G1 CYCLINS CONTROL THE RETINOBLASTOMA GENE-PRODUCT GROWTH-REGULATION ACTIVITY VIA UPSTREAM MECHANISMS

Inactivation of the retinoblastoma gene product (pRb) occurs concomita nt with the appearance of its hyperphosphorylated form in mid to late G(1). Multiple cyclin/CDK complexes are implicated in the cell cycle p hosphorylation of pRb. Using in vivo expression systems, we show that cyclins A, E, D1, D2, and D3 each function to phosphorylate and inacti vate pRb. In vivo, G1 cyclin/kinase complexes enhance the phosphorylat ion of pRb, and these effects of cyclin/kinases on pRb can be overcome by the addition of p21, a wide spectrum inhibitor of G1 kinases. Kina ses associated with cyclins A, E, and D1 phosphorylate pRb indistingui shably in vivo, according to proteolytic maps. Although cyclin D1 has been reported to bind to pRb directly, requiring the pRb-binding motif LXCXE, a mutant D1 lacking the pRb-binding motif remains able to phos phorylate pRb in vivo and in vitro and is also able to reverse the gro wth-inhibitory properties of pRb in intact cells. Finally, coexpressio n of G1 cyclins and kinases represses pRb-mediated growth inhibition i n Saos-2 cells. The multiplicity of mechanisms for pRb phosphorylation and inactivation suggests that several pathways exist for the regulat ion of pRb by phosphorylation.