Post-translational modifications that occur on p53 include ubiquitination, sumoylation, phosphorylation, acetylation, and neddylation all of which are covalent linkages that serve to regulate either protein levels or protein activity (Fig. 15.1). They provide layers of regulation that, when combined, offer virtually endless combinations of nuanced regulatory possibilities. The life of p53 exists in a sea of regulatory enzymes all competing for interaction time with the protein upon appropriate activation. The fact that p53 is so highly regulated at the post-translational level underscores its central importance in cellular homeostasis.
A: Ubiquitination p53 was first shown to be ubiquitinated by a cellular factor that associated with the viral E6 protein in papilloma-virus-infected cells (Scheffner et al., 1993). It was later determined to be predominantly degraded through the ubiquitin-proteosomal pathway (Chowdary et al., 1994; Maki et al., 1996). However, in normal cells Mdm2 (Hdm2 in humans; hereafter referred to as Mdm2) acts as the predominant regulator of p53 protein levels by implementing its E3 ligase activity on p53 (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997). Additionally, Mdm2 can inhibit the transactivation ability of p53 (Momand et al., 1992). Maintaining p53 at low levels is critical for cellular homeostasis in unstressed conditions. p53 assists in its own regulation by driving the gene expression of mdm2 in a negative feedback loop, effectively guaranteeing minimal effects when not needed (Wu et al., 1993). Mdm2 was first discovered as an extrachromsomal amplification present in several mouse tumors and is itself a highly regulated protein.
The quick response needed from p53 upon cellular stress requires this autoregulatory feedback loop to be blocked efficiently and effectively for sufficient amounts of the protein to accumulate. Logistically, this can occur either by a) disruptive modifications occurring on p53, b) disruptive modifications on Mdm2, c) protein-protein interactions, or d) inhibition of mdm2 gene expression. Although the latter has been proposed as one mechanism for p53 stability, the fact that p53 continues to drive Mdm2 expression, coupled with the abundance of information supporting the first two hypotheses, suggest post-translational events as the predominant mechanisms. The vast number of mechanisms used by the cell to regulate both p53 and Mdm2 suggests the true physiologic importance they have in maintaining genomic integrity in resting cells. Indeed, p53 homozygous null mutant mice develop tumor formation within 6 months of age and the removal of mdm2 gene expression leads to embryonic lethality in mice (Donehower et al., 1992; Jones et al., 1995; Montes de Oca Luna et al., 1995). Importantly, crossing both nullizygous mice yields a normal, viable p53/mdm2 double-knockout mouse. These findings indicate that Mdm2 is not only in the same genetic pathway as p53, but that it can also be viewed as one of the most important regulators of p53 function. Because of its intimate association with p53, Mdm2 provides an important regulatory level and point of attack for quickly stabilizing and activating the transactivation activity of p53.
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