Identification of a bipartite focal adhesion localization signal in RhoU/Wrch‐1, a Rho family GTPase that regulates cell adhesion and migration

Abstract

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/ Wrch-1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N-and C-terminal domains. The N-terminal extension promotes binding to SH3 (Src homology 3)-domain-containing adaptors, whereas the C-terminal extension mediates membrane targeting through palmitoylation of its non-conventional CAAX box. RhoU/Wrch-1 possesses transforming activity, which is negatively regulated by its N-terminal extension and depends on palmitoylation.Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c-Srcexpressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N-terminal extension and the palmitoylation site were dispensable, whereas the C-terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked-down. Conclusions.Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C-terminal domains are critical for these functions.

Rho GTPases in osteoclasts: Orchestrators of podosome arrangement

“…In these cells, RhoU induces Pyk2-and Src-dependent stress fiber dissolution. RhoU binding to Pyk2 requires its proline rich region, but similarly to what we found (Ory et al, 2007), RhoU-dependent effects on the actin cytoskeleton also requires an intact effector binding site (Ruusala and Aspentröm, 2007).…”

“…This localization is dependent on the ability of RhoU to bind to effectors since a RhoU mutant unable to bind the Cdc42/Rac interaction domain of PAK is only found on vesicles (Ory et al, 2007;HB, unpublished data). Like many components of podosomes, RhoU is also found in focal 8 adhesions where it alters their dynamics and, in consequence, the migration rate of fibroblasts (Ory et al, 2007;Chuang et al, 2007). The C-terminal extension of RhoU and residues critical for effector binding are required for targeting RhoU to focal adhesions (Ory et al, 2007).…”

“…RhoU is related to Rac and Cdc42 and like them, it binds and activates PAK1. But RhoU possesses additional domains at its N-and C-termini: RhoU N-terminal extension is involved in binding to SH3-containing proteins such as Nck1 and Grb2 (Saras et al, 2004;Shutes et al, 2004) whereas the C-terminal extension participates to the correct subcellular localization of RhoU (Ory et al, 2007). Its subcellular localization is also in part driven by the addition of palmitoyl motif rather than a prenyl group, which is usually added on the CAAX box of Rho GTPases (Berzat et al, 2005).…”

“…Like many components of podosomes, RhoU is also found in focal 8 adhesions where it alters their dynamics and, in consequence, the migration rate of fibroblasts (Ory et al, 2007;Chuang et al, 2007). The C-terminal extension of RhoU and residues critical for effector binding are required for targeting RhoU to focal adhesions (Ory et al, 2007). Whether RhoU affects podosome organization of osteoclasts remains to be addressed but the fact that RhoU may activate the RhoA pathway (Saras et al, 2004) and localizes to osteoclast podosomes (Ory et al, 2007) identifies it as an attractive candidate for the control of podosome arrangement in osteoclasts.…”

The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

“…We previously showed that Wrch1 associated with adhesion structures: it localized to focal adhesion in fibroblasts and to podosomes in osteoclasts (Ory et al, 2007). We (Ory et al, 2007) and others (Chuang et al, 2007) also reported that Wrch1 regulated focal adhesion assembly and cell migration in fibroblasts and epithelial cells.…”