G1 cyclins control the retinoblastoma gene product growth regulation activity via upstream mechanisms.
Inactivation of the retinoblastoma gene product (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, D1, D2, and D3 each function to phosphorylate and inactivate pRb. In vivo, G1 cyclin/kinase complexes enhance the phosphorylation 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. Kinases associated with cyclins A, E, and D1 phosporylate pRb indistinguishably 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 phosphorylate pRb in vivo and in vitro and is also able to reverse the growth-inhibitory properties of pRb in intact cells. Finally, coexpression of G1 cyclins and kinases represses pRb-mediated growth inhibition in Saos-2 cells. The multiplicity of mechanisms for pRb phosphorylation and inactivation suggests that several pathways exist for the regulation of pRb by phosphorylation.
Horton
L E
LE
Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Qian
Y
Y
Templeton
D J
DJ
D002455:Cell Division; D002460:Cell Line; D019938:Cyclin D1; D016213:Cyclins / Q000378:metabolism*; D016193:G1 Phase; D005786:Gene Expression Regulation; D006801:Humans; D015513:Oncogene Proteins / Q000378:metabolism*; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism; D014616:Vaccinia virus
D016213:Cyclins; D015513:Oncogene Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1