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

Abstract

To investigate the role of serine/threonine autophosphorylation of protein kinase C-δ (PKC-δ), we mutated serine 643 of PKC-δ to an alanine residue (PKC-δS643A). Two different expression vectors containing PKC- δS643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells. In vitro autophosphorylation assays demonstrated 65-83% reduction in autophosphorylation of PKC-δS643A in comparison to wild type PKC-δ (PKC- δWT). The enzymatic activity of PKC-δS643A mutant as measured by phosphorylating the PKC-δ pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PKC-δWT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-δS643A when compared with PKC-δWT, further substantiating that serine 643 is phosphorylated in vivo. Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-δS645A were not altered in comparison to PKC-δWT, indicating that mutagenesis did not affect the structural integrity of the mutant protein. 12-O-Tetradecanoyl-phorbol-13-acetate-mediated monocytic differentiation of 32D cells overexpressing PKC-δS643A mutant protein was impaired in comparison to that of PKC-δWT transfectant. Taken together, our results demonstrate that serine 643 of PKC-δ is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function.