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 suppre ssor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16(INK4a) have be en identified in > 100 melanoma-prone families. To predict the melanoma ris k 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 relati onship 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 pl 6(INK4a) mutations (Arg24Pro, Ala36Pro, Met53Ile, and Val126Asp) were compa red by use of inducible expression in stably transfected melanoma cells, de ficient in expression of the endogenous protein, and compared with their ab ility to bind CDK4. Results: The cell cycle-inhibitory activity of all of the mutants was profo undly reduced, and partially retained capacity for CDK4 binding in function al assays did not correlate with significant preservation of cell cycle-reg ulatory 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.