A melanoma-predisposing germline CDKN2A mutation with functional significance for both p16 and p14ARF
Introduction
The CDKN2A locus on human chromosome 9p21 contains two overlapping tumor suppressor genes CDKN2A (INK4a) and ARF that encode two distinct cell cycle regulatory proteins, p16 and p14ARF (p19ARF in mouse) [1], [2], [3], [4]. The regular p16 protein is encoded by exon 1α, 2 and 3. An alternative first exon (1β) is located ∼22 kb upstream of the CDKN2A coding sequence. Transcripts initiating from exon 1β are spliced onto the common exon 2 and exon 3, giving rise to an alternative mRNA that encodes p14ARF in a different reading frame [2], [5], [6].
The p16 protein inhibits cyclin D1-dependent kinases CDK4 and CDK6 and thus prevents phosphorylation of the pRb protein. Hypophopsphorylated pRb blocks progression into the S phase of the cell cycle [1], [7]. Mutant p16 proteins that are unable to form stable complexes with CDK4/6 have therefore lost their ability to control cell cycle progression and cell proliferation [9]. In contrast to p16, ARF is induced by illegitimate oncogene activation [8], [10] and acts mainly in the p53 pathway by inhibiting the p53 antagonist MDM2 [3], [4], [11]. ARF blocks MDM2-mediated nuclear export of p53 and inhibits the E3 ubiquitin ligase activity of MDM2 [12], [13], [14], although the precise biochemical mechanisms remain unclear [10], [15]. Human p14ARF is localized predominantly in the nucleolus but also in the nucleoplasm [16] and the protein contains two nuclear and nucleolar localization signals (NLS, NoLS), in part overlapping with the MDM2 binding domain (Fig. 1A) [11], [13], [17], [18], [19].
Hypermethylation or homozygous deletion of the CDKN2A locus occurs frequently in human tumors. The unique organization of this locus complicates the analysis of the roles of p16 and p14ARF [20]. Approximately 10% of cutaneous melanomas occur in families with hereditary predisposition [21], [22]. In a proportion of melanoma kindreds, germline mutations that affect exon 1α or 2 of CDKN2A have been identified [23], [24], [25]. To date, two germline alterations of the p14ARF-specific exon 1β have been described. One is a deletion in a family characterized by multiple melanomas and neural system tumors [26], and the other is a 16 bp insertion in a family with multiple primary melanoma [27]. Due to the dual utilization of exon 2, some mutations in this exon alter both p16 and p14ARF amino acid sequences [24]. The effect of such mutations on p14ARF function is unclear. The cell cycle inhibitory function of the p14ARF protein as well as a nuclear localization signal has been localized to the N-terminal region encoded by exon 1β [11], [17]. The C-terminal nucleolar localization domain encoded by exon 2 is not essential for p53 stabilization or nuclear localization, but it appears to potentiate the activity of human p14ARF [13], [18], [28].
Recently, we reported a CDKN2A 24 bp exon 2 deletion in a patient with multiple primary cutaneous melanomas who belongs to a kindred with hereditary melanoma predisposition [29]. The in-frame deletion of eight amino acids (Δ62–69) is localized at the end of the second conserved ankyrin repeat in p16. Functional analysis by an in vitro binding assay showed that the mutant p16 protein is functionally abnormal [29]. The 24 bp deletion causes an in-frame deletion of residues 77–84 in p14ARF. This disrupts an arginine-rich cluster motif (residues 80–90) in the C-terminus that confers nuclear and nucleolar localizations [17]. To further clarify the possible role of this 24 bp deletion in melanoma development, we have studied the cellular distribution and function of the p14ARFΔ77–84 and p16Δ62–69 mutant proteins.
Section snippets
Patient and family history
The patient with the germline CDKN2A 24 bp deletion was identified in the course of screening for CDKN2A germline mutations in patients with multiple primary melanomas [29]. The patient belongs to a family with hereditary predisposition for melanoma and dysplastic naevus syndrome. He is a Caucasian male of 60 years, who was investigated since he had had three primary melanomas excised. The first two melanomas were diagnosed at the age of 50 years. Both were invasive superficial spreading
Intracellular localization of p14ARFΔ77–84 and stabilization of p53
To investigate the localization of wt p14ARF and p14ARFΔ77–84, we expressed these proteins in MCF7 breast cancer, U2OS osteosarcoma and 397 melanoma cells, all of which lack endogenous p14ARF. We distinguished between transfectants with nucleolar localization and cells that had a more diffuse staining throughout the nucleus and the cytoplasm. Some residual nucleolar p14ARF staining remained in a few transfectants with nucleoplasmic and cytoplasmic staining. The wt p14ARF protein was distinctly
Discussion
According to the current view, ARF induces p53 in response to oncogenic signaling by inhibiting the MDM2 protein that targets p53 for degradation in cytoplasmic proteasomes. ARF can probably antagonize MDM2 through several mechanisms, including inhibition of the E3 ubiquitin ligase activity of MDM2 and prevention of MDM2-mediated nuclear export of p53 [13], [14]. Under certain circumstances, ARF sequesters MDM2 in nucleoli [32], but the significance of this for induction of p53 is still a
Acknowledgments
This study was supported by grants from the Cancer Society in Stockholm, the Gustaf V Jubilee Fund, the Swedish Cancer Society (Cancerfonden), and the Research Funds of the Karolinska Institute.
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These authors contributed equally to this work.