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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Cyclin-Dependent Kinases / antagonists & inhibitors*
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Cyclins / antagonists & inhibitors*
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DNA-Binding Proteins / antagonists & inhibitors
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Electrophoretic Mobility Shift Assay
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Inositol Phosphates* / metabolism
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Inositol Phosphates* / pharmacology
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Inositol Phosphates* / physiology
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Protein Binding
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Recombinant Proteins / metabolism
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Recombinant Proteins / pharmacology
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Repressor Proteins / antagonists & inhibitors*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / antagonists & inhibitors*
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Substrate Specificity
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Transcription Factors / antagonists & inhibitors
Substances
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Cyclins
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DNA-Binding Proteins
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Inositol Phosphates
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PHO4 protein, S cerevisiae
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PHO80 protein, S cerevisiae
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PHO81 protein, S cerevisiae
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Recombinant Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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inositol heptakisphosphate
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Cyclin-Dependent Kinases
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PHO85 protein, S cerevisiae
Associated data
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PubChem-Substance/26740886
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PubChem-Substance/26740887
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PubChem-Substance/26740888
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PubChem-Substance/26740889
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PubChem-Substance/26740890
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PubChem-Substance/26740891