RhoG GTPase Controls a Pathway That Independently Activates Rac1 and Cdc42Hs

Abstract

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth

“…The answer to these questions will require identi®cation of the extracellular signals that stimulate TC10, as well as the use of dominant negative GTPases to determine whether the function of one GTPase is dependent on the activation of another. At this time, the possibility of a cascade incorporating TC10, at least in regard to ®lopodial induction, seems likely since the ability of yet another family member, RhoG, to stimulate ®lopodial and lamellipodial formation requires CDC42 and Rac, respectively (Gauthier-Rouviere et al, 1998). On the other hand, the inability of TC10 to induce lamellipodia or stress ®bers, both in our assay and in a similar assay with Swiss 3T3 cells (Neudauer et al, 1998), suggests that Rac may not be downstream of TC10 in a cascade regulating cytoskeletal rearrangements.…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…A functional correlation between microtubules and Rho activity could also be drawn from studies on another exchange factor, TrioGEF that activates RhoG [52]. RhoG is an activator of Rac and Cdc42 that can promote membrane protrusion [53]. Neither RhoG nor TrioGEF binds directly to microtubules, but their activities on Rac-and Cdc42-dependent protrusion and localization to the cell periphery are dependent on an intact microtubule network [53].…”

“…RhoG is an activator of Rac and Cdc42 that can promote membrane protrusion [53]. Neither RhoG nor TrioGEF binds directly to microtubules, but their activities on Rac-and Cdc42-dependent protrusion and localization to the cell periphery are dependent on an intact microtubule network [53]. This might imply that microtubule motors provide a link between Rac, Cdc42 and Rho and/or their activating GEFs and microtubules to transport activated Rho proteins to protrusions.…”

“…This might imply that microtubule motors provide a link between Rac, Cdc42 and Rho and/or their activating GEFs and microtubules to transport activated Rho proteins to protrusions. Since kinesin (a predominantly plus end directed motor family) and its interactor, kinectin are required for RhoG mediated actin assembly [54], and RhoG binds kinectin [53], kinesin motors may transport activated RhoG to sites proximal to cell adhesions. Interestingly, the Rac GEF, ASEF is activated upon binding to APC, a plus end binding protein that surfs the growing tip of microtubules [55], perhaps providing a means to target active Rac to membranes during cell adhesion [56].…”

Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations

“…Tensile strain decreased the amount of membrane associated Rho GTPase by 70%. Other workers have shown there is a functional relationship between both Rho GTP'ase, GEF protein, and microtubules that requires an intact microtubule cytoskeleton [52,54,55]. The fact that both proteins lost their localisation to the cell periphery upon microtubule depolymerisation suggested that a microtubule dependent transport process was involved [52,54,55].…”