Identification of cdk2 binding sites on the p27Kip1 cyclin-dependent kinase inhibitor.
A cdk2 binding domain on p27Kip1 located within the sequence of amino acids 53-85 was further characterized by generating a series of point mutations within amino acid residues 62-75. Two regions, FDF (residues 62-64) and GXY (residues 72 and 74), were identified within the beta hairpin region of p27Kip1. Mutations within these regions essentially completely inhibited the binding to in vitro translated cdk2 and cdk2/cyclin E complexes formed in vitro or in vivo. The p27Kip1 GST-fusion protein of the point mutation that replaces phenylalanine at residue 64 to alanine (F64A) showed approximately twofold less inhibition of cdk2 kinase activity. The cellular response to the introduction of the F64A mutant form of p27Kip1 was compared to that of p27Kip1 wild type by transfecting HeLa cells with constructs of full length sense and antisense coding sequences. Overexpression of the F64A mutant form of p27Kip1 bound significantly lower levels of cdk2 as compared to wild type and did not affect the cdk2 related kinase activity of the transfected HeLa cells. Overexpression of wild type p27Kip1 resulted in a reduction of the level of cdk2 kinase activity and effectively suppressed the growth of the transfected HeLa cells.
Kwon
T K
TK
Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Maryland 21224, USA.
Nordin
A A
AA
D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002455:Cell Division; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases