Activation of the epidermal growth factor receptor signal transduction pathway stimulates tyrosine phosphorylation of protein kinase C delta.
The expression of an oncogenic rasHa gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C delta 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 rasHa 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-rasHa expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGF alpha and the G protein activator aluminum fluoride (AlF4-), we determined if TGF alpha or AlF4- could induce tyrosine phosphorylation of PKCdelta. Treatment of primary keratinocyte cultures for 4 days with TGFalpha induced tyrosine phosphorylation of PKCdelta, whereas AlF4- only slightly stimulated PKCdelta tyrosine phosphorylation. The PKCdelta that was tyrosine-phosphorylated in response to TGFalpha had reduced activity compared with the nontyrosine-phosphorylated PKCdelta. Treatment of keratinocytes expressing a normal epidermal growth factor receptor (EGFR) with TGFalpha or epidermal growth factor for 5 min induced PKCdelta tyrosine phosphorylation. This acute epidermal growth factor treatment did not induce tyrosine phosphorylation of PKCdelta in keratinocytes isolated from waved-2 mice that have a defective epidermal growth factor receptor. In addition, the level of PKCdelta tyrosine phosphorylation in v-rasHa-transduced keratinocytes from EGFR null mice was substantially lower than in v-rasHa transduced wild type cells, suggesting that activation of the EGFR is important for PKC delta tyrosine phosphorylation in ras transformation. However, purified EGFR did not phosphorylate recombinant PKC delta 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 PKCdelta 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 PKCdelta translocation in contrast to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which induced both translocation and tyrosine phosphorylation of PKCdelta. This suggests that TGFalpha-induced tyrosine phosphorylation of PKC delta results from the activation of a tyrosine kinase rather than physical association of PKCdelta with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKCdelta 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 PKCdelta in keratinocytes.
Denning
M F
MF
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
Dlugosz
A A
AA
Threadgill
D W
DW
Magnuson
T
T
Yuspa
S H
SH
D000483:Alleles; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000096:biosynthesis / Q000502:physiology*; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D011905:Genes, ras; D007527:Isoenzymes / Q000378:metabolism*; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008817:Mice, Mutant Strains; D016296:Mutagenesis; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D017607:Aluminum Compounds; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016211:Transforming Growth Factor alpha; C050992:tetrafluoroaluminate; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src); D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D005459:Fluorides