Several noncontiguous domains of CDK4 are involved in binding to the P16 tumor suppressor protein.
Cyclin-dependent kinase 4 (CDK4) is a key molecule in the regulation of cell cycle progression at the G1-S phase restriction point. Its activity is specifically regulated by p16 (also known as p16/CDKN2A, p16(INK4a), and MTS1), a tumor suppressor frequently altered in human cancers. A specific mutation in CDK4 codon 24 (Arginine to Cysteine) prevents p16 binding and thus inhibition by p16. This mutated CDK4 acts as a dominant oncogene and has been found in both sporadic and familial melanoma. To study the effects of other mutations in CDK4, we generated a panel of 18 CDK4 mutants using Charged-to-Alanine scanning mutagenesis, and investigated the p16-binding capacity of these mutants to identify novel sites involved in p16 binding. The mutant CDK4 proteins were generated by direct coupled transcription-translation in vitro and tested for binding to p16 using a p16-GST fusion protein. Several mutants demonstrated loss of p16 binding. In addition to the previously identified codon 24 mutants, alterations in and around codon 22, 25, 97, and 281 all showed loss of p16 binding capacity. These results indicate that several noncontiguous amino acid sequences on CDK4 are required for binding to p16, which suggests the existence of multiple sites of interaction with p16. Since p16-binding deficient CDK4 has oncogenic potential, these mutations may be present in melanomas or other human neoplasms.
Department of Radiotherapy, Academic Medical Center, Amsterdam, The Netherlands.
H M
HM
Ceha
I
I
Nasser
R H
RH
Medema
R J
RJ
Slebos
D001120:Arginine / Q000235:genetics; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics
D019941:Cyclin-Dependent Kinase Inhibitor p16; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D001120:Arginine; D005982:Glutathione Transferase; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D003545:Cysteine