Abstract
Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.
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Abbreviations
- PBD:
-
p21-binding domain of protein kinase Pak1
- GST:
-
glutathion-S-transferase
- coIP:
-
coimmunoprecipitation
References
Boureux A., Vignal E., Faure S., Fort P. 2007. Evolution of the Rho family of Ras-like GTPases in eukaryotes. Mol. Biol. Evol. 24, 203–216.
Hodge R.G., Ridley A.J. 2017. Regulation and functions of RhoU and RhoV. Small GTPases. 30, 1–8.
Aspenstrom P. 2017. Fast-cycling Rho GTPases. Small GTPases 20, 1391365.
Jaffe A.B., Hall A. 2005. Rho GTPases: Biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21, 247–269.
Aronheim A., Broder Y.C., Cohen A., Fritsch A., Belisle B., Abo A. 1998. Chp, a homologue of the GTPase Cdc42Hs, activates the JNK pathway and is implicated in reorganizing the actin cytoskeleton. Curr. Biol. 8, 1125–1128.
Aspenstrom P., Fransson A., Saras J. 2004. Rho GTPases have diverse effects on the organization of the actin filament system. Biochem. J. 377, 327–337.
Shepelev M.V., Korobko I.V. 2012. Pak6 protein kinase is a novel effector of an atypical Rho family GTPase Chp/RhoV. Biochemistry (Moscow). 77, 26–32.
Weisz Hubsman M., Volinsky N., Manser E., Yablonski D., Aronheim A. 2007. Autophosphorylationdependent degradation of Pak1, triggered by the Rhofamily GTPase, Chp. Biochem. J. 404, 487–497.
Tay H.G., Ng Y.W., Manser E. 2010. A vertebrate-specific Chp-PAK-PIX pathway maintains E-cadherin at adherens junctions during zebrafish epiboly. PLoS One. 5, e10125.
Shepelev M.V., Chernoff J., Korobko I.V. 2011. Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation. Small GTPases. 2, 17–26.
Benard V., Bokoch G.M. 2002. Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345, 349–359.
Shutes A., Berzat A.C., Cox A.D., Der C.J. 2004. Atypical mechanism of regulation of the Wrch-1 Rho family small GTPase. Curr. Biol. 14, 2052–2056.
Chenette E.J., Mitin N.Y., Der C.J. 2006. Multiple sequence elements facilitate Chp Rho GTPase subcellular location, membrane association, and transforming activity. Mol. Biol. Cell. 17, 3108–3121.
Lamarche N., Tapon N., Stowers L., Burbelo P.D., Aspenstrom P., Bridges T., Chant J., Hall A. 1996. Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade. Cell. 87, 519–529.
Elliot-Smith A.E., Mott H.R., Lowe P.N., Laue E.D., Owen D. 2005. Specificity determinants on Cdc42 for binding its effector protein ACK. Biochemistry. 44, 12373–12383.
Abo A., Qu J., Cammarano M.S., Dan C., Fritsch A., Baud V., Belisle B., Minden A. 1998. PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia. EMBO J. 17, 6527–6540.
Ching Y.P., Leong V.Y., Wong C.M., Kung H.F. 2003. Identification of an autoinhibitory domain of p21-activated protein kinase 5. J. Biol. Chem. 278, 33621–33624.
Ruusala A., Aspenstrom P. 2008. The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics. Mol. Cell. Biol. 28, 1802–1814.
Ory S., Brazier H., Blangy A. 2007. Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration. Biol. Cell. 99, 701–716.
Brady D.C., Alan J.K., Madigan J.P., Fanning A.S., Cox A.D. 2009. The transforming Rho family GTPase Wrch-1 disrupts epithelial cell tight junctions and epithelial morphogenesis. Mol. Cell. Biol. 29, 1035–1049.
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Original Russian Text © I.V. Korobko, M.V. Shepelev, 2018, published in Molekulyarnaya Biologiya, 2018, Vol. 52, No. 4, pp. 692–698.
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Korobko, I.V., Shepelev, M.V. Mutations in the Effector Domain of RhoV GTPase Impair Its Binding to Pak1 Protein Kinase. Mol Biol 52, 598–603 (2018). https://doi.org/10.1134/S002689331804009X
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DOI: https://doi.org/10.1134/S002689331804009X