Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.
The autophosphorylation sites of the beta 2 isozyme of protein kinase C (PKC) were recently identified as Ser-16/Thr-17 near the NH2 terminus, Thr-314/Thr-324 in the hinge region, and Thr-634/Thr-641 near the COOH terminus [Flint, A.J., Paladini, R.D. & Koshland, D.E. (1990) Science 294, 408-411]. To define the role of autophosphorylation we constructed three site-directed mutants of PKC beta 1 isozyme in which each pair of phosphorylatable residues is changed to alanine. Wild-type PKC beta 1 and the mutant proteins were transiently overexpressed in COS cells, resulting in at least a 20-fold increase in [3H]phorbol 12,13-dibutyrate binding compared with control transfectants. Enzyme assays of PKC partially purified from transfected cells indicated at least a 5-fold increase in PKC activity upon expression of the wild-type protein or the NH2-terminal and hinge mutants. In contrast, no increased activity was detected upon expression of the COOH-terminal mutant. Immunoblot analysis using a beta isoform-specific antibody showed that wild-type, NH2-terminal mutant, and hinge mutant proteins are similarly distributed between the Triton-soluble and insoluble fractions. In contrast, the COOH-terminal mutant protein is largely Triton-insoluble. Immunoblot analysis also indicated that this mutant is resistant to down-regulation upon chronic exposure of cells to phorbol ester. Moreover, RNA blot analysis showed that overexpression of wild-type PKC but not of the COOH-terminal mutant enhances phorbol ester induction of c-FOS and c-JUN mRNA. Our results indicate that (i) alteration in the NH2-terminal and hinge autophosphorylation sites has no effect on PKC function by the criteria examined and (ii) the COOH-terminal autophosphorylation sites are critical for PKC function and possibly subcellular localization in COS cells.
Zhang
J
J
Molecular Pharmacology Section, Schering-Plough Research Institute, Kenilworth, NJ 07033-0539.
Wang
L
L
Petrin
J
J
Bishop
W R
WR
Bond
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015536:Down-Regulation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate