Phosphorylation of p53 by IκB kinase 2 promotes its degradation by β-TrCP

Abstract

Functional inactivation of p53 and constitutive activation of the NF-B pathway has been associated with several human cancers. In this study, we show that I B kinase 2 (IKK2/IKK␤), which is critical for NF-B activation, also phosphorylates p53. Phosphorylation of p53 at serines 362 and 366 by IKK2 leads to its recruitment to and ubiquitination by ␤-TrCP1. Degradation of ubiquitinated p53 is independent of Mdm2, because it occurs in both wild-type and Mdm2 ؊/؊ cells. SiRNA-mediated reduction in the levels of ␤-TrCP1 and other members of the SCF ␤؊TrCP1 E3 ubiquitin ligase complex or overexpression of a dominant negative form of ␤-TrCP1 enhances p53 stability. Substitutions at Ser-362 and 366 of p53 by alanines (p53 AA) result in reduced phosphorylation of p53 by IKK2, decreased association with ␤-TrCP1, and thus increased stability of p53 and expression of p53 target genes such as p21, altering the G1 phase of the cell cycle. Our results identify IKK2 and ␤-TrCP1 as novel regulators of the p53 pathway and suggest that blocking of IKK2 and ␤-TrCP1 could be a means of regulating p53 stability and thereby modulating its biological activity.␤-TrCP1 ͉ DNA damage ͉ IKK2 ͉ p53 stability N F-B is a family of transcription factors crucial for several biological processes involved in innate and adaptive immunity, inflammation, cell survival, and cancer (1). More than 150 different types of stimuli, including proinflammatory cytokines, bacterial/ viral infection, and stress, activate NF-B-dependent transcription of target genes. Activation of the I B kinase (IKK) complex is a prerequisite for NF-B activation. Once activated, IKK phosphorylates serines 32 and 36 of I B proteins (2), followed by ubiquitination by ␤-TrCP1, an E3 ubiquitin ligase, which targets them for proteosomal degradation (3). IKK2 has been shown to phosphorylate other cellular and viral proteins, such as ␤-catenin, I B␤, I B , p65, HIV-VPU, and SRC-3; this phosphorylation regulates the steady-state levels and transcriptional activity of these substrates (4-7). Approximately 30% of cellular proteins contain covalently bound phosphate, even though only 500-600 protein kinases likely are encoded by the human genome (8). Therefore, it is conceivable that, like many other kinases, IKK2 also has additional substrates, and that each protein phosphorylated by IKK2 in turn can be a substrate of other kinases.The p53 tumor suppressor plays a crucial role in the development of many cancers and is frequently mutated or deleted in human cancers (9, 10). In the absence of stress, endogenous p53 levels are very low, because of the constant recruitment of p53 to Mdm2, an E3 ubiquitin ligase that inhibits its transcriptional activity and targets it for proteosomal degradation (11,12). In response to DNA damage and other cellular stresses, the p53 protein levels are up-regulated and its activities are induced. The inhibition of Mdm2 binding to p53 and increases in p53 transcriptional activity involve the phosphorylation and stabilization of p53.Human p53 has been reported ...

IκB Kinase β Promotes Cell Survival by Antagonizing p53 Functions through ΔNp73α Phosphorylation and Stabilization

“…A model can be proposed in which the activation of the mechanisms responsible for IKK␣ or IKK␤ nuclear accumulation favor apoptosis or proliferation via stabilization of TAp73 or ⌬Np73␣, respectively. Interestingly, an additional study has recently shown that IKK␤ phosphorylates p53 at two specific serines, promoting its ubiquitination and degradation by ␤-TrCP1 and independently of MDM2 and NF-B (31). Taking into consideration our data and those of Xia's study (31), IKK␤ appears to modulate p53 transcriptional activity through two independent mechanisms, the first promoting its phosphorylation and destabilization and the second inducing phosphorylation and accumulation of the p53 antagonist ⌬Np73␣.…”

Conditional ablation of Ikkb inhibits melanoma tumor development in mice

“…Ikkβ may play a role in the regulation of p53-mediated gene expression, through a mechanism whereby phosphorylated p65 stably associates with p53 at p53 responsive promoters, resulting in an inhibition of p53-mediated transcriptional activity (66,67). Alternatively, Ikkβ may directly phosphorylate p53 on serines 362 and 366 to promote its turnover (68). Here, we show that Ikkb knockout in melanocytes increased p53 protein levels.…”

Inflammation and p53: A Tale of Two Stresses

“…Another relevant study showed that IKKβ, a major component and mediator of the conventional NF-κB pathway, can phosphorylate p53, leading to its ubiquitination and degradation independent of Mdm2. 50 Thus, it is possible that IKK, which is constitutively activated by extrinsic mediators of chronic inflammation (cytokines), might cause long-term inhibition of p53 activity. Products of NF-κB-responsive genes can also suppress p53 function by acting against its downstream targets: For example, NF-κB-induced anti-apoptotic factors can neutralize the effects of pro-apoptotic products induced by p53.…”