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

Abstract

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␣-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 3 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.

Alteration of vascular permeability in burn injury

“…It has also been shown that some PKC isozymes, such as PKCa, could activate RhoA by inducing rapid phosphorylation of GDP dissociation inhibitor (GDI), indicating that RhoA would be one of the important substrates of PKC. 45,46 Our previous study also suggested that NOS was a potent substrate of PKC, while inhibition of nitric oxide synthase (NOS) with specific blocker NG-monomethyl-L-arginine (L-NMMA) greatly attenuated the hyperpermeability effect of PMA, indicating that PKC may alter endothelial permeability by directly acting on endothelial structural proteins and/or indirectly by modulating activity of common signaling protein NOS, as can be seen in Fig. 8B.…”

“…As mentioned above, RhoA might be one of the important substrates of PKC. 44,45 These data re-stress the pivotal role of Rho/ROCK pathway in the modulation of endothelial barrier function. In contrast, while PKC also restrained the formation of stress fiber in burned-serum treated endothelial cells, the inhibition of PKC with Ro-31-7549 did not attenuate the phosphorylation of p38 after burned plasma administration, implying the p38-independent effect of PKC on endothelial morphological regulation.…”

eIF5

“…In addition, both yeast and mammalian eIF5 have been shown to be phosphorylated by Casein Kinase II. 37,38 While regulation by phosphorylation has been demonstrated for a number of other GDIs [39][40][41] it is not yet clear if eIF5-GDI is regulated by changes in post-translational modification.…”

Mechanisms Regulating Endothelial Permeability

“…2,12,17 In an unperturbed endothelium, IEJs dynamically open to allow the passage of small molecules and inflammatory cells for tissue homeostasis and immune surveillance. 2,8,9,12,14 Proinflammatory agonists such as thrombin, vascular endothelial growth factor (VEGF), and platelet-activating factor, [19][20][21][22] by binding to their receptors, disorganize IEJs, leading to increase in endothelial permeability. Thus, to understand how tissue-fluid homeostasis is modulated under normal conditions and pathological processes, we must understand the signaling mechanisms that regulate IEJs.…”