Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration.
BACKGROUND
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-Src-expressing 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.
Ory
Stéphane
S
CRBM CNRS UMR5237, 1919 route de Mende, 34293 Montpellier cedex 5, France.
Brazier
Hélène
H
Blangy
Anne
A
D000818:Animals; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D022001:Focal Adhesions / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010010:Osteoclasts / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019061:src-Family Kinases
D011518:Proto-Oncogene Proteins; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; C432668:RHOU protein, human; D020741:rho GTP-Binding Proteins