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An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein

Nature Structural & Molecular Biology volume 17, pages 1051–1057 (2010)Cite this article

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Abstract

The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking.

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Figure 1: Rb880–892 is necessary and sufficient for PP1c association.
Figure 2: Structure of the Rb870–882–PP1c complex.
Figure 3: The RbC KLRF docking sequence is required for efficient dephosphorylation by PP1c.
Figure 4: PP1c inhibits Cdk2-CycA activity toward RbC.
Figure 5: PP1c inhibits Cdk inactivation of Rb independently of phosphatase activity.
Figure 6: Abundant Rb–PP1c complexes during PP1c-dependent growth arrest.

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Acknowledgements

The authors acknowledge the staff at Beamline 5.0.1 of the Advanced Light Source (Lawrence Berkeley National Laboratories) and are grateful to N. Dyson (Massachusetts General Hospital) for CMV-CycA and T. Pawson (MSHRI, Toronto) for the CMV-myc-PP1c plasmid. The Rb 4.1 hybridoma developed by J. Sage was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the US National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biology. This work is supported by grants from the Canadian Institutes of Health Research (MOP89765 to F.A.D.) and the US National Institutes of Health (R01CA132685 to S.M.R.). A.H. is supported by a US National Institutes of Health training grant (T32GM008646). M.C. acknowledges the Canadian Institutes of Health Research for an M.D./Ph.D. studentship award and the CaRTT training program. A.H. and S.M.R. thank the Santa Cruz Cancer Benefit Group for their support. F.A.D. thanks the Canadian Cancer Society for a Research Scientist Award. S.M.R. is a Pew Scholar in the Biomedical Sciences.

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Authors and Affiliations

  1. Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California, USA

    Alexander Hirschi

  2. and Department of Biochemistry, London Regional Cancer Program, Children's Health Research Institute, University of Western Ontario, London, Ontario, Canada

    Matthew Cecchini & Frederick A Dick

  3. Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA

    Rachel C Steinhardt, Michael R Schamber & Seth M Rubin

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  1. Alexander Hirschi
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Contributions

A.H., M.C., F.A.D. and S.M.R. all designed aspects of the study; A.H., M.C., R.C.S., M.R.S. and S.M.R. performed experiments; all authors analyzed data; F.A.D. and S.M.R. wrote the manuscript.

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Correspondence to Seth M Rubin.

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Hirschi, A., Cecchini, M., Steinhardt, R. et al. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein. Nat Struct Mol Biol 17, 1051–1057 (2010). https://doi.org/10.1038/nsmb.1868

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