ACTIVATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR SIGNAL-TRANSDUCTION PATHWAY STIMULATES TYROSINE PHOSPHORYLATION OF PROTEIN-KINASE-C-DELTA

The expression of an oncogenic ras(Ha) gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C delta and inhibits its enzymatic activity (Denning, M. F., Dlugosz, A. A., Howe tt, M. K., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). K eratinocytes expressing an activated ras(Ha) gene secrete transforming growth factor alpha (TGF alpha) and have an altered response to diffe rentiation signals involving protein kinase C (PKC), Because the neopl astic phenotype of v-ras(Ha) expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGF alpha and the G prote in activator aluminum fluoride (AlF4-), we determined if TGF alpha or AlF4- could induce tyrosine phosphorylation of PKC delta. Treatment of primary keratinocyte cultures for 4 days with TGF alpha induced tyros ine phosphorylation of PKC delta, whereas AlF4- only slightly stimulat ed PKC delta tyrosine phosphorylation. The PKC delta that was tyrosine -phosphorylated in response to TGF alpha had reduced activity compared with the nontyrosine-phosphorylated PKC delta. Treatment of keratinoc ytes expressing a normal epidermal growth factor receptor (EGFR) with TGF alpha or epidermal growth factor for 5 min induced PKC delta tyros ine phosphorylation, This acute epidermal growth factor treatment did not induce tyrosine phosphorylation of PKC delta in keratinocytes isol ated from waved-2 mice that have a defective epidermal growth factor r eceptor, In addition, the level of PKC delta tyrosine phosphorylation in v-ras(Ha)-transduced keratinocytes from EGFR null mice was substant ially lower than in v-ras(Ha) transduced mild type cells, suggesting t hat activation of the EGFR is important for PKC delta tyrosine phospho rylation in ras transformation. However, purified EGFR did not phospho rylate recombinant PKC delta in vitro, whereas members of the Src fami ly (c-Src, c-Fyn) and membrane preparations hom keratinocytes did, Fur thermore, clearing c-Src or c-Fyn from keratinocyte membrane lysates d ecreased PKC delta tyrosine phosphorylation, and c-Src and c-Fyn isola ted from keratinocytes treated with TGF alpha had increased kinase act ivity, Acute or chronic treatment with TGF alpha did not induce signif icant PKC delta translocation in contrast to the phorbol ester 12-O-te tradecanoylphorbol-13-acetate, which induced both translocation and ty rosine phosphorylation of PKC delta. This suggests that TGF alpha-indu ced tyrosine phosphorylation of PKC delta results from the activation of a tyrosine kinase rather than physical association of PKC delta wit h a membrane-anchored tyrosine kinase, Taken together, these results i ndicate that PKC delta activity is inhibited by tyrosine phosphorylati on in response to EGFR-mediated signaling and activation of a member o f the Src kinase family may be the proximal tyrosine kinase acting on PKC delta in keratinocytes.