CANCER-ASSOCIATED MIS-SENSE AND DELETION MUTATIONS IMPAIR P16(INK4) CDK INHIBITORY ACTIVITY

The p16(INK4) gene is a candidate tumour-suppressor gene which maps to the genomic locus 9p21, and mutations of this gene are associated wit h melanoma and other cancers. Biochemical studies suggest that p16(INK 4) mediates its effects by specifically inhibiting the GI cyclin-depen dent kinases CDK4 and CDK6, thereby regulating progression through GI into S phase of the cell cycle. To evaluate the functional effects of mutations in p16(INK4) which have been observed in primary cancers and cancer cell lines, we constructed a series of deletion mutants compri sing amino acid regions 9-72, 9-131, 73-131 and 73-156; a mis-sense mu tation identified in melanoma (Arg87Pro); and the polymorphism Ala 148 Thr and investigated their ability to inhibit cyclin D1/CDK4 kinase ac tivity in vitro. Removal of 25 amino acids from the carboxy terminus o f p16(INK4) (9-131) had little impact on its inhibitory activity. In c ontrast, deletion of the 65 N-terminal amino acids comprising the firs t and second ankyrin repeats of p16(INK4) (73-131) abolished its inhib itory activity. The carboxy (73-156) and amino terminal (9-72) fragmen ts of p16(INK4) also failed to inhibit cyclin D1/CDK4 activity. These results indicate that the core region (73-131) as well as amino acids N-terminal of this sequence are important, whereas sequences C-termina l of amino acid 131 are less important for the inhibitory activity of this molecule. The melanoma-associated Arg87Pro mutation resulted in l oss of inhibitory activity, whereas the Ala148Thr polymorphic variant was as effective as the alanine variant of p16(INK4) in inhibiting D1/ CDK4 kinase activity. Binding assays revealed that inhibition was inva riably associated with p16(INK4) binding to CDK4. Hence, our studies i ndicate that minor perturbations in p16(INK4) primary structure can le ad to loss of its inhibitory activity, possibly contributing to oncoge nesis in numerous cell types. (C) 1996 Wiley-Liss, Inc.