Cancer-associated mutations at the INK4a locus cancel cell cycle arrest by p16 INK4a but not by the alternative reading frame protein p19 ARF

Abstract

The INK4a gene, one of the most frequently disrupted tumor suppressor loci in human cancer, encodes two unrelated proteins, p16 INK4a and p19 ARF , each of which is capable of inducing cell cycle arrest. Splicing of alternative first exons (1α vs. 1β) to a common second exon within INK4a generates mRNAs in which exon 2 sequences are translated 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, because many such mutations reside in exon 2, they also affect the alternative reading frame (ARF) protein. To determine whether such mutations disrupt p19 ARF function, we introduced naturally occurring missense mutations into mouse INK4a exon 2 sequences and tested mutant p16 INK4a and p19 ARF proteins for their ability to inhibit cell cycle progression. Six p19 ARF point mutants remained fully active in mediating cell cycle arrest in NIH 3T3 fibroblasts, whereas two of the corresponding mutations within p16 INK4a resulted in complete loss of activity. Analysis of p19 ARF deletion mutants indicated that the unique aminoterminal domain encoded by exon 1β was both necessary and sufficient for inducing G 1 arrest. Therefore, cancer-associated mutations within exon 2 of the INK4a gene specifically target p16 INK4a , and not p19 ARF , for inactivation.