CDK1 phosphorylates ULK1-ATG13 complex to regulate mitotic autophagy and Taxol chemosensitivity

Abstract

ULK1-ATG13 is the most upstream autophagy initiation complex that is phosphorylated by mTORC1 and AMPK to induce autophagy in asynchronous conditions. However, the phospho-regulation and function of ULK1-ATG13 in mitosis and cell cycle remains unknown. Here we show that ULK1-ATG13 complex is differentially regulated throughout the cell cycle. Notably, in mitosis, both ULK1 and ATG13 are highly phosphorylated by CDK1/cyclin B, the key cell cycle machinery. Combining mass spectrometry and site-directed mutagenesis, we found that CDK1-induced ULK1-ATG13 phosphorylation positively regulates mitotic autophagy and Taxol chemosensitivity, and some phosphorylation sites occur in cancer patients. Moreover, double knockout 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 machineries of autophagy and mitosis, illustrate the mitotic autophagy regulation mechanism, but also provide ULK1-ATG13 as potential targets for cancer therapy. Immunoprecipitation and Western blotsThe procedure was instructed as previously [68]. Most immunoprecipitation experiments were conducted in HEK-293T derived cell lines due to higher expression level for the exogenous protein. Briefly, HEK-293T cells stably expressing GFP-3FLAG or mULK1-3FLAG were lysed with M-PER supplemented with protease inhibitors and phosphatase inhibitors and centrifuged at 4°C 14,000g for 10 min. The supernatant mixed with pre-incubated Protein G Dynabeads and FLAG antibody at 4°C for 12 h and washed three times with lysis buffer. Then the immunoprecipitate was denatured in 1×SDS-PAGE buffer at 95°C for 7 min and subjected to SDS-PAGE and immunoblotting or Coomassie brilliant blue staining. Apoptosis by flow cytometryCells treated with Taxol combine with or without chloroquine were subjected to apoptosis assay according to the manufacturer instructions (BD Biosciences). Briefly, cells were trypsinized and washed twice with ice cold PBS, and stained with PI and/or FITC-Annexin V in apoptosis buffer for 30 min. The FITC and PE channel were used for flow cytometry detection and the data were analyzed by FlowJo. Cell cycle synchronizationVarious cell synchronization methods are used in this paper, which have been used previously. Briefly, a double-thymidine (2.5 μM) block arrested cells in G1/S border and cells progress through S, G2 and M phase after release. A double-thymidine or single-thymidine block in combination with nocodazole (100 ng/mL) or STLC (5 μM) treatment arrested cells in prometaphase or prophase. A double-thymidine block in combination with RO-3306 (10 μM) treatment arrested cells in late G2 phase and progressed into mitosis after RO-3306 washout for three times with prewarmed PBS. Cell cycle analysis by flow cytometry/FACSCells for cell cycle analysis were trypsinized with 0.25% Trypsin/EDTA. After washing with ice-cold PBS twice, cells were fixed with -20°C 75% ethanol overnight and then stained with PI/RN...