Biologic and biochemical analyses of p16(INK4a) mutations from primary tumors

Background: Point mutations in the tumor suppressor gene p16(INK4a) (also k nown as p16, CDKN2, MTS1, and INK4a) are found in many tumor types, Because the function of the products of these naturally occurring mutants has not been fully explored, we investigated the functional activities of a wide ra nge of naturally occurring p16 mutant proteins. Methods: Sixteen cancer-ass ociated p16 mutant proteins, resulting from missense mutations, were charac terized for their ability to bind and inhibit the cyclin-dependent kinases (CDK4 and CDK6) and to induce cell cycle arrest in G(1) phase. Results/ Con clusions: Among 16 mutants analyzed, nine had detectable functional defects . Three mutants (D84V, D84G, and R87P) had defects in CDK binding, kinase i nhibition, and cell cycle arrest. The corresponding mutations are located i n the third ankyrin repeat in a highly conserved region believed to form th e CDK binding cleft. Three mutants (P48L, D74N, and R87L) had defects in ki nase inhibition and cell cycle arrest, Among the 10 mutants with normal CDK binding and inhibitory activity, three mutants (N71S, R80L, and H83Y) had defects only in their ability to induce cell cycle arrest. Thus, p16 mutant proteins that retain CDK4 and CDK6 binding may have more subtle functional defects, All nine mutations leading to functional impairments mapped to th e central portion of the p16 protein. Ankyrin repeats II and III appear mor e critical to p16 function, and mutations in ankyrin repeats I and IV are l ess likely to disrupt p16 function.