No abstract

Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.

BackgroundAutophagy is a bulk degradation pathway for long-lived proteins, protein aggregates, and damaged organelles. ULK1 protein kinase and Vps34 lipid kinase are two key autophagy regulators that are critical for autophagosome biogenesis. However, it isn’t fully understood how ULK1 regulates Vps34, especially in the context of disease. Polyglutamine expansion in huntingtin (Htt) causes aberrant accumulation of the aggregated protein and disrupts various cellular pathways including autophagy, a lysosomal degradation pathway, underlying the pathogenesis of Huntington’s disease (HD). Although autophagic clearance of Htt aggregates is under investigation as therapeutic strategy for HD, the precise mechanism of autophagy impairment remains poorly understood. Moreover, in-vivo assays of autophagy have been particularly challenging due to lack of reliable and robust molecular biomarkers.MethodWe generated anti-phosphorylated ATG14 antibody to determine ATG14-mediated autophagy regulation; we employed Huntington’s disease (HD) genetic cell models and animal models as well as autophagy reporter animal model to understand autophagy signaling and regulation in vivo. We applied biochemical analysis and molecular biology approaches to dissect the alteration of autophagy kinase activity and regulation.ResultsHere, we demonstrate that ULK1 phosphorylates ATG14 at serine 29 in an mTOR-dependent manner. This phosphorylation critically regulates ATG14-Vps34 lipid kinase activity to control autophagy level. We also show that ATG14-associated Vps34 activity and ULK1-mediated phosphorylation of ATG14 and Beclin 1 are compromised in the Q175 mouse model of Huntington’s disease. Finally, we show that ATG14 phosphorylation is decreased during general proteotoxic stress caused by proteasomal inhibition. This reduction of the specific phosphorylation of ATG14 and Beclin 1 is mediated, in part, by p62-induced sequestration of ULK1 to an insoluble cellular fraction. We show that increased ULK1 levels and phosphor-mimetic mutant ATG14 facilitate the clearance of polyQ mutant in cells.ConclusionOur study identifies a new regulatory mechanism for ATG14-Vps34 kinase activity by ULK1, which can be used as valuable molecular markers for in-vivo autophagic activity as well as potential therapeutic target for the clearance of polyglutamine disease protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0141-0) contains supplementary material, which is available to authorized users.