Molecular evolution allows bypass of the requirement for activation loop phosphorylation of the Cdc28 cyclin-dependent kinase.
Molecular evolution allows bypass of the requirement for activation loop phosphorylation of the Cdc28 cyclin-dependent kinase.
复制标题
分子进化允许绕过 Cdc28 细胞周期蛋白依赖性激酶激活环磷酸化的要求。
DOI：
10.1128/mcb.18.5.2923
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发表时间：
1998
影响因子：
5.3
通讯作者：
Levine,K
中科院分区：
文献类型：
--
作者：
Cross,FR;Levine,K
许多蛋白激酶在活化环中通过磷酸化调节，这是酶活性所需的。谷氨酸可以取代某些磷酸化激活蛋白中的磷酸苏氨酸，但这种在酵母细胞凋亡蛋白依赖性激酶（Cdk）Cdc 28 p激活环磷酸化位点的取代（T169 E）阻断了生物学功能和蛋白激酶活性。使用易错DNA扩增循环，然后选择连续更高水平的功能，我们确定了突变版本的Cdc 28 p-T169 E具有高生物活性。突变体Cdc 28 p的酶和生物活性基本上正常调节细胞周期蛋白，和突变体支持正常的细胞周期进程和调节。因此，控制酵母细胞周期不需要Cdc 28 p被循环磷酸化和去磷酸化。这些CDC 28突变体允许在Cak 1 p（磷酸化Cdc 28 p-T169的基本激酶）缺失的情况下存活，这表明T169磷酸化是Cak 1 p的唯一基本功能。一些生长缺陷仍然存在于携带突变型CDC 28基因的cak 1 cdc 28菌株中，这与CAK 1额外的非必需作用一致。
Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in theSaccharomyces cerevisiaecyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity. The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation. Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated. TheseCDC28mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p. Some growth defects remain in suppressedcak1 cdc28strains carrying the mutantCDC28genes, consistent with additional nonessential roles forCAK1.