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 pro tein kinase C-delta (PMC-delta), we mutated serine 643 of PKC-delta to an alanine residue (PKC-delta S643A). Two different expression vector s containing PKC-delta S643A mutant cDNAs were transfected and express ed in 32D myeloid progenitor cells. In vitro autophosphorylation assay s demonstrated 65-83% reduction in autophosphorylation of PKC-delta S6 43A 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 m ore than 70% in comparison to that of PRC-delta WT. In vivo labeling a nd subsequent two-dimensional phosphopeptide analysis demonstrated tha t at least one phosphopeptide was absent in PKC-delta S643A when compa red with PRC-delta WT, further substantiating that serine 643 is phosp horylated in vivo, Localization and 12-O-tetradecanoylphorbol-13-aceta te-dependent translocation and tyrosine phosphorylation of PKC-delta S 643A were not altered in comparison to PRC-delta WT, indicating that m utagenesis did not affect the structural integrity of the mutant prote in, 12-O-Tetradecanoylphorbol-13-acetate-mediated monocytic differenti ation of 32D cells overexpressing PKC-delta S643A mutant protein was i mpaired in comparison to that of PRC-delta WT transfectant, Taken toge ther, oar results demonstrate that serine 643 of PRC-delta is a major autophosphorylation site, and phosphorylation of this site plays an im portant role in controlling its enzymatic activity and biological func tion.