Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

Young Sam Lee; Kexin Huang; Florante A. Quiocho; Erin K. O'Shea

doi:10.1038/nchembio.2007.52

Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

Young Sam Lee, Kexin Huang, Florante A. Quiocho, Erin K. O'Shea

Molecular and Cellular Biochemistry

Producción científica: Article › revisión exhaustiva

135 Citas (Scopus)

Resumen

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 IP ₇-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP₇ action.

Idioma original	English
Páginas (desde-hasta)	25-32
Número de páginas	8
Publicación	Nature Chemical Biology
Volumen	4
N.º	1
DOI	https://doi.org/10.1038/nchembio.2007.52
Estado	Published - ene 2008

Nota bibliográfica

Funding Information:
We thank J. York (Duke University Medical School) for plasmids encoding Vip1 and hIP6K, I. Carter-O’Connell for protein preparation, O’Shea lab members for helpful discussions and comments on the manuscript, and D. Kahne (Harvard University) for access to equipment. This work was supported by the Helen Hay Whitney Foundation (Y.S.L.), Welch Foundation grant Q0581 (F.A.Q), US National Institutes of Health grant R01 GM051377, the David and Lucile Packard Foundation and the Howard Hughes Medical Institute (E.K.O.).

Financiación

We thank J. York (Duke University Medical School) for plasmids encoding Vip1 and hIP6K, I. Carter-O’Connell for protein preparation, O’Shea lab members for helpful discussions and comments on the manuscript, and D. Kahne (Harvard University) for access to equipment. This work was supported by the Helen Hay Whitney Foundation (Y.S.L.), Welch Foundation grant Q0581 (F.A.Q), US National Institutes of Health grant R01 GM051377, the David and Lucile Packard Foundation and the Howard Hughes Medical Institute (E.K.O.).

Financiadores	Número del financiador
National Institutes of Health (NIH)
David and Lucile Packard Foundation
Howard Hughes Medical Institute
National Institute of General Medical Sciences DP2GM119177 Sophie Dumont National Institute of General Medical Sciences	R01GM051377
Welch Foundation	Q0581
Helen Hay Whitney Foundation

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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title = "Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate",

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 IP 7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action.",

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N2 - 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 IP 7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action.

AB - 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 IP 7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action.

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Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

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Nota bibliográfica

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