Absence of p16 ^INK4a and truncation of ARF tumor suppressors in chickens

Abstract

The INK4b-ARF-INK4a locus on human chromosome 9p21 (Human Genome Organization designation CDKN2B-CDKN2A), and the corresponding locus on mouse chromosome 4, encodes three distinct products: two members of the INK4 cyclin-dependent kinase inhibitor family and a completely unrelated protein, ARF, whose carboxyl-terminal half is specified by the second exon of INK4a but in an alternative reading frame. As INK4 proteins block the phosphorylation of the retinoblastoma gene product and ARF protects p53 from degradation, the locus plays a key role in tumor suppression and the control of cell proliferation. To gain further insights into the relative importance of INK4a and ARF in different settings, we have isolated and characterized the equivalent locus in chickens. Surprisingly, although we identified orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a. Moreover, the reading frame for chicken ARF does not extend into exon 2, because splicing occurs in a different register to that used in mammals. The resultant 60-aa product nevertheless shares functional attributes with its mammalian counterparts. As well as indicating that the locus has been subject to dynamic evolutionary pressures, these unexpected findings suggest that in chickens, the tumor-suppressor functions of INK4a have been compensated for by other genes.

Binding to nucleophosmin determines the localization of human and chicken ARF but not its impact on p53

“…We initially investigated the localization of chicken p7 ARF by transient expression of epitope-tagged versions of the protein (Kim et al, 2003). When expressed in primary chicken embryo fibroblasts (CEFs), GFP-tagged p7 ARF (GFP/ARF Ch ) did not accumulate in the nucleolus but showed heterogeneous speckled staining of the nucleoplasm (Figure 1a).…”

“…Moreover, GFP/ARF Ch showed a different staining pattern (both nucleolar and nucleoplasmic) when expressed in human U20S cells (Kim et al, 2003). We therefore investigated the distribution of HA-and FLAG-tagged versions of ARF Ch in CEFs.…”

“…Reports of a direct interaction between mammalian ARF and NPM (Itahana et al, 2003;Bertwistle et al, 2004;Brady et (Kim et al, 2003).…”

“…Although the genomic arrangement of the INK4a-ARF locus is broadly conserved in different species, the extent of the alternative reading frame varies. For example, the chicken orthologue of the protein, p7 ARF , has no contribution from exon 2 because the splicing of exon 1b to exon 2 occurs in a different register to that used in mammals, and translation terminates abruptly at the end of exon 1b (Kim et al, 2003). We have therefore argued that the evolutionary pressure for exon sharing is unlikely to reflect the composition of the ARF protein and that its functional attributes are most likely specified by the sequences encoded by exon 1b (Gil and Peters, 2006).…”

“…In so doing, ARF stabilizes p53 resulting in the activation of p53 target genes, including that of the CDK inhibitor p21 CIP1 , and MDM2 itself. Importantly, these properties are retained in chicken p7 ARF (Kim et al, 2003) and in truncated versions of mammalian ARF from which the exon 2-derived sequences have been removed (Quelle et al, 1997;Stott et al, 1998;Zhang et al, 1998). However, when visualized by immunofluorescence, human and mouse ARF are predominantly found in the nucleolus (Quelle et al, 1995;Stott et al, 1998) while both p53 and MDM2 are regarded as nucleoplasmic proteins that shuttle between the nucleus and cytoplasm Roth et al, 1998).…”