Reproductive Endocrinology Center Department of Obstetrics,
Gynecology and Reproductive Sciences University of California
School of Medicine San Francisco, California 94143 Departments
of Cellular and Molecular Pharmacology and Medicine (T.I.)
University of California San Francisco, California 94143
Technologies Research and Development (W.J.F.) Chiron Corp. Emeryville, California 94608
Laboratoire de
Biologie Moléculaire et de Génie Génétique
(J.M.) Université de Liège B-4000 Sart Tilman,
Belgium
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The actions of both bFGF and VEGF are mediated by receptors of the tyrosine kinase family (12, 13). One of the potential pathways leading from tyrosine kinase receptors to the activation of the mitogen-activated protein kinase (MAPK) involves Shc-Grb2-Sos-Ras-Raf-MEK-MAPK. Binding of agonists results in 1) receptor dimerization, 2) activation of a tyrosine kinase, 3) autophosphorylation of the receptor, and 4) recruitment of adapter molecules via Src homology domains (SH2) such as Shc or Grb2 (14, 15). Grb2, through its SH2 domain, can associate with the tyrosine-phosphorylated Shc (16) or can bind directly to growth factor receptors and mediate, through its SH3 domains, the recruitment of Son of sevenless (Sos) to the membrane (17) and activates Ras. Ras, located at the plasma membrane, associates with and stimulates the serine/threonine kinase Raf-1, which phosphorylates and activates the dual-specificity threonine/tyrosine kinase MEK (MAPK kinase). MEK, in turn, phosphorylates p44 and p42 MAPK, resulting in increased expression of early response genes and stimulation of cell proliferation. In contrast, Ras inactivation is regulated by a Ras-guanosine triphosphatase (GTPase)-activating protein (Ras-GAP), which stimulates the hydrolysis of GTP-bound Ras to a GDP-bound state (18). The balance between the activities of Sos and Ras-GAP regulates the activation state of Ras.
These signaling pathways have been partially confirmed for the action of VEGF. In porcine aortic endothelial cells overexpressing KDR, the human homolog of Flk-1, occupancy of the receptor promoted the association and phosphorylation of Shc and the induction of Shc-Grb2 complex formation (19). In aortic endothelial cells, treatment with VEGF resulted in tyrosine phosphorylation of several proteins including Ras-GAP (20). We previously reported, in BBE cells, that VEGF activated Raf-1 (21) and MAPK (22).
Signaling mechanisms that turn off cell proliferation include an increase in cAMP levels, a process mediated through the activation of cAMP-dependent protein kinase A (PKA) (23, 24). Consistent with previous observations (23, 24, 25), we demonstrated that, in BBE cells, PKA blocked VEGF-induced MAPK pathway at the level of Raf-1 (21). We also showed that 16K PRL inhibited the mitogenic action of both bFGF and VEGF on BBE cells (6, 7). Currently, the signaling pathway for the antiproliferative action of 16K hPRL has been partially described (22). A high-affinity membrane receptor that was neither the PRL, bFGF, nor VEGF receptor was identified by ligand binding studies (26). In addition, we previously demonstrated that 16K hPRL inhibited VEGF-induced phosphorylation and activation of MAPK by acting downstream to the autophosphorylation of Flk-1 (22).
In the highly differentiated BBE cells, we now show that 16K hPRL blocks VEGF-induced Raf-1 activity. The blockade of Raf-1 by 16K hPRL was not dependent on the increase of cAMP levels and the subsequent activation of PKA, but rather on the inhibition of the GTP-bound form of Ras. Consistent with the inactivation of Ras, translocation of Raf-1 to the plasma membrane was blocked as shown by cell fractionation and immunofluorescence studies. The negative effect of 16K hPRL on the VEGF-induced activation of Ras did not involve an upstream signaling event including tyrosine phosphorylation of Flk-1 or association of the activated Flk-1 with Shc/Grb2/Ras-GAP complexes. The inhibition of Ras by 16K hPRL was correlated with a further increase in phosphorylation of Flk-1-associated 120-kDa proteins comigrating with Ras-GAP. These findings represent a potential mechanism for the inhibition of the MAPK signaling cascade activated by VEGF.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment of the plasma membrane fractions with VEGF induced a similar
2-fold stimulation, over the control levels, of the phosphorylation of
the substrates for the endogenous Raf-1 and exogenous MEK and GST-ERK
(Fig. 1, A and B). A potent inhibitory
effect of 16K hPRL was observed on the VEGF-induced activation of Raf-1
and the distal kinases in the signaling cascade; indeed, after addition
of 16K hPRL, Raf-1, MEK, and MAPK activities were totally inhibited and
returned to basal levels (Fig. 1
, A and B). VEGF also induced a 7-fold
increase of Raf-1 phosphorylation, which was completely blocked by 16K
hPRL (Fig. 1A
). The finding that inhibition of phosphorylation of Raf-1
is greater than the inhibition of kinase activity suggests that all of
the phosphorylated residue(s) on Raf-1 may not be involved in the Raf-1
kinase activity.
|
|
16K hPRL Prevents VEGF-Induced Raf-1 Translocation to the Plasma
Membrane
It has been recently demonstrated that an essential step in
activation of Raf-1 is its translocation to the plasma membrane (27, 30, 31). To determine whether VEGF and 16K hPRL could modulate the
subcellular localization of Raf-1, normalized protein content in the
cytosolic (S100) and plasma membrane (P100) fractions were separated by
electrophoresis, and the presence of Raf-1 was detected by protein
immunoblotting. Figure 4A clearly shows that in untreated cells, the
majority of Raf-1 was in the cytosolic fraction; only 10% of the
protein was detected in the plasma membrane fraction. In
VEGF-stimulated cells, 50% of Raf-1 was translocated to the plasma
membrane (Fig. 3A
). In contrast, Raf-1
was more abundant in the cytosolic fraction from cells cotreated with
VEGF and 16K hPRL. The addition of 16K hPRL dramatically inhibited
(75%) VEGF-induced Raf-1 translocation compared with unstimulated
cells. In cells treated with 16K hPRL alone, as in control cells, Raf-1
was more abundant in the cytosolic fraction. Similar results were also
obtained in BBE cells stimulated with bFGF in the presence or absence
of 16K hPRL (data not shown).
|
|
16K hPRL Inhibits VEGF-Activated Ras
Several recent reports have strongly suggested that the principal
function of activated Ras in Raf-1 activation is the recruitment of
Raf-1 to the plasma membrane (for review, see Ref. 32). Therefore,
because we observed a blockade of VEGF-induced Raf-1 translocation to
the plasma membrane in the presence of 16K hPRL, we next studied the
effect of VEGF and 16K hPRL on Ras activation in BBE cells (Fig. 4, A and B). In the absence of VEGF, a
small fraction (20%) of Ras was in the GTP-bound state. Treatment with
VEGF stimulated a 3-fold increase in the amount of GTP bound to Ras.
The addition of 16K hPRL reduced the increase in the Ras GTP-bound
state stimulated by VEGF by nearly 80%, while 16K hPRL alone had no
effect (Fig. 4B
). Thus, the inhibitory effect of 16K hPRL on
VEGF-induced conversion of Ras to the activated GTP-bound state
correlates with the blockade of Raf-1 translocation by 16K hPRL.
Effect of 16K hPRL Upstream of Ras Activation
We have previously demonstrated that 16K hPRL had no effect on
VEGF-induced tyrosine phosphorylation of Flk-1 on BBE cells (22). To
investigate the effect of 16K hPRL on proteins that link Flk-1 to the
Ras cascade, BBE cell lysates were immunoprecipitated with an
anti-Flk-1 antibody. The first half of Flk-1-immunoprecipitated
proteins was separated by SDS-PAGE and immunoblotted with an
antiphosphotyrosine antibody. As shown in Fig. 5A, VEGF treatment induced the tyrosine phosphorylation of several
Flk-1-associated proteins including proteins migrating at 200, 160,
120, 66, 52, 46, and 44 kDa. Consistent with our previous observations,
addition of 16K hPRL specifically inhibited the VEGF-induced tyrosine
phosphorylation of the 44 kDa protein without affecting the
phosphorylation level of the 160-kDa protein, identified as MAPK and
phospholipase C-
, respectively (22).
|
The immunoprecipitation with anti-Flk-1 polyclonal antiserum followed
by immunodetection with an anti-Shc monoclonal antiserum revealed that
the three Shc isoforms of 46, 52, and 66 kDa were constitutively
associated with Flk-1 (Fig. 5D). Although the association of the Shc
isoforms with Flk-1 was only slightly increased by VEGF, VEGF
dramatically stimulated the tyrosine phosphorylation of the proteins
(Fig. 5
, A and D). Phosphorylation of the 46- and 52-kDa isoforms of
Shc protein was previously reported in porcine aortic endothelial cells
overexpressing KDR (19). VEGF-induced phosphorylation or association of
Shc isoforms with Flk-1 was not affected by addition of 16K hPRL (Fig. 5
, A and D). 16K hPRL alone had no effect on the association of the
three Shc isoforms with Flk-1 (Fig. 5D). Interestingly, the 46-kDa
Shc isoform appeared to be phosphorylated and associated to Flk-1
even in the absence of ligand (Fig. 5
, A and D).
Stimulation with VEGF resulted in the association of Grb2 with
Flk-1 as shown in Fig. 5E. This association might occur via the binding
of Grb2 to the phosphotyrosines on Flk-1 or those on the 52- and 66-kDa
isoforms of Shc. The binding of Grb2 with Flk-1 was not modified by the
addition of 16K hPRL (Fig. 5E
).
Effect of VEGF and 16K hPRL on Grb2-Sos Complex
In many types of unstimulated cells, the SH3 domains of Grb2 bind
to the proline-rich carboxy-terminal domain of Sos (33, 34). We
therefore asked whether Grb2 and Sos were associated in BBE cells and
whether this association was affected by VEGF and/or 16K hPRL
treatment. In the absence of any treatment, immunoprecipitation of BBE
cell lysates with a Grb2 polyclonal antibody resulted in the
coimmunoprecipitation of Sos as revealed by Western blotting using a
Sos polyclonal antibody (Fig. 6A).
Proteins were equally loaded in all four groups (Fig. 6A
, lower
panel). Five minutes after VEGF stimulation, a 30% decrease in
Grb2-immunoprecipitated Sos protein was observed (Fig. 6A
). The
simultaneous treatment with 16K hPRL prevented a VEGF effect on
Grb2-Sos dissociation. 16K hPRL alone had no effect on Grb2-Sos complex
compared with the control (Fig. 6A
).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We have recently found that in BBE cells, as in other cell types
(23, 24, 25), high levels of cAMP block mitogen-induced Raf-1 activation
(21). We also provided evidence that increased levels of cAMP mimic
the inhibitory effect of 16K hPRL on mitogen-induced BBE cell
proliferation and activation of Raf-1 (21). Treatment with 16K hPRL
increased cAMP levels in a time-dependent manner; however, this action
does not appear to involve a direct activation of AC.
Although 16K hPRL stimulated intracellular levels of cAMP, the assumed
activation of PKA did not appear to play a major role in the
suppression of VEGF- induced Raf-1 activity. This conclusion is
based on the finding that treatment with H89 failed to reverse the
action of 16K hPRL. However, the possibility exists for pleiotropy in
the signaling pathway which, under certain circumstances, permits
activation of PKA to participate in the inhibitory action of 16K
hPRL (Fig. 7).
Because Raf-1 activation depends on its recruitment to the plasma membrane by activated Ras (27, 36), we examined the effect of VEGF and 16K hPRL on Raf-1 subcellular localization. The current data are the first to show that stimulation with VEGF resulted in the translocation of Raf-1 to the plasma membrane in BBE cells. Importantly, VEGF-induced translocation of Raf-1 was inhibited by 75% after 16K hPRL treatment. Thus, the inhibitory effect of 16K hPRL on Raf-1 translocation was consistent with the blockade of Ras activation. Indeed, the stimulation of Ras activity by VEGF was suppressed by 16K hPRL.
Upstream of Ras, activation of tyrosine kinase receptors rapidly leads
to the phosphorylation of Shc adapter proteins (Fig. 7). In BBE cells,
the three isoforms of Shc were constitutively associated with Flk-1.
VEGF-induced activation of Flk-1 resulted in a modest recruitment of
Shc to the receptor and in a major increase in tyrosine phosphorylation
of the 52- and 66-kDa isoforms of Shc. Furthermore, the association of
Grb2 with Flk-1 was totally dependent on VEGF stimulation. This
association might be the result of interactions between the SH2 domain
of Grb2 and the phosphotyrosine residues on Flk-1 and/or on the 52- and
66-kDa Shc isoforms. Similar results were recently reported in porcine
aortic endothelial cells overexpressing KDR (19); however, the
constitutive association of Shc isoforms to Flk-1 appears to be unique
to BBE cells. In addition, our data showed that treatment with 16K hPRL
did not affect the VEGF-dependent phosphorylation and association of
Shc with Flk-1 and the recruitment of Grb2.
In unstimulated cells, the SH3 domains of Grb2 direct the binding to the proline-rich carboxy-terminal region of Sos. Thus, growth factor-induced tyrosine phosphorylation of Shc results in the formation of a Shc/Grb2/Sos complex. Sos is then targeted to the plasma membrane and, through its exchange activity, converts Ras-GDP to active Ras GTP-bound state. Grb2-Sos dissociation occurs through phosphorylation of Sos, as it was reported for other growth factors (34). We demonstrated, as in other cell types (33, 34), that Grb2 and Sos form a complex in BBE cells. VEGF treatment for 5 min caused a partial dissociation (30%) of the complex and a concomitant increase of Sos phosphorylation. The addition of 16K hPRL inhibited the VEGF-induced disruption of the complex and Sos phosphorylation. As for other mitogens, the mechanism underlying this dissociation is still not known. However, it has been reported that MAPK and/or MEK were the phosphorylating kinases involved in this negative feedback mechanism (35, 37, 38). Consistent with this hypothesis, 16K hPRL, which inhibits VEGF-induced activation of MAPK and MEK, could account for the blockade of Grb2/Sos complex dissociation in response to VEGF. Since removal of this feedback mechanism by 16K hPRL does not result in the maintenance of Ras in a GTP-bound active state, it appears that 16K hPRL is inhibiting Ras activation downstream of the Grb2-Sos complex, in close proximity to Ras. However, the possibility exists that these results reflect the effects of both VEGF and 16K hPRL on total Grb2-Sos complex within the cell and not only on that bound to Flk-1. Since we were unable to detect Sos after Flk-1 immunoprecipitation, we hypothesize that only a small fraction of Grb2-Sos complex was associated with Flk-1, as reported for the epidermal growth factor receptor (39). If that is the case, we expect that the effect of 16K hPRL on the Grb2-Sos complex bound to Flk-1 might not be detected.
Ras has a very low intrinsic GTPase activity, and its inactivation is dependent on Ras-GAP. Ras-GAP functions as a negative regulator by accelerating the conversion of active GTP-bound Ras to the inactive GDP-bound state (Ref. 40 ; for review, see Ref. 41). In the amino-terminal portion, Ras-GAP contains two SH2 domains flanking one SH3 domain, and the GTPase catalytic activity is within the carboxy-terminal domain. The SH2 domains of Ras-GAP were shown to interact with activated growth factor receptor kinases, e.g. the epidermal growth factor and the platelet- derived growth factor receptors (42). Previous studies have demonstrated that tyrosine 457 (Tyr 457) in bovine Ras-GAP is the major phosphorylation site (43). In addition to Tyr 457, serine and threonine phosphorylation sites were identified, but their localization remains unclear (43). VEGF treatment of BBE cells resulted in an increase in tyrosine phosphorylation of proteins migrating at 120 kDa. Interestingly, the level of phosphorylation of these proteins, comigrating with Ras-GAP, was further increased by addition of 16K hPRL. One hypothesis, consistent with these observations, is that the inhibition of Ras activation by 16K hPRL is mediated through the hyperphosphorylation of Ras-GAP. However, the effect of tyrosine phosphorylation on Ras-GAP activity is unclear (42).
In conclusion, the modulation of VEGF signaling by 16K hPRL appears to be mediated via the inhibition of Ras activation. This study demonstrates, for the first time, the ability of a regulatory factor to inhibit the activation of Ras. Since Ras is generally acknowledged to be a critical event in mitogen-stimulated cell proliferation (27, 36), the antagonism of VEGF-induced Ras activation by 16K hPRL represents a novel and potentially important mechanism for the regulation of angiogenesis. These studies raise a number of interesting questions. One issue is whether the ability of Sos to activate Ras is prevented by a mechanism other than its dissociation from Flk-1-bound Grb2-Sos complex. Indeed, phosphatidylinositol 4,5-diphosphate has recently been shown to bind and inhibit Sos activity (44). Second, the relationship between the phosphorylation level and the activity of Ras-GAP remains to be clarified. If the phosphorylation of Ras-GAP increases its ability to hydrolyze GTP, this could represent a mechanism for the maintenance of Ras in the inactive state by 16K hPRL. Alternatively, regulation of Ras-GAP activity could occur through formation of a complex with other Ras-GAP-associated proteins such as p62 and p190 (45). Studies are currently being conducted to address these issues.
The initial signaling events in the mediation of the action of 16K hPRL on the MAPK-signaling pathway are still unknown. Although all of the effects on the MAPK cascade are observed only after treatment with VEGF or bFGF (22), direct effects of 16K hPRL have been observed. Treatment of BBE cells with 16K hPRL increases the level of expression of the PAI-1 gene (46) and activates apoptosis, resulting in increased DNA fragmentation (47). We have shown that 16K hPRL activates the caspase cascade, but again the initial signaling events are yet to be discovered. Obviously the signaling events mediating these pleiotropic actions of 16K hPRL will only be understood after the cloning of the receptor.
![]() |
MATERIAL AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
BBE cells were isolated as previously described (50). The cells
were grown and serially passaged in low-glucose DMEM supplemented with
10% calf serum (CS), 2 mM L-glutamine, and
antibiotics (100 U of penicillin/streptomycin per ml and 2.5 mg of
fungizone per ml). Recombinant human bFGF (Promega Corp.,
Madison, WI) was added (1 ng/ml) to the cultures every other day.
Experiments were initiated with confluent cells between passages 5 and
13.
Cell Stimulation and Preparation of Cell Extracts
Confluent BBE cells were dispersed and plated at a density of
5 x 105 cells per 60-mm culture plate (one plate per
condition) in DMEM containing 1 ng/ml bFGF. Twenty four hours after
plating, cells were serum starved in DMEM containing 0.5% CS for
48 h. Cells were left untreated or treated with 1 nM
recombinant human VEGF165 (N. Ferrara, Genentech, Inc., South San Francisco, CA), 1 nM VEGF plus 1
nM 16K hPRL, or 1 nM 16K hPRL for 5 min at 37
C, to reach a maximal stimulation of MAPK activity as we previously
demonstrated (22). For experiments testing the effect of H89, a
specific inhibitor of PKA (29), cells were incubated with different
concentrations of H89 for 60 min before stimulation with the above
factors. Incubations were terminated by aspiration of the medium, two
washes with ice-cold PBS, and addition of 200 µl of lysis buffer as
previously described (22).
Cell Fractionation
After incubation with the relevant agonists, cells were washed
twice with cold PBS, scraped on ice into 500 µl of lysis buffer [10
mM Tris-HCl (pH 7.5), 25 mM NaF, 5
mM MgCl2, 1 mM EGTA, 1
mM dithiothreitol (DTT), 100 µM sodium
vanadate, 10 µg/ml soybean trypsin inhibitor (Sigma Chemical Co., St. Louis, MO), 2 mM leupeptin, and 0.14 U of
aprotinin per ml]. After 15 min on ice to allow swelling, cells were
homogenized with 80 strokes in a Dounce homogenizer. Cell nuclei were
removed by centrifugation at 4 C for 5 min at 3,000 x
g. Supernatants were centrifuged at 4 C for 30 min at
100,000 x g. The supernatants (S100 fractions) were
removed and stored for further analysis. The pellets were washed twice
(centrifugation at 4 C for 15 min at 100,000 x g) in
500 µl of lysis buffer containing 500 mM NaCl, and once
in lysis buffer to remove any MAPK and MEK activity associated with the
pellet (27). The washed pellets were resuspended in 200 µl of lysis
buffer containing 1% NP-40 and incubated on ice for 15 min, and the
NP40-soluble fractions (P100 fractions) were stored for further
analysis.
Assay for Raf-1 Activity
Raf-1 was immunoprecipitated from equal quantities of protein
from cell lysates and S100- and NP40-soluble fractions (P100 fractions)
from control or stimulated BBE cells. Ten microliters of the specific
Raf-1 polyclonal antiserum (Raf-1 (C12), Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to samples for
1216 h at 4 C, followed by protein A-Sepharose beads for the last 60
min. Immune complexes were washed twice in lysis buffer containing 1%
NP-40 and once in kinase buffer [50 mM Tris (pH 7.5), 100
mM NaCl, 10 mM MgCl2, 1
mM DTT, and 100 µM sodium vanadate]. To
assay Raf-1 kinase activity in a linked in vitro assay,
precipitates were resuspended in 10 µl kinase buffer and incubated
for 20 min at 30 C with 0.5 µg of recombinant baculovirus-expressed
catalytically active MEK, 1 µg of GST-ERK1 [S. Macdonald, Onyx
Pharmaceuticals Inc., Richmond, CA; (28, 51)], and 10 µM
ATP. Then 10 µl of kinase buffer containing 50 µM ATP,
320 µg of MBP, and 5 µCi of [-32P]ATP were added
to each sample and incubated for 10 min at 30 C. Both assays were
terminated by the addition of hot 4 x SDS-PAGE sample buffer
followed by boiling for 5 min. Reaction products were resolved by
SDS-PAGE (12% gels). Gels were dried and subjected to autoradiography.
The radioactivity incorporated into MEK, GST-ERK, and MBP was
quantitated by phosphorimaging.
cAMP Studies
For cAMP studies, cells were washed with 3 ml of fresh medium
and then cultured with various concentrations of IBMX
(Calbiochem, La Jolla, CA) for 90 min. Cells were
challenged with 1 nM 16K hPRL for the indicated times. At
the end of the incubation period, the medium was discarded, the cells
were lysed in 0.25 ml ice-cold 0.1 N HCl and immediately
frozen on dry ice. Subsequently, the cells were sonicated (10 sec),
incubated at 4 C for 48 h, and centrifuged (2,000 x
g for 30 min), and the supernatants were analyzed for cAMP
by RIA (52). All samples from an experiment were analyzed in the
same assay. The limit of detection was 10 fmol/ml, and the intraassay
coefficient of variation was less than 3%.
AC Assay
Membranes were prepared by nitrogen cavitation as previously
described (21, 53). Membranes (0.3 mg/ml) were incubated for 15 min at
30 C in a volume of 50 µl containing 50 mM Tris (pH 8), 1
mM EDTA, 2.5 mM MgCl2, 2
mM ß-mercaptoethanol, 1 µg/ml BSA, 10 mM
creatine phosphate (Sigma Chemical Co.), 100 U of creatine
phosphokinase per ml (Sigma Chemical Co.), 1
mM cAMP (Sigma Chemical Co.); 0.4
mM ATP, 0.1 µCi of [3H]cAMP (2540
Ci/mmol; DuPont NEN, Boston, MA), 4 µCi of
[-32P]ATP (3,000 Ci/mmol; DuPont NEN),
and various activators. Reactions were terminated by addition of 1 ml
of 0.5% SDS, and cAMP was isolated as described (54). Under these
conditions, the rate of cAMP synthesis was constant during the time of
incubation.
Subcellular Localization of Raf-1
Confluent BBE cell cultures were dispersed and plated at 125,000
cells per chamber slide precoated with 100 µg/ml
poly-L-lysine in 1 ml of incubation media containing 1
ng/ml bFGF. Twenty four hours after plating, cells were serum starved
in DMEM containing 0.5% CS for 48 h. Cells were left untreated or
stimulated for 5 min with 1 nM VEGF, 1 nM 16K
hPRL, or both factors for 5 min at 37 C. Cells were then fixed for 15
min in ice-cold methanol, washed in PBS, and permeabilized in PBS
containing 0.1% saponin for 20 min. After blocking with 5% donkey
serum in PBS, cells were incubated with anti-Raf-1 polyclonal antiserum
(1:100) for 60 min. After washing, they were incubated with
rhodamine-donkey antirabbit Ig (1:100) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in 5% donkey serum in PBS,
washed, and mounted. Immunofluorescence was performed with a
Zeiss-Axiophot microscope (Carl Zeiss, Thornwood, NY).
Determination of GTP-Bound Ras
Confluent BBE cells were dispersed and plated at a density of
500,000 cells per 60-mm culture plate (one plate per condition) in DMEM
containing 1 ng/ml bFGF. Twenty four hours after plating, cells were
serum starved in DMEM containing 0.5% CS for 36 h and then
exposed for 12 h with 0.8 mCi/ml of
[32P]-orthophosphate (PO42-) in
3 ml of phosphate-free DMEM. Cells were either left untreated or
stimulated with 1 nM VEGF, 1 nM 16K hPRL, or
both factors in duplicate for 5 min, and incubations were terminated by
washing with ice-cold Tris-buffered saline. Cells were lysed in lysis
buffer [0.5% NP-40, 50 mM Tris-HCl (pH 7.5), 20
mM MgCl2, 150 mM NaCl, 1%
aprotinin, 1 mM Pefabloc] and Ras was immunoprecipitated
with anti-Ras monoclonal antibody (Y13259, 1:10 dilution; Santa Cruz Biotechnology, Inc.) precoupled to protein A-Sepharose by
rabbit antirat Ig for 60 min at 4 C (55). Immunoprecipitates were
washed three times in lysis buffer and three times in washing buffer
[50 mM Tris-HCl (pH 7.5), 20 mM
MgCl2, 150 mM NaCl]. Ras-associated
guanylnucleotides were eluted with 20 mM EDTA, 20
mM DTT, 4% SDS, 0.5 mM GDP, 0.5 mM
GTP for 5 min at 68 C. Eluted GDP and GTP were resolved on
polyethyleneimine cellulose plates (J.T. Baker, Inc., Phillipsburg,
NJ) by TLC using 0.75 M
KH2PO4 (pH 3.4) as the solvent. Labeled
nucleotides were visualized by autoradiography, and the radioactivity
in GTP and GDP was determined by phosphorimaging. Results are as
(2/3 x GTP/GDP + 2/3 x GDP) x100.
Immunoprecipitation
Total cell lysates (450 µg protein) from control or stimulated
BBE cells were immunoprecipitated for 12 h at 4 C with 0.5 µg of
antimouse Flk-1 rabbit polyclonal antiserum (gift from Dr. N. Ferrara,
Genentech, Inc.); 4 µg of antihuman Grb2-NT rabbit
polyclonal antiserum (Upstate Biotechnology, Inc., Lake
Placid, NY); or 4 µg of antimouse Sos1 rabbit polyclonal antiserum
(Upstate Biotechnology, Inc.. Immune complexes were
purified with protein A-Sepharose and washed three times in lysis
buffer. Precipitates were subjected to Western blot analysis.
Western Blot Analysis
P100 and S100 fractions (normalized for protein content)
or immunoprecipitates were resolved by SDS-PAGE (6, 7.5, 8, 12%) gels
and transferred to Immobilon-P membranes (Millipore Corp.,
Bedford, MA). Western blots were probed with the following antibodies:
anti-Raf-1 polyclonal antiserum (1:1,000 dilution); anti-Flk-1 rabbit
polyclonal antiserum (1:500 dilution); antihuman Shc mouse monoclonal
antiserum (1:500 dilution, Santa Cruz Biotechnology, Inc.); antihuman Shc rabbit polyclonal antibody (1:1,000
dilution, Upstate Biotechnology, Inc.; anti-Ash/Grb2 mouse
monoclonal antiserum (1:1,000 dilution, Upstate Biotechnology, Inc.; antihuman GAP (Ras-GAP) mouse monoclonal antibody (1:1,000
dilution); antimouse Sos1 rabbit polyclonal antiserum (1:250 dilution);
or antiphosphotyrosine mouse monoclonal antiserum (4G10, 1:1,000
dilution, Upstate Biotechnology, Inc.. Western blots were
incubated with the appropriate antibody and then washed in
Tris-buffered saline containing 0.05% (vol/vol) Tween 20.
Antigen-antibody complexes were detected with horseradish
peroxidase-coupled secondary antibodies and the enhanced
chemiluminescence reagent (DuPont NEN). The blots were
exposed to Reflection NEF films (DuPont NEN). Western
blots were "stripped" for reprobing with additional antibodies by
incubation for 30 min at 22 C in a buffer containing 0.2 M
glycine (pH 2.5) followed by two washes in PBS. Autoradiographs of the
different Western blots were analyzed by densitometry and quantified
using Intelligent Quantifier (Bio Image, Ann Arbor, MI) software.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
This work was supported by Human Frontier Science Grant RG-479/94-M and UC/Chiron STAR Grant.
1 The first two authors contributed equally to this work.
2 Current address: Institut de Pharmacologie Moléculaire et
Cellulaire, UPR 411 Centre Nationale de la Recherche Scientifique,
Sophia-Antipolis, 06050 Valbonne, France.
3 Current address: Fourth Department of Internal Medicine, 3-28-6
Mejirodai, Bunkyo-ku, Tokyo 112-8688, Japan.
Received for publication November 9, 1998. Revision received January 27, 1999. Accepted for publication February 19, 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|