Jiaxin Niu

G Protein βγ Subunits Stimulate p114RhoGEF, a Guanine Nucleotide Exchange Factor for RhoA and Rac1 Regulation of Cell Shape and Reactive Oxygen Species Production

Abstract— Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of G&bgr;&ggr; subunits, transducin. We have determined that G&bgr;&ggr; subunits but not G&agr; subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that G&bgr;&ggr; subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, G&bgr;&ggr; subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that G&bgr;&ggr; subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production.

Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation

Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.

RhoGDI-1 Modulation of the Activity of Monomeric RhoGTPase RhoA Regulates Endothelial Barrier Function in Mouse Lungs

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1−/− mice. We observed that basal endothelial permeability in lungs of RhoGDI-1−/− mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1−/− lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1–mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

Functional significance of protein kinase A activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA.

Endothelin-1 (ET1) and ATP stimulate contraction and hypertrophy of vascular smooth muscle cells (VSMC) by activating diverse signalling pathways. In this study, we show that in VSMC, ET1 and ATP stimulate transient and sustained activation of protein kinase A (PKA), respectively. Using a dominant negative PKA mutant (PKA-DN), we examined the functional significance of PKA activation in the signalling of ET1 and ATP. Overexpression of PKA-DN did not alter the ET1- or ATP-induced phosphorylation of the extracellular signal-regulated protein kinase, Erk2. ATP stimulated a profound, PKA-dependent activation of cAMP-response element (CRE), whereas the effect of ET1 was negligible. Both ET1 and ATP stimulated serum response factor (SRF)-dependent gene expression. Overexpression of PKA-DN potentiated the effects of ET1 and ATP on SRF activity, whereas stimulation of PKA by isoproterenol, forskolin or by overexpression of the PKA catalytic subunit decreased SRF activity. These data demonstrate that (i) PKA negatively regulates SRF activity and (ii) ET1 and ATP stimulate opposing pathways, whose balance determines the net activity of SRF.

A Novel Mechanism of G Protein-dependent Phosphorylation of Vasodilator-stimulated Phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) α-thrombin induced phosphorylation of VASP. Specific inhibition of Gα13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Gα13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-κB signaling pathway. α-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Gα13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Gα13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IκB and pretreatment with the proteasome inhibitor MG-132 abolished Gα13- and α-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Gα13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IκB, release of PKA catalytic subunit from the complex with IκB and NF-κB, and subsequent phosphorylation of VASP.

Zyxin is involved in thrombin signaling via interaction with PAR-1 receptor

Protease‐activated receptor 1 (PAR‐1) mediates thrombin signaling in human endothelial cells. As a G‐protein‐coupled receptor, PAR‐1 transmits thrombin signal through activation of the heterotrimeric G proteins, Gi, Gq, and G12/13. In this study, we demonstrated that zyxin, a LIM‐domain‐containing protein, is involved in thrombin‐mediated actin cytoskeleton remodeling and serum response element (SRE)‐ dependent gene transcription. We determined that zyxin binds to the C‐terminal domain of PAR‐1, providing a possible mechanism of involvement of zyxin as a signal transducer in PAR‐1 signaling. Data showing that disruption of PAR‐1‐zyxin interaction inhibited thrombin‐induced stress fiber formation and SRE activation supports this hypothesis. Similarly, depletion of zyxin using siRNA inhibited thrombin‐induced actin stress fiber formation and SRE‐dependent gene transcription. In addition, depletion of zyxin resulted in delay of endothelial barrier restoration after thrombin treatment. Notably, down‐regulation of zyxin did not affect thrombin‐induced activation of RhoA or Gi, Gq, and G12/13 heterotrimeric G proteins, implicating a novel signaling pathway regulated by PAR‐1 that is not mediated by G‐proteins. The observation that zyxin targets VASP, a partner of zyxin in regulation of actin assembly and dynamics, to focal adhesions and along stress fibers on thrombin stimulation suggests that zyxin may participate in thrombin‐induced cytoskeletal remodeling through recruitment of VASP. In summary, this study establishes a crucial role of zyxin in thrombin signaling in endothelial cells and provides evidence for a novel PAR‐1 signaling pathway mediated by zyxin.—Han, J., Liu, G., Profirovic, J., Niu. J., Voyno‐Yasenetskaya, T. Zyxin is involved in thrombin signaling via interaction with PAR‐1 receptor. FASEB J.23, 4193‐4206 (2009). www.fasebj.org

24 ZYXIN IS INVOLVED IN THROMBIN SIGNALING VIA INTERACTION WITH PROTEASE-ACTIVATED RECEPTOR 1.

Introduction Thrombin-induced increase in endothelial monolayer permeability depends on reorganization of cytoskeletal structures. Protease-activated receptor 1 (PAR-1) is the main receptor responsible for thrombin signaling in endothelial cells. Zyxin, a low-abundance phosphoprotein, serves as a molecular adaptor that mediates the productive juxtaposition of protein partners. Zyxin may participate in aspects of cytoskeletal assembly and dynamics by recruiting components of actin assembly machinery to specific sites in the cells. The subcellular distribution of zyxin is critical to its functions. In human umbilical vein endothelial cells (HUVECs), thrombin stimulation induces zyxin targeting to actin stress fibers. We proposed that zyxin participates in thrombin induced cytoskeletal reorganization by interaction with PAR-1. Methods Coimmunoprecipitation and GST fusion protein pull-down assay were performed to identify the domains of zyxin to interact with carboxyl-terminal PAR-1 in COS-7 cells. Confocal immunofluorescence microscopy was utilized to observe the subcellular distribution of zyxin, PAR-1, actin stained with appropriate antibodies against these proteins. Zyxin-specific siRNA duplexes are generated to knock down the expression of endogenous zyxin. Results We showed that PAR-1 interacted with zyxin both in vitro and in vivo. We have shown that LIM domains 1 and 2 of zyxin interacted with C-terminal domain of PAR-1. In human umbilical vein endothelial cells (HUVECs), thrombin promotes zyxin targeting to actin stress fibers. Down-regulation of zyxin using siRNA and disruption of the interaction between zyxin and PAR-1 resulted in reduced cellular responses to thrombin stimulation: actin stress fiber formation and serum response element (SRE)-dependent gene transcription. Zyxin-specific siRNA and C terminus of zyxin overexpression did not inhibit thrombin-induced RhoA activity, which plays a key role in thrombin-induced stress fiber formation and SRE activity and knockdown of endogenous zyxin could still attenuate the active RhoA-induced SRE activity, suggesting that zyxin may regulate thrombin-induced actin reorganization and SRE activity in an RhoA-independent manner. These results suggest that zyxin is involved in thrombin signaling by direct interaction with PAR-1.

4 INCREASED ENDOTHELIAL PERMEABILITY IN RHOGDI ALPHA NULL MICE DUE TO THE ACTIVATION OF RHOA SIGNALING CASCADE

Changes in the endothelial permeability that are essential for vascular repair or inflammatory responses may also contribute to pathological conditions such as vascular leakage, septic shock, edema, atherosclerosis, hypertension, or cancer. Rho family GTPases have been implicated in the regulation of endothelial permeability via action on both actin cytoskeletal organization and the integrity of intercellular junctions. Regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs) prevent activation of RhoA, Rac1, and Cdc42. We studied the lung microcirculation to address the in-vivo role of RhoGDIα signaling in endothelial barrier regulation. We found that endothelial permeability in the lungs of RhoGDIα null mice was two-fold higher than that in the lungs of wild type mice. Perfusion of the mice lungs with the ROCK kinase inhibitor Y-27632 restored endothelial permeability to control level. The activity of RhoA and Rac1, but not Cdc42 was increased in RhoGDIα knock-out mice. Interestingly, myosin light chain 2 and cofilin were hyperphosphorylated, indicating the involvement of the RhoA signaling cascade. Thus, we concluded that regulation of RhoA and Rac1 activity via RhoGDIα has barrier protective effect in endothelium.