Abstract
Nur77 is a member of the NR4A subfamily of nuclear receptors and has been shown to regulate various biological processes such as apoptosis and inflammation. Here, we show that Nur77 ubiquitination is mediated by the tripartite motif 13 (Trim13), a RING-type E3 ubiquitin ligase. The interaction between Nur77 and Trim13 was confirmed by co-immunoprecipitation. Moreover, we found that Lys539 in Nur77 ubiquitination is targeted for Trim13, which leads to Nur77 degradation. The Trim13-mediated ubiquitination of Nur77 was optimal in the presence of the E2 enzyme UbcH5. Importantly, in addition to Trim13-mediated ubiquitination, the stability of Nur77 was also regulated by casein kinase 2α (CK2α). Pharmacological inhibition of CK2 markedly increased Nur77 levels, whereas overexpression of CK2α, but not its inactive mutant, dramatically decreased Nur77 levels by promoting Nur77 ubiquitination. CK2α phosphorylated Ser154 in Nur77 and thereby regulated Nur77 protein levels by promoting its ubiquitin-mediated degradation. Importantly, we also show that degradation of Nur77 is involved in TNFα-mediated IL-6 production via CK2α and Trim13. Taken together, these results suggest that the sequential phosphorylation and ubiquitination of Nur77 controls its degradation, and provide a therapeutic approach for regulating Nur77 activity through the CK2α-Trim13 axis as a mechanism to control the inflammatory response.
Highlights
Nur[77], known as NGF1B, TR3, or NR4A1, was the first member of the NR4A family to be identified as a gene induced by NGF in PC12 cells[1]
We show that phosphorylation of Nur[77] at serine 154 (S154) by casein kinase 2α (CK2α) triggers Nur[77] ubiquitination by Trim[13] and subsequent degradation, resulting in the control IL-6 production in response to TNFα signaling
TNFα-induced Nur[77] proteins were degraded faster in tripartite motif 13 (Trim13)- transfected cells than in vector-transfected cells (Fig. 8d). These results suggest that both CK2α and Trim[13] were involved in degradation of Nur[77] mediated by TNFα
Summary
Nur[77], known as NGF1B (nerve growth factor 1B), TR3, or NR4A1 (nuclear receptor subfamily 4 group member 1), was the first member of the NR4A family to be identified as a gene induced by NGF in PC12 cells[1]. It has been reported that Nur[77] levels are regulated by both transcriptional and post-transcriptional mechanisms[11,12]. Similar to the case of E3 ligase, the identity of the kinase that regulates Nur[77] ubiquitination is unclear. Given that both of these reversible covalent modifications have defined roles in altering the function of proteins it is inevitable that these two processes can have both positive and negative effects on each other[20]. The aim of the present study was to identify the specific E3 ligase required for Nur[77] ubiquitination and to examine whether CK2 facilitates Nur[77] degradation by promoting its phosphorylation. We show that phosphorylation of Nur[77] at serine 154 (S154) by CK2α triggers Nur[77] ubiquitination by Trim[13] and subsequent degradation, resulting in the control IL-6 production in response to TNFα signaling
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