ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

Abstract

Unc-51-like autophagy activating kinase 1 (ULK1)-autophagy-related 13 (ATG13) is the most upstream autophagy initiation complex that is phosphorylated by mammalian targetof-rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) to induce autophagy in asynchronous conditions. However, their phospho-regulation and functions in mitosis and cell cycle remain unknown. Here we show that ULK1-ATG13 complex is differentially regulated throughout the cell cycle, especially in mitosis, in which both ULK1 and ATG13 are highly phosphorylated by the key cell cycle machinery cyclin-dependent kinase 1 (CDK1)/cyclin B. Combining mass spectrometry and site-directed mutagenesis, we found that CDK1-induced ULK1-ATG13 phosphorylation promotes mitotic autophagy and cell cycle progression. Moreover, double knockout (DKO) of ULK1 and ATG13 could block cell cycle progression and significantly decrease cancer cell proliferation in cell line and mouse models. Our results not only bridge the mutual regulation between the core machinery of autophagy and mitosis but also illustrate the positive function of ULK1-ATG13 and their phosphorylation by CDK1 in mitotic autophagy regulation.

ULK1 inhibitor induces spindle microtubule disorganization and inhibits phosphorylation of Ser10 of histone H3

“…SBI-0206965/MRT68921-induced spindle microtubule disorganization and pH3(S10) inhibition resulted in prolonged mitotic duration and chromatin condensation delay Autophagy regulators were shown to modulate mitosis [30][31][32][33][34], but the effect of ULK1 inhibitor on mitosis is unknown, which hampered the comprehensive understanding of ULK1 inhibitor's therapeutic potential. HeLa cells stably expressing GFP-Tubulin and RFP-H2B released from thymidine were treated with the widely used ULK1 inhibitors, SBI-0206965 or MRT68921, to examine their effects on mitosis.…”

“…HeLa cells expressing GFP-Tubulin-red fluorescent protein (RFP)-H2B were a gift of Timothy Mitchison from Harvard Medical School and maintained in DMEM complete medium as described earlier with 500 ngÁmL À1 G418. ULK1 knockout (KO) HeLa/HEK-293T cells were constructed as described previously [34]. In brief, after 12 h transfection, HeLa or HEK-293T cells transfected with the plasmid PX458 subcloned with ULK1-guide RNA were diluted into 0.5 cell/100 lL in a 96-well plate.…”

“…Cell-cycle synchronization was performed as described previously [34]. In brief, cells were plated at 25% confluence 24 h before thymidine block assay.…”

“…Autophagy has been reported to be closely related to cell cycle or mitosis [23,[27][28][29], which indicates a potential combinatorial therapy with mitotic drugs for tumors dependent on autophagy for survival. Although autophagy regulators such as ULK1, AMPK, ATG13 and p62 were shown to regulate mitosis [30][31][32][33][34], whether ULK1/2 inhibitors are involved in mitosis is unknown. Herein we found that ULK1 inhibitors SBI-0206965 or MRT68921 induced striking spindle microtubule fragmentation and polymerization and chromatin condensation delay, which induced polyploidy formation and mitotic entry delay, respectively.…”

Mitotic phosphorylation of the ULK complex regulates cell cycle progression

“…Two papers (one by Li and colleagues in this issue and another recently published in Molecular Cell by Odle and colleagues) reveal that CDK1 phosphorylates the autophagy machinery [26,27] (Fig 1). In these works, both groups observe up-shifted bands of ULK1 and ATG13 in mitotic cell lysates.…”