The ineffectiveness of WGEF shRNA in N1E-115 cells may be due to the low level of expression of WGEF protein in the cells, compared with those of p114-RhoGEF and Lfc proteins

The ineffectiveness of WGEF shRNA in N1E-115 cells may be due to the low level of expression of WGEF protein in the cells, compared with those of p114-RhoGEF and Lfc proteins. Wnt-3aC and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells. INTRODUCTION The Wnt family of secreted proteins play crucial AGK2 roles in numerous developmental and physiological processes by regulating cell proliferation, migration, morphogenesis, and polarity formation (Logan and Nusse, 2004 ). Wnt signals are transduced via at least three distinct pathways: a canonical -cateninCdependent pathway and two noncanonical -cateninCindependent pathways, comprising a planar cell polarity (PCP) pathway and a Ca2+ pathway (Veeman (Shulman gastrulation and the planar polarized orientation of stereocilia of sensory hair cells in the mammalian inner ear (Jones and Chen, 2007 ; Seifert and Mlodzik, 2007 ). The Dvl-mediated activation of RhoA and ROCK is also involved in Wnt-3aCinduced neurite retraction in rat PC-12 pheochromocytoma and mouse N1E-115 neuroblastoma cells (Kishida gastrulation (Tanegashima WGEF can bind to Dvl and Daam1, and depletion of WGEF caused axis elongation defects and inhibition of convergent extension in embryos (Tanegashima luciferase cDNA from pGL4.10 (Promega, Madison, WI) and the partial (400 base pairs) cDNA of each Rho-GEF, prepared by RT-PCR from total RNA of N1E-115 cells, into the test was applied. In all cases, p 0.05 was considered statistically significant. RESULTS Expression Profile of Rho-GEFs in N1E-115 Cells The human genome encodes at least 69 Dbl-like Rho family GEFs, of which 16 members were reported to exhibit GEF activity preferentially activating RhoA (Rossman embryos (Tanegashima and mammalian cells demonstrated that Dvl mediates Wnt-induced RhoA activation (Strutt gastrulation (Tanegashima embryos, but not in N1E-115 cells. Accordingly, Wnt/Dvl-induced RhoA activation appears to be mediated by distinct Rho-GEFs in a cell typeCdependent manner. The ineffectiveness of WGEF shRNA in N1E-115 cells may be due to the low level of expression of WGEF protein in the cells, compared with those of p114-RhoGEF and Lfc proteins. Alternatively, WGEF may require additional factors (that exist in embryo cells, but not in N1E-115 cells) to function. Further studies are required to understand the mechanisms governing the cell type-specific functions of Rho-GEFs in the Wnt-PCP pathway. Lfc is a microtubule-associated Rho-GEF, which is thought to mediate the cross-talk between microtubules and the actin cytoskeleton (Ren (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10-02-0095) on August 25, 2010. REFERENCES Adler P. N. Planar signaling and morphogenesis in Drosophila. Dev. Cell. 2002;2:525C535. [PubMed] [Google Scholar]Aurandt J., Vikis H. G., Gutkind J. S., Ahn N., Guan K. L. The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor, LARG. Proc. Natl. Acad. Sci. USA. 2002;99:12085C12090. [PMC free article] [PubMed] [Google Scholar]Birkenfeld J., Nalbant P., Bohl B. P., Pertz O., Hahn K. M., Bokoch G. M. GEF-H1 AGK2 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases. Dev. Cell. 2007;12:699C712. [PMC free article] [PubMed] [Google Scholar]Blomquist A., Schw?rer G., Schablowski H., Psoma A., Lehnen M., Jakobs K. H., Rmenapp U. AGK2 Identification and characterization of a novel Rho-specific guanine nucleotide exchange factor. Biochem. J. 2000;352:319C325. [PMC free article] [PubMed] [Google Scholar]Etienne-Manneville S., Hall A. Rho GTPases in cell biology. Nature. 2002;420:629C635. [PubMed] [Google Scholar]Fukuhara S., Chikumi H., Gutkind J. S. Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho. FEBS Lett. 2000;485:183C188. [PubMed] [Google Scholar]Habas R., Dawid I. B., He X. Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev. 2003;17:295C309. [PMC free Rabbit Polyclonal to MINPP1 article] [PubMed] [Google Scholar]Habas R., Kato Y., He X. Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell. 2001;107:843C854. [PubMed] [Google Scholar]Jalink K., AGK2 van Corven E. J., Hengeveld T., Morii N., Narumiya S., Moolenaar.