Reconstitution of protein kinase C alpha function by the protein kinase C beta-I carboxy terminus.
The Ca(2+)- and phospholipid-dependent Ser/Thr kinase protein kinase C (PKC) plays important roles in the transduction of cellular signals. Various PKC isoforms exist in mammalian cells which share conserved and variable regions as defined by cDNA sequence comparisons. To test whether carboxyl (C) terminal sequences of distinct isoforms can complement each other to yield functional chimeric molecules, we have constructed a PKC chimera in which amino acids 595-672 at the C-terminus of bovine PKC alpha (a) were replaced with the corresponding C-terminal amino acids (598-671) of rat PKC beta-I (b) to yield the chimera alpha/beta-I (ab). The chimera was then characterized biochemically and functionally, and compared with the parental isoforms. Since structure/function analysis of PKC in mammalian experimental systems is complicated by multiple PKC isoforms and by cellular complexity, we stably introduced the PKC constructs into the yeast Saccharomyces cerevisiae, a simple, lower eukaryote with a short doubling time and well established molecular genetics. In yeast, the faithfully expressed PKCab chimera and normal PKC isoforms bound radiolabelled phorbol ester and were recognized on immunoblots by PKC-specific antibodies. The chimera phosphorylated substrate peptides in a PMA- and Ca(2+)-dependent manner, and, upon activation, increased the cell doubling time and the rate of Ca2+ uptake into cells. In addition, PKCab displayed characteristics distinct from normal PKCb, but virtually indistinguishable from normal PKCa. Our findings indicate the reconstitution of PKCa function by the PKCb carboxyl terminus.
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Section on Molecular Biology, Joslin Diabetes Center, Boston, MA 02215.
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D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018076:DNA, Complementary; D007527:Isoenzymes; D010446:Peptide Fragments; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium