Cytotoxic Necrotizing Factor 1 from Escherichia coli and Dermonecrotic Toxin from Bordetella bronchiseptica Induce p21rho-dependent Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin in Swiss 3T3 Cells

Hadriano M Lacerda,Gill D Pullinger,Alistair J Lax,Enrique Rozengurt

doi:10.1074/jbc.272.14.9587

Abstract

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.

Highlights

The mechanism of action of bacterial toxins has provided novel insights into the control of cellular regulatory processes, including signal transduction and cell proliferation
The results presented here show for the first time that CNF1 and DNT, both intracellularly acting bacterial toxins, induce tyrosine phosphorylation of multiple substrates in Swiss 3T3 cells
Our results suggest that p125fak and paxillin tyrosine phosphorylation could play a role in the signaling pathways stimulated by CNF1 and DNT

Summary

Introduction

The mechanism of action of bacterial toxins has provided novel insights into the control of cellular regulatory processes, including signal transduction and cell proliferation. CNF and DNT induce actin reorganization and multinucleation in several cell types [37,38,39,40] but their effects on signal transduction pathways, specially on p125fak and paxillin tyrosine phosphorylation have not been examined. We report that CNF1 and DNT stimulate tyrosine phosphorylation of p125fak and paxillin and induce a concomitant increase in the formation of actin stress fibers and in the assembly of focal adhesion plaques in Swiss 3T3 cells.

Results

Conclusion