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. 2024 Apr;300(4):105778.
doi: 10.1016/j.jbc.2024.105778. Epub 2024 Feb 21.

SUMO modifies GβL and mediates mTOR signaling

Sophia Louise Lucille Park  1 , Uri Nimrod Ramírez-Jarquín  1 , Neelam Shahani  1 , Oscar Rivera  1 , Manish Sharma  1 , Preksha Sandipkumar Joshi  1 , Aayushi Hansalia  1 , Sunayana Dagar  1 , Francis P McManus  2 , Pierre Thibault  3 , Srinivasa Subramaniam  4
Affiliations

SUMO modifies GβL and mediates mTOR signaling

Sophia Louise Lucille Park et al. J Biol Chem. 2024 Apr.

Abstract

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GβL (G protein β-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GβL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GβL but not SUMOylation-defective KR mutant GβL promotes mTOR signaling in GβL-depleted cells. Taken together, we report for the very first time that SUMO modifies GβL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

Keywords: SUMO interactive motif; SUMO isoforms; SUMO mechanism; amino acid stimulation; kinase signaling; lysine-site regulation; nutrient signaling; post-translational modification; protein–protein interaction.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
GβL is the primary mTOR complex component that is SUMOylated.A, representative Western blot of Ni–NTA enrichment of proteins in denaturing conditions and corresponding input from HEK293 cells transfected with His-SUMO1 and HA-GβL in full media conditions, showing an enrichment of HA-GβL high molecular weight conjugates with His-SUMO1 but not His-SUMO1 amino acid (AA) (conjugation-defective mutant). B–D, Ni–NTA enrichment of His-SUMO1 conjugates associated with FLAG-mTOR (B), myc-Raptor or myc-Rictor (C), or FLAG-Pras40 (D) as in A. E, Ni–NTA enrichment of His-SUMO1, 2, and 3 conjugates associated with HA-GβL (indicated by S∗ GβL) and corresponding inputs as in A. GβL, G protein β-subunit–like protein; HEK293, human embryonic kidney 293 cell line; mTOR, mechanistic target of rapamycin; Ni, nickel; NTA, nitrilotriacetic acid; Raptor, regulatory protein associated with mTOR; Rictor, rapamycin-insensitive companion of mTOR; SUMO, small ubiquitin–like modifier.
Figure 2
Figure 2
GβL is SUMOylated at multiple lysines.A, primary sequence domain structure of GβL with predicated SUMO-modification sites for KR mutagenesis. B, 3D representation of the mTOR–GβL interface demonstrating that all lysines on GβL are surface exposed and accessible. SUMO consensus sites are shown in orange, nonconsensus sites in yellow, mTOR in blue, and GβL in tan. The interface was modeled using the reconstructed density from Protein Data Bank (ID: 5FLC). C, Western blot analysis of Ni–NTA denaturing pull down and corresponding input from HEK293 cells transfected with His-SUMO1 and WT or single KR mutant HA-GβL plasmids. D and E, Western blot analysis as in C from HEK293 cells transfected with HA-GβL KR mutant plasmids as indicated. F, SUMO-conjugated lysine identification on HA-GβL by LC–MS/MS (depicted with ∗) and corresponding location in the domain structure. GβL, G protein β-subunit–like protein; HEK293, human embryonic kidney 293 cell line; MS, mass spectrometry; mTOR, mechanistic target of rapamycin; Ni, nickel; NTA, nitrilotriacetic acid; SUMO, small ubiquitin–like modifier.
Figure 3
Figure 3
In vitro SUMOylation of GβL. In vitro SUMOylation reactions were performed with RanGAP1 or GβL, E1, and E2 in the presence and absence of ATP (5 mM) or Rhes (200 ng) as indicated. A and B, in vitro SUMOylation of recombinant GST-RanGAP1 (A) or GST-GβL (B), detected using anti-GST-HRP. C, in vitro SUMOylation of purified recombinant His-GβL, detected using anti-GβL. D, in vitro SUMOylation on beads from HA immunoprecipitates from HEK293 cells transfected with HA-GβL, detected using anti-HA. GβL, G protein β-subunit–like protein; GST, glutathione-S-transferase; HEK293, human embryonic kidney 293 cell line; HRP, horseradish peroxidase; SUMO, small ubiquitin–like modifier.
Figure 4
Figure 4
mTORC1 activity is altered in SUMO1−/−MEFs.A, representative Western blot showing indicated phosphorylation of mTORC1 substrates in WT (Sumo1+/+) and Sumo1 KO (Sumo1−/−) primary MEFs grown in F12 media starved of amino acids (−AA) and stimulated with 3 mM l-leucine (+Leu). B, quantification of indicated proteins from A. Error bars represent mean ± SEM, ∗∗p < 0.01 by one-way ANOVA/Tukey’s multiple comparison test, #p < 0.05 by Student’s t test. MEF, mouse embryonic fibroblast; mTOR, mechanistic target of rapamycin.
Figure 5
Figure 5
mTOR activity in SUMO-depleted striatal neuronal cells.A, representative Western blot showing indicated signaling proteins in striatal control CRISPR- (control) or SUMO1/2/3-depleted (SUMO1/2/3Δ) cells in full media, deprived of amino acids (AAs) in Krebs buffer (−AA), and starved and stimulated with 3 mM leucine (+Leu). B–F, quantification of indicated proteins from A. G, cell proliferation assay using cell counting kit-8 (CCK-8) assay. Error bar represents mean ± SEM, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA/Tukey’s multiple comparison test. mTOR, mechanistic target of rapamycin; SUMO, small ubiquitin–like modifier.
Figure 6
Figure 6
SUMO influences mTOR complex formation in striatal neuronal cells.A, immunoprecipitation of mTOR with mTOR IgG or control IgG and Western blotting for endogenous Raptor, Rictor, or GβL from striatal control CRISPR- or SUMO1/2/3-depleted (SUMO1/2/3Δ) cells in AA-starved (Krebs) and stimulated with 3 mM leucine (+Leu). B, quantification of indicated protein interactions from A. Error bar represents mean ± SEM, ∗∗∗∗p < 0.001 by Student’s t test. AA, amino acid; GβL, G protein β-subunit–like protein; IgG, immunoglobulin G; mTOR, mechanistic target of rapamycin; Raptor, regulatory protein associated with mTOR; Rictor, rapamycin-insensitive companion of mTOR; SUMO, small ubiquitin–like modifier.
Figure 7
Figure 7
Effect of SUMOylation-defective GβL on mTOR signaling.A, representative Western blot showing indicated signaling proteins in striatal control CRISPR- (control) or GβL-depleted (GβLΔ) cells in full media, deprived of amino acids (AAs) in Krebs buffer (−AA), and starved and stimulated with 3 mM leucine (+Leu). B, quantification of indicated proteins in full media from A. Error bar represents mean ± SEM, ∗∗∗∗p < 0.0001 by Student’s t test. C–H, representative confocal immunofluorescence images and quantification (n = 13–30) of the signal intensity of pS6S235/236 (C and D), pAktS473 (E and F), and mTOR (G and H) in GβLΔ cells expressing HA-GβL-WT or HA-GβL-KR mutant in AA-starved (Krebs, –AA) or starved and stimulated with 3 mM leucine (+Leu). DAPI was used for nuclear stain. Error bar represents mean ± SEM, ∗∗∗∗p < 0.001 by one-way ANOVA/Tukey’s multiple comparison test. DAPI, 4′,6-diamidino-2-phenylindole; GβL, G protein β-subunit–like protein; mTOR, mechanistic target of rapamycin; SUMO, small ubiquitin–like modifier.
Figure 8
Figure 8
Schematic depiction of role of SUMOylation in the regulation of mTORC1/2 signaling. Our model predicts that there are three possible ways that SUMO modification may influence mTORC1 and mTORC2 signaling. (1) mTORC assembly, (2) mTOR substrate phosphorylation, and (3) intracellular localization of mTORC. In addition to GβL, it is possible that one or more additional components of the mTOR and other non-mTOR complex regulators (depicted as question mark [?]) can be modified by SUMO and influence complex assembly and activation. mTOR, mechanistic target of rapamycin; mTORC, mTOR complex; SIM, SUMO-interacting motif; SUMO, small ubiquitin–like modifier.
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References

    1. Liu G.Y., Sabatini D.M. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 2020;21:183–203. - PMC - PubMed
    1. Seeler J.S., Dejean A. SUMO and the robustness of cancer. Nat. Rev. Cancer. 2017;17:184–197. - PubMed
    1. Yau T.Y., Molina O., Courey A.J. SUMOylation in development and neurodegeneration. Development. 2020;147 - PMC - PubMed
    1. Wu Z., Huang R., Yuan L. Crosstalk of intracellular post-translational modifications in cancer. Arch. Biochem. Biophys. 2019;676 - PubMed
    1. Gwinn D.M., Shackelford D.B., Egan D.F., Mihaylova M.M., Mery A., Vasquez D.S., et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell. 2008;30:214–226. - PMC - PubMed

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