Functional impairment of melanoma-associated p16^INK4a mutants in melanoma cells despite retention of cyclin-dependent kinase 4 binding

Purpose: Melanoma-associated germ-line mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16^INK4a have been identified in >100 melanoma-prone families. To predict the melanoma risk for carriers of specific mutations, it is useful to test the function of the mutant proteins in biochemical assays; however, it is unclear how well these results correlate with their cellular effects. We examined the relationship between loss of CDK binding by mutant proteins and various measures of cellular growth in melanoma cells. Experimental Design: The cellular activities of four melanoma-associated p16^INK4a mutations (Arg24Pro, Ala36Pro, Met53Ile, and Val126Asp) were compared by use of inducible expression in stably transfected melanoma cells, deficient in expression of the endogenous protein, and compared with their ability to bind CDK4. Results: The cell cycle-inhibitory activity of all of the mutants was profoundly reduced, and partially retained capacity for CDK4 binding in functional assays did not correlate with significant preservation of cell cycle-regulatory function. Conclusion: Testing of p16^INK4a interactions with CDKs in protein-binding assays is an unreliable predictor of mutant p16^INK4a function in cells. In addition to exhibiting reduced stability, these mutant proteins may also be defective in interaction with cellular targets other than CDKs.