Nonreceptor tyrosine kinase ABL1 regulates lysosomal acidification by phosphorylating the ATP6V1B2 subunit of the vacuolar-type H+-ATPase.

The vacuolar-type H⁺-ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. Its activity and assembly are tightly controlled by multiple pathways, of which phosphorylation-mediated regulation is poorly understood. In this report, we show that in response to starvation stimuli, the nonreceptor tyrosine kinase ABL1 directly interacts with ATP6V1B2, a subunit of the V₁ domain of the V-ATPase, and phosphorylates ATP6V1B2 at Y68. Y68 phosphorylation in ATP6V1B2 facilitates the recruitment of the ATP6V1D subunit into the V₁ subcomplex of V-ATPase, therefore potentiating the assembly of the V₁ subcomplex with the membrane-embedded V₀ subcomplex to form the integrated functional V-ATPase. ABL1 inhibition or depletion impairs V-ATPase assembly and lysosomal acidification, resulting in an increased lysosomal pH, a decreased lysosomal hydrolase activity, and consequently, the suppressed degradation of lumenal cargo during macroautophagy/autophagy. Consistently, the efficient removal of damaged mitochondrial residues during mitophagy is also impeded by ABL1 deficiency. Our findings suggest that ABL1 is a crucial autophagy regulator that maintains the adequate lysosomal acidification required for both physiological conditions and stress responses.Abbreviation: ANOVA: analysis of variance; Baf A1: bafilomycin A1; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CRK: CRK proto-oncogene, adaptor protein; CTSD: cathepsin D; DMSO: dimethylsulfoxide; EBSS: Earle's balanced salt solution; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; GST: glutathione S-transferase; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PD: Parkinson disease; PLA: proximity ligation assay; RFP: red fluorescent protein; WT: wild-type.