Identification of serine 643 of protein kinase C-delta as an important autophosphorylation site for its enzymatic activity

To investigate the role of serine/threonine autophosphorylation of protein kinase C-delta (PMC-delta), we mutated serine 643 of PKC-delta to an alanine residue (PKC-delta S643A). Two different expression vectors containing PKC-delta S643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells. In vitro autophosphorylation assays demonstrated 65-83% reduction in autophosphorylation of PKC-delta S643A in comparison to wild type PKC-delta (PKC-delta WT). The enzymatic activity of PKC-delta S643A mutant as measured by phosphorylating the PKC-delta pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PRC-delta WT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-delta S643A when compared with PRC-delta WT, further substantiating that serine 643 is phosphorylated in vivo, Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-delta S643A were not altered in comparison to PRC-delta WT, indicating that mutagenesis did not affect the structural integrity of the mutant protein, 12-O-Tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells overexpressing PKC-delta S643A mutant protein was impaired in comparison to that of PRC-delta WT transfectant, Taken together, oar results demonstrate that serine 643 of PRC-delta is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function.