Identification of CDK4 sequences involved in cyclin D1 and p16 binding.
Activation of CDK4 is regulated, in part, by its association with a D-type cyclin. Conversely, CDK4 activity is inhibited when it is bound to the cyclin-dependent kinase inhibitor, p16(INK4A). To investigate the molecular basis of the interactions between CDK4 and cyclin D1 or p16(INK4A) we performed site-directed mutagenesis of CDK4. The interaction was examined using in vitro translated wild type and mutant CDK4 proteins and bacterially expressed cyclin D1 and p16 fusion proteins. As mutational analysis of CDC2 suggests that its cyclin binding domain is primarily located near its amino terminus, the majority of the mutations constructed in CDK4 were located near its amino terminus. In addition, CDK4 residues homologous to CDC2 sites involved in Suc1 binding were also mutated. Our analysis indicates that cyclin D1 and p16 binding sites are overlapping and located primarily near the amino terminus. All CDK4 mutations that resulted in decreased p16 binding capability also diminished cyclin D1 binding. In contrast, amino-terminal sequences were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding.
Coleman
K G
KG
Department of Biomolecular Drug Discovery/Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Wautlet
B S
BS
Morrissey
D
D
Mulheron
J
J
Sedman
S A
SA
Brinkley
P
P
Price
S
S
Webster
K R
KR
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015513:Oncogene Proteins / Q000378:metabolism*; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins
D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016213:Cyclins; D004791:Enzyme Inhibitors; D015513:Oncogene Proteins; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D019938:Cyclin D1; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases