Cancer-associated mis-sense and deletion mutations impair p16(INK4) CDK inhibitory activity

Abstract

The p16(INK4) gene is a candidate tumour-suppressor gene which maps to the genomic locus 9p21, and mutations of this gene are associated with melanoma and other cancers. Biochemical studies suggest that p16(INK4) mediates its effects by specifically inhibiting the G1 cyclin-dependent kinases CDK4 and CDK6, thereby regulating progression through G1 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 comprising amino acid regions 9-72, 9-131, 73-131 and 73-156; a mis-sense mutation identified in melanoma (Arg87Pro); and the polymorphism Ala148Thr and investigated their ability to inhibit cyclin D1/CDK4 kinase activity in vitro. Removal of 25 amino acids from the carboxy terminus of p16(INK4) (9-131) had little impact on its inhibitory activity. In contrast, deletion of the 65 N-terminal amino acids comprising the first and second ankyrin repeats of p16(INK4) (73-131) abolished its inhibitory activity. The carboxy (73-156) and amino terminal (9-72) fragments 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-terminal of amino acid 131 are less important for the inhibitory activity of this molecule. The melanoma-associated Arg87Pro mutation resulted in loss 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 invariably associated with p16(INK4) binding to CDK4. Hence, our studies indicate that minor perturbations in p16(INK4) primary structure can lead to loss of its inhibitory activity, possibly contributing to oncogenesis in numerous cell types.