Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2

Abstract

contributed equally to this workIn response to environmental stress, the p53 phosphoprotein is stabilized and activated to inhibit cell growth. p53 stability and activity are negatively regulated by the murine double minute (Mdm2) oncoprotein in an autoregulatory feedback loop. The inhibitory effect of Mdm2 on p53 has to be tightly regulated for proper p53 activity. Phosphorylation is an important level of p53 regulation. In response to DNA damage, p53 is phosphorylated at several N-terminal serines. In this study we examined the role of Ser20, a potential phosphorylation site in human p53, in the regulation of p53 stability and function. Substitution of Ser20 by Ala (p53-Ala20) significantly increases the susceptibility of human p53 to negative regulation by Mdm2 in vivo, as measured by apoptosis and transcription activation assays. Mutation of Ser20 to Ala renders p53 less stable and more prone to Mdm2-mediated degradation. While the in vitro binding of p53 to Mdm2 is not increased by the Ala20 mutation, the same mutation results in a markedly enhanced binding in vivo. This is consistent with the conclusion that phosphorylation of Ser20 in vivo attenuates the binding of wild-type p53 to Mdm2. Peptides bearing nonphosphorylated Ser20 or Ala20 compete with p53 for Mdm2 binding, while a similar peptide with phosphorylated Ser20 does not. This implies a critical role for Ser20 in modulating the negative regulation of p53 by Mdm2, probably through phosphorylationdependent inhibition of p53-Mdm2 interaction.

Mechanisms of switching on p53: a role for covalent modification?

“…Based on current evidence, serine 15 phosphorylation has pleiotropic e ects on p53 activity, which may cooperate with other modi®cations of p53. Both serine 20 phosphorylation and threonine 18 phosphorylation (which has yet to be shown to occur in vivo) have been demonstrated to weaken signi®cantly the interaction of p53 with MDM2 in vitro (Bottger et al, 1999;Unger et al, 1999). Moreover, loss of serine 20 makes p53 more susceptible to MDM2 inhibition as measured by p53 transactivation and apoptosis functions (Unger et al, 1999), providing biological data which supports the model.…”

Dual-specificity phosphatase 26 is a novel p53 phosphatase and inhibits p53 tumor suppressor functions in human neuroblastoma

“…Many serine residues within the N-and C-terminal regions of p53 are phosphorylated in response to stress conditions such as hypoxia and DNA damage (Kruse and Gu, 2009). Both Ser20 and Ser37 phosphorylation on the p53 protein has been shown to have an important role in p53 stability and downstream apoptotic function (Chehnab et al, 1999;Unger et al, 1999;Li et al, 2006;Amano et al, 2009).…”

Crippling p53 activities via knock-in mutations in mouse models

“…Genotoxic or oncogenic stresses stabilize p53 through post-translational modifications such as phosphorylation. Phosphorylation at the aminoterminus disrupts interaction with Mdm2 and phosphorylation at the carboxyl terminus increases oligomerization (Hupp and Lane, 1994;Sakaguchi et al, 1997;Shieh et al, 1997;Chehab et al, 1999;Craig et al, 1999;Unger et al, 1999a). Lysines at the carboxyl terminus are also modified by ubiquitination, acetylation, neddylation, sumoylation and methylation (Gu and Roeder, 1997;Gostissa et al, 1999;Rodriguez et al, 1999Rodriguez et al, , 2000Chuikov et al, 2004;Harper, 2004;Xirodimas et al, 2004;Friedler et al, 2005).…”