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Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.

ABSTRACT: The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner.

SUBMITTER: Marei H

PROVIDER: S-EPMC4759627 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.

Marei Hadir H Carpy Alejandro A Woroniuk Anna A Vennin Claire C White Gavin G Timpson Paul P Macek Boris B Malliri Angeliki A

Nature communications 20160218

The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influen ...[more]

PMID: 26887924

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Project description:Macrophages are important immune sentinels that detect and clear pathogens and initiate inflammatory responses through the activation of surface receptors, including Toll-like receptors (TLRs). Activated TLRs employ complex cellular trafficking and signalling pathways to initiate transcription for inflammatory cytokine programs. We have previously shown that Rab8a is activated by multiple TLRs and regulates downstream Akt/mTOR signalling by recruiting the effector PI3Kγ, but the guanine nucleotide exchange factors (GEF) canonically required for Rab8a activation in TLR pathways is not known. Using GST affinity pull-downs and mass spectrometry analysis, we identified a Rab8 specific GEF, GRAB, as a Rab8a binding partner in LPS-activated macrophages. Co-immunoprecipitation and fluorescence microscopy showed that both GRAB and a structurally similar GEF, Rabin8, undergo LPS-inducible binding to Rab8a and are localised on cell surface ruffles and macropinosomes where they coincide with sites of Rab8a mediated signalling. Rab nucleotide activation assays with CRISPR-Cas9 mediated knock-out (KO) cell lines of GRAB, Rabin8 and double KOs showed that both GEFs contribute to TLR4 induced Rab8a GTP loading, but not membrane recruitment. In addition, measurement of signalling profiles and live cell imaging with the double KOs revealed that either GEF is individually sufficient to mediate PI3Kγ-dependent Akt/mTOR signalling at macropinosomes during TLR4-driven inflammation, suggesting a redundant relationship between these proteins. Thus, both GRAB and Rabin8 are revealed as key positive regulators of Rab8a nucleotide exchange for TLR signalling and inflammatory programs. These GEFs may be useful as potential targets for manipulating inflammation. Abbreviations: TLR: Toll-like Receptor; OCRL: oculocerebrorenal syndrome of Lowe protein; PI3Kγ: phosphoinositol-3-kinase gamma; LPS: lipopolysaccharide; GEF: guanine nucleotide exchange factor; GST: glutathione S-transferases; BMMs: bone marrow derived macrophages; PH: pleckstrin homology; GAP: GTPase activating protein; ABCA1: ATP binding cassette subfamily A member 1; GDI: GDP dissociation inhibitor; LRP1: low density lipoprotein receptor-related protein 1.

Project description:Aims/hypothesisRho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells.MethodsCo-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA).ResultsWestern blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016.Conclusions/interpretationOur data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

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Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.

Publications

Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.

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