Inactivation of the small GTPase Rho disrupts cellular attachment and induces adhesion-dependent and adhesion-independent apoptosis.
Rho small GTPases regulate a variety of cellular signaling pathways involved in cell growth and transformation. In this study, we examined potential roles for Rho in adhesion-dependent and -independent pathways regulating apoptosis. Rho GTPases are specifically inactivated by exoenzyme C3 (C3) of Clostridium botulinum. Using a novel Sindbis virus-based gene expression system, we created a double subgenomic recombinant (dsSIN:C3) capable of expressing active C3 in intact cells. Infection of L929 fibroblasts with dsSIN:C3 caused essentially complete ADP-ribosylation of intracellular Rho within 1 h. dsSIN:C3-infected cells also became rounded within 1-2 h and detached by 5 h post-infection. Infection of L929 in suspension with dsSIN:C3 disrupted the ability for normal cellular attachment and spreading. Infection of primary cell explants of chicken embryo fibroblasts (CEF) and rat aortic smooth muscle cells (RSM) with dsSIN:C3 caused cytoskeletal effects similar to those seen in L929. We also observed that C3 markedly decreased the basal phosphorylation state of focal adhesion kinase (FAK). Most intriguingly, we found that dsSIN-based expression of C3 or loss of function mutants of Rho could each induce apoptosis and, in RSM, this effect was observed to be adhesion-independent. Rho GTPases, therefore, appear to regulate signal pathways that are required for cell survival and growth that are separate from, but likely overlap with, Rho-dependent pathways involved in cellular adhesion.
Bobak
D
D
Department of Medicine, Beirne Carter Center for Immunology Research, University of Virginia School of Medicine, Charlottesville 22908, USA.
Moorman
J
J
Guanzon
A
A
Gilmer
L
L
Hahn
C
C
D036002:ADP Ribose Transferases / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000502:physiology; D017209:Apoptosis / Q000502:physiology*; D001905:Botulinum Toxins; D002448:Cell Adhesion / Q000502:physiology; D002642:Chick Embryo; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D005347:Fibroblasts / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D007668:Kidney / Q000166:cytology; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology
D011994:Recombinant Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins