Quantitative analyses reveal the importance of regulated Hdmx degradation for P53 activation

Abstract

P53 regulates numerous downstream targets to induce cell cycle arrest, senescence, apoptosis, and DNA repair in response to diverse stresses. Hdm2 and Hdmx are critical negative regulators of P53 because Hdm2 regulates P53 abundance, and both can antagonize P53 transactivation. Modest changes in Hdm2 or Hdmx abundance affect P53 regulation, yet quantitative information regarding their endogenous intracellular concentrations and subcellular distributions during a stress response are lacking. We analyzed these parameters in normal and cancer cells after DNA damage. Our data show that the nuclear abundance of Hdm2 and Hdmx relative to P53 limits P53 activity in cells growing in culture. Upon DNA damage, P53 nuclear abundance increases, whereas Hdm2 and Hdmx stability decreases, which greatly limits their ability to antagonize P53, regardless of their levels. These data indicate that the damage-activated switch in Hdm2 ubiquitin ligase preference from P53 to itself and Hdmx is central to P53 activation. The mechanisms by which P53 is activated after DNA damage have been widely studied (1). Two models that incorporate key roles for damage-activated kinases have emerged to explain how Hdm2-and Hdmx-mediated inhibition of P53 is overcome. The first proposes that activation of the ATM kinase leads to a kinase cascade resulting in phosphorylation of highly conserved serine and threonine residues in P53 within and flanking the Hdm2/ Hdmx-binding region (6). This induces a conformational change in P53, leading to Hdm2 dissociation, P53 stabilization and accumulation, and binding of P53 transcriptional coactivators (7,8). Histone acetyl transferase binding acetylates P53 C-terminal lysines and chromatin and promotes transactivation (9). The situation is likely more complex, because P53 in which highly conserved C-terminal lysines are replaced by arginines has basal and stress-induced stability and activity comparable with wildtype P53 (10, 11). Additionally, stabilization of P53 in the absence of detectable N-and C-terminal phosphorylation engenders full P53 activation (12). Thus, posttranslational P53 modifications fine-tune P53 transcription responses but do not act as on-off switches.A second model is supported by accumulating evidence that damage-activated kinases also phosphorylate Hdm2 to switch its E3 ligase specificity from P53 to itself and Hdmx (13-17). In part, the switch in substrate specificity is mediated by posttranslational modifications of Hdm2 and Hdmx that promote dissociation of the deubiquitinating enzyme HAUSP from Hdm2 and Hdmx (18). This increases Hdm2 and Hdmx degradation, resulting in P53 stabilization, accumulation, and transcriptional activation. Whether increasing Hdm2 levels is required for efficient Hdmx degradation after DNA damage remains unclear (13, 19). Together these data suggest a model in which destabilization and degradation of Hdm2 and Hdmx are critical for P53 stabilization and activation.Subtle perturbations in Hdm2 and Hdmx stoichiometry profoundly alter P53 activity and tumor sup...

A single-molecule characterization of p53 search on DNA

“…This estimate is consistent with about an hour for initial expression of downstream genes (25). Moreover this reasoning suggests that the latent p53 with a lifetime of about 20 min and its slow oligomerization kinetics (26,27) is unlikely to yield significant occupancy in tetrameric form at hundreds of target promoters.…”

“…Finally, we are able to relate our single-molecule measurements to published in vivo fluorescence recovery studies (22,23) and p53 copy-number measurements (25) to calculate the time it takes p53 to find a specific site (e.g., p21) on DNA (see SI Text).…”

Mdm2 and Mdm4 Loss Regulates Distinct p53 Activities

“…On the basis of structural analysis, Kostic et al (41) proposed that Mdm2 and Mdm4 heterodimerization via their respective RING domains contributes to enhanced Mdm2 activity. DNA damage also redirects the ligase activity of Mdm2 from p53 to itself and Mdm4, facilitating p53 activation (42). Therefore, cellular context becomes important as the ratios of Mdm2, Mdm4, and p53 might favor Mdm4-p53, Mdm2-p53, or Mdm4-Mdm2 complexes, resulting in p53 stabilization or degradation.…”

MdmX is a substrate for the deubiquitinating enzyme USP2a

“…In addition, DNA damage destabilizes Mdm2 by enhancing its proteasomal degradation (Stommel and Wahl, 2004). The destabilization of these two repressors of p53 contributes to p53 activation (Wang et al, 2007).…”