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Methods to Study the Unique SOCS Box Domain of the Rab40 Small GTPase Subfamily

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Rab GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2293))

Abstract

Despite the critical role of Rab GTPases for intracellular transport, the vast majority of proteins within this family remain poorly characterized, including the Rab40 subfamily. Often recognized as atypical Rabs, the Rab40 family of proteins are unlike any other small GTPase because they contain a C-terminal suppressor of cytokine signaling (SOCS) box. It is well established that this SOCS domain in other proteins mediates an interaction with the scaffold protein Cullin5 in order to form a E3 ubiquitin ligase complex critical for protein ubiquitylation and turnover. Although the function of SOCS/Cullin5 complexes has been well defined in several of these other proteins, this is not yet the case for the Rab40 family of proteins. We have previously shown that the Rab40b family member plays an important role during three-dimensional (3D) breast cancer cell migration. To further this knowledge, we began to investigate the SOCS-dependent role of Rab40b during cell migration. Here, we describe an unbiased approach to identify potential Rab40b/Cullin5 substrates. We anticipate that this method will be useful for studying the function of other Rab40 family members as well as other SOCS box containing proteins.

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References

  1. Stenmark H, Olkkonen VM (2001) The Rab GTPase family. Genome Biol 2:REVIEWS3007–7. https://doi.org/10.1186/gb-2001-2-5-reviews3007

    Article  Google Scholar 

  2. Coppola U, Ristoratore F, Albalat R, D’Aniello S (2019) The evolutionary landscape of the Rab family in chordates. Cell Mol Life Sci 76:4117–4130. https://doi.org/10.1007/s00018-019-03103-7

    Article  PubMed  CAS  Google Scholar 

  3. Jékely G (2003) Small GTPases and the evolution of the eukaryotic cell. BioEssays 25:1129–1138. https://doi.org/10.1002/bies.10353

    Article  PubMed  CAS  Google Scholar 

  4. Surkont J, Pereira-Leal JB (2016) Are there Rab GTPases in archaea? Mol Biol Evol 33:1833–1842. https://doi.org/10.1093/molbev/msw061

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Klöpper TH, Kienle N, Fasshauer D, Munro S (2012) Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis. BMC Biol 10:71–17. https://doi.org/10.1186/1741-7007-10-71

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117. https://doi.org/10.1038/35052055

    Article  PubMed  CAS  Google Scholar 

  7. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Publ Group 10:513–525. https://doi.org/10.1038/nrm2728

    Article  CAS  Google Scholar 

  8. Pfeffer SR (2001) Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol 11:487–491. https://doi.org/10.1016/s0962-8924(01)02147-x

    Article  PubMed  CAS  Google Scholar 

  9. Grosshans BL, Ortiz D, Novick P (2006) Rabs and their effectors: achieving specificity in membrane traffic. Proc Natl Acad Sci U S A 103:11821–11827. https://doi.org/10.1073/pnas.0601617103

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Schwartz SL, Cao C, Pylypenko O et al (2008) Rab GTPases at a glance. J Cell Sci 121:246–246. https://doi.org/10.1242/jcs.03495

    Article  CAS  Google Scholar 

  11. Hutagalung AH, Novick PJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119–149. https://doi.org/10.1152/physrev.00059.2009

    Article  PubMed  CAS  Google Scholar 

  12. Pereira-Leal JB, Seabra MC (2001) Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 313:889–901. https://doi.org/10.1006/jmbi.2001.5072

    Article  PubMed  CAS  Google Scholar 

  13. Pereira-Leal JB, Seabra MC (2000) The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily 1 1Edited by M. Yaniv. J Mol Biol 301:1077–1087. https://doi.org/10.1006/jmbi.2000.4010

    Article  PubMed  CAS  Google Scholar 

  14. Pylypenko O, Hammich H, Yu I-M, Houdusse A (2018) Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity. Small GTPases 9:22–48. https://doi.org/10.1080/21541248.2017.1336191

    Article  PubMed  CAS  Google Scholar 

  15. Kasahara M, Naruse K, Sasaki S et al (2007) The medaka draft genome and insights into vertebrate genome evolution. Nature 447:714–719. https://doi.org/10.1038/nature05846

    Article  PubMed  CAS  Google Scholar 

  16. Hilton DJ, Richardson RT, Alexander WS et al (1998) Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U S A 95:114–119. https://doi.org/10.1073/pnas.95.1.114

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Hilton DJ (1999) Negative regulators of cytokine signal transduction. Cell Mol Life Sci 55:1568–1577. https://doi.org/10.1007/s000180050396

    Article  PubMed  CAS  Google Scholar 

  18. Okumura F, Joo-Okumura A, Nakatsukasa K, Kamura T (2016) The role of cullin 5-containing ubiquitin ligases. Cell Div 11:1–16. https://doi.org/10.1186/s13008-016-0016-3

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Petroski MD, Deshaies RJ (2005) Function and regulation of cullin–RING ubiquitin ligases. Nat Rev Mol Cell Biol 6:9–20. https://doi.org/10.1038/nrm1547

    Article  PubMed  CAS  Google Scholar 

  20. Linossi EM, Nicholson SE (2012) The SOCS box-adapting proteins for ubiquitination and proteasomal degradation. IUBMB Life 64:316–323. https://doi.org/10.1002/iub.1011

    Article  PubMed  CAS  Google Scholar 

  21. Kile BT, Schulman BA, Alexander WS et al (2002) The SOCS box: a tale of destruction and degradation. Trends Biochem Sci 27:235–241

    Article  CAS  Google Scholar 

  22. Mahrour N, Redwine WB, Florens L et al (2008) Characterization of Cullin-box sequences that direct recruitment of Cul2-Rbx1 and Cul5-Rbx2 modules to Elongin BC-based ubiquitin ligases. J Biol Chem 283:8005–8013. https://doi.org/10.1074/jbc.M706987200

    Article  PubMed  CAS  Google Scholar 

  23. Kamura T, Sato S, Haque D et al (1998) The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes Dev 12:3872–3881

    Article  CAS  Google Scholar 

  24. Kamura T, Maenaka K, Kotoshiba S et al (2004) VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18:3055–3065. https://doi.org/10.1101/gad.1252404

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Kim YK, Kwak MJ, Ku B et al (2013) Structural basis of intersubunit recognition in elongin BC-cullin 5-SOCS box ubiquitin-protein ligase complexes. Acta Cryst D69:1587–1597. https://doi.org/10.1107/S0907444913011220

    Article  CAS  Google Scholar 

  26. Dart AE, Box GM, Court W et al (2015) PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion. J Cell Biol 211:863–879. https://doi.org/10.1083/jcb.201501072

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Bedoyan JK, Schaibley VM, Peng W et al (2012) Disruption of RAB40AL function leads to Martin–Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder. J Med Genet 49:332–340. https://doi.org/10.1136/jmedgenet-2011-100575

    Article  PubMed  CAS  Google Scholar 

  28. Ołdak M, Ruszkowska E, Pollak A et al (2014) A note of caution on the diagnosis of Martin-Probst syndrome by the detection of the p.D59G mutation in the RAB40AL gene. Eur J Pediatr 174:693–696. https://doi.org/10.1007/s00431-014-2452-x

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Ołdak M, Ścieżyńska A, Młynarski W et al (2014) Evidence against RAB40ALBeing the locus for Martin-Probst X-linked deafness-intellectual disability syndrome. Hum Mutat 35:1171–1174. https://doi.org/10.1002/humu.22620

    Article  PubMed  CAS  Google Scholar 

  30. Jacob A, Jing J, Lee J et al (2013) Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells. J Cell Sci 126:4647–4658. https://doi.org/10.1242/jcs.126573

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Jacob A, Linklater E, Bayless BA et al (2016) The role and regulation of Rab40b-Tks5 complex during invadopodia formation and cancer cell invasion. J Cell Sci 129:4341–4353. https://doi.org/10.1242/jcs.193904

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Myat MM, Louis D, Mavrommatis A et al (2019) Regulators of cell movement during development and regeneration in drosophila. Open Biol 9:180245–180210. https://doi.org/10.1098/rsob.180245

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Lee RHK, Iioka H, Ohashi M et al (2007) XRab40 and XCullin5 form a ubiquitin ligase complex essential for the noncanonical Wnt pathway. EMBO J 26:3592–3606. https://doi.org/10.1038/sj.emboj.7601781

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Yatsu A, Shimada H, Ohbayashi N, Fukuda M (2015) Rab40C is a novel Varp-binding protein that promotes proteasomal degradation of Varp in melanocytes. Biol Open 4:267–275. https://doi.org/10.1242/bio.201411114

    Article  PubMed  PubMed Central  Google Scholar 

  35. Rodriguez-Gabin AG, Almazan G, Larocca JN (2004) Vesicle transport in oligodendrocytes: probable role of Rab40c protein. J Neurosci Res 76:758–770. https://doi.org/10.1002/jnr.20121

    Article  PubMed  CAS  Google Scholar 

  36. Tan R, Wang W, Wang S et al (2013) Small GTPase Rab40c associates with lipid droplets and modulates the biogenesis of lipid droplets. PLoS One 8:e63213–e63211. https://doi.org/10.1371/journal.pone.0063213

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Tandon N, Thakkar K, LaGory E et al (2018) Generation of stable expression mammalian cell lines using lentivirus. Bio-Protocol 8:1–6. https://doi.org/10.21769/BioProtoc.3073

    Article  Google Scholar 

  38. Mellacheruvu D, Wright Z, Couzens AL et al (2013) The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nat Methods 10:730–736. https://doi.org/10.1038/nmeth.2557

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgments

We thank the University of Colorado School of Medicine Biological Mass Spectrometry Facility, specifically Monika Dzieciatkowska for performing the mass spectrometry and proteomic analyses. This work was supported by NIHT32GM008730 to EDD, NIHT32CA174648 to EL, and NIH1R01GM122768 to RP.

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA

    Emily D. Duncan, Ezra Lencer, Erik Linklater & Rytis Prekeris

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  1. Emily D. Duncan
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  2. Ezra Lencer
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  3. Erik Linklater
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  4. Rytis Prekeris
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Correspondence to Rytis Prekeris .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Guangpu Li

  2. Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA

    Nava Segev

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Duncan, E.D., Lencer, E., Linklater, E., Prekeris, R. (2021). Methods to Study the Unique SOCS Box Domain of the Rab40 Small GTPase Subfamily. In: Li, G., Segev, N. (eds) Rab GTPases. Methods in Molecular Biology, vol 2293. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1346-7_11

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  • DOI: https://doi.org/10.1007/978-1-0716-1346-7_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1345-0

  • Online ISBN: 978-1-0716-1346-7

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