p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.
The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases.
Woo
M S
MS
Sánchez
I
I
Dynlacht
B D
BD
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D016213:Cyclins / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050704:E2F4 Transcription Factor; D006131:Growth Inhibitors; D006801:Humans; D046911:Macromolecular Substances; D009036:Moths; D009687:Nuclear Proteins / Q000502:physiology*; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011506:Proteins; D011994:Recombinant Proteins; D056465:Retinoblastoma-Binding Protein 1; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000502:physiology
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; D006131:Growth Inhibitors; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; C493844:RBL1 protein, human; C493837:RBL2 protein, human; D011994:Recombinant Proteins; D056465:Retinoblastoma-Binding Protein 1; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D050680:Transcription Factor DP1; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases