Protein kinase C in yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product.
The Saccharomyces cerevisiae PKC1 gene encodes a homolog of mammalian protein kinase C (Levin, D. E., Fields, F.O., Kunisawa, R., Bishop, J.M., and Thorner, J. (1990) Cell 62, 213-224). A protein of 150 kDa is recognized by a polyclonal antiserum raised against a trpE-Pkc1 fusion protein. In subcellular fractionations, Pkc1p associates with the 100,000 x g particulate fraction. This association is resistant to extraction with high salt concentrations, alkali buffer, or nonionic detergents, suggesting that Pkc1p may be associated with a large protein complex. Pkc1p modified at its COOH terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ sequence tag) was able to fully restore the growth defects of a pkc1ts strain at restrictive temperature. ZZ-tagged Pkc1p was partially purified by chromatography on DEAE-Sepharose, followed by IgG-Sepharose. In vitro, Pkc1p phosphorylates the pseudosubstrate peptide and myelin basic protein, but not histones. Replacing an isoleucine with an arginine 2 amino acids COOH-terminal of the acceptor serine in the substrate peptide resulted in a 10-fold decrease of Km. Pkc1p activity was independent of cofactors such as phospholipids, diacylglycerol, and Ca2+, known to activate several mammalian protein kinase C isoenzymes, making it a rather distantly related member of the protein kinase C superfamily.
Antonsson
B
B
Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
Montessuit
S
S
Friedli
L
L
Payton
M A
MA
Paravicini
G
G
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D010455:Peptides; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D011493:Protein Kinase C