Analysis of Activated GAPs and GEFs in Cell Lysates

Garcı́a-Mata, Rafael; Wennerberg, Krister; Arthur, William T.; Noren, Nicole K.; Ellerbroek, Shawn M.; Burridge, Keith

doi:10.1016/s0076-6879(06)06031-9

Cited by 184 publications

(255 citation statements)

References 26 publications

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“…Affinity Precipitation Assay for Active GEF-H1-GST-G17A-RhoA, a mutant with high affinity for activated GEFs, was described previously (31). The assay was performed as described previously (32) with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

Role of Guanine Nucleotide Exchange Factor-H1 in Complement-mediated RhoA Activation in Glomerular Epithelial Cells

Mouawad

Aoudjit

Jiang

et al. 2014

Journal of Biological Chemistry

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Background: Disruption of the actin cytoskeleton-dependent structural integrity of glomerular epithelial cells (GEC) leads to glomerular dysfunction. Results: We have identified molecular mechanisms through which the injury-causing complement complex induces cytoskeletal changes in GEC. Conclusion: Complement-induced activation of the ERK/GEF-H1/RhoA pathway protects GEC from cell death. Significance: This study furthers our understanding of mechanisms underlying GEC foot process effacement and functional impairment in immune mediated glomerular diseases.

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Section: Methodsmentioning

confidence: 99%

Role of Guanine Nucleotide Exchange Factor-H1 in Complement-mediated RhoA Activation in Glomerular Epithelial Cells

Mouawad

Aoudjit

Jiang

et al. 2014

Journal of Biological Chemistry

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“…Active RhoGEFs were trapped using a modified nucleotide-free GSTRhoA 17A pulldown assay (García-Mata et al, 2006). In brief, mouse tails were harvested at the indicated days after wounding, deboned, and snap frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

“…Because RhoA is persistently activated during epidermal wound healing in 4OHT-treated K14-CreER T2 ; CD98hc fl/fl mice, we sought to identify its upstream active RhoGEFs during wound repair. Therefore, we performed unbiased in vivo nucleotide-free GST-RhoA 17A pulldowns on lysates from wounded mouse tails to trap and identify active RhoA-specific GEFs during wound repair (García-Mata et al, 2006;Dubash et al, 2007). In brief, GST-RhoA 17A precipitates were resolved by SDS-PAGE and differentially regulated bands were excised from the gel and analyzed by trypsin digestion coupled to LC-ESI-MS/MS.…”

Section: Cd98hc-deficient Epidermis Displays a Strong In Vivo C-src Amentioning

confidence: 99%

CD98hc (SLC3A2) regulation of skin homeostasis wanes with age

Boulter¹,

Estrach²,

Errante³

et al. 2013

J Cell Biol

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Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal 1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports.

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“…To perform the pulldown assay with nucleotide-free Rac1 mutant (G15ARac1), HUVECs were lysed and processed as described in Garcia-Mata et al [16]. Briefly, cells were lysed in a buffer consisting of 1% Triton X-100, 20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , and protease inhibitors.…”

Section: Precipitation Of Activated Gefs With Recombinant Rac1 Proteinmentioning

confidence: 99%

“…This technique is based on the concept that GEFs bind small GTPases with high affinity when the GTPase is in a transitional nucleotide-free state [16,26]. As such, the nucleotide-free Rac1 mutant G15ARac1 has higher affinity for binding active GEFs, and our laboratory has used this mutant previously to bind and identify active GEFs [16].…”

Section: Exchange Factor Vav2 Couples Vegf Signaling To Rac1mentioning

confidence: 99%

VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

Garrett

Buul

Burridge

2007

Experimental Cell Research

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152

132

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Vascular endothelial growth factor (VEGF) signaling is critical for both normal and diseaseassociated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the Rho family of small GTPases. As a member of this family, Rac1 promotes VEGF-induced endothelial cell migration by stimulating the formation of lamellipodia and membrane ruffles. To form these membrane protrusions, Rac1 is activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP. The goal of this study was to identify the GEF responsible for activating Rac1 in response to VEGF stimulation. We have found that VEGF stimulates biphasic activation of Rac1 and for these studies we focused on the peak of activation that occurs at 30 min. Inhibition of VEGFR-2 signaling blocks VEGF-induced Rac1 activation. Using a Rac1 nucleotide-free mutant (G15ARac1), which has a high affinity for binding activated GEFs, we show that the Rac GEF Vav2 associates with G15ARac1 after VEGF stimulation. Additionally, we show that depleting endothelial cells of endogenous Vav2 with siRNA prevents VEGF-induced Rac1 activation. Moreover, Vav2 is tyrosine phosphorylated upon VEGF treatment, which temporally correlates with Rac1 activation and requires VEGFR-2 signaling and Src kinase activity. Finally, we show that depressing Vav2 expression by siRNA impairs VEGF-induced endothelial cell migration. Taken together, our results provide evidence that Vav2 acts downstream of VEGF to activate Rac1.

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Analysis of Activated GAPs and GEFs in Cell Lysates

Cited by 184 publications

References 26 publications

Role of Guanine Nucleotide Exchange Factor-H1 in Complement-mediated RhoA Activation in Glomerular Epithelial Cells

Role of Guanine Nucleotide Exchange Factor-H1 in Complement-mediated RhoA Activation in Glomerular Epithelial Cells

CD98hc (SLC3A2) regulation of skin homeostasis wanes with age

VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

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