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.
Mesh Terms:
Cyclin-Dependent Kinases, Cyclins, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Inositol Phosphates, Protein Binding, Recombinant Proteins, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Substrate Specificity, Transcription Factors
Cyclin-Dependent Kinases, Cyclins, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Inositol Phosphates, Protein Binding, Recombinant Proteins, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Substrate Specificity, Transcription Factors
Nat. Chem. Biol.
Date: Jan. 01, 2008
PubMed ID: 18059263
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