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Cited by 70Protein Kinase Regulation and GTPase SignalingReceptor Mechanisms and SignalingMicrotubule and mitosis dynamics

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

Open access
Meiri, David, Marshall, Christopher B., Mokady, Daphna, LaRose, Jose, Mullin, Michael, Gingras, Anne-Claude, Ikura, Mitsuhiko, Rottapel, Robert·2014·Nature Communications
DOI 10.1038/ncomms5857
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Abstract

The RhoGEF GEF-H1 can be sequestered in an inactive state on polymerized microtubules by the dynein motor light-chain Tctex-1. Phosphorylation of GEF-H1 Ser885 by PKA or PAK kinases creates an inhibitory 14-3-3-binding site. Here we show a new mode of GEF-H1 activation in response to the G-protein-coupled receptor (GPCR) ligands lysophosphatidic acid (LPA) or thrombin that is independent of microtubule depolymerization. LPA/thrombin stimulates disassembly of the GEF-H1:dynein multi-protein complex through the concerted action of Gα and Gβγ. Gα binds directly to GEF-H1 and displaces it from Tctex-1, while Gβγ binds to Tctex-1 and disrupts its interaction with the dynein intermediate chain, resulting in the release of GEF-H1. Full activation of GEF-H1 requires dephosphorylation of Ser885 by PP2A, which is induced by thrombin. The coordinated displacement of GEF-H1 from microtubules by G-proteins and its dephosphorylation by PP2A demonstrate a multistep GEF-H1 activation and present a unique mechanism coupling GPCR signalling to Rho activation.

AI snapshot

Written from the paper’s full text.

TL;DR

This study identifies a multistep, microtubule-depolymerization-independent mechanism by which GPCR ligands LPA/thrombin activate the RhoGEF GEF-H1: Gα12/13 binds GEF-H1 and displaces Tctex-1, Gβγ binds Tctex-1 and disrupts its dynein interaction, and PP2A dephosphorylates Ser885. Full activation requires both G-protein subunit actions and transient PP2A-mediated dephosphorylation.

Key findings

  • LPA/thrombin released GEF-H1 from microtubules without microtubule depolymerization: GEF-H1-eGFP microtubule correlation was 0.67 (n=91) in untreated cells, and LPA induced focal adhesion colocalization with vinculin at 0.81 (n=74). Taxol did not block thrombin-induced Ser885 dephosphorylation or LPA/thrombin-induced GEF-H1 relocalization/stress fibres, but did block nocodazole effects.
  • Gβγ bound Tctex-1 and disrupted the GEF-H1:Tctex-1 interaction; a Gβ mutant (R48A, R49A, R52A) pulled down less Tctex-1, Gβγ expression redistributed GEF-H1 and induced stress fibres in wild-type but not GEF-H1-knockout MEFs, and Gβγ relieved Tctex-1-mediated inhibition of GEF-H1 exchange activity in a βARKct-sensitive manner.
  • Active Gα13 bound directly to the N-terminal region of GEF-H1, with amino acids 1–240 sufficient for optimal binding; Gα13QL reduced Tctex-1 and 14-3-3 association with GEF-H1, antagonized Tctex-1 inhibition of exchange activity, and Gα-bound GEF-H1 was dephosphorylated at Ser885. Inhibiting both Gα and Gβ was required to block thrombin/LPA-induced GEF-H1 release or activation.
  • PP2A associated with GEF-H1 through B′ regulatory subunits and the GEF-H1 PH domain; recombinant PP2Ac dephosphorylated 14-3-3-binding sites within 10 min, and thrombin induced transient Ser885 dephosphorylation at 5 min that returned by 10 min, with maximal GEF-H1:PP2A interaction at 5 min and undetectable interaction after 20 min.

Methods

  • Cell-based assays used HEK293T cells, GEF-H1−/− MEFs, and wild-type MEFs, with transfections, serum starvation, and treatments including LPA (1 μM, 30 min), thrombin (3 U ml−1, 1 h or time course), nocodazole (20 μM, 10 min), and Taxol (10 μM, 24 h).
  • Main analyses included immunoprecipitation/western blot, confocal immunofluorescence and live-cell imaging, image correlation and focal adhesion counts, real-time NMR GEF assays with 15N-labeled RhoA, affinity purification–mass spectrometry of Flag-tagged PP2A subunits, and in vitro PP2A phosphatase assays.
  • Statistics used paired Student’s t-tests, at least three independent experiments, P<0.05, and correlation coefficients from ≥60 images across three independent experiments.

Limitations

  • Stated in the paper: the precise mechanisms by which G-protein subunits trigger GEF-H1:PP2A assembly remain to be established, and the detailed Gα12/13–GEF-H1 binding interface awaits further structural studies.
  • Evident from the text: many conclusions rely on overexpression in HEK293T cells or MEFs and on purified/recombinant proteins, with no in vivo animal model or endogenous GPCR knockout validation described.
  • Evident from the text: one reported effect—Gα13QL alone increasing GEF-H1 exchange activity without Tctex-1 overexpression—did not reach statistical significance, supporting Tctex-1 displacement as the major mechanism.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/ncomms5857

Cites

44 of 44 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

13 of 23 cited sources checked · 5 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Co-expression of the Gbg subunits relieved Tctex-1-mediated inhibition of GEF-H1 exchange activity.

    “As previously reported, Tctex-1 potently inhibited the exchange activity of full-length GEF-H1 (refs 6,17), whereas co-expression of the Gbg subunits relieved this inhibitory effect (Fig. 2e).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [6] Meiri, D. et al. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. Mol. Cell 45, 642–655 (2012). source
    • cites [17] Marshall, C. B. et al. Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts. Methods 57, 473–485 (2012). source
  • Premisecitation

    The dynein light-chain Tctex-1 tethers GEF-H1 to polymerized microtubules.

    “We have shown that the dynein light-chain Tctex-1 tethers GEF-H1 to polymerized microtubules 6 , where it is poised to undergo D-AKAP-1dependent phosphorylation of Ser885 by PKA.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [6] Meiri, D. et al. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. Mol. Cell 45, 642–655 (2012). source
  • Premisecitation

    Nocodazole-induced microtubule depolymerization potently activates GEF-H1.

    “Nocodozole-induced microtubule depolymerization potently activates GEF-H1 (refs 5-7), thus GEF-H1 activity during mitosis may be a consequence of the change in microtubule dynamics during the reorganization of stable interphase microtubules into the mitotic spindle 36 .”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [5] Krendel, M., Zenke, F. T. & Bokoch, G. M. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton. Nat. Cell Biol. 4, 294–301 (2002). source
    • cites [6] Meiri, D. et al. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. Mol. Cell 45, 642–655 (2012). source

      In “Polymerized Microtubules Are Required for Tctex-1-Mediated Inhibition of Lfc” (page not provided), the paper reports that nocodazole increased Lfc/GEF-H1 exchange activity 1.5-fold in lysates of Lfc-overexpressing cells. This supports activation after microtubule depolymerization, but does not establish that the activation was potent or that the measured effect applies beyond those cells. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [7] Callow, M. G., Zozulya, S., Gishizky, M. L., Jallal, B. & Smeal, T. PAK4 mediates morphological changes through the regulation of GEF-H1. J. Cell Sci. 118, 1861–1872 (2005). source
  • Interpretationown data

    GEF-H1 activation results from disruption of the GEF-H1:dynein complex by G-protein subunits in the absence of microtubule depolymerization.

    “Our results suggest a model where GEF-H1 activation results from disruption of the GEF-H1:dynein complex by G-protein subunits in the absence of microtubule depolymerization 6,7 .”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [6] Meiri, D. et al. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. Mol. Cell 45, 642–655 (2012). source

      The Discussion identifies the dynein light chain Tctex-1 as anchoring Lfc (GEF-H1) to polymerized microtubules, and reports that Tctex-1 represses its exchange activity. It does not show G-protein subunits disrupting that complex or activating GEF-H1 without microtubule depolymerization; searches for those mechanisms found no supporting passage. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [7] Callow, M. G., Zozulya, S., Gishizky, M. L., Jallal, B. & Smeal, T. PAK4 mediates morphological changes through the regulation of GEF-H1. J. Cell Sci. 118, 1861–1872 (2005). source
  • Premisecitation

    Microtubule-dependent inhibition of GEF-H1 requires the assembly of a protein complex including Tctex-1, dynein intermediate chain (DIC) and 14-3-3 proteins.

    “We have recently shown that microtubule-dependent inhibition of GEF-H1 requires the assembly of a protein complex including Tctex-1, dynein intermediate chain (DIC) and 14-3-3 proteins 6 .”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [6] Meiri, D. et al. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. Mol. Cell 45, 642–655 (2012). source

      The Discussion (page unavailable) says that Lfc inhibition depends on a complex containing Tctex-1 and dynein intermediate chain, and that intact microtubules are required. It also reports that phosphorylation creates a 14-3-3 binding site that inhibits Lfc activity, but does not establish that 14-3-3 proteins are required members of the Tctex-1–DIC complex. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • May combine two claims. 25
  • Classified as probably not a substantial claim. 14

  • May combine two claims.

    Premisecitation

    Phosphorylation of GEF-H1 Ser885 by PKA creates a high-affinity 14-3-3-binding site that inhibits GEF-H1 exchange activity.

    “Phosphorylation of Ser885 creates a high-affinity 14-3-3-binding site that inhibits the exchange activity of GEF-H1 (ref.3).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Meiri, D. et al. Modulation of Rho guanine exchange factor Lfc activity by protein kinase A-mediated phosphorylation. Mol. Cell. Biol. 29, 5963–5973 (2009). source

  • Classified as probably not a substantial claim.

    Premisecitation

    The Gb subunit binds to Tctex-1 and competes with DIC for binding to Tctex-1, regulating assembly of Tctex-1 into the dynein motor complex.

    “Sachdev et al. 15 showed that the Gb subunit binds to Tctex-1 and competes with DIC for binding to Tctex-1, regulating assembly of Tctex-1 into the dynein motor complex 14,15 .”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [15] Sachdev, P. et al. G protein beta gamma subunit interaction with the dynein light-chain component Tctex-1 regulates neurite outgrowth. Embo J. 26, 2621–2632 (2007). source
    • cites [14] Takesono, A. et al. Receptor-independent activators of heterotrimeric G-protein signaling pathways. J. Biol. Chem. 274, 33202–33205 (1999). source

      The Results and Discussion identify AGS2 as Tctex1 and report that it directly bound brain Gβγ. They suggest a possible role for G-protein subunits in dynein function, but do not report competition with DIC or show that this binding regulates Tctex1 assembly into the dynein complex. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    GEF-H1 is required for LPA/thrombin-induced stress fibre formation and focal adhesion formation following stimulation with tumour necrosis factor-a, LPA or thrombin.

    “GEF-H1 is required for LPA/thrombin-induced stress fibre formation, and focal adhesion formation following stimulation with tumour necrosis factor-a, LPA or thrombin 2,3,6 .”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [2] Birukova, A. A. et al. GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction. Am. J. Physiol. Lung Cell. Mol. Physiol. 290, L540–L548 (2006). source
    • cites [3] Meiri, D. et al. Modulation of Rho guanine exchange factor Lfc activity by protein kinase A-mediated phosphorylation. Mol. Cell. Biol. 29, 5963–5973 (2009). source

      The Introduction identifies Lfc as GEF-H1 and says its depletion impairs actin reorganization after LPA or thrombin; the Results directly show that Lfc depletion prevents LPA-induced stress fibres. The Results also say Lfc modulates focal-adhesion assembly downstream of LPA, but the passages do not support the claim about focal-adhesion formation after tumour necrosis factor-α or thrombin stimulation. Page numbers were not available in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [6] Meiri, D. et al. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. Mol. Cell 45, 642–655 (2012). source

      The Results and Discussion report that Lfc (GEF-H1) is required for LPA-induced stress fibres and focal adhesions in mouse embryonic fibroblasts, and the Discussion describes a role in LPA/thrombin-induced stress fibre formation. The paper also examines focal adhesions after thrombin stimulation, but the passages found do not address focal adhesion formation after tumour necrosis factor-α stimulation (searched: tumour necrosis factor, TNF-α). (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Recombinant full-length GEF-H1 protein expressed and purified from insect cells bound to recombinant Ga13, indicating that active Ga can bind directly to GEF-H1 in vitro.

    “Recombinant full-length GEF-H1 protein expressed and purified from insect cells 6 bound to recombinant Ga 13 , indicating that active Ga can bind directly to GEF-H1 in vitro (Supplementary Fig. 4a).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [6] Meiri, D. et al. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. Mol. Cell 45, 642–655 (2012). source

      The passage on full-length Lfc/GEF-H1 (labeled Abstract in our copy; page unavailable) describes assays in HEK293T cell lysates and regulation by Tctex-1, not binding to Gα13. Searches for Gα13 binding, purified protein from insect cells, and Supplementary Fig. 4a found no passage establishing the claimed direct interaction. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    PKA phosphorylates and inhibits GEF-H1 by creating a 14-3-3-binding site.

    “In a previous publication, we found that PKA phosphorylates and inhibits GEF-H1 by creating a 14-3-3-binding site 3 , which strongly suggests that a cellular phosphatase(s) is involved in the activation of GEF-H1.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Meiri, D. et al. Modulation of Rho guanine exchange factor Lfc activity by protein kinase A-mediated phosphorylation. Mol. Cell. Biol. 29, 5963–5973 (2009). source

  • May combine two claims.

    Findingown data

    The level of GEF-H1 Ser885 phosphorylation was strongly correlated with association with 14-3-3 and Tctex-1 and inversely correlated with Ga13 association.

    “We observed that the level of GEF-H1 Ser885 phosphorylation was strongly correlated with association with 14-3-3 and Tctex-1 and inversely correlated with Ga 13 association (Fig. 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ser885 phosphorylation was detected in starved cells, but decreased after 5 min of thrombin treatment.

    “Ser885 phosphorylation was detected in starved cells, but decreased after 5 min of thrombin treatment (Fig. 7b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GEF-H1 was readily recovered with both the catalytic and scaffolding subunits of PP2A, but only co-purified with members of the B0 regulatory family PPP2R5A, PPP2R5B and PPP2R5E.

    “While GEF-H1 was readily recovered with both the catalytic and the scaffolding subunits, it only co-purified with members of one of the regulatory (B) families, the B 0 regulatory subunits PPP2R5A, PPP2R5B and PPP2R5E (Fig. 6a), suggesting specificity in the regulatory subunit selection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Taxol suppresses nocodazole but not LPA-stimulated redistribution of GEF-H1 from the microtubule to the cytoplasm.

    “(b) Taxol suppresses nocodazole but not LPA-stimulated redistribution of GEF-H1 from the microtubule to the cytoplasm.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither the rgRGS domain nor the bARKct peptide alone were sufficient to block the relocalization of GEF-H1 by thrombin, but expression of both inhibitory proteins had a potent blocking effect on GEF-H1 relocalization.

    “We observed that neither the rgRGS domain nor the bARKct peptide alone were sufficient to block the relocalization of GEF-H1 by thrombin, however, the expression of both inhibitory proteins had a potent blocking effect on GEF-H1 relocalization (Fig. 5d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Amino acids 1-240 of GEF-H1 are sufficient for optimal binding to endogenous Ga12, while shorter deletions within this region markedly reduced Ga binding.

    “Coimmunoprecipitation experiments with a series of Flag-tagged, N-terminal expressed GEF-H1 fragments demonstrated that amino acids 1-240 of GEF-H1 are sufficient for optimal binding to endogenous Ga 12 while shorter deletions within this region markedly reduced Ga binding (Fig. 4b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of either rgRGS or bARKct alone had little impact on thrombin's ability to disrupt the GEF-H1-DIC interaction, but co-expression of both rgRGS and bARKct blocked the disruptive effect of thrombin on the GEF-H1-DIC complex.

    “Expression of either rgRGS or bARKct alone had little impact on thrombin's ability to disrupt the GEF-H1-DIC interaction, however, co-expression of both rgRGS and bARKct blocked the disruptive effect of thrombin on the GEF-H1-DIC complex (Fig. 5c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Taxol suppresses nocodazole- but not LPA- or thrombin-induced activation of GEF-H1 exchange activity.

    “(e) Taxol suppresses nocodazole-but not LPA-or thrombin-induced activation of GEF-H1 exchange activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of the Gbg subunits was sufficient to induce actin-based stress fibres in wild-type MEFs but not in GEF-H1 knockout MEFs.

    “We found that expression of the Gbg subunits was sufficient to induce actin-based stress fibres in wild-type MEFs but not in GEF-H1 knockout MEFs (Fig. 2d), reinforcing the idea that Gbg plays a role in inducing stress fibre formation through GEF-H1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In untreated cells, GEF-H1-eGFP was closely associated with the microtubule array (correlation coefficient 0.67, n=91), whereas after 5 min of LPA or thrombin treatment GEF-H1-eGFP moved into a largely cytoplasmic distribution.

    “In untreated cells, GEF-H1-eGFP was closely associated with the microtubule array (correlation coefficient of 0.67, n ¼ 91), whereas after 5 min of LPA or thrombin treatment GEF-H1-eGFP moved into a largely cytoplasmic distribution (Fig. 1b-d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the absence of Tctex-1 overexpression, GEF-H1 exchange activity increased when Ga13QL was coexpressed, but this trend did not reach statistical significance.

    “In the absence of Tctex-1 overexpression, GEF-H1 exchange activity increased when Ga 13QL was coexpressed, however, this trend did not reach statistical significance, supporting the hypothesis that displacement of Tctex-1 is the major mechanism by which Ga 13 activates GEF-H1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    PP2A and GEF-H1 form a dynamic complex in response to thrombin, which is temporally correlated to dephosphorylation of the GEF-H1 negative regulatory site Ser885, loss of dynein interaction and redistribution from the microtubule array.

    “These results show that PP2A and GEF-H1 form a dynamic complex in response to thrombin, which is temporally correlated to dephosphorylation of the GEF-H1 negative regulatory site Ser885, loss of dynein interaction and redistribution from the microtubule array.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All statistical analyses were derived from 60 or more images from three independent experiments for each treatment condition.

    “All statistical analyses were derived from 60 or more images from three independent experiments for each treatment condition.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Following thrombin stimulation, the PP2A phosphatase associates with GEF-H1 and dephosphorylates the negative regulatory 14-3-3-binding site, Ser885.

    “In addition, we have discovered that following thrombin stimulation, the PP2A phosphatase associates with GEF-H1 and dephosphorylates the negative regulatory 14-3-3-binding site, Ser885.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning

    Error bars represent s.d. of at least three independent replicates, and P values derived from pairwise t-tests are indicated.

    “Error bars represent s.d. of at least three independent replicates, and P values derived from pairwise t-tests are indicated.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The amount of GEF-H1 in each assay was standardized by eGFP fluorescence of the lysate.

    “The amount of GEF-H1 in each assay was standardized by eGFP fluorescence of the lysate measured by a Shimadzu RF-5301PC spectrofluorophotometer.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All images in each set of experiments were acquired with the same microscope sensitivity settings.

    “All images in each set of experiments were acquired with the same microscope sensitivity settings.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Two biological replicate analyses were performed on each of the baits.

    “Two biological replicates analyses were performed on each of the baits.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All images compared within each figure panel were acquired on the same day, with identical staining conditions, gain and contrast setting, and same magnification, except Fig. 1c.

    “All images compared within each figure panel were acquired on the same day, with identical staining conditions, gain and contrast setting, and same magnification (except Fig. 1c).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    To compare endogenous cellular GEF activity, total cellular protein in lysates was standardized using the Bradford assay.

    “To compare endogenous cellular GEF activity, total cellular protein in lysates was standardized using the Bradford assay.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Ga binding to GEF-H1 involves an intrinsically disordered region in the N-terminal portion of GEF-H1, which may undergo binding-coupled folding.

    “These results strongly argue that Ga binding to GEF-H1 involves an intrinsically disordered region in the N-terminal portion of GEF-H1, which may undergo binding-coupled folding.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Experiments were performed at least three times, and means with P<0.05 were considered statistically significant.

    “Experiments were performed at least three times, and means with Po0.05 were considered statistically significant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Gbg can disrupt the interaction of GEF-H1 with the dynein motor complex leading to its displacement from the microtubule array and activation of its exchange activity.

    “These data demonstrate that Gbg can disrupt the interaction of GEF-H1 with the dynein motor complex leading to its displacement from the microtubule array and activation of its exchange activity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ga12/13 subunits directly activate a class of RhoGEFs including p115-RhoGEF, PDZ-RhoGEF, LARG and Lbc-RhoGEF that contain Ga-binding RH domains.

    “Ga 12/13 subunits directly activate a class of RhoGEFs including p115-RhoGEF, PDZ-RhoGEF, LARG and Lbc-RhoGEF 11,12 that contain Ga-binding RH domains.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] Fukuhara, S., Chikumi, H. & Gutkind, J. S. RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho? Oncogene 20, 1661–1668 (2001). source
    • cites [12] Dutt, P., Jaffe, A. B., Merdek, K. D., Hall, A. & Toksoz, D. Galphaz inhibits serum response factor-dependent transcription by inhibiting Rho signaling. Mol. Pharmacol. 66, 1508–1516 (2004). source

  • May combine two claims.

    Interpretationown data

    Active Ga12/13 binds to a region encompassing the first 240 amino acids of GEF-H1 and stimulates its intrinsic exchange activity.

    “GEF-H1 lacks a canonical RGS domain, nevertheless, in this study, we have shown that active Ga 12/13 binds to a region encompassing the first 240 amino acids of GEF-H1 and stimulates its intrinsic exchange activity.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Paired Student's t-tests were performed to determine statistical significance between samples.

    “Paired Student's t-tests (ref.39) were performed to determine statistical significance between samples.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites ref.39

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The correlation coefficient (R) measuring co-localization was determined from three independent experiments.

    “The correlation coefficient (R) measuring co-localization was determined from three independent experiments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    GEF-H1 physically interacts with PP2A, which can dephosphorylate 14-3-3-binding sites in vitro.

    “Together, these data demonstrate that GEF-H1 physically interacts with PP2A, which can dephosphorylate 14-3-3-binding sites in vitro.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Fourteen negative control runs were performed using cells expressing the FLAG tag fused to a green fluorescent protein.

    “Fourteen negative control runs were also performed that consisted of cells expressing the FLAG tag fused to a green fluorescent protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Either Ga or Gb is sufficient to trigger the displacement of GEF-H1 from the microtubules.

    “These results provide evidence that either Ga or Gb is sufficient to trigger the displacement of GEF-H1 from the microtubules.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Either Ga or Gb is sufficient to induce GEF-H1 activation.

    “However co-expression of rgRGS and bARKct together blocked the ability of LPA to relieve Tctex-1 inhibition of GEF-H1, further suggesting that either Ga or Gb is sufficient to induce GEF-H1 activation.”
    Quote in the paperSubstantial claim
36 of 36

  • The paper relies on this without support.

    LPA and thrombin signal to cells through activation of the LPA receptors and protease-activated GPCRs, respectively.

    “LPA and thrombin signal to cells through activation of the LPA receptors and protease-activated GPCRs, respectively.”

  • The paper relies on this without support.

    The GEF-H1-eGFP fusion protein behaves like endogenous GEF-H1 in localization assays.

    “GEF-H1-knockout murine embryonic fibroblasts (MEFs) were transfected with GEF-H1enhanced green fluorescent protein (eGFP) and visualized by confocal microscopy before and after treatment.”

  • The paper relies on this without support.

    Overexpression of tagged proteins does not create non-physiological interactions that invalidate the conclusions.

    “We confirmed that Gb bound to Tctex-1 in 293T cells co-expressing Gb 12 isoform, Tctex-1 and GEF-H1, while a Gb variant (Gb mut ) bearing three mutations (R48A, R49A and R52A) in the Tctex-1binding site pulled down significantly less Tctex-1 (Fig. 2a), consistent with results reported by Sachdev et al. 15”
3 of 3

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 5c gives conflicting GEF-H1 construct tags

    Minor

    The Results identify the GEF-H1 construct used for Figure 5c as Flag-tagged, but the figure caption says cells were transfected with Myc-GEF-H1.

  • Figure 4b identifies different Ga subunits

    Minor

    The Results describe the Figure 4b binding experiment as involving endogenous Ga 12, while the figure caption identifies the measured subunit as endogenous Ga 13.

  • Figure 6a uses two names for the normalization subunit

    Minor

    The caption calls the PP2A scaffolding A subunit PPP1R1A, but elsewhere identifies the same normalization subunit as PPP2R1A.

3 of 3

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Authors
Meiri, D., Marshall, C. B., Mokady, D., LaRose, J., Mullin, M., Gingras, A.-C., Ikura, M., & Rottapel, R.
Affiliations
  • Technion – Israel Institute of Technology (IL)
  • University Health Network (CA)
  • Mount Sinai Hospital (CA)
  • St. Michael's Hospital (CA)
Corresponding author
Rottapel, Robert
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2014-09-11
Volume
5
Issue
1
Pages
4857-4857
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/ncomms5857
OpenAlex
W2029393686
Semantic Scholar
45aa028afe506595e5f204b5e154c8f9bbc06762
Cited by
70
References
43
Citations by year
2026: 9 · 2025: 6 · 2024: 4 · 2023: 3 · 2022: 1 · 2021: 9 · 2020: 5 · 2019: 2 · 2018: 5 · 2017: 11 · 2016: 6 · 2015: 8 · 2014: 1
FWCI
3.55
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Amgen
  • Cancer Research Society
  • Ontario Institute for Cancer Research
  • Canadian Institutes of Health Research (MOP 84314)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
Keywords
GEF-H1, RhoA, G protein-coupled receptors, G protein-coupled receptor signaling, microtubules, Tctex-1, dynein light chain, Gβγ, G protein alpha subunit, lysophosphatidic acid, thrombin, PP2A dephosphorylation, 14-3-3 binding
MeSH terms
Animals, Dyneins, Lysophospholipids, Microtubules, Phosphorylation, Thrombin, Mice, Knockout, Receptors, G-Protein-Coupled, GTP-Binding Protein alpha Subunits, G12-G13, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, 14-3-3 Proteins, Mice, p21-Activated Kinases, Rho Guanine Nucleotide Exchange Factors

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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