Antonsson B, Montessuit S, Friedli L, Payton M A, Paravicini G
Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
J Biol Chem. 1994 Jun 17;269(24):16821-8.
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.
酿酒酵母PKC1基因编码一种哺乳动物蛋白激酶C的同源物（莱文，D.E.，菲尔兹，F.O.，久泽，R.，毕晓普，J.M.，和索纳，J.（1990年）《细胞》62卷，213 - 224页）。一种针对trpE - Pkc1融合蛋白产生的多克隆抗血清能识别出一种150 kDa的蛋白质。在亚细胞分级分离中，Pkc1p与100,000×g的颗粒组分相关联。这种关联对高盐浓度、碱性缓冲液或非离子去污剂的提取具有抗性，这表明Pkc1p可能与一个大的蛋白质复合物相关联。在其COOH末端用两个金黄色葡萄球菌蛋白A IgG结合片段（ZZ序列标签）重复修饰的Pkc1p能够在限制温度下完全恢复pkc1ts菌株的生长缺陷。带有ZZ标签的Pkc1p通过在DEAE - 琼脂糖上进行层析，然后在IgG - 琼脂糖上进行层析而部分纯化。在体外，Pkc1p使假底物肽和髓鞘碱性蛋白磷酸化，但不使组蛋白磷酸化。在底物肽中，将受体丝氨酸的COOH末端第2个氨基酸处的异亮氨酸替换为精氨酸，导致Km降低了10倍。Pkc1p的活性不依赖于已知可激活几种哺乳动物蛋白激酶C同工酶的辅因子，如磷脂、二酰基甘油和Ca2 +，这使其成为蛋白激酶C超家族中一个亲缘关系较远的成员。
