TEMPERATURE-SENSITIVE MUTANTS OF P16CDKN2 ASSOCIATED WITH FAMILIAL MELANOMA

Altered expression or function of the p16(CDKN2) tumor suppressor gene on chromosome 9p21 occurs in a wide range of human tumors, and mutati ons in the gene have been shown to segregate with familial predisposit ion to malignant melanoma. We have used a variety of assays to examine the functional properties of tumor-associated alleles, including eigh t premature termination mutants, eight missense mutants, and three iso forms of p16 initiated at different amino-terminal methionine codons. The amino- and carboxy-terminal domains of the protein, outside the an kyrin-like repeats, appeared to be dispensable, but the majority of th e premature termination mutations led to loss of function. Of the miss ense mutations tested, four displayed clear loss of function whereas t wo behaved like the wild type under all conditions tested. The remaini ng two mutations, a G-to-W mutation at position 101 (G101W) and V126D, both of which are associated with familial melanoma, were found to be temperature sensitive for binding to Cdk4 and Cdk6 in vitro, for inhi biting cyclin D1-Cdk4 in a reconstituted pRb-kinase assay, and for inc reasing the proportion of G(1)-phase cells following transfection. The se findings clarify previous disparities and argue strongly that p16(C DKN2) is a bona fide tumor suppressor associated with familial melanom a.