vascular endothelial cell growth factor activates creb by … · 2001-05-02 · lindsey d. mayo1,...

Vascular endothelial cell growth factor activates CREB by signaling through the

KDR receptor tyrosine kinase

Running Title: VEGF activation of CREB

Lindsey D. Mayo1, Kelly M. Kessler1, Roxana Pincheira1, Robert S. Warren2 and

David B. Donner1

1Department of Microbiology and Immunology and the Walther Oncology

Center, Indiana University School of Medicine, Indianapolis, IN 462021, and the

2Department of Surgery, University of California, San Francisco, CA 941432

* Corresponding author:

Dr. David B. Donner

The Walther Oncology Center

Indiana University School of Medicine

1044 West Walnut Street

Indianapolis, IN 46202.

TEL: (317) 278-2155. FAX: (317) 274-7592. Email: [email protected].

+Abbreviations: VEGF, vascular endothelial cell growth factor; PlGF, placental

growth factor; HUVEC, human umbilical vein endothelial cells; CREB, CRE

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 2, 2001 as Manuscript M102932200 by guest on June 9, 2020

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binding protein; p-CREB, phosphorylated CREB; α-CREB, CREB antibody;

ATF-1, activating transcription factor-1; α-ATF-1, ATF-1 antibody; MAPK,

mitogen activated protein kinase; PKC, protein kinase C; electrophoretic mobility

shift, EMSA; MOPS, [3-(N-Morpholino) propanesulfonic acid].

Vascular endothelial cell growth factor (VEGF) plays a crucial role in the

development of the cardiovascular system and in promoting angiogenesis

associated with physiological and pathological processes. While a great deal is

known of the cytoplasmic signaling pathways activated by VEGF, much less is

known of the mechanisms through which VEGF communicates with the nucleus

and alters the activity of transcription factors. Binding of VEGF to the

KDR/Flk1 receptor tyrosine kinase induces phosphorylation of the CREB

transcription factor on serine-133 and increases CREB DNA binding and

transactivation. p38 MAP kinase/MSK-1 and protein kinase C/p90 RSK

pathways mediate CREB phosphorylation. Confocal microscopy shows that

VEGF induced phosphorylation of nuclear CREB is blocked by pharmacological

inhibition of protein kinase C and p38 mitogen-activated protein kinase

signaling. Thus, KDR/Flk1 uses multiple pathways to transmit signals into the

nucleus where CREB becomes activated. These results suggest that CREB may

play a role in alterations of gene expression important to angiogenesis.

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Angiogenesis plays an important role in embryonic development, wound

healing and organ regeneration (1,2). Angiogenesis also contributes to

pathologies dependent on neovascularization, among which are tumor growth,

diabetes mellitus, ischemic ocular diseases, and rheumatoid arthritis. Vascular

endothelial cell growth factor (VEGF)1is an endothelial cell specific mitogen that

promotes many other events necessary for angiogenesis (1,3-6). VEGF induces

endothelial cell proliferation and movement, remodeling of the extracellular

matrix, formation of capillary tubules, and vascular leakage. VEGF plays a role

in the development of the cardiovascular system and in the physiology of normal

vasculature (3,7-11). VEGF is produced by many types of tumor cells and, by

promoting vascularization, plays a significant role in the progression of cancer

(12-14).

VEGF exerts its actions by binding to two cell surface receptors, KDR (the

human homolog of Flk1) and Flt1 (15-20), structurally similar to members of the

platelet-derived growth factor receptor family (21). Flk1 and Flt1 are essential

for fetal angiogenesis and mouse embryos null for either receptor die in utero

between days 8.5 and 9.5 (22,23). Hematopoietic and endothelial cell

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development are impaired in Flk1 null mutant mice (23), whereas there is

overgrowth of endothelial cells and disorganized blood vessels in Flt1 null

mutant mice (22). The phenotypes of Flk1 and Flt1 deficient embryos shows that

each plays a distinct role in angiogenesis and suggests that KDR/Flk1 and Flt1

utilize distinct signaling cascades to elicit responses.

Flk1 is abundant on the proliferating endothelial cells of vascular sprouts

of embryonic and early postnatal brain, but is reduced in adult brains in which

endothelial cell proliferation has ceased (17). Experiments with mutant forms of

VEGF that selectively bind one or the other VEGF receptor show that Flk1/KDR

promotes endothelial cell proliferation and survival (24). The induction of

chemotaxis and procoagulant activity by VEGF and placental growth factor

(PlGF), a VEGF homolog that binds Flt1, suggests that these responses are

mediated, at least in part, by Flt1 (25-27). Thus, the functions of VEGF are

segregated between its two receptors.

In previous studies we demonstrated that VEGF promotes the tyrosine

phosphorylation of signaling molecules that contain SH2 domains, and this

process is associated with endothelial cell proliferation (28). By comparing

signaling events induced by VEGF or PlGF, in the absence or presence of SU5416,

a KDR/Flk1 antagonist (29), we identified signaling molecules that interact with

and promote or inhibit KDR/Flk1 action (30). The present study finally

identifies pathways through which cytoplasmic signaling pathways downstream

of KDR/Flk1 transmit signals into the nucleus. VEGF promotes phosphorylation

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and activation of the cyclic AMP responsive element binding protein by

activation of p38 mitogen-activated protein kinase/MSK-1 and protein kinase

C/p90RSK signaling pathways downstream of KDR. As CREB is a transcription

factor that plays a important role in promoting proliferation and cellular

adaptive responses (31), a mechanism has been defined through which

VEGF/KDR may allow endothelial cells to respond to changes in their

environment through alterations of gene expression.

EXPERIMENTAL PROCEDURES

Materials-Human recombinant VEGF was a gift from Genentech (South

San Francisco, CA). SU5416 was a gift from SUGEN, Inc. (South San Francisco,

CA). Placenta growth factor and VEGF-D were from R & D Laboratories

(Minneapolis, MN). Antibodies to phosphorylated CREB/ATF-1,

phosphorylated MSK-1, phosphorylated and non-phosphorylated p38 MAPK

and MAPK were from Cell Signaling Technology (Beverly, MA). Anti-MAPKAP

kinase 2 and recombinant ATF-1 were from Santa Cruz Biotechnology (Santa

Cruz, CA). Anti-MAPKAP kinase 3 was from Upstate Biotechnology (Lake

Placid, NY). PGAL4-CREB was from Stratagene (La Jolla, CA) and the GAL4

luciferase reporter construct was a kind gift of Dr. Lawrence Quilliam (Indiana

University School of Medicine). GF109203X was from LC Laboratories (Boston,

MA). All other inhibitors were from Calbiochem (San Diego, CA) or Sigma, Inc.

(St. Louis, MO).

Cell Culture and Western blotting-HUVEC were grown on 0.2% gelatin-

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coated tissue culture plates in endothelial cell growth medium (Clonetics, San

Diego CA.) under 5% CO2 at 370C. For VEGF stimulation, the cells were

cultured in endothelial cell basal medium (Clonetics) that was supplemented

with 1% bovine serum albumin. After 16 hours, the cells were treated with

various inhibitors as described in the figure legends and then stimulated with 1

nM VEGF for 10 minutes. Cells were harvested into lysis buffer (50 mM Hepes

pH 7.0, 150 mM NaCl, 10% glycerol, 1.2% Triton X-100, 1.5 mM MgCl2, 1 mM

EGTA, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM sodium

orthovanadate) that was supplemented with 1 mM PMSF, 0.15 units/ml

aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A and incubated on ice for 30

minutes, being vortexed every 10 min. Fifty micrograms from each sample was

fractionated by SDS-PAGE and then transferred to PVDF membranes. The

membranes were blocked in 5% nonfat dried milk in PBST. Antibodies used to

probe the Western blots were p-ERK, ERK, p38MAPK, MAPK, pCREB and

CREB (Cell Signaling Tech., Beverly, MA). Proteins on the Western blots were

detected using the enhanced chemiluminescent detection system.

In vitro kinase assays-Whole cell lysates were prepared from HUVEC

treated with medium or VEGF for 10 min.as described above. In independent

experiments, MAPKAP kinase-2, MAPKAP kinase-2, or MSK-1 were

immunoprecipitated from 500 µg of whole cell lysate (2 h incubations at 4oC

using 2 µg of an antibody specific to each kinase). Protein A/G agarose was

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added and incubation continued for 1 hour. After centrifugation, the pellets

were washed with kinase assay buffer (20 mM MOPS, pH 7.2, 25 mM, β-glycerol

phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 75 mM MgCl2, 1 mM

dithiothreitol) and this procedure was repeated twice. The washed pellet was

incubated for 10 min at 37oC in 25 µl of kinase assay buffer modified to contain

50 µM ATP and 1 µg of recombinant ATF-1. Immunoprecipitated proteins were

fractionated on 8% polyacrylamide gels and Western blots were probed with

antibodies to p-CREB/ATF-1 or p-MSK-1.

Electrophoretic Mobility Shift and Gene Reporter Assays-Nuclear protein

extracts were prepared using the NE-PER kit by cell lysis and centrifugation

according to the directions of the manufacturer (Pierce, Inc.). The CRE and

mutant CRE double stranded DNA oligonucleotides were purchased from Santa

Cruz Biotechnology, Inc. The CRE oligonucleotide was end labeled with γ 32P

using the T4 polynucleotide kinase and purified by passage through G25 spin

column. To initiate formation of DNA-protein complexes six micrograms of

each nuclear extract, binding buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 50

mM NaCl, 1 mM MgCl, 1 mM EDTA, 5 mM DTT, and 5% glycerol), and 1µg poly

dIdC in a final volume of 20 µl were incubated on ice for 10 min. and then 5 ng of

end-labeled oligonucleotide was added to the reaction which was continued for

20 additional minutes. The reaction mixture was fractionated on a 5 % native

polyacrylamide gel (1.5 hours, 180 V). The gel was dried, and exposed to film.

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For gene reporter assays, cells were transiently cotransfected with β-

galactosidase and CMV-Gal4-CREB (a plasmid consisting of the DNA binding

domain of Gal4 and the transactivation domain of CREB) and pGal4-Tk-Luc (an

expression vector for the luciferase reporter gene driven by the enhancerless

thymidine kinase promoter linked to four copies of the Gal4 regulatory

sequence). After 24 h, luciferase activity was assayed in cell lysates and

normalized to β-gal expression.

Confocal microscopy-HUVEC incubated with vehicle, SB 202190 or

GF109203X for 1 h and then with VEGF for 10 minutes were fixed, permeated

with 0.1% Triton X-100 and blocked with 2% bovine serum albumin. Anti-

phosphoCREB was added followed by a Texas red conjugated sheep anti-rabbit

secondary antibody. The nucleus was detected by staining with nuclear dye Syto

16. Exictation of the stains was performed on a Bio Rad MRC 1024

Krypton/Argon laser confocal imaging system.

RESULTS

Experiments were conducted to determine whether VEGF induces CREB

phosphorylation and transactivation and if so to identify the receptor through

which this effect is mediated. HUVEC were stimulated with VEGF and a

Western blot prepared from cell lysates was probed with an antibody directed

towards CREB phosphorylated on serine-133. VEGF induced CREB

phosphorylation in a time-dependent manner (Fig. 1A). Due to the structural

similarities between CREB and ATF-1 (31), the phospho-CREB antibody also

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recognizes phosphorylated serine-63 of ATF-1. The phosphorylation kinetics

CREB and ATF-1 increased in parallel. To evaluate which VEGF receptor was

responsible for inducing CREB and ATF-1 phosphorylation, HUVEC were

stimulated with VEGF or PlGF, which only binds Flt1 (26,27). VEGF, but not

PlGF, induced CREB phosphorylation, showing that KDR induced this event

(Fig. 1B). Additionally, SU5416, a low molecular weight KDR antagonist (29),

abrogated VEGF induced CREB and ATF-1 phosphorylation. To further exclude

the possibility that Flt1 contributes to activation of CREB, VEGF-D, another

member of the VEGF family does not bind Flt1 (32), was tested and found to

induce phosphorylation of CREB and ATF-1 (Fig. 1C).

Phosphorylation of serine 133 in CREB does not alter the DNA binding of

CREB to CRE, but increases its association with adapter proteins such as the

CREB-binding protein, leading to activation of transcription. Therefore, the

demonstration that treatment of HUVEC with VEGF leads to phosphorylation of

CREB downstream of KDR next led us to evaluate whether CREB transcriptional

activity was increased by VEGF. A gene reporter assay in which HUVEC were

transfected with pGal4-CREB and a Gal4 luciferase reporter construct

demonstrated that VEGF induces a 2.5-fold stimulation of CREB transcriptional

activation (Fig. 1D).

Experiments were next conducted to identify the signaling pathways that

mediated CREB phosphorylation. By probing Western blots of HUVEC with

antibodies to activated MAPK (ERK1 and 2) and p38 MAPK, we found that

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VEGF activates these each of these kinases, maximal activation occurring after 10

min (Figs. 2A and B). To test whether activation of MAPK family members was

mediated by signaling through KDR, HUVEC were incubated in the absence or

presence of SU5416 and then stimulated with VEGF or PlGF. The KDR

antagonist abrogated VEGF activation of p38 MAPK (Figure 3A) and ERK1 and

ERK2 (Figure 3B), indicating that the MAPKs are activated through KDR

signaling. This result is consistent with the inability of PlGF to activate p38

MAPK or MAPK (Figs. 3A and B).

To determine whether MAPK, or p38 MAPK, plays a role in CREB

phosphorylation, HUVEC were treated with PD98059, a MEK inhibitor which

blocks MAPK activation (33), or SB202190, an inhibitor of p38 MAPK (34), and

then with VEGF. CREB phosphorylation induced by VEGF was unaffected by

inhibition of MAPK by PD98059. However, p38 MAPK inhibition partly

suppressed CREB phosphorylation (Fig. 4A), showing that this member of the

MAPK family plays a role in VEGF activation of CREB.

We next examined whether other kinases contribute to CREB activation by

VEGF. HUVEC were treated with wortmannin, an inhibitor of

phosphatidylinositol 3-kinase (PI 3-kinase), H89, an inhibitor of protein kinase

A (PKA), K252A, an inhibitor of CaM kinase II, or GF109203X, an inhibitor of

protein kinase C (PKC)(35). PD98059 and SB202190 were included in the

experiment as positive and negative controls. LY294002, H89, and K252A did

not impair CREB phosphorylation induced by VEGF, whereas GF109203X partly

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inhibited phosphorylation (Fig. 4B). Treatment of cells with GF109203X and

SB202190 showed that inhibition of PKC and p38 MAPK had no effect on basal

CREB phosphorylation, but blocked VEGF-induced phosphorylation (Fig. 4C).

To identify the downstream kinase that mediated CREB phosphorylation

induced by PKC, HUVEC were incubated with GF102203X or SB202190 and then

with VEGF (Fig. 4D). A Western blot prepared from cell lysates was probed with

an antibody to phosphorylated (active) p90 RSK, a CREB kinase downstream of

PKC. VEGF promoted p90 RSK phosphorylation and this was inhibited by

GF102203X, but not SB202190, suggesting that a PKC/p90 RSK pathway is a

component of the mechanism through which VEGF induces CREB

phosphorylation.

MAPKAP kinase 2, MAPKAP kinase 3 and MSK-1 are CREB and ATF-1

kinases activated by p38 MAPK (36-39). As antibodies to phosphorylated forms

of each of these CREB kinases are not available, their activation was determined

by an in vitro kinase assay in which ATF-1 was the substrate. HUVEC were

stimulated with vehicle or VEGF and then MAPKAP kinase 2, MAPKAP kinase

3, or MSK-1 were immunoprecipitated from cell lysates and incubated with

ATF-1. A Western blot was probed with an antibody to phosphorylated

CREB/ATF-1 and, of the three kinases, VEGF activated only MSK1 (Fig. 4E).

HUVEC were next incubated with GF102203X or SB202190, to inhibit PKC and

p38 MAPK activation, respectively, and then stimulated with VEGF. MSK1

immunoprecipitated from cell lysates was incubated with ATF-1 and a Western

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blot probed with antiphospho-CREB/ATF-1 and then with antiphospho-MSK-

1. VEGF induced phosphorylation of ATF-1 and MSK-1 and this was blocked

by inhibition of p38 MAPK but not PKC (Fig. 4F). This result suggests that

activation of p38 MAPK/MSK1 is a component of a signaling cascade through

which VEGF induces CREB phosphorylation.

Confocal microscopy was conducted to establish a bridge between

phosphoryation of CREB on serine 133 and subsequent DNA binding of the

transcription factor. HUVEC were stimulated with VEGF or treated with

inhibitors of PKC and p38 MAPK and then with VEGF. Confocal microscopy

established that nuclear phosphorylation of CREB was induced by VEGF and

this was inhibited by pharmacological blockade of the p38 MAPK and PKC

signaling cascades (Fig. 5).

CREB binds to the specific sequence, 5´-TGFCGTCA-3´, known as

CRE. An electrophoretic mobility shift assay (EMSA) showed that stimulation of

HUVEC with VEGF induced the formation of a protein-DNA complex. The

protein-DNA complex was lost upon addition of control but not mutant CRE.

Furthermore, supershifting with antibodies directed against CREB, c-fos and

ATF2 showed that only CREB was a component of the retarded DNA-protein

complex induced by VEGF (Fig. 6A). To establish that KDR was the receptor

required for induction of CREB/DNA binding, HUVEC were treated with

SU5416 before incubation with VEGF. CREB DNA binding was absent after

exposure of HUVEC to SU5416, demonstrating that KDR is required for

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activation of CREB (Fig. 6B).

DISCUSSION

Angiogenesis requires the coordinated expression of gene products that

allow endothelial cells to alter their environment, proliferate, migrate and

intubate (2). Physiological processes, such as wound healing, or pathological

processes, such as tumor growth, require that the cells involved switch from a

quiescent to an angiogenic phenotype (2). This process, called the angiogenic

switch, is characterized by increased production of angiogenic mediators, such as

VEGF and its receptors, decreased production of inhibitors of angiogenesis, such

as thrombospondin, or both types of events (8,9,11,17,40-42). VEGF induced

angiogenesis can be exploited to perfuse tissues isolated by blocked blood

vessels, and inhibition of VEGF and its receptors blocks the progression of cancer

(1,12,19,43,44).

Results from this and previous studies are summarized in the model in

Figure 7, which shows that by signaling through KDR/Flk1, VEGF induces

activation of MAPK, p38 MAPK, Akt, and tyrosine phosphorylation of

phospholipase Cγ and focal adhesion kinase (30,45,46). FAK is associated with

cell migration whereas PI 3-kinase/AKT and MAPK signaling promote cell

survival (47). MAPK also promotes the proliferative response of endothelial cells

to VEGF (30). KDR/Flk1 binds a novel adaptor protein, VRAP, which may

shuttle proteins that contain SH2 domains to the receptor (48) and the SHP-1

protein tyrosine phosphatase (49). The level of SHP-1 associated with

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KDR/Flk1 is augmented by tumor necrosis factor, indicating that the activity of

this receptor and its association with signaling proteins is regulated by cytokines

(49).

Stimulation of porcine aortic endothelial cells overexpressing KDR/Flk1

or Flt1 with VEGF induces association of the former, but not the latter, receptor

with Shc, Grb2, Nck, SHP-1 and SHP-2 (50,51). MAPK activation, changes of

cell morphology, actin reorganization, membrane ruffling, chemotaxis and cell

proliferation are also induced by signaling through KDR/Flk1. These

distinctions between KDR/Flk1 and Flt1 were not observed in studies with NIH

3T3 cells transfected with these receptors (52,53,57) or in experiments in which

affigel-immobilized Flt1 was used to precipitate proteins from cell lysates (54).

However, the weight of evidence from experiments with indicates that

KDR/Flk1 is a pleiotropic inducer of VEGF action in the endothelium, whereas

the role of Flt1 needs to be more clearly resolved. Thus, considerable insight has

been gained into cytoplasmic events initiated by VEGF in support of its activities.

Far less is known of how VEGF induces changes of gene expression that underlie

angiogenesis.

CREB is a 43-kDa member of the CREB/CREM/ATF family of nuclear

transcription factors that was first identified as a mediator of cyclic AMP-

induced gene expression (55). Activation of CREB is mediated by

phosphorylation of serine-133 (56), which augments CREB induced gene

transcription (57). Peptide hormones, growth factors, neuronal activity, UV

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irradiation and cross-linking of surface IgG activate CREB (38,57-61). This

diversity of signal initiation, and the different signaling systems through which

these cellular stressors act, indicates that serine-133 in CREB may be a substrate

for a multiplicity of protein kinases. Mitogen-induced phosphorylation

commences with stimulation of guanine nucleotide exchange factors, such as

SOS, which activates Ras. Ras induces activation of the Raf serine-threonine

kinase, an upstream activator of MAP kinase kinases (MEKs). Downstream

targets of the MEKs are the MAP kinases ERK1 and ERK2, which in turn can play

a role in activation of ribosomal S6 kinase pp90rsk, a CREB kinase (62). p38

MAPK and its downstream effectors MAPKAP-K2 and 3 are components of an

alternative pathway leading to CREB activation (36-39). Other kinases that

activate CREB are MSK1, a relative of p90RSK, which is activated by ERKs,

calcium calmodulin-dependent kinases, and protein kinase C (61,63-65).

The present study has shown that VEGF increases phosphorylation of

nuclear CREB on serine-133 and increases CREB DNA binding and

transactivation. Thus, three independent lines of experimentation support the

conclusion that VEGF activates CREB. The ability of VEGF and VEGF-D, but not

PlGF, to induce CREB phosphorylation shows that it is by signaling through

KDR that CREB activation is mediated. This conclusion is supported by the

ability of SU5416 to block CREB activation by VEGF. Thus, only one of the two

VEGF receptors plays a role in the induction of CREB activity.

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ERK1 and ERK2, PI 3-kinase, PKA, and CAM kinase II were excluded as

mediators of VEGF-induced CREB phosphorylation based on the inability of

PD98059, LY294002, H89 and K252A, respectively, to inhibit VEGF-induced

CREB phosphorylation. Inhibition of p38 MAPK by SB202190 partly inhibited

VEGF-induced activation of CREB, as did GF109203X, an inhibitor of PKC.

Complete blockade of CREB activation was observed when p38 MAPK and PKC

were inhibited. These results show that p38 MAPK and PKC mediate CREB

phosphorylation downstream of KDR.

CREB plays an important role in cell proliferation, differentiation,

adaptive responses and metabolism (31,66). In mice, CREB is required for the

generation of a normal repertoire of T cell lineages during development and the

absence of CREB leads to dwarfism and cardiac myopathy in the adult (67-69).

CREB family members are important for learning and memory and contribute to

the neuronal adaptation to drugs of abuse (70,71). Our observations now suggest

that by mediating cellular responses to VEGF, CREB is likely to play an

important role in endothelial cell function and blood vessel development.

This work was supported by NIH grant CA 73023 to DBD and NIH grant

CA84018 to RSW. LDM was supported by Hematology-Oncology Training grant

T32 DK07519 from NIH.

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Figure legends

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Fig. 1. CREB phosphorylation and activation induced by VEGF is mediated by

signaling through KDR. A. HUVEC were incubated with 1 nM VEGF for various

times and then a Western blot prepared from cell lysates was probed with anti-

pCREB (top) or anti-CREB (bottom). B. HUVEC were incubated with medium

or 1 µM SU5416 for 1 h and then with 1 nM VEGF or PlGF for 10 min. A Western

blot was probed with anti-pCREB (top) or anti-CREB (bottom). C. HUVEC

were incubated with medium 1 nM VEGF-D for 10 min. A Western blot was

probed with anti-pCREB (top) or anti-CREB (bottom). D. CREB reporter activity

in cells treated with vehicle or VEGF was assayed.

Fig. 2. p38 MAPK and MAPK activation by VEGF. HUVEC were treated with

VEGF for various times and Western blots were prepared from cell lysates. A. A

Western blot was probed with an anti-phospho-p38 MAPK (top) and then with

anti-p38 MAPK (bottom). B. A Western blot was probed with anti-pMAPK

(top) and then with anti-MAPK (bottom).

Fig. 3. p38 MAPK and MAPK are activated by signaling through KDR. HUVEC

incubated in the absence or presence of 1 µM SU5416 were treated with VEGF.

A. A Western blot was probed with anti-phospho-p38 MAPK (top) and then with

anti-p38 MAPK (bottom). B. A Western blot was probed with anti-MAPK (top)

and then with anti-MAPK (bottom).

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Fig. 4. Signaling pathways used by VEGF to promote CREB phosphorylation. A.

HUVEC were treated with PD98059 or SB202190 for 1 h and then incubated in

the absence or presence of 1 nM VEGF for 10 min. CREB phosphorylation was

assayed by Western blot analysis. B. HUVEC were treated with medium, 25 µM

PD98059, 20 µM Ly294002, 25 µM SB202190, 20 µM H89, 100 nM K252A, 1 µM

GF109203X or GF109203X and SB202190 and then with VEGF for 10 min. CREB

phosphorylation was assayed by Western blot analysis. C. HUVEC were

incubated with medium, SB202190, or GF109203X and then treated with medium

or VEGF. CREB phosphorylation was assayed Western blotting. D. HUVEC

were incubated with medium, SB202190 or GF109203X and then with medium or

VEGF. p90 RSK phosphorylation was assayed by probing a Western blot with

anti-phospho RSK . E. An in vitro kinase assay using ATF-1 as substrate was

used to test for activation of MAPKAP kinase 2, MAPKAP kinase 3, and MSK-1,.

These kinases were immunoprecipitated from lysates of control or VEGF-

treated HUVEC and a Western blot was probed with anti- phosphorylated

CREB/ATF1. F. HUVEC were incubated with GF109203X or SB202190 and then

with vehicle or VEGF. MSK1 was immunoprecipitated from cell lysates and a

Western blot was probed with antibodies to phosphorylated CREB/ATF1 and

phosphorylated MSK-1.

Fig. 5. Confocal microscopy. HUVEC were treated with vehicle or SB202190

together with GF109203X for 1 hour and then stimulated with VEGF. Confocal

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microscopy was used to detect and localize phosphorylated CREB/ATF-1.

Fig. 6. Binding of CREB to DNA. A. Nuclear extracts of HUVEC treated with

vehicle or VEGF were incubated with a 100-fold excess of control or mutant CRE

before CREB DNA binding was assayed by EMSA. Supershifting with anti-

CREB (αCREB), anti-fos (αfos), or anti-ATF2 (αATF2) established the

component(s) of the retarded complex. B. HUVEC were treated with medium or

SU5416 for 1 h and then with VEGF. Nuclear extracts were used for EMSA.

Fig. 7. A model of KDR/Flk1 signaling.

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DonnerLindsey D. Mayo, Kelly M. Kessler, Roxana Pincheira, Robert S. Warren and David B.

receptor tyrosine kinaseVascular endothelial cell growth factor activates CREB by signaling through the KDR

published online May 2, 2001J. Biol. Chem.

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