CD40 plays an important role in immune responses by activating the c-Jun N-terminal protein kinase (JNK) and NF-κB pathways; however, the precise mechanisms governing the spatiotemporal activation of these two signaling pathways are not fully understood. Here, using four different TRAF2-deficient cell lines (A20.2J, CH12.LX, HAP1, and mouse embryonic fibroblasts [MEFs]) reconstituted with wild-type or phosphorylation mutant forms of TRAF2, along with immunoprecipitation, immunoblotting, gene expression, and immunofluorescence analyses, we report that CD40 ligation elicits TANK-binding kinase 1 (TBK1)-mediated phosphorylation of TRAF2 at Ser-11. This phosphorylation interfered with the interaction between TRAF2's RING domain and membrane phospholipids and enabled translocation of the TRAF2 complex from CD40 to the cytoplasm. We also observed that this cytoplasmic translocation is required for full activation of the JNK pathway and the secondary phase of the NF-κB pathway. Moreover, we found that in the absence of Ser-11 phosphorylation, the TRAF2 RING domain interacts with phospholipids, leading to the translocation of the TRAF2 complex to lipid rafts, resulting in its degradation and activation of the noncanonical NF-κB pathway. Thus, our results provide new insights into the CD40 signaling mechanisms whereby Ser-11 phosphorylation controls RING domain-dependent subcellular localization of TRAF2 to modulate the spatiotemporal activation of the JNK and NF-κB pathways.

中文翻译：

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TRAF2 Ser-11 磷酸化促进 CD40 复合物的胞质易位以调节下游信号通路。


CD40 通过激活 c-Jun N 末端蛋白激酶 (JNK) 和 NF-κB 通路，在免疫反应中发挥重要作用；然而，控制这两种信号通路时空激活的精确机制尚不完全清楚。在这里，使用用野生型或磷酸化突变形式的 TRAF2 重建的四种不同的 TRAF2 缺陷细胞系（A20.2J、CH12.LX、HAP1 和小鼠胚胎成纤维细胞 [MEF]），以及免​​疫沉淀、免疫印迹、基因表达，和免疫荧光分析，我们报告 CD40 连接引发 TANK 结合激酶 1 (TBK1) 介导的 TRAF2 Ser-11 磷酸化。这种磷酸化干扰了 TRAF2 的 RING 结构域和膜磷脂之间的相互作用，并使 TRAF2 复合物从 CD40 易位到细胞质。我们还观察到这种细胞质易位是 JNK 通路和 NF-κB 通路第二阶段完全激活所必需的。此外，我们发现，在 Ser-11 磷酸化缺失的情况下，TRAF2 RING 结构域与磷脂相互作用，导致 TRAF2 复合物易位至脂筏，导致其降解并激活非经典 NF-κB 通路。因此，我们的结果为 CD40 信号传导机制提供了新的见解，其中 Ser-11 磷酸化控制 TRAF2 的 RING 结构域依赖性亚细胞定位，从而调节 JNK 和 NF-κB 通路的时空激活。