Biochemistry
ARF expression is regulated by oncogenic signaling. Aberrant mitogenic stimulation, such as by MYC or Ras (protein), will increase its expression, as will an amplification of mutated p53 or Mdm2, or p53 loss. ARF can also be induced by enforced E2F expression. Although E2F expression is increased during the cell cycle, ARF expression probably is not because the activation of a second, unknown transcription factor might be needed to prevent an ARF response to transient E2F increases. ARF is negatively regulated by Rb-E2F complexes and by amplified p53 activation. Aberrant growth signals also increase smARF expression.
ARF is a highly basic (pI>12) and hydrophobic protein. Its basic nature is attributed to its arginine content; more than 20% of its amino acids are arginine, and it contains little or no lysine. Due to these characteristics, ARF is likely to be unstructured unless it is bound to other targets. It reportedly complexes with more than 25 proteins, although the significance of each of these interactions is not known. One of these interactions results in sumoylating activity, suggesting that ARF may modify proteins to which it binds. The SUMO protein is a small ubiquitin-like modifier, which is added to lysly ε-amino groups. This process involves a three-enzyme cascade similar to the way ubiquitylation occurs. E1 is an activating enzyme, E2 is a conjugation enzyme, and E3 is a ligase. ARF associates with UBC9, the only SUMO E2 known, suggesting ARF facilitates SUMO conjugation. The importance of this role is unknown, as sumoylation is involved in different functions, such as protein trafficking, ubiquitylation interference, and gene expression changes.
The half-life of ARF is about 6 hours, while the half-life of smARF is less than 1 hour. Both isoforms are degraded in the proteasome. ARF is targeted for the proteasome by N-terminus ubiquitylation. Proteins are usually ubiquinated at lysine residues. Human ], however, does not contain any lysines, and mouse p19Arf only contains one lysine. If the mouse lysine is replaced with arginine, there is no effect on its degradation, suggesting it is also ubiquinated at the N-terminus. This adds to the uniqueness of the ARF proteins, because most eukaryotic proteins are acetylated at the N-terminus, preventing ubiquination at this location. Penultimate residues affect the efficiency of acetylation, in that acetylation is promoted by acidic residues and inhibited by basic ones. The N-terminal amino acid sequences of p19Arf (Met-Gly-Arg) and p14ARF (Met-Val-Arg) would be processed by methionine aminopeptidase but would not be acetylated, allowing ubiquination to proceed. The sequence of smARF, however, predicts that the initiating methionine would not be cleaved by methionine aminopeptidase and would probably be acetylated, and so is degraded by the proteasome without ubiquination.
Full-length nucleolar ARF appears to be stabilized by NPM. The NPM-ARF complex does not block the N-terminus of ARF, but likely protects ARF from being accessed by degradation machinery. The mitochondrial matrix protein p32 stabilizes smARF. This protein binds various cellular and viral proteins, but its exact function is unknown. Knocking down p32 dramatically decreases smARF levels by increasing its turnover. The levels of p19Arf are not affected by p32 knockdown, and so p32 specifically stabilizes smARF, possibly by protecting it from the proteasome or from mitochondrial proteases.
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