(Received for publication, December 2, 1996, and in revised form, February 21, 1997)
From the Department of Biological Sciences, Hunter
College of the City University of New York, New York, New York 10021 and the ¶ Section of Biochemistry, Molecular and Cell Biology,
Cornell University, Ithaca, New York 14853
In response to the kinase activity of v-Src there
is an increase in the membrane association of the novel protein kinase
C (PKC) isoform PKC (Zang, Q., Frankel, P., and Foster, D. A. (1995) Cell Growth Differ. 6, 1367-1373). We report here
that in v-Src-transformed cells PKC
co-immunoprecipitates with
v-Src and is phosphorylated on tyrosine. The tyrosine-phosphorylated PKC
had reduced enzymatic activity relative to the
non-tyrosine-phosphorylated PKC
from v-Src-transformed cells. The
association between Src and PKC
was dependent upon both an active
Src kinase and membrane association. The association between c-Src
Y527F and PKC
was substantially enhanced by mutating a PKC
phosphorylation site at Ser-12 in Src to Ala indicating that PKC
phosphorylation of Src at Ser-12 destabilizes the interaction, possibly
in a negative feedback loop. These data demonstrate that upon
recruitment of PKC
to the membrane in v-Src-transformed cells there
is the formation of a Src·PKC
complex in which PKC
becomes
phosphorylated on tyrosine and down-regulated.
Protein kinase C (PKC)1 has been
implicated in a wide variety of signaling mechanisms (1, 2). There are
several isoforms of PKC that fall into three major categories based on
differential Ca2+ and lipid requirements. The ,
,
, and
PKC isoforms are predominant in fibroblasts (3, 4). The
conventional
PKC isoform requires both Ca2+ and
diacylglycerol (DG). The novel
and
isoforms require DG but not
Ca2+, and the atypical
isoform is insensitive to both
DG and Ca2+. The activation of several transcriptional
promoters by the oncogenic tyrosine kinase v-Src is dependent upon PKC
(5-7). We recently reported that in both murine and rat fibroblasts
transformed by the oncogenic tyrosine kinase v-Src there is an
increased membrane association of the
and
but not the
or
PKC isoforms (4). Since the
and
PKC isoforms both belong to
the Ca2+-independent class of PKC, the preferential
increase in membrane association of the
over the
isoform could
not be explained by Ca2+ and suggested that regulation of
this class of PKC isoform involved more than simply elevating DG
levels.
The selective increase in membrane association of the over the
isoform of PKC in v-Src-transformed cells was also surprising because
of previous reports that overexpression of PKC
inhibits cell
proliferation and that overexpression of PKC
enhances cell growth
(8, 9). These observations suggested the possibility that PKC
might
have a different effect in v-Src-transformed cells than in the
non-transformed parental cells. Alternatively, membrane association of
PKC
in v-Src-transformed cells may not correlate with an activation
of its kinase activity since it has been demonstrated that PKC isoforms
and
can affect phospholipase D (10, 11) and phosphatidate
phosphohydrolase (12) activity independent of the kinase activity of
the
and
isoforms respectively.
Tyrosine phosphorylation of PKC in response to several different
stimuli has recently been reported (13-16). The biological significance of the tyrosine phosphorylation of PKC
is unclear. It
has been reported that tyrosine-phosphorylated PKC
has a reduced
kinase activity in Ras-transformed cells (13). Similarly, epidermal
growth factor receptor activation also resulted in a decrease in the
kinase activity of tyrosine-phosphorylated PKC
(16). In contrast,
PKC
that was phosphorylated on tyrosine by either Fyn or the
insulin receptor in vitro had elevated kinase activity (14).
In response to antigen activation of the IgE receptor, PKC
becomes
tyrosine-phosphorylated, and phosphorylation apparently alters its
substrate specificity (15). Thus, the effect of tyrosine
phosphorylation on PKC
activity is apparently complex and may
involve other cellular factors.
The tyrosine kinase(s) responsible for PKC phosphorylation are not
known. In vitro studies have shown that PKC
can be phosphorylated by Src family and receptor tyrosine kinases (14, 17). In
this report, we describe a functional interaction between Src and PKC
in cells transformed by v-Src.
3Y1 and v-Src-transformed 3Y1 rat fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum (Life Technologies, Inc.) as described previously (4). In some cases, 12-O-tetradecanoylphorbol-13-acetate (TPA) was added at 200 nM for 30 min to activate PKC or 800 nM for 24 h to deplete cells of PKC.
Transfections and Plasmid Vectors3Y1 cells were plated at
a density of 105 cells/100-mm dish 18 h prior to
transfection. Transfections were performed using LipofectAMINE reagent
(Life Technologies, Inc.) according to the vendor's instructions. The
plasmid expression vectors contained the G418 resistance marker, and
transfected cultures were selected in 400 ng/ml G418 for 8-10 days at
37 °C. At that time colonies were examined for morphology, picked,
and expanded for additional analysis. The c-Src mutants transfected
into 3Y1 cells are as follows: c-Src Y527F has a mutation of Tyr to Phe
at position 527 (18); c-Src Y527F/S12A has an additional change at
Ser-12 to Ala (19); the LN mutation has 4 additional amino acids at the
amino terminus (MAAA) (20) and was placed in the c-Src Y527F context as
described for the S12A mutation (19); the SH2 deletion of c-Src
Y527F-SH2 has a disruption of the SH2 domain in which amino acids
148-187 have been deleted (21), and this mutation was placed in the
c-Src 527 context as with the LN and S12A mutations (19). All Src
constructs were in the pEVX expression vector (22, 23).
Anti-phosphotyrosine monoclonal antibody (4G10)
(Upstate Biotechnology) was used for Western blots, and monoclonal
anti-phosphotyrosine (PY20) (Transduction Laboratories) was used for
immunoprecipitations. For Src, a monoclonal antibody from Oncogene
Sciences was used for Western blots, and a monoclonal antibody from
Upstate Biotechnology was used for immunoprecipitations. For PKC , a
polyclonal antibody obtained from Life Technologies, Inc. was used for
Western blots and a polyclonal antibody obtained from Calbiochem was
used for immunoprecipitations. Protein-tyrosine phosphatase 1B was
obtained from Upstate Biotechnology.
Cells grew to approximately 85% confluence in 150-mm culture dishes and then were shifted to Dulbecco's modified Eagle's medium containing 0.5% serum for 24 h. Cells were washed three times with ice-cold isotonic buffer (phosphate-buffered saline (PBS), 136 mM NaCl, 2.6 mM KCl, 1.4 mM KH2PO4, 4.2 mM Na2HPO4, pH 7.2). For subcellular fractionation, cells from 150-mm dishes were washed and then scraped into 1 ml of homogenization buffer (20 mM Tris-HCl, pH 7.5, 5 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 2 mM dithiothreitol, 200 µM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cells were then disrupted with 20 strokes in a Dounce homogenizer (type B pestle), and the lysate was centrifuged at 100,000 × g for 1 h. The supernatant was collected as the cytosolic fraction. The membrane pellet was suspended in the same volume of homogenization buffer with 1% Triton X-100. After incubation for 30 min at 4 °C, the suspension was centrifuged at 100,000 × g for 1 h. The supernatant was collected as the membrane fraction. For whole cell lysates, cells were treated with 1 ml of homogenization buffer containing 1% Triton X-100 followed by centrifugation at 100,000 × g for 1 h. The supernatant was collected and used as the whole cell lysate.
ImmunoprecipitationCell lysates or cell fractions prepared as described above were incubated with appropriate antibodies at 4 °C overnight. Antigen-antibody complexes were recovered using protein A-agarose beads (Santa Cruz Biotechnology). For immunoprecipitations with mouse monoclonal antibodies, rabbit anti-mouse IgG was added to the lysates for an additional hour of incubation prior to recovery with protein A. The immunoprecipitates were washed three times with immunoprecipitation wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5% Nonidet P-40, 1% Triton X-100).
Western Blot AnalysisSamples were normalized to contain equal amounts of protein in the total cell lysates or from cytosolic and membrane fractions prior to immunoprecipitation. The immunoprecipitated samples were subjected to SDS-polyacrylamide gel electrophoresis using an 8% acrylamide separating gel followed by transfer to nitrocellulose as described previously (4, 24). After blocking at 4 °C overnight with 5% nonfat dry milk in PBS buffer, nitrocellulose filters were incubated with appropriate primary antibodies. Depending on the origin of the primary antibodies, either anti-mouse or anti-rabbit IgG was used for detection using the ECL system (Amersham Corp.) or the super signal system (Pierce).
Assay of in Vitro PKC ActivityPKC activity of
tyrosine-phosphorylated and non-tyrosine-phosphorylated PKC was
determined according to protocols described by Denning et
al. (13). Cell lysates from v-Src-transformed cells were
immunoprecipitated with anti-phosphotyrosine antibody, and
phosphotyrosine-containing proteins were recovered with protein A-agarose beads. The supernatant was used as the source of
non-tyrosine-phosphorylated PKC
. The anti-phosphotyrosine
immunoprecipitate pellet was resuspended in homogenization buffer
containing 30 mM phenylphosphate to release the
tyrosine-phosphorylated proteins. The antibodies were recovered by
centrifugation, and the supernatant was used as the source of
tyrosine-phosphorylated PKC
. Both the tyrosine-phosphorylated and
non-tyrosine-phosphorylated preparations were then
immunoprecipitated with anti-PKC
antibody. The immunoprecipitates
were washed three times with immunoprecipitation buffer and twice with
20 mM HEPES, pH 7.5, and 10 mM
MgCl2 followed by resuspension in 100 µl of kinase buffer
(20 mM HEPES, pH 7.5, 10 mM MgCl2,
1 mM dithiothreitol, 1 mg/ml histone type IIIS, 60 µg/ml
phosphatidylserine, and TPA at 1 µM if included).
[
-32P]ATP (10 µCi, 3000 Ci/mmol) was present at 100 µM. PKC activity was then determined as described
previously (24). The PKC
levels in the assays was determined by
Western blot analysis, and activity was normalized to these levels.
In v-Src-transformed 3Y1 cells, the isoform of PKC is
preferentially associated with the membrane relative to the parental 3Y1 cells (4). It was recently reported that PKC
can be
phosphorylated on tyrosine (13-16) and that Src family kinases can
phosphorylate PKC
on tyrosine in vitro (14, 17). We
therefore investigated tyrosine phosphorylation of PKC
in
v-Src-transformed 3Y1 rat fibroblasts, where the expression of v-Src
results in increased membrane association of PKC
. 3Y1 cells and
v-Src-transformed 3Y1 cells were lysed and subjected to
immunoprecipitation with antibodies against either phosphotyrosine
(Tyr(P)) or PKC
. The immunoprecipitates were then subjected to
Western blot analysis using either anti-Tyr(P) or anti-PKC
antibody. As shown in Fig. 1A, anti-Tyr(P)
antibody precipitated a protein from v-Src-transformed 3Y1 cells that
could be recognized by the anti-PKC
antibody, and reciprocally, the
80-kDa protein precipitated by the anti-PKC
antibody from the
v-Src-transformed cells was recognized by the anti-Tyr(P) antibody.
These results were observed only in the v-Src-transformed cells. As
expected, PKC depletion by prolonged treatment with phorbol ester
abolished precipitation of PKC
by the anti-Tyr(P) antibody, and
treatment with phenyl phosphate (a phosphotyrosine analog) abolished
precipitation of PKC
by anti-Tyr(P) antibody. As expected, the
peptide used to generate the PKC
antibody abolished the ability of
the anti-PKC
antibody to precipitate PKC
.
To establish that the data shown in Fig. 1A was not due to
contamination with a co-precipitating tyrosine-phosphorylated 80-kDa protein, we repeated the experiments using denatured cell lysates in
which protein-protein interactions were disrupted. As shown in Fig.
1B, the same results as observed in Fig. 1A were
obtained using lysates that were treated with 1% SDS and heated at
100 °C for 10 min prior to immunoprecipitation. We concluded that
PKC is tyrosine-phosphorylated in v-Src-transformed 3Y1 cells.
Tyrosine phosphorylation of PKC isoforms
and
was not detected
in similar experiments (data not shown), suggesting that the
v-Src-induced tyrosine phosphorylation is specific for the
isoform
of PKC.
We demonstrated previously that there is an
increased membrane association of PKC in v-Src-transformed cells
(4). Therefore we wished to determine whether the
tyrosine-phosphorylated PKC
is preferentially membrane-bound.
v-Src-transformed cells were fractionated into membrane and cytosolic
fractions, and lysates from each fraction were immunoprecipitated with
anti-PKC
antibody and subjected to Western blot analysis using
anti-Tyr(P) or anti-PKC
antibody. As shown in Fig.
2, when the anti Tyr(P) antibody was used to identify
the PKC
, the majority of PKC
(~70%) was associated with the
membrane fraction. In contrast, when the PKC
antibody was used
there was an excess of PKC
(~60%) in the cytosolic fraction. As
a control, cells were stimulated with TPA for 30 min to shift all of
the PKC
to the membrane fraction. These data indicate that the
tyrosine-phosphorylated PKC
is preferentially associated with the
membrane.
Tyrosine-phosphorylated PKC
Tyrosine phosphorylation of PKC has been reported to both enhance (14) and reduce (13, 16) the kinase
activity of PKC
. We therefore compared the kinase activity of
tyrosine-phosphorylated and non-tyrosine-phosphorylated PKC
.
Sequential immunoprecipitation with anti-Tyr(P) and anti-PKC
antibodies was used to separate tyrosine-phosphorylated and
non-tyrosine-phosphorylated PKC
isolated from v-Src-transformed
cells as described under "Experimental Procedures." We then
examined the in vitro kinase activity as described
previously (24). As shown in Fig. 3, the kinase activity of the tyrosine-phosphorylated PKC
was reduced to about 33% of
the non-tyrosine-phosphorylated PKC
for both basal and TPA-induced enzymatic activity. Consistent with tyrosine phosphorylation having an
inhibitory effect on the kinase activity of PKC
, treatment of the
tyrosine-phosphorylated PKC
with protein-tyrosine phosphatase 1B
restored the kinase activity to about 70% of that observed in the
non-tyrosine-phosphorylated PKC
(Fig. 3). These data suggest that
tyrosine phosphorylation of PKC
reduces the enzymatic activity of
PKC
in v-Src-transformed cells.
PKC
Since v-Src was shown
previously to be able to phosphorylate PKC directly in
vitro (17), we further explored the possibility that PKC
may
be a substrate of v-Src in vivo by examining it for an
association between PKC
and v-Src. The results of
co-immunoprecipitation experiments are shown in Fig. 4.
When cell lysates were immunoprecipitated with v-Src antibody and then
Western-blotted with anti-PKC
antibody, PKC
was detected in
v-Src immunoprecipitates from v-Src-transformed 3Y1 cells, but not the
parental 3Y1 cells. In the reciprocal experiment, where anti-PKC
immunoprecipitates were Western-blotted with anti-v-Src antibody, the
PKC
antibody co-precipitated v-Src protein. v-Src was not detected
in anti-PKC
immunoprecipitates (Fig. 4). The amount of v-Src in the
anti-PKC
immunoprecipitates is estimated to be about 1-2% of the
total v-Src, and the amount of PKC
in the anti-v-Src precipitates
is also estimated to be about 1-2% of the total PKC
.
Interaction between PKC
To further
investigate the interaction between Src and PKC , we characterized
the interaction between PKC
and Src in cells overexpressing c-Src
and several c-Src mutants (Fig. 5A). Cell lines that overexpress the c-Src genes were established and expression levels of the c-Src proteins were determined by Western blot analysis (Fig. 5B). We first examined the interaction between PKC
and c-Src and an activated mutant of c-Src that has the Tyr at 527 converted to Phe (c-Src Y527F) (18, 20). As shown in Fig. 5C, very little Src protein was present in anti-PKC
immunoprecipitates from cells overexpressing c-Src. Consistent with
this observation, little or no tyrosine phosphorylation of PKC
was
detected in the c-Src-overexpressing cells (Fig. 5C). In
contrast, activated c-Src Y527F was associated with PKC
, and PKC
was tyrosine-phosphorylated, although not quite to the level
observed in cells expressing v-Src. However, c-Src Y527F was as active
as v-Src in inducing tyrosine phosphorylation of PKC
if TPA was
added to stimulate membrane association of PKC
.
We also investigated the effect of a mutation at Ser-12, a
phosphorylation site for PKC (25). As shown, changing Ser-12 of c-Src
527 to Ala (c-Src 527-S12A) substantially enhanced the association
between PKC and Src and the level of tyrosine phosphorylation of
PKC
. A mutation to the SH2 domain of c-Src 527 had little or no
effect upon either tyrosine phosphorylation of PKC
or the association between Src and PKC
(Fig. 5C). Lastly we
examined the effect of an amino-terminal modification of c-Src 527 that prevents membrane association but not kinase activity. This mutant protein (c-Src 527-LN) failed to associate with PKC
and did not
stimulate tyrosine phosphorylation of PKC
. These data indicate that
the interaction between Src and PKC
requires both Src tyrosine kinase activity and membrane localization. Phosphorylation of Src at
Ser-12 may lead to the dissociation of a Src·PKC
complex, since a
mutation at this site increased the Src-PKC
interaction.
We have demonstrated that in cells transformed by v-Src, PKC is phosphorylated on tyrosine and is associated with v-Src. This
interaction requires active, membrane-localized Src kinase. The
association between Src and PKC
was not significantly affected by
SH2 deletion but was greatly enhanced by a mutation to the PKC
phosphorylation site on Src at Ser-12. The tyrosine-phosphorylated PKC
had reduced kinase activity relative to the
non-tyrosine-phosphorylated PKC
. We previously reported that PKC
becomes preferentially associated with the membrane in response to
the kinase activity of v-Src (4). The increase in membrane association
of PKC isoforms has been widely used to demonstrate PKC isoform
activation. The finding here that tyrosine phosphorylation of PKC
inhibits its kinase activity suggests that regulation of novel PKC
isoforms involves more than DG-mediated recruitment to the
membrane.
It was previously reported that PKC could be phosphorylated on
tyrosine in response to phorbol esters that activate PKC (14). However,
in 3Y1 cells and in 3Y1 cells overexpressing wild type c-Src or
activated c-Src that was not membrane-localized (c-Src Y527F-LN), we
did not see an increase in PKC
tyrosine phosphorylation in response
to TPA. On the other hand, in cells expressing activated membrane-bound
c-Src Y527F, we did detect a TPA-induced increase in PKC
tyrosine
phosphorylation. These data suggest that tyrosine phosphorylation of
PKC
in response to TPA is dependent upon an active membrane-bound
tyrosine kinase and is consistent with the hypothesis that TPA-induced
tyrosine phosphorylation of PKC
is a secondary effect of
TPA-induced membrane localization.
Overexpression of PKC has previously been reported to inhibit cell
growth (8). Our previous observation that PKC
became membrane-associated in response to the mitogenic stimuli of v-Src (4)
was surprising since membrane association of PKC isoforms has been
widely used to imply activation. The finding here that PKC
becomes
phosphorylated and has a reduced kinase activity in v-Src-transformed
cells is perhaps consistent with the previous reports that PKC
is
an inhibitor of cell growth. The increased DG levels observed in
response to v-Src (26) may reflect a requirement for activation of the
PKC isoform, which also becomes membrane-bound in response to v-Src
(4). PKC
has been reported to phosphorylate Raf, which contributes
to the activation of Raf (27). Since Raf is required for transformation
by v-Src (28), it is possible that activation of PKC
and
phosphorylation of Raf is required for the mitogenic signals activated
by the tyrosine kinase activity of v-Src. The increased DG needed for
PKC
activation may be causing the PKC
recruitment to the
membrane. However, since PKC
is inhibitory for mitogenic signals,
there may be a mechanism whereby tyrosine phosphorylation, which
correlates well with mitogenic signals, results in down-regulation of
the enzymatic activity of PKC
.
Although PKC becomes membrane-associated in v-Src-transformed
cells, there is no change in the subcellular distribution of the
PKC isoform, which is also a DG-dependent
Ca2+-independent PKC isoform (4). The preferential increase
in membrane association of PKC
over PKC
observed in
v-Src-transformed cells suggests that there may be some functional
significance for the observed membrane association of PKC
in
response to v-Src. Several recent reports have suggested
kinase-independent roles for PKC isoforms (10-12). It is possible that
increased membrane association of PKC
and down-regulation of its
enzymatic activity indicate a kinase-independent function for PKC
.
Alternatively, Src could be a critical substrate for PKC
and that
upon phosphorylation of Ser-12 there is a reciprocal tyrosine
phosphorylation that serves as a negative feedback control mechanism
for PKC
. A mutation to c-Src at Ser-12 was previously shown to be
required for the enhanced responsiveness to
-adrenergic agonists in
cells overexpressing c-Src (29). Thus, the interaction between Src and
PKC
may also be important for regulating other indirect effects of
Src.
The effect of the Ser-12 mutant on both association and tyrosine
phosphorylation further supports the hypothesis that PKC is a
direct substrate of Src. The dependence of the association on an active
kinase suggests that interaction occurs only when Src has been
activated. It is still not clear as to what role(s) c-Src plays in cell
physiology, and while the data presented here with cells overexpressing
activated forms of Src do not prove that PKC
is a normal cellular
target of c-Src, the data do show that PKC
could be regulated by
Src or perhaps a related Src family kinase. Perhaps more importantly,
the data presented here in cells transformed by v-Src demonstrate that
v-Src can associate with and down-regulate a protein kinase that has
been strongly associated with inhibiting cell growth. The ability to
down-regulate this inhibitory PKC isoform may be important for the
transforming ability of v-Src.