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首页> 外文期刊>Journal of Molecular Biology >A Distal, High-affinity Binding Site on the Cyclin-CDK Substrate Pho4 is Important for its Phosphorylation and Regulation.
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A Distal, High-affinity Binding Site on the Cyclin-CDK Substrate Pho4 is Important for its Phosphorylation and Regulation.

机译：Cyclin-CDK底物Pho4上的远端，高亲和力结合位点对其磷酸化和调节至关重要。

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摘要

Cyclins and cyclin-dependent kinases (CDKs) are key components of signaling pathways essential for cell growth and survival. The cyclin-CDK Pho80-Pho85 inactivates the transcription factor Pho4 in budding yeast by phosphorylating it on five sites. We isolated seven single amino acid substitutions outside of the phosphorylation sites that cause Pho4 to be constitutively active. The substitutions decrease the amount of Pho4 phosphorylation in vivo, and they increase the apparent K(M) of the in vitro phosphorylation reaction by an order of magnitude but do not alter k(cat) substantially. These data suggest that the substituted residues are part of a cyclin-CDK-binding site that is distal to the phosphorylation sites. Further analysis revealed that all of Pho4 variants were phosphorylated by Pho80-Pho85 in a more distributive manner than the wild-type protein, further supporting the idea that binding at a distal, high-affinity binding site is important in determining the processivity of Pho4 phosphorylation. In addition, computational modeling of the Pho4 phosphorylation reactions shows that the K(D) of binding between the Pho4 mutants and Pho80-Pho85 increases, confirming that the mutations are located in a relatively high-affinity "docking site" for the kinase. Interestingly, the K(D) derived from the in vitro data correlates well with the strength of the in vivo phenotypes, demonstrating that the in vitro data are relevant to the in vivo regulation of Pho4.
机译：细胞周期蛋白和细胞周期蛋白依赖性激酶（CDK）是细胞生长和存活所必需的信号传导途径的关键组成部分。细胞周期蛋白CDK Pho80-Pho85通过在五个位点磷酸化芽芽酵母中的转录因子Pho4使其失活。我们分离了导致Pho4具有组成型活性的磷酸化位点之外的七个单氨基酸取代。取代减少了体内Pho4磷酸化的量，并且它们使体外磷酸化反应的表观K（M）增加了一个数量级，但基本上没有改变k（cat）。这些数据表明，取代的残基是细胞周期蛋白-CDK-结合位点的一部分，其位于磷酸化位点的远端。进一步的分析表明，所有Pho4变体都比野生型蛋白以更分散的方式被Pho80-Pho85磷酸化，这进一步支持了在远端，高亲和力结合位点的结合对于确定Pho4磷酸化的持续性很重要的观点。 。此外，Pho4磷酸化反应的计算模型表明，Pho4突变体与Pho80-Pho85之间的结合K（D）增加，证实了该突变位于激酶的相对较高亲和力的“对接位点”。有趣的是，源自体外数据的K（D）与体内表型的强度具有很好的相关性，表明体外数据与Pho4的体内调控有关。

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