Abstract
The small GTPase RhoA participates in actin and microtubule machinery, cell migration and invasion, gene expression, vesicular trafficking and cell cycle, and its dysregulation is a determining factor in many pathological conditions. Similar to other Rho GTPases, RhoA is a key component of the wound-healing process, regulating the activity of different participating cell types. RhoA gets activated upon binding to guanine nucleotide exchange factors (GEFs), which catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTPase-activating proteins (GAPs) mediate the exchange of GTP to GDP, inactivating RhoA, whereas guanine nucleotide dissociation inhibitors (GDIs) preserve the inactive pool of RhoA proteins in the cytosol. RhoA and Rho GEF activation is detected by protein pull-down assays, which use chimeric proteins with Rhotekin and G17A mutant RhoA as “bait” to pull down active RhoA and RhoA GEFs, respectively. In this chapter, we describe an optimized protocol for performing RhoA and GEF pull-down assays.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ridley AJ (2015) Rho GTPase signalling in cell migration. Curr Opin Cell Biol 36:103–112. https://doi.org/10.1016/j.ceb.2015.08.005
Kim JG, Islam R, Cho JY et al (2018) Regulation of RhoA GTPase and various transcription factors in the RhoA pathway. J Cell Physiol 233(9):6381–6392. https://doi.org/10.1002/jcp.26487
Zahra FT, Sajib MS, Ichiyama Y et al (2019) Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis. Sci Rep 9(1):11666. https://doi.org/10.1038/s41598-019-48053-z
Desai LP, Aryal AM, Ceacareanu B et al (2004) RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 287(6):L1134–L1144. https://doi.org/10.1152/ajplung.00022.2004
Jackson B, Peyrollier K, Pedersen E et al (2011) RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes. Mol Biol Cell 22(5):593–605. https://doi.org/10.1091/mbc.E09-10-0859
Mikelis CM, Palmby TR, Simaan M et al (2013) PDZ-RhoGEF and LARG are essential for embryonic development and provide a link between thrombin and LPA receptors and Rho activation. J Biol Chem 288(17):12232–12243. https://doi.org/10.1074/jbc.M112.428599
Michaelson D, Silletti J, Murphy G et al (2001) Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J Cell Biol 152(1):111–126. https://doi.org/10.1083/jcb.152.1.111
van Unen J, Reinhard NR, Yin T et al (2015) Plasma membrane restricted RhoGEF activity is sufficient for RhoA-mediated actin polymerization. Sci Rep 5:14693. https://doi.org/10.1038/srep14693
Garcia-Mata R, Boulter E, Burridge K (2011) The 'invisible hand': regulation of RHO GTPases by RHOGDIs. Nat Rev Mol Cell Biol 12(8):493–504. https://doi.org/10.1038/nrm3153
Reid T, Furuyashiki T, Ishizaki T et al (1996) Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J Biol Chem 271(23):13556–13560. https://doi.org/10.1074/jbc.271.23.13556
Watanabe G, Saito Y, Madaule P et al (1996) Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho. Science 271(5249):645–648. https://doi.org/10.1126/science.271.5249.645
Ren XD, Kiosses WB, Schwartz MA (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18(3):578–585. https://doi.org/10.1093/emboj/18.3.578
van Buul JD, Geerts D, Huveneers S (2014) Rho GAPs and GEFs: controling switches in endothelial cell adhesion. Cell Adhes Migr 8(2):108–124. https://doi.org/10.4161/cam.27599
Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6(2):167–180. https://doi.org/10.1038/nrm1587
Erickson JW, Cerione RA (2004) Structural elements, mechanism, and evolutionary convergence of Rho protein-guanine nucleotide exchange factor complexes. Biochemistry 43(4):837–842. https://doi.org/10.1021/bi036026v
Schmidt A, Hall A (2002) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev 16(13):1587–1609. https://doi.org/10.1101/gad.1003302
Dvorsky R, Ahmadian MR (2004) Always look on the bright site of Rho: structural implications for a conserved intermolecular interface. EMBO Rep 5(12):1130–1136. https://doi.org/10.1038/sj.embor.7400293
Ridley AJ, Paterson HF, Johnston CL et al (1992) The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70(3):401–410. https://doi.org/10.1016/0092-8674(92)90164-8
Guilluy C, Dubash AD, Garcia-Mata R (2011) Analysis of RhoA and rho GEF activity in whole cells and the cell nucleus. Nat Protoc 6(12):2050–2060. https://doi.org/10.1038/nprot.2011.411
Garcia-Mata R, Wennerberg K, Arthur WT et al (2006) Analysis of activated GAPs and GEFs in cell lysates. Methods Enzymol 406:425–437. https://doi.org/10.1016/S0076-6879(06)06031-9
Nunes KP, Rigsby CS, Webb RC (2010) RhoA/Rho-kinase and vascular diseases: what is the link? Cell Mol Life Sci 67(22):3823–3836. https://doi.org/10.1007/s00018-010-0460-1
Luo W, Liu CT, Yang QH et al (2014) New angle of view on the role of rho/rho kinase pathway in human diseases. Iran J Allergy Asthma Immunol 13(6):378–395
Fortin Ensign SP, Mathews IT, Symons MH et al (2013) Implications of Rho GTPase Signaling in Glioma cell invasion and tumor progression. Front Oncol 3:241. https://doi.org/10.3389/fonc.2013.00241
Rattan S, Phillips BR, Maxwell PJ (2010) RhoA/Rho-kinase: pathophysiologic and therapeutic implications in gastrointestinal smooth muscle tone and relaxation. Gastroenterology 138(1):13–18. e11-13. https://doi.org/10.1053/j.gastro.2009.11.016
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (NCI) R15CA231339 and Texas Tech University Health Sciences Center (TTUHSC) Office of Research. The funders had no role in study design, decision to write, and preparation of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Sajib, M.S., Zahra, F.T., Akwii, R.G., Mikelis, C.M. (2021). Identification of Rho GEF and RhoA Activation by Pull-Down Assays. In: Das, H. (eds) Wound Regeneration. Methods in Molecular Biology, vol 2193. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0845-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0845-6_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0844-9
Online ISBN: 978-1-0716-0845-6
eBook Packages: Springer Protocols