The nuclear protein p34SEI-1 regulates the kinase activity of cyclin-dependent kinase 4 in a concentration-dependent manner.
Previous studies have shown that p34(SEI-1), also known as TRIP-Br1, is involved in cell cycle regulations by interacting with a number of important proteins including CDK4. However, the detailed mechanism and structural basis of the interaction remains to be determined. We report the use of in vitro studies to address these problems. First, it was shown that p34(SEI-1) binds to CDK4 directly, and the binding does not compete directly with p16. In the presence of p16, a quaternary complex is formed between p34(SEI-1), CDK4, cyclin D2, and p16. Second, it was found that p34(SEI-1) activates the kinase activity of CDK4 at lower concentrations (reaching the maximum at 500 nM) but inhibits the same activity at higher concentrations, implying that p34(SEI-1)-mediated CDK4 activation is dose-dependent. Again, the effects of p34(SEI-1) and p16 are independent of each other. Third, it was shown that p34(SEI-1) possesses a LexA-mediated transactivation activity. Finally, a set of truncation mutants were used to dissect the structural elements responsible for the different functions of p34(SEI-1). The results indicate that the fragment 30-160 can bind, activate, and inhibit CDK4; the fragment 30-132 can bind and activate but does not inhibit CDK4, while the fragment 30-88 cannot bind, activate, or inhibit but retains the LexA-mediated transactivation activity.
Li
Junan
J
Surgery Department, The Ohio State University, Columbus, Ohio 43210, USA.
Melvin
W Scott
WS
Tsai
Ming-Daw
MD
Muscarella
Peter
P
D020816:Amino Acid Motifs; D017487:Ankyrins / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001667:Binding, Competitive; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D056742:Cyclin D2; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000037:antagonists & inhibitors / Q000737:chemistry; D050763:Cyclin-Dependent Kinase Inhibitor p18; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D016213:Cyclins / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D016356:Gene Products, tax / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015513:Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D012697:Serine Endopeptidases / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors; D015533:Transcriptional Activation; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism; D000099160:LexA Repressor Protein
D017487:Ankyrins; D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D056742:Cyclin D2; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D018844:Cyclin-Dependent Kinases; D016213:Cyclins; D004791:Enzyme Inhibitors; D016356:Gene Products, tax; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D010446:Peptide Fragments; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins; D012697:Serine Endopeptidases; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000099160:LexA Repressor Protein; C080336:CCND2 protein, human; C493971:CDKN2C protein, human; C403591:PSMD10 protein, human; C402464:SERTAD1 protein, human; C495900:CDK4 protein, human