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Originally published In Press as doi:10.1074/jbc.M111618200 on January 25, 2002

J. Biol. Chem., Vol. 277, Issue 14, 12318-12323, April 5, 2002
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Src Family Kinases Phosphorylate Protein Kinase C delta  on Tyrosine Residues and Modify the Neoplastic Phenotype of Skin Keratinocytes*

Elizabeth JoseloffDagger , Christophe CataissonDagger , Heather AamodtDagger , Henrietta OcheniDagger , Peter BlumbergDagger , Alan J. Kraker§, and Stuart H. YuspaDagger

From the Dagger  Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, NCI, Bethesda, Maryland 20892 and the § Cancer Pharmacology, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105-2430

Received for publication, December 5, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein kinase C delta  (PKC delta ) is tyrosine-phosphorylated and catalytically inactive in mouse keratinocytes transformed by a ras oncogene. In several other model systems, Src kinases are upstream regulators of PKC delta . To examine this relationship in epidermal carcinogenesis, v-ras transformed mouse keratinocytes were treated with a selective Src kinase inhibitor (PD 173958). PD 173958 decreased autophosphorylation of Src, Fyn, and Lyn kinases and prevented tyrosine phosphorylation of the Src kinase substrate p120. PD 173958 also prevented PKC delta  tyrosine phosphorylation and activated PKC delta  as detected by membrane translocation. Expression of keratinocyte differentiation markers increased in PD 173958-treated v-ras-keratinocytes, and fluid-filled domes emerged, indicative of tight junction formation. Antisense PKC delta  or bryostatin 1 inhibited dome formation, while overexpression of PKC delta  in the presence of PD 173958 enhanced the formation of domes. Plasmids encoding phenylalanine mutants of PKC delta  tyrosine residues 64 and 565 induced domes in the absence of PD 173958, while phenylalanine mutants of tyrosine residues 52, 155, and 187 were inactive. Thus, Src kinase mediated post-translational modification of PKC delta  on specific tyrosine residues in ras-transformed mouse keratinocytes inactivates PKC delta  and contributes to alterations in the differentiated phenotype and tight junction formation associated with neoplasia.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The elucidation of specific genetic changes associated with early events in human cancer pathogenesis has focused efforts to relate these changes to particular characteristics of the neoplastic phenotype. The mouse skin carcinogenesis model lends itself well to these analyses. Activating mutations of the ras gene family are frequently detected in human squamous cell carcinomas, and expression of an oncogenic ras gene is sufficient to initiate benign tumor formation in mouse skin. The clearly defined genetic and phenotypic stages in the evolution of squamous tumors on mouse skin provide an opportunity to evaluate mechanisms of carcinogenesis in a stage-specific manner. We have used an in vitro model for initiation of skin carcinogenesis whereby we introduce an oncogenic v-ras into normal primary keratinocytes via retroviral transduction (1). Initiated cells in this model display the hyperproliferative phenotype, express an altered pattern of differentiation markers, and resist terminal cell death, which are all characteristics of benign squamous papillomas (2). Since tumor formation in terminally differentiating tissues requires inhibition of terminal cell death, understanding the mechanisms involved may suggest novel targets for rational therapy of these tumors. Our earlier studies had implicated PKC as a regulator of keratinocyte terminal differentiation, and studies with v-ras initiated keratinocytes specifically identified PKC delta  as being inactivated by tyrosine phosphorylation in papilloma cells (3-5).

Direct evidence that PKC delta  was involved in a keratinocyte death pathway came from studies in which PKC delta , overexpressed in neoplastic keratinocytes by an adenoviral vector, induced apoptosis associated with PKC delta  translocation to mitochondria and reduction in mitochondrial membrane potential (6). These results were subsequently confirmed for PKC delta  in other tumor model systems (7, 8). In human keratinocytes, PKC delta  participates in apoptosis induced by ultraviolet light (9). Furthermore, PKC delta  acts as a lamin kinase leading to phosphorylation and proteolysis needed for the disassembly of the nuclear lamina, a step that occurs during apoptosis (10). Together these findings support a role for PKC delta  as a death inducer and tumor suppressor, a hypothesis consistent with the finding that transgenic mice that overexpress PKC delta  in the skin are resistant to chemically induced skin tumor formation (11).

In several model systems, tyrosine phosphorylation is involved in regulating PKC delta  activity with resultant increase or decrease in catalytic activity dependent on cell type and phosphorylation site (12, 13). Signaling through the insulin-like growth factor-1 or PDGF1 receptor or exposure to H2O2 increases both tyrosine phosphorylation and catalytic activity of PKC delta  (14-16). PKC delta  is a substrate for activated c-Src and v-Src in vivo, and in several systems this interaction inactivates PKC delta  catalytic function and causes rapid degradation of the PKC (17, 18). Furthermore, overexpression of PKC delta  reverses v-src transformation of rat colonic epithelial cells (19). Src family kinases phosphorylate PKC delta  on tyrosine residues in vitro, and these kinases are activated in v-ras-transformed keratinocytes (14, 20, 21). Src family kinases are also activated in epidermal tumors (22), and targeting a human c-Src protein to the basal epidermis in transgenic mice causes spontaneous skin tumor formation (23). In this study, we used a selective pyrido[2,3-d]pyrimidine Src family tyrosine kinase inhibitor, PD 173958 (24, 25), to evaluate the interaction of Src kinases and PKC delta  in the neoplastic phenotype produced by v-ras initiation of normal mouse keratinocytes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Primary mouse keratinocytes were isolated from newborn BALB/c epidermis and seeded at a density of 5 × 106 cells per 60-mm dish in Ca2+- and Mg2+-free minimal essential medium (Invitrogen), supplemented with 8% Chelex treated fetal bovine serum (Gemini Bioproducts) and 0.05 mM Ca2+ as described previously (26). Cells were cultured for 3 days prior to infection with a replication-defective retrovirus encoding v-rasHa as described in Roop et al. (1). To induce dome formation, v-rasHa-transduced primary keratinocytes (v-ras-keratinocytes) were cultured for 24 h in medium containing 1.4 mM Ca2+ prior to the addition of the pyrido[2,3-d]pyrimidine tyrosine kinase inhibitor PD 173958 for 24 h. The dishes were fixed with 10% formalin, stained with 0.36% rhodamine, and domes were quantified using a dissecting microscope.

Immunoblot Analysis-- Cultured cells were lysed in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 20 µM leupeptin, 200 µM NaVO3, and 10 mM NaF. Proteins were quantified by the Bradford method (Bio-Rad) and separated by 7.5% SDS-PAGE followed by immunoblotting. PKC delta  polyclonal antibody was used at a 1:30000 dilution (R & D Systems), and mouse monoclonal anti-Tyr(P) antibody (PY99) and rabbit polyclonal anti-p120 (S-19) were used at 1:1000 dilution (Santa Cruz Biotechnology). Detection of differentiation markers, keratins 1 and 10, loricrin, and filaggrin, has been described previously (27).

Reverse Transcriptase-PCR Analysis-- RNA was isolated from cultured keratinocytes with TRIzol (Invitrogen). For cDNA synthesis, 2 µg of total RNA were reverse-transcribed using Superscript II reverse transcriptase (Invitrogen). PCR amplifications were performed in a volume of 20 µl using Platinum PCR SuperMix (Invitrogen). The primers used for this analysis were: glyceraldehyde-3-phosphate dehydrogenase forward sequence, ATG GTG AAG GTC GGT GTG AAC G; glyceraldehyde-3-phosphate dehydrogenase reverse sequence, ACC TGG TCC TCA GTG TAGC CCA; loricrin forward sequence, TAC CTG GCC GTG CAA GTA AG; loricrin reverse sequence, AAC AGG ATA CAC CTT GAG CGA C; filaggrin forward sequence, CAA GAT CAG GCT CAG GAG GAA G; filaggrin reverse sequence, GCA GGA ACA GAA TTG CAA ACA C. To avoid saturation or plateau effect of amplification, PCR was limited to a total of 20 cycles for glyceraldehyde-3-phosphate dehydrogenase and 30 cycles for filaggrin and loricrin. Each reaction was performed from three independent experiments.

Immunoprecipitation-- Primary control or v-ras-keratinocytes were seeded in 10-cm dishes and treated with the kinase inhibitor PD 173958 for 5 h. Cells were lysed in the same buffer mix for immunoblotting but containing 50 mM Tris, pH 7.5. Lysates were pre-cleared by rotating with 20 µl of G/A PLUS agarose (Santa Cruz Biotechnology) for 1 h at 4 °C. The immunoprecipitation reactions consisted of 0.7 mg of protein lysate, 1 µg of antibody, and 20 µl of G/A PLUS agarose. The antibody and protein were incubated with rotation for 2 h at 4 °C prior to the addition of the agarose beads. The immunoprecipitation proceeded at 4 °C overnight, and beads were washed six times with radioimmune precipitation buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% deoxycholic acid. The immunoprecipitate was resolved by SDS-PAGE and immunoblotting. The antibodies used for immunoprecipitation of Src kinase family members and tyrosine-phosphorylated proteins were purchased from Santa Cruz Biotechnology. Immunoprecipitation studies were repeated in three independent experiments.

Transfection-- For transient transfection of v-ras-keratinocytes using LipofectAMINE PLUS reagent (Invitrogen), cells were cultured for 48 h post-viral infection. PLUS reagent was diluted 1:50 in serum-free 0.05 mM Ca2+ medium, and the LipofectAMINE reagent was diluted 1:33 in serum-free 0.05 mM Ca2+ medium. Diluted PLUS was incubated with 4 µg of plasmid DNA for 15 min at room temperature prior to the addition of the diluted lipid reagent. Cells were transfected under serum-free conditions for 6 h and then placed in complete 0.05 mM Ca2+ medium. The plasmid DNAs used for the tyrosine mutants of PKC delta  have been described previously (28). The dominant-negative PKC delta  plasmid was a generous gift from Susan Jaken (29).

Cell Viability Assay-- Cell viability was determined using the CellTiter 96 (Promega) kit. Cells were seeded in six-well dishes at a concentration of 2.5 × 106 cells per well. Cells were cultured for 3 days prior to v-rasHa infection. PD 173958 was added at doses of 0.25, 0.5, or 1 µM overnight in 3 ml of 0.05 mM Ca2+ medium. 300 µl/well of dye solution was added for 4 h followed by 3 ml of the stop solution. After 1 h of incubation, absorbency of the samples was determined at 570 nm. As a control for cell death, wells were freeze-thawed for several repetitions prior to the addition of the MTT dye. Each treatment was performed in duplicate, and the experiment was repeated independently four times.

Subcellular Fractionation-- To examine membrane translocation of PKC delta  as an indication of kinase activation, particulate and cytosolic protein fractions were prepared for immunoblotting. After treatment with varying doses of PD 173958, keratinocytes were lysed in a buffer containing 20 mM Hepes, pH 7.4, 250 mM sucrose, 150 mM NaCl, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 8 µg/ml aprotinin, 20 µM leupeptin, 200 µM NaVO3, and 10 mM NaF. Cell lysates were sonicated and centrifuged for 1 h at 4 °C at 100,000 × g. The soluble protein fraction and the membrane protein pellet were resuspended in the lysis buffer with 1% Triton X-100 added. Protein was quantified by the Bradford method and ~4-5 µg of particulate protein and 4-6 µg of cytosolic protein were separated by 7.5% SDS-PAGE and analyzed by immunoblotting for PKC delta .

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PD 173958 Inhibits Tyrosine Phosphorylation of Src Kinases-- At concentrations of 0.25 to 10 µM the pyrido[2,3-d]pyrimidine tyrosine kinase inhibitor PD 173958 was not toxic to v-ras-keratinocytes when assayed by the MTT assay (Fig. 1A and not shown). In these same cells, low doses of the inhibitor decreased tyrosine phosphorylation of proteins with the molecular weight of ~60 kDa somewhat selectively (Fig. 1B) and specifically reduced tyrosine phosphorylation of p120, a known substrate for Src kinase activity (Fig. 1C). To confirm a specific effect of PD 173958 on Src activity, the individual Src kinases were immunoprecipitated from protein lysates of treated v-rasHa keratinocytes and analyzed by immunoblotting for tyrosine phosphorylation. Three of the Src kinases, Src, Fyn, and Lyn, were tyrosine-phosphorylated in neoplastic keratinocytes. Tyrosine phosphorylation of all three Src kinases was reduced in PD 173958 treated cells in a dose-dependent manner (Fig. 2). Thus, PD 173958 appears to be a selective compound to reduce activity of these enzymes in neoplastic keratinocytes.


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  		Fig. 1.   PD 173958 is nontoxic and inhibits tyrosine phosphorylation. A, the viability of primary v-ras-keratinocytes treated with PD 173958 was analyzed by MTT assay, using freeze-thawed cells as a control. Cell viability was measured in duplicate cultures after 18 h of treatment, and the experiment was repeated four times. B, protein tyrosine phosphorylation in neoplastic keratinocytes treated with PD 173958 was analyzed by immunoblotting. Total protein was extracted from v-ras-keratinocytes treated with increasing concentrations of PD 173958 and quantified by Bradford assay. 20 µg of total protein was separated by SDS-PAGE, and transfer filters were immunoblotted with an anti-phosphotyrosine antibody. C, reduced tyrosine phosphorylation of p120 isolated from inhibitor treated neoplastic keratinocytes. v-ras-keratinocytes were treated with varying doses of PD 173958 for 5 h. The Src kinase substrate p120 was immunoprecipitated (IP), isolated by SDS-PAGE, and tyrosine phosphorylation determined by immunoblotting (IB) with an anti-phosphotyrosine antibody. Shown is a representative of three independent experiments.


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  		Fig. 2.   PD 173958 inhibits Src kinase activity in neoplastic keratinocytes. v-ras-keratinocytes were treated with various doses of PD 173958 for 5 h. Using specific antibodies, Src, Fyn, and Lyn proteins were immunoprecipitated from cell lysates and analyzed by immunoblotting with an anti-phosphotyrosine antibody and specific Src family antibodies. Shown is a representative of three independent experiments.

Inhibiting Src Kinase Activity Reduces Tyrosine Phosphorylation and Activates PKC delta -- While overall PKC delta  protein levels were unchanged, a specific dose-dependent decrease in tyrosine phosphorylation of PKC delta  was detected in neoplastic keratinocytes as a result of inhibiting Src kinases (Fig. 3A). Inhibitor treatment did not alter the low levels of tyrosine phosphorylation detected for PKC alpha  or PKC epsilon . The decrease in tyrosine phosphorylation corresponded with activation of PKC delta  as indicated by translocation of the enzyme almost entirely to the membrane fraction in treated v-ras-keratinocytes (Fig. 3B). This translocation was likely to be mediated by the elevated levels of diacylglycerol measured in v-ras-keratinocytes (30). These results indicate that Src, Fyn, or Lyn are the essential kinases that tyrosine phosphorylate and inactivate PKC delta  in neoplastic keratinocytes. While Abl kinase can also tyrosine-phosphorylate PKC delta  under some circumstances (31) and the closely related PD180970 is reported to inhibit Abl kinase in vitro (32), Abl kinase tyrosine phosphorylation was not increased in v-ras-keratinocytes, and constitutive tyrosine phosphorylation was not decreased at doses of PD 173958 below 1 µM (data not shown).


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  		Fig. 3.   PD 173958 inhibits tyrosine phosphorylation and causes translocation of PKC delta . A, PKC alpha , delta , and epsilon  were immunoprecipitated from v-ras-keratinocytes treated with PD 173958 for 5 h and tyrosine phosphorylation determined by immunoblotting with an anti-phosphotyrosine antibody. B, v-ras-keratinocytes were treated with varying doses of PD 173958 for 5 h, cell lysates fractionated into membrane and cytosolic fractions, and each fraction was analyzed for PKC delta  by SDS-PAGE and immunoblotting.

Treatment with the Src Kinase Inhibitor Results in PKC delta -dependent Formation of Domes and Increased Expression of Markers of Differentiation in Vitro-- Treatment with 0.5 µM PD 173958 caused v-ras-keratinocytes to form multiple domes randomly across the plate when cultured in 1.4 mM Ca2+ (Fig. 4A). The number of domes forming was dependent on the concentration of PD 173958, and domes did not form when neoplastic cells were maintained in 0.05 mM Ca2+ medium. Domes also formed when normal keratinocytes were treated with the inhibitor and cultured in medium with 1.4 mMCa2+, and these appeared to be even larger than domes forming in v-ras keratinocytes (Fig. 4A). In the absence of overlying medium, the domes could be seen to retain fluid (Fig. 4A, inset). By inverted phase contrast microscopy, domes were identical to those reported to be PKC-dependent fluid-filled hemicysts in LLC-PK1 pig kidney cortical cells (33) (Fig. 4B). Dome formation was dependent on PKC delta  activity as quantified by counting the number of domes in formalin fixed and rhodamine-stained dishes (Fig. 4C). Transient transfection with a dominant-negative plasmid for PKC delta  or treatment with 1 nM bryostatin, a concentration that specifically inhibits PKC delta  (34), prevented dome formation in PD 173958-treated v-ras-keratinocytes (Fig. 4D). Furthermore, transfection of a GFP-PKC delta  fusion plasmid (6) enhanced formation of domes in the presence of the inhibitor.


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  		Fig. 4.   Treatment with PD 173958 induces dome formation in keratinocytes cultured in 1.4 mM Ca2+ medium. A, digital photograph of living normal keratinocytes or formalin-fixed and rhodamine-stained v-ras-keratinocytes treated with 0.5 µM PD 173958 or vehicle for 48 h in the presence of 1.4 mM Ca2+ medium. The inset shows fluid inside the domes visualized by retention of phenol red supplemented culture medium after aspiration and phosphate-buffered saline washing of culture dishes. B, phase contrast morphology of v-ras-keratinocytes treated with PD 173958 and cultured in 1.4 mM Ca2+ showing the formation of domes (phase contrast ×40). C, the number of fixed and stained domes formed in v-ras-keratinocytes in the presence or absence of PD 173958 and additional treatment with 1 nM bryostatin or by transient transfection with plasmids encoding PKC delta  or dominant-negative PKC delta  (DN PKCdelta ).

To determine whether inhibition of Src kinases and restoration of PKC delta  activity influenced the differentiation state of v-ras-keratinocytes, PD 173958-treated cultures were exposed to 0.05 or 1.4 mM Ca2+ medium for various times and examined for expression of differentiation markers by immunoblotting (Fig. 5A). Inhibition of Src kinases caused a slight increase in the expression of loricrin and filaggrin in normal keratinocytes, and PD 173958 substantially enhanced expression in v-ras-keratinocytes, both at the protein and RNA levels (Fig. 5B). Furthermore, inhibition of Src activity increased the expression of keratins 1 and 10 in the neoplastic cells, where v-ras transformation has a suppressive influence on expression of these markers.


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  		Fig. 5.   PD 173958 increases expression of differentiation markers in v-ras-keratinocytes cultured in 1.4 mM Ca2+ medium. A, total cell extracts were analyzed through immunoblotting with specific antibodies for the expression of early and late markers of differentiation at 24, 48, and 72 h after the addition of PD 173958 (1 µM) in 0.05 or 1.4 mM Ca2+ media. SDS lysates from control (mock-infected) and v-ras-keratinocytes were normalized through densitometry of the Ponceau S-stained membrane after transfer from the polyacrylamide gel and rerun a second time to obtain equal loading in each lane. These results are representative of three independent experiments. B, transcripts for loricrin and filaggrin were quantified in v-ras-keratinocytes by reverse transcriptase-PCR at 48 h after addition of PD 173958 (1 µM) and 1.4 mM Ca2+.

Tyrosine Residues 64 and 565 Are Important Residues for Regulation of PKC delta  Activity-- Since dome formation is quantitative and dependent on PKC delta  activity, we used the formation of domes as an assay to map tyrosine residues that are functionally important for PKC delta  activation in v-ras-keratinocytes. PKC delta  plasmids encoding specific phenylalanine mutants in tyrosine residues where biological activity is preserved (28) were transiently transfected into v-ras-keratinocytes in the absence of PD 173958 (Table I). After switching to 1.4 mM Ca2+ medium, dome formation was quantified on formalin-fixed and rhodamine-stained dishes. The single mutants Y64F (C2-like domain) and Y565F (catalytic domain) produced domes while Y52F, Y187F, and Y155F did not. Combinations of a mutant that induces dome formation with a mutant that did not (Y52F), or inclusion of all five mutations in the same construct, were still permissive for dome formation, indicating that the sites that did not induce dome formation did not have a suppressive influence on dome formation. These results indicate that in neoplastic epidermal cells, tyrosine residues 64 and 565 are critical sites for PKC delta  tyrosine phosphorylation and inactivation by Src kinases.

                              
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Table I
Tyrosine residues 64 and 565 are targets for PKC delta  tyrosine phosphorylation
Five tyrosine residues of PKC delta  were mutated to phenylalanine, and plasmids containing either all five mutations (PKC delta  V) or individual mutations were transiently transfected into v-ras-keratinocytes without exposure to PD 173958. Rhodamine-stained formalin fixed foci were counted after 48 h in 1.4 mM Ca2+ medium. Three independent transfections were performed with values indicating 4-12 domes per dish (+) and indicating no domes observed (-).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tyrosine phosphorylation of PKC delta  is emerging as an important theme in signal transduction in a variety of cellular systems (12, 13). The consequence of this modification appears to be cell type and stimulus specific. For example, tyrosine-phosphorylated PKC delta  resulting from activation of the IgE receptor in rat basophilic leukemia cells or v-Src transformation of rat fibroblasts has diminished catalytic activity for selective substrates (17, 35). In contrast, phosphotyrosine-modified PKC delta  isolated from H202-treated C0S-7 cells, ceramide-treated HeLa cells, or PDGF-treated 32D cells has increased catalytic activity (15, 16, 36). While the underlying mechanisms that determine these responses are not apparent, recent data suggest that the tyrosine residue that is phosphorylated is important in the biological readout (16, 28).

Cross-talk among Src kinases and PKC delta , first reported from in vitro kinase reactions (20, 37), is now recognized as an important signaling complex in several biological processes. Activated Src phosphorylates PKC delta  on tyrosine 311 in murine fibroblasts leading to degradation of PKC delta  that is required for growth stimulation by v-Src or PDGF (18). Inactivation of PKC delta  by v-Src in rat fibroblasts is required for transformation and defines a reciprocal interaction between serine phosphorylation on Src and tyrosine phosphorylation on PKC delta  leading to down-regulation of the PKC (17). In PDGF-treated C6 glioma cells, tyrosine phosphorylation of PKC delta  by Fyn kinase is required for inhibition of glutamine synthetase, a marker of differentiation (28).

We have previously reported the tyrosine phosphorylation and catalytic inactivation of PKC delta  in response to transformation of primary mouse keratinocytes by oncogenic Ras or activation of the EGF receptor (5, 37). In this model, tyrosine phosphorylation of PKC delta  was associated with a block in terminal differentiation as implied by studies with staurosporine, a kinase inhibitor that both prevented PKC delta  tyrosine phosphorylation and induced PKC-dependent terminal cell death (5). We now present direct evidence that c-Src, c-Fyn, and c-Lyn are activated in ras-transformed keratinocytes and mediate PKC delta  tyrosine phosphorylation. It is likely that all three Src kinases are involved in the modification of PKC delta , since previous studies have shown that clearing Src or Fyn from keratinocyte membranes reduces tyrosine phosphorylation of recombinant PKC delta  in vitro (37), and Lyn phosphorylates tyrosine residue 565 in vitro, a likely target of modification in the keratinocyte system (38). When Src kinase activity is inhibited by a Src kinase-selective inhibitor, PKC delta  tyrosine phosphorylation is prevented and PKC delta  translocates from the cytoplasmic to membrane compartment, indicating it has been activated. The active PKC delta  induces the neoplastic population to form domes and express terminal differentiation markers when challenged by 1.4 mM Ca2+ as a differentiation inducer. Studies with transfected PKC delta  tyrosine mutants implicate residues 64 and 565 as critical targets for inactivation of PKC delta  by tyrosine phosphorylation in v-ras-transformed keratinocytes.

Previous studies have shown that PKC delta  translocates to membrane compartments in normal mouse keratinocytes induced to differentiate by calcium (4), and PKC delta  levels increase as hyperproliferative neonatal epidermis matures into the adult more differentiated phenotype (39). Furthermore, overexpression of PKC delta  in human keratinocytes causes growth arrest and induction of transglutaminase 1, a key enzyme in terminal differentiation (40). PKC delta  also activates the promoter sequences of involucrin and SPRR1B, both important components of cornified envelopes, the terminal mature phenotype of normal keratinocytes (41, 42). These results are consistent with our current findings that preventing PKC delta  inactivation by Src kinase-mediated tyrosine phosphorylation in neoplastic keratinocytes is associated with increased gene expression of markers of late differentiation, loricrin and filaggrin, in response to 1.4 mM Ca2+ medium. However, v-ras-keratinocytes did not continue through terminal cell death in this model. This partial differentiation response may result from a requirement for Fyn activity in conjunction with PKC eta  to express the fully differentiated phenotype as recently reported by Cabodi et al. (43). In the presence of PD 173958, this activity would be inhibited.

The PKC delta -dependent formation of domes may be another manifestation of a differentiation program. Significant dome formation required both PD 173958 and 1.4 mM Ca2+ in cells transformed by v-ras. Large domes also formed in normal keratinocytes treated with PD 173958 and 1.4 mM Ca2+, possibly reflecting an inhibition of PKC delta  tyrosine phosphorylation detected in differentiating keratinocytes in vitro and in vivo (44). Dome formation was dependent on PKC delta  activity as it was reduced by PKC delta  inhibition and increased by active PKC delta . Furthermore, domes were induced in the absence of PD 173958 by transfecting PKC delta  tyrosine phosphorylation-defective mutants Y64F and Y565F. The domes forming in our study appear to be identical to those studied extensively by Mullin and collaborators in LLC-PK1 pig kidney epithelial cells (33). In that model, dome formation represents a differentiated phenotype in which functionally intact tight junctions form at confluence in the presence of high calcium medium. These intact tight junctions reduced transepithelial permeability producing fluid-filled blisters. Both PKC delta  and PKC alpha  activation increased transepithelial permeability and disrupted dome formation (45, 46). In keratinocytes, the action of PKC on dome formation seems to be reversed. This may reflect the different functions of the two tissues, where in one case secretion and absorption are required, and in the other barrier formation is needed. Thus, the formation of tight junctions may be an important keratinocyte differentiation function regulated by PKC delta  and disturbed by oncogenic Ras transformation (47, 48). Furthermore, Src kinases disrupt cell-cell adherens junctions in normal and neoplastic human keratinocytes (49), possibly contributing further to a defective differentiation program in keratinocyte transformation. Since PKC delta  also phosphorylates the alpha 6beta 4 integrin complex, reducing keratinocyte attachment to substrate (50), the inhibition of Src kinase in v-ras-keratinocytes may increase tight junction and adherens junction binding, and PKC delta  may induce cell detachment from the substratum to form domes. The stratified detached epithelial sheet forming the dome may be analogous to the suprabasal layers of the epidermis that are detached from a basement membrane. PKC delta  tyrosine phosphorylation detected in suprabasal differentiating normal keratinocytes may serve to regulate the extent of tight junction formation depending on the physiological requirements of the dynamic epidermis.

From the existing literature, it is difficult to define a unifying functional consequence of PKC delta  tyrosine phosphorylation (12, 13). For example, tyrosine phosphorylation at residue 311 in H202-treated C0S-7 cells or by Lck in vitro activates catalytic activity (16), while modification at residue 311 by Src kinase inactivates catalytic activity and promotes degradation of PKC delta  (18). In various cell systems or in vitro assays, tyrosine residues 52, 155, 187, and 565 have been implicated as targets for phosphorylation, and growth factor receptors (insulin-like growth factor receptor 1, PDGF receptor), cell surface antigen receptors (IgE, B cell antigen receptor), or nonreceptor tyrosine kinases (v-Src, c-Src, c-Lyn, Lck) as proximate kinases. When phenylalanine mutants at residues 155 and 187 were introduced independently into C6 glioma cells, each mutant had a distinct functional consequence indicating that the specific tyrosine residue phosphorylated may determine the divergence of signaling processes (28). Our studies support this concept. Preventing phosphorylation at tyrosine 64 in the regulatory domain and 565 in the kinase domain induces dome formation in the absence of Src kinase inhibition. Thus, these sites are likely targets for Src kinase mediated catalytic inactivation of PKC delta  in v-ras-keratinocytes. In contrast, phenylalanine substitutions at tyrosine residues 52, 155, and 187 were without effect.

Confirmation of these modification sites and identification of others that may also be involved will require additional structural approaches. However, the specificity of the results with mutant constructs indicates potential strategies to overcome the terminal differentiation defect in neoplastic keratinocytes. Previous studies have demonstrated the pro-apoptotic influence of overexpressed PKC delta  in v-ras-keratinocytes and tumor-derived keratinocyte cell lines from murine and human sources (6). Transgenic targeting of PKC delta  to mouse epidermis inhibits tumor formation (11). Preliminary studies indicate that adenoviral encoded PKC delta  can prevent tumor growth when injected into the tumor mass in situ.2 By using biologically active PKC delta  mutants where tyrosine phosphorylation at crucial residues is prevented or by combined treatment with native PKC delta  and selective Src kinase inhibitors, it might be possible to increase the antitumor activity of a PKC delta -based therapy. While this strategy might be at first confined to mouse models, published studies on human tumors or human tumor cells suggest that activating PKC delta  in the tumor mass may have broader applicability (51-53).

    ACKNOWLEDGEMENTS

We are grateful to Dr. Susan Jaken for the generous gift of the dominant-negative PKC delta  plasmid and Bettie Sugar for outstanding editorial assistance.

    FOOTNOTES

* 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.

To whom correspondence should be addressed: Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, NCI, 37 Convent Dr., MSC-4255, Bldg. 37, Rm. 3B25, Bethesda, MD 20892. Tel.: 301-496-2162; Fax: 301-496-8709; E-mail: yuspas@dc37a.nci.nih.gov.

Published, JBC Papers in Press, January 25, 2002, DOI 10.1074/jbc.M111618200

2 L. Li and S. H. Yuspa, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PDGF, platelet-derived growth factor; PKC, protein kinase C; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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