Structural consequences of tumor-derived mutations in p16INK4a probed by limited proteolysis

The cyclin-dependent kinase inhibitor p16(INK4a), (hereafter p16) functions as a multiple tumor suppressor. Mutations in p16, which are distributed throughout the entire protein, have been identified in a variety of human cancers and cancer-derived cell lines. It is unclear how tumor-derived mutations disrupt the structure and function of p16, especially since many of these mutations are located far away from the cyclin-dependent kinase binding site. In this study, we investigated the effect of two tumor-derived mutations, P81L and V126D, on the structure of p16 by limited proteolysis. The proteolytic products were characterized by gel electrophoresis, HPLC, and mass spectrometry. Our results show that the N-terminal half of p16 is significantly more sensitive to proteolysis in both tumor-derived mutant proteins than in the wild type, suggesting that this region is particularly unstable. Interestingly, the N-terminal half of p16 contains many residues that are important for cyclin-dependent kinase binding. Thus, our results provide a structural mechanism by which tumor-derived mutations inactivate the function of p16 and suggest that stabilization of the N-terminal region could be a useful strategy for future therapeutic development.