Cdk2-dependent phosphorylation and functional inactivation of the pRB-related p130 protein in pRB(-), p16(INK4A)(+) tumor cells

The retinoblastoma family proteins pRB, p107, and p130 are phosphorylated a nd released from E2Fs in the late G(1) phase of the cell cycle. This phosph orylation is thought to contribute to the derepression of E2F-responsive ge nes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 act ivity is inhibited by p16(INK4A) in most pRB(-) cells suggests that p107 an d p130 may be underphosphorylated and remain associated with E2Fs during G( 1)-S progression in cells that lack pRB. To examine this, we evaluated the cell cycle-dependent phosphorylation and E2F binding abilities of p107 and p130 in pRB(-), p16(+) Saos-2 osteosarcoma cells. p130, but not p107, was p hosphorylated and released from E2F-4 in late G(1) and S phase cells, altho ugh p130 phosphorylation differed qualitatively in these and other pRB(-), p16(+) cells as compared with pRB(+), p16(-) cell types. p130 phosphorylati on occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyc lin E- and Cdk2-associated kinase activity, and was prevented by expression of dominant negative Cdk2. Moreover, dominant negative Cdk2 prevented the dissociation of endogenous p130-E2F-4 complexes and inhibited E2F-4-depende nt transcription. These findings show that p130 can be phosphorylated and f unctionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family pro teins.