The ubiquitin pathway provides an efficient, structured, and reversible mechanism for controlling a number of cellular processes, including signaling, protein degradation, trafficking, DNA repair, and apoptosis [2
]. The process of ubiquitination is an enzymatically orchestrated event that involves E1 activating enzymes, E2 conjugating enzymes, and E3 ubiquitin ligases [4
]. Together, these enzymes function in a cascading event to attach an ubiquitin molecule, which is an evolutionarily conserved protein that consists of 76 amino acids, to a lysine residue on a target substrate. Ubiquitin is linked to lysine residues of the target substrate directly (monoubiquitination) or to another ubiquitin protein (polyubiquitination) though a covalent isopeptide bond. Monoubiquitination acts as an important signaling event for the regulation of a number of proteins, whereas polyubiquitination of at least 4 ubiquitins serves as a signal for degradation by the 26S proteosome.
An important control mechanism for p53 regulation is through ubiquitination. It has been shown that p53 ubiquitination is highly dynamic and reversible, with monoubiquitination and polyubiquitation playing important and distinct roles in the functions of p53. The first indication that p53 was regulated by the ubiquitination pathway was by the identification of the human papilloma virus (HPV) E6-associated cellular protein E6AP [5
]. The HPV E6 protein commandeers E6AP to reduce p53 levels as a mechanism for replicating in the host cell. Shortly thereafter, Mdm2 was identified as a cellular factor that ubiquitinates and degrades p53 in the absence of exogenous factors [6
]. A number of studies have shown that Mdm2 is the predominant and critical E3 ubiquitin ligase for p53 and mediates p53 ubiquitination through a RING domain. Together, p53 and Mdm2 function in a negative feedback loop, with p53 driving the transcription of mdm2
during times of normal homeostasis and maintaining low levels of p53 protein. However, upon DNA damage or other type of cellular stress event, p53 protein levels rise due to both a disruption of p53-mediated mdm2
transcription and the post-translational inhibition of Mdm2 function. The direct importance of these interactions was highlighted with the generation of mdm2
null mice, which exhibit embryonic lethality at day E6.5. Interestingly, the lethality is completely rescued in a double knockout of mdm2
A number of mechanisms have been described that inhibit the p53-Mdm2 interaction during cellular stress, suggesting that this interplay is critical for regulating the balance of p53 protein at any given time in the cell. Phosphorylation of p53 at Ser15 and Ser20 in response to DNA damage and other types of cellular stress by ATM, ATR, DNA-PK, Chk1, and Chk2 is thought to abrogate the Mdm2-p53 protein-protein interaction and thereby stabilize p53 [13
]. Moreover, the acetylation of p53, which is a process that is critically important for transcriptional activity, occurs on the same C-terminal lysine residues as ubiquitination, and therefore this enzymatic process can compete with and block ubiquitination to induce p53 stabilization. Indirect mechanisms of p53 stabilization exist as well. The tumor suppressor p14ARF can stabilize p53 by binding to and preventing Mdm2 from physically interacting with p53 [11
]. Although p14ARF is known to be a nucleolar protein, the Mdm2 sequestration and subsequent p53 activation has been shown to occur both inside and outside of the nucleolus [14
]. It has also been shown that p14ARF can block Mdm2 in response to aberrant oncogenes, which provides the cell with a response mechanism for the activation of p53 to this type of cellular stress [12
Mdm2 ubiquitinates p53 at six key lysine residues located at the C-terminus of the protein, including K370, K372, K373, K381, K382, and K386 () [16
]. Knock-in studies, where the lysines are replaced with arginines (the so-called p53-6KR mutant) in vivo, have shown that p53 expression levels are not dramatically altered, suggesting that while these lysines are important for the regulation and function of p53, they are not sufficient for degradation [18
]. The in vivo half-life of the p53-7KR mutant, which is the murine equivalent of the p53-6KR mammalian mutant, was also shown to be similar to wild-type p53 [19
]. These findings suggested that alternative sites on p53 may be important for stability. More recently, it was shown that p53 can also be ubiquitinated in vitro within the DNA binding domain as well [20
]. When this domain was removed, the overall ubiquitination and stability of p53 decreased, though these sites we also not sufficient for complete p53 degradation.
Overview of major ubiquitination, neddylation, and sumoylation sites of p53. The major sites are shown with their corresponding modifying enzymes. Ub, Ubiquitination; Ne, Neddylation; Su, Sumoylation.
Another structurally related protein to Mdm2, MdmX, adds a layer of complexity to p53 regulation. Although MdmX has sequence homology to Mdm2 and possesses a RING domain, it does not have E3 ligase activity for p53. Instead, MdmX was shown to repress p53-mediated transcriptional activation [21
]. Interestingly, mdmx
null mice are embryonic lethal, but can be rescued by crossing with p53
null mice [22
]. More recently, animal studies have aimed to address the intricate functions of Mdm2 and MdmX on p53. In one study, mutant knockout mice were generated that lacked p53
together with either mdm2
]. A temperature sensitive p53 mutant was then reintroduced into the mice, which allowed for a detailed in vivo analysis of p53 in the selective absence of Mdm2 or MdmX. Interestingly, a differential effect was observed, where loss of mdm2 promoted p53-dependent activation of apoptosis-related genes, while loss of MdmX promoted p53-dependent activation of cell cycle arrest genes. This study provided the first in vivo analysis on the specific effects of these proteins on p53 activity, and suggests distinct functions for these two proteins. MdmX and Mdm2 both bind to the promoters of p53-responsive genes and form a 3 protein complex with p53 by interacting with the transactivation domain [28
]. This interaction has been shown to inhibit p53-mediated transcription of some p53 target genes [30
]. In addition, although both Mdm2 and MdmX have important effects on p53 function, in vivo evidence suggests that only Mdm2 has an effect on p53 protein levels [31
]. Nevertheless, additional in vivo studies of MdmX in relation to Mdm2 and p53 will help identify the true physiological mechanisms at play in p53 regulation.