Kuo JC, Han X, Hsiao CT, et al. Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for beta-Pix in negative regulation of focal adhesion maturation

Cell Biology and Physiology Centre, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland 20892, USA.
Nature Cell Biology (Impact Factor: 19.68). 03/2011; 13(4):383-93. DOI: 10.1038/ncb2216
Source: PubMed


Focal adhesions undergo myosin-II-mediated maturation wherein they grow and change composition to modulate integrin signalling for cell migration, growth and differentiation. To determine how focal adhesion composition is affected by myosin II activity, we performed proteomic analysis of isolated focal adhesions and compared protein abundance in focal adhesions from cells with and without myosin II inhibition. We identified 905 focal adhesion proteins, 459 of which changed in abundance with myosin II inhibition, defining the myosin-II-responsive focal adhesion proteome. The abundance of 73% of the proteins in the myosin-II-responsive focal adhesion proteome was enhanced by contractility, including proteins involved in Rho-mediated focal adhesion maturation and endocytosis- and calpain-dependent focal adhesion disassembly. During myosin II inhibition, 27% of proteins in the myosin-II-responsive focal adhesion proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in focal adhesions, establishing that focal adhesion protein recruitment is also negatively regulated by contractility. We focused on the Rac guanine nucleotide exchange factor β-Pix, documenting its role in the negative regulation of focal adhesion maturation and the promotion of lamellipodial protrusion and focal adhesion turnover to drive cell migration.

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Available from: Xuemei Han, Jun 23, 2014
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    • "Cytoplasmic STAT3 could bind to and thereby inhibit Rac1-specific GEF b-PIX, and normalize Rac1 activity (Teng et al., 2009). GEF b-PIX plays a key role in the negative regulation of focal adhesion maturation and promotion of lamellipodial protrusion formation, thus driving cell migration (Kuo et al., 2011). Collectively, it shows that Rac1/STAT3 activation is involved in fibroblast proliferation and migration , with different mechanisms of STAT3 regulation, and a negative feedback loop to inactivate Rac1. "
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    ABSTRACT: The Rho family of small GTPases forms a 20-member family within the Ras superfamily of GTP-dependent enzymes that are activated by a variety of extracellular signals. The most well known Rho family members are RhoA (Ras homolog gene family, member A), Cdc42 (cell division control protein 42), and Rac1 (Ras-related C3 botulinum toxin substrate 1), which affect intracellular signaling pathways that regulate a plethora of critical cellular functions, such as oxidative stress, cellular contacts, migration, and proliferation. In this review, we describe the current knowledge on the role of GTPase Rac1 in the vasculature. Whereas most recent reviews focus on the role of vascular Rac1 in endothelial cells, in the present review we also highlight the functional involvement of Rac1 in other vascular cells types, namely, smooth muscle cells present in the media and fibroblasts located in the adventitia of the vessel wall. Collectively, this overview shows that Rac1 activity is involved in various functions within one cell type at distinct locations within the cell, and that there are overlapping but also cell type–specific functions in the vasculature. Chronically enhanced Rac1 activity seems to contribute to vascular pathology; however, Rac1 is essential to vascular homeostasis, which makes Rac1 inhibition as a therapeutic option a delicate balancing act.
    Journal of Pharmacology and Experimental Therapeutics 08/2015; 354(2):91-102. DOI:10.1124/jpet.115.223610 · 3.97 Impact Factor
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    • "The accelerated cell migration after reggie downregulation and the interaction with CAP (c-cbl-associated protein), a component of FAs, which is known to bind reggie-2 (Kimura et al., 2001; Kioka et al., 2002; Langhorst et al., 2008a), suggested that reggies might also play a role in FA formation (Schmidt and Dikic, 2005). Indeed, a proteomic analysis previously recognized that reggie- 1 and reggie-2 are part of the FA complex (Kuo et al., 2011). In addition, the downregulation of reggie and overexpression of a dominant negative (DN) reggie-1 construct disturbed the localization of overexpressed prion protein in structures resembling FAs (Schrock et al., 2009), affected Src and FAK and impaired axon growth (Langhorst et al., 2008a; Munderloh et al., 2009). "
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    ABSTRACT: Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and β1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled β1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or β1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and β1-integrins to FAs to regulate FA formation and membrane dynamics. Copyright © 2015 Elsevier GmbH. All rights reserved.
    European Journal of Cell Biology 07/2015; DOI:10.1016/j.ejcb.2015.07.003 · 3.83 Impact Factor
    • "Similar forces have been shown to regulate the ECM elasticity-dependent control of MSC cell fate via YAP/TAZ [9]. Interestingly, GO enrichment analysis of proteins identified by MS from MSC adhesion complexes demonstrated a significant enrichment of LIM domain-containing proteins (Supplementary Data), as previously observed for adhesion complexes isolated from fibroblasts [15] [16]. In total, 24 LIM domain proteins were identified in the dataset, 21 of which were at least two-fold enriched to FN (Supplementary Data). "
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    ABSTRACT: Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision-making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force-sensing, we sought to catalogue MSC integrin-associated adhesion complex composition. Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by mass spectrometry. Identified proteins were interrogated by comparison to a literature-based reference set of cell adhesion-related components and using ontological and protein-protein interaction network analyses. Adhesion complex-specific proteins in MSCs were identified that consisted predominantly of cell adhesion-related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain-containing proteins in MSC adhesion complexes were highlighted, which may act as force-sensing components. These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    PROTEOMICS - CLINICAL APPLICATIONS 07/2015; DOI:10.1002/prca.201500033 · 2.96 Impact Factor
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