MCB Accepts, published online ahead of print on 16 July 2007

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Mol. Cell. Biol. doi:10.1128/MCB.00523-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

GDI-1 Phosphorylation Switch at Serine 96 Induces RhoA Activation and Increase Endothelial Permeability

Nebojsa Knezevic, Arun Roy, Barbara Timblin, Maria Konstantoulaki, Tiffany Sharma, Asrar B. Malik, and Dolly Mehta^*

Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois

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 (HPAEC) transduced with the full-length (FL) GDI-1. Expression of a GDI-1 mutant (C-GDI) containing the C-terminus (amino acids 69-204) also prevented RhoA activation whereas further deletions failed to alter RhoA activation. We observed that PKC-mediated phosphorylation of the GDI C-terminus at at Ser96 reduced the affinity of GDI-1 for RhoA, and thereby enabled RhoA activation. Rendering GDI-1 phospho-defective by Ser96 Ala substitution rescued the GDI-1's inhibitory activity towards RhoA, but did not alter the thrombin-induced activation of other RhoGTPases Rac1 or Cdc42. Phospho-defective GDI-1 mutant also suppressed myosin light chain phosphorylation, actin stress fiber formation, and increased endothelial permeability induced by thrombin. In contrast, expressing phospho-mimicking S96D-GDI-1 mutant induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of phosphorylation of GDI-1 C-terminus 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.

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