©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the Epidermal Growth Factor Receptor Signal Transduction Pathway Stimulates Tyrosine Phosphorylation of Protein Kinase C (*)

(Received for publication, August 30, 1995; and in revised form, October 24, 1995)

Mitchell F. Denning (1)(§) Andrzej A. Dlugosz (1)(¶) David W. Threadgill (**) Terry Magnuson (**) Stuart H. Yuspa (1)(§§)

From the Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892-4255 and the Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106-4955

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The expression of an oncogenic ras gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C and inhibits its enzymatic activity (Denning, M. F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H.(1993) J. Biol. Chem. 268, 26079-26081). Keratinocytes expressing an activated ras gene secrete transforming growth factor alpha (TGFalpha) and have an altered response to differentiation signals involving protein kinase C (PKC). Because the neoplastic phenotype of v-ras expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGFalpha and the G protein activator aluminum fluoride (AlF(4)), we determined if TGFalpha or AlF(4) could induce tyrosine phosphorylation of PKC. Treatment of primary keratinocyte cultures for 4 days with TGFalpha induced tyrosine phosphorylation of PKC, whereas AlF(4) only slightly stimulated PKC tyrosine phosphorylation. The PKC that was tyrosine-phosphorylated in response to TGFalpha had reduced activity compared with the nontyrosine-phosphorylated PKC. Treatment of keratinocytes expressing a normal epidermal growth factor receptor (EGFR) with TGFalpha or epidermal growth factor for 5 min induced PKC tyrosine phosphorylation. This acute epidermal growth factor treatment did not induce tyrosine phosphorylation of PKC in keratinocytes isolated from waved-2 mice that have a defective epidermal growth factor receptor. In addition, the level of PKC tyrosine phosphorylation in v-ras-transduced keratinocytes from EGFR null mice was substantially lower than in v-ras transduced wild type cells, suggesting that activation of the EGFR is important for PKC tyrosine phosphorylation in ras transformation. However, purified EGFR did not phosphorylate recombinant PKC in vitro, whereas members of the Src family (c-Src, c-Fyn) and membrane preparations from keratinocytes did. Furthermore, clearing c-Src or c-Fyn from keratinocyte membrane lysates decreased PKC tyrosine phosphorylation, and c-Src and c-Fyn isolated from keratinocytes treated with TGFalpha had increased kinase activity. Acute or chronic treatment with TGFalpha did not induce significant PKC translocation in contrast to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which induced both translocation and tyrosine phosphorylation of PKC. This suggests that TGFalpha-induced tyrosine phosphorylation of PKC results from the activation of a tyrosine kinase rather than physical association of PKC with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKC activity is inhibited by tyrosine phosphorylation in response to EGFR-mediated signaling and activation of a member of the Src kinase family may be the proximal tyrosine kinase acting on PKC in keratinocytes.


INTRODUCTION

The protein kinase C (PKC) (^1)family of serine/threonine protein kinases is a central component of phospholipase-coupled growth factor receptor signaling pathways(1) . The enzymatic activities of several PKC isoforms are regulated by the allosteric activators diacylglycerol and Ca, which are elevated in response to growth factor receptor activation(2) . The G protein Ras is also a component of the mitogen-activated protein kinase signaling pathways for growth factors such as epidermal growth factor (EGF) and platelet derived growth factor (PDGF). In addition, PKCalpha can directly affect the mitogen-activated protein kinase pathway by phosphorylating and stimulating the autokinase activity of Raf-1(3) . Ras can act either as a regulator or effector of PKC function, but the molecular mechanisms involved are unclear(4, 5, 6, 7, 8) .

In epidermal keratinocytes, Ras can have both positive and negative effects on PKC signaling. Neoplastic mouse keratinocytes expressing an activated ras allele have increases in phosphatidylinositol turnover, diacylglycerol levels, and calcium-dependent PKC activity(9, 10) . Activation of PKCalpha, the only calcium-dependent PKC isoform expressed in keratinocytes, is correlated with the expression of granular layer differentiation markers in normal keratinocytes and in v-ras expressing keratinocytes, where granular layer differentiation markers are up-regulated(10, 11) . Keratinocytes expressing a v-ras oncogene also have decreased Ca-independent PKC activity and have a block in their ability to commit to Ca and TPA-induced terminal differentiation(10, 12, 13, 14) . The block in differentiation response may be due to the tyrosine phosphorylation and inactivation of PKC because the kinase inhibitor staurosporine blocks PKC tyrosine phosphorylation and induces terminal differentiation of neoplastic keratinocytes(5) . In addition, v-ras transformation of keratinocytes up-regulates epidermal growth factor receptor (EGFR) ligand expression and induces the secretion of transforming growth factor alpha (TGFalpha)(15, 16, 17) . Several studies have indicated that increased secretion of TGFalpha can substitute for ras mutations in the altered response to differentiation signals (17) and can initiate skin carcinogenesis(18, 19) .

In this study, we further characterized the signal transduction pathways regulating PKC tyrosine phosphorylation in murine keratinocytes. We demonstrate that TGFalpha is able to induce tyrosine phosphorylation of PKC resulting in inhibition of its activity. Although tyrosine phosphorylation of PKC in intact cells requires a functional EGFR, it is not directly mediated by EGFR and may involve a member of the Src kinase family. Tyrosine phosphorylation of PKC by the EGFR pathway demonstrates a perturbation of PKC signaling by TGFalpha that may contribute to the process of neoplastic transformation in epithelial cells.


EXPERIMENTAL PROCEDURES

Materials

Purified EGFR was from Promega, and Src and Fyn kinases were purchased from Upstate Biotechnology Inc. PKC isozymes were produced in SF9 insect cells using a baculovirus expression system as described previously (20) and were kindly provided by Drs. Marcelo G. Kazanietz and Peter M. Blumberg of the Laboratory of Cellular Carcinogenesis and Tumor Promotion at the National Cancer Institute.

Cell Culture

Primary keratinocytes were isolated from newborn BALB/c, waved-2 mice, and EGFR +/+ or -/- newborn mice(21) . The waved-2 mice were obtained from Jackson Laboratories. The phenotype of the wa-2/wa-2 mice were distinguished from the +/+ and +/wa-2 mice by the curly whiskers observed in the wa-2/wa-2 newborn mice (22) . The keratinocytes were cultured in Eagle's minimal essential medium containing 8% Chelex-treated (Bio-Rad) fetal bovine serum with the final Ca concentration adjusted to 0.05 mM as described(23) . The EGFR genotype of newborn mice was determined by polymerase chain reaction amplification of tail DNA as described previously(21) . Primary keratinocytes from the EGFR +/+ and -/- mice (21) were cultured in 0.05 mM Ca-containing medium with 1 ng/ml keratinocyte growth factor for 2-3 days to stimulate proliferation of the EGFR -/- keratinocytes. After reaching approximately 80% confluence, the cells were cultured without keratinocyte growth factor for 3-4 days before harvesting. For the introduction of a v-ras gene into cells, keratinocytes were exposed to 4 µg/ml polybrene in the absence or the presence of a replication-defective retrovirus containing the v-ras gene and cultured 4-6 days before use(24) .

Immunoblotting and Immunoprecipitations

For the extraction of proteins, the cells were washed twice in cold phosphate-buffered saline, scraped into immunoprecipitation lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM EGTA, 1 mM NaVO(3), 10 µM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), and separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose electrophoretically, blocked with 5% nonfat dry milk, and stained with the indicated primary antibodies as described previously(5, 11) . The immunoblots were incubated with antibodies specific for PKC at a 1:500 dilution (Calbiochem) or phosphotyrosine at 1 µg/ml (clone 4G10, Upstate Biotechnology Inc.). Immunoblotted proteins were detected using the Renaissance Chemiluminescence Reagents (DuPont NEN) with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) at 1:5000 dilution. The immunoblots were quantitated using a Molecular Dynamics Personal Densitometer.

Immunoprecipitations were performed as described previously(5) . Briefly, the cells were washed with phosphate-buffered saline and scraped into immunoprecipitation lysis buffer, and equal amounts of protein were immunoprecipitated with 20 µl of protein A/G PLUS-Agarose (Santa Cruz Biotechnology) and either 2.5 µg anti-phosphotyrosine antibody or 0.5-1 µl of anti-PKC antibody. The c-Src and c-Fyn immunoprecipitations were performed with 2 µg of antibody from Santa Cruz Biotechnology, Inc. (sc-19 and sc-16 respectively) and 50 µl protein A-Sepharose from Sigma. For some phosphotyrosine immunoprecipitations, an agarose-conjugated anti-phosphotyrosine monoclonal antibody from Upstate Biotechnology Inc. was used. For immunoblotting, the immunoprecipitates were washed three times with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) and boiled in 20 µl of SDS sample buffer(25) .

In Vitro Kinase Assays

Tyrosine kinase reactions were performed in a volume of 20-30 µl containing 50 mM Tris, pH 7.5, 10 mM MgCl(2), 2 mM MnCl(2), 100 µM phenylmethylsulfonyl fluoride, 100 µM NaVO(3), and 1 mM NaF. PKC isozymes from baculovirus-infected Sf-9 cells were included as substrates for either Src or Fyn kinase (10 units) or keratinocyte membranes (25 µg of protein). The keratinocyte membranes were prepared from normal primary keratinocytes by scraping the cells into hypotonic lysis buffer (1 mM HEPES, pH 7.5, 5 mM MgCl(2), and 100 µM phenylmethylsulfonyl fluoride) and spinning at approximately 50 times g for 5 min at 4 °C. The supernatant was then spun in a microcentrifuge for 20 min, and the membrane pellet resuspended in assay buffer and used as a source of tyrosine kinase. The reactions were started by adding 25 µM ATP and incubated for 10 min at 30 °C. Tyrosine phosphorylation was assessed by immunoblotting with an anti-phosphotyrosine antibody. For the Src and Fyn immunoprecipitation kinase assays, the immunoprecipitates were washed twice with immunoprecipitation lysis buffer, twice with assay buffer (20 mM HEPES, pH 7.4, 3 mM MnCl(2), and 5 mM MgCl(2)), and incubated in 30 µl of assay buffer containing 10 µM ATP for 10 min at 30 °C. The Sepharose beads were washed once in immunoprecipitation lysis buffer and analyzed by immunoblotting with an anti-phosphotyrosine antibody.

PKC assays of tyrosine-phosphorylated and nontyrosine-phosphorylated PKC were performed as described previously with minor modifications (5) . Primary keratinocytes were cultured in 10 ng/ml TGFalpha for 4 days to induce the tyrosine phosphorylation of a fraction (50%) of PKC. Lysates were prepared (n = 3) and immunoprecipitated with an anti-phosphotyrosine antibody for 2.5 h. The phosphotyrosine-cleared lysates were taken for immunoprecipitation of nontyrosine-phosphorylated PKC. Both phosphorylated and nonphosphorylated samples were immunoprecipitated for PKC overnight in the presence of 30 mMp-nitrophenylphosphate to release phosphotyrosine containing proteins from the anti-phosphotyrosine antibody. The immunoprecipitates were washed once with immunoprecipitation lysis buffer and twice in 50 mM Tris-HCl, pH 7.4, and resuspended in 25 µl of Tris-HCl and 5 mM 2-mercaptoethanol. PKC assays were performed on the immunoprecipitated PKC in the presence or the absence of 1 µM TPA as described by Nakadate et al.(26) except that the PKC substrate peptide [ser] (Life Technologies, Inc.) was the phosphate acceptor, and 20%/80% phosphatidylserine/phosphatidylcholine vesicles were the phospholipid. After the enzyme reaction, the agarose beads were spun, and 30 µl of supernatant was spotted for scintillation counting. The PKC remaining on the beads was boiled in SDS sample buffer and analyzed by anti-PKC immunoblot to correct for differences in the amount of PKC in the assay.


RESULTS

TGFalpha Induces Tyrosine Phosphorylation of PKC in Normal Keratinocytes

Because treatment of primary keratinocyte cultures with TGFalpha and the nonspecific G protein activator AlF(4) elicits many phenotypic changes characteristic of v-ras transformation(9) , we examined whether the tyrosine phosphorylation of PKC was also induced by these pharmacological agents. Culturing primary keratinocytes in the presence of 10 ng/ml TGFalpha for 4 days induced tyrosine phosphorylation of PKC (27-fold), whereas a 4-day AlF(4) (1 µM AlCl(3) and 1 mM NaF) treatment induced tyrosine phosphorylation only slightly (Fig. 1A). The combined treatment of TGFalpha and AlF(4) resulted in a more than additive increase in PKC tyrosine phosphorylation (48-fold).


Figure 1: Tyrosine phosphorylation of PKC in response to TGFalpha. In A, primary mouse keratinocytes were cultured in the presence of 10 ng/ml TGFalpha or 1 µM AlCl(3), 1 mM NaF (AlF(4)) for 4 days, and lysates (700 µg protein) were immunoprecipitated with an anti-phosphotyrosine antibody. The immunoprecipitates were immunoblotted with an anti-PKC antibody. In B, 10 ng/ml of TGFalpha was added to cultures of primary keratinocytes for the indicated times, and lysates (550 µg of protein) were prepared for PKC immunoprecipitation. The immunoprecipitates were immunoblotted against an anti-phosphotyrosine antibody and restained with an anti-PKC antibody. Similar results were obtained in one additional experiment.



Because TGFalpha was a more effective inducer of PKC tyrosine phosphorylation than AlF(4), we further analyzed TGFalpha-induced PKC tyrosine phosphorylation. The kinetics of TGFalpha-induced PKC tyrosine phosphorylation were biphasic (Fig. 1B). Tyrosine phosphorylation of PKC was increased approximately 8-fold after a 5-min TGFalpha treatment (10 ng/ml), returned toward basal levels by 1-2 h, and increased again from 4 and 6 h (9-11-fold), remaining elevated (4-fold) even after 24 h of TGFalpha treatment. The total levels of PKC did not change appreciably during this time. Acute and chronic treatment with EGF also induced PKC tyrosine phosphorylation (see Fig. 3A).


Figure 3: Mutant alleles of the EGFR inhibit PKC tyrosine phosphorylation. A, primary keratinocytes were isolated from mice having a wild type EGFR (BALB/c, +/+ and +/wa-2) and from waved-2 mice homozygous for the mutant EGFR (wa-2/wa-2). The keratinocytes were cultured and infected with v-ras for 5 days (v-ras), treated with 10 ng/ml EGF for 4 days (EGF 4D), or 5 min (EGF 5 M), and PKC was immunoprecipitated from cellular lysates (750 µg of protein). The immunoprecipitates were immunoblotted with an anti-phosphotyrosine antibody (Anti p-Tyr) and anti-PKC antibody (Anti PKC). Similar results were obtained in two additional experiments. B, primary keratinocytes were isolated from mice having a wild type EGFR (+/+) and from EGFR null mice (-/-). The keratinocytes were initially cultured in 1 ng/ml keratinocyte growth factor for 3 days, switched to unsupplemented medium, and either treated with 100 nM TPA for 10 min (TPA) or infected with v-ras for 4 days (ras). Cellular lysates were immunoprecipitated with anti-phosphotyrosine antibody (p-Tyr IP), and gel separated total lysates (Total) were immunoblotted for the detection of total PKC levels with the anti-PKC antibody. A similar result was observed in one additional experiment.



TGFalpha-induced Tyrosine Phosphorylation of PKC Inhibits Its Activity

The tyrosine-phosphorylated form of PKC isolated from ras expressing keratinocytes is not activated by the phorbol ester TPA(5) . To determine the effect of TGFalpha-induced tyrosine phosphorylation on PKC enzymatic activity, we performed assays on tyrosine-phosphorylated and nontyrosine-phosphorylated PKC isolated from keratinocytes treated for 4 days with 10 ng/ml TGFalpha (Fig. 2). Similar to the results for v-ras, the tyrosine-phosphorylated PKC from TGFalpha-treated cells had much lower constitutive activity, and TPA caused only a modest increase in activity. In fact, the specific activity of the tyrosine-phosphorylated PKC assayed in the presence of 1 µM TPA was substantially lower than the basal specific activity of the nontyrosine-phosphorylated PKC.


Figure 2: Enzymatic activity of tyrosine-phosphorylated PKC. Primary keratinocytes were cultured for 4 days in medium containing 10 ng/ml TGFalpha, and the lysates (n = 3) were immunoprecipitated sequentially with an anti-phosphotyrosine antibody followed by an anti-PKC antibody. PKC activity was assayed in the tyrosine-phosphorylated and nonphosphorylated fractions in the presence or the absence of 1 µM TPA. For calculating PKC-specific activities, the amount of PKC in each assay tube was normalized by immunoblotting with an anti-PKC antibody. Similar results were obtained in two additional experiments.



Tyrosine Phosphorylation of PKC Requires a Functional EGF Receptor

To determine the contribution of the EGFR to PKC modification, keratinocytes were isolated from waved-2 mice that have a mutated EGFR with decreased tyrosine kinase activity(22) . Fig. 3A shows that tyrosine phosphorylation of PKC was induced by v-ras transduction and acute (5 min) or chronic (4 day) treatment with EGF in keratinocytes from BALB/c and phenotypically normal waved-2 mice (+/+ and +/wa-2). However, acute EGF treatment (5 min) did not induce tyrosine phosphorylation of PKC in the waved-2 keratinocytes (wa-2/wa-2), whereas chronic treatment or v-ras did. This is consistent with the defective but not inactive EGFR previously documented in the waved-2 strain(22) .

Tyrosine phosphorylation of PKC was also assessed in keratinocytes isolated from mice harboring a disrupted EGFR gene(21) . The immunoblots in Fig. 3B show that PKC was tyrosine-phosphorylated to a similar extent in both EGFR +/+ and -/- keratinocytes (1200 and 1700% increase, respectively) in response to acute TPA treatment. In contrast, v-ras transduction was less effective at inducing PKC tyrosine phosphorylation in the EGFR -/- keratinocytes than in the +/+ cells of the same genetic background (60 and 250% increase, respectively). There was no difference in the amount of total PKC between the EGFR +/+ and -/- keratinocytes. These genetic data strongly support the involvement of the EGFR in the v-ras-induced PKC tyrosine phosphorylation.

In Vitro Tyrosine Phosphorylation of PKC by Src Family Kinases but Not EGFR

The ability of EGF or TGFalpha to rapidly induce the tyrosine phosphorylation of PKC suggests that the EGFR directly phosphorylates PKC. To test this hypothesis, we performed in vitro phosphorylation reactions using EGFR, c-Src, c-Fyn, or a membrane fraction from keratinocytes as the tyrosine kinase and recombinant PKC as the phosphate acceptor (Fig. 4A). As detected by phosphotyrosine immunoblotting, purified EGFR did not tyrosine phosphorylate PKC either in the total reaction mixture or in the PKC immunoprecipitate. The EGFR did autophosphorylate, indicating that it was enzymatically active. The tyrosine-phosphorylated band at the M(r) of the EGFR in the PKC immunoprecipitates has never been detected in PKC immunoprecipitates from cell lysates and may associate with PKC only under these in vitro conditions. As shown in the PKC immunoprecipitate for c-Src and in the total blot for c-Fyn, both kinases were able to tyrosine phosphorylate PKC, consistent with previous work demonstrating the phosphorylation of PKC by c-Src and c-Fyn in vitro(27, 28) . A crude membrane fraction from normal keratinocytes also mediated tyrosine phosphorylation of PKC (see PKC IP in Fig. 4A). Although the tyrosine-phosphorylated form of PKC has been previously localized to the membrane fraction(5, 29) , the tyrosine-phosphorylated PKC in the membrane reaction mixture is not endogenous PKC formed in vivo because the amount of tyrosine-phosphorylated PKC is very low in normal keratinocytes (see Fig. 1, Fig. 3, and Fig. 5).


Figure 4: Tyrosine phosphorylation of PKC by Src family kinases. In A, recombinant PKC from baculovirus-infected Sf-9 insect cells was incubated alone or with purified EGFR, c-Src, c-Fyn, or a membrane fraction from untreated normal keratinocytes in tyrosine kinase assay buffer as indicated. Total reaction mixtures and PKC immunoprecipitated from the reactions were analyzed for phosphotyrosine by immunoblotting with anti-phosphotyrosine antibody. In B, 10 µg of membrane protein from untreated keratinocytes were incubated with the indicated clearing antibody followed by protein A-Sepharose, and the cleared lysate was added to recombinant PKC for the tyrosine kinase assay. The reaction mixtures were immunoprecipitated for phosphotyrosine and stained with an anti-PKC antibody. In C, primary keratinocytes were treated with 10 ng/ml TGFalpha for 5 min (5M) or 5 days (5D) as indicated, and cellular lysates immunoprecipitated with nonspecific rabbit IgG, c-Src, or c-Fyn antibodies. The immunoprecipitates were incubated with tyrosine kinase assay buffer (``Experimental Procedures''), and the phosphorylated proteins were analyzed by immunoblotting with an anti-phosphotyrosine antibody. Kinase refers to the band corresponding to either Src or Fyn kinase. IP, immunoprecipitation.




Figure 5: Subcellular distribution of PKC after TGFalpha or TPA treatment. In A, keratinocytes were treated with 10 ng/ml TGFalpha for either 2 min or 4 days and either fractionated into soluble and particulate fractions or lysates prepared and immunoprecipitated with phosphotyrosine antibody. The fractionated and immunoprecipitated samples were immunoblotted with anti-PKC antibody. In B, keratinocytes were treated with 100 nM TPA for 10 min, and PKC translocation and tyrosine phosphorylation were assessed as described for A. IP, immunoprecipitation.



To determine if either c-Src or c-Fyn was present in the keratinocyte membranes and was phosphorylating PKC, we immunoprecipitated c-Src or c-Fyn from a solubilized membrane fraction and tested if this cleared lysate could phosphorylate PKC on tyrosine. Fig. 4B shows that removal of c-Src or c-Fyn from the membrane lysate significantly decreased the PKC tyrosine kinase activity compared with the nonspecific rabbit IgG cleared lysate. The tyrosine kinase activity of c-Src and c-Fyn immunoprecipitated from keratinocytes treated with TGFalpha for 5 min or 5 days is shown in Fig. 4C. The autophosphorylation and heterophosphorylation of multiple proteins by both c-Src and c-Fyn was increased by acute and chronic TGFalpha treatment. Kinase activity was determined by densitometry of the phosphotyrosine immunoblots. C-Src kinase activity increased 1.42 ± 0.35-fold and 1.66 ± 0.34-fold (mean ± standard deviation, n = 2) after acute and chronic TGFalpha treatment. The effect of TGFalpha on c-Fyn kinase activity was greater, increasing 2.05 ± 0.35-fold and 3.26 ± 0.50-fold after acute and chronic TGFalpha (n = 3). Immunoprecipitation with nonspecific IgG recovered no detectable tyrosine kinase activity (Fig. 4C). In addition, the recovery of tyrosine kinase activity in c-Src and c-Fyn immunoprecipitates was completely blocked by adding an excess of the peptide that the antibodies were raised against (data not shown). PKC was not present in c-Src and c-Fyn immunoprecipitates when assayed by immunoblot (data not shown). These results indicate that keratinocyte c-Src and c-Fyn can phosphorylate PKC and that both of these tyrosine kinases are activated in response to TGFalpha treatment of keratinocytes.

Translocation Is Not Required for Tyrosine Phosphorylation of PKC

Treatment of cells with certain growth factors is able to cause translocation of PKC isozymes, including PKC(29, 30, 31) . Because translocation is associated with PKC tyrosine phosphorylation(29, 31, 32) , we measured the subcellular distribution of PKC following TGFalpha treatment. Fig. 5A shows that there was no significant increase in the proportion of particulate PKC following a 2-min or 4-day exposure to 10 ng/ml TGFalpha. Both of these treatments induced tyrosine phosphorylation of PKC as shown by the phosphotyrosine immunoprecipitation and PKC immunoblot in Fig. 5A. As a positive control for translocation, the phorbol ester TPA was used to translocate PKC (Fig. 5B). TPA induced both PKC translocation and tyrosine phosphorylation as described previously(27, 29, 33) . These results indicate that translocation of PKC is not a prerequisite for TGFalpha-induced PKC tyrosine phosphorylation.

In Vitro Tyrosine Phosphorylation of Other PKC Isoforms

We examined the ability of c-Src and c-Fyn to tyrosine phosphorylate recombinant PKCs alpha, beta1, , , , , and in vitro. Analysis of the total kinase reaction mixtures in Fig. 6A indicated that PKCalpha, beta1, and were not appreciably phosphorylated by either c-Src or c-Fyn but tyrosine-phosphorylated proteins corresponding to the M(r) of PKC, , , and were detected. To verify tyrosine phosphorylation of PKC, , , and , phosphotyrosine-containing proteins were immunoprecipitated from the kinase assays and the immunoprecipitates immunoblotted for the indicated PKC isozyme. Fig. 6B confirms that c-Src and c-Fyn phosphorylate PKC. The phosphotyrosine immunoprecipitations revealed that PKC, , and were phosphorylated by c-Src and to a lesser extent by c-Fyn. These results suggest that PKC, , and may also be tyrosine-phosphorylated under conditions where Src family kinases are activated in cells. However, in three independent experiments, we were unable to detect tyrosine phosphorylation of other PKC isoforms in TGFalpha-treated cells,demonstrating the specificity of this phosphorylation for the isoform of PKC in vivo (data not shown).


Figure 6: In vitro tyrosine phosphorylation of PKC isozymes. In A, recombinant PKC isozymes from Sf-9 cells were incubated with either c-Src or c-Fyn kinase as described under ``Experimental Procedures,'' and the total reaction mixtures were analyzed for phosphotyrosine by immunoblotting. To identify the location of each PKC isozyme, the blot was stripped and restained for individual PKC isoforms. Similar results were obtained in two additional experiments. In B, total tyrosine kinase reaction mixtures of the indicated PKC isozyme either alone or with c-Src or c-Fyn were immunoprecipitated with an anti-phosphotyrosine antibody. The immunoprecipitates were immunoblotted with PKC antibodies specific for PKC, , , or as indicated.




DISCUSSION

In this report, we demonstrate that TGFalpha treatment induces tyrosine phosphorylation of PKC and inhibits its activity. In view of the high level of TGFalpha produced by transformed mouse keratinocytes (15, 16) , these results provide a mechanism for the tyrosine phosphorylation of PKC induced by oncogenic ras(5) . Studies using keratinocytes from mice with mutant alleles of EGFR revealed that the EGFR is required for TGFalpha-induced tyrosine phosphorylation of PKC, but biochemical analysis indicates that the EGFR does not phosphorylate PKC directly. We demonstrate that c-Src and c-Fyn kinases become activated after TGFalpha treatment, and PKC, as well as PKC, , and , are substrates for c-Src and c-Fyn kinases in vitro.

Activation of Src family kinases in response to EGFR-ligand interaction has been reported previously in other cell types(34, 35) . Several possible mechanisms have been proposed for the activation of Src family tyrosine kinase by EGFR. The Src SH2 domain can bind to activated, tyrosine-phosphorylated EGFR(36, 37) , and Src itself has been shown to phosphorylate the EGFR and create a Src SH2 docking site(35) . C-Src can also bind to tyrosine-phosphorylated Neu, which can be transphosphorylated by activated EGFR(36, 38) . Finally, signaling molecules downstream from the EGFR such as p21Gap and Raf-1 associate with Src family members, illustrating that direct association with the EGFR is not necessary for Src activation(39, 40) .

The mutant mouse strain waved-2 harbors a point mutation in the EGFR, which decreases its tyrosine kinase activity >90%(22) , consistent with the lack of PKC tyrosine phosphorylation in waved-2 keratinocytes after acute EGF treatment. In contrast, v-ras keratinocytes from waved-2 mice have tyrosine-phosphorylated PKC, suggesting that EGFR ligands secreted by transformed keratinocytes are responsible for the PKC tyrosine phosphorylation, just as chronic (4 days) treatment with EGF is capable of inducing PKC phosphorylation in waved-2 keratinocytes.

The role of EGFR ligands in v-ras-stimulated PKC tyrosine phosphorylation was strongly supported by experiments using EGFR deficient keratinocytes (Fig. 3B). The increase in PKC tyrosine phosphorylation in the EGFR -/- v-ras keratinocytes was only 35% ± 17% (mean ± standard deviation, n = 2) of that induced in the EGFR +/+ v-ras keratinocytes of the same strain. In the same experiments, the induction of PKC tyrosine phosphorylation by TPA was almost identical in the EGFR +/+ and -/- keratinocytes (18.9-fold and 18.4-fold, respectively), indicating that the EGFR kinase is not essential for PKC tyrosine phosphorylation, that the EGFR null keratinocytes are competent to respond to other external stimuli, and that TPA targets PKC through an independent pathway. These studies establish the critical role for EGFR signal transduction in altering PKC tyrosine phosphorylation and subsequent enzymatic activity in Ras-transformed keratinocytes. Nevertheless, transduction of EGFR -/- keratinocytes with v-ras induced a small increase in tyrosine phosphorylation of PKC, indicating that other pathways may contribute to Ras-induced PKC tyrosine phosphorylation. For example, stimulation of phosphatidylinositol turnover by AlF(4) slightly elevated PKC tyrosine phosphorylation in normal keratinocytes (Fig. 1A), and inositol phosphate metabolism is up-regulated in v-ras-transformed keratinocytes(9, 41) .

Tyrosine phosphorylation of PKC has been reported in the promyeloid cell line 32D and NIH-3T3 fibroblasts treated with the tumor promoter TPA or PDGF(27, 29) . PKC tyrosine phosphorylation may also play a role in the IgE receptor signaling pathway of mast cells (32) and in saliva production by parotid acinar cells in response to substance P or the muscarinic agonist carbachol(31) . However, the published effects of tyrosine phosphorylation on PKC activity are ambiguous. Tyrosine-phosphorylated PKC isolated from ras-transformed (5) and TGFalpha-treated keratinocytes (Fig. 2) has reduced activity. Li et al. observed increased PKC activity in the membrane fraction of 32D/PDGF-betaR/PKC- or NIH-3T3/PKC- cells treated with either TPA or PDGF to increase the level of membrane-associated tyrosine-phosphorylated PKC(29) . However, this activity may be due to nontyrosine-phosphorylated PKC or other PKC isozymes that translocate to the membrane fraction after TPA treatment. In vitro tyrosine phosphorylation of PKC by Fyn, insulin receptor, or PDGF receptor also resulted in a <2-fold increase in PKC activity (27) , but the relevance to tyrosine phosphorylation in intact cells is unknown because differences in substrates or assay conditions can influence the activity of PKC. For example, the activity of tyrosine-phosphorylated PKC in response to activation of the IgE receptor showed decreased activity toward its physiological substrate, the FcRI chain, and increased activity toward myelin basic protein(32) . Li et al. have also generated a mutant PKC that was constitutively phosphorylated on tyrosine and catalytically inactive, further supporting an association between tyrosine phosphorylation and inhibition of PKC activity(42) . PKC can be tyrosine-phosphorylated on more than one site, and distinct phosphorylation sites may regulate activity differently (43) . (^2)To date, most experiments where the tyrosine-phosphorylated PKC is isolated from intact cells support a role for tyrosine phosphorylation in the inhibition of PKC activity (5, 32, 42) .

Translocation of PKC in response to EGF is observed in some cell types(44) , but not in others ( Fig. 5and (31) ), and our results indicate that translocation is not a prerequisite for tyrosine phosphorylation. In unstimulated mouse keratinocytes, 30-50% of the PKC is localized to the particulate fraction(11, 33) . Furthermore, as shown in Fig. 4(A and B), c-Src and c-Fyn constitute the major PKC tyrosine kinase activity in the membrane fraction of keratinocytes where tyrosine-phosphorylated PKC is localized(5, 29) . Thus, a pool of particulate-associated PKC exists in keratinocytes that can become tyrosine-phosphorylated upon activation of the appropriate kinase.

Specialized functions for individual PKC isozymes have been identified in several cell types(45) . PKC is involved in myeloid differentiation (46) and secretion in basophilic RBL-2H3 cells(47) . PKC also regulates cell cycle progression in CHO cells (48) and growth arrest in fibroblasts(49, 50) . In primary mouse keratinocytes, PKC translocates in response to Ca-induced differentiation(11) . Moreover, TPA-induced keratinocyte differentiation is inhibited by concentrations of bryostatin 1 (10-1000 nM) that protect PKC from down-regulation (33) . Thus, activation of PKC may be important for commitment to keratinocyte terminal differentiation. Both v-ras and EGFR ligands modify keratinocyte differentiation in vitro, and EGFR activation reproduces a subset of phenotypic alterations characteristic of neoplastic v-ras keratinocytes(9, 14, 17) . Therefore, the common target of PKC tyrosine phosphorylation for v-ras and EGFR ligands may be relevant to the phenotypic alterations in epidermal neoplasia.

In addition to PKC, c-Src and c-Fyn phosphorylate other PKC isoforms (, , and ) in vitro, but we have not detected tyrosine phosphorylation of any isoforms except PKC in intact cells. The in vivo substrate specificity of c-Src and c-Fyn may be determined by the subcellular distribution of the kinases and substrates as well as direct physical associations. Thus, PKC isoforms such as PKC and , which are readily phosphorylated in vitro by c-Src and c-Fyn, may not be accessible to the appropriate tyrosine kinase in living cells. As can be seen from Fig. 4C, c-Src and c-Fyn have a different pattern of associated proteins, and these could influence their substrate specificity.

This report defines a novel connection between tyrosine kinase signaling and the PKC family of enzymes, which may have important functional consequences for epithelial growth, differentiation, and carcinogenesis. The EGFR and PKC pathways are two major signaling systems for epidermal keratinocytes. A better understanding of these signal transduction pathways and cross-talk between the different kinase cascades provides new insight for the design of drugs to treat diseases involving this cell type.


FOOTNOTES

*
A preliminary description of this report was presented at the 1995 American Association for Cancer Research Meeting. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Northwestern University Medical School, Dept. of Pathology W127, 303 E. Chicago Ave., Chicago, IL 60611-3008.

Present address: Laboratory of Tumor Virus Biology, National Cancer Institute, Bldg. 41, Rm. C111, 41 Library Dr., MSC 5055, Bethesda, MD 20892-5055.

§§
To whom reprint requests should be sent: Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bldg. 37, Rm. 3B24, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-496-2162; Fax: 301-496-8709.

(^1)
The abbreviations used are: PKC, protein kinase C; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; EGFR, EGF receptor; TGF, transforming growth factor.

(^2)
Z. Szallasi and M. F. Denning, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Enzo Calautti and G. Paolo Dotto at the Cutaneous Biology Research Center for many helpful discussions. We also thank Dr. Zoltan Szallasi for critical reading of this manuscript.


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