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.
Lee
Young-Sam
YS
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.
Huang
Kexin
K
Quiocho
Florante A
FA
O'Shea
Erin K
EK
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