AKT-mediated phosphorylation of ATG4B impairs mitochondrial activity and enhances the Warburg effect in hepatocellular carcinoma cells

Phosphorylation is a major type of post-translational modification, which can influence the cellular physiological function. ATG4B, a key macroautophagy/autophagy-related protein, has a potential effect on the survival of tumor cells. However, it is still unknown as to the role of ATG4B phosphorylation in cancers. In this study, we identified a novel phosphorylation site at Ser34 of ATG4B induced by AKT in HCC cells. The phosphorylation of ATG4B at Ser34 had little effect on autophagic flux, but promoted the Warburg effect including the increase of L-lactate production and glucose consumption, and the decrease of oxygen consumption in HCC cells. The Ser34 phosphorylation of ATG4B also contributed to the impairment of mitochondrial activity including the inhibition of F₁Fo-ATP synthase activity and the elevation of mitochondrial ROS in HCC cells. Moreover, the phosphorylation of ATG4B at Ser34 enhanced its mitochondrial location and the subsequent colocalization with F₁Fo-ATP synthase in HCC cells. Furthermore, recombinant human ATG4B protein suppressed the activity of F₁Fo-ATP synthase in MgATP submitochondrial particles from patient-derived HCC tissues <i>in vitro</i>. In brief, our results demonstrate for the first time that the phosphorylation of ATG4B at Ser34 participates in the metabolic reprogramming of HCC cells via repressing mitochondrial function, which possibly results from the Ser34 phosphorylation-induced mitochondrial enrichment of ATG4B and the subsequent inhibition of F₁Fo-ATP synthase activity. Our findings reveal a noncanonical working pattern of ATG4B under pathological conditions, which may provide a scientific basis for developing novel strategies for HCC treatment by targeting ATG4B and its Ser34 phosphorylation.

磷酸化是一类关键的翻译后修饰类型，可调控细胞生理功能。ATG4B作为与巨自噬/自噬相关的核心蛋白，对肿瘤细胞存活具有潜在调控作用。然而，ATG4B磷酸化在癌症中的具体作用仍未明确。本研究在肝细胞癌（hepatocellular carcinoma, HCC）细胞中鉴定出AKT诱导的ATG4B Ser34位点新型磷酸化修饰。ATG4B Ser34位点磷酸化对自噬流影响微弱，但可促进肝癌细胞的瓦伯格效应，具体表现为L-乳酸生成与葡萄糖消耗增加，以及氧消耗降低。ATG4B Ser34磷酸化还会损伤线粒体功能，具体包括抑制F₁Fo-ATP合酶活性，以及升高肝癌细胞内的线粒体活性氧水平。此外，ATG4B Ser34磷酸化可增强其线粒体定位，并进一步与F₁Fo-ATP合酶发生共定位。进一步实验发现，重组人ATG4B蛋白在体外可抑制患者来源肝癌组织的MgATP亚线粒体颗粒中F₁Fo-ATP合酶的活性。综上，本研究首次证实，ATG4B Ser34位点磷酸化通过抑制线粒体功能参与肝癌细胞的代谢重编程，其潜在机制为Ser34磷酸化诱导ATG4B在线粒体富集，进而抑制F₁Fo-ATP合酶活性。本研究揭示了病理状态下ATG4B的非经典作用模式，可为通过靶向ATG4B及其Ser34磷酸化开发肝癌治疗新策略提供科学依据。