TRIM27 cooperates with STK38L to inhibit ULK1‐mediated autophagy and promote tumorigenesis

Article7 June 2022free access Source DataTransparent process TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis Yi Yang Yi Yang orcid.org/0000-0001-8697-3062 The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Contribution: Data curation, Formal analysis, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Yifu Zhu Yifu Zhu orcid.org/0000-0002-1625-692X The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Contribution: Data curation, Formal analysis, ​Investigation, Methodology Search for more papers by this author Shuai Zhou Shuai Zhou orcid.org/0000-0001-9065-4777 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Contribution: Data curation, ​Investigation, Methodology Search for more papers by this author Peipei Tang Peipei Tang orcid.org/0000-0002-1233-2833 Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai’an, China Contribution: Data curation, ​Investigation, Methodology Search for more papers by this author Ran Xu Ran Xu Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai’an, China School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia Contribution: ​Investigation, Methodology Search for more papers by this author Yuwei Zhang Yuwei Zhang orcid.org/0000-0002-7410-9047 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Contribution: Resources, Formal analysis, Visualization Search for more papers by this author Dongping Wei Dongping Wei Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China Contribution: Resources, Validation, Methodology Search for more papers by this author Jian Wen Jian Wen orcid.org/0000-0002-2297-4346 Department of Breast Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, China Contribution: Resources, Formal analysis Search for more papers by this author Rick F Thorne Rick F Thorne orcid.org/0000-0001-7882-7081 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia Contribution: Formal analysis, Validation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Xu Dong Zhang Xu Dong Zhang orcid.org/0000-0001-9457-8003 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia Contribution: Resources, Formal analysis, Writing - review & editing Search for more papers by this author Jun-Lin Guan Jun-Lin Guan orcid.org/0000-0001-8720-8338 Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Contribution: Conceptualization, Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Lianxin Liu Corresponding Author Lianxin Liu [email protected] orcid.org/0000-0002-3535-6467 The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Contribution: Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Project administration Search for more papers by this author Mian Wu Corresponding Author Mian Wu [email protected] orcid.org/0000-0002-2714-0500 The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Contribution: Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Project administration Search for more papers by this author Song Chen Corresponding Author Song Chen [email protected] orcid.org/0000-0001-6412-5740 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai’an, China Contribution: Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, ​Investigation, Visualization, Project administration, Writing - review & editing Search for more papers by this author Yi Yang Yi Yang orcid.org/0000-0001-8697-3062 The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Contribution: Data curation, Formal analysis, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Yifu Zhu Yifu Zhu orcid.org/0000-0002-1625-692X The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Contribution: Data curation, Formal analysis, ​Investigation, Methodology Search for more papers by this author Shuai Zhou Shuai Zhou orcid.org/0000-0001-9065-4777 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Contribution: Data curation, ​Investigation, Methodology Search for more papers by this author Peipei Tang Peipei Tang orcid.org/0000-0002-1233-2833 Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai’an, China Contribution: Data curation, ​Investigation, Methodology Search for more papers by this author Ran Xu Ran Xu Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai’an, China School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia Contribution: ​Investigation, Methodology Search for more papers by this author Yuwei Zhang Yuwei Zhang orcid.org/0000-0002-7410-9047 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Contribution: Resources, Formal analysis, Visualization Search for more papers by this author Dongping Wei Dongping Wei Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China Contribution: Resources, Validation, Methodology Search for more papers by this author Jian Wen Jian Wen orcid.org/0000-0002-2297-4346 Department of Breast Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, China Contribution: Resources, Formal analysis Search for more papers by this author Rick F Thorne Rick F Thorne orcid.org/0000-0001-7882-7081 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia Contribution: Formal analysis, Validation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Xu Dong Zhang Xu Dong Zhang orcid.org/0000-0001-9457-8003 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia Contribution: Resources, Formal analysis, Writing - review & editing Search for more papers by this author Jun-Lin Guan Jun-Lin Guan orcid.org/0000-0001-8720-8338 Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Contribution: Conceptualization, Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Lianxin Liu Corresponding Author Lianxin Liu [email protected] orcid.org/0000-0002-3535-6467 The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Contribution: Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Project administration Search for more papers by this author Mian Wu Corresponding Author Mian Wu [email protected] orcid.org/0000-0002-2714-0500 The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Contribution: Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Project administration Search for more papers by this author Song Chen Corresponding Author Song Chen [email protected] orcid.org/0000-0001-6412-5740 Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai’an, China Contribution: Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, ​Investigation, Visualization, Project administration, Writing - review & editing Search for more papers by this author Author Information Yi Yang1,†, Yifu Zhu1,†, Shuai Zhou2, Peipei Tang3, Ran Xu3,4, Yuwei Zhang2, Dongping Wei5, Jian Wen6, Rick F Thorne2,4, Xu Dong Zhang2,4, Jun-Lin Guan7, Lianxin Liu *,1, Mian Wu *,1,2 and Song Chen *,2,3 1The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China 2Translational Research Institute, Henan Provincial and Zhengzhou City Key Laboratory of Non-coding RNA and Cancer Metabolism, Henan International Join Laboratory of Non-coding RNA and Metabolism in Cancer, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China 3Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai’an, China 4School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia 5Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China 6Department of Breast Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, China 7Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA † These authors contributed equally to this work *Corresponding author. Tel: +86 551 62283477; E-mail: [email protected] *Corresponding author. Tel: +86 551 63606264; E-mail: [email protected] *Corresponding author. Tel: +86 137 70397497; E-mail: [email protected] The EMBO Journal (2022)41:e109777https://doi.org/10.15252/embj.2021109777 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Autophagy represents a fundamental mechanism for maintaining cell survival and tissue homeostasis in response to physiological and pathological stress. Autophagy initiation converges on the FIP200-ATG13-ULK1 complex wherein the serine/threonine kinase ULK1 plays a central role. Here, we reveal that the E3 ubiquitin ligase TRIM27 functions as a negative regulatory component of the FIP200-ATG13-ULK1 complex. TRIM27 directly polyubiquitinates ULK1 at K568 and K571 sites with K48-linked ubiquitin chains, with proteasomal turnover maintaining control over basal ULK1 levels. However, during starvation-induced autophagy, TRIM27 catalyzes non-degradative K6- and K11-linked ubiquitination of the serine/threonine kinase 38-like (STK38L) kinase. In turn, STK38L ubiquitination promotes its activation and phosphorylation of ULK1 at Ser495, rendering ULK1 in a permissive state for TRIM27-mediated hyper-ubiquitination of ULK1. This cooperative mechanism serves to restrain the amplitude and duration of autophagy. Further evidence from mouse models shows that basal autophagy levels are increased in Trim27 knockout mice and that Trim27 differentially regulates tumorigenesis and metastasis. Our study identifies a key role of STK38L-TRIM27-ULK1 signaling axis in negatively controlling autophagy with relevance established in human breast cancer. Synopsis While ULK1 is essential for autophagy initiation, the mechanisms controlling its expression levels and function remain unclear. This report discloses how E3 ubiquitin ligase TRIM27 suppresses ULK1 protein expression to restrain autophagy through dual ubiquitination events, differentially impacting tumorigenesis. E3 ligase TRIM27 negatively regulates autophagy flux under basal and starvation conditions. TRIM27 mediates ubiquitination of ULK1 via K48-linked chains, inducing its proteasomal degradation. Starvation induces TRIM27 ubiquitination and activation of STK38L kinase via K6-linked chains. STK38L phosphorylates ULK1, providing a permissive state for increased ULK1 ubiquitination by TRIM27. Ablation of TRIM27 in the PyMT model inhibits murine mammary tumor initiation but promotes metastasis. Introduction Autophagy is a conserved metabolic recycling pathway whereby proteins, insoluble aggregates, and dysfunctional organelles are engulfed and degraded by lysosomes (Mizushima & Komatsu, 2011). Dysregulated autophagy can lead to a variety of diseases such as cancer and neurodegenerative disorders. Autophagy occurs at low levels under nutrient-rich conditions where there is constitutive recycling of biomaterials to maintain energy homeostasis (Levine & Kroemer, 2008). Conversely, autophagic flux is increased following stress conditions such as limiting nutrients, hypoxia and growth factor deprivation in order to support cellular activities and maintain cell viability. Autophagy is a sequential process (Nakatogawa et al, 2009; Yang & Klionsky, 2009) and among autophagy-related genes, the serine/threonine kinase ULK1 plays a central role in autophagy initiation through a complex formed with ATG13, FIP200, and ATG101 (Ganley et al, 2009; Hosokawa et al, 2009a, 2009b; Mercer et al, 2009; Mizushima, 2010). In yeast, Atg1 complex formation is dynamically regulated by the Tor1 complex, leading to inhibition or initiation of autophagy in a nutrient-dependent manner. However in mammals, ULK1 constitutively associates with ATG13 and FIP200 irrespective of nutrient conditions and the presence of this complex per se is not sufficient to initiate autophagy (Ganley et al, 2009; Hosokawa et al, 2009a; Wong et al, 2013). This predicates that other factors are involved in initiating autophagy. Indeed, under stress conditions, ULK1 activation is not only induced by AMPK-mediated phosphorylation but also by mTOR inhibition which leads to the removal of inhibitory mTOR sites on ULK1 (Ganley et al, 2009; Hosokawa et al, 2009a; Kim et al, 2011). Such activation permits ULK1 to phosphorylate downstream targets in the Beclin1 complex to trigger the autophagy cascade (Russell et al, 2013). A number of studies have established that ULK1 is essential for autophagy initiation during starvation, but paradoxically, recent reports have suggested that ULK1 is rapidly degraded upon nutrient-induced starvation (Allavena et al, 2016; Liu et al, 2016; Nazio et al, 2016). Relatively few studies have disclosed the precise mechanisms involved and how the ULK1 complex is regulated is still largely unknown. TRIM27 (tripartite motif-containing 27) belongs to the tripartite motif (TRIM) proteins, a family of more than 70 genes with conserved domain architectures (Hatakeyama, 2017). Most members contain an N-terminal RING-finger domain, the signature domain for E3 ubiquitin ligases, along with one or two B-box zinc finger domains followed by a coiled-coil domain (Watanabe & Hatakeyama, 2017). TRIM proteins are collectively implicated in a broad range of physiopathological processes, including cancer, immunological diseases, and neurological disorders. Different TRIM family proteins have been variously linked to autophagy where they most often have been shown to influence key upstream pathways that converge on autophagy initiation (Di Rienzo et al, 2020). For example, TRIM37 affects the mTOR pathway to inhibit autophagy (Wang et al, 2018), whereas similarly, the control of intracellular Ca2+ levels by TRIM19 also restrains autophagy through effects on AMPK activity (Missiroli et al, 2016). In some instances, the ubiquitin ligase activity of TRIM proteins directs key targets toward proteasomal degradation as occurs with TRIM28 in the turnover of AMPK (Pineda et al, 2015). Nevertheless, TRIM protein-mediated ubiquitination does not always result in proteasomal degradation as seen with TRIM50 which activates autophagy through the non-degradative modification of Beclin-1 (Fusco et al, 2018). Moreover, not all mechanisms linking TRIM proteins with autophagy have been fully clarified, in particular those directly acting on key autophagy components. Specifically regarding TRIM27, different reports have revealed its functions in cell proliferation, apoptosis, and innate immunity (Zaman et al, 2013; Zheng et al, 2015; Ma et al, 2016); however, its involvement in autophagy has not been reported. Here, we reveal a regulatory nexus between TRIM27 and the autophagy initiation complex that is critical for controlling autophagy under basal and starvation conditions. We show basal levels of autophagy are maintained through TRIM27-mediated ubiquitination and proteasomal degradation of ULK1. Following autophagy induction, TRIM27 ubiquitinates and activates the serine/threonine kinase STK38L which then phosphorylates ULK1, delivering ULK1 in a permissive state for hyper-ubiquitination by TRIM27. We further demonstrate increased basal autophagy in Trim27 knockout mice and establish physiological relevance in the context of breast cancer. Results Identification of TRIM27, a novel binding partner of the FIP200-ATG13-ULK1 complex To discover regulators of the FIP200-ATG13-ULK1 complex, we used an unbiased mass spectrometry approach to recover endogenous FIP200-interacting proteins in mouse embryonic fibroblasts (MEF) under control versus starvation conditions. Using FIP200 knockout MEF cells to filter out non-specific interactors, we identified interactions between FIP200 and previously reported partners including ULK1, ATG13, ATG101 and CCPG1 (Hosokawa et al, 2009b; Wong et al, 2013; Smith et al, 2018) (Fig 1A and Appendix Table S1). An additional 21 novel candidates were selectively retrieved under starvation conditions including two E3 ubiquitin ligases, TRIM27 and NEDD4. Since NEDD4 has previously been implicated in autophagy regulation (Lin et al, 2017; Sun et al, 2017), we focused on verifying the role of TRIM27. Figure 1. TRIM27 binds directly to ULK1 and this binding is regulated by nutritional starvation A. Venn diagram of FIP200 binding proteins identified in MEF following FIP200 co-immunoprecipitation analysis under normal versus starvation conditions. B. Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and the indicated combinations of empty HA-vector, HA-tagged FIP200, ULK1, or ATG13 before immunoprecipitation with anti-HA antibodies. Immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to Western blotting against anti-HA or Flag. C–F. WT (C), FIP200 knockout (KO) (D), ULK1/2 knockout (E) MEF cells or ATG13 knockout 4T1 cells (F) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G (IgG) or anti-TRIM27 before blotting against the indicated endogenous forms of TRIM27, FIP200, ULK1, ATG13, and USP7. G. Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and either full length ULK (HA-ULK1 1–1,051) or the indicated various truncated forms of ULK1 designated HA-ULK1 1–828, HA-ULK1 1–600, and HA-ULK1 1–500; AA, amino acids. H. Co-immunoprecipitation assays in HeLa cells stably expressing HA-ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated. I. HeLa cells stably expressing Flag-TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium. Anti-Flag immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to blotting against Flag and ULK1. J. Purified recombinant GST or GST-TRIM27 was incubated with recombinant HA-ULK1 and bound ULK1 analyzed by Western blotting. K. Schematic diagram of TRIM27 binding to ULK1-ATG13-FIP200 complex in normal or starvation conditions. Data information: For B–J, the experiments were performed at least three times independently with similar results. Source data are available online for this figure. Source Data for Figure 1 [embj2021109777-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint To characterize whether TRIM27 bound to FIP200 discretely or as part of the ULK complex, we reconstructed interactions between ectopically expressed Flag-TRIM27 with individually expressed HA-tagged forms of FIP200, ULK1 and ATG13. Notably, we found Flag-TRIM27 was co-immunoprecipitated with exogenous FIP200, ULK1 and ATG13 (Fig 1B), suggesting their interactions occurred as a complex. These findings were verified in the endogenous context where TRIM27 coprecipitated FIP200, ULK1 and ATG13 in nutrient-starved MEF (Fig 1C). Repeating the assay in FIP200 knockout cells showed endogenous TRIM27 still bound to ULK1 and ATG13 (Fig 1D and Appendix Fig S1A). Moreover, TRIM27 failed to coprecipitate FIP200 and ATG13 in ULK1/2 double knockout MEF cells, although the previously reported interactor USP7 was recovered (Figs 1E and Appendix Fig S1B). Lastly, TRIM27 was shown to bind to ULK1 in the absence of ATG13 (Figs 1F and Appendix Fig S1C). Together, these data suggested that TRIM27 is associated with the FIP200-ATG13-ULK1 complex via interaction with ULK1. Next, we performed domain mapping to better define the TRIM27-ULK1 interaction. Interactions were assessed by reciprocal immunoprecipitation between truncated forms of epitope-tagged ULK1 and TRIM27, respectively. Notably, while the N-terminal 1–600 amino acids (AA) of ULK1 interacted with TRIM27, 1–500AA did not, thereby localizing the TRIM27-interaction sequence between 500 and 600AA (Fig 1G). Conversely, the coiled-coil domain but not the RING-BOX domain of TRIM27 was required for its interaction with ULK1 (Appendix Fig S1D). Lastly, given that TRIM27 bound ULK1 in MEF cells under starvation, as independent verification, we then confirmed the association in human HeLa cells, in that HA-ULK1 bound to more endogenous TRIM27 in response to starvation (Figs 1H and Appendix Fig S1E). Moreover, the interaction between ULK1 and TRIM27 was gradually increased upon starvation but was reduced when the starved cells were replenished with normal growth medium (Fig 1I). Additionally, the interaction between recombinant forms of TRIM27 and ULK1 could be readily observed in glutathione S-transferase (GST) pulldown assays (Fig 1J). Taken together, our data indicate that TRIM27 binds to components of FIP200-ATG13-ULK1 complex via ULK1 and its binding to ULK1 was induced in response to starvation conditions (Fig 1K). TRIM27 is an E3 ubiquitin ligase targeting ULK1 We next evaluated whether TRIM27 served to ubiquitinate ULK1 using His-based ubiquitination assays in HEK293T cells. Indeed, co-expression of TRIM27 selectively promoted the ubiquitination of ULK1, but not FIP200 or ATG13 (Figs 2A and EV1A and B). Depletion of endogenous TRIM27 significantly decreased ULK1 ubiquitination (Fig 2B) and as anticipated, the RING-finger domain of TRIM27 was required for ULK1 ubiquitination (Fig EV1C). Moreover, GST-TRIM27 readily ubiquitinated recombinant HA-ULK1 in vitro (Fig 2C). We also compared the relative ability of TRIM27 to ubiquitinate ULK1 against NEDD4L, another E3 ligase previously shown to target ULK1 (Nazio et al, 2016) and its homologue NEDD4 (Yang & Kumar, 2010). Interestingly, TRIM27 mediated comparatively higher levels of ULK1 ubiquitination than either NEDD4L or NEDD4 (Appendix Fig S2A). Notably, knockdown of TRIM27 did not affect interactions between ULK1 and NEDD4L or NEDD4 (Appendix Fig S2B and C) suggesting TRIM27 does not intrinsically compete with NEDD4L or NEDD4 for ULK1 binding. Thus, together these data establish ULK1 as a direct substrate of TRIM27 E3 ligase. Figure 2. TRIM27 promotes ULK1 degradation via K48-linked ubiquitination of ULK1 at K568/K571 A, B. Ubiquitination assays in HEK293T cells after co-transfection with HA-ULK1 and His-Ub along with Flag-TRIM27 (A) or after knockdown of TRIM27 using independent shRNAs (shTRIM27-1 and -2) (B). Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA, Flag or His as indicated. C. In vitro ubiquitination assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1 and His-Ub in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST. D, E. Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs (D), or HEK293T cells transfected with empty Flag vector (−) or Flag-TRIM27 after treatment with or without 10 μM MG132 for 4 h (E). F. Fluorescence microscopy image of MEFs with stable expression of mCherry-ULK1 and GFP-TRIM27 after treating cells with DMSO (MOCK) or 10 μM MG132 for 4 h. Scale bar, 10 μm. G–J. HA-ULK1 levels (G, H), or endogenous ULK1 levels (I, J) in 50 μg/ml cycloheximide (CHX)-treated HEK293T cells with or without co-expression of Flag-TRIM27 (G, H) or TRIM27 RNAi (I, J). Western blots against HA-ULK1 and actin control (G, I) were subjected to densitometric quantitation (H, J). K, L. Ubiquitination assays were performed in HEK293T cells as per (A, B) after co-transfection with full length HA-ULK1 or the indicated truncated constructs (K) or double (K568/K571R) or single (K568R, K571R) lysine substitution mutants (L) in the presence or absence of Flag-TRIM27. M. Ubiquitination of HA-ULK1 measured as per (A, B) in cells co-expressing Flag-TRIM27 with either His-Ub WT, His-Ub K48, or His-Ub K63, respectively. N. In vitro ubiquitination assays performed as per (C) against affinity-purified HA-ULK1 WT or the HA-ULK1 K568/K571R mutant in the presence or absence of GST-TRIM27. O. ULK1/2 knockout MEFs stably expressing the indicated HA-ULK1 constructs were subjected to Western blot analysis against HA or actin before (0 h) and after treatment with CHX for 12 h. Data