PHOSPHORYLATION OF A SPECIFIC CDK SITE IN E2F-1 AFFECTS ITS ELECTROPHORETIC MOBILITY AND PROMOTES PRB-BINDING IN-VITRO

The E2F transcription factor family participates in growth control pre sumably through transcriptional activation of genes that promote entry into S phase. E2F activity is believed to be controlled across the ce ll cycle by association with various cellular proteins, including the product of the retinoblastoma gene (pRB). We find that E2F-1 proteins are heterogeneously phosphorylated in insect cells, as a result of whi ch they migrate as a doublet on SDS-polyacrylamide gels. This electrop horetic shift is shown to be dependent upon specific phosphorylation o f E2F-1 on serine-375 (S375), near the pRB-binding site. Phosphorylati on on S375 also occurs in human cells. E2F-1 was most efficiently phos phorylated on this residue by cyclin A/cdc2 kinase, and to a lesser ex tent by cyclin A/cdk2, irrespective of the presence of the pRB-related p107 protein. Phosphorylation of E2F-1 on S375 greatly enhanced its a ffinity of PRE in vitro. These results suggest a novel way of regulati ng E2F-1 activity, namely by cell-cycle-dependent phosphorylation of t his transcription factor.