Mdm-2 binding and TAFII31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21

Abstract

Analyses of five wild-type p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 after treatment with ionizing (IR) or ultraviolet (UV) radiation. Importantly, Thr18 phosphorylation correlated with induction of the p53 downstream targets p21 Waf1/Cip1 (p21) and Mdm-2, suggesting a transactivation enhancing role. Thr18 phosphorylation has been shown to abolish side-chain hydrogen bonding between Thr18 and Asp21, an interaction necessary for stabilizing alpha -helical conformation within the transactivation domain. Mutagenesisderived hydrogen bond disruption attenuated the interaction of p53 with the transactivation repressor Mdm-2 but had no direct effect on the interaction of p53 with the basal transcription factor TAF II 31. However, prior incubation of p53 mutants with Mdm-2 modulated TAF II 31 interaction with p53, suggesting Mdm-2 blocks the accessibility of p53 to TAF II 31. Consistently, p53-null cells transfected with hydrogen bond disrupting p53 mutants demonstrated enhanced endogenous p21 expression, whereas p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. We conclude disruption of intramolecular hydrogen bonding between Thr18 and Asp21 enhances p53 transactivation by modulating Mdm-2 binding, facilitating TAF II 31 recruitment.

Graded enhancement of p53 binding to CREB-binding protein (CBP) by multisite phosphorylation

“…Phosphorylation of p53 at Thr18 is a major negative effector of HDM2 binding, functioning as an on/off switch that controls the interaction between the p53 TAD and the N-terminal domain of HDM2 (9,20). Mechanistically, Thr18 phosphorylation disrupts specific side chain interactions that stabilize the helical structure of the p53 TAD in the HDM2-bound state (28). In contrast, phosphorylation acts as a rheostat to regulate binding of the p53 TAD to CBP, with the affinity for the various CBP domains increasing in a graded manner as successive phosphoryl groups are added.…”

“…4. Single-site phosphorylation at Thr18 inhibits the interaction with the N-terminal region of HDM2, thereby stabilizing p53 against polyubiquitination (9) and relieving HDM2-mediated repression of the p21 gene (28). Mechanistically, activation of p21 expression is likely to occur by recruitment of a limited amount of CBP/p300, primarily through enhanced binding of the HDM2-free, Thr18-phosphorylated p53 TAD to TAZ2; the K d decreases from 55 nM for binding of the p53:HDM2 complex to TAZ2 (28) to 6.5 nM for binding of the pThr18 TAD (Table 1).…”

Expression of the VRK (vaccinia‐related kinase) gene family of p53 regulators in murine hematopoietic development

“…Some of the p53 phosphorylations take place in its N-terminus [5,19], in a region that is required for binding to its negative regulator mdm-2/hdm-2 [20], preventing their interaction and thus resulting in an increase in p53 stability. The phosphorylation of Thr-18 is the main regulator of the p53m dm2 interaction [21,22]. Recently some new kinases that phosphorylate p53 in its mdm-2 binding site have been reported [5,6].…”

TAF9b (Formerly TAF9L) Is a Bona Fide TAF That Has Unique and Overlapping Roles with TAF9

“…TAF9 was shown to bind to the Nterminal region of p53, a region that is also required for interaction with the oncoprotein Mdm2. It has been shown that overexpression of TAF9 inhibits Mdm2-mediated ubiquitination of p53 and increases p53 levels and that TAF9-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts (9,24).Crystal structures demonstrate that the amino-terminal portions of Drosophila TAF9 and TAF6 adopt a canonical histone fold (HF) configuration consisting of two short ␣-helices flanking a long central ␣-helix. In the crystal structure, the dTAF9/ TAF6 HF complex exists as a heterotetramer, resembling the (H3/H4) 2 heterotetrameric core of the histone octamer, suggesting that TFIID may contain a histone octamer-like substructure (65).…”

“…TAF9 was shown to bind to the Nterminal region of p53, a region that is also required for interaction with the oncoprotein Mdm2. It has been shown that overexpression of TAF9 inhibits Mdm2-mediated ubiquitination of p53 and increases p53 levels and that TAF9-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts (9,24).…”