 94: 1680- 1685.
Fauman. E. B. and M. A. Saper. 1996. Structure and function of the protein tyrosine phosphatases. Trends Biochem.Sci. 2 1 :4 13-4 17.
Li. L. and J. E. Dixon. 2000. Form, function. and regulation of protein tyrosine phosphatases and their involvement in human diseases. Setnin.lrnrnuno1. 1275-84.
Craven. S. E., A. E. El Husseini, and D. S. Bredt. 1999. Synaptic targeting of the postsynaptic density protein PSD-95 mediatrd by lipid and protein motifs. Neriron 22497-509.
Cnven, S. E. and D. S. Bredt. 1998. PD2 proteins organize synaptic signaling pathways. Ce11 93:395498.
Georgescu. M. M., K. H. Kirsch. T. Akagi. T. Shishido, and H. Hanafusa. 1999. The tumor- suppressor activity of PTEN is regulated by its carboxyl-terminal region. Proc. NdAcadSci. U.S.A 96: 10 182-10 187.
Wu. X.. K. Hepner. S. Castelino-Prabhu. D. Do. M. B. Kaye. X. J. Yuan. J. Wood, C. Ross, C. L. Sawyers. and Y. E. Whang. 2000. Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-3. Proc.N(zt1.Acnd.Sci.U.S.A 97:42334338.
Wu, Y.. D. Dowbenko. S. Spencer. R. Laura. J. Lee. Q. Gu, and L. A. Lasky. 2000. interaction of the tumor suppressor PTENMMAC with ri PDZ domain of MAGI3. a novel membrane- associated guanylate kinase [In Proccss Citation]. J. Biol. Chem. 2752 1477-2 1485.
Vazquez. F.. S. Ramaswamy. N. Nakamura, and W. R. Sellers. 2000. Phosphorylation of the PTEN tail regulates protein stability and function. Mol.Cell Biol. 20:5010-5018.
Wang. S. 1.. J. Puc. J. Li. J. N. Bruce, P. Cairns. D. Sidransky, and R. Parsons. 1997. Somatic mutations of PTEN in glioblastoma multiforme. Cancer Rrs. 57:4 1834 186.
Wymnnn. M. P. and L. Pirola. 1998. Structure and function of phosphoinositide 3-kinases. Biochitn. Biophys.Acta 1436: 127- 150.
Fniman. D. A.. R. E. hkyers. and L. C. Cantley. 1998. Phosphoinositide kinases. Anntc. Rev. Biochenr. 67:48 1 -507:48 1-507.
Hiles. 1. D.. M. Otsu. S. Volinia. M. f . Fry. 1. Gout. R. Dhand. G. Panayotou. F. Ruiz-hrrea. A. Thompson. and N. F. Totty. 1992. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cd1 70:419-129.
Otsu. M.. 1. Hiles. 1. Gout. M. J. Fry. F. Ruiz-Larrea. G. Panayotou. A. Thompson. R. Dhand. J. Hsuan. and N. Totty. 1991. Characterimion of two 85 kd proteins that associate with receptor tyrosine kinases. rniddle-TIpp6Oc-src comp lexss, and PI3-kinase. Cr11 659 1 - 104.
Inukai. K.. M. Anai. E. Van Breda, T. Hosaka. H. Katagiri, M. Funaki. Y. Fukushima. T. Ogihara, Y. Yazaki. Kikuchi. Y. Oka. and T. Asano. 1996. A novel 55-kDa regulatory subunit for phosphatidylinositoI 3-kinase stnicturally similx to p55PiK 1s genented by alternative splicing of the p85alpha gene. J. Biol. Chem. 37 153 17-5320.
inukai, K.. M. Funaki. T. Ogihara. H. Katagiri, A. Kanda. M. Anai. Y. Fukushim, T. Hosaka. M. Suzuki. B. C. Shin, K. Takata, Y. Yazaki, M. Kikuchi, Y. Oka, and T. Asano. 1997. p8Saipha gene genentes three isoforms of reguiatory subunit for phosphatidylinositol 3-kinase (PI 3-Kinase). p50alpha, p55alpha. and p85alpha. with different PI 3-kinase activity elevating responses to insulin. J. Biol. Chem. 2727873-7882.
80. Chan, T. O., S. E. Rittenhouse, and P. N. Tsichlis. 1999. AKTfPKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositidedependent phosphorylation. Annu. Rev.Biochem. 68:965-10 14:965-10 13.
Kapeller. R. and L. C. Cantley. 1994. Phosphatidylinositol3-kinase. Bioessays 16565-576.
McGlade, C. J., C. Ellis, M. Reedijk, D. Anderson, G. Mbarnalu, A. D. Reith, G. Panayotou. P. End, A. Bernstein, and A. Kazlauskas. 1992. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors. Mol.Cel1 Biol. l2:99 1-997.
von Willebrand, M.. G. Baier. C. Couture, P. Bum. and T. Mustelin. 1994. Activation of phosphatidylinositol-3-kinase in Jurkat T cells depends on the presence of the p561ck tyrosine kinase. Errr. J. lmmrtnol. 24:234-238.
Kapeller. R.. K. V. Prasad. O. Janssen. W. Hou, B. S. Schaffhausen. C. E. Rudd. and L. C. Cantley. 1993. Identification of two SH3-binding motifs in the regulatory subunit of phosphatidy linositol3-kinase. J. Biol. Chem. 269: 1937- 1933.
Vogel. L. B. and D. J. Fujita. 1993. The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes. Mol. CeII Biol. 13:7408-74 17.
Rodriguez-Viciana. P.. P. H. Warne. R. Dhand. B. Vanhaesebrwck. 1. Gout, M. J. Fry. M. D. Waterfield, and J. Downward. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras f see cornments]. Nmue 370:527-532.
Bruyns. E.. A. Marie-Cardine. H. Kirchgessner. K. Sagolla. A. Shevchenko, M. Mann. F. Autschbach. A. Bensussan. S. Meuer. and B. Schnven. 1998. T ce11 receptor ( K R ) interacting molecule (TRM). a novel disulfide-linked dimer associated with the TCR-CD3-zeta complex. recruits inuacelluIar signaling proteins to the pIasma membrane. J.E.rp.Med. 188:561-575.
Zhang. W.. J. Sloan-Lancaster. J. Kitchen. R. P. Trible. and L. E. Samelson. 1998. LAT: the ZAP-70 tyrosine kinase substnte that links T ceIl receptor ro cellular activation. Cell92:83-92.
Dhand. R.. 1. Hiles, G. Panayotou. S. Roche. M. I. Fry. 1. Gout. N. F. Totty. O. Truong, P. Vicendo, and K. Yonezawa. 1994. PI 3-kinase is a dual specificity enzyme: autoregulation by an intnnsic protein-serine kinase activity. EMBO J. 13522-533.
Ahmed. N. N.. H. L. Grimes, A. Bellacosa. T. O. Chan. and P. N. Tsichlis. 1997. Transduction of interleukin-2 antiapoptotic and prolifentive signals via Akt protein kinase. Proc.Nati.Acnd.Sci. U.S.A 943627-3632.
Kulik. G.. A. Klippel. and M. J. Weber. 1997. Antiapoptotic signalling by the insulin-like growth factor 1 receptor. phosphatidylinositol3-kinase, and Akt. Mol.Cell Biol. 17: 1595-1606.
Khwaja. A.. P. Rodrigurz-Viciana. S. Wrnnstrom. P. H. Wme. and J. Downward. 1997. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J. 162783-2793.
Kennedy. S. G.. A. J. Wagner. S. D. Conzen. I. Jordan. A. Bellacosa. P. N. Tsichlis. and N. Hay. 1997. The PI 3-kinase1Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev. 11:701-713.
Bottocdey. M. J.. K. Salim. and G. Panayotou. 1998. Phospholipid-binding protein domains. Biochim,Biophys.Acta 1436:165-183.
Lemmon, M. A.. K. M. Ferguson. and J. Schlessinger. 1996. PH domains: diverse sequences with a common fold recmit signaling moIecuIes to the ce11 surface. CeII 85:621-624.
Blomberg, N., E. Banldi, M. Nilges, and M. Saraste. 1999. The PH superfold: a stmctunl scaffold for multiple functions. Trends Biochern.Sci. 2 4 4 1 4 5 .
Andjelkovic, M., D. R. Alessi, R. Meier, A. Fernandez, N. J. Lamb, M. Frech, P. Cron, P. Cohen, J. M. Lucocq, and B. A. Hemmings. 1997. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 2723 15 15-3 1524.
Alessi. D. R.. M. Andjelkovic, B. Caudwell, P. Cron. N. Morrice, P. Cohen. and B. A. Hemmings. 1996. Mechanism of activation of protein kinase B by insulin and IGF- 1. EMBO J. l5:6541-655 1.
Andjelkovic. M.. T. Jakubowicz. P. Cron. X. F. Ming, J. W. Han, and B. A. Hemmings. 1996. Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PWPKB) promoted by serum and protein phosphatase inhibitors. Proc. N d . Acad. Sci. U.S.A 93: 5 699-5 704.
Franke. T. F.. D. R. Kaplan. L. C. Cantley. and A. Toker. 1997. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3.4-bisphosphate [see comments]. Science 275:665-668.
Klippel. A.. W. M. Kavanaugh. D. Pot. and L. T. Williams. 1997. A specific product of phosphatidylinositol 3-kinase directly activates the prorein kinase Akt through its pleckstrin homology domain. !Mol. C d Biol. l73338-3U.
Stephens. L.. K. Anderson, D. Stokoe, H. Erdjument-Bromage. G. F. Painter. A. B. Holmes, P. R. Gaffney, C. B. Reese, F. McCormick. P. Tempst. J. Coadwell, and P. T. Hawkins. 1998. Protein kinase B kinases that mediate phosphatidy1inositoI 3.4.5-trisphosphatedependent activation of protein kinase B [sec comments]. Science 279:7 10-7 14.
Alessi. D. R.. S. R. James. C. P. Downes. A. B. Holmes, P. R. Gaffney. C. B. Reese, and P. Cohen. 1997. Characterization of a 3-phosphoinositidedependent protein kinase which phosphory latrs and activates protein kinase Bal pha. Ctirr. Biol. 7: 26 1-269.
Stokoe. D., L. R. Stephens. T. Copeland, P. R. Gaffney. C. B. Reese. G. F. Painter. A. B. Holmes. F. McCorrnick. and P. T. Hawkins. 1997. DuaI role of phosphritidylinositol-3.45- trisphosphate in the activation of protein kinase B (see cornments]. Science 277567-570.
Delcornmenne. M.. C. Tan. V. Gray. L. Rue. J. Woodgett. and S. Dedhar. 1998. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-Iinked kinase. Proc.Nar1.Acnd.Sci.U.S.A 95: 1121 1-1 1216.
Persad. S.. S. Attwell. V. Gray. M. Delcomrnenne. A. Troussard. J. Sanghen. and S. Dedhar. 2000. Inhibition of integrin-linked kinase ( C M ) suppresses activation of protein kinase BtAkt and induces ce11 cycle arrest and apoptosis of ETEN-mutant prostate cancer cells. Proc. Nd. Acnd. Sci. LI. S A 97:3207-32 12.
Balendran. A.. A. Casamayor. M. Derik. A. Paterson. P. Gaffney. R. Cunie, C. P. Downes, and D. R. Alessi. 1999. PDKl acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of P m . Curr-Biol. 9:393404.
Chao, D. T. and S. J. Korsmeyer. 1998. BCL-2 family: regulators of ce11 death. Annu. Rrv. Immiinol. 16:3954 19:395419.
Zha. J.. H. Harada. E. Yang. J. lockel, and S. J. Korsmeyer. 1996. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L) [see cornments]. Ceil 87:6 19-628.
Muslin. A. J.. J. W. Tanner, P. M. Allen, and A. S. Shaw. 1996. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Celi 84889-897.
del Peso, L., M. Gonzalez-Garcia. C. Page, R. Herrera. and G. Nunez. 1997. Interleukin-3- induccd phosphorylation of BAD through the protein kinase Akt. Science 378:687-689.
Datta, S. R., W. Dudek. X. Tao. S. Masters, H. Fu, Y. Gotoh, and M. E. Greenberg. 1997. Akt phosphorylation of BAD couples survival signals to the ceil-intrinsic death mchinery. Ce11 9l:23 1-241.
Kennedy, S. G., E. S. Kandel, T. K. Cross. and N. Hay. 1999. AktProtein kinase B inhibits ce11 death by preventing the release of cytochrome c from rnitochondria. Mol.Cel1 Biol. 195800- 58 IO.
Hinton, H. J. and M. J. Welham. 1999. Cytokine-induced protein kinase B activation and Bad phosphorylation do not correlate with cell survival of hemopoietic cells. J.lmmuno1. 162:7002- 7009.
Harada. H., B. Becknell. M. WiIm, M. Mann, L. J. Huang, S. S. Taylor, J. D. Scott. and S. 1. Korsrne yer. 1999. Phosphory lation and inactivation of BAD by mitoc hondria-anchored protein kinase A. Mol.Cell3:J l3-EL
Scheid, M. P.. K. M. Schubert. and V. Duronio. 1999. Regdation of bad phosphorylation and association with Bcl-x(L) by the MAPKErk kinase. J.Biol.Chrrn. 274:3 1108-3 1 1 13.
Bonni. A.. A. Brunet. A. E. West. S. R. Datta. M. A. Takasu. and M. E. Greenberg. 1999. Cell survival prornoted by the Ras-MAPK signalhg pathway by transcriptiondependent and - independent mechanisms [see comments]. Science 286: 1358- 1362.
Cardone. M. H.. N. Roy. H. R. Stennicke. G. S. Salvesen, T. F. Franke. E. Stanbndge. S. Frisch. and J. C. Reed. 1998. Rcgulation of ceIl death protease caspase-9 by phosphorylation [see comments 1. Science 282: 13 18- 132 1.
Li, P., D. Nijhawan. 1. Budihardjo. S. M. Srinivrisula. M. Ahmad. E. S. Alnemri, and X. Wang. 1997. Cytochrome c and dATPdependent formation of Apaf-lkaspase-9 complex initiates an apoptotic protease cascade. CeIl 9 1:479489.
Zou. H., Y. Li. X. Liu. and X. Wang. 1999. An APAF-1.cytochrome c multimeric cornplex is ri
functional apoptosome that activates procaspase-9. J. Biol. Clirrn. 274: 1 1549- 1 1556.
Fujita. E.. A. Jinbo. H. Matuzaki. H. Konishi. U. Kikkawa. and T. Momoi. 1999. Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9. Biochem. Biophvs. Res. Cortintun. 264550-555.
Ogg, S., S. Paradis. S. Gottlieb. G. 1. Patterson, L. Lee. H. A. Tissenbaum. and G. Ruvkun. 1997. The Fork head transcription factor DM-16 transduces insulin-like metabolic and longevity signals in C. elegans. Natwe 389:994-999.
Brunet. A.. A. Bonni, M. J. Zigrnond. M. 2. Lin. P. Juo. L. S. Hu, M. J. Anderson. K. C. Arden. I. Blenis. and M. E. Greenberg. 1999. Akt prornotes ceil sun.iva1 by phosphorylating and inhibiting a Forkhead transcription factor. Ce11 962357-868.
Biggs, W. H.. m. J. Meisenhelder, T. Hunter, W. K. Cavenee, and K. C. Arden. 1999. Protein kinase BIAkt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR 1. Proc.Natl.Acad.Sci. U.S.A 96742 1-7426.
Tang, E. D.. G. Nunez, F. G. Barr. and K. L. Gum. 1999. Negative regulation of the forkhead transcription factor FKHR by Akt. J. Biol. Chem. 274: 1674 1- 16746.
Guo, S., G. Rena, S. Cichy, X. He. P. Cohen, and T. Unterman. 1999. Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-l promoter activity through a conserved insulin response sequence. 3-Biol. Cheni. 274: 17 184- 17 192.
127. Kops, G. J., N. D. de Ruiter, A. M. Vries-Smits, D. R. Powell. J. L. Bos, and B. M. Bwgering. 1999. Direct controi of the Forkhead transcription factor A l 3 by protein kinase B. Nancre 398:630-634.
128. Takaishi, H.. H. Konishi, H. Matsuzrtki, Y. Ono. Y. Shirai, N. Saito, T. Kitamura, W. Ogawa, M. Kasuga, U. Kikkawa. and Y. Nishizukii. 1999. Regulation of nuclear translocation of forkhead transcription factor M X by protein kinase B. Proc. Natl.AcadSci. U.S.A 96: 1 1836- 11841.
129. Du. K. and M. Montrniny. 1998. CREB is a regulatory target for the protein kinase AkflKB. J. Biol. Chem. 273:32377-32379.
130. Barton, K., N. Muthusamy, M. Chanyangam. C. Fischer. C. Clendenin, and J. M. Leiden. 1996. Defective thymocyte proliferation and IL-2 production ir. transgenic mice expressing a dominant-negative form of CREB. Nnnire 3793 1-85.
131. Datta, S. R., A. Brunet. and M. E. Greenberg. 1999. Cellular surviviil: a play in three Akts. Gertes Dev. l3:29Oj-2927.
132. Kane, L. P.. V. S. Shapiro. D. Stokoe. and A. Weiss. 1999. Induction of NF-kappa by the AktlPKB kinase. Curr. Biol. 9:60 1-604.
133. Ozes, O. N.. L. D. Mayo. J. A. Gustin. S. R. Pfeffer. L. M. Pfeffer. and D. B. Donner. 1999. NF-kappa activation by turnour necrosis factor requires the Akt serine-threonine kinase [see comments1. Nature 40 1:82-85.
134. Kandel. E. S. and N. Hay. 1999. The regulation and activitiss of the multifunctional serinefthreonine kinase AktPKB. Erp. Ce11 Res. 253:2 10-219.
135. Cross, D. A.. D. R. Alessi, P. Cohen, M. Andjelkovich, and B. A. Hemmings. 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nntlire 378:785-789.
136. Kohn, A. D.. S. A. Summers. M. J. Birnbaum. and R. A. Roth. 1996. Expression of ri
constitutively active Akt Serlïhr kinase in 3T3-LI adipocytes stimuistes glucose uptake and glucose transporter 4 translocation. J. Biol. Chem. 27 1 :3 1373-3 1378.
137. Pap, M. and G. M. Cooper. 1998. RoIr of glycogen synthase kinase-3 in the phosphatidy linositol 3-KinasdAkt ccll survival pathway. J. Biol. Chem. 273: 19919- 19932.
138. Beals, C. R.. C. M. Sheridan. C. W. Turck, P. Gardner. and G. R. Crabtree. 1997. Nuclear cxport of NF-ATc enhanced by giycogen synthase kinase-3. Science 275: 1930- 1934.
139. Goid, M. R.. M. P. Scheid. L. Santos, M. Drtng-Lawson. R. A. Roth, L. Matsuuchi. V. Duronio, and D. L. Krebs. 1999. The B cell antigen receptor activates the Akt (protein kinase B)/glycogen synthase kinase3 signaling pathway via phosphatidylinositol 3-kinase. J.lmnnino1. 163: 1894-1905.
110. Davies. M. A.. Y. Lu. T. Sano. X. Fang, P. Tang. R. LaPushin. D. Koul. R. Bookstein. D. Stokoe. W. K. Yung. G. B. Mills, and P. A. Steck. 1998. Adenovirril transgene expression of MMACPTEN in hurnan glioma c d s inhibits Akt activation md induces anoikis [published erratum appears in Cancer Res 1999 Mar 1:59(5): 1 1671. Cancer Res. 5852854290.
141. Lu. Y.. Y. 2. Lin, R. LaPushin. B. Cuevas. X. Fang. S. X. Yu. M. A. Davies, H. Khan. T. Furui, M. Mao. R. Zinner. M. C. Hung. P. Steck. K. Siminovitch. and G. B. Mills. 1999. The PTEN/MMAC l m P turnor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 18:7034-7M5.
142. Ramaswamy, S.. N. Nakamun. F. Vazquez. D. B. Batt. S. Perera, T. M. Roberts, and W. R. SelIers. 1999. Regulation of G1 progression by the PTEN tumor suppressor protein is linked to
inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc. NatL.Acad.Sci. U.S.A 96:2 110-2 1 15.
143. Li. D. M. and H. Sun. 1998. PTEN/MMACl/TEPl suppresses the tumorigenicity and induces G 1 cell cycle arrest in human gIioblastoma cells. Proc. Natl.Acad.Sci. U.S.A 95: 15406- 154 1 1.
144. Furnari, F. B., H. Lin. H. S. Huang, and W. K. Cavenee. 1997. Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc.Nat1.Acad.Sci.U.S.A 94: 1247% 12384.
145. Sun, H., R. Lesche, D. M. Li, J. Liliental, H. Zhang, J. Gao, N. Gavrilova, B. MuelIer, X. Liu, and H. Wu. 1999. PTEN modulates ce11 cycle progression and ce11 survival by regulating phosphatidylinositol 3.3.5.-trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl.Acnd.Sci. U.5.A 96:6 199-6204.
146. Davies, M. A., D. Koul. H. Dhesi. R. Berman, T. J . McDonnell. D. McConkey, W. K. Yung, and P. A. Steck. 1999. Regulation of AktIPKB activity, cellular growth. and apoptosis in prostate carcinoma cells by MMAC/PTEN. Cancer Res. 59235 1-2356.
147. Kops, G. J. and B. M. Burgering. 1999. Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling. J.Mol. Med 77:656-665.
148. Guarente, L., G. Ruvkun, and R. Amasino. 1998. Aging, life span, and senescence. Proc. Natl.Acad.Sci. U.S.A 95: 1 1033- 1 1036.
149. GiI, E. B.. L. E. MaIone, L. X. Liu, C. D. Johnson, and J. A. Lees. 1999. Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene. Proc. Natl.Acad.Sci. U.S.A 962925-2930.
150. Mihayiova, V. T.. C. 2. Borland. L. Manjarrez. M. J. Stem. and H. Sun. 1999. The PTEN tumor suppressor hornolog in Caenorhabditis elegans regulates longevity and dauer formation in an insulin receptor-like signaling pathway. Proc.Nntl.Acad.Sci. U.SA 96:7427-7432.
15 1. Rouault, J. P.. P. E. Kuwrtbara, O. M. Sinilnikovri. L. Duret, D. Thierry-Mieg, and M. Billaud. 1999. Regulation of dauer larva development in Caenorhabditis elegans by daf-18. a homologue of the tumour suppressor PTEN. Citrr. Biol. 9:329-332.
152. Ogg, S. and G. Ruvkun. 1998. The C. elegans PTEN homolog, DAF-18. acts in the insulin receptor-Iike metabolic signaling pathway. Mol.Cell2:587-893.
153. Ward. S. G., C. H. June. and D. Olive. 1996. PI 3-kinase: a pivota1 pathway in T-ce11 activation? Immnnol. Todq 17: 187- 197.
154. Borlado, L. R., C. Redondo, B. Alvarez. C. Jimenez. L. M. Criado. J. Flores, M. A. Marcos, A. Mutinez. D. Balornenos. and A. C. Carrera. 2000. hcreased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo. FASEB 3. 14:895-903.
155. Campbell. K. S. 1999. Signal transduction from the B celI antigen-receptor. Curr. Opin. lmmrinol. 1 1 256-264.
156. Fruman. D. A., S. B. Snapper, C. M. YbalIe. L. Davidson, J. Y. Yu, F. W. Alt, and L. C. Cantley. 1999. tmpaired B cell development and proliferation in absence of phosphoinositide 3- kinase p85alpha. Science 283:393-397.
157. Wang, X., A. Gjorloff-Wingren, M. Saxena. N. Pathan, J. C. Reed. and T. Mustelin. 2000. The tumor suppressor PTEN regulates T ce11 survival and antigen receptor signaling by acting as a phosphatidy linositol 3-phosphatase. J. Imm~tnal. 163: 1934- 1939.
Genot, E. M., C. Arrieumerlou, G. Ku, B. M. Burgering, A. Weiss, and 1. M. Kramer. 2000. The T-ceIl receptor regulates k t (protein kinase B) via a pathway involving Racl and phosphatidylinositide 3-kinase. Mol-Ce11 Biol. 205469-5478.
Jones, R. G., M. Parsons, M. Bonnard, V. S. Chan, W. C. Yeh, J. R. Woodgett, and P. S. Ohashi. 3000. Protein kinase B regulates T lymphocyte survival, nuclear factor h p p d activation, and Bcl-X(L) levels in vivo. J.Erp. Mrd 19 1: 1721- 1734.
Mok. C. L., G. Gil-Gomez. O. Williams. M. Coles, S. Taga, M. Tolaini. T. Nonon, D. Kioussis, and H. J. Brady. 1999. Bad can act as a key regulator of T ce11 apoptosis and T ce11 development. J.&. Med. 189575586.
Shan. X., M. J. Czar, S. C. Bunnell. P. Liu. Y. Liu. P. L. Schwartzberg. and R. L. Wange. 2000. Deficiency of PTEN in jurkat T cclls causes constitutive localization of itk to the plasma membrane and hyperresponsivrnsss to CD3 stimulation [In Process Citation]. Mol.Cell Biof. 20:6945-6957.
Pani, G. and K. A. Sirninovitch. 1997. Protein tyrosine phosphatase roles in the regulation of lymphocyte signaling. Clin.frrtmttnol. Imrniinopathol. 84: 1 - 16.
Zhang. J.. A. K. Somani. and K. A. Siminovitch. 2000. Rolss of the SHP-1 tyrosine phosphatase in rhe negative regulation of ce11 signalling [u1 Process Citation]. Semin.Immrino1. 12361-378.
Tamura, M.. J . Gu. E. H. Danen, T. Trikino, S. Miyamoto, and K. M. Yamüda. 1999. PTEN interactions with focal adhesion kinase rind suppression of the zxuacellular matrixdependent phosphatidylinositol3-kinase/Akt ce11 surviviil pathway. J. BioLChern. 274:20693-20703.
Rodriguez-Fernandez, J. L.. M. Gornez, A. Luque. N. Hogg. F. Sanchez-Madrid, and C. Cabanas. 1999. The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-nch tyrosine kinase 2 in T lymphocytes. Mol.Biol.Cel1 10: 189 1- 1907.
Tamura, M.. J. Gu. H. Tran, rind K. M. Yamada. 1999. PTEN gene and integrin signaling in cancer. J. Natl. Cancer Inst. 9 1 : 1820- 1828.
Tsuchida. M.. S. J. Knechtle. and M. M. Harnawy. 1999. CD23 ligation induces tyrosine phosphorylation of Pyk2 but not Fak in Jurkat T cells. J. BioKhem. 274:6735-6740.
Tsuchida. M.. E. R. Manthei. T. Alam. S. J. Knechtle. and M. M. Hamüwy. 2000. Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases. J. Biol. Chem. 275: 1344- L 350.
Schlaepfer. D. D., S. K. Hanks. T. Hunter. and G. P. van der. 1994. Integrin-mediated signal transduction linked to Ras pathway by GRBî, binding to focaI adhesion h a s e . Natrtre 372786-79 1.
Sonoda, Y.. Y. Matsumoto, M. Funakoshi, D. Yamamoto. S. K. Hanks. and T. Kasaham. 2000. Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a humn leukemic ce11 line. HL-60. J.Biol. Chem. 275: 16309- 163 15.
Gu. J., M. Tamura. and K. M. Yarnada. 1998. Tumor suppressor FEN inhibits inteben- and growth factor-mediated mitogen-activated protein ( M M ) kinase signaling pathways. J.Cell Biol. 133: 1375-1 383.
173. Neel. B. G. 1997. Role of phosphatases in lymphocyte activation. Curr. 0pin.Imm~rnol. 9~405- 430.
173. Nogales. E. 2000. STRUCTURAL INSIGHTS iNTO MICROTUBILE FUNCTION. Annu. Rev. Biochern. 69: 277-302:277-302.
174. Ratner. S.. W. S. Sherrod, and D. Lichlyter. 1997. Microtubule retraction into the uropod and its rote in T ce11 polarization and rnotility. J.Irtintrrnof. 159: 1063- 1067.
175. Offiinga, R. and B. E. Bierer. 1993. Association of CD2 with tubulin. Evidence for a role of the cytoskeleton in T ce11 activation. J. Biol.Chern. 268:49794988.
176. Huby. R. D.. G. W. Carlile, and S. C. Ley. 1995. interactions between the protein-tyrosine kinase ZAP-70, the proto-oncoprotein Vav, and tubulin in Jurkat T cells. J.Biol.Cheni. 270:3024 1-30234.
177. Ley, S. C.. W. Verbi. D. J. Pappin. B. Druker. A. A. Davies. and M. J. Crumpton. 1994. Tyrosine phosphorylation of alpha tubulin in humn T lymphocytes. Eitr.J.lmmrtnol. 74:99- 106.
178. Roobol, A.. 2. P. Sahyoun. and M. J. Carden. 1999. Selected subunits of the cytosolic choperonin associate with microtubules assembled in vitro. J. Biol. Chem. 2742408-24 15.
179. Srivastava. M. and H. B. Pollrtrd. 1999. Molecular dissection of nucleolin's role in growth and ceIl proliferrition: new insights. FASEB J. 13: 19 1 1-1922
180. Ginisty. H.. H. Sicard. B. Roger. and P. Bouvet. 1999. Structure and functions of nucleolin. J. C d Sci. 1 1 ?:76 1-77?.
18 1. Serrador. J. M.. J. L. Alonso-Lebrero, M. A. del Pozo. H. Furthmayr. R. Schwartz-Aibisz, J. Calvo, F. Lozano, and F. Sanchez-Madrid. 1997. Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during ceIl polarization. J.Cell Biol. 138: 1409- 1473.
183. Semdor, J. M., M. Nieto, and F. Sanchez-Madrid. 1999. Cytoskeletal remangement during migration and activation of T lymphocytes. Trerids Ce11 Biol. 928-233.
183. Vaesen. M.. S. BarnikoI-Watanabe, H. Gotz. L. A. Awni, T. Cole, B. Zimmermann. H. D. Kratzin. and N. Hilschmann. 1994. Purification and chruacterization of two putative HLA class II associated proteins: P M I and PHAPII. Biol.Chem. Huppe Sqler 375: 1 13- 126.
184. Li. M.. A. Makkinje. and 2. Damuni. 1996. Molecular identification of I lPEA. a novel potent heat-stable inhibitor protein of protein phosphatase ?A. Biochernistry 356998-7003.
185. Cuevas. B.. Y. Lu. S. Wm. R. Kumar. I. Zhang, K. A. Siminovitch. and G. B. Mills. 1999. SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase phosphorylation and activity. J.Biol.Chem. 374:27583-27589.