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首页> 外文期刊>Molecular and Cellular Biology >Reciprocal Activation by Cyclin-Dependent Kinases 2 and 7 Is Directed by Substrate Specificity Determinants outside the T Loop
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Reciprocal Activation by Cyclin-Dependent Kinases 2 and 7 Is Directed by Substrate Specificity Determinants outside the T Loop

机译：T细胞外的底物特异性决定子指导细胞周期蛋白依赖性激酶2和7的相互激活。

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Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.
机译：细胞周期蛋白依赖性激酶7（CDK7）是后生CDK激活激酶（CAK）的催化亚基，其通过T环中保守的苏氨酸残基的磷酸化来激活CDK，例如CDC2和CDK2。 CDK7的完全激活需要与阳性调节亚基，细胞周期蛋白H结合，并在其自身T环的170位保守苏氨酸残基的磷酸化。我们显示，CDK7的苏氨酸170在体外被其靶标CDC2和CDK2磷酸化，这也使CDK7 T环中的丝氨酸164磷酸化，该位点完全匹配其共有的磷酸化位点。相反，CDK4和CDK7本身都不能在体外磷酸化CDK7 T环。 CDC2或CDK2和CDK7彼此磷酸化的能力，但并不自身磷酸化，这意味着每种激酶可以区分密切相关的序列，并可以识别与其通常优选位点不同的底物位点。为了了解这种矛盾的底物特异性的基础，我们构建了一个嵌合的CDK，将CDK7的T环嫁接到CDK2的主体上。令人惊讶的是，杂化酶CDK2-7被细胞周期蛋白A依赖性方式有效地被CDK7激活，而完全未被CDK2激活。此外，CDK2-7磷酸化了野生型CDK7，但没有磷酸化。我们的研究结果表明，T环的主要氨基酸序列在确定细胞周期蛋白依赖性CAK对其CDK底物的特异性方面仅起次要作用，并且涉及T环以外序列的蛋白质相互作用会影响底物积极和消极的特异性。

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