The INK4a/ARF locus encodes two cell cycle regulatory proteins, the cyclin-dependent kinase inhibitor, p16 INK4a , and the p53 activator, p14 ARF . Germline mutations in this locus are associated with melanoma susceptibility in 20 -40% of multiple case melanoma families. Many of these mutations specifically impair p16INK4a , whereas mutations uniquely targeting p14 ARF are rare. Nevertheless, the importance of p14 ARF has not been excluded because more than 40% of INK4a/ARF alterations affect p16INK4a and p14 ARF . We now report that p14 ARF is functionally impaired in melanoma kindreds carrying INK4a/ARF mutations. Of the seven INK4a/ARF mutations tested, three altered the subcellular distribution of p14 ARF and diminished the ability of p14 ARF to activate the p53 pathway. This work establishes the importance of p14 ARF in melanoma predisposition.The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered sequences in human cancer. It is mutated, deleted, or hypermethylated in many tumors with a frequency ranging from 30% in esophageal tumors to ϳ60% in gliomas and mesotheliomas (1). Inherited mutations involving this locus are also associated with melanoma predisposition in 20 -40% of multiple case melanoma families (2). The dual coding capacity of the INK4a/ARF locus may account for its high disruption rate in human tumors. This locus encodes two distinct proteins, which are translated in different reading frames from alternatively spliced transcripts (3). The ␣ transcript (exons 1␣, 2, and 3) encodes the p16INK4a cyclin-dependent kinase (CDK) 1 inhibitor, whereas the ␤ transcript (exons 1␤, 2, and 3) specifies the alternative product, p14 ARF (3-5). Both INK4a/ARF-encoded proteins maintain cell cycle control. p16INK4a regulates G 1 -phase exit by interacting with CDK4 and CDK6 to inhibit the phosphorylation of the retinoblastoma protein (pRb) (6), whereas p14 ARF interacts with hdm2 to prevent the degradation of p53 (7-10). The interaction of p14 ARF with hdm2 inhibits the ubiquitin-protein isopeptide ligase activity of hdm2 (11,12) and prevents the nuclear export of p53 (13,14). Although p53 stabilization is the best understood function of ARF, recent evidence confirms that p14 ARF also functions in the pRb pathway. The growth inhibitory activity of ARF remains active when the p53 pathway is inactivated but can be suppressed by inactivation of both the pRb and p53 pathways (15). The various functions of p14 ARF presumably reflect its multiple binding partners. In addition to hdm2, p14ARF interacts with the pRb-associated transcription factors E2F 1, -2, and -3, the protein phosphatase subunit, spinophilin, and the DNA enzyme topoisomerase I (16 -19). Although all the functional details have not been determined, p14ARF is activated in response to abnormal oncogenic stimuli, including E1A, MYC, and oncogenic Ras, and therefore must play an important role in sensitizing transformed cells to either growth arrest or apoptosis (20 -24).Although the INK4a/ARF locus encodes two pote...

The INK4a/ARF locus encodes two distinct tumour suppressors, p16 INK4a and p14ARF, that regulate cell cycle progression via the pRB and p53 pathways, respectively. The ARF protein inhibits hdm2 activity, leading to the stabilization of the p53 tumour suppressor and cell cycle inhibition. The amino-terminal domain of human p14ARF and of the mouse homologue, p19ARF, is su cient for these e ects. This domain is also su cient for the nucleolar localization of the mouse ARF protein. In contrast, we show that the human ARF protein requires two arginine rich domains, one in the amino-and the other in the carboxy-terminus, for nucleolar targeting. The amino-terminal nucleolar-targeting domain of p14ARF is also important for ARF-hdm2 binding and cell cycle inhibition. The carboxy-terminal p14ARF nucleolar localization domain lies within the shared INK4a/ARF exon 2, and is mutated in a small number of melanoma-prone kindreds. The INK4a/ARF exon2-mutations could a ect the function of both the p16 INK4a and p14ARF tumour suppressors. Oncogene (2000) 19, 2978 ± 2985.