Determination of GTP loading on Rho

Ren, Xiang‐Dong; Schwartz, Martin A.

doi:10.1016/s0076-6879(00)25448-7

Cited by 324 publications

(300 citation statements)

References 10 publications

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“…2N). By contrast, Vangl2 overexpression did not affect the activation state of Rac1 and RhoA, as demonstrated by pulldown assays with GST-PAK1 or GST-rhotekin, effectors that binds to activated Rac1 or RhoA, respectively (Akasaki et al, 1999;Ren and Schwartz, 2000) ( Fig. 2O,P).…”

Section: Journal Of Cell Science 123 (3)mentioning

confidence: 88%

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

Lindqvist¹,

Horn²,

Bryja

et al. 2010

Journal of Cell Science

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The Wnt planar cell polarity (Wnt/PCP) pathway signals through small Rho-like GTPases to regulate the cytoskeleton. The core PCP proteins have been mapped to the Wnt/PCP pathway genetically, but the molecular mechanism of their action remains unknown. Here, we investigate the function of the mammalian PCP protein Vang-like protein 2 (Vangl2). RNAi knockdown of Vangl2 impaired cell-cell adhesion and cytoskeletal integrity in the epithelial cell lines HEK293T and MDCK. Similar effects were observed when Vangl2 was overexpressed in HEK293T, MDCK or C17.2 cells. The effects of Vangl2 overexpression could be blocked by knockdown of the small GTPase Rac1 or by dominant-negative Rac1. In itself, knockdown of Rac1 impaired cytoskeletal integrity and reduced cell-cell adhesion. We found that Vangl2 bound and re-distributed Rac1 within the cells but did not alter Rac1 activity. Moreover, both transgenic mouse embryos overexpressing Vangl2 in neural stem cells and loop-tail Vangl2 loss-of-function embryos displayed impaired adherens junctions, a cytoskeletal unit essential for neural tube rigidity and neural tube closure. In vivo, Rac1 was re-distributed within the cells in a similar way to that observed by us in vitro. We propose that Vangl2 affects cell adhesion and the cytoskeleton by recruiting Rac1 and targeting its activity in the cell to adherens junctions.

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Section: Journal Of Cell Science 123 (3)mentioning

confidence: 88%

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

Lindqvist¹,

Horn²,

Bryja

et al. 2010

Journal of Cell Science

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“…RhoGTP levels in Jurkat cells were assessed by using a Rhotekin Rho-binding domain (RBD) affinity precipitation assay (Ren and Schwartz, 2000). Briefly, after serum-starvation overnight, 1 ϫ 10 7 cells were stimulated with SDF1␣ (100 nM) for the indicated times.…”

Section: Rho Activation Assaymentioning

confidence: 99%

“…The reaction was stopped by adding 5ϫ lysis buffer at 4°C (1ϫ lysis buffer: 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal, 10 mM MgCl 2 , 1 mM EDTA, and 10% glycerol). Cell lysates were prepared and the level of RhoGTP in the lysates was determined by using Rhotekine-RBD bound to glutathione-Sepharose beads as described previously (Ren and Schwartz, 2000).…”

Section: Rho Activation Assaymentioning

confidence: 99%

RACK1 Regulates Directional Cell Migration by Acting on Gβγ at the Interface with Its Effectors PLCβ and PI3Kγ

Chen

Lin

Shin

et al. 2008

MBoC

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Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein ␤␥ (G␤␥) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with G␤␥ and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains G␤␥-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKC␤ and Src. Rather, RACK1 selectively inhibits G␤␥-stimulated phosphatidylinositol 3-kinase ␥ (PI3K␥) and phospholipase C (PLC) ␤ activity, due to the competitive binding of RACK1, PI3K␥, and PLC␤ to G␤␥. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with G␤␥-dependent activation of key effectors critical for chemotaxis.

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“…Determination of activated cellular Rho and MLC phosphorylation. The amount of activated cellular Rho was determined by precipitation with a fusion protein consisting of glutathione-S-transferase (GST) and the Rhobinding domain of rhotekin (GST-RBD) 26 . Platelets were stimulated with 10 nM of U46619 for 10 s, lysed and subjected to GST-rhotekin pull-down.…”

Section: Conditional Inactivation Of the Gna13 Gene See Supplementarmentioning

confidence: 99%

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Moers

Nieswandt

Maßberg

et al. 2003

Nat Med

232

236

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Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke 1,2 . Platelet activators such as adenosine diphosphate, thrombin or thromboxane A 2 (TXA 2 ) activate receptors that are coupled to heterotrimeric G proteins 1,3 . Activation of platelets through these receptors involves signaling through G q , G i and G z (refs. 4-6). However, the role and relative importance of G 12 and G 13 , which are activated by various platelet stimuli 7-9 , are unclear. Here we show that lack of Gα 13 , but not Gα 12 , severely reduced the potency of thrombin, TXA 2 and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Gα 13 deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G 13 -mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.

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Determination of GTP loading on Rho

Cited by 324 publications

References 10 publications

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

Vang-like protein 2 and Rac1 interact to regulate adherens junctions

RACK1 Regulates Directional Cell Migration by Acting on Gβγ at the Interface with Its Effectors PLCβ and PI3Kγ

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

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