摘要：

The expression of an oncogenic ras(Ha) gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C δ 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(Ha) gene secrete transforming growth factor α (TGFα) and have an altered response to differentiation signals involving protein kinase C (PKC). Because the neoplastic phenotype of v-ras(Ha) expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGFα and the G protein activator aluminum fluoride (AlF/), we determined if TGFα or AlF/could induce tyrosine phosphorylation of PKCδ. Treatment of primary keratinocyte cultures for 4 days with TGFα induced tyrosine phosphorylation of PKCδ, whereas AlF/only slightly stimulated PKCδ tyrosine phosphorylation. The PKCδ that was tyrosine.phosphorylated in response to TGFα had reduced activity compared with the nontyrosine-phosphorylated PKCδ. Treatment of keratinocytes expressing a normal epidermal growth factor receptor (EGFR) with TGFα or epidermal growth factor for 5 min induced PKCδ tyrosine phosphorylation. This acute epidermal growth factor treatment did not induce tyrosine phosphorylation of PKCδ in keratinocytes isolated from waved-2 mice that have a defective epidermal growth factor receptor. In addition, the level of PKCδ tyrosine phosphorylation in v-ras(Ha)-transduced keratinocyte from EGFR null mice was substantially lower than in v-ras(Ha) transduced wild type cells, suggesting that activation of the EGFR is important for PKCδ tyrosine phosphorylation in ras transformation. However, purified EGFR did not phosphorylate recombinant PKCδ 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 PKCδ tyrosine phosphorylation, and c-Src and c-Fyn isolated from keratinocytes treated with TGFα had increased kinase activity. Acute or chronic treatment with TGFα did not induce significant PKCδ translocation in contrast to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which induced both translocation and tyrosine phosphorylation of PKCδ. This suggests that TGFα-induced tyrosine phosphorylation of PKCδ results from the activation of a tyrosine kinase rather than physical association of PKCδ with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKCδ 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 PKCδ in keratinocytes.

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