Saccharomyces cerevisiae PKC1 encodes a protein kinase C (PKC) homolog with a substrate specificity similar to that of mammalian PKC.
Saccharomyces cerevisiae PKC1 encodes a protein kinase C (PKC) homolog with a substrate specificity similar to that of mammalian PKC.
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发表时间：
1994-06
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通讯作者：
M. Watanabe;Cheau-Yun Chen;D. E. Levin
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作者：
M. Watanabe;Cheau-Yun Chen;D. E. Levin
芽殖酵母酿酒酵母的PKC 1基因编码哺乳动物蛋白激酶C（PKC）的α、β和γ同种型的同源物，其对于细胞生长是必需的。PKC 1功能的丧失导致细胞溶解缺陷，这是由于细胞壁构建的缺陷。在这项研究中，Pkc1p被修改在其COOH末端与流感病毒血凝素表位，并通过SDS-聚丙烯酰胺凝胶电泳检测到作为一个145-和150-kDa的双联体时，在酵母细胞中过度生产。Pkc1p显示内在的Ser/Thr蛋白激酶活性在体外，具有类似的哺乳动物PKC所描述的底物特异性。具体地，优选的底物在相对于靶位点的位置-3处具有精氨酸并且在位置+2处具有碱性残基。催化失活的错义突变体的Pkc1p未能补充的pkc1 δ突变体，这表明蛋白激酶活性是必需的Pkc1p的生物学功能。野生型Pkc1p和失活形式都被分离为磷蛋白，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通常刺激哺乳动物PKC所需的活化辅因子。然而，突变失能的pseudosubstrate网站的Pkc1p导致的组成型激活的酶，在体内和体外，这表明Pkc1p通常是由一种机制类似的哺乳动物的同行。Pkc1p的表观分子量和底物特异性，以及其未能响应激活辅因子，表明这种酶是不同于以前从芽殖酵母中纯化的酶，其具有类似于哺乳动物PKC的酶性质。
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.