Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rae is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rae GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF. (c) 2007 Elsevier Inc. All rights reserved.