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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Stenmark H, Olkkonen VM (2001) The Rab GTPase family. Genome Biol 2:REVIEWS3007–7. https://doi.org/10.1186/gb-2001-2-5-reviews3007
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
Jékely G (2003) Small GTPases and the evolution of the eukaryotic cell. BioEssays 25:1129–1138. https://doi.org/10.1002/bies.10353
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
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
Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117. https://doi.org/10.1038/35052055
Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Publ Group 10:513–525. https://doi.org/10.1038/nrm2728
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
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
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
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
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
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
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
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
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
Hilton DJ (1999) Negative regulators of cytokine signal transduction. Cell Mol Life Sci 55:1568–1577. https://doi.org/10.1007/s000180050396
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
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
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
Kile BT, Schulman BA, Alexander WS et al (2002) The SOCS box: a tale of destruction and degradation. Trends Biochem Sci 27:235–241
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1346-7_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1345-0
Online ISBN: 978-1-0716-1346-7
eBook Packages: Springer Protocols