BIOCHEMICAL AND MUTAGENIC ANALYSIS OF THE MELANOMA TUMOR-SUPPRESSOR GENE-PRODUCT P16

P16 was originally discovered by its ability to interact with CDK4 and to specifically inhibit the catalytic activity of the CDK4/D1 kinase. Increased attention has focused on the p16 gene because of its locati on on chromosome 9p21, a region involved in chromosomal rearrangements in a large number of tumor types. The p16 gene is also mutated in a l arge number of tumor cell lines and primary tumor cells. Furthermore, linkage analysis studies suggest that the p16 gene is involved in fami lial melanoma susceptibility. Due to the oncogenic potential of mutati ons in this tumor suppressor, it is important to identify and characte rize those mutations which alter p16 activity. We have performed a sys tematic analysis of melanoma associated p16 mutants and of mutants gen erated in charge to Ala mutagenesis. Using microtiter plate assays to measure both p16-cdk4 binding and cdk4/D1 kinase activity, we show her e that the melanoma associated mutants are defective, as are some of t he Ala mutants. These results support the idea that p16 mutation, via its deregulation of the cdk4/D1 pathway, is of biological significance in the development of melanoma. Furthermore, we have defined a region within the p16 molecule in which changes are likely to result in a de fective protein.