Cdk2-dependent phosphorylation and functional inactivation of the pRB-related p130 protein in pRB(-), p16INK4A(+) tumor cells.
The retinoblastoma family proteins pRB, p107, and p130 are phosphorylated and released from E2Fs in the late G(1) phase of the cell cycle. This phosphorylation is thought to contribute to the derepression of E2F-responsive genes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 activity is inhibited by p16(INK4A) in most pRB(-) cells suggests that p107 and 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 phosphorylated and released from E2F-4 in late G(1) and S phase cells, although p130 phosphorylation differed qualitatively in these and other pRB(-), p16(+) cells as compared with pRB(+), p16(-) cell types. p130 phosphorylation occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyclin 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-dependent transcription. These findings show that p130 can be phosphorylated and functionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family proteins.
Cheng
L
L
Departments of Medicine and Pathology, and the Institute of Cancer Genetics, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Rossi
F
F
Fang
W
W
Mori
T
T
Cobrinik
D
D
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000378:metabolism*; D002453:Cell Cycle; D016895:Culture Media, Serum-Free; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D050704:E2F4 Transcription Factor; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000172:deficiency*; D050717:Retinoblastoma-Like Protein p130; D016196:S Phase; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D019927:Cyclin E; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D050704:E2F4 Transcription Factor; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D050717:Retinoblastoma-Like Protein p130; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases