Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation duringactivated Bcellsapoptosis

Here, we decipher the molecular mechanisms bridging B cell activation and apoptosis mediated by post-translational modification(PTM). We found that O-GlcNAcase inhibition enhances B cell activation and apoptosisinduced by B cell receptor (BCR)cross-linking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identified313O-GlcNAcylation-dependent phosphositeson 224 phosphoproteins.Among these phosphoproteins, temporal regulation ofthe O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggersapoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1, identified as the major O-GlcNAc site on Lsp1 by both electron-transfer dissociationand collision-induced dissociationtandem mass spectrometry,was a prerequisite for the recruitment of its kinase, PKC-β1, to induce S243 phosphorylation, leading to ERK activation and down-regulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation.

本研究解析了翻译后修饰（post-translational modification, PTM）所介导的B细胞活化与细胞凋亡之间的分子机制。我们发现，O-连接N-乙酰葡糖胺糖苷酶（O-GlcNAcase）抑制可增强B细胞受体（B cell receptor, BCR）交联诱导的B细胞活化与细胞凋亡。本研究针对受刺激的小鼠原代B细胞中蛋白质O-连接N-乙酰葡糖胺糖基化（O-GlcNAcylation）与磷酸化的功能互作开展蛋白质组规模分析，共鉴定得到224个磷酸化蛋白质上的313个O-GlcNAcylation依赖型磷酸化位点。在上述磷酸化蛋白质中，淋巴细胞特异性蛋白1（lymphocyte-specific protein-1, Lsp1）的O-连接N-乙酰葡糖胺糖基化与磷酸化的时序调控，是触发活化B细胞凋亡的关键分子开关。经电子转移解离（electron-transfer dissociation, ETD）与碰撞诱导解离（collision-induced dissociation, CID）串联质谱共同鉴定，Lsp1的主要O-连接N-乙酰葡糖胺糖基化位点为其丝氨酸209（S209）位点；该修饰是其激酶蛋白激酶C-β1（PKC-β1）招募的先决条件，可诱导丝氨酸243（S243）位点发生磷酸化，进而激活细胞外调节蛋白激酶（ERK），并下调BCL-2与BCL-xL的蛋白表达水平。综上，本研究证实了Lsp1关键的翻译后修饰互作，其可传递B细胞受体交联后启动细胞凋亡的信号。