Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

TAX1BP1 is a selective macroautophagy/autophagy receptor that inhibits NFKB and RIGI-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as SQSTM1/p62 and OPTN are phosphorylated by the kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that TBK1 and IKBKE/IKKi function redundantly to phosphorylate TAX1BP1 and regulate its autophagic turnover through canonical macroautophagy. TAX1BP1 phosphorylation promotes its localization to lysosomes, resulting in its degradation. Additionally, we found that during vesicular stomatitis virus infection, TAX1BP1 is targeted to lysosomes in an ATG8-family protein-independent manner. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 controls its MAVS aggrephagy function. Together, our data support a model whereby TBK1 and IKBKE license TAX1BP1-selective autophagy function to inhibit MAVS and RLR signaling. <b>Abbreviations:</b> ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2: calcium binding and coiled-coil domain 2; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; IFN: interferon; IκB: inhibitor of nuclear factor kappa B; IKK: IκB kinase; IRF: interferon regulatory factor; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; NFKB: nuclear factor kappa B; OPTN: optineurin; Poly(I:C): polyinosinic-polycytidylic acid; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SDD-AGE: semi-denaturing detergent-agarose gel electrophoresis; SeV: Sendai virus; SLR: SQSTM1-like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF: TNF receptor associated factor; VSV: vesicular stomatitis virus; ZnF: zinc finger.

TAX1BP1（Tax1结合蛋白1，Tax1 binding protein 1）是一种选择性巨自噬/自噬受体，可抑制核因子κB（NFKB，nuclear factor kappa B）与RIG-I样受体（RLR，RIGI-like receptor）信号通路，从而防止过度炎症反应并维持体内稳态。诸如SQSTM1/p62（sequestosome 1）与OPTN（optineurin）这类选择性自噬受体，可被激酶TBK1（TANK结合激酶1，TANK binding kinase 1）磷酸化，以激活其选择性自噬功能。然而，在基础状态或RNA病毒感染期间，TAX1BP1是否受TBK1或其他激酶调控，目前尚不明确。本研究发现，TBK1与IKBKE/IKKi功能冗余，可磷酸化TAX1BP1，并通过经典巨自噬调控其自噬周转过程。TAX1BP1的磷酸化可促进其向溶酶体定位，最终引发自身降解。此外，本研究发现，在水疱性口炎病毒（VSV，vesicular stomatitis virus）感染过程中，TAX1BP1可通过不依赖ATG8家族蛋白的方式被靶向运输至溶酶体。进一步研究表明，TAX1BP1在线粒体抗病毒信号蛋白（MAVS，mitochondrial antiviral signaling protein）聚集体的清除中发挥关键作用，而TAX1BP1的磷酸化可调控其介导的MAVS聚集体自噬功能。综上，本研究数据支持如下模型：TBK1与IKBKE赋予TAX1BP1选择性自噬功能，从而抑制MAVS与RLR信号通路。
<b>缩写说明：</b>ATG（autophagy related）：自噬相关基因；BafA1（bafilomycin A1）：巴弗洛霉素A1；CALCOCO2（calcium binding and coiled-coil domain 2）：钙结合卷曲螺旋结构域2；GFP（green fluorescent protein）：绿色荧光蛋白；IFA（indirect immunofluorescence assay）：间接免疫荧光试验；IFN（interferon）：干扰素；IκB（inhibitor of nuclear factor kappa B）：核因子κB抑制剂；IKK（IκB kinase）：IκB激酶；IRF（interferon regulatory factor）：干扰素调节因子；KO（knockout）：基因敲除；LAMP1（lysosomal associated membrane protein 1）：溶酶体相关膜蛋白1；LIR（LC3-interacting region）：LC3相互作用区域；MAP1LC3/LC3（microtubule associated protein 1 light chain 3）：微管相关蛋白1轻链3；MAVS（mitochondrial antiviral signaling protein）：线粒体抗病毒信号蛋白；MEF（mouse embryonic fibroblast）：小鼠胚胎成纤维细胞；MOI（multiplicity of infection）：感染复数；IKBKG/NEMO（inhibitor of nuclear factor kappa B kinase regulatory subunit gamma）：核因子κB激酶调节亚基γ；NFKB（nuclear factor kappa B）：核因子κB；OPTN（optineurin）：视神经蛋白；Poly(I:C)（polyinosinic-polycytidylic acid）：聚肌胞苷酸；RB1CC1/FIP200（RB1 inducible coiled-coil 1）：RB1诱导性卷曲螺旋1；RIGI（RNA sensor RIG-I）：RNA感受器RIG-I；RLR（RIGI-like receptor）：RIG-I样受体；SDD-AGE（semi-denaturing detergent-agarose gel electrophoresis）：半变性去污剂琼脂糖凝胶电泳；SeV（Sendai virus）：仙台病毒；SLR（SQSTM1-like receptor）：SQSTM1样受体；SQSTM1（sequestosome 1）：隔离体1；TAX1BP1（Tax1 binding protein 1）：Tax1结合蛋白1；TBK1（TANK binding kinase 1）：TANK结合激酶1；TNF（tumor necrosis factor）：肿瘤坏死因子；TRAF（TNF receptor associated factor）：TNF受体相关因子；VSV（vesicular stomatitis virus）：水疱性口炎病毒；ZnF（zinc finger）：锌指结构域。