©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vascular Endothelial Cell Growth Factor Promotes Tyrosine Phosphorylation of Mediators of Signal Transduction That Contain SH2 Domains
ASSOCIATION WITH ENDOTHELIAL CELL PROLIFERATION (*)

(Received for publication, September 27, 1994; and in revised form, December 20, 1994)

Danqun Guo (1)(§) Qing Jia (1) Ho-Yeong Song (1)(¶) Robert S. Warren (2) David B. Donner (1)(**)

From the  (1)Department of Physiology and Biophysics and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and the (2)Department of Surgery, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Vascular endothelial cell growth factor (VEGF), an endothelial cell-specific mitogen that plays an important role in angiogenesis, promotes the tyrosine phosphorylation of at least 11 proteins in bovine aortic endothelial cells (BAEC). Proteins immunoprecipitated from lysates of control- and VEGF-stimulated BAEC with antisera to phospholipase C- (PLC-) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P. Evaluation of the Western blots with antisera to phosphotyrosine demonstrated that PLC- and two proteins (100 and 85 kDa) that associate with PLC- were phosphorylated in response to VEGF. By using antisera specific to other mediators of signal transduction that contain SH2 domains for immunoprecipitation, it was demonstrated that VEGF promotes phosphorylation of phosphatidylinositol 3-kinase, Ras GTPase activating protein (GAP), and the oncogenic adaptor protein NcK. Proteins of M(r) consistent with the VEGF receptors Flt-1 and Flk-1/KDR were also tyrosine phosphorylated in stimulated cells. Tyrosine-phosphorylated Nck, PLC-, and two GAP-associated proteins, p190 and p62, were in GAP immunoprecipitates of VEGF-stimulated BAEC, and tyrosine-phosphorylated NcK was in phosphatidylinositol 3-kinase immunoprecipitates. These observations suggest that VEGF promotes formation of multimeric aggregates of VEGF receptors with proteins that contain SH2 domains and activate various signaling pathways. VEGF-promoted proliferation of endothelial cells and tyrosine phosphorylation of SH2 domain containing signaling molecules were inhibited by the tyrosine kinase inhibitor genistein.


INTRODUCTION

Angiogenesis, the formation of new blood vessels by sprouting from pre-existing endothelium, is a significant component of a wide variety of biological processes, including embryonic vascular development and differentiation, wound healing, organ regeneration, and pathological processes including tumorigenesis(1, 2) . The proliferation of capillary endothelial cells, migration of capillary tubules, and extracellular matrix degradation are important steps in angiogenesis. A variety of growth factors are associated with this process, including tumor necrosis factor, epidermal growth factor, transforming growth factor, angiogenin, and prostaglandin E(2)(1, 2) . However, these factors are believed to induce angiogenesis indirectly. Other growth factors important to angiogenesis, such as acidic fibroblast growth factor, basic fibroblast growth factor, and plateletderived growth factor, are mitogens for a large number of cell types(1, 2) . Vascular endothelial cell growth factor (VEGF)(^1)(3, 4) , also known as vascular permeability factor because of its ability to induce vascular leakage in guinea pig skin, is unique in being an endothelial cell-specific mitogen(5, 6, 7, 8) . VEGF is produced by normal and transformed cells (9, 10) and plays a significant role in the physiology of normal vasculature and in tumor-induced angiogenesis(11, 12, 13, 14, 15, 16) , which makes it important to understand the mechanisms through which this mitogen promotes cell proliferation.

The first step in VEGF action is binding to either of two receptor protein tyrosine kinases, Flk-1/KDR or Flt-1(17, 18, 19, 20, 21) . Signaling by such receptors initiates with activation of the intrinsic tyrosine kinase followed by autophosphorylation of tyrosine residues in the cytoplasmic domain of the receptor(22) . Such tyrosine-phosphorylated receptors are recognized by cytoplasmic signaling molecules that connect the activated receptor to transduction cascades and promote cellular responses(23) . The signaling molecules contain a conserved sequence of approximately 100 amino acids called the Src homology region 2 (the SH2 domain)(24) , which directs their interaction with growth factor receptors phosphorylated on tyrosine residues(25) . The specificity of the interaction is defined by the amino acids surrounding the phosphotyrosine and the amino acid sequence of the SH2 domain. However, this model is untested insofar as VEGF is concerned, and the downstream signaling molecules activated by this mitogen have not been identified.

In the present study, VEGF-induced tyrosine phosphorylations in cultured bovine aortic endothelial cells (BAEC) have been characterized. We report that this mitogen promotes the tyrosine phosphorylations of numerous proteins, among which are four proteins that contain SH2 domains: PLC-, GAP, PI-3 kinase, and NcK; inhibition of these phosphorylation reactions is associated with diminished ability of VEGF to promote cell proliferation.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human VEGF was purchased from R & D Systems (Minneapolis, MN). Horseradish peroxidase-conjugated monoclonal antiphosphotyrosine antibody (RC20) and agarose-conjugated monoclonal antiphosphotyrosine antibody (PY20) were from Transduction Laboratories (Lexington, KY). Monoclonal antisera to PLC-, NcK, and GAP and polyclonal antiserum to PI-3 kinase were from Upstate Biotechnology, Inc.

Cell Culture

BAEC (a gift from Dr. Eric Jaffee, Cornell University Medical College, NY) were grown on gelatin-coated tissue culture plates in medium 199 containing 20% fetal bovine serum, 20 mM HEPES, pH 7.4, and 80 units/ml penicillin and streptomycin. For experiments testing the ability of VEGF to promote tyrosine phosphorylations, near confluent cells were incubated in serum-free medium 199 for 16-18 h, washed once with serum-free medium 199, and cultured in this medium in the absence or presence of 1 nM VEGF for 5 min at 37 °C.

Immunoprecipitation and Immunoblotting

For immunoprecipitations with antisera to phosphotyrosine, NcK, and PI-3 kinase, 1 times 10^7 BAEC were lysed by incubation in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.2% Triton X-100, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM sodium orthovanadate for 20 min at 4 °C. For immunoprecipitation using antisera to GAP and PLC-, 2 times 10^7 BAEC were used, and EGTA and EDTA were excluded from the lysis buffer. Cell lysates were clarified by centrifugation (12,000 times g, 15 min, 4 °C). Supernatants were shaken with antisera for 2 h at 4 °C, after which 50 µl of a slurry of protein G-plus/protein A-agarose was added, and incubation was continued for 2 h at 4 °C. The incubate was centrifuged for 5 s, and the agarose was washed with lysis buffer and recentrifuged. This process was repeated three times, after which Laemmli medium was added to the agarose, which was heated at 100 °C for 5 min. Immunoprecipitated proteins were fractionated on 7.5% acrylamide gels and blotted to Immobilon-P (Millipore) during an overnight transfer. The immunoblots were blocked by incubation in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 1% bovine serum albumin for 1 h. The immunoblots were then incubated with TBST containing 1% bovine serum albumin and the primary antibody for 1-2 h at room temperature while being constantly shaken, washed three times with TBST, and then incubated with anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase for 1 h. The enhanced chemiluminescent detection system (ECL) was used for protein detection. To probe immunoblots with a second antiserum, membranes were stripped by incubation in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol for 30 min at 50 °C. The blots were then incubated with antisera and processed as described above.

Evaluation of Antibody Specificity

Proteins immunoprecipitated from lysates of BAEC using antisera to PLC-, GAP, PI-3 kinase, or Nck were fractionated by SDS-PAGE and blotted to Immobilon-P. The Western blots were incubated with antisera to PLC-, GAP, PI-3 kinase, or Nck. The antisera to PLC-, GAP, and PI-3 kinase each recognized a single protein of M(r) corresponding to PLC-, GAP, and PI-3 kinase, respectively. The antisera to Nck recognized two proteins, Nck and PLC-, which was expected as these proteins contain a common antigenic epitope that is probably near their SH3 domains(26) .


RESULTS

To determine whether tyrosine kinase activation is a component of VEGF signaling, phosphoproteins from control- and VEGF-stimulated BAEC were immunoprecipitated with antisera to phosphotyrosine, fractionated by SDS-PAGE, and analyzed by Western blotting using antiphosphotyrosine antibodies. As illustrated in Fig. 1A (leftpanel), incubation of BAEC with 1 nM VEGF for 5 min promoted the tyrosine phosphorylation of proteins of M(r) 200, 120, 80, 70, and 47. By extending the exposure time of film to the Western blot, it was possible to additionally detect VEGF-stimulated phosphorylation of M(r) 145, 130, 62, 52, 42, and 32 proteins (Fig. 1A, rightpanel). The M(r) values of several protein substrates for VEGF-stimulated phosphorylation were consistent with those of signaling molecules implicated in the actions of other angiogenic factors, such as PDGF and EGF. Among these were 145-kDa PLC-, 120-kDa GAP, the 85-kDa subunit of PI-3 kinase, and the 47-kDa oncogene ncK, which were investigated further.


Figure 1: VEGF-promoted tyrosine phosphorylations. A, BAEC were treated with 1 nM VEGF for 5 min at 37 °C. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates using PTyr Ab, fractionated by SDS-PAGE, and transferred to Immobilon-P. Western blots were incubated with pTyr Ab, and the ECL system was used to visualize proteins. B, proteins in lysates from control- and VEGF-treated cells were immunoprecipitated with anti-PLC- Ab and then treated as described under A. C, the membrane from B was stripped and reblotted with anti-PLC- Ab. D, proteins in lysates from control- and VEGF-treated cells were immunoprecipitated with anti-NcK mAb and then treated as described under A. E, the membrane from D was stripped and reblotted with anti-NcK Ab.



To determine whether PLC- is involved in VEGF signaling, serum-starved BAEC were incubated with 1 nM VEGF for 5 min at 37 °C. PLC- in cell lysates was then immunoprecipitated and separated from other proteins by SDS-PAGE. The phosphorylation state of PCL- from control and VEGF-treated cells was evaluated by Western blotting using antiphosphotyrosine antibodies. As illustrated by Fig. 1B, phosphorylation of 145-kDa PLC-, and two proteins (100 and 85 kDa) known to associate with PLC-(27) , was stimulated by VEGF. Also phosphorylated in response to VEGF were a 200-kDa protein, which is probably Flk-1(18, 20, 21) , and NcK, a 47-kDa protein that contains an epitope recognized by some antisera to PLC- (27) . Phosphorylation of NcK in response to treatment of BAEC with VEGF was confirmed using antisera to NcK for immunoprecipitation, after which Western blots of SDS-PAGE-fractionated proteins were probed with antiphosphotyrosine antisera (Fig. 1D). These experiments showed that phosphorylation of Nck, two associated proteins (85 and 75 kDa), and probably Flk-1 was increased by VEGF. The 55-kDa protein detected by Western blotting was the IgG heavy chain of the NcK antiserum. Control experiments (Fig. 1, C and E) validated that equal amounts of PLC- and NcK were present in lysates of control- and VEGF-treated cells, showing that the increased signal detected by phosphotyrosine antibodies on Western blots did result from augmented phosphorylation.

We next investigated whether another SH2 containing protein, GAP is a substrate for VEGF-promoted phosphorylation. Western blots of proteins from anti-GAP immunoprecipitates were probed with antiphosphotyrosine antibodies, revealing that phosphorylation of 120-kDa GAP, a 110-kDa GAP degradation product(28) , and two GAP-associated proteins, p190 (29) and p62(30) , was stimulated by VEGF (Fig. 2A), comparable amounts of GAP being present in lysates of control- and VEGF-treated cells (Fig. 2B). The phosphorylation of 155-, 145-, and 47-kDa proteins in GAP immunoprecipitates was also augmented by VEGF; these may be Flt-1(17) , PLC-, and NcK, respectively. Coimmunoprecipitation of NcK was demonstrated by stripping the Western blot shown in Fig. 2A, which was probed with antisera to NcK (Fig. 2C). In a separate experiment, a Western blot of proteins from anti-GAP immunoprecipitates was probed with PLC- Ab (Fig. 2D), which led to the detection of tyrosine-phosphorylated PLC- in immunoprecipitates of VEGF-treated but not control cells.


Figure 2: Tyrosine phosphorylation of GAP and associated proteins. A, proteins in lysates from control- and VEGF-treated cells were immunoprecipitated with anti-GAP Ab, fractionated by SDS-PAGE, and transferred to Immobilon-P, which was probed with PTyr Ab. B, the membrane from A was stripped and reblotted with anti-GAP Ab. C, the membrane from B was stripped and reblotted with anti-NcK Ab. D, proteins in lysates from control- (lane1) and VEGF-treated cells (lane2) were immunoprecipitated with anti-GAP Ab, fractionated by SDS-PAGE, and blotted to Immobilon-P, which was probed with anti-PLC- Ab. Lane3, proteins (30 µg) in a cell lysate were fractionated by SDS-PAGE and then blotted to Immobilon-P, which was probed with anti-PLC- Ab.



Another possible substrate for VEGF-promoted phosphorylation suggested by immunoprecipitations with antiphosphotyrosine antibodies (Fig. 1A) is PI-3 kinase. To test this, BAEC were stimulated with VEGF, and PI-3 kinase was immunoprecipitated (Fig. 3A) from cell lysates containing equal amounts of the protein (Fig. 3B). After SDS-PAGE, Western blots were probed with anti-phosphotyrosine antibodies to permit evaluation of the effect of VEGF on the phosphorylation of PI-3 kinase. Phosphorylation of the 85- and 110-kDa subunits of PI-3 kinase, and a 190-kDa protein, which may correspond to a complex of the 110- and 85-kDa proteins, was increased by VEGF. The 47-kDa phosphoprotein substrate for VEGF was confirmed as NcK by stripping the Western blot shown in Fig. 3A and then reprobing with NcK antisera (Fig. 3C).


Figure 3: Tyrosine phosphorylation of PI-3 kinase and associated proteins. A, proteins in lysates from control- and VEGF-treated cells were immunoprecipitated using anti-PI-3 kinase Ab, fractionated by SDS-PAGE, and blotted to Immobilon-P, which was probed with PTyr Ab. B, the membrane from A was stripped and reblotted with anti-PI-3 kinase Ab. C, the membrane from B was stripped and reblotted with anti-NcK Ab.



Finally, we determined whether tyrosine phosphorylations promoted by VEGF could be related to the proliferative response elicited by this mitogen from BAEC. As shown in Fig. 4A, the tyrosine kinase inhibitor genistein suppressed the ability of VEGF to promote tyrosine phosphorylations. Fig. 4B shows that the diminished tyrosine phosphorylations were accompanied by an attenuation in the ability of VEGF to promote a proliferative response from BAEC.


Figure 4: Relationship of tyrosine phosphorylations to endothelial cell proliferation. A, serum-starved BAEC were incubated in the absence (lanes1 and 2) or presence (lanes3 and 4) of 2 µg/ml genistein for 16 h at 37 °C. Cells were then incubated in the absence (lanes1 and 3) or presence (lanes2 and 4) of 1 nM VEGF for 5 min at 37 °C and lysed. Proteins in cell lysates were immunoprecipitated with PTyr Ab, fractionated by SDS-PAGE, and blotted to Immobilon-P, which was probed with PTyr Ab. B, BAEC were seeded at a density of 2 times 10^4 cells/35-mm plate. After 24 h, 1.5 ng/ml VEGF (filledsymbols) and 2 µg/ml genistein (unfilledsymbols) were added to the cultures (day 0). Cell number was then assayed using a Coulter counter at the indicated days. Results are expressed as the mean of triplicate determinations, and the experiment was repeated twice with similar results.




DISCUSSION

Activation of growth factor receptors promotes a cascade of intracellular phosphorylations that ultimately produces cellular responses. Tyrosine phosphorylations are most particularly associated with the proliferative response of cells to mitogens, which led us to investigate whether VEGF might promote such reactions. The covalent modification of specific tyrosyl residues in growth factor receptors and in signaling molecules is a form of cellular communication (22, 23, 24, 25) . A conserved domain of about 100 amino acids, the SH2 domain, specifically recognizes and binds to phosphotyrosine, thereby promoting interactions of activated receptors and signaling molecules with one another(23, 24, 25) . The amino acid motif about the phosphotyrosine and within the SH2 domain lends this process of proteinprotein interaction specificity. The present study demonstrated that the endothelial cell-specific mitogen, VEGF, promotes an array of tyrosine phosphorylations in BAEC. We have identified four signaling molecules that contain SH2 domains (PLC-, GAP, NcK, and PI-3 kinase) as among the substrates for VEGF-promoted phosphorylations. By attenuating tyrosine phosphorylations induced by VEGF using the tyrosine kinase inhibitor genistein, it was possible to diminish the growth-stimulatory effect of VEGF on BAEC, thereby establishing a relationship between these processes.

VEGF stimulates endothelial cell growth and angiogenesis, increases vascular permeability, induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration(3, 4, 31) . The role of the phosphorylated signaling molecules, identified in the present study, in cellular responses to VEGF is not known but may be tentatively inferred by analogy to other growth factor receptor systems, particularly that for PDGF. Activated PLC- hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate and diacylglycerol, which stimulate calcium release and activate protein kinase C, respectively(32) . PI-3 kinase is a heterodimer composed of 85-kDa regulatory and 110-kDa catalytic subunits that promotes phosphorylation of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-diphosphate on the D3 position of the inositol ring(33) . GAP is a negative regulator of p21, a downstream target of many receptors with intrinsic tyrosine kinase activity(34) . NcK is an oncogenic protein composed of one SH2 and three SH3 domains that may couple cell surface receptors to downstream effectors that regulate cellular responses induced by receptor activation(27) . When the PI-3 kinase or PLC- binding sites on PDGF receptors are mutated and the mutant receptors are expressed in epithelial cells, PDGF-induced mitogenesis is diminished(35, 36, 37) . The role of GAP in mitogenesis is uncertain as DNA synthesis is normal in cells expressing a PDGF receptor mutant that lacks the GAP binding site (35, 36, 37) . PLC-, PI-3 kinase, and GAP each modulate PDGF-induced chemotaxis (38) and PLC- may additionally play a role in permeabilization of the endothelium by activating protein kinase C, which promotes this process(39) . Thus, VEGF promotes phosphorylation of signaling molecules associated with the production of various second messengers, activation of multiple signal transduction pathways, and induction of diverse types of cellular responses.

Stimulation of BAEC with VEGF promotes formation of multimeric aggregates of signaling molecules. Immunoprecipitation of proteins from VEGF-treated BAEC with anti-PLC- Ab or anti-NcK Ab resulted in identification not only of phosphorylated PLC- and NcK but also two phosphoproteins (100 and 85 kDa) that associate with PLC-(27) . Immunoprecipitates with anti-GAP Ab resulted in identification of phosphorylated GAP, two GAP-associated proteins (p190 and p62)(29, 30) , PLC-, and NcK. p62 shows significant sequence similarity to two types of RNA binding proteins and may play a role in mRNA processing (30) . p190 contains motifs found in all GTPases and a segment that is nearly identical to a transcriptional repressor(29) . This suggests that GAPbulletp190 complexes can transduce signals from p21 to the nucleus, thereby affecting the expression of specific cellular genes.

VEGF binds to either of two receptors, 160-kDa Flt-1 (17) or 180-210-kDa Flk-1(18, 20, 21) , which is the mouse homolog of KDR. Flk-1 mRNA is abundantly expressed in proliferating endothelial cells of vascular sprouts and branching vessels of embryonic and early postnatal brain but is dramatically reduced in adult, nonproliferating brain(21) . Correlation of the temporal and spatial expression pattern of Flk-1 to sites of VEGF action is consistent with a role for this receptor in vasculogenesis and angiogenesis. Expression of Flt-1 in Xenopuslaevis oocytes produced a system in which VEGF induced calcium release(17) . Thus, each VEGF receptor may play a role in promoting cellular responses. In the present study, we detected tyrosine-phosphorylated proteins of M(r) appropriate to both Flt-1 and Flk-1 when anti-PLC- Ab and anti-GAP mAb were used to immunoprecipitate proteins from lysates of VEGF-stimulated cells. Coimmunoprecipitation of GAP with NcK, and PLC- and PI-3 kinase with NcK suggests that groups of SH2 containing signaling molecules simultaneously bind to and are activated by tyrosine-phosphorylated VEGF receptors. Preliminary experiments in which an antibody raised to a GST-Flk-1 fusion protein was used to immunoprecipitate proteins from control- and VEGF-treated cells suggest that PLC-, GAP, NcK, PI-3 kinase, and other proteins associate with the Flk-1 tyrosine kinase. (^2)However, unequivocal identification of the downstream mediators of VEGF action that associate with Flk-1/KDR and Flt-1 and the affinity with which such interactions occur must await further studies. These variables are likely to determine which cellular responses are mediated by each type of VEGF receptor.

After submission of the present study, Waltenberger et al.(40) described experiments with porcine aortic endothelial cells (PAEC), which do not express VEGF receptors, transfected with cDNAs for KDR or Flt-1. In neither PAEC/KDR nor PAEC/Flt-1 was VEGF able to induce tyrosine phosphorylation of PLC-, nor did either receptor appear to bind PI-3 kinase or GAP, although their activation downstream of Flt-1 and KDR was not ruled out. One explanation for the different signaling events detected in the BAEC used in the present study and in the transfected PAEC is that the latter may lack components of signaling pathways necessary for particular transduction events. Alternatively, the transfected receptors may not have appropriately aggregated or coupled with other signaling molecules despite their presence in PAEC.

In summary, the present study demonstrated that tyrosine phosphorylations are a component of the signaling system used by VEGF to promote responses. Four signaling molecules that contain SH2 domains, PLC-, NcK, GAP, and PI-3 kinase, are tyrosine phosphorylated upon stimulation of BAEC with VEGF. In other systems, these putative mediators of VEGF action have been associated with altered permeability of cell monolayers to macromolecules, chemotaxis, and cell growth. Tyrosine phosphorylations promoted by VEGF and the ability of VEGF to promote the proliferation of BAEC were reduced in parallel by genistein relating these processes. These observations constitute a considerable step toward defining postreceptor mechanisms that transduce VEGF receptor binding into cellular responses.


FOOTNOTES

*
This work was supported by a grant from the Indiana affiliate of the American Diabetes Association (to D. B. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a predoctoral fellowship from the Indiana affiliate of the American Heart Association.

Supported by a postdoctoral fellowship from the Indiana affiliate of the American Heart Association.

**
To whom correspondence and reprint requests should be addressed: Dept. of Physiology and Biophysics and The Walther Oncology Center, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202. Tel.: 317-278-2155; Fax: 317-274-3318.

(^1)
The abbreviations used are: VEGF, vascular endothelial cell growth factor; BAEC, bovine aortic endothelial cells; PLC-, phospholipase C-; PI-3 kinase, phosphatidylinositol 3-kinase; GAP, GTPase activating protein; PTyr Ab, antisera to phosphotyrosine; PAGE, polyacrylamide gel electrophoresis; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; PAEC, porcine aortic endothelial cells.

(^2)
D. Guo and D. B. Donner, unpublished observations.


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