SUBSTRATE-SPECIFICITY OF PROTEIN-KINASE-C - USE OF SYNTHETIC PEPTIDES CORRESPONDING TO PHYSIOLOGICAL SITES AS PROBES FOR SUBSTRATE RECOGNITION REQUIREMENTS
SUBSTRATE-SPECIFICITY OF PROTEIN-KINASE-C - USE OF SYNTHETIC PEPTIDES CORRESPONDING TO PHYSIOLOGICAL SITES AS PROBES FOR SUBSTRATE RECOGNITION REQUIREMENTS
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DOI：
10.1111/j.1432-1033.1986.tb10139.x
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发表时间：
1986-11-17
期刊：
影响因子：
--
通讯作者：
HUNTER, T
中科院分区：
文献类型：
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作者：
WOODGETT, JR;GOULD, KL;HUNTER, T
尽管 Ca2+/磷脂依赖性蛋白激酶（蛋白激酶 C）在体外具有广泛的底物特异性，但该酶在体内的混杂性明显较低。迄今为止，只有少数蛋白质被确定为该蛋白激酶的生理底物。为了确定完整细胞中这种底物选择性的基础，我们使用与四种已知生理底物的磷酸化位点相对应的合成肽探测了蛋白激酶C的底物一级序列要求。我们还鉴定了鸡肌肉乳酸脱氢酶的乙酰化 N 末端丝氨酸作为该蛋白激酶的体外磷酸化位点。这些比较研究表明，在体内，该酶对可磷酸化残基的 C 端一个碱性残基表现出偏好，如序列：Ser/Thr-Xaa-Lys/Arg，其中 Xaa 通常是不带电荷的残基。目标氨基酸 N 端和 C 端的额外碱性残基可增强磷酸化的 Vmax 和 Km 参数。基于蛋白激酶 C 的生理磷酸化位点的肽都不是 cAMP 依赖性蛋白激酶的有效底物，这强调了这两种多效性蛋白激酶的不同位点识别选择性。几种合成肽的有利动力学参数，加上它们对蛋白激酶 C 磷酸化的选择性，将有助于在组织和细胞提取物中存在其他蛋白激酶的情况下对该酶进行测定。
Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrate in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhbiits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.