RhoE Regulates Actin Cytoskeleton Organization and Cell Migration

ABSTRACT

The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.

ACKNOWLEDGMENTS

We are grateful to Ritu Garg for excellent cell culture assistance, to Robert Harris and Thomas Eichholtz (Glaxo Wellcome) for the gift of the R4.2 cell line, and to Alan Entwistle for assistance with confocal laser scanning microscopy and image processing. We thank Emma Cannell for characterizing the r13, r15, r16, and r20 clones, Nick Totty for laser desorption mass spectrometric analysis of recombinant RhoE, and Jeff Settleman for advice and discussion of results. We also thank Ignacio Perez-Roger for help with the sequence analysis software.

This work was supported by European Communities grant ERBCHBGCT940702.

ADDENDUM IN PROOF

After submission of this paper, the sequences of Rho6,Rho7, and Rho8 were published (C. D. Nobes et al., J. Cell Biol. 141:1–11, 1998) and were renamedRnd1, Rnd2, and Rnd3, respectively. The protein encoded by Rnd3 is identical to RhoE, except that it has an extra 15 amino acids at the N terminus.