ARF Tumor Suppressor - Background

Background

The ARF tumor suppressor transcript was first identified in humans in 1995, and its protein product confirmed in mice that same year. Its gene locus is on the short arm of chromosome 9 in humans, and on a corresponding location on chromosome 4 in mice. It is located near the genes for the tandem repeats INK4a and INK4b, which are 16 kDa (p16INK4a) and 15 kDa (p15INK4b) proteins, respectively. These INK4 proteins directly inhibit the cyclin D-dependent kinases CDK4 and CDK6. There are other INK4 genes on other chromosomes, however these are not linked to cancer, and so their functions are not likely to be overlapping. An important cyclin-dependent substrate is the retinoblastoma protein Rb, which is phosphorylated in late gap 1 phase (G1 phase), allowing G1 exit. The Rb protein limits cell proliferation by blocking the activity of E2F transcription factors, which activate the transcription of genes needed for DNA replication. When Rb is phosphorylated by cyclin D and E-dependent kinases during the G1 phase of the cell cycle, Rb can not block E2F-dependent transcription, and the cell can progress to the DNA synthetic phase(S phase). Therefore, INK4a and INK4b serve as tumor suppressors by restricting proliferation though the inhibition of the CDKs responsible for Rb phosphorylation.

In addition to the INK4a protein, the unrelated protein, ARF, is transcribed from an alternate reading frame at the INK4a/ARF locus. INK4a and ARF mRNA each consist of three exons. They share exons 2 and 3, but there are two different exon 1 transcripts, α and β. Exon 1β (E1β) is intercalated between the genes for INK4a and INK4b. Although exon 1α (E1α) and E1β are about the same in terms of content and size, the 5’ AUG (start codon) of exon 1β has its own promoter and opens an alternative reading frame in exon 2, hence the name ARF (ARF exon 3 is not translated). Because of this, INK4a and ARF have unrelated amino acid sequences despite overlapping coding regions, and have distinct functions. This dual use of coding sequences is not commonly seen in mammals, making ARF an unusual protein. When the ARF β-transcript was found, it was thought that it probably would not encode a protein. In humans, ARF is translated into the 14kDa, 132 amino acid ] protein, and in mice, it is translated into the 19kDa, 169 amino acid p19Arf. The E1β protein segment of mouse and human ARF are 45% identical, with an overall ARF identity of 50%, compared to a 72% identity between mouse and human INK4a E1α segment, and a 65% overall identity.

Although the INK4a and ARF proteins are structurally and functionally different, they are both involved in cell cycle progression. Together, their broad inhibitory role may help counter oncogenic signals. As mentioned above, INK4a inhibits proliferation by indirectly allowing Rb to remain associated with E2F transcription factors. ARF is involved in p53 activation by inhibiting Mdm2 (HDM2 in humans). Mdm2 binds to p53, inhibiting its transcriptional activity. Mdm2 also has E3 ubiquitin ligase activity toward p53, and promotes its exportation from the cell nucleus to the cytoplasm for degradation. By antagonizing Mdm2, ARF permits the transcriptional activity of p53 that would lead to cell cycle arrest or apoptosis. A loss of ARF or p53, therefore, would give cells a survival advantage.

The function of ARF has primarily been attributed to its Mdm2/p53 mechanism. ARF does, however, also inhibit proliferation in cells lacking p53 or p53 and Mdm2. It has recently been found that one of ARF’s p53-independent functions involves its binding to nucleophosmin/B23 (NPM). NPM is an acidic ribosomal chaperone (protein) involved in preribosomal processing and nuclear exportation independent of p53, and oligomerizes with itself and p19ARF. Nearly half of p19ARF is found in NPM-containing complexes with high molecular mass (2 to 5 MDa). Enforced expression of ARF retards early 47S/45S rRNA precursor processing and inhibits 32S rRNA cleavage. This suggests that p19ARF can bind to NPM, inhibiting rRNA processing. ARF-null cells have increased nucleolar area, increased ribosome biogenesis, and a corresponding increase in protein synthesis. The larger size resulting from more ribosomes and protein is not associated with increased proliferation, however, and this ARF-null phenotype occurs even though the normal basal levels of Arf are usually low. Knocking down ARF with siRNA to exon 1β results in increased rRNA transcripts, rRNA processing, and ribosome nuclear export. The unrestrained ribosome biogenesis seen when NPM is not bound to ARF does not occur if NPM is also absent. Although the induction of ARF in response to oncogenic signals is considered to be of primary importance, the low levels of ARF seen in interphase cells also has a considerable effect in terms of keeping cell growth in check. Therefore, the function of basal level ARF in the NPM/ARF complex appears to be to monitor steady-state ribosome biogenesis and growth independently of preventing proliferation.