Byrne Meghan, Miller Nicole, Springer Michael, O'Shea Erin K
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, 600 16th Street, Genentech Hall Room S472D, San Francisco, CA 94143-2240, USA.
J Mol Biol. 2004 Jan 2;335(1):57-70. doi: 10.1016/j.jmb.2003.10.035.
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.
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通路的关键组成部分。在芽殖酵母中，细胞周期蛋白-CDK Pho80-Pho85通过在五个位点磷酸化转录因子Pho4使其失活。我们在磷酸化位点之外分离出七个单氨基酸取代，这些取代导致Pho4组成型激活。这些取代降低了体内Pho4的磷酸化水平，并且使体外磷酸化反应的表观K(M)增加了一个数量级，但对k(cat)没有实质性影响。这些数据表明，被取代的残基是磷酸化位点远端的细胞周期蛋白-CDK结合位点的一部分。进一步分析表明，所有Pho4变体被Pho80-Pho85磷酸化的方式比野生型蛋白更分散，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之间结合的K(D)增加，证实这些突变位于激酶的相对高亲和力“对接位点”。有趣的是，从体外数据得出的K(D)与体内表型的强度密切相关，表明体外数据与Pho4的体内调节相关。
