Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth
Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth
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SMURF1 泛素化 UVRAG 可促进自噬体成熟并抑制肝细胞癌生长
DOI：
10.1080/15548627.2019.1570063
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发表时间：
2019-07-03
期刊：
影响因子：
13.3
通讯作者：
Zhang, Zhiyong
中科院分区：
文献类型：
--
作者：
Feng, Xing;Jia, Yanyan;Zhang, Zhiyong
UVRAG（UV radiation resistance associated）是哺乳动物巨自噬/自噬的重要调控因子，与BECN 1、PIK 3C 3和RUBCN相互作用。MTORC 1对UVRAG的磷酸化在营养丰富的条件下负调节自噬体成熟。然而，UVRAG泛素化是如何调节的仍然是未知的。在这里，我们报告说，UVRAG是泛素化的SMURF 1在赖氨酸残基517和559，这减少了UVRAG与RUBCN的协会，促进自噬体成熟。然而，去泛素化酶ZRANB 1特异性地从UVRAG切割SMURF 1诱导的K29和K33连接的多聚泛素链，从而增加UVRAG与RUBCN的结合并抑制自噬通量。我们还证明了CSNK 1A 1介导的UVRAG在Ser 522处的磷酸化通过PPxY基序破坏了SMURF 1与UVRAG的结合，并阻断了UVRAG泛素化介导的自噬体成熟。有趣的是，ZRANB 1在Thr 35和Ser 209残基被CSNK 1A 1磷酸化，这种磷酸化激活了其去泛素化活性。重要的是，我们提供了体外和体内证据，证明UVRAG在赖氨酸残基517和559处的泛素化或通过D4476（一种CSNK 1A 1抑制剂）阻止Ser 522磷酸化，增强了EGFR的溶酶体降解，从而显著抑制了肝细胞癌（HCC）的生长。此外，在HCC患者中，UVRAG S522磷酸化水平与ZRANB 1 T35/S209磷酸化水平和不良预后相关。这些发现确定了一种新的分子机制，UVRAG的泛素化和磷酸化调节其在自噬体成熟和HCC生长中的功能，鼓励进一步研究其潜在的治疗意义。缩略语：美国电话：自噬相关; BafA1：巴弗洛霉素A1; BECN 1：1; CHX：环己酰亚胺; CSNK1A1/CK1α：酪蛋白激酶1 α 1; CQ：氯喹; DUB：去泛素化酶; EBSS：Earle平衡盐溶液; EGF：表皮生长因子; GFP：绿色荧光蛋白; GST：谷胱甘肽S-转移酶; HBSS：Hanks平衡盐溶液; HCC：肝细胞癌; MAP 1 LC 3 B/LC 3：微管相关蛋白1轻链3 β; MEFs：小鼠胚胎成纤维细胞; mRFP：单体红色荧光蛋白; PIK 3C 3/VPS 34：磷脂酰肌醇3-激酶催化亚基3型; PTM：翻译后修饰; RUBCN：rubicon自噬调节因子; siRNA：小干扰RNA; SMURF 1：SMAD特异性E3泛素蛋白连接酶1; SQSTM 1：螯合体1; Ub-AMC：泛素-7-酰胺基-4-甲基香豆素：荧光底物; UVRAG：UV辐射抗性相关; ZRANB 1/TRABID：锌指RANBP 2型含1
ABSTRACT UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications. Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; BECN1: beclin 1; CHX: cycloheximide; CSNK1A1/CK1α: casein kinase 1 alpha 1; CQ: chloroquine; DUB: deubiquitinase; EBSS: Earle’s balanced salt solution; EGF: epidermal growth factor; GFP: green fluorescent protein; GST: glutathione S-transferase; HBSS: Hanks balanced salts solution; HCC: hepatocellular carcinoma; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryo fibroblasts; mRFP: monomeric red fluorescent protein; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTMs: post-translational modifications; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; Ub-AMC: ubiquitin-7-amido-4-methylcoumarin: a fluorogenic substrate; UVRAG: UV radiation resistance associated; ZRANB1/TRABID: zinc finger RANBP2-type containing 1