Cumulative Effect of Phosphorylation of pRB on Regulation of E2F Activity

Abstract

The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule.

ACKNOWLEDGMENTS

This work was supported in part by the National Cancer Institute of Canada with funds from the Terry Fox Run, the Medical Research Council of Canada, and Apotex Inc. through the University-Industry Program.

We thank Paul Hamel, Eldad Zachsenhaus, and Rod Bremner for helpful discussions. V. Brown especially thanks Sanja Pajovic for technical help with the gel mobility shift assays, P. Hamel for the gift of ΔBXHA, Δp34HA, ΔP1,2HA, ΔP3,4HA, and ΔP1,2,3,4HA, and E. Zachsenhaus for the gift of ΔK11.