ULK1 (
unc-51 like autophagy activating kinase 1) is a core component at multiple steps of canonical macroautophagy/autophagy. The activity of ULK1 is tightly regulated by several post-translational modifications, including ubiquitination, yet the
deubiquitinase (DUB) responsible for its reversible deubiquitination has not been described. Here, we identified USP1 (
ubiquitin specific peptidase 1) as a key player in the modulation of ULK1 K63-linked deubiquitination. Moreover, both USP1 depletion and its chemical inhibition by
pimozide are coupled to a reduction of ULK1 in
Triton X-100 soluble cellular lysates, and its compartmentalization to a fraction that can be solubilized in 5 M
urea. In USP1-depleted cells this fraction is also enriched in SQSTM1 (sequestosome 1), the aggresome marker HDAC6 (
histone deacetylase 6), and the prototype of USP1 targets FANCD2 (FA complementation group D2). Consistently, in USP1-depleted and
pimozide-treated cells, ULK1 forms
protein aggregates enriched in SQSTM1, as detected by both immummunofluorescence and co-immunoprecipitation studies. Notably, depletion of USP1 inhibits canonical autophagic flux and promotes an alternative route leading to lysosomal-mediated degradation of SQSTM1. Our findings reveal a novel function of the USP1-ULK1 axis as a modulator of the switch between canonical and unconventional autophagy. Further, we provide the first evidence supporting the existence of a subset of
breast tumors co-expressing ULK1 and MAP1LC3B (
microtubule associated protein 1 light chain 3 beta)
proteins. Because the USP1 inhibitor
pimozide affects
breast cancer cell growth, targeting USP1 in those
tumors relying on autophagy for growth might prove to be a convenient therapeutic strategy. Abbreviations: ATG13: autophagy related 13; BECN1:
beclin 1; BZ:
bortezomib; CAPN1:
calpain 1; DUB:
deubiquitinase; FANCI: FA complementation group I; FANCD2: FA complementation group D2; FZR1: fizzy and cell division cycle 20 related 1; HDAC6:
histone deacetylase 6; MAP1LC3B:
microtubule associated protein 1 light chain 3 beta; PMZ:
pimozide; SH3GLB1: SH3 domain containing GRB2 like, endophilin B1; SQSTM1: sequestosome 1;
TRAF6:
TNF receptor associated factor 6; ULK1:
unc-51 like autophagy activating kinase 1; USP1:
ubiquitin specific peptidase 1; WDR48: WD repeat domain 48.