Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate.
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, Faculty of Arts and Sciences Center for Systems Biology, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA.
Young-Sam
YS
Lee
Kexin
K
Huang
Florante A
FA
Quiocho
Erin K
EK
O'Shea
D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000037:antagonists & inhibitors*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D024202:Electrophoretic Mobility Shift Assay; D007295:Inositol Phosphates / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012097:Repressor Proteins / Q000037:antagonists & inhibitors*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000037:antagonists & inhibitors
D016213:Cyclins; D004268:DNA-Binding Proteins; D007295:Inositol Phosphates; C060292:PHO4 protein, S cerevisiae; C066824:PHO80 protein, S cerevisiae; C081227:PHO81 protein, S cerevisiae; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C402123:inositol heptakisphosphate; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae