Abstract
BackgroundThe Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains.ResultsHere we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference.ConclusionCollectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in the regulation of cytokinesis, but instead may regulate centrosome duplication. The kinesin-like motor protein KIF3B may modulate the ARHGEF10-RhoA pathway through the binding to ARHGEF10.
Highlights
The Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves
Involvement of ARHGEF10 in the regulation of mitotic spindle formation As a first step to clarify the function of ARHGEF10 at a molecular level, we attempted to identify substrate Rho family GTPases for putative guanine nucleotide exchange factors (GEFs) activity of ARHGEF10 by a pull-down assay (Figure 1B)
RhoA was accumulated in the midbody in ARHGEF10-knockdown tripolar as well as control cells(Figure 4F). These results suggest that occurrence of multiple centrosomes in ARHGEF10-knockdown cells may not be due to abnormalities incytokinesis
Summary
The Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. The function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. Centrosomes coordinate the cytoplasmic microtubule network serving as the major microtubule-organizing centers in mammalian cells [1,2,3]. A single centrosome consists of a pair of centrioles surrounded by the amorphous pericentriolar material (PCM). During M phase of the cell cycle, two centrosomes orchestrate the assembly of bipolar mitotic spindles, which is prerequisite for accurate chromosome segregation. Aberrations in centrosome numbers, which frequently occur in aggressive human tumors, almost certainly represent one crucial cause of missegregation of chromosomes, leading to a phenotype termed genetic instability [3]. The rigorous control of the duplication cycle of the centrosome is vital for the cell. Our understanding of the regulation of the centrosome cycle remains incomplete
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