Dataset Information

Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

ABSTRACT: The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.

SUBMITTER: Marei H

PROVIDER: S-EPMC4968972 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

Marei Hadir H Carpy Alejandro A Macek Boris B Malliri Angeliki A

Cell cycle (Georgetown, Tex.) 20160506 15

The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstrea ...[more]

PMID: 27152953

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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.

Dataset Information

Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

Publications

Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

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