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 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.

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Figure 1: Reversible regulation of Pho80-Pho85-Pho81-MD by IP7.
Figure 2: Characterization of IP7 binding to Pho80-Pho85-Pho81-MD.
Figure 3: Enzyme kinetic analysis of IP7-mediated Pho80-Pho85-Pho81-MD inactivation.
Figure 4: Additional interaction between Pho81-MD and Pho80-Pho85.
Figure 5: Dissection of Pho81-MD into binding and inhibitory segments.
Figure 6: Model of Pho80-Pho85 regulation by 4/6-IP7 and Pho81.

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Acknowledgements

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.).

Author information

Y.-S.L. and E.K.O. designed experiments, Y.-S.L. carried out experiments, Y.-S.L. and E.K.O. interpreted data and wrote the manuscript. K.H. and F.A.Q. designed Pho81 peptides and carried out preliminary experiments to study Pho80-Pho85 binding.

Correspondence to Erin K O'Shea.

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Lee, Y., Huang, K., Quiocho, F. et al. Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate. Nat Chem Biol 4, 25–32 (2008) doi:10.1038/nchembio.2007.52

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