CANCER-ASSOCIATED MUTATIONS AT THE INK4A LOCUS CANCEL CELL-CYCLE ARREST BY P16(INK4A) BUT NOT BY THE ALTERNATIVE READING FRAME PROTEIN P19(ARF)

The INK4a gene, one of the most frequently disrupted tumor suppressor loci in human cancer, encodes two unrelated proteins, p16(INK4a) and p 19(ARF), each of which is capable of inducing cell cycle arrest, Splic ing of alternative first exons (1 alpha vs, 1 beta) to a common second exon within INK4a generates mRNAs in which exon 2 sequences are trans lated in two different reading frames, One of the products, the cyclin D-dependent kinase inhibitor p16(INK4a), is functionally inactivated by mutations or deletions in a wide variety of cancers, However, becau se many such mutations reside in exon 2, they also affect the alternat ive reading frame (ARF) protein, To determine whether such mutations d isrupt p19(ARF) function, we introduced naturally occurring missense m utations into mouse INK4a exon 2 sequences and tested mutant p16(INK4a ) and p19(ARF) proteins for their ability to inhibit cell cycle progre ssion, Six p19(ARF) point mutants remained fully active in mediating c ell cycle arrest in NIH 3T3 fibroblasts, whereas two of the correspond ing mutations within p16(INK4a) resulted in complete loss of activity, Analysis of p19(ARF) deletion mutants indicated that the unique amino terminal domain encoded by exon 1 beta was both necessary and sufficie nt for inducing G(1) arrest, Therefore, cancer-associated mutations wi thin exon 2 of the INK4a gene specifically target p16(INK4a), and not p19(ARF), for inactivation.