Epidermal growth factor receptor phosphorylates protein kinase C δ at tyr332 to form a trimeric complex with p66Shc in the H2O2-stimulated cells

Protein kinase C (PKC) delta is phosphorylated at Tyr311 and Tyr332 and its catalytic activity is enhanced in the H2O2-stimulated cells, but the enzymes that recognize these tyrosine residues, especially Tyr332, have been remained to be clarified. The analysis of the endogenous proteins in COS-7 cells revealed that PKC delta binds to p66Shc, an adaptor protein containing two phosphotyrosine-binding domains, in a manner dependent on its tyrosine phosphorylation upon H2O2 stimulation. The studies using the mutated PKC delta clarified that PKC delta associates with p66Shc through the phosphorylated Tyr332 residue. Epidermal growth factor (EGF) receptor was detected in the anti-p66Shc immunoprecipitate prepared from the H2O2-stimulated cells, and this receptor-type tyrosine kinase phosphorylated PKCS at Tyr332 in vitro. PKCS was, however, not tyrosine phosphorylated in the EGF-stimulated cells, whereas H2O2-induced tyrosine phosphorylation of PKC delta and its association with p66Shc were strongly suppressed by EGF receptor kinase inhibitors such as AG1478 and PD153035. These results indicate that EGF receptor phosphorylates PKC delta at Tyr332 in the H2O2-stimulated but not in the growth-factor treated cells, and suggest that PKCS in the complex with p66Shc and EGF receptor may play a role in the stress-signalling pathway.