GDI-1 phosphorylation switch at serine 96 induces RhoA activation and increased endothelial permeability.
We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Calpha-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 --> Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.
Knezevic
Nebojsa
N
Department of Pharmacology, College of Medicine, The University of Illinois, 835 S Wolcott Avenue, Chicago, IL 60612, USA.
Roy
Arun
A
Timblin
Barbara
B
Konstantoulaki
Maria
M
Sharma
Tiffany
T
Malik
Asrar B
AB
Mehta
Dolly
D
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology; D012694:Serine / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000032:analysis / Q000378:metabolism*
D020730:Guanine Nucleotide Dissociation Inhibitors; C066737:RHOA protein, human; D049452:Green Fluorescent Proteins; D012694:Serine; D008156:Luciferases; D051571:Protein Kinase C-alpha; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D000409:Alanine