Cell cycle regulation of retinoblastoma protein phosphorylation.

The product of the retinoblastoma susceptibility gene (Rb) is a substrate of the cell cycle-regulated cdc2 and cdk kinases. The Rb protein is phosphorylated from S through M phases of the cell cycle and is dephosphorylated in G1. In in vivo phosphorylated Rb protein, we have found ten phosphotryptic peptides, all of which could be phosphorylated by cdc2 kinase, p34cdc2, in vitro. The sites of phosphorylation for eight of the ten peptides have been mapped and they conform to the known p34cdc2 phosphorylation consensus. Although the activated p34cdc2 in mitotic cells is the major phosphorylation enzyme for Rb, the Rb kinase activity of p34cdc2 is not activated at G1/S transition. A cyclin A/p33 complex is activated at G1/S. We have assembled active cyclin B1/p34cdc2 complex in insect cells. The insect cell-derived kinase complex phosphorylates histone H1 well but exhibits a poor Rb kinase activity. These results indicate that the retinoblastoma protein is phosphorylated by distinct cyclin/kinase complexes in the cell cycle and suggest a regulation of the substrate specificity of the p34cdc2/cyclin complex.