Distinct phosphorylation events regulate p130- and p107-mediated repression of E2F-4.
The "pocket proteins" pRb (retinoblastoma tumor suppressor protein), p107, and p130 regulate cell proliferation via phosphorylation-sensitive interactions with E2F transcription factors and other proteins. We previously identified 22 in vivo phosphorylation sites in human p130, including three sites selectively targeted by cyclin D-Cdk4(6) kinases. Here we assessed the effects of alanine substitution at the individual or combined Cdk4(6)-specific sites in p130, compared with homologous sites in p107 (Thr(369)/Ser(650)/Ser(964)). In U-2-OS cells, the triple p107(DeltaCdk4)* mutant strongly inhibited E2F-4 activity and imposed a G(1) arrest resistant to cyclin D1 coexpression. In contrast, the p130(DeltaCdk4) mutant still responded to cyclin D1, suggesting the existence of additional phosphorylation sites critical for E2F-4 regulation. Extensive mutagenesis, sensitive E2F reporter assays, and cell cycle analyses allowed the identification of six such residues (serines 413, 639, 662, 1044, 1080, and 1112) that, in addition to the Cdk4-specific sites, are necessary and sufficient for the regulation of E2F-4 and the cell cycle by p130. Surprisingly, 12 of the in vivo phosphorylation sites seem dispensable for E2F regulation and probably modulate other functions of p130. These results further elucidate the complex regulation of p130 and provide a molecular mechanism to explain the differential control of p107 and p130 by cyclin-dependent kinases.
Farkas
Thomas
T
Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49, Copenhagen DK-2100, Denmark.
Hansen
Klaus
K
Holm
Karin
K
Lukas
Jiri
J
Bartek
Jiri
J
D001665:Binding Sites; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D019938:Cyclin D1 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050704:E2F4 Transcription Factor; D005434:Flow Cytometry; D016193:G1 Phase; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008954:Models, Biological; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D050704:E2F4 Transcription Factor; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D011506:Proteins; C493844:RBL1 protein, human; C493837:RBL2 protein, human; D011993:Recombinant Fusion Proteins; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D014157:Transcription Factors; D019938:Cyclin D1; D005982:Glutathione Transferase