Cumulative effect of phosphorylation of pRB on regulation of E2F activity

The product of the retinoblastoma susceptibility gene, pRB, is a nuclear ph osphoprotein that controls cell growth by binding to and suppressing the ac tivities of transcription factors such as the E2F family. Transactivation a ctivity is inhibited when E2F is bound to hypophosphorylated pRB and releas ed when pRB is phosphorylated by cyclin-dependent kinases (CDKs), To determ ine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F i nteraction, mutant PRE 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 reg ulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phos phate groups on the RE molecule.