Abstract

Macroautophagy/autophagy is an evolutionarily conserved degradation pathway wherein cytoplasmic components are sequestered within double-membrane autophagosomes for lysosomal delivery. The initiation of autophagy is governed by autophagy-related (ATG) proteins, with the ULK1 kinase complex serving as the most upstream regulator. However, how ULK1 senses and integrates metabolic signals via post-translational modifications remains poorly understood. Here, we discover that ULK1 undergoes lactylation at lysine 46, catalyzed by the mitochondrial aminoacyl-tRNA synthetase AARS2, in response to autophagic stimuli. This modification promotes ULK1 kinase activity, leading to enhanced and selective phosphorylation of its downstream substrate ATG14 at Ser29, thereby activating the class III PtdIns3K complex and facilitating autophagosome biogenesis. Furthermore, we demonstrate that AARS2-mediated ULK1 lactylation drives autophagic flux and promotes tumor metastasis in clear cell renal cell carcinoma (ccRCC), and that a cell-penetrating peptide targeting K46 lactylation suppresses ccRCC progression in vitro and in vivo. Our study identifies lactylation as a novel regulatory mechanism controlling autophagy initiation and suggests that targeting AARS2-mediated ULK1 lactylation could be a potential strategy for treating ccRCC.Abbreviations: AARS2: alanyl-tRNA synthetase 2, mitochondrial; ATG14: autophagy related 14; Baf.A1: bafilomycin A1; ccRCC: clear cell renal cell carcinoma; co-IP: co-immunoprecipitation; CPP: cell-penetrating peptide; EBSS: Earle’s balanced salt solution; HIF: hypoxia-inducible factor; K46la: lactylation at lysine 46; Kla: lysine lactylation; KO: knockout; LDHA: lactate dehydrogenase A; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; PtdIns3K: class III phosphatidylinositol 3-kinase; PTM: post-translational modification; SQSTM1/p62: sequestosome 1; TCGA-KIRC: The Cance