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Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Siddiqui MR, Komarova YA, Vogel SM, Gao X, Bonini MG, Rajasingh J, Zhao YY, Brovkovych V, Malik AB - J. Cell Biol. (2011)

Bottom Line: We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation.Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A.Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

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Nitration of p190A induces RhoA activation in endothelial cells. (a) p190A was co-immunoprecipitated and precipitates were probed for phosphotyrosine (PY20) and p190A; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Human microvascular endothelial cells (dermal) overexpressing HA-tagged p190A, p190B, or p190A mutants Y1105F and Y1087F were treated with SIN-1. Nitration of endogenous p190A and exogenously expressed proteins was determined with 3-N antibody. Exogenous proteins were co-immunoprecipitated with anti-HA antibody; n = 3. (c) Live imaging of a genetically encoded FRET-based RhoA biosensor. Representative YFP and FRET/CFP ratio confocal images. Pixel intensities of ratio images were scaled from 0 to 5 and color-coded as indicated on the left. Bar, 10 µm. (d) FRET/CFP emission ratio; mean and SEM are as in Fig. 1 e; n = 13; *, P < 0.01. (e) RhoA-GTP was pulled down with Rhotekin-RBD beads. Resultant precipitates and 5% of cell extracts were probed for RhoA; results of two independent experiments are shown; n = 4. Molecular mass standards are indicated next to the gel blots in kilodaltons.
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fig3: Nitration of p190A induces RhoA activation in endothelial cells. (a) p190A was co-immunoprecipitated and precipitates were probed for phosphotyrosine (PY20) and p190A; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Human microvascular endothelial cells (dermal) overexpressing HA-tagged p190A, p190B, or p190A mutants Y1105F and Y1087F were treated with SIN-1. Nitration of endogenous p190A and exogenously expressed proteins was determined with 3-N antibody. Exogenous proteins were co-immunoprecipitated with anti-HA antibody; n = 3. (c) Live imaging of a genetically encoded FRET-based RhoA biosensor. Representative YFP and FRET/CFP ratio confocal images. Pixel intensities of ratio images were scaled from 0 to 5 and color-coded as indicated on the left. Bar, 10 µm. (d) FRET/CFP emission ratio; mean and SEM are as in Fig. 1 e; n = 13; *, P < 0.01. (e) RhoA-GTP was pulled down with Rhotekin-RBD beads. Resultant precipitates and 5% of cell extracts were probed for RhoA; results of two independent experiments are shown; n = 4. Molecular mass standards are indicated next to the gel blots in kilodaltons.

Mentions: Nitration compromises protein function by altering tyrosine phosphorylation (Gow et al., 1996). The ability of p190A to hydrolyze RhoA is regulated by c-Src and FAK phosphorylation of p190A (Chang et al., 1995; Holinstat et al., 2006). Therefore, we tested whether nitration alters tyrosine phosphorylation of p190A, which could thereby inhibit its GAP activity. We found a marked decrease in p190A phosphorylation in Cav-1−/− MLVECs (Fig. 3 a). We next addressed whether Y1105, the phosphorylation site critical for p190A activity (Roof et al., 1998), might be targeted for nitration. Mutation of Y1105, but not Y1087, to phenylalanine (Y→F) prevented the chemically induced nitration of p190A by 3-morpholinosydnonimine (SIN-1; Fig. 3 b). SIN-1 also induced nitration of endogenous and transiently expressed Wt p190A but not of p190RhoGAP-B (Fig. 3 b).


Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Siddiqui MR, Komarova YA, Vogel SM, Gao X, Bonini MG, Rajasingh J, Zhao YY, Brovkovych V, Malik AB - J. Cell Biol. (2011)

Nitration of p190A induces RhoA activation in endothelial cells. (a) p190A was co-immunoprecipitated and precipitates were probed for phosphotyrosine (PY20) and p190A; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Human microvascular endothelial cells (dermal) overexpressing HA-tagged p190A, p190B, or p190A mutants Y1105F and Y1087F were treated with SIN-1. Nitration of endogenous p190A and exogenously expressed proteins was determined with 3-N antibody. Exogenous proteins were co-immunoprecipitated with anti-HA antibody; n = 3. (c) Live imaging of a genetically encoded FRET-based RhoA biosensor. Representative YFP and FRET/CFP ratio confocal images. Pixel intensities of ratio images were scaled from 0 to 5 and color-coded as indicated on the left. Bar, 10 µm. (d) FRET/CFP emission ratio; mean and SEM are as in Fig. 1 e; n = 13; *, P < 0.01. (e) RhoA-GTP was pulled down with Rhotekin-RBD beads. Resultant precipitates and 5% of cell extracts were probed for RhoA; results of two independent experiments are shown; n = 4. Molecular mass standards are indicated next to the gel blots in kilodaltons.
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fig3: Nitration of p190A induces RhoA activation in endothelial cells. (a) p190A was co-immunoprecipitated and precipitates were probed for phosphotyrosine (PY20) and p190A; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Human microvascular endothelial cells (dermal) overexpressing HA-tagged p190A, p190B, or p190A mutants Y1105F and Y1087F were treated with SIN-1. Nitration of endogenous p190A and exogenously expressed proteins was determined with 3-N antibody. Exogenous proteins were co-immunoprecipitated with anti-HA antibody; n = 3. (c) Live imaging of a genetically encoded FRET-based RhoA biosensor. Representative YFP and FRET/CFP ratio confocal images. Pixel intensities of ratio images were scaled from 0 to 5 and color-coded as indicated on the left. Bar, 10 µm. (d) FRET/CFP emission ratio; mean and SEM are as in Fig. 1 e; n = 13; *, P < 0.01. (e) RhoA-GTP was pulled down with Rhotekin-RBD beads. Resultant precipitates and 5% of cell extracts were probed for RhoA; results of two independent experiments are shown; n = 4. Molecular mass standards are indicated next to the gel blots in kilodaltons.
Mentions: Nitration compromises protein function by altering tyrosine phosphorylation (Gow et al., 1996). The ability of p190A to hydrolyze RhoA is regulated by c-Src and FAK phosphorylation of p190A (Chang et al., 1995; Holinstat et al., 2006). Therefore, we tested whether nitration alters tyrosine phosphorylation of p190A, which could thereby inhibit its GAP activity. We found a marked decrease in p190A phosphorylation in Cav-1−/− MLVECs (Fig. 3 a). We next addressed whether Y1105, the phosphorylation site critical for p190A activity (Roof et al., 1998), might be targeted for nitration. Mutation of Y1105, but not Y1087, to phenylalanine (Y→F) prevented the chemically induced nitration of p190A by 3-morpholinosydnonimine (SIN-1; Fig. 3 b). SIN-1 also induced nitration of endogenous and transiently expressed Wt p190A but not of p190RhoGAP-B (Fig. 3 b).

Bottom Line: We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation.Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A.Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

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