Article abstract
Nature Chemical Biology 4, 25 - 32 (2008)
Published online: 25 November 2007 | doi:10.1038/nchembio.2007.52
Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate
Young-Sam Lee1, Kexin Huang2, Florante A Quiocho2 & Erin K O'Shea1
Abstract
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.
- Howard Hughes Medical Institute, Harvard University, Department of Molecular and Cellular Biology and Department of Chemistry and Chemical Biology, Faculty of Arts and Sciences Center for Systems Biology, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA.
- Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Room T-517, Houston, Texas 77030, USA.
Correspondence to: Erin K O'Shea1 e-mail: erin_oshea@harvard.edu
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