Biochemical and Biophysical Research Communications
Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation
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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