An Overlapping Kinase and Phosphatase Docking Site Regulates Activity of the Retinoblastoma Protein

Insights into the molecular mechanisms that regulate the phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are fundamental to understanding the control of cell proliferation. While much focus has been placed upon regulation of Cyclin-dependent kinase (Cdk) activity towards Rb, less is known about Rb dephosphorylation catalyzed by the major Rb phosphatase, protein phosphatase-1 (PP1). Using x-ray crystallography, we have determined the crystal structure of a PP1:Rb peptide complex to 3.2A that reveals an overlapping kinase and phosphatase docking site. Kinetic assays show that Cdk and PP1 docking to Rb are mutually exclusive and that this docking site is required for efficient dephosphorylation, as well as phosphorylation of Rb. Cell cycle arrest assays demonstrate that the ability of PP1 to compete with Cdks is sufficient to retain Rb activity and block cell cycle advancement. These results establish a novel mechanism for the regulation of Rb phosphorylation state in which kinase and phosphatase compete for substrate docking.