CLONING AND CHARACTERIZATION OF MURINE P16(INK4A) AND P15(INK4B) GENES

Progression through the G1 phase of the cell cycle is regulated in part by the D-type cyclin-dependent kinases, cdk4 and cdk6. Genes encoding two specific inhibitors of these kinases, human p16((INK4A/MTSI)) and p15((INK4B/MTS2)), map to a region of common cytogenetic abnormalities on chromosome 9p21. The murine cognates of these genes were isolated and identified as mouse p16(INK4a) and p15(INK4b) based on their homology to their human counterparts and their selective transcriptional induction by SV40T-antigen and TGF-beta, respectively. Both genes map to position C3-C6 on mouse chromosome 4, in a region syntenic with human chromosome 9p. Amplification of polyadenylated mRNA by polymerase chain reactions revealed no expression of mouse p16(INK4a) in many normal tissues, whereas p15(INK4b) was expressed ubiquitously. Like human p16(INK4a), mouse p16(INK4a) binds specifically to cdk4 and cdk6 in vitro and inhibits the phosphorylation of the retinoblastoma protein, pRb, by each of these cyclin D-dependent kinases. In mouse MEL erythroleukemia cells, p16(INK4a) associates preferentially with cdk6 under conditions where cdk4 and cdk6 are coexpressed at equivalent levels. Expression vectors encoding human or mouse p16(INK4a) caused G1 phase arrest in NIH3T3 fibroblasts, and cyclin D1- and cdk4-dependent pRb kinase activities were inhibited in the p16(INK4a)-arrested cells.