Association between v-Src and Protein Kinase C delta  in v-Src-transformed Fibroblasts*

(Received for publication, December 2, 1996, and in revised form, February 21, 1997)

Qun Zang Dagger , Zhimin Lu Dagger , Marcello Curto Dagger §, Nancy Barile Dagger , David Shalloway and David A. Foster Dagger par

From the Dagger  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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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 delta  (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 delta  co-immunoprecipitates with v-Src and is phosphorylated on tyrosine. The tyrosine-phosphorylated PKC delta  had reduced enzymatic activity relative to the non-tyrosine-phosphorylated PKC delta  from v-Src-transformed cells. The association between Src and PKC delta  was dependent upon both an active Src kinase and membrane association. The association between c-Src Y527F and PKC delta  was substantially enhanced by mutating a PKC phosphorylation site at Ser-12 in Src to Ala indicating that PKC delta  phosphorylation of Src at Ser-12 destabilizes the interaction, possibly in a negative feedback loop. These data demonstrate that upon recruitment of PKC delta  to the membrane in v-Src-transformed cells there is the formation of a Src·PKC delta  complex in which PKC delta  becomes phosphorylated on tyrosine and down-regulated.


INTRODUCTION

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 alpha , delta , epsilon , and zeta  PKC isoforms are predominant in fibroblasts (3, 4). The conventional alpha  PKC isoform requires both Ca2+ and diacylglycerol (DG). The novel delta  and epsilon  isoforms require DG but not Ca2+, and the atypical zeta  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 alpha  and delta  but not the epsilon  or zeta  PKC isoforms (4). Since the delta  and epsilon  PKC isoforms both belong to the Ca2+-independent class of PKC, the preferential increase in membrane association of the delta  over the epsilon  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 delta  over the epsilon  isoform of PKC in v-Src-transformed cells was also surprising because of previous reports that overexpression of PKC delta  inhibits cell proliferation and that overexpression of PKC epsilon  enhances cell growth (8, 9). These observations suggested the possibility that PKC delta  might have a different effect in v-Src-transformed cells than in the non-transformed parental cells. Alternatively, membrane association of PKC delta  in v-Src-transformed cells may not correlate with an activation of its kinase activity since it has been demonstrated that PKC isoforms alpha  and epsilon  can affect phospholipase D (10, 11) and phosphatidate phosphohydrolase (12) activity independent of the kinase activity of the alpha  and epsilon  isoforms respectively.

Tyrosine phosphorylation of PKC delta  in response to several different stimuli has recently been reported (13-16). The biological significance of the tyrosine phosphorylation of PKC delta  is unclear. It has been reported that tyrosine-phosphorylated PKC delta  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 delta  (16). In contrast, PKC delta  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 delta  becomes tyrosine-phosphorylated, and phosphorylation apparently alters its substrate specificity (15). Thus, the effect of tyrosine phosphorylation on PKC delta  activity is apparently complex and may involve other cellular factors.

The tyrosine kinase(s) responsible for PKC delta  phosphorylation are not known. In vitro studies have shown that PKC delta  can be phosphorylated by Src family and receptor tyrosine kinases (14, 17). In this report, we describe a functional interaction between Src and PKC delta  in cells transformed by v-Src.


EXPERIMENTAL PROCEDURES

Cells and Cell Culture Conditions

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 Vectors

3Y1 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-Delta 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).

Antibodies

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 delta , 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.

Cell Lysate Preparation and Subcellular Fractionation

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.

Immunoprecipitation

Cell 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 Analysis

Samples 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 Activity

PKC activity of tyrosine-phosphorylated and non-tyrosine-phosphorylated PKC delta  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 delta . 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 delta . Both the tyrosine-phosphorylated and non-tyrosine-phosphorylated preparations were then immunoprecipitated with anti-PKC delta  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). [gamma -32P]ATP (10 µCi, 3000 Ci/mmol) was present at 100 µM. PKC activity was then determined as described previously (24). The PKC delta levels in the assays was determined by Western blot analysis, and activity was normalized to these levels.


RESULTS

PKC delta  Is Tyrosine-phosphorylated in v-Src-transformed 3Y1 Cells

In v-Src-transformed 3Y1 cells, the delta  isoform of PKC is preferentially associated with the membrane relative to the parental 3Y1 cells (4). It was recently reported that PKC delta  can be phosphorylated on tyrosine (13-16) and that Src family kinases can phosphorylate PKC delta  on tyrosine in vitro (14, 17). We therefore investigated tyrosine phosphorylation of PKC delta  in v-Src-transformed 3Y1 rat fibroblasts, where the expression of v-Src results in increased membrane association of PKC delta . 3Y1 cells and v-Src-transformed 3Y1 cells were lysed and subjected to immunoprecipitation with antibodies against either phosphotyrosine (Tyr(P)) or PKC delta . The immunoprecipitates were then subjected to Western blot analysis using either anti-Tyr(P) or anti-PKC delta  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 delta  antibody, and reciprocally, the 80-kDa protein precipitated by the anti-PKC delta  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 delta by the anti-Tyr(P) antibody, and treatment with phenyl phosphate (a phosphotyrosine analog) abolished precipitation of PKC delta  by anti-Tyr(P) antibody. As expected, the peptide used to generate the PKC delta  antibody abolished the ability of the anti-PKC delta  antibody to precipitate PKC delta .


Fig. 1. PKC delta  is tyrosine-phosphorylated in v-Src-transformed 3Y1 cells. A, cell lysates were generated from either 3Y1 cells or 3Y1 cells transformed by v-Src (3Y1-v-Src). Cell lysates (containing 1.5 mg of total protein) were immunoprecipitated (IP) with a control mouse serum or antibodies raised against Tyr(P) (P-Tyr) or PKC delta , and immune complexes were recovered with protein A-agarose and subjected to Western blot (WB) analysis using antibodies against either Tyr(P) (P-Tyr) or PKC delta  as shown. 3Y1-vSrc - PKC, cells were depleted of PKC by prolonged treatment with TPA (800 nM, 24 h). 3Y1-vSrc + delta  pep, the peptide against which the PKC delta  antibody had been raised was included in the immunoprecipitation to neutralize the anti-PKC delta  antibody. 3Y1-vSrc + pNPP, 30 mM phenylphosphate was included to neutralize the anti-phosphotyrosine antibody. 3Y1 Lysate (containing 20 µg total protein) was loaded prior to immunoprecipitation. B is identical to A except that the lysates were denatured (D) by treatment with 1% SDS and boiled for 10 min prior to immunoprecipitation.
[View Larger Version of this Image (22K GIF file)]

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 delta  is tyrosine-phosphorylated in v-Src-transformed 3Y1 cells. Tyrosine phosphorylation of PKC isoforms alpha  and epsilon  was not detected in similar experiments (data not shown), suggesting that the v-Src-induced tyrosine phosphorylation is specific for the delta  isoform of PKC.

Tyrosine-phosphorylated PKC delta  Associates Preferentially with the Membrane Fraction

We demonstrated previously that there is an increased membrane association of PKC delta  in v-Src-transformed cells (4). Therefore we wished to determine whether the tyrosine-phosphorylated PKC delta  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 delta  antibody and subjected to Western blot analysis using anti-Tyr(P) or anti-PKC delta  antibody. As shown in Fig. 2, when the anti Tyr(P) antibody was used to identify the PKC delta , the majority of PKC delta  (~70%) was associated with the membrane fraction. In contrast, when the PKC delta  antibody was used there was an excess of PKC delta  (~60%) in the cytosolic fraction. As a control, cells were stimulated with TPA for 30 min to shift all of the PKC delta  to the membrane fraction. These data indicate that the tyrosine-phosphorylated PKC delta  is preferentially associated with the membrane.


Fig. 2. Tyrosine-phosphorylated PKC delta  is primarily associated with the membrane fraction. Lysates from membrane (M) and cytosolic (C) cell fractions were prepared as described under "Experimental Procedures" and immunoprecipitated with anti-PKC delta  antibody. The immunoprecipitates were then subjected to Western blot analysis using anti-PKC delta  or anti-Tyr(P) (anti-P-Tyr) antibodies as indicated. Cells were either treated (TPA) or were untreated (Con.) with 100 nM TPA for 30 min prior to preparation of subcellular fractions. The relative amounts of PKC delta  in the different fractions was determined by densitometer scanning of autoradiographs.
[View Larger Version of this Image (44K GIF file)]

Tyrosine-phosphorylated PKC delta  from v-Src-transformed Cells Has Reduced Enzymatic Activity

Tyrosine phosphorylation of PKC delta  has been reported to both enhance (14) and reduce (13, 16) the kinase activity of PKC delta . We therefore compared the kinase activity of tyrosine-phosphorylated and non-tyrosine-phosphorylated PKC delta . Sequential immunoprecipitation with anti-Tyr(P) and anti-PKC delta  antibodies was used to separate tyrosine-phosphorylated and non-tyrosine-phosphorylated PKC delta 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 delta  was reduced to about 33% of the non-tyrosine-phosphorylated PKC delta  for both basal and TPA-induced enzymatic activity. Consistent with tyrosine phosphorylation having an inhibitory effect on the kinase activity of PKC delta , treatment of the tyrosine-phosphorylated PKC delta  with protein-tyrosine phosphatase 1B restored the kinase activity to about 70% of that observed in the non-tyrosine-phosphorylated PKC delta  (Fig. 3). These data suggest that tyrosine phosphorylation of PKC delta  reduces the enzymatic activity of PKC delta  in v-Src-transformed cells.


Fig. 3. Tyrosine-phosphorylated PKC delta  from v-Src-transformed cells has reduced enzymatic activity. Cell lysates were subjected to immunoprecipitation with anti-phosphotyrosine antibody. Tyrosine-phosphorylated proteins were recovered with protein A-agarose and then eluted with phenyl phosphate as described under "Experimental Procedures." PKC delta  was then precipitated with anti-PKC delta  antibody from both the eluted tyrosine-phosphorylated proteins and the supernatant of the anti-phosphotyrosine immunoprecipitation. PKC delta  activity was determined in the presence (+) and absence (-) of TPA (1 µM). Treatment with protein-tyrosine phosphatase 1B (PTP1B) was performed after recovery with anti-PKC delta  antibody and was for 30 min according to the vendor's instructions. PKC delta  was then recovered from the PTP1B assay mixture by centrifugation of the antibody-bound PKC delta . PKC delta  kinase activity was determined by phosphorylation of histone type IIIS as described under "Experimental Procedures." Kinase activity values were normalized to PKC delta  levels in the immunoprecipitates as determined by Western blot analysis. Relative PKC delta  activity represents the PKC delta  activity normalized to the activity present in the non-phosphorylated PKC delta  in the absence of TPA which was given a value of 100%. Error bars represent the range of values for at least two experiments where values varied by less than 10%.
[View Larger Version of this Image (21K GIF file)]

PKC delta  Associates with v-Src

Since v-Src was shown previously to be able to phosphorylate PKC delta  directly in vitro (17), we further explored the possibility that PKC delta  may be a substrate of v-Src in vivo by examining it for an association between PKC delta  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 delta  antibody, PKC delta  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 delta  immunoprecipitates were Western-blotted with anti-v-Src antibody, the PKC delta  antibody co-precipitated v-Src protein. v-Src was not detected in anti-PKC epsilon  immunoprecipitates (Fig. 4). The amount of v-Src in the anti-PKC delta  immunoprecipitates is estimated to be about 1-2% of the total v-Src, and the amount of PKC delta  in the anti-v-Src precipitates is also estimated to be about 1-2% of the total PKC delta .


Fig. 4. PKC delta  associates with v-Src. Lysates from 3Y1 or v-Src-transformed 3Y1 cells (3Y1-vSrc) were immunoprecipitated (IP) with either anti-v-Src, anti-PKC delta , or anti-PKC epsilon  antibody and then subjected to Western blot analysis using the anti-PKC delta  and anti-v-Src antibodies as shown. The 3Y1 and 3Y1-vSrc lysates were subjected to Western blot analysis without prior immunoprecipitation and represented 2% of the lysate used for the immunoprecipitations.
[View Larger Version of this Image (29K GIF file)]

Interaction between PKC delta  and Src Mutants

To further investigate the interaction between Src and PKC delta , we characterized the interaction between PKC delta  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 delta  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 delta  immunoprecipitates from cells overexpressing c-Src. Consistent with this observation, little or no tyrosine phosphorylation of PKC delta  was detected in the c-Src-overexpressing cells (Fig. 5C). In contrast, activated c-Src Y527F was associated with PKC delta , and PKC delta  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 delta  if TPA was added to stimulate membrane association of PKC delta .


Fig. 5. PKC delta  tyrosine phosphorylation and PKC delta  association with Src in 3Y1 cells expressing c-Src and mutants of c-Src. Cell lines expressing wild type or mutant c-Src were established as described under "Experimental Procedures." The Src genes used are shown schematically in A. c-Src Y527F (cSrc527F) has a mutation of Tyr to Phe at position 527 that activates the tyrosine kinase. c-Src Y527F-S12A (cSrc527F-12A), in addition to the change at Tyr-527, has Ser-12 changed to Ala. c-Src Y527F-LN (cSrc527F-LN) has 4 additional amino acids at the amino terminus (MAAA) that prevent myristoylation and membrane association. c-Src Y527F/Delta SH2 (cSrc527F-Delta SH2) has the activating Tyr-527 mutation and a disruption of the SH2 domain in which amino acids 148-187 have been deleted. B, expression levels of the Src proteins in the transfected cell lines was analyzed by Western blot analysis. C, lysates from 3Y1 cells and 3Y1 cells expressing v-Src, c-Src, and the c-Src mutants were immunoprecipitated (IP) with anti-PKC delta  antibody and then subjected to Western blot (WB) analysis using anti-phosphotyrosine and anti-Src antibodies as shown. The phosphotyrosine analysis was also performed upon cells that had been treated with TPA (100 nM) for 30 min prior to lysis of cells.
[View Larger Version of this Image (23K GIF file)]

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 delta  and Src and the level of tyrosine phosphorylation of PKC delta . A mutation to the SH2 domain of c-Src 527 had little or no effect upon either tyrosine phosphorylation of PKCdelta or the association between Src and PKC delta  (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 delta  and did not stimulate tyrosine phosphorylation of PKC delta . These data indicate that the interaction between Src and PKC delta  requires both Src tyrosine kinase activity and membrane localization. Phosphorylation of Src at Ser-12 may lead to the dissociation of a Src·PKC delta  complex, since a mutation at this site increased the Src-PKC delta  interaction.


DISCUSSION

We have demonstrated that in cells transformed by v-Src, PKC delta  is phosphorylated on tyrosine and is associated with v-Src. This interaction requires active, membrane-localized Src kinase. The association between Src and PKCdelta 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 delta  had reduced kinase activity relative to the non-tyrosine-phosphorylated PKC delta . We previously reported that PKC delta  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 delta  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 delta  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 delta  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 delta  tyrosine phosphorylation. These data suggest that tyrosine phosphorylation of PKC delta  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 delta  is a secondary effect of TPA-induced membrane localization.

Overexpression of PKC delta  has previously been reported to inhibit cell growth (8). Our previous observation that PKC delta  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 delta  becomes phosphorylated and has a reduced kinase activity in v-Src-transformed cells is perhaps consistent with the previous reports that PKC delta  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 alpha  PKC isoform, which also becomes membrane-bound in response to v-Src (4). PKC alpha  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 alpha  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 alpha  activation may be causing the PKC delta  recruitment to the membrane. However, since PKC delta  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 delta .

Although PKC delta  becomes membrane-associated in v-Src-transformed cells, there is no change in the subcellular distribution of the epsilon  PKC isoform, which is also a DG-dependent Ca2+-independent PKC isoform (4). The preferential increase in membrane association of PKC delta  over PKC epsilon  observed in v-Src-transformed cells suggests that there may be some functional significance for the observed membrane association of PKC delta  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 delta  and down-regulation of its enzymatic activity indicate a kinase-independent function for PKC delta . Alternatively, Src could be a critical substrate for PKC delta  and that upon phosphorylation of Ser-12 there is a reciprocal tyrosine phosphorylation that serves as a negative feedback control mechanism for PKC delta . A mutation to c-Src at Ser-12 was previously shown to be required for the enhanced responsiveness to beta -adrenergic agonists in cells overexpressing c-Src (29). Thus, the interaction between Src and PKC delta  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 delta  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 delta  is a normal cellular target of c-Src, the data do show that PKC delta  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.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA46677 (to D. A. F.) and CA32317 (to D. S.), Council for Tobacco Research Grant 3075 (to D. A. F.), American Cancer Society Grant BE-243 (to D. A. F.), and Research Centers in Minority Institutions Award RR-03037 from the Division of Research Resources, National Institutes of Health to Hunter College.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Recipient of a fellowship from the Associazione Italiana Ricera Sul Cancro.
par    To whom correspondence should be addressed: Dept. of Biological Sciences, Hunter College of the City University of New York, 695 Park Ave., New York, NY 10021. Tel.: 212-772-4075; Fax: 212-772-5227; E-mail: foster{at}genectr.hunter.cuny.edu.
1   The abbreviations used are: PKC, protein kinase C; DG, diacylglycerol; Tyr(P), phosphotyrosine; TPA, 12-O-tetradecanoylphorbol-13-acetate; SH2, Src homology 2; PBS, phosphate-buffered saline.

REFERENCES

  1. Nishizuka, Y. (1992) Science 258, 607-614 [Medline] [Order article via Infotrieve]
  2. Nishizuka, Y. (1995) FASEB J. 9, 484-496 [Abstract/Free Full Text]
  3. Borner, C., Guadagno, S. N., Fabbro, D., and Weinstein, I. B. (1992) J. Biol. Chem. 267, 12892-12899 [Abstract/Free Full Text]
  4. Zang, Q., Frankel, P., and Foster, D. A. (1995) Cell Growth Differ. 6, 1367-1373 [Abstract]
  5. Alexandropoulos, K., Qureshi, S. A., and Foster, D. A. (1993) Oncogene 8, 803-807 [Medline] [Order article via Infotrieve]
  6. Qureshi, S. A., Joseph, C. K., Rim, M.-H., Maroney, A., and Foster, D. A. (1991) Oncogene 6, 995-999 [Medline] [Order article via Infotrieve]
  7. Qureshi, S. A., Alexandropoulos, K., Rim, M., Joseph, C. K., Bruder, J. T., Rapp, U. R., and Foster, D. A. (1992) J. Biol. Chem. 267, 17635-17639 [Abstract/Free Full Text]
  8. Mischak, H., Goodnight, J., Kolch, W., Martiny-Baron, G., Schaechtle, C., Kazanietz, M. G., Blumberg, P. M., Pierce, J. H., and Mushinski, J. F. (1993) J. Biol. Chem. 268, 6090-6096 [Abstract/Free Full Text]
  9. Borner, C., Ueffing, M., Jaken, S., Parker, P. J., and Weinstein, I. B. (1995) J. Biol. Chem. 270, 78-86 [Abstract/Free Full Text]
  10. Conricode, K. M., Brewer, K. A., and Exton, J. H. (1992) J. Biol. Chem. 267, 7199-7202 [Abstract/Free Full Text]
  11. Singer, W. D., Brown, H. A., Jiang, X., and Sternweis, P. C. (1996) J. Biol. Chem. 271, 4504-4510 [Abstract/Free Full Text]
  12. Jiang, Y., Lu, Z., Zang, Q., and Foster, D. A. (1996) J. Biol. Chem. 271, 29529-29532 [Abstract/Free Full Text]
  13. Denning, M. F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081 [Abstract/Free Full Text]
  14. Li, W., Mischak, H., Yu, J.-C., Wang, L.-M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994) J. Biol. Chem. 269, 2349-2352 [Abstract/Free Full Text]
  15. Haleem-Smith, H., Chang, E.-Y., Szallasi, Z., Blumberg, P. M., and Rivera, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9112-9116 [Abstract]
  16. Denning, M. F., Dlugosz, A. A., Threadgill, D. W., Magnuson, T., and Yuspa, S. H. (1996) J. Biol. Chem. 271, 5325-5331 [Abstract/Free Full Text]
  17. Gschwendt, M., Kielsbassa, K., Kittstein, F., and Marks, F. (1994) FEBS Lett. 347, 85-89 [CrossRef][Medline] [Order article via Infotrieve]
  18. Kmiecik, T. E., and Shalloway, D. (1987) Cell 49, 65-73 [Medline] [Order article via Infotrieve]
  19. Yaciuk, P., Choi, J. K., and Shalloway, D. (1989) Mol. Cell. Biol. 9, 2453-2463 [Medline] [Order article via Infotrieve]
  20. Bagrodia, S., Taylor, S. J., and Shalloway, D. (1993) Mol. Cell. Biol. 13, 1464-1470 [Abstract]
  21. Seidel-Dugan, C., Meyer, B. E., Thomas, S. M., and Brugge, J. S. (1992) Mol. Cell. Biol. 12, 1835-1845 [Abstract]
  22. Johnson, P. L., Coussens, P. M., Danko, A. V., and Shalloway, D. (1995) Mol. Cell. Biol. 5, 1073-1083
  23. Kriegler, M., Perez, C. F., Hardy, C., and Botchan, M. (1984) Cell 38, 483-491 [Medline] [Order article via Infotrieve]
  24. Joseph, C. K., Qureshi, S. A., Wallace, D. W., and Foster, D. A. (1992) J. Biol. Chem. 267, 1327-1330 [Abstract/Free Full Text]
  25. Gould, K. L., Woodgett, J. R., Cooper, J. A., Buss, J. E., Shalloway, D., and Hunter, T. (1985) Cell 42, 849-857 [Medline] [Order article via Infotrieve]
  26. Song, J., Pfeffer, L. M., and Foster, D. A. (1991) Mol. Cell. Biol. 11, 4903-4908 [Medline] [Order article via Infotrieve]
  27. Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993) Nature 364, 249-252 [CrossRef][Medline] [Order article via Infotrieve]
  28. Qureshi, S. A., Joseph, C. K., Gupta, R., Hendrickson, M., Song, J., Bruder, J. T., Rapp, U. R., and Foster, D. A. (1993) Biochem. Biophys. Res. Commun. 192, 969-975 [CrossRef][Medline] [Order article via Infotrieve]
  29. Moyers, J. S., Bouton, A. H., and Parsons, S. J. (1993) Mol. Cell. Biol. 13, 2391-2400 [Abstract]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.