Phosphorylation of the retinoblastoma protein by cdk2.

Abstract

The retinoblastoma gene product (the RB protein) is phosphorylated In a cell cycle-dependent manner and this modification is believed to be important for cells to progress through the cell cycle. We found that purified cdk2 (cyclin-dependent kinase/cell division kinase 2) can phosphorylate the RB protein in vitro at the sites phosphorylated in the cell. The timing ofactivation ofcdk2 in the cell cycle was similar (3)(4)(5). The RB protein also interacts specifically with several cellular proteins, including transcription factor E2F and the protooncogene product Myc (6-10). Thus, the normal function of the RB protein may involve formation of complexes with cellular proteins. Recently, microinjection of the RB protein into cells early in the G1 phase of the cell cycle was found to inhibit progression to the S phase, suggesting that the RB protein regulates cell proliferation by restricting cell cycle progression at a specific point in the G1 phase (11). The function of the RB protein is thought to be regulated mainly by phosphorylation. The RB protein has been shown to be phosphorylated in a cell cycle-dependent manner, being underphosphorylated in Go/Gj and highly phosphorylated during the G1 to S transition (12-16). Interestingly, only underphosphorylated RB protein can form a complex with large T antigen (17) and transcription factor E2F (7). The underphosphorylated RB protein is tightly associated with the nucleus, whereas the hyperphosphorylated form is easily extracted under the same conditions, suggesting that the underphosphorylated RB protein interacts tightly with nuclear proteins (18,19). These findings suggest that underphosphorylated RB protein inhibits transition ofcells from G, to S and that phosphorylation is a regulatory event leading to inactivation of the growth-suppressing activity of the RB protein.The RB protein possesses a number of consensus sequences for phosphorylation by cdc2 kinase. In fact, cdc2 kinase phosphorylates the RB protein in vitro at the same sites as those phosphorylated in living cells, and the amino acid sequences of the five sites phosphorylated in the cells were recently shown to correspond closely to the consensus sequence for phosphorylation by cdc2 kinase (20-23). However, cdc2 kinase is known to be active in the late G2 and M phases (24), whereas the RB protein becomes highly phosphorylated at about the G1 to S transition. On the other hand, although in yeast a single cdc2 kinase regulates both the G, to S and G2 to M transitions (24)

Co-amplification and overexpression of CDK4, SAS and MDM2 occurs frequently in human parosteal osteosarcomas

“…CDK2 is similar to CDK4 in that it phosphorylates pRb at the G1/S transition leading to cell cycle progression, and could therefore provide tumor cells with a proliferative advantage (Akiyama et al, 1992;Tsai et al, 1993;Demetrick et al, 1994). In contrast to CDK4, we did not observe CDK2 ampli®cation or overexpression in any OSA, including those samples with ampli®cation of other 12q13-15 genes.…”

BCL-6 is phosphorylated at multiple sites in its serine- and proline-clustered region by mitogen-activated protein kinase (MAPK) in vivo

“…at 48C for 20 min, as described previously (Akiyama et al, 1992). Lysates were incubated with the required antibodies for 1 h at 48C and the resulting immunocomplexes were adsorbed to protein G-Sepharose (Pharmacia), washed thoroughly with solubilizing bu er and subjected to SDS ± PAGE.…”

The role of p27Kip1 in gamma interferon-mediated growth arrest of mammary epithelial cells and related defects in mammary carcinoma cells

“…Which cyclin D isoform is the most active and/ or abundant appears to be dependent in part on cell type speci®city (Ewen et al, 1993a and references therein;Sherr, 1995). Cdk2 in complex with cyclin E, and later cyclin A, may also participate in maintaining the hyperphosphorylation of Rb in later stages of G 1 and S phase when the complexes are active, as Rb is a substrate for these complexes both in vitro (Akiyama et al, 1992) and in vivo (Hinds et al, 1992;Dowdy et al, 1993;. Evidence has been presented indicating that cdk2/cyclin E-speci®c phosphorylation of Rb, apart from cdk4/6/cyclin D activity, may not merely maintain Rb hyperphosphorylation but is essential for entry into S phase (Hatakeyama et al, 1994).…”