a The affiliated Hospital of Guilin Medical University , Guangxi Key Laboratory of Brain and Cognitive Neuroscience, Guangxi Neurological Diseases Clinical Research Center , Guilin , Guangxi , China.
b Rutgers Cancer Institute of New Jersey , Rutgers University , New Brunswick , New Jersey , USA.
Autophagy. 2019 Jul;15(7):1130-1149. doi: 10.1080/15548627.2019.1570063. Epub 2019 Jan 27.
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 and 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. ATG: autophagy related; BafA: bafilomycin A; 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; PIK3C3VPS34: 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.
UVRAG（与紫外线辐射抗性相关）通过与 BECN1、PIK3C3 和 RUBCN 相互作用，是哺乳动物大自噬/自噬的重要调节剂。在营养丰富的条件下，MTORC1 对 UVRAG 的磷酸化负调控自噬体成熟。然而，UVRAG 泛素化的调节方式仍不清楚。在这里，我们报告 UVRAG 在赖氨酸残基 517 和 559 处被 SMURF1 泛素化，这降低了 UVRAG 与 RUBCN 的结合，并促进自噬体成熟。然而，去泛素酶 ZRANB1 特异性地从 UVRAG 上切割 SMURF1 诱导的 K29 和 K33 连接的多泛素链，从而增加 UVRAG 与 RUBCN 的结合并抑制自噬通量。我们还证明，CSNK1A1 介导的 UVRAG 丝氨酸 522 磷酸化通过 PPxY 基序破坏 SMURF1 与 UVRAG 的结合，并阻止 UVRAG 泛素化介导的自噬体成熟。有趣的是，ZRANB1 被 CSNK1A1 在 Thr35 和 Ser209 残基上磷酸化，这种磷酸化激活了其去泛素酶活性。重要的是，我们提供了和证据表明，UVRAG 在赖氨酸残基 517 和 559 处的泛素化或通过 CSNK1A1 抑制剂 D4476 防止丝氨酸 522 磷酸化，增强 EGFR 的溶酶体降解，从而显著抑制肝癌（HCC）的生长。此外，UVRAG S522 磷酸化水平与 ZRANB1 T35/S209 磷酸化水平相关，并与 HCC 患者的不良预后相关。这些发现确定了 UVRAG 泛素化和磷酸化调节其在自噬体成熟和 HCC 生长中功能的新分子机制，鼓励进一步研究其潜在的治疗意义。
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