Lee Young-Sam, Huang Kexin, Quiocho Florante A, O'Shea Erin K
Howard Hughes Medical Institute, Harvard University, Department of Molecular and Cellular Biology, Faculty of Arts and Sciences Center for Systems Biology, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA.
Nat Chem Biol. 2008 Jan;4(1):25-32. doi: 10.1038/nchembio.2007.52. Epub 2007 Nov 25.
When Saccharomyces cerevisiae cells are starved of inorganic phosphate, the Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) is inactivated by the Pho81 CDK inhibitor (CKI). The regulation of Pho80-Pho85 is distinct from previously characterized mechanisms of CDK regulation: the Pho81 CKI is constitutively associated with Pho80-Pho85, and a small-molecule ligand, inositol heptakisphosphate (IP7), is required for kinase inactivation. We investigated the molecular basis of the IP7- and Pho81-dependent Pho80-Pho85 inactivation using electrophoretic mobility shift assays, enzyme kinetics and fluorescence spectroscopy. We found that IP7 interacts noncovalently with Pho80-Pho85-Pho81 and induces additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site. Using synthetic peptides corresponding to Pho81, we define regions of Pho81 responsible for constitutive Pho80-Pho85 binding and IP7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action.
当酿酒酵母细胞缺乏无机磷酸盐时，Pho80-Pho85细胞周期蛋白-细胞周期蛋白依赖性激酶（CDK）会被Pho81 CDK抑制剂（CKI）灭活。Pho80-Pho85的调控不同于先前已表征的CDK调控机制：Pho81 CKI与Pho80-Pho85组成型结合，并且激酶失活需要一种小分子配体——肌醇七磷酸（IP7）。我们使用电泳迁移率变动分析、酶动力学和荧光光谱法研究了IP7和Pho81依赖性Pho80-Pho85失活的分子基础。我们发现IP7与Pho80-Pho85-Pho81非共价相互作用，并诱导Pho81与Pho80-Pho85之间产生额外的相互作用，从而阻止底物进入激酶活性位点。使用与Pho81对应的合成肽，我们确定了Pho81中负责与Pho80-Pho85组成型结合以及IP7调节的相互作用和抑制的区域。这些发现扩展了我们对细胞周期蛋白-CDK调控机制以及IP7作用的生化机制的理解。
