Regulation of novel protein kinase C ε by phosphorylation

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKCε isoform, these are Thr&'' in the activation loop, Thr("! in the turn motif and Ser(#* in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKCε kinase activity in controlling the phosphorylation of Thr&'' and Ser(#*. In NIH 3T3 fibroblasts, PKCε migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr&'' and Ser(#*. Cells transfected with an active PDK-1 allele also resulted in increased PKCε Thr&'' and Ser(#* phosphorylation, whereas an active myristoylated PKCε mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKCε were not phosphorylated at

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