Growth Arrest Cellular Senescence Apoptosis

Fig.15.3 A model for p53 transcriptional activation. (A) DNA damage signaling causes Mdm2-p53 disassociation and post-translational events leading to p53 activation. (B) The cell chooses a particular cell fate through known and unknown mechanisms. (C) If the DNA repair is not complete or is not repairable, the cell may choose G1 arrest, apoptosis, or cellular senescence as a fate. (D) On the other hand, if DNA repair is complete the cell may choose to re-enter the cell cycle through recruitment of Sir2a and/or PID/HDAC1 to deacetylate p53 and shut down p53-dependent transcription. (E) p53 is then regulated at low levels by Mdm2 until the next DNA damage event.

locus, a chromosomal region frequently mutated in cancer (Ruas and Peters, 1998). Today the locus is known to code for two genes: piand p 14ARF, which function to activate the Rb and p53 pathways, respectively. ARF functions upstream of Mdm2 and blocks its activity on p53, a hypothesis strengthened by the tumorigenicity of ARF knockout mice (Lowe and Sherr, 2003). Still, its exact mechanistic function remains unclear. ARF is capable of sequestering Mdm2 in the nucleolus, though more recent data show that ARF can stabilize p53 independent of Mdm2 relocalization (Llanos et al., 2001). ARF can also directly inhibit the enzymatic function of Mdm2 in vitro, however in vivo it can only reduce polyubiquitinated forms of p53 and shows no effect on Mdm2 self-ubiquitination (Honda and Yasuda, 1999; Midgley et al., 2000; Xirodimas et al., 2001). Based on these observations, as well as other data, several working hypotheses of ARF-mediated p53 stabilization can be drawn. ARF may physically sequester Mdm2 away from p53, ARF can directly inhibit Mdm2 enzymatic activity, or ARF possesses distinct and unique functions aside from those proposed that are Mdm2 independent. It is quite likely that all are correct, and ARF has the capability of multi-tasking several functions that have direct or indirect implications on p53 stability.

The nucleolus, possessing ill-defined structural features and entry requirements, is nevertheless an up-and-coming star of complexity contained within the nucleoplasm. In addition to its role as a required exit port for mRNA transcripts ready for translation, it is also becoming an important link between protein synthesis and the p53-Mdm2 pathway (Horn and Vousden, 2004). ARF resides within the nucleolus, and its recently documented interaction with the nucleolar protein B23 has implicated ARF as a link between ribosomal biogenesis and p53 (Bertwistle et al., 2004; Itahana et al., 2003). ARF promotes the polyubiquitination and degradation of B23, and by doing so, may prevent it from acting as a required rRNA processing enzyme needed for proper cellular proliferation. In this regard, ARF seems to serve a dual role as a tumor suppressor, acting on both B23 and the Mdm2-p53 pathway to stop cell growth and proliferation. ARF is clearly not the only player in this novel offshoot of the pathway, and recent evidence has shown Mdm2 may be involved in ribosomal biogenesis. Mdm2 was previously shown to interact with ribosomal protein L5 suggesting that

Mdm2 was involved in some aspect of protein translation (Marechal et al., 1994). More recently it has been shown the Mdm2 can also interact with ribosomal protein Lll and that this protein can inhibit Mdm2's activity on p53 (Lohrum et al., 2003; Zhang et al., 2003). Lll, as well as L5, may then act as sensors of nucleolar stress that can inhibit Mdm2 activity so that p53 can be efficiently stabilized. While it appears that no individual interaction is an exclusive mechanism for p53 stabilization and activation, it is quite likely that the cell utilizes a balance of pathways under various stress responses to sufficiently activate the protein (Ashcroft et al., 2000).

MdmX is yet another protein that has an intricate and poorly understood involvement in p53 regulation. The embryonic lethal phenotype of MdmX null embryos and the failure to rescue this phenotype when crossed with the p53 null mice clearly places it as an important negative regulator of p53 during embryonic development. Still, its physiologic function and the question of whether they hold true in all cell types remains to be seen. MdmX possesses structural similarities with Mdm2, and though it has a C-terminal RING domain, does not possess an in vivo ability to ubiquitinate and degrade p53. MdmX can stabilize p53, as polyubiquitinated forms of p53 readily accumulate within the nucleus (Jackson and Berberich, 2000; Stad et al., 2001). However, when the ratio of MdmX:Mdm2 is low, these proteins cooperatively decrease p53 levels (Gu et al., 2002; Iwakuma and Lozano, 2003). It has been shown that MdmX can act as a transcriptional repressor suggesting another possible physiologic role for MdmX (Kadakia et al., 2002; Wunderlich et al., 2004; Yam et al., 1999) MdmX imparts a negative affect on p53 acetylation, possibly through inhibition of p300/CBR This observation has also been supported by an increased level of acetylated p53 in mdmx- mutant cells. Regardless of the mechanisms, MdmX may rival the functional importance of Mdm2, considering that it is found upregulated in many tumors expressing wildtype p53.

In addition, some proteins can stabilize p53 in an Mdm2-independent manner. Calpain 1, p-catenin, and JNK have all been shown to stabilize p53 independently of Mdm2 (Damalas et al., 1999; Fuchs et al., 1998; Kubbutat and Vousden, 1997). NQOl, an NADH quinone oxidoreductase, stabilizes p53 by regulating its interaction with the 20S proteosome and represents a ubiquitin-independent pathway for p53 regulation (Asher et al., 2005). The protein Sin3a can also bind to and stabilize p53 on promoters of genes targeted for transcriptional repression in response to DNA damage

(Zilfou et al., 2001). This spatial interaction is thought to extend p53 promoter association.

A: Cytoplasmicp53

Compartmentalization of the cell into distinct structural components provides physical isolation of proteins from one another and adds an additional layer of protein regulation. In the case of p53, its activity as a potent transcription factor is in part regulated by sequestering it away from its targets in the nucleus during times of homeostasis. The importance of this mechanism is underscored by its exploitation by several viruses. The cytomegalovirus and adenovirus type 12 both maintain p53 in the cytoplasm, presumably as a way to ensure continued cell proliferation (Kovacs et al., 1996; Zhao and Liao, 2003).

The cellular protein PARC (p53-associated, Parkin-like cytoplasmic protein) has recently been isolated from stable p53 cytoplasmic protein complexes in unstressed cells (Nikolaev et al., 2003). PARC acts as a cytoplasmic anchor for p53 by physically binding to and sequestering it in the cytoplasm. Indeed, during unstressed conditions p53 is found diffusely dispersed throughout the cytoplasm of cells. Several neuroblastoma cell lines also show high expression of PARC and abnormally distributed cytoplasmic wildtype p53 that fails to respond to DNA damage signals. When PARC was specifically ablated using siRNA in these tumor cells, the p53-mediated DNA damage response was restored. These findings are hopeful when considering novel chemotherapeutic targets, as neuroblastomas represent the most common extracranial malignancy in children, often with poor prognosis (Kastan and Zambetti, 2003).

Moving p53 away from its transcriptional targets seems to be a critical requirement for inhibiting its function, and Mdm2 may play an important role in this regard. Interestingly, recent evidence suggests monoubiquitination is a critical signal for nuclear export, and Mdm2 is capable of inducing both monoubiquitination and polyubiquitination on p53 (Li et al., 2003). When Mdm2 is low, it catalyzes monoubiquitination of p53 that is effectively exported out of the nucleus for degradation and/or further modifications in the cytoplasm. When Mdm2 levels are high, p53 is quickly polyubiquitinated within the nucleus and degraded by nuclear proteasomes. One question that is quickly raised is why this type of regulation is needed. It has been known for some time that proteasomes exist in both the nucleus and cytoplasm, and therefore some type of regulation is clearly needed to prevent rapid degradation of proteins in close contact. It also has been shown that during the late stages of DNA damage, a time where p53 has accomplished its jobs and needs to be quickly shut down, that Mdm2 protein levels are very high. While p53 drives Mdm2 expression through a negative feedback loop, the Mdm2-p53 interaction is known to be blocked during cellular stress and DNA damage. A high level of Mdm2 at the end of the DNA damage response would allow the cell to quickly degrade p53 through polyubiquitination as soon as the mechanisms for p53 stabilization are reversed.

The reasons for moving monoubiquitinated p53 into the cytoplasm are less clear. Perhaps this mechanism removes p53 from a region where its actions have large consequences on the fate of the cell. Another intriguing possibility is that monoubiquitinated p53 is acted upon by cellular factors present in the cytoplasm for diverse functions other than transcriptional activation.

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