Phosphorylation-dependent and -independent functions of p130 cooperate to evoke a sustained G1 block.
The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb and p130 operate through interactions with cellular proteins, including the E2F transcription factors. While such interactions are controlled by phosphorylation of multiple sites of pRb, regulation of p130 remains poorly understood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4(6) [Cdk4(6)] specific phosphorylations, which appear critical for controlling the growth-restraining activity of p130. When expressed in U2OS cells, the phosphorylation-deficient mutant p130(Delta)(CDK4), in which the Cdk4 specific sites were mutated to alanine residues, imposed a more sustained G1 arrest than a constitutively active pRb(Delta)(CDK), known to repress all cellular E2F activity. Experiments using p130(Delta)(Cdk4) and another phosphorylation-deficient mutant, p130(PM19A), with 19 phosphorylation sites mutated, revealed that the p130-imposed G1 block reflects cooperative growth-suppressive effects of phosphorylation-regulated E2F binding and phosphorylation-independent sequestration of cyclin E(A)-Cdk2 through the N-terminal cyclin binding motif of p130.
Hansen
K
K
Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark.
Farkas
T
T
Lukas
J
J
Holm
K
K
Rönnstrand
L
L
Bartek
J
J
D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D056741:Cyclin D; D019927:Cyclin E / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016193:G1 Phase / Q000502:physiology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050717:Retinoblastoma-Like Protein p130; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056741:Cyclin D; D019927:Cyclin E; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D010750:Phosphoproteins; D011506:Proteins; C493837:RBL2 protein, human; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050717:Retinoblastoma-Like Protein p130; D050680:Transcription Factor DP1; D014157:Transcription Factors; D018844:Cyclin-Dependent Kinases