(Received for publication, September 27, 1994; and in revised form, December 20, 1994)
From the
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
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.
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(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)(
)(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.
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 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
145, 130, 62, 52, 42, and 32 proteins (Fig. 1A, rightpanel). The M
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 10
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.
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 GAP
p190 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 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. (
)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.