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Molecular and Cellular Biology, September 2007, p. 6323-6333, Vol. 27, No. 18
0270-7306/07/$08.00+0     doi:10.1128/MCB.00523-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

GDI-1 Phosphorylation Switch at Serine 96 Induces RhoA Activation and Increased Endothelial Permeability{triangledown}

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

Received 26 March 2007/ Returned for modification 27 April 2007/ Accepted 5 July 2007

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 C{alpha}-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.

* Corresponding author. Mailing address: Department of Pharmacology, College of Medicine, The University of Illinois, 835 S Wolcott Avenue, Chicago, IL 60612. Phone: (312) 355-0236. Fax: (312) 996-1225. E-mail: dmehta{at}uic.edu

{triangledown} Published ahead of print on 16 July 2007.

Molecular and Cellular Biology, September 2007, p. 6323-6333, Vol. 27, No. 18
0270-7306/07/$08.00+0     doi:10.1128/MCB.00523-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mong, P. Y., Wang, Q. (2009). Activation of Rho Kinase Isoforms in Lung Endothelial Cells during Inflammation. J. Immunol. 182: 2385-2394 [Abstract] [Full Text]  
  • Qiao, J., Holian, O., Lee, B.-S., Huang, F., Zhang, J., Lum, H. (2008). Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA. Am. J. Physiol. Cell Physiol. 295: C1161-C1168 [Abstract] [Full Text]  
  • Komarova, Y. A., Mehta, D., Malik, A. B. (2007). Dual Regulation of Endothelial Junctional Permeability. Sci Signal 2007: re8-re8 [Abstract] [Full Text]  


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