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PLC -Erk Axis in VEGF-induced eIF4E Phosphorylation and Protein Synthesis in

Renal Epithelial Cells

Meenalakshmi M Mariappan§, Duraisamy Senthil

§, Kavithalakshmi S. Natarajan

§,

Goutam Ghosh Choudhury§#

, and Balakuntalam S Kasinath§#

#South Texas Veterans Healthcare System, GRECC, O’Brien Kidney Research Center,

§Department of Medicine, University of Texas Health Science Center, San Antonio, TX

Running Title: VEGF-induced protein synthesis mediated by PLC and Erk

Address correspondence to: B. S. Kasinath, MD

Department of Medicine

Mail Code 7882

University of Texas Health Science Center

7703 Floyd Curl Drive

San Antonio, TX 78248

Phone: 210-567-4707

Fax: 210-567-4712

Email: Kasinath@uthscsa.edu

JBC Papers in Press. Published on May 26, 2005 as Manuscript M504861200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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PLC -ERK AXIS IN VEGF-INDUCED EIF4E PHOSPHORYLATION AND

PROTEIN SYNTHESIS IN RENAL EPITHELIAL CELLSMeenalakshmi M Mariappan

§, Duraisamy Senthil

§, Kavithalakshmi S. Natarajan

§,

Goutam Ghosh Choudhury§#

, and Balakuntalam S. Kasinath§#

#South Texas Veterans Healthcare System, GRECC, O’Brien Kidney Research Center,

§Department of Medicine, University of Texas Health Science Center, San Antonio, TX

VEGF increases protein synthesis and induces

hypertrophy in renal tubular epithelial cells

(Senthil et al., 2003). We examined the role of

Erk1/2 MAP kinase in protein synthesis induced

by VEGF. VEGF st imulated Erk

phosphorylation that was required for induction

of protein synthesis. VEGF-induced Erk

activation was not dependent on PI 3-kinase

act ivat ion but required sequent ia l

phosphorylation of type 2 VEGF receptor, PLC

and c-Src, as demonstrated by inhibitors

SU1498, U73122 and PP1, respectively. c-Src

phosphorylation was inhibited by U73122,

indicating it was downstream of PLC . Studies

with PP1/2 showed that phosphorylation of c-Src

was required for tyrosine phosphorylation of

Raf-1, an upstream regulator of Erk. VEGF also

stimulated phosphorylation of Pyk-2; VEGF-

induced phosphorylation of Pyk2, c-Src and Raf-

1 could be abolished by BAPTA-AM,

demonstrating requirement for induction of

intracellular calcium currents. We examined the

downstream events following phosphorylation of

Erk. VEGF stimulated phosphorylation of Mnk1

and eIF4E, and induced Mnk1 to shift from

cytoplasm to the nucleus upon phosphorylation.

VEGF-induced phosphorylation of Mnk1 and

eIF4E required phosphorylation of PLC , c-Src

and Erk. Expression of dominant negative Mnk1

abrogated eIF4E phosphorylation and protein

synthesis induced by VEGF. VEGF-stimulated

protein synthesis could be blocked by inhibition

of PLC by chemical inhibitor or expression of a

dominant negative construct. Our data

demonstrate that VEGF stimulated protein

synthesis is Erk-dependent and requires the

activation of VEGFR2, PLC , c-Src, Raf, Erk

pathway. VEGF also stimulates Erk-dependent

phosphorylation of Mnk1 and eIF4E, crucial

events in the initiation phase of protein

translation.

The response of a cell to injurious stimuli

includes increase in cell protein synthesis

(hypertrophy), cell division (hyperplasia) or

apoptosis. Of these processes, the mechanisms

underlying cell hypertrophy have not been well

understood. Regulation of cellular protein synthesis

occurs at the initiation phase of protein translation

(1). One of the main events in initiation phase is

phosphorylation of eukaryotic initiation factor 4E

(eIF4E) binding protein (4E-BP1, 15-20 kDa) (2).

Phosphorylation of this repressor protein results in

dissolution of eIF4E-4E-BP1 complex, releasing the

eIF4E to bind to the mRNA cap and promote

translation initiation (3). Increase in eIF4E

phosphorylation is commonly seen during the

initiation phase and has been attributed to Erk-1/-2

type MAP kinase (4). However, Erk is not the direct

kinase for eIF4E; it activates Mnk1/-2 kinase,

which, in turn, phosphorylates ser209 on eIF4E

(5,6). Agonists that induce Mnk1 phosphorylation

have not been well studied. We examined whether

VEGF regulates this important kinase.

Functional effects of VEGF as an

angiogenic agent and as a key regulator of

endothelial cell functions have been well

documented. Our recent observations have shown

that VEGF increases protein synthesis and induces

hypertrophy in renal proximal tubular epithelial

(MCT) cells, suggesting a role for VEGF in non-

angiogenic processes in non-endothelial cells (7).

Previously, we have also shown that VEGF

stimulation of protein synthesis and hypertrophy in

MCT cells recruit PI 3-kinase-Akt axis to induce

phosphorylation of 4E-BP1 (7). In that study, the

role of Erk in VEGF-induced protein synthesis was

not examined. Increase in Erk phosphorylation in

the renal cortex of mice with type 2 diabetes occurs

simultaneously with the onset of kidney

hypertrophy that mostly involves renal tubular

epithelial cells (8). Coinciding with Erk

phosphorylation and kidney hypertrophy, VEGF

expression in the kidney is increased (7), and anti-

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VEGF antibody reverses kidney enlargement (9).

However, whether VEGF recruits Erk pathway in

regulation of protein synthesis and renal cell

hypertrophy is not known. Also unknown is the

identity of upstream kinases that regulate Erk

activity in response to VEGF in MCT cells. In this

study, we tested the hypothesis that PLC- , an

important kinase that binds to type 2 VEGF

receptor (VEGFR2), plays a regulatory role in

VEGF-induced Erk activation and protein synthesis.

Downstream of Erk, we also studied whether VEGF

regulated Mnk1 phosphorylation and its target,

eIF4E.

Methods

Materials. Dulbecco's Modified Eagle's Medium

(DMEM), penicillin and streptomycin were

purchased from Gibco-Invitrogen (Carlsbad, CA).

Fetal bovine serum was purchased from Hyclone

Laboratories Inc. VEGF-165 recombinant protein,

and, anti-actin antibody were purchased from

Sigma-Aldrich (St. Louis, MO). Anti-

phosphotyrosine antibody 4G10 was purchased

from Upstate Inc (Charlottesville, Virginia).

Antibody against Mnk1 and anti v-Src antibody

were bought from Santa Cruz Biotechnology (Santa

Cruz, CA) and Oncogene Research Products (San

Diego, CA), respectively. All other antibodies were

purchased from Cell Signaling (Beverly, MA). 35

S-

labeled methionine was purchased from Perkin-

Elmer (Boston, MA). ECL chemiluminescent

substrate reagent was from Pierce Biotechnology

Inc (Rockford, IL). PP1 and PP2 Analog, SU1498,

LY294002 and BAPTA/AM were purchased from

Calbiochem-EMD Biosciences (San Diego, CA).

As PP1 is no longer available we employed PP2 as

an inhibitor of c - Src. U73122 was from Biomol

International L.P. (Plymouth Meeting, PA) and

U0126 was purchased from Promega Life Sciences

(Madison, WI).

Cell culture (7 ). SV-40 immortalized murine

proximal tubular epithelial (MCT) cells (kindly

provided by Dr. Eric Neilson, Vanderbilt

University, Nashville, TN) were grown in DMEM

containing 7% fetal bovine serum, 5 mmol/L

glucose, 100 u/ml penicillin, 100 µg/ml

streptomycin and 2 mM glutamine. MCT cells

express in vivo properties of proximal tubular

epithelial cells (10). The cells were grown to 90%

confluence and then growth-arrested for 18 hours in

serum-free DMEM before experiments.

Immunoblotting (7). Equal amounts of protein

from control and VEGF-treated cells were separated

by SDS-PAGE and transferred to a nitrocellulose

membrane. After transfer overnight at 4°C, the

membrane was blocked in TBS, pH 7.2, containing

0.1% Tween 20 (TBST) for 1 hour. Membrane was

then washed and probed with primary antibody for

3 hours. After extensive washing in TBST, the

membrane was incubated with horseradish

peroxidase conjugated secondary antibody (Jackson

ImmunoResearch Laboratories,Inc, West

Grove,PA). Proteins were visualized by

chemiluminescence using ECL reagent. Images of

the bands were scanned by reflectance scanning

densitometry and the intensity of the bands was

quantified using NIH Image software.

Protein synthesis measurement (11). Serum-

starved cells were incubated with 20 ng/ml of

VEGF-165 in the presence of 10 µCi/ml of [35

S]-

methionine for 2 hours with or without the

respective inhibitors. Cells were then washed in

phosphate buffered saline (PBS) and lysed in

radioimmunoprecipitation assay (RIPA) buffer.

Equal amount of protein (20 µg) was spotted onto

the 3 mm filter paper (Whatman International,

Maidstone, UK). Filters were washed three times by

boiling for 1 minute in 10% trichloroacetic acid

(TCA) containing 0.1 g/L methionine before

determining radioactivity.

Erk1/2 MAP kinase (Erk) activity assay (12).

Equal amounts of cell lysates (200 µg) were

immunoprecipitated with Erk1/2 antibody. Protein

A-agarose beads were added and incubated at 4°C

for 1 hour. The beads were then washed and the Erk

kinase assay was performed in kinase assay buffer

in the presence of 50 µmol cold ATP, 1 µCi of

[32

P]-ATP and 5 µg of myelin basic protein (MBP)

at 30°C for 30 minutes. The reaction was arrested

by the addition of an equal volume of 2X sample

buffer. Phosphorylated bands were detected by

SDS-PAGE followed by autoradiography. In some

experiments, immunoblotting with anti-phospho-

Erk antibody was done to assess Erk

phosphorylation.

Immunofluorescence microscopy. Cells were

seeded in 8-well chamber slides. Semi-confluent

cells were serum-starved for 18 hours and treated

with VEGF (20 ng/ml) for different time periods.

Cells were washed with PBS, fixed and incubated

with rabbit anti-phospho-Mnk1 antibody followed

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by staining with Cy3-conjugated donkey anti-rabbit

secondary antibody (Chemicon International,

Temecula, CA). PhosphoMnk1 was visualized with

a confocal laser microscopy system (Olympus

Fluoview 500). The confocal images were analyzed

using Fluoview software to determine the

localization of phospho-Mnk.

For quantification of phosphoMnk1 nuclear

localization, cells were grown in chamber slides,

individual chambers representing one time point.

Cells were viewed under uniform magnification

(40X). For each time point, the number of cells

showing nuclear localization of phosphoMnk1 per

total number of cells in a field of fixed area

containing a minimum of 150 cells, was counted

and converted to percentage. Finally, these values

were compared with that of value at time zero

(control). The experiment was performed three

times and composite data were converted into

graphic form and analyzed by ANOVA.

Transfection studies (13). Plasmid constructs

containing dominant negative mutants of Src and

PLC (PLCz) were employed for transient

transfection in MCT cells using lipofectamine and

lipo-plus reagent (Invitrogen Life Technologies

Inc., Calrsbad, CA), as described earlier. PCR 295

expression vector containing dominant negative c-

Src was kindly provided by Dr. T. Yoneda,

University of Texas Health Science Center, San

Antonio, TX. The kinase inactivation mutation in c-

Src consisted of Lys295 to Met295. PLCz is a

dominant-negative fragment of PLC gene which

encodes the Z region (PCI region, PLC inhibitor

region) containing the SH2 and SH3 domains (aa

517-901) of this enzyme (14). The pXf/PLCz

expression was driven by SV40 early promoter.

pEBG-3X eukaryotic expression vector containing

dominant-interfering Mnk1 mutant (pEBG-T2A2

containing Thr197Ala/Thr202Ala mutants (5)) was

a kind gift from Dr J. Cooper (Fred Hutchinson

Cancer Research Center, Seattle, WA).

Statistics. All values are expressed as mean ± SE

obtained from at least three independent

experiments. Statistical analysis was performed

using one-way analysis of variance (ANOVA) for

comparison between multiple groups; p values of

p<0.05 were considered significant.

Results

VEGF-induced protein synthesis is Erk-

dependent in MCT cells

We investigated whether VEGF induced

Erk phosphorylation and if VEGF-induced protein

synthesis was Erk-dependent since Erk1/2 MAP

kinase occupies a central position in transducing

signals from extra-cellular milieu to the nucleus.

VEGF promoted Erk phosphorylation within 2 min

that lasted nearly 15 min with peak phosphorylation

at 10 min (Fig. 1, panel A). VEGF significantly

stimulated protein synthesis in MCT cells that was

abrogated by pre-incubation of cells with U0126, an

inhibitor of MEK, the direct upstream kinase of Erk

(Fig. 1, panel B). VEGF induction of protein

synthesis is, thus, dependent on stimulation of Erk.

We initiated the search for upstream

regulators of VEGF-induced Erk activation. We

started with PI 3-kinase as insulin- and IGF-1-

induced Erk activation in MCT cells is PI 3-kinase

dependent (12,13). We have previously shown that

VEGF induces PI 3-kinase activity in these cells

(7). Pre-incubation with LY294002, a selective

inhibitor of PI 3-kinase, did not affect VEGF-

induced Erk activity, although it was abolished by

U0126 (Fig. 1, panel C). These observations

suggest that VEGF-induced Erk activation is

independent of PI 3-kinase but dependent on MEK

activation.

Having excluded PI 3-kinase, we sought to

identify other kinases that could act as upstream

effectors of Erk activation by VEGF. We selected

phospholipase C (PLC ) and c-Src as potential

regulators as they are membrane-associated and

interact with VEGF receptors (15,16).

Type 2 VEGF receptor (VEGFR2) stimulates

PLC and c-Src to regulate Erk phosphorylation

Immunoblotting cell lysates with anti-

phosphotyrosine antibody following VEGF

incubation for 10 min showed increased protein

tyrosine phosphorylation of several proteins,

including those of approximate sizes 220 kD, 180

kD, 150 kD, 110 kD, 90 kD, 60 kD and 30 kD. The

180 kD, 150 kD and 60 kD bands may represent

VEGFR2, PLC and c-Src, respectively. Pre-

incubation with SU1498, a selective VEGFR2

inhibitor, abolished or markedly inhibited most of

these changes implying VEGFR2 phosphorylation

was required for these tyrosine phosphorylation

events (Fig. 2A).

VEGF stimulated tyrosine phosphorylation

of PLC and c-Src rapidly within 5 minutes (Fig. 2,

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panels B and C). VEGFR2 is believed to mediate

most of the biological effects of VEGF. Pre-

treatment of cells with SU-1498 abolished VEGF-

induced PLC and c-Src phosphorylation (Fig. 2,

panels D and E), suggesting involvement of

VEGFR2. Next, we examined the sequence of

phosphorylation of PLC and c-Src by VEGF.

U73122, a PLC inhibitor, blocked phosphorylation

of c-Src, demonstrating that the latter is

downstream of PLC in VEGFR2 signaling events

in MCT cells (Fig. 2, panel F).

We investigated whether PLC plays a role

in regulating VEGF-induced Erk activation. VEGF-

stimulated Erk activation was inhibited by U-

73122, the PLC inhibitor (Fig. 3, panel A). This

was further confirmed by expressing dominant

negative constructs of PLC (PLCz). VEGF-

stimulated Erk activation was abolished in cells

transfected with PLCz, but not in control cells

transfected with vector alone (Fig. 3, panel B).

Although it appears as though expression of PLCz

reduced Erk activity only upon addition of VEGF,

densitometric analysis of bands from 3 experiments

did not reveal a significant difference in band

intensity between VEGF+PLCz and untreated

control groups (bars 1 Vs 4 in Fig. 3, panel B).

We next examined the role of c-Src in

VEGF induction of Erk. PP1, a c-Src inhibitor,

abolished VEGF induced Erk phosphorylation (Fig.

4, panel A). Further confirmation was obtained by

employing a kinase-inactive dominant negative

construct of c-Src (Fig. 4, panel B). Raf is an

upstream kinase in Erk pathway (17,18). VEGF

stimulated Raf phosphorylation at Tyr340, 341

within 5 min that lasted for nearly 15 min (Fig. 4,

panel C). This was completely inhibited by PP2, a

c-Src inhibitor (Fig. 4, panel D), indicating

Tyr340/341 phosphorylation of Raf was c-Src

dependent. The Src inhibitor, at this concentration

at this concentration, did not affect VEGF-induced

PLC phosphorylation, which is VEGFR2-

dependent (See Supplementary fig. S1).

VEGF induces phosphorylation of Pyk2, c-Src

and Erk in a Ca-dependent manner

VEGF increases intracellular calcium transients

through VEGFR2 (19) that correlate with

phosphorylation of PLC (20). Activation of PLC

has been linked to the production of inositol 1,4,5,

tris-phosphate and diacylglycerol, leading to

increased intracellular calcium mobilization and

PKC activation, respectively. A read-out of Ca

signaling is the phosphorylation of Pyk2, a proline-

rich, Ca++

-dependent non-receptor tyrosine kinase.

Pyk2 phosphorylation was studied using a phospho-

specific antibody that detects phosphorylation on

tyr402. Incubation with VEGF rapidly induced

phosphorylation of Pyk2 that lasted for nearly 30

min (Fig. 5, panel A). We next examined the role of

Ca++

in Erk activation induced by VEGF. Chelation

of intracellular Ca++

by BAPTA/AM abolished

VEGF-induced phosphorylation of Pyk2, Src and

Erk (Fig. 5, panels B, C and D), confirming that

Ca++

transients are required for VEGF-induced

phosphorylation of Erk and its upstream regulators.

The data described above established the

upstream regulators of VEGF-induced Erk

activation along the lines of VEGFR2, PLC ,

Pyk2/c-Src, Raf, Erk. We next sought to gain

insight into the possible mechanism downstream of

Erk activation related to events of regulatory

importance in protein synthesis.

VEGF induces phosphorylation of Mnk1

As discussed before, phosphorylation of 4E-BP1

releases eIF4E from the dimeric complex. 4E-BP-1

phosphorylation is required for VEGF-induced

protein synthesis and hypertrophy in MCT cells;

this process is PI 3-kinase- and Akt-dependent (7).

Following dissolution of eIF4E-4E-BP1 complex,

free eIF4E binds to a scaffolding protein eIF4G and

is phosphorylated by MAP kinase integrating

kinases (Mnk1/2) that also bind eIF4G at the C-

terminus (21). Mnk1, a 52 kDa protein, is activated

by phosphorylation at Thr197 by Erk1/2. As Erk

regulation of protein synthesis could involve Mnk

and eIF4E, we examined whether VEGF regulates

their phosphorylation. VEGF rapidly stimulated

phosphorylation of Mnk1 within 5 min and the

effect lasted for nearly 30 min (Fig. 6, panel A).

These data suggest that VEGF activates a

downstream target of Erk. Mnk1 shuttles between

the cytoplasm and the nucleus (22); however,

agonists that stimulate nuclear shift of Mnk1 have

not been extensively identified. Therefore, we

examined if VEGF regulates shift of Mnk1 into the

nucleus. As seen in Fig. 6, panel B, we observed

increased immunofluorescence corresponding to

Mnk1 phosphorylation in the cytoplasm within 2

minutes with perinuclear localization at 5 min.

Nuclear translocation was observed at 15 min,

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coinciding with maximum phosphorylation of the

protein (Fig. 6, panel A). Figure 6B shows the

scoring for nuclear localization of phosphoMnk1.

There was a progressive increase in nuclear

immunofluorescence that peaked at 15 min

following treatment of the cells with VEGF (p<0.01

at 5 and 15 min). To our knowledge, this is the first

demonstration of VEGF regulation of Mnk1

phosphorylation and its localization in the cell. We

examined the upstream regulators of VEGF-

induced Mnk1 phosphorylation. U73122, the PLC

inhibitor, PP2, the c-Src inhibitor, and U0126, the

MEK inhibitor, all abolished VEGF-stimulated

Mnk1 phosphorylation (Fig. 7, panels A, B and C),

identifying PLC, c-Src and MEK (immediately

upstream of Erk) as kinases that control Mnk1

phosphorylation.

VEGF induces phosphorylation of eIF4E.

Our next aim was to test whether VEGF induced

phosphorylation of eIF4E, the Mnk1 substrate.

VEGF stimulated phosphorylation of eIF4E

robustly within 5 min and the effect lasted for about

30 min (Fig. 8, panel A). U73122, PP2, and U0126

completely inhibited VEGF-induced

phosphorylation of eIF4E (Fig. 8, panels B, C and

D). Thus, VEGF-induced phosphorylation of eIF4E

is regulated via the PLC , c-Src and Erk axis.

DN-Mnk1 expression inhibits eIF4E

phosphorylation and protein synthesis induced

by VEGF

We next investigated the involvement of Mnk1 in

VEGF-stimulated protein synthesis by expressing

dominant negative Mnk1 in which the Thr197 and

202 are mutated to Ala. Both VEGF-induced eIF4E

phosphorylation and protein synthesis were

abrogated by expression of mutant Mnk1 (Fig. 9,

panels A and B). These data demonstrate that

VEGF-induced protein synthesis requires Mnk1

phosphorylation.

PLC activation is required for VEGF-induced

enhanced protein synthesis. We have established

the role of PLC in activation of Erk to increase

phosphorylation of eIF4E, a regulator of protein

synthesis initiation. We directly examined whether

PLC activation, the proximal event in Erk

activation, is required for VEGF induction of

protein synthesis. VEGF-induced protein synthesis

in MCT cells was completely abrogated when the

cells were pre-treated with U73122 (Fig. 10, panel

A). Further confirmation was obtained employing

cells transfected to express PLCz, the dominant

negative construct of PLC (Fig. 10, panel B).

Fig.11 summarizes the proposed sequence of

signaling events induced by VEGF leading to

protein synthesis in MCT cells.

Discussion

In this report we demonstrate that VEGF

augments protein synthesis in an Erk

phosphorylation-dependent manner. Our data show

that VEGF-induced Erk phosphorylation is

regulated by sequential phosphorylation of

VEGFR2, PLC , and c-Src. We report for the first

time that VEGF stimulation of Erk phosphorylation,

in turn, results in phosphorylation of Mnk1 and its

substrate eIF4E. Phosphorylation of Mnk1 is

associated with its shift from the cytoplasm to the

nucleus. Our results provide new evidence for a role

for PLC in VEGF-induced eIF4E phosphorylation

and protein synthesis.

VEGF stimulation of protein synthesis in MCT

cells is mediated via activation of VEGFR2 (7).

Pathways regulating phosphorylation of 4E-BP1

and eIF4E seem to diverge downstream of

VEGFR2, the former being dependent on PI 3-

kinase-Akt activation (7) whereas, as reported here,

e IF4E phosphory la t ion requi res Erk

phosphorylation. As Erk phosphorylation was

independent of PI 3-kinase, our observations

suggest distinct roles for PI 3-kinase and Erk

pathways in regulation of crucial events that control

initiation phase of protein translation. PLC

phosphorylation by VEGFR2 is the most proximal

event identified in eIF4E phosphorylation in this

study. PLC binds to tyr1175 in the cytoplasmic

domain of VEGFR2 (23). Its role in regulation of

protein translation has not been studied in depth.

U73122, an inhibitor of PLC, has been shown to

abolish cardiac myocyte hypertrophy induced by

norepinephrine (24), although it appears not

required in angiotensin II-induction of protein

synthesis in cardiac fibroblasts (25). Thus,

involvement of PLC in protein synthesis appears to

be cell- and agonist-specific. In several previous

studies, involvement of PLC was not directly

studied but suspected by the use of its inhibitor, and

changes in eIF4E were not addressed. Our study

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demonstrates for the first time, a link between PLC

phosphorylation and eIF4E phosphorylation for

inducing protein synthesis in cells stimulated with

VEGF.

Phosphorylation of PLC is followed by

generation of inositol 1,4,5, tris-phosphate and

diacylglycerol, which are known to stimulate

mobilization of intracellular calcium and protein

kinase C, respectively. Ca++

transients appear to be

important for VEGF induction of Erk

phosphorylation as BAPTA/AM abolished this

effect. In MCT cells, VEGF stimulation of c-Src

requires activation of PLC . c-Src has been

identified as a mediator of angiotensin II-

stimulation of protein synthesis in smooth muscle

cells grown from resistance arteries from

hypertensive patients (26). Additionally, c-Src is

involved in phosphorylation of RNA binding

protein hnRNP-K that regulates translation of select

mRNAs (27). Our observations identify c-Src as

one of the upstream regulators of VEGF-induced

phosphorylation of Mnk1 and eIF4E. Pyk2, a

proline-rich non-receptor tyrosine kinase, is

activated in a Ca++

-dependent manner, resulting in

its interaction with activated c-Src (28).

Phosphorylation of tyr402 of Pyk2 leads to binding

and activation of c-Src in PC12 cells (29). Pyk2

phosphorylation is required for angiotensin II-

induced eIF4E phosphorylation and protein

synthesis in vascular smooth muscle cells (30,31).

Pyk2 may serve as a bridge between PLC -c-Src

axis and the canonical Raf-MEK-Erk pathway (29).

Activated Raf is phosphorylated on Ser338 (32).

However, for full activation of Raf-1, c-Src

mediated phosphorylation of Tyr340/341 is

necessary (33,34). Our data suggest that VEGF

stimulation of Erk pathway involves Raf-1 tyrosine

phosphorylation by c-Src. It is well known that Raf-

1 directly phosphorylates and activates the protein

kinase MEK.

eIF4E regulates the initiation phase of protein

synthesis that is regarded as the rate-limiting step

(35). In general, translation of most capped mRNAs

in eukaryotes is facilitated by eIF4E, the cap

binding protein (36). In response to signals that

promote protein synthesis, 4E-BP1 is

phosphorylated which results in dissolution of its

heterodimeric complex with eIF4E (37). Once freed

from 4E-BP1, eIF4E associates with eIF4G and

eIF4A to form the eIF4F complex and binds to the

m7 cap of the mRNA. Erk has been implicated in

eIF4E phosphorylation on Ser209; the

physiologically important site (38) although it is not

the direct kinase as eIF4E lacks the proline residues

around Ser209 involved in Erk recognition. It is

now established that Erk phosphorylates Mnk1,

which serves as the kinase for eIF4E (5,6). Binding

studies have shown that Mnk1 associates with C

terminus of eIF4G, the scaffolding protein, which

has binding sites for eIF4E and eIF4A, and forms

the anchor for eIF4F complex. Bulk of

phosphorylation of eIF4E by Mnk1 occurs

following their binding to eIF4G (21). Mnk1 carries

a basic amino acid-rich nuclear import signal in its

N-terminus that determines its shift from the

cytoplasm to the nucleus (22). Our observations

show that while phosphorylation of Mnk1 following

VEGF incubation starts while the protein is in the

cytoplasm, the peak in phosphorylation appears to

occur when the protein has shifted to the nucleus.

The relevance of Mnk1 nuclear shift to cellular

protein synthesis needs to be elucidated.

The functional significance of eIF4E

phosphorylation is also unclear. Initially, in vitro

studies showed that phosphorylation of eIF4E

augmented the avidity of the molecule for the

mRNA cap and promoted the efficiency of its

translation (39). In Drosophila, expression of an

eIF4E phosphorylation mutant resulted in stunted

development, suggesting phosphorylation of eIF4E

facilitated cell protein synthesis (40). However,

opposing views have recently been expressed.

Phosphorylation of eIF4E is thought to cause

electrostatic repulsion from phosphate groups on

the mRNA cap and inhibit its cap binding (41,42).

The precise timing of eIF4E phosphorylation

relative to mRNA cap binding is not known. If

phosphorylation of eIF4E were to occur after

binding of the factor to the mRNA cap, anionic

repulsion could serve to disengage eIF4E from the

mRNA. The association and dissociation reactions

between eIF4E and mRNA are known to be rapid in

the range of 0.1 sec (43) and could be facilitated by

eIF4E phosphorylation. Although present in

cytoplasm, a significant fraction of cellular eIF4E is

present in the nucleus and may participate in

nuclear protein translation (44) and export of select

mRNAs e.g., cyclin D1, from the nucleus (45,46).

Role of eIF4E phosphorylation in these processes is

not yet known.

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Stimulation of protein synthesis in renal

epithelial (MCT) cells by VEGF has implications

for kidney hypertrophy, which may occur

physiologically, after removal of one kidney (9), or,

pathologically, in early phase of diabetes (7,47).

Neutralizing antibodies against VEGF ameliorate

early renal dysfunction in experimental diabetes

including renal hypertrophy (9,48). These data

implicate VEGF in the pathogenesis of kidney

hypertrophy in diabetes that mostly involves renal

epithelial cells (49). VEGF could have constructive

effects on the kidney in a different setting. In

thrombotic glomerular disease, VEGF

administration helps restore renal architecture (50).

Knowledge of signaling intermediaries that regulate

VEGF-induction of protein synthesis in renal cells

should help facilitate modulation of this process to

advantage in renal disease states.

Acknowledgements: These studies were supported

by grants from the NIH (BSK, GGC-DK55815 &

DK50190), American Diabetes Association (BSK),

Veterans Administration (BSK, GGC), the Juvenile

Diabetes Research Foundation (GGC), and the

National Kidney Foundation of South and Central

Texas (MMM). We acknowledge the staff at the

Cellular and Structural Biology Department at

UTHSC San Antonio for providing the confocal

microscopy instruments. GGC is a recipient of the

Veterans Administration Career Scientist Award.

Abbreviations used are: IGF-1, insulin-like

growth factor 1; PAGE, polyacrylamide gel

electrophoresis; PP1, 4-Amino-1-tert-butyl-3-(1¢-

naphthyl)pyrazolo[3,4-d]pyrimidine; PP2, 4-amino-

5-(4-chlorophenyl)-7-(t-

butyl)pyrazolo[3,4,d]pyrimidine; PCI domain,

phospholipase C inhibitory domain; PI 3-kinase,

phosphoinositide-3-kinase; PLC- 1, phospholipase

C-gamma one; SH2, SH3, Src homology 2 and 3;

VEGF, vascular endothelial growth factor;

VEGFR2, vascular endothelial growth factor

receptor 2

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Figure legends

Fig 1.

Panel A. VEGF stimulates Erk phosphorylation. Serum-deprived cells were incubated with VEGF (20 ng/ml).

Equal amounts of protein from the cell lysates were fractionated on a 12.5% gel. Immunoblotting was performed

using 1:2000 dilution of an antibody that specifically detects phospho-Erk. The blots were stripped and re-probed

with an Erk antibody to assess loading. A representative blot from more than 3 experiments is shown.

Densitometric quantification of data from 3 experiments is shown in the graph *p<0.01 VEGF Vs control.

Panel B. VEGF stimulation of protein synthesis is Erk-dependent. Quiescent MCT cells were incubated with

or without VEGF (20 ng/ml) simultaneously in the presence of [35

S]-methionine for 2 hours with or without pre-

incubation with 25 µM U0126, a MEK inhibitor, for 30 min. Incorporation of radiolabel into TCA-precipitable

protein was taken as a measure of protein synthesis and expressed as percentage of control. Composite data from 3

experiments are shown in a graph; *p<0.001 VEGF Vs control, **p<0.001 for VEGF+U0126 Vs VEGF, by

ANOVA.

Panel C. VEGF-induced Erk activation is independent of PI 3-kinase activation. MCT cells were incubated

with or without VEGF (20 ng/ml) for 10 min with or without pre-incubation with LY294002 (25 µM) or U0126

(25 µM) for 30 min. Equal amounts of cell lysates (100 µg) were immunoprecipitated with an anti-Erk-1/-2-type

MAP kinase antibody. Erk kinase activity was determined in the immunoprecipitates by an in vitro kinase assay

using myelin basic protein (MBP) as the substrate. The samples were fractionated by 15% SDS-PAGE and

phosphorylation of MBP visualized by autoradiography. Immunoblotting with total Erk antibody was done to

assess loading and band intensities were measured by densitometry. A representative blot from 3 individual

experiments is shown. The graph presents composite densitometric data from 3 experiments with controls

expressed as 100%; *p<0.001 VEGF or VEGF+LY294002 Vs control, **p<0.001 VEGF+U0126 Vs VEGF, by

ANOVA.

Fig 2.

Panel A. VEGF-induced tyrosine phosphorylation of proteins is dependent on VEGFR2 tyrosine

phosphorylation. Serum-starved MCT cells were incubated with VEGF (20 ng/ml) for 10 min with or without

pre-incubation with SU1498, the selective VEGFR2 inhibitor. Equal amounts of cell lysates were resolved on

SDS-PAGE and immunoblotted with phospho-tyrosine antibody. A representative blot from 3 independent

experiments is shown. The bottom panel shows immunoblot analysis of the same samples with anti-actin antibody.

Panels B, C. VEGF stimulates phosphorylation of phospholipase C (PLC ) (B) and c-Src kinase (C).

Serum-starved MCT cells were incubated with VEGF (20 ng/ml) for the time periods indicated. Equal amounts of

cell lysates were resolved on SDS-PAGE and immunoblotted with phospho-specific antibodies for PLC and c-

Src; loading was assessed by immunoblotting with antibodies against PLC and Src, respectively. A representative

blot from 3 independent experiments for each kinase is shown.

Panels D, E. VEGF-induced phosphorylation of PLC and c-Src is mediated through VEGFR2. Cells were

incubated with or without VEGF for 10 min after pre-incubation for 30 min with or without 10 µM SU1498, a

VEGFR2-selective inhibitor. Equal amounts of cell lysates were fractionated on SDS-PAGE and immunoblotted

with the phospho-specific antibodies for PLC and c-Src; loading was assessed by immunoblotting with antibodies

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against PLC and Src, respectively. A representative blot from 3 independent experiments for each kinase is

shown.

Panel F. c-Src phosphorylation is PLC -dependent. Cells were pre-treated with or without U73122 (5 µM), a

PLC inhibitor, prior to incubation for 10 min with or without VEGF 20 ng/ml. Western blot analysis was

performed with the cell lysates using phospho-Src antibody. The lower panel represents immunoblot analyses of

the same samples with Src antibody to assess loading. A representative blot from 3 independent experiments is

shown.

Fig 3.

VEGF-stimulated Erk activation is PLC -dependent in MCT cells

Panel A. Cell lysates prepared from cells, treated with or without VEGF (20 ng/ml) after pre-treatment with or

without U73122 (5 µM), a PLC inhibitor, were run on SDS-PAGE, transferred and immunoblotted with phospho-

Erk antibody. Loading was assessed by stripping the blot and re-probing with an Erk antibody. A representative

blot from 4 independent experiments is shown. The graph shows quantification of results from 4 different

experiments. *p<0.01 VEGF Vs control, **p<0.01 VEGF Vs VEGF+U73122 by ANOVA.

Panel B. MCT cells were transfected with 1 µg of PLCz (a dominant negative construct of PLC ) per well or an

empty vector without the construct as control and allowed to grow for 48 hours. Cells were treated with or without

VEGF (20 ng/ml) for 10 min and Erk phosphorylation was assessed as described in panel A. A representative blot

from 3 experiments is shown. Densitometric analysis of the bands is presented in a graph. *p<0.01 VEGF Vs

control, **p<0.01 VEGF Vs VEGF+PLCz by ANOVA.

Fig 4.

VEGF-induced Erk phosphorylation is dependent on c-Src

Panel A. Cells were treated with or without PP1, a Src inhibitor, for 30 min prior to incubation with or without

VEGF (20 ng/ml). Erk phosphorylation was examined as described in panel A. A representative blot from 4

experiments is shown. The graph shows quantification of composite data from 3 experiments. *p<0.01 VEGF Vs

control, **p<0.01 VEGF Vs VEGF+PP1 by ANOVA.

Panel B. Cells were transfected with a plasmid carrying a c-Src dominant negative (DN-Src) construct or an

empty vector before incubation with or without VEGF for 10 min. Cell lysates were fractionated on SDS-PAGE

and Erk phosphorylation was assessed as described in panel A. A representative blot from 3 experiments is

shown. The graph shows quantification of composite data from 3 experiments. *p<0.01 VEGF Vs control,

**p<0.01 VEGF Vs VEGF+DN Src by ANOVA.

Panel C. VEGF stimulates Raf-1 phosphorylation that is c-Src-dependent. MCT cells were incubated with

or without VEGF for 10 min. Western blot analysis was done with phospho-specific antibodies for Tyr340/341

of Raf. The blots were stripped and re-probed with Raf-1 antibody (bottom panel) to assess loading. A

representative blot from three experiments is shown.

Panel D. MCT cells were pre-treated with or without PP2 analog, a Src inhibitor, prior to incubation with or

without VEGF (20ng/ml) for 10 min. Cell lysates were fractionated by SDS-PAGE gel and immunoblotted with

phospho-specific Raf antibody. Blots were stripped and re-probed with Raf-1 antibody to assess loading (bottom

panel). A representative blot from 3 experiments is shown.

Fig 5.

VEGF induces tyrosine phosphorylation of Pyk2, and, Ca++

-dependence of VEGF activation of kinases.

Panels A. MCT cells were incubated with or without VEGF for 10 min. Immunoblot analysis was done with anti-

phospho-specific antibodies for Pyk2. The blots were stripped and re-probed with Pyk2 antibody (bottom panel) to

assess loading. A representative blot from three experiments is shown.

Panels B, C, and D. Serum-starved MCT cells were pre-treated with BAPTA/AM (100 µM) for 30 min followed

by incubation with or without VEGF (20 ng/ml). Equal amounts of cell lysates were fractionated by SDS PAGE

and immunoblotted with phospho-specific antibodies for Pyk2 (B), c-Src (C) and Erk (D). The lower panel in each

section shows immunoblotting with antibodies against Pyk2, c-Src and Erk, respectively, done to assess loading.

Representative blots from three experiments are shown for each kinase. The graph shows quantification of

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composite data from 3 experiments on BAPTA/AM abolition of Erk activation by VEGF in panel D. *p<0.01

VEGF Vs Control, **p<0.01 VEGF Vs VEGF+BAPTA/AM by ANOVA.

Fig 6.

VEGF induces phosphorylation of (Mnk1) and promotes its shift from cytoplasm into nucleus.

Panel A. Serum-deprived MCT cells were stimulated with VEGF 20 ng/ml. The lysates were fractionated by SDS

PAGE and immunoblotted with phospho-specific antibodies for Mnk1. Probing with Mnk1 antibody (lower panel)

was performed to assess loading. A representative blot from 3 experiments is shown.

Panel B. Quiescent MCT cells were immunostained with anti-phospho Mnk1 antibody with or without incubation

with VEGF (20ng/ml), for various time durations. Immunofluorescent staining was performed as described in

Methods and the images were analyzed by confocal microscopy. Control cells showed little staining for

phosphorylated Mnk1. After incubation with VEGF for 2 min, intense staining for phospho Mnk1 in the

cytoplasm is seen followed by peri-nuclear staining at 5 min. The nuclear staining is maximal at 15 min and gets

dispersed by about 30 min. The graph represents percent of cells that showed nuclear localization of phospho-

Mnk1 at individual time points following VEGF treatment. Cells at time zero served as control. Equal numbers of

cells were plated in 8-chambered slides; after treatment with VEGF, cells were viewed at uniform magnification

of 40X and counted for phospho-Mnk1 nuclear localization. Composite data from 3 experiments are shown in a

graph (p<0.01 time zero Vs 5 min and 15 min, by ANOVA).

Fig 7.

Panels A, B, and C. VEGF-induced Mnk1 phosphorylation is dependent on PLC , c-Src and Erk. Cells

were serum-starved overnight and subjected to pre-treatment with or without U73122 (5 µM), a PLC inhibitor (A),

PP2 (10 µM), a Src inhibitor (B), and U0126 (25 µM), a MEK inhibitor (C), prior to incubation with or without

VEGF 20 ng/ml for 10 min. Equal amounts of cell lysates were run on a SDS-PAGE and immunoblotted with

phospho-specific antibody for Mnk1. The lower panels of A, B and C show the blots probed with antibody against

Mnk1 to assess loading. Representative blots from 3 experiments with each kinase inhibitor are shown.

Fig 8.

VEGF stimulates eIF4E phosphorylation that is dependent on PLC , c-Src and Erk.

Panel A. Serum-deprived MCT cells were stimulated with VEGF 20 ng/ml. The lysates were fractionated by SDS

PAGE and immunoblotted with a phospho-specific antibody for eIF4E. Loading was assessed by probing with

eIF4E antibody (lower panel). A representative blot from 3 experiments is shown for each kinase.

Panels B, C, and D. Quiescent cells were subjected to pre-treatment with or without U73122 (5 µM), a PLC

inhibitor (B), PP2 analog (10 µM), a Src inhibitor (C) and U0126 (25 µM), a MEK inhibitor (D), prior to

incubation with or without VEGF 20 ng/ml for 10 min. Equal amounts of cell lysates were run on a SDS-PAGE

and immunoblotted with phospho-specific antibody for eIF4E. The lower panels of B, C and D show the blots

probed with eIF4E antibody to assess loading. Representative blots from 3 experiments with each kinase inhibitor

are shown.

Fig. 9.

Mnk1 mediates eIF4E phosphorylation and is needed for VEGF-induced protein synthesis

Panel A. MCT cells were transfected with a plasmid carrying DN-Mnk1 (pEBG-T2/A2) or empty vector and

allowed to grow for 48 hours. Phosphorylation of eIF4E was assessed by immunoblotting as described in Fig. 8A.

A representative blot from 3 experiments is shown.

Panel B. MCT cells were transfected with dominant negative Mnk1 or empty vector as described in panel A, and

treated with or without VEGF prior to addition of [35

S]-methionine and incubated for a total of 2 hours. Equal

amount of protein from the lysates were taken for the estimation of [35

S]-label incorporation into TCA-precipitable

protein and expressed as percentage of control (Mean±SE). Data from 3 experiments are shown in a graph;

*p<0.01 for VEGF Vs vector-transfected control, **p<0.01 for VEGF Vs VEGF-treated DN-Mnk1 expressing

cells, by ANOVA.

Fig. 10.

VEGF-stimulated protein synthesis is dependent on phosphorylation of PLC .

Panel A. Quiescent cells were stimulated with VEGF simultaneously in the presence of [35

S]-methionine for 2

hours with or without pre-incubation with 5 µM U73122, a PLC inhibitor, for 30 min. Incorporation of radiolabel

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into TCA-precipitable protein was taken as a measure of protein synthesis and expressed as percentage of control

(Mean ± SE). Composite data from 3 experiments are shown in a graph; *p<0.01 VEGF Vs control, **p<0.01 for

VEGF+U73122 Vs VEGF, by ANOVA.

Panel B. MCT cells transfected with empty plasmid vector or with PLCz, a plasmid carrying a dominant negative

construct of PLC , were incubated with VEGF in the presence of [35

S]-methionine for 2 hours. Protein synthesis

was measured as in Panel A. The values are expressed as percentage of control from an average of 3 individual

experiments; *p<0.01 VEGF Vs control, **p<0.01 for VEGF+U73122 Vs VEGF, by ANOVA.

Fig 11.

Erk-1/-2 MAP kinase mediated signaling mechanism involved in VEGF-induced protein synthesis.

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Figure 1

0 2 5 10 15 30 60050100150200250300350

Time in minutes

P E

rk/T

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rk

(% o

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VEGF(20 ng/ml)

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U0126

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– – – – + +– – + + – –LY (25 µM)

U0126 (25 µM)

MBP

Erk

C

0

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Figure 2

A B

Phospho-PLCγ

VEGF(20 ng/ml)Time in minutes

PLCγ

0 5 10 15 30 60

VEGF (20 ng/ml) U73122 (5 µM)

Phospho-Src

Src

_ + _ +_ _ ++

F

_ + _ +_ _ ++

E

Phospho-Src

Src

VEGF(20 ng/ml)

SU1498 (10 µM)

D

Phospho-PLCγ

PLCγ

VEGF(20 ng/ml)_ + _ +_ _ ++SU1498 (10 µM)

220kD

97kD

66kD

46kD

VEGF(20 ng/ml) _ + _ +_ _ ++SU1498 (10 µM)

Actin

C

Phospho-Src

Src

0 5 10 15 30 60VEGF(20 ng/ml)Time in minutes

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Figure 3

Phospho-Erk

VEGF (20 ng/ml)

Erk

U73122 (10 µM)

_ + _ +_ _ ++

A

0

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VEGF (20 ng/ml)

Raf-1

C

Figure 4

D

_ + _ +_ _ ++

Phospho-Raf

VEGF (20 ng/ml)

Raf-1

PP2(10 µM)

A

VEGF(20 ng/ml)

Phospho-Erk

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0

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Figure 5

B

Phospho-Pyk2

Pyk2

VEGF (20 ng/ml)

BAPTA/AM (100 µM)

_ + _ +_ _ ++A

VEGF (20 ng/ml)

Phospho-Pyk2

Pyk2

0 5 10 15 30 60

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Phospho-Src

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AFigure 6

Phospho-Mnk

VEGF (20 ng/ml)Time in minutes 0 5 10 15 30 60

Total Mnk

2 min 5 min

15 min 30 min

0 minB

0 2 5 15 30 0

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A

Phospho-Mnk

U73122 (5 µM) VEGF(20 ng/ml) _ + _ +

_ _ ++

C

Phospho-Mnk

VEGF(20 ng/ml) U0126 (25 µM)

_ + _ +_ _ ++

Figure 7

B

Phospho-Mnk

PP2 (10 µM)VEGF(20 ng/ml) _ + _ +

_ _ ++

Total Mnk

Total Mnk

Total Mnk

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Figure 8

B VEGF (20 ng/ml)

Phospho-eIF4E

eIF4E

U73122 (5 µM)

_ + _ +_ _ ++

D VEGF(20 ng/ml)

Phospho-eIF4E

eIF4E

U0126 (25 µM)

_ + _ +_ _ ++

C VEGF (20 ng/ml)

eIF4E

Phospho-eIF4E

PP2 (10 µM)

_ + _ +_ _ ++

Phospho-eIF4E

eIF4E

VEGF (20 ng/ml)Time in minutes 0 5 10 15 30 60

A

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Figure 9

VEGF (20 ng/ml)

DN-MnkVector

_ _ + ++ +

_ _++_ _

Phospho-eIF4E

eIF4E

A

B

VEGF (20 ng/ml)

DN-MnkVector

_ _ + ++ +

_ _++_ _0

20

40

60

80

100

120

140

160

180

[35S

] -M

et.

Inco

rp. %

of

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Figure 10

A

0

20

40

60

80

100

120

140

160

_ VEGF (20 ng/ml)

U73122 (5 µM)+ _ +

_ _ + +

[35S

] -M

et.

Inco

rp. %

of

con

tro

l

B

0

50

100

150

200

250

300

350

400

VEGF (20 ng/ml)

PLCzVector

+_+

_ _ + +

_

+ + _ _

[35S

] -M

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Inco

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Figure 11

Pyk2

c-Src PP2Src-DN

Raf MEK

Erk-1/-2

Mnk-1/-2

eIF4E

Protein synthesis

U0126

VEGF + VEGF RECEPTOR 2

PLCγ

SU1498

U73122 PLCz

BAPTA/AM

Mnk-DN

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PP2(10 µM) - - +VEGF (20ng/ml) - + +

Phospho-PLCγ

PLCγ

Supplementary figure 1. (S1)

Cells were incubated with or without VEGF for 10 min after pre-incubation with PP2 for 30 min. Equal amounts of cell lysates were

fractionated on SDS PAGE and immunoblotted with phospho-specific antibody against PLCγ. Loading was assessed by immunoblotting

with an antibody against PLCγ. PP2 did not inhibit VEGF-induced PLCγ phosphorylation.

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Goutam Ghosh Choudhury and Balakuntalam S. KasinathMeenalakshmi Malini Mariappan, Duraisamy Senthil, Kavithalakshmi S. Natarajan,

renal epithelial cells-Erk axis in VEGF-induced eIF4E phosphorylation and protein synthesis inγPLC

published online May 26, 2005J. Biol. Chem. 

  10.1074/jbc.M504861200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2005/06/14/M504861200.DC1

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