Allosteric Interactions by p53 mRNA Govern HDM2 E3 Ubiquitin Ligase Specificity under Different Conditions

Abstract

c HDM2 and HDMX are key negative regulatory factors of the p53 tumor suppressor under normal conditions by promoting its degradation or preventing its trans activity, respectively. It has more recently been shown that both proteins can also act as positive regulators of p53 after DNA damage. This involves phosphorylation by ATM on serine residues HDM2(S395) and HDMX(S403), promoting their respective interaction with the p53 mRNA. However, the underlying molecular mechanisms of how these phosphorylation events switch HDM2 and HDMX from negative to positive regulators of p53 is not known. Our results show that these phosphorylation events reside within intrinsically disordered domains and change the conformation of the proteins. The modifications promote the exposition of N-terminal interfaces that support the formation of a new HDMX-HDM2 heterodimer independent of the C-terminal RING-RING interaction. The E3 ubiquitin ligase activity of this complex toward p53 is prevented by the p53 mRNA ligand but, interestingly, does not affect the capacity to ubiquitinate HDMX and HDM2. These results show how ATM-mediated modifications of HDMX and HDM2 switch HDM2 E3 ubiquitin ligase activity away from p53 but toward HDMX and itself and illustrate how the substrate specificity of HDM2 E3 ligase activity is regulated.T he activity of the RING-E3 ubiquitin ligase HDM2 is tightly regulated by posttranslational modifications and protein-protein interactions that control its subcellular localization and interacting partners (1, 2). The best-characterized activity of HMD2 is the N-terminal interaction between its hydrophobic pocket and the conserved BOX-I domain of p53, which under normal cellular conditions promotes p53 polyubiquitination. The nonredundant HDM2 paralogue HDMX (also called HDM4) is, like HDM2, upregulated in human cancers (3)(4)(5). Despite the similarity with the C-terminal RING domain of HDM2 (approximately 75%), HDMX does not harbor E3 ubiquitin ligase activity toward p53, and its negative activity is instead linked to suppression of p53 trans activity. Mice lacking either mdm2 (the hdm2 mouse orthologous) or mdmx (the hdmx mouse orthologous) die early during development in a p53-dependent manner (6-8). It has been shown more recently that HDMX assists HDM2-mediated degradation of p53 by stabilizing HDM2 via a C-terminal RING-RING interaction and also by promoting HMD2-mediated polyubiquitination of p53 in the cytoplasm (9, 10).The ataxia telangiectasia mutated (ATM) kinase is a key regulator of p53 activity during the double-stranded DNA damage response that helps to induce p53 expression and activity. The direct and indirect phosphorylation of residues within the p53 BOX-I domain of p53 prevents the interaction with HDM2 and is linked to an increase in p53 activity (11-13). The phosphorylation on HDM2 at serine 395 (394 in mouse MDM2) is crucial for p53 stabilization, and animals carrying the S394A mutation have an impaired response to irradiation (14). The corresponding mutation in the human protein prevents ...

Fanconi Anemia Signaling and Cancer

“…Subsequently, phosphorylated H2AX (γH2AX) interacts with MDC1 (mediator of DNA damage checkpoint protein-1) at the BRCT region [45], which leads to the expansion of the similar processes (the recruitment of more ATM–MRN complexes and further H2AX phosphorylation). Activated ATM then phosphorylates downstream targets, in particular, ATM phosphorylates Chk2 at T68 [46–49], p53 at S15 and S20 [50, 51], and HDM2 at S395 [46, 52] to initiate G1 arrest. However, phosphorylation of NBS1 at S343 [53], FANCD2 at S222 [32], FANCS (BRCA1) at S1387 [54], and SMC1at S957 and S966 [55], which also depends upon ATM, leads to an S-phase arrest; accordingly, phosphorylated FANCS (BRCA1) at S1423 [55, 56], hRad17 at S635 and S645 [57] and others cause a G2 arrest.…”

Dual function of MDM2 and MDMX toward the tumor suppressors p53 and RB

“…The Ser403 near to the RING domain of MDMX is ATM-dependently phosphorylated following DNA damage; this event promotes the binding of MDMX to p53 mRNA and acts as an RNA chaperone to properly fold the p53 mRNA, optimizing the correct recognition for MDM2; together the two proteins enhance p53 translation ensure proper cellular response [10]. Evidence supports the idea that MDM2 and MDMX are partners and work in collaboration through the formation of a heterodimer to down-regulate p53 under normal conditions [51, 52], but also to up-regulate p53 after DNA damage [53] (Table 1). …”

HDM2 and HDMX Proteins in Human Cancer

“…Proteíny HDM2 a HDMX sú po fosforylácii schopné efektívnejšie viazať p53 mRNA a prerušuje sa interakcia s proteínom p53 [15][16][17]. Ubikvitinačná aktivita pro-teínu HDM2 je presmerovaná na samotný proteín HDM2 a HDMX, čím dochádza k ich odbúravaniu [18].…”

“…Takto fosforylované proteíny HDM2 a HDMX majú zvýšenú afinitu voči p53 mRNA [15,17,88]. Fosforylácia a zároveň väzba s p53 mRNA znemožnia ich schopnosť viazať p53 proteín, a tak dochádza k aktivácii p53 dráhy [18]. Fosforylácia taktiež mení ubikvitinačnú aktivitu proteínu HDM2, pretože namiesto ubikvitinácie p53, proteín HDM2 ubikvitinuje sám seba a proteín HDMX [18].…”

“…Fosforylácia a zároveň väzba s p53 mRNA znemožnia ich schopnosť viazať p53 proteín, a tak dochádza k aktivácii p53 dráhy [18]. Fosforylácia taktiež mení ubikvitinačnú aktivitu proteínu HDM2, pretože namiesto ubikvitinácie p53, proteín HDM2 ubikvitinuje sám seba a proteín HDMX [18]. Fosforylácie v týchto miestach boli skúmané aj na myších modeloch a bolo ukázané, že skutočne hrajú úlohu v odpovedi na poškodenie DNA [89,90].…”