Autophagy receptor p62/SQSTM1 signals a complex network that links autophagy-lysosomal system to proteasome. Phosphorylation of p62 on Serine 349 (P-Ser349 p62) is involved in a cell protective, antioxidant pathway. We have shown previously that P-Ser349 p62 occurs and is rapidly degraded during human synovial fibroblasts autophagy. In this work we observed that fingolimod (FTY720), used as a medication for multiple sclerosis, induced coordinated expression of p62, P-Ser349 p62 and inhibitory TFEB form, phosphorylated on Serine 211 (P-Ser211 TFEB), in human synovial fibroblasts. These effects were mimicked and potentiated by proteasome inhibitor MG132. In addition, FTY720 induced autophagic flux, LC3B-II up-regulation, Akt phosphorylation inhibition on Serine 473 but down-regulated TFEB, suggesting stalled autophagy. FTY720 decreased cytoplasmic fraction contained TFEB but induced TFEB in nuclear fraction. FTY720-induced P-Ser211 TFEB was mainly found in membrane fraction. Autophagy and VPS34 kinase inhibitor, autophinib, further increased FTY720-induced P-Ser349 p62 but inhibited concomitant expression of P-Ser211 TFEB. These results suggested that P-Ser211 TFEB expression depends on autophagy. Overexpression of GFP tagged TFEB in HEK293 cells showed concomitant expression of its phosphorylated form on Serine 211, that was down-regulated by autophinib. These results suggested that autophagy might be autoregulated through P-Ser211 TFEB as a negative feedback loop. Of interest, overexpression of p62, p62 phosphorylation mimetic (S349E) mutant and phosphorylation deficient mutant (S349A) in HEK293 cells markedly induced P-Ser211 TFEB. These results showed that p62 is involved in regulation of TFEB phosphorylation on Serine 211 but that this involvement does not depend on p62 phosphorylation on Serine 349.

中文翻译：

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丝氨酸 211 上的 TFEB 磷酸化由人滑膜成纤维细胞中的自噬和 HEK293 细胞中的 p62/SQSTM1 过表达诱导

自噬受体 p62/SQSTM1 发出一个复杂的网络信号，该网络将自噬-溶酶体系统与蛋白酶体联系起来。丝氨酸 349 (P-Ser349 p62) 上 p62 的磷酸化参与细胞保护、抗氧化途径。我们之前已经表明 P-Ser349 p62 在人类滑膜成纤维细胞自噬过程中发生并迅速降解。在这项工作中，我们观察到芬戈莫德 (FTY720)，用作多发性硬化症的药物，在人滑膜成纤维细胞中诱导 p62、P-Ser349 p62 和抑制性 TFEB 形式的协调表达，在丝氨酸 211 (P-Ser211 TFEB) 上磷酸化。这些作用被蛋白酶体抑制剂 MG132 模拟和加强。此外，FTY720 诱导自噬通量、LC3B-II 上调、Akt 磷酸化抑制丝氨酸 473 但下调 TFEB，表明自噬停滞。FTY720 减少细胞质部分含有 TFEB，但在核部分诱导 TFEB。FTY720 诱导的 P-Ser211 TFEB 主要存在于膜组分中。自噬和 VPS34 激酶抑制剂 autophinib 进一步增加了 FTY720 诱导的 P-Ser349 p62，但同时抑制了 P-Ser211 TFEB 的表达。这些结果表明 P-Ser211 TFEB 表达依赖于自噬。HEK293 细胞中 GFP 标记的 TFEB 的过表达显示其磷酸化形式在丝氨酸 211 上的伴随表达，这被 autophinib 下调。这些结果表明自噬可能通过 P-Ser211 TFEB 作为负反馈回路进行自动调节。有趣的是，在 HEK293 细胞中 p62、p62 磷酸化模拟物 (S349E) 突变体和磷酸化缺陷突变体 (S349A) 的过表达显着诱导了 P-Ser211 TFEB。