Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells
醣啟動了調控活化B細胞存亡之樞紐

Nat Commun. 2016, 7, 12526.
Wu JL, Wu HY, Tsai DY, Chiang MF, Chen YJ, Gao S, Lin CC, Lin CH, Khoo KH, Chen YJ, Lin KI

Crosslinking of B-cell receptor (BCR) sets off an apoptosis programme, but the underlying pathways remain obscure. Here we decipher the molecular mechanisms bridging B-cell activation and apoptosis mediated by post-translational modification (PTM). We find that O-GlcNAcase inhibition enhances B-cell activation and apoptosis induced by BCR crosslinking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identifies 313 O-GlcNAcylation-dependent phosphosites on 224 phosphoproteins. Among these phosphoproteins, temporal regulation of the O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggers apoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1 is a prerequisite for the recruitment of its kinase, PKC-β1, to induce S243 phosphorylation, leading to ERK activation and downregulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation.

Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells

O連結乙醯葡萄氨糖修飾是細胞中的一種後轉錄調控修飾作用,在細胞中此種修飾是經由OGT酵素在蛋白質的絲氨酸和蘇氨酸進行催化完成,另一個酵素OGA則負責將此修飾移除。由於此種修飾是發生在絲氨酸和蘇氨酸上,其在細胞中所參與的許多作用被認為是藉由影響磷酸化修飾來達成。因為B細胞經由受體接合而活化的過程中會產生大量的磷酸化修飾訊息傳遞,因此本研究的目標為探討及了解O連結乙醯葡萄氨糖修飾如何對於B細胞的活化及其伴隨的細胞凋亡作用產生影響。研究中我們發現O連結乙醯葡萄氨糖修飾的累積會加強B細胞的活化和細胞凋亡反應。接著,藉由磷酸化蛋白質體的比較分析我們找出224對於O連結乙醯葡萄氨糖修飾敏感的磷酸化蛋白 (313位點)。從磷酸化蛋白質體結果中我們發現一個對O連結乙醯葡萄氨糖修飾敏感的Lsp1蛋白並在後續的實驗中證實其會被O連結乙醯葡萄氨糖修飾在絲氨酸209位點上。接著,藉由觀察Lsp1蛋白的O連結乙醯葡萄氨糖修飾和磷酸化在B細胞活化過程的程度變化,發現Lsp1蛋白的磷酸化修飾之增加發生在O連結乙醯葡萄氨糖修飾化的增加之後。實驗也證明,O連結乙醯葡萄氨糖修飾對於絲氨酸243位點的磷酸化是必須的。在利用Lsp1蛋白不同的後轉譯修飾突變進行功能性分析時,我們觀察到Lsp1蛋白的O連結乙醯葡萄氨糖修飾和絲氨酸243位點的磷酸化均對於B細胞的細胞凋亡作用是重要的。在機制研究上,實驗結果發現Lsp1蛋白的O連結乙醯葡萄氨糖修飾是藉由增加Lsp1蛋白和負責絲氨酸243位點的磷酸化之激酶,PKC-β1的接合量來達成促進下游和細胞凋亡相關的訊息傳遞體之活化及降低抗細胞凋亡蛋白BCL-2和BCL-xL的表現來達成促進細胞凋亡的作用。綜合上述,我們的實驗結果闡述了蛋白O連結乙醯葡萄氨糖修飾和磷酸化修飾之間的交互作用決定了B細胞受受體接合而活化後的細胞凋亡命運。