Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)

Taro Chaya,Satoshi Shibata,Yasunori Tokuhara,Wataru Yamaguchi,Hiroshi Matsumoto,Ichiro Kawahara,Mikihiko Kogo,Yoshiharu Ohoka,Shinobu Inagaki

doi:10.1074/jbc.m111.236810

Taro Chaya, Satoshi Shibata + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m111.236810

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Journal: Journal of Biological Chemistry	Publication Date: Aug 1, 2011
Citations: 23	License type: cc-by

Affiliation: Osaka University

Abstract

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

Highlights

When our initiating methionine is at residue Met-1, that selected by Mohl et al is at residue Met-87, and they studied an ARHGEF10 protein starting at residue Ala-112 [16]
We showed that ARHGEF10 has a negatively regulatory region in the N terminus and that the T332I mutant is a constitutively active ARHGEF10 mutant for RhoA, B, and C
The mutants that induced cell contraction had higher guanine nucleotide exchange factors (GEFs) activity than the wt, suggesting that cell contraction observed in this study reflects the GEF activity of ARHGEF10 mutants

Summary

Introduction

Expressions of HA-RhoA and GFP-ARHGEF10 in observed on the number and localization of centrosome of HEK293T cells (data not shown). These data suggest that the N-terminal region of ARHGEF10 functions as the domain required for repressing its GEF activity for RhoA, RhoB, and/or RhoC. To examine the effect of expression of this mutant for cell morphology, HEK293T cells were transfected with the plasmid encoding GFP-ARHGEF10 wt, ⌬N, or ⌬NЈ, and GFP images of living cells were analyzed using fluorescence microscopy.

Results

Conclusion