It is well accepted that integrin-derived signals regulate many cellular processes including growth, survival, and cytoskeletal dynamics (Cordes, 2006
; Delon and Brown, 2007
; Reddig and Juliano, 2005
; Schwartz and Assoian, 2001
). Rho GTPases are important components of this signaling and regulate actin polymerization and organization through multiple pathways. These pathways include activation of the Arp2/3 complex by Rac and Cdc42, activation of formin proteins through Rho, and activation of myosin phosphorylation through ROCK and Rho. Rac was proposed to be the main regulator of lamellipodial protrusions that mediate cell migration and spreading (Hall, 2005
); Rac is also important for cell proliferation and resistance to apoptosis (Ruggieri et al., 2001
; Vidali et al., 2006
). The mechanism by which integrins trigger Rac activation is of great interest because of its relevance to abnormal cell migration and survival in pathologies including cancer and metastasis, developmental and immune disorders.
Recent publications suggested that RhoG is a critical mediator of integrin signaling to Rac during cell spreading and migration. This conclusion was based on dramatic inhibition of adhesion-induced cell spreading and migration by DN mutants of RhoG and its downstream partner ELMO, and the behavior of a RhoG knockdown clone in HeLa cells. By contrast, we found that efficient knockdown of RhoG did not substantially affect cell spreading or migration. We also found that RhoG was not detectably activated by adhesion to fibronectin and did not contribute significantly to activation of Rac1 during cell adhesion to fibronectin.
The inconsistencies between the effects of shRNA-mediated RhoG depletion and effects of DN RhoG mutants are most likely to be due to non-specific effects of the DN mutants. DN mutants of Rho proteins such as N17 RhoG may bind and sequester multifunctional GEFs that normally interact with other GTPases. Indeed, Trio, Kallirin, VAV1, VAV2, VAV3 and PLEKHG6 are all able to interact with both Rac and RhoG (Abe et al., 2000
; Blangy et al., 2000
; D'Angelo et al., 2007
; Movilla and Bustelo, 1999
; Rabiner et al., 2005
; Schuebel et al., 1998
; Tybulewicz et al., 2003
; Wennerberg et al., 2002
). Furthermore the observation that effects of constitutively active Q61L RhoG were reduced by T17N RhoG (Wennerberg et al., 2002
) supports the notion that this dominant negative has broader specificity. Similar findings of nonspecific effects by DN constructs have been reported for other Rho family GTPases (Wang and Zheng, 2007
). Similarly, overexpressed fragments of ELMO may interact with other target proteins.
The reason for reduced spreading and migration in the stable RhoG knockdown Hela clone (Katoh et al., 2006
) is less clear. We noticed, however, that HeLa cells are remarkably heterogeneous in their rate of spreading on fibronectin. It is therefore not surprising that individual clones would spread at different rates. Testing additional RhoG deficient clones would help to resolve the discrepancy.
While physiological signals upstream of RhoG are only beginning to be identified (see below), the effects of its active mutant on the actin cytoskeleton are well documented (Gauthier-Rouviere et al., 1998
; Katoh et al., 2006
; Katoh and Negishi, 2003
; Katoh et al., 2000
). There is, however, a disagreement regarding the mechanism of actin regulation by RhoG. While some studies indicated that active RhoG induced formation of ruffles via Rac (Gauthier-Rouviere et al., 1998
; Katoh and Negishi, 2003
), others suggested a parallel, Rac-independent pathway (Prieto-Sanchez and Bustelo, 2003
; Wennerberg et al., 2002
). These investigations tested the effects of DN Rac on the phenotype induced by active RhoG. To address these issues, we used conditional Rac knockout cells, an approach that should be more specific than overexpression of DN constructs (Vidali et al., 2006
). We found that RhoG can affect the cytoskeleton through both Rac-dependent and -independent pathways. The Rac-dependent pathway was evident at low expression of active RhoG, whereas the Rac-independent pathway required higher expression. The functionality of a Rac-independent pathway for induction of actin rearrangements by RhoG is supported by a study in Salmonella Typhimurium
(Patel and Galan, 2006
). Salmonella independently activates Rac through bacterial GEF SopE and RhoG through bacterial protein SopB. RhoG activation by SopB is mediated by SGEF, a host GEF for RhoG. While a SopE-deficient Salmonella strain could not activate Rac, it was still able to induce SopB-dependent actin rearrangements leading to SGEF and RhoG mediated bacterial uptake. Furthermore, SopB could function also in the absence of Rac, suggesting that RhoG can contribute to bacterial uptake independently of Rac.
However, the mechanism of RhoG-induced actin rearangements in the absence of Rac is still not clear. Rac induces actin polymerization by activation of the WAVE complex, which in turn leads to Arp2/3 complex activation (Ridley et al., 2003
). Rac effectors IRSp53 and Sra1/PIR121 have been reported to mediate the activation of Wave complex by Rac (Ridley, 2006
). Rac can also affect actin polymerization through its effector PAK (Jaffe and Hall, 2005
). In spite of sequence similarity of RhoG to Rac, neither IRSp53 nor PAK was found to interact with RhoG (Wennerberg et al., 2002
). We considered that Sra1 might mediate RhoG effect on actin. However, we found that, in contrast to Rac, only weak, nucleotide-independent binding of RhoG to Sra-1 could be detected. Thus, despite their close homology, RhoG does not appear to affect actin through known Rac effectors. The mechanism of Rac-independent actin regulation by RhoG will require further investigation.
In contrast to conclusions based on DN Rac mutants, conditional knockout of Rac1 in fibroblasts (Vidali et al., 2006
) and double knockout of Rac1 and Rac2 in macrophages (Wheeler et al., 2006
) showed that Rac is not absolutely essential for migration. Our data demonstrate that RhoG is an important mediator of this Rac-independent cell migration in fibroblasts. It therefore seems plausible that RhoG may mediate migration in response to specific stimuli or under specific conditions, however, its physiological role in migration remains unknown. We did find that serum stimulation induced mild and transient activation of RhoG (unpublished data), however, the effects were weak enough that it was difficult to obtain a statistically significant effect. RhoG might also be regulated by signals derived from cellular events involved in cytokinesis or membrane trafficking.
While this work was in progress, publications suggested that RhoG participates in phagocytosis (deBakker et al., 2004
; Nakaya et al., 2006
), endothelial cup formation (van Buul et al., 2007
) and macropinocytosis (D'Angelo et al., 2007
; Ellerbroek et al., 2004
). It is interesting that these functions, at least in culture, involve localized actin rearrangements at the dorsal membrane. A restricted spectrum of RhoG-dependent functions is more consistent with the lack of developmental abnormalities in RhoG knockout mice, as opposed to the model claiming a non-redundant role of RhoG for developmentally essential processes such as integrin mediated signaling and migration.