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 (PKC-delta), we mutated serine 643 of PKC-delta to an alanine residue (PKC-deltaS643A). Two different expression vectors containing PKC-deltaS643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells. In vitro autophosphorylation assays demonstrated 65-83% reduction in autophosphorylation of PKC-deltaS643A in comparison to wild type PKC-delta (PKC-deltaWT). The enzymatic activity of PKC-deltaS643A mutant as measured by phosphorylating the PKC-delta pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PKC-deltaWT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-deltaS643A when compared with PKC-deltaWT, further substantiating that serine 643 is phosphorylated in vivo. Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-deltaS643A were not altered in comparison to PKC-deltaWT, 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-deltaS643A mutant protein was impaired in comparison to that of PKC-deltaWT transfectant. Taken together, our results demonstrate that serine 643 of PKC-delta is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function.
Li
W
W
Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. Liwe@dc37a.nci.nih.gov
Zhang
J
J
Bottaro
D P
DP
Pierce
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002454:Cell Differentiation / Q000187:drug effects; D018076:DNA, Complementary; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018076:DNA, Complementary; D007527:Isoenzymes; D012694:Serine; C497259:Prkcd protein, mouse; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate