Watanabe M, Chen C Y, Levin D E
Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205.
J Biol Chem. 1994 Jun 17;269(24):16829-36.
The PKC1 gene of the budding yeast Saccharomyces cerevisiae encodes a homolog of the alpha, beta, and gamma isoforms of mammalian protein kinase C (PKC) that is essential for cell growth. Loss of PKC1 function results in a cell lysis defect that is due to a deficiency in cell wall construction. In this study, Pkc1p was modified at its COOH terminus with the influenza virus hemagglutinin epitope and was detected by SDS-polyacrylamide gel electrophoresis as a 145- and 150-kDa doublet when overproduced in yeast cells. Pkc1p displayed intrinsic Ser/Thr protein kinase activity in vitro, possessing a substrate specificity similar to that described for mammalian PKC. Specifically, preferred substrates possess an arginine at position -3 and a basic residue at position +2 relative to the target site. A catalytically inactive missense mutant of Pkc1p failed to complement a pkc1 delta mutant, suggesting that protein kinase activity is required for the biological function of Pkc1p. Both wild-type Pkc1p and the inactive form were isolated as phosphoproteins, indicating that Pkc1p is phosphorylated in vivo by another protein kinase. In vitro protein kinase activity of Pkc1p was not dependent on activating cofactors normally required for stimulation of mammalian PKC. However, mutational incapacitation of the pseudosubstrate site of Pkc1p resulted in constitutive activation of the enzyme, both in vivo and in vitro, suggesting that Pkc1p is normally regulated by a mechanism similar to that of its mammalian counterparts. The apparent molecular mass and substrate specificity of Pkc1p, together with its failure to respond to activating cofactors, suggest that this enzyme is distinct from an enzyme purified previously from budding yeast that has enzymatic properties similar to those of mammalian PKC.
出芽酵母酿酒酵母的PKC1基因编码哺乳动物蛋白激酶C（PKC）的α、β和γ亚型的同源物，该同源物对细胞生长至关重要。PKC1功能的丧失导致细胞裂解缺陷，这是由于细胞壁构建缺陷所致。在本研究中，Pkc1p在其COOH末端用流感病毒血凝素表位进行了修饰，当在酵母细胞中过量表达时，通过SDS-聚丙烯酰胺凝胶电泳检测为145 kDa和150 kDa的双峰。Pkc1p在体外表现出内在的丝氨酸/苏氨酸蛋白激酶活性，具有与哺乳动物PKC相似的底物特异性。具体而言，相对于靶位点，优选的底物在-3位具有精氨酸，在+2位具有碱性残基。Pkc1p的催化无活性错义突变体无法互补pkc1δ突变体，表明蛋白激酶活性是Pkc1p生物学功能所必需的。野生型Pkc1p和无活性形式均作为磷蛋白被分离出来，表明Pkc1p在体内被另一种蛋白激酶磷酸化。Pkc1p的体外蛋白激酶活性不依赖于刺激哺乳动物PKC通常所需的激活辅因子。然而，Pkc1p假底物位点的突变失活导致该酶在体内和体外均组成性激活，表明Pkc1p通常通过与其哺乳动物对应物相似的机制进行调节。Pkc1p的表观分子量和底物特异性，以及其对激活辅因子无反应，表明该酶与先前从出芽酵母中纯化的一种酶不同，后者具有与哺乳动物PKC相似的酶学性质。
