Pasteurella multocida Toxin-induced Activation of RhoA Is Mediated via Two Families of Gα Proteins, Gαq and Gα12/13

Joachim H.C Orth,Simona Lang,Masatoshi Taniguchi,Klaus Aktories

doi:10.1074/jbc.m507203200

Joachim H.C Orth, Simona Lang + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m507203200

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Abstract

Pasteurella multocida toxin (PMT) is a potent mitogen, which is known to activate phospholipase Cbeta by stimulating the alpha-subunit of the heterotrimeric G protein G(q). PMT also activates RhoA and RhoA-dependent pathways. Using YM-254890, a specific inhibitor of G(q/11), we studied whether activation of RhoA involves G proteins other than G(q/11). YM-254890 inhibited PMT or muscarinic M3-receptor-mediated stimulation of phospholipase Cbeta at similar concentrations in HEK293m3 cells. In these cells, PMT-induced RhoA activation and enhancement of RhoA-dependent luciferase activity were partially inhibited by YM-254890. In Galpha(q/11)-deficient fibroblasts, PMT induced activation of RhoA, increase in RhoA-dependent luciferase activity, and increase in ERK phosphorylation. None of these effects were influenced by YM-254890. However, RhoA activation by PMT was inhibited by RGS2, RGS16, lscRGS, and dominant negative G(13)(GA), indicating involvement of Galpha(12/13) in the PMT effect on RhoA. In Galpha(12/13) gene-deficient cells, PMT-induced stimulation of RhoA, luciferase activity, and ERK phosphorylation were blocked by YM-254890, indicating the involvement of G(q). Infection with a virus harboring the gene of Galpha(13) reconstituted the increase in RhoA-dependent luciferase activity by PMT even in the presence of YM-254890. The data show that YM-254890 is able to block PMT activation of Galpha(q) and indicate that, in addition to Galpha(q), the Galpha(12/13) G proteins are targets of PMT.

Highlights

The 146-kDa Pasteurella multocida toxin (PMT)2 has diverse biological effects, including potent mitogenic and osteolytic activity [1]
To get further hints for the types of G proteins involved, we studied the effects of various regulators of G protein signaling (RGS) on PMTinduced increase in luciferase activity
PMT acts on G␣q but not on G␣11, both proteins are ϳ89% identical [4]

Summary

Introduction

The 146-kDa Pasteurella multocida toxin (PMT)2 has diverse biological effects, including potent mitogenic and osteolytic activity [1]. We studied the activation of RhoA by PMT, using the Rhobinding domain of rhotekin in pull-down assays with G␣12/13- and G␣q/ 11-deficient cells. In G␣q/11-deficient cells, PMT increased pull-down of the RhoA-rhotekin-Rho-binding domain complex, indicating activation of the GTPase.

Results

Conclusion