Gi Cyclins Control the Retinoblastoma Gene Product Growth Regulation Activity via Upstream Mechanisms1

Inactivation of the retinoblastoma gene produd (pRb) occurs concomitant with the appearance of its hyperphosphorylated form in mid to late G1 . Multiple cyclin/CDK complexes are implicated in the cell cycle phosphorylation of pRb. Using in vivo expression systems, we show that cyclins A, E, , D2, and D3 each function to phosphorylate and inactivate pRb. In vivo, Gi cyclin/kinase complexes enhance the phosphorylation of pRb, and these effeds of cyclin/kinases on pRb can be overcome by the addition of p21 , a wide spedrum inhibitor of Gi kinases. Kinases associated with cyclins A, E, and Dl phosphorylate pRb indistinguishably in vivo, according to proteoiytic maps. Although cyclin Dl has been reported to bind to pRb diredly, requiring the pRb-binding motif LXCXE, a mutant Dl lacking the pRb-binding motif remains able to phosphorylate pRb in vivo and in vitro and is also able to reverse the growth-inhibitory properties of pRb in intad cells. Finally, coexpression of Gi cyclins and kinases represses pRb-mediated growth inhibition in Saos-2 cells. The multiplicity of mechanisms for pRb phosphorylation and inadivation suggests that several pathways exist for the regulation of pRb by phosphorylation.