The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)–Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner.
The authors declare no competing or financial interests.
antiARHGEF10 was produced by T.T. Localization analysis of ARHGEF10 and Rab6 and establishment of stably expressing cells were performed by T.K. and A.Y. ARHGEF10 mutants were constructed by T.C. and Y.T. Analysis of the cells expressing ARHGEF10 mutants were performed by C.T. Transwell invasion assays were performed by T.H. Immunoprecipitation assays were performed by M.S. GEF assays for Rab8 and blind tests were performed by A.Y. S.I. planned and supervised the experiments. S.S. wrote manuscript and arranged figures.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.186817.supplemental
- Received January 29, 2016.
- Accepted August 13, 2016.
- © 2016. Published by The Company of Biologists Ltd