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The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response

Nature Cell Biology volume 15, pages 1220–1230 (2013)Cite this article

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Abstract

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively engaging two components of the exocyst complex, EXO84 and SEC5. RALB employs SEC5 to trigger innate immunity signalling, whereas RALB–EXO84 interaction induces autophagocytosis. How this differential interaction is achieved molecularly by the RAL GTPase remains unknown. We found that whereas GTP binding turns on RALB activity, ubiquitylation of RALB at Lys 47 tunes its activity towards a particular effector. Specifically, ubiquitylation at Lys 47 sterically inhibits RALB binding to EXO84, while facilitating its interaction with SEC5. Double-stranded RNA promotes RALB ubiquitylation and SEC5–TBK1 complex formation. In contrast, nutrient starvation induces RALB deubiquitylation by accumulation and relocalization of the deubiquitylase USP33 to RALB-positive vesicles. Deubiquitylated RALB promotes the assembly of the RALB–EXO84–beclin-1 complexes driving autophagosome formation. Thus, ubiquitylation within the effector-binding domain provides the switch for the dual functions of RALB in autophagy and innate immune responses.

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Figure 1: The deubiquitylase USP33 is an interacting partner of the RAL GTPases.
Figure 2: Ubiquitylation of RALB at Lys 47 determines a choice of RALB to interact with SEC5 or EXO84.
Figure 3: USP33 modulates interactions between RALB and the exocyst proteins by regulating RALB ubiquitylation at Lys 47.
Figure 4: RALB ubiquitylation at Lys 47 activates the TBK1 pathway.
Figure 5: USP33 triggers RALB–EXO84–beclin-1 complex formation in response to nutrient starvation.
Figure 6: USP33 regulates autophagy.

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References

  1. Colicelli, J. Human RAS superfamily proteins and related GTPases. Sci. STKE 2004, re13 (2004).

    PubMed  PubMed Central  Google Scholar 

  2. Bodemann, B. O. & White, M. A. Ral GTPases and cancer: linchpin support of the tumorigenic platform. Nat. Rev. Cancer 8, 133–140 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Feig, L. A. Ral-GTPases: approaching their 15 minutes of fame. Trends Cell Biol. 13, 419–425 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Moskalenko, S. et al. The exocyst is a Ral effector complex. Nat. Cell Biol. 4, 66–72 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Moskalenko, S. et al. Ral GTPases regulate exocyst assembly through dual subunit interactions. J. Biol. Chem. 278, 51743–51748 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Sugihara, K. et al. The exocyst complex binds the small GTPase RalA to mediate filopodia formation. Nat. Cell Biol. 4, 73–78 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Chen, X. W., Leto, D., Chiang, S. H., Wang, Q. & Saltiel, A. R. Activation of RalA is required for insulin-stimulated Glut4 trafficking to the plasma membrane via the exocyst and the motor protein Myo1c. Dev. Cell 13, 391–404 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Nozaki, S., Ueda, S., Takenaka, N., Kataoka, T. & Satoh, T. Role of RalA downstream of Rac1 in insulin-dependent glucose uptake in muscle cells. Cell Signal. 24, 2111–2117 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Kashatus, D. F. et al. RALA and RALBP1 regulate mitochondrial fission at mitosis. Nat. Cell Biol. 13, 1108–1115 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chien, Y. et al. RalB GTPase-mediated activation of the IkappaB family kinase TBK1 couples innate immune signaling to tumor cell survival. Cell 127, 157–170 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Bodemann, B. O. et al. RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 144, 253–267 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shi, C. S. et al. Activation of autophagy by inflammatory signals limits IL-1beta production by targeting ubiquitinated inflammasomes for destruction. Nature Immunol. 13, 255–263 (2012).

    Article  CAS  Google Scholar 

  13. Fenwick, R. B. et al. The RalB-RLIP76 complex reveals a novel mode of ral-effector interaction. Structure 18, 985–995 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fenwick, R. B. et al. Solution structure and dynamics of the small GTPase RalB in its active conformation: significance for effector protein binding. Biochemistry 48, 2192–2206 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Jin, R. et al. Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase. EMBO J. 24, 2064–2074 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nicely, N. I., Kosak, J., de Serrano, V. & Mattos, C. Crystal structures of Ral-GppNHp and Ral-GDP reveal two binding sites that are also present in Ras and Rap. Structure 12, 2025–2036 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Eyckerman, S. et al. Design and application of a cytokine-receptor-based interaction trap. Nat. Cell Biol. 3, 1114–1119 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Lievens, S., Peelman, F., De Bosscher, K., Lemmens, I. & Tavernier, J. MAPPIT: a protein interaction toolbox built on insights in cytokine receptor signaling. Cytokine Growth Factor Rev. 22, 321–329 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Lievens, S. et al. Array MAPPIT: high-throughput interactome analysis in mammalian cells. J. Proteome Res. 8, 877–886 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Lievens, S., Vanderroost, N., Defever, D., Van der Heyden, J. & Tavernier, J. ArrayMAPPIT: a screening platform for human protein interactome analysis. Methods Mol. Biol. 812, 283–294 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Brymora, A., Valova, V. A., Larsen, M. R., Roufogalis, B. D. & Robinson, P. J. The brain exocyst complex interacts with RalA in a GTP-dependent manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene. J. Biol. Chem. 276, 29792–29797 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Neyraud, V. et al. RalA and RalB proteins are ubiquitinated GTPases, and ubiquitinated RalA increases lipid raft exposure at the plasma membrane. J. Biol. Chem. 287, 29397–29405 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, Z. et al. Identification of a deubiquitinating enzyme subfamily as substrates of the von Hippel-Lindau tumor suppressor. Biochem. Biophys. Res. Commun. 294, 700–709 (2002).

    Article  CAS  Google Scholar 

  24. Li, Z. et al. Ubiquitination of a novel deubiquitinating enzyme requires direct binding to von Hippel-Lindau tumor suppressor protein. J. Biol. Chem. 277, 4656–4662 (2002).

    Article  CAS  Google Scholar 

  25. Fukai, S., Matern, H. T., Jagath, J. R., Scheller, R. H. & Brunger, A. T. Structural basis of the interaction between RalA and Sec5, a subunit of the sec6/8 complex. EMBO J. 22, 3267–3278 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Baker, R. et al. Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Nat. Struct. Mol. Biol. 20, 46–52 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for thesimulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zavodszky, M. I., Stumpff-Kane, A. W., Lee, D. J. & Feig, M. Scoring confidence index: statistical evaluation of ligand binding mode predictions. J. Comput. Aided Mol. Des. 23, 289–299 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chien, Y. & White, M. A. Characterization of RalB–Sec5–TBK1 function in human oncogenesis. Methods Enzymol. 438, 321–329 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Shenoy, S. K. et al. Beta-arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2. Proc. Natl Acad. Sci. USA 106, 6650–6655 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Thorne, C., Eccles, R. L., Coulson, J. M., Urbe, S. & Clague, M. J. Isoform-specific localization of the deubiquitinase USP33 to the Golgi apparatus. Traffic 12, 1563–1574 (2011).

    Article  CAS  Google Scholar 

  32. Croteau, N. J., Furgason, M. L., Devos, D. & Munson, M. Conservation of helical bundle structure between the exocyst subunits. PloS One 4, e4443 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Dikic, I., Wakatsuki, S. & Walters, K.J. Ubiquitin-binding domains—from structures to functions. Nat. Rev. Mol. Cell Biol. 10, 659–671 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Moerkerke, B. & Goetghebeur, E. Selecting ‘significant’ differentially expressed genes from the combined perspective of the null and the alternative. J. Comput. Biol. 13, 1513–1531 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Sablina, A. A. et al. The tumor suppressor PP2A Abeta regulates the RalA GTPase. Cell 129, 969–982 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the VIB (A.A.S.), Swiss Bridge Award (A.A.S.), a PhD-student fellowship of the Research Foundation of Flanders (FWO) (M.S.), the KU Leuven Onderzoekstoelage Grant 13/097 (S.V.S.), FWO Project G.0709.12 (S.V.S.), FWO Project G.0864.10 (J.T.), and the Group-ID Multidisciplinary Research Partnership of Ghent University and the Belgian government, Interuniversity Attraction Poles Project P6/36 (J.T.).

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Authors and Affiliations

  1. VIB Center for the Biology of Disease, VIB, 3000 Leuven, Belgium

    Michal Simicek, Peter Kalev, Maria Francesca Baietti & Anna A. Sablina

  2. Department of Human Genetics, KU Leuven, Herestraat 49, 3000 Leuven, Belgium

    Michal Simicek, Peter Kalev, Maria Francesca Baietti & Anna A. Sablina

  3. Department of Medical Protein Research, VIB, 9000 Ghent, Belgium

    Sam Lievens, Mathias Laga, Kris Gevaert & Jan Tavernier

  4. Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, Belgium

    Sam Lievens, Mathias Laga, Kris Gevaert & Jan Tavernier

  5. Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49, 3000 Leuven, Belgium

    Dmytro Guzenko & Sergei V. Strelkov

  6. Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center, Kashirskoye Shosse 24, 115478 Moscow, Russia

    Vasily N. Aushev

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Contributions

M.S. and A.A.S. conceived of the study. M.S. performed most of the experiments; S.L. and J.T. designed and performed the MAPPIT screen; M.L. and K.G. performed the mass spectrometry analysis; D.G. and S.V.S. carried out the Rosetta docking; P.K. and M.F.B. assisted with the fluorescence microscopy. V.N.A. assisted with ubiquitylation experiments. A.A.S. wrote the manuscript. All authors discussed results and commented on the manuscript.

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Correspondence to Anna A. Sablina.

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Integrated supplementary information

Supplementary Figure 1 The interactions of Ral GTPases with interacting partners as detected in the MAPPIT assay.

a, The MAPPIT assay detected interactions between RalA and its downstream targets. wt-RalA or RalA-G23V baits were introduced into 293T cells together with the indicated preys. The results are expressed as a mean of normalized luciferase activity ± s.d. (leptin-treated cells vs leptin-untreated cells) for five independent experiments. Expression of Flag-tagged preys was verified by immunoblotting. RNF41 prey that binds to the bait receptor itself was used to evaluate expression of the Ral baits. b, MAPPIT assay detected interactions between RalB and its downstream targets. wt-RalB or RalB-G23V baits were introduced into 293T cells together with the indicated preys. The results of the MAPPIT assay are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells) ± sd for five independent experiments. c, Validation of the RalA interacting partners identified in the high-throughput MAPPIT screen. RalA-G23V or eDHFR (a negative control) were used as baits in 293T cells. Interactors identified in Figure 1B were used as preys. The results of the MAPPIT assay are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells) for two independent experiments.

Supplementary Figure 2 Modulation of USP33 or Sec5 expression by specific shRNAs.

Lentiviral shRNAs against USP33 or Sec5 were purchased from Sigma-Aldrich. HEK TE cells infected with the indicated constructs were selected by puromycin for 3 days. a, USP33 expression was assessed by immunoblotting with anti-USP33 antibody. shRNA-TRCN000004442 (refers in the paper as shUSP33-1) induced massive cell death, probably due to off-target effect. shRNA-TRCN000004445 (refers in the paper as shUSP33-2 or shUSP33) was used for the most experiments. b, Silent substitutions in the USP33 sequence targeted by shUSP33 (TRCN0000004445). c, Sec5 expression was assessed by immunoblotting with anti-Sec5 antibody. shRNA-TRCN0000116102 (refers in the paper as shSec5-1) and shRNA-TRCN0000116103 (refers in the paper as shSec5-2) was used for the experiments.

Supplementary Figure 3 USP20 does not affect ubiquitination of the Ral GTPases.

a, Lentiviral shRNAs against USP20 were purchased from Sigma-Aldrich. b, Suppression of USP20 expression does not affect ubiquitination of either RalA, or RalB. 6xHis-tagged ubiquitin and Flag-RalA-G23V or Flag-RalB-G23V mutants were introduced into 293T cells stably expressing shGFP or shUSP20. Ubiquitinated Ral proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RalA or RalB. Suppression of USP20 expression was confirmed by immunoblotting using anti-USP20 antibody. c, USP20 overexpression does not affect ubiquitination levels of the Ral proteins. 6xHis-tagged ubiquitin and Flag-RalA-G23V or Flag-RalB-G23V mutants were introduced into 293T cells expressing empty vector (V) or HA-tagged USP20. Ubiquitinated Ral proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RalA or RalB. Overexpression of USP20 expression was confirmed by immunoblotting using anti-HA antibody.

Supplementary Figure 4 Characterization of ubiquitinated RalB.

a, Mass Spec sequence coverage of TAP-purified ubiquitinated RalB. b, Detection of ubiquitinated RalB by immunoblotting analysis using antibodies specific to RalB. Ubiquitinated RalB was purified by Co2+ metal affinity chromatography after transfection with 6xHis-ubiquitin. c, USP33 suppression increases levels of ubiquitinated RalB. Flag-tagged RalB was overexpressed in 293T expressing shGFP or shUSP33. GTP-bound RalB was purified using RalBP1-RalBD agarose and detected by antibody specific to RalB. d, Ubiquitinated RalB interacts with Sec5. Mass spectrometry analysis revealed several exocyst components co-purified with ubiquitinated RalB.

Supplementary Figure 5 USP33 regulates the function of RalB but not RalA.

a, USP33 does not affect RalA activity. The activity of RalA was determined in 293T cells expressing the indicated constructs by RalBP1-RBD binding assay. b, USP33 does not affect interaction between RalA and Sec5. Flag-tagged RalA was overexpressed in 293T cells expressing the indicated constructs and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-Sec5 antibodies. c, The MAPPIT assay determined interactions between RalB and Sec5 after stable suppression or overexpression of USP33. The MAPPIT RalB-G23V bait together with the Sec5 prey were transiently overexpressed in 293T cells shGFP and shUSP33 (left histogram) or empty vector (V) and HA-USP33 (right histogram). The results of the MAPPIT assay are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells) ± sd for three independent experiments.

Supplementary Figure 6 Intracellular localization of RalB and the exocyst proteins.

a, Suppression of USP33 does not affect RalB subcellular localization. RalB-G23V-EGFP was overexpressed in HeLa cells expressing shLuciferase or shUSP33. Cells were imaged by GFP fluorescence. Scale bar 10 μm. b, Endogenous RalB co-localizes with Sec5 and Exo84. HA-Sec5 was overexpressed in HeLa cells. Cells were immunostained using anti-RalB, anti-HA, and anti-Exo84. Scale bar 10 μm.

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Simicek, M., Lievens, S., Laga, M. et al. The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response. Nat Cell Biol 15, 1220–1230 (2013). https://doi.org/10.1038/ncb2847

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