Inhibition of Ribosome Biogenesis by p14(ARF)
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Inhibition of Ribosome Biogenesis by p14(ARF)
CDKN2A (Cyclin Dependent Kinase Inhibitor-2A) belongs to the CDKN family which show specificity for G1 phase CDKs (Cyclin-Dependent Kinases) and such binding results in block exit from G1 into S phase. CDKN2A, CDK4, CcnD (Cyclin-D) and Rb (Retinoblastoma) are components of the same growth-regulatory pathway that functions to control progression through G1 into S phase. CDKN2A is sometimes referred to as p16(INK4A) (Ref.1). Like p16(INK4A), ARFs (Alternative Reading Frames), or transcript variants of p16(INK4A) encodes p14(ARF), a tumor suppressor but its action is distinct from that of p16(INK4A). p16(INK4A) acts on the Rb pathway by inhibiting CDK4, whereas, p14(ARF) acts on the p53 pathway by interacting with MDM2 (Mouse Double Minute-2 Homolog), thereby blocking degradation of p53 and resulting in p53 stabilization and activation. Further more, binding of p14(ARF) to MDM2 results in sequestration of MDM2 in the nucleolus. This prevents shuttling of MDM2 between nucleus and cytoplasm (to where it must export p53 for degradation), thereby leaving p53 free within the nucleus to transactivate transcription of responsive genes. Interestingly, p14(ARF) is required for p53 activation by proliferative signals such as Myc (v-Myc Avian Myelocytomatosis Viral Oncogene Homolog) but not for activation in response to ultraviolet or ionizing radiation. In addition p14(ARF) is under the control of E2F1 (E2F Transcription Factor-1), thus providing an additional mechanism whereby cells that have deregulated G1 progression and elevated pool of free E2F, are subject to G1 arrest. p14(ARF) function is lost in cancers like leukemias, lymphomas, glioblastomas etc (Ref.1 & 2).

The tumor suppressor p14(ARF) plays an important role as an inhibitor of the MDM2-mediated degradation of p53. The human ARF that is, p14(ARF) forms homo-oligomers that are stabilized by disulphide links. The stability of these oligomers is favoured by oxidizing agents in a reversible fashion and involves all three cysteine residues in p14(ARF). The N-terminus (Amino terminus) of p14(ARF) interferes with the ability of the protein to form multimeric complexes. p14(ARF) activity is linked to its oligomerization and is sensitive to the redox status of the cell. Oxidative stress affects p14(ARF) oligomerization (Ref.2). Tumor promotion is associated with an altered redox status, and it is known that cancer cells produce an excess of ROS (Reactive Oxygen Species). The oligomeric forms of p14(ARF)  are sensitive to reducing agents. All three cysteines in p14(ARF)  are involved in the stabilization of the oligomers (Ref.3). p14(ARF) binds to MDM2 and promotes its rapid degradation. This interaction is mediated by the Exon1Beta-encoded N-terminal domain of p14(ARF) and a C-terminal region of MDM2. MDM2 degradation by p14(ARF) is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of p14(ARF)-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed p14(ARF) to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Normally, p53 protein is kept at a low concentration in a cell by its relatively short half-life. Several types of cellular stresses, particularly DNA damage, result in a rapid increase in p53 concentration. The p53 protein is significantly destabilized by the over expression of MDM2, leading to the postulation that MDM2 plays a role in maintaining low levels of p53 in normal cells as well as in removing p53 during recovery from stress-induced arrest through a feedback regulatory loop. In the case of DNA double-strand breaks produced by Gamma-irradiation, p14(ARF) plays at most a redundant, if not a minor, role in mediating p53 accumulation (Ref.1 & 4).

The 5.8S rRNA (5.8S (5.8 Svedberg Unit) Ribosomal RNA) plays an important role in ribosome translocation. The cytoplasmic ribosomes of eukaryotes contain one additional integral RNA component, the 5.8S rRNA, which forms an RNA-RNA complex with the 25-28S rRNA of the large ribosomal subunit The 5.8S rRNA plays a critical role in protein elongation (Ref.5). The 5.8S rRNA forms a covalent linkage with tumor suppressor p53 protein and plays a functional role in protein elongation. The 5.8S rRNA is an important target in the control of ribosome function during cell differentiation and oncogenesis (Ref.6). The covalent linkage of the 5.8S rRNA to p53 implies that p53 and the RNA are be two integral components of a single functional entity. p53 is involved in one or more of the following mechanisms: regulation of the transcription of ribosomal RNA genes, regulation of the processing of the rRNA precursor to give 5.8S rRNA, regulation of the transport of both p53 and 5.8S rRNA from the nucleus and translational control. p53 binds specifically to a region upstream of the transcription start site for the human ribosomal gene cluster (rDNA or Ribosomal DNA). In addition, wild-type p53 activates transcription from promoters bearing the rDNA. One possible meaning for the p53-5.8S rRNA linkage is that 5.8S rRNA, through its binding to p53, is a feedback regulator (positive or negative) of its own synthesis. The p53-5.8S rRNA linkage is also involved in the cytoplasmic transport of both molecules. Another implication of the p53-5.8S rRNA linkage is that p53 is involved in processing either a major or a minor portion of the rRNA transcript to produce 5.8S rRNA. 5.8S rRNA is a nuclear cleavage product of the >45S rRNA transcript. It is transcribed from multicopy rRNA genes in a cell cycle-dependent manner, and it is an essential substituent of the ribosome. 5.8S rRNA forms hydrogen bonds with 28S RNA in both the nuclear ribosomal precursor and in the cytoplasmic 60S ribosomal subunit. However, 5.8S rRNA also binds the RPS9 (Ribosomal Protein-S9) and RPS13 (Ribosomal Protein-S13) proteins of the 40S ribosomal subunit, forms the ribosomal subunit interface and participates in the ribosomal-A (Aminoacyl) site. The p53 is associated with cytoplasmic ribosomes and the p53-5.8S rRNA complex is involved in translational control and ribosome biogenesis, under the influence of TFs (Transcription Factors) and RNA PolI (RNA Polymerase-I) (Ref.5 & 7). 

p14(ARF) is also induced upon hyper-proliferative signaling by oncogenes such as Myc and E2F1. Therefore, p14(ARF) activation work as an important fail-safe mechanism, because efficient oncogenic transformation by these factors occur only when the p14(ARF)-MDM2-p53 pathway is inactivated. There are several regulators of the p14(ARF) promoter. Of these a number are most likely indirect regulators, such as Myc, Twist1 (Twist Homolog-1), Abl1 (v-Abl Abelson’s Murine Leukemia Viral Oncogene Homolog-1), etc. However, some of them potentially are direct regulators, like p53 and E2F1. Among several disease-implicated regulators of p14(ARF) includes the T-Box genes; TBx2 (T-Box-2), which resides on an amplicon in primary breast tumors and TBx3, which is mutated in the human developmental disorder Ulnar-Mammary syndrome. The consensus T-Box and a C-terminal conserved repression domain are essential for p14(ARF) repression. TBx2 potently down-regulate the p14(ARF)  tumor suppressor, thereby causing efficient immortalization of primary fibroblasts (Ref.2 & 8). Twist1 is known to block myogenic differentiation. It plays multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis and interfering with the p53 tumor suppressor pathway. Loss of anchorage dependence is a hallmark of tumor cells, and the ability to promote anchorage-independent growth is a common property of oncogenes. Twist1 has the ability to promote anchorage-independent growth (Ref.9). 

The p14(ARF)-p53 tumor suppressor pathway plays a critical role in cell-cycle checkpoint control and apoptosis, whereas Rho family small GTPases are key regulators of actin structure and cell motility. The endogenous PI3K (Phosphatidylinositde-3 Kinase) and Rac1 (Ras-Related C3 Botulinum Toxin Substrate-1) activities are required for p14(ARF) and p53-regulated migration. A functional relationship between an established tumor suppressor pathway and  a signaling module that controls actin structure and cell motility show that p14(ARF) and p53 negatively regulate cell migration by suppression of PI3K and Rac1 activities through Rac1 pathway (Ref.10 & 11). The ARF-INK4A mutations have a negative impact on the outcome of cancer. Disruptions of the ARF-INK4A locus contribute to chemoresistance in human tumors. However, it is noteworthy that p53 mutations are strongly associated with highly aggressive tumors and chemoresistance in human hematologic malignancies. These mutations have important implications for understanding the clinical behavior of human tumors (Ref.11).