RhoGTPases are molecular switches that regulate cytoskeleton dynamics both spatially and temporally. At the cell front Rac and Cdc42 stimulate the formation of protrusions, whereas RhoA at the rear induces retraction through its effector kinase ROCK. Mutual antagonism between Rac and RhoA often contributes to the maintenance of polarity in migrating cells. This month in Nature Cell Biology, Ohta et al. identify FilGAP, a novel ROCK-regulated GTPase-activating protein (GAP) that limits Rac activity in lamellae.

FilGAP expression in Swiss 3T3 cells abolishes Rac- and Cdc42-mediated formation of membrane ruffles and filopodia, but not RhoA-dependent formation of stress fibres, in response to extracellular signals. Furthermore, expression of FilGAP noticeably reduces the levels of Rac-GTP isolated from cell lysates, indicating that FilGAP specifically inactivates Rac, and to a lesser extent Cdc42, both in vitro and in cells.

FilGAP binds through its coiled-coil domain to the C-terminus of the actin crosslinking protein filamin A (FLNa). Co-expression of FilGAP and FLNa, but not a FLNa construct lacking the FilGAP-binding domain, reduces the levels of active Rac in cells, suggesting that FLNa is an upstream regulator of FilGAP. However, this effect could not be replicated in vitro, so the authors investigated whether FLNa was important for targeting FilGAP to particular locations within cells. As predicted, treatment with epidermal growth factor (EGF) induced an accumulation of both FLNa and FilGAP in lamellae. They also found that small-interference RNA (siRNA)-mediated depletion of FilGAP, or expression of a FilGAP mutant lacking GAP activity, strongly stimulated lamellae formation. Together, these findings suggest that FLNa targets FilGAP to particular sites in the cortex to suppress lamellae formation.

To shed further light on the regulation of FilGAP, Ohta et al. examined whether FilGAP affects RhoA/ROCK-dependent downregulation of Rac. Depletion of endogenous FilGAP by siRNA prevented the decrease in active Rac caused by the expression of constitutively active ROCK. Moreover, ROCK was found to phosphorylate and stimulate FilGAP's activity, further supporting a model in which FilGAP is required for ROCK-dependent Rac inactivation.

In summary, this study provides new insights into the mechanism by which RhoA antagonises Rac activity in lamellae. While FLNa ensures FilGAP is positioned at the appropriate location in the cell cortex, ROCK (and potentially other factors) activate FilGAP by phosphorylation, and limit the extent of lamellae formation. By ensuring that the main site of lamellae formation remains at the front of the cell, FilGAP could be important for achieving directed migration.

Author: Monica Hoyos-Flight