THE CONSENSUS MOTIF FOR PHOSPHORYLATION BY CYCLIN D1-CDK4 IS DIFFERENT FROM THAT FOR PHOSPHORYLATION BY CYCLIN A E-CDK2/

Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in ph osphorylation of the retinoblastoma protein (pRB) during the G(1)/S tr ansition of the cell cycle. However, it is unclear why several Cdks ar e needed and how they are different from one another. We found that th e consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for pho sphorylation by cyclin A/E-Cdk2 using various synthetic peptides as su bstrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RP PTLS(780)PIP- that contained a part of the sequence of pRB, while cycl ins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against p hospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser78O in pRB w as phosphorylated in the GI phase in a cell cycle-dependent manner, Fu rthermore, we found that pRB phosphorylated at Ser780 cannot bind to E 2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin, A/E Cdk2 p hosphorylate different sites of pRB in vivo.