Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation

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Abstract

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac 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 Rac 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.

Section snippets

Materials and methods

Materials and chemicals. cDNA encoding mouse STEF was obtained as described previously [16]. Rabbit polyclonal anti-STEF and anti-GST antibodies were produced as described previously [17]. Monoclonal anti-GFP antibody was obtained from Roche Diagnostics (Mannheim, Germany). Y-27632 was provided by Mitsubishi Pharma Co. (Osaka, Japan). A rabbit polyclonal antibody against STEF phosphorylated at Thr1662 was produced against the phosphopeptide Cys-Lys1657-Arg-Gly-Lys-Gly-phosphoThr1662

Suppression of STEF-induced Rac activity by Rho-kinase

To examine whether Rho-kinase inhibits STEF-induced Rac activation in vivo, we transfected GFP-Rac1 into COS7 cells and monitored the GFP-Rac1 activity by the GST-PAK-CRIB pull-down assay (Fig. 1). Coexpression of GFP-STEF-FL increased the amount of GTP-bound GFP-Rac1 in COS7 cells, whereas coexpression of GFP-Rho-kinase-CAT with GFP-STEF-FL reduced the amount of GTP-bound GFP-Rac1 in COS7 cells. Thus, the results of the pull-down assay suggest that Rho-kinase suppresses the STEF-induced Rac1

Discussion

Cell polarity controls many aspects of cellular processes, such as tissue development, chemotaxis, and wound healing. Rho family GTPases are key regulators of cell polarity in a variety of cell types, including epithelial cells, neutrophils, and neuronal cells [2], [21], [22]. For example, Rac and Cdc42 regulate actin polymerization and membrane protrusion at the leading edge of the cells during cell migration, while Rho regulates contraction at the rear [23]. It is generally accepted that, for

Acknowledgments

We thank Dr. T. Watanabe and Dr. S. Wang for helpful discussion and preparation of some materials, Mrs. T. Ishii for secretarial assistance. This research was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and the Japan Society for the Promotion of Science (JSPS); a Grant-in-Aid for Creative Scientific Research from JSPS; The 21st Century Centre of Excellence Program from MEXT; and the Program

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