The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia

AbstractCell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia [1], these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins, targets of Abl tyrosine kinases [2–4], have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation [5]. Here, we describe the unique localization of Abi proteins in living, motile cells. We show that Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. We identify the targeting domain as the homologous N terminus of these two proteins. Our findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility

Abl Family Kinases Regulate Endothelial Barrier Function <i>In Vitro</i> and in Mice

<div><p>The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg), as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR), Kit, colony stimulating factor 1 receptor (CSF1R), and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial <i>Abl</i> knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability <i>in vivo</i>. Using both Abl/Arg-specific pharmacological inhibition and endothelial <i>Abl</i> knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both <i>in vitro</i> and <i>in vivo</i>. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca²⁺ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use of Abl kinase inhibitors may have potential for the treatment of disorders involving pathological vascular leakage.</p> </div

Abl tyrosine kinases are required for infection by Shigella flexneri

Infection by the opportunistic bacterial pathogen Shigella flexneri stimulates tyrosine phosphorylation of host cell proteins, but the kinases involved and their effects on the regulation of cell signaling pathways during bacterial entry remain largely undefined. Here, we demonstrate a requirement for the Abl family of tyrosine kinases during Shigella internalization. Family members Abl and Arg are catalytically activated upon Shigella infection, accumulate at the site of bacterial entry, and are required for efficient bacterial uptake, as internalization is blocked upon targeted deletion of these kinases or treatment with a specific pharmacological inhibitor. We identify the adapter protein Crk as a target for Abl kinases during Shigella uptake, and show that a phosphorylation-deficient Crk mutant significantly inhibits bacterial uptake. Moreover, we define a novel signaling pathway activated during Shigella entry that links Abl kinase phosphorylation of Crk to activation of the Rho family GTPases Rac and Cdc42. Together, these findings reveal a new role for the Abl kinases, and suggest a novel approach to treatment of Shigella infections through inhibition of host cell signaling pathways

Abl kinase activity is required for VEGF- and thrombin-induced disruption of endothelial adherens junctions.

<p>(<b>A</b>) Staining of HMVEC monolayers for the adherens junction marker VE-cadherin (green) following treatment with VEGF (100ng/mL, 30 minutes), with or without imatinib pre-treatment (10μM). (<b>B</b>) VE-cadherin staining (red) of HMVEC monolayers treated with thrombin (1U/mL, 5 minutes), +/- imatinib. (<b>C</b>) VE-cadherin staining (red) of VEGF or thrombin-treated HMVECs, with or without GNF-2 pre-treatment (15μM). VEGF and thrombin treatment induced formation of inter-endothelial cell gaps (arrows) and destabilization of endothelial cell-cell junctions (“zig-zag” VE-cadherin staining pattern, arrowheads), which were reduced by pre-treatment with Abl kinase inhibitors.</p

Loss of Abl kinase function decreased endothelial barrier dysfunction <i>in</i><i>vitro</i>.

<p>(<b>A</b>) Evaluation of endothelial monolayer permeability, as assessed by passage of fluorescein-labeled dextran (molecular weight 40kDa) through HMVEC monolayers grown on Transwells, following treatment with VEGF (100ng/mL) with or without imatinib pre-treatment (10μM). Data shown are mean fluorescence of samples collected from bottom Transwell chambers at the indicated times post-VEGF treatment, +/- SD of three replicates per treatment. Data are representative of 3 independent experiments. (<b>B</b>) Quantification of inhibition of endothelial monolayer permeability to fluorescein-labeled dextran by imatinib (10μM) or GNF-2 (15μM). Endothelial barrier dysfunction was induced by treatment of HMVECs with VEGF (100ng/mL, 120 minutes), thrombin (1U/mL, 30 minutes) or histamine (100μM, 60 minutes). Values are expressed relative to permeability induction in vehicle-treated cells (UT). Data are presented as means +/- SEM (n=3). (<b>C</b>) Quantification of VEGF-induced endothelial permeability of HMVECs expressing either control or Abl miRNAs, with or without re-expression of miRNA-resistant, wild-type murine Abl (mAbl-WT). Values are expressed relative to VEGF-induced permeability in control miRNA-expressing cells. Data are presented as means +/- SEM (n=3). (<b>D</b>) Assessment of Abl protein levels following miRNA expression in HMVECs, with or without re-expression of miRNA-resistant Abl. *P<0.05; **P<0.01; ***P<0.001.</p

Abl Kinases Regulate Actin Comet Tail Elongation via an N-WASP-Dependent Pathway

Microbial pathogens have evolved diverse strategies to modulate the host cell cytoskeleton to achieve a productive infection and have proven instrumental for unraveling the molecular machinery that regulates actin polymerization. Here we uncover a mechanism for Shigella flexneri-induced actin comet tail elongation that links Abl family kinases to N-WASP-dependent actin polymerization. We show that the Abl kinases are required for Shigella actin comet tail formation, maximal intracellular motility, and cell-to-cell spread. Abl phosphorylates N-WASP, a host cell protein required for actin comet tail formation, and mutation of the Abl phosphorylation sites on N-WASP impairs comet tail elongation. Furthermore, we show that defective comet tail formation in cells lacking Abl kinases is rescued by activated forms of N-WASP. These data demonstrate for the first time that the Abl kinases play a role in the intracellular motility and intercellular dissemination of Shigella and uncover a new role for Abl kinases in the regulation of pathogen motility

Abl kinases are required for VEGF-induced vascular permeability <i>in</i><i>vivo</i>.

<p>(<b>A</b>) Evaluation of vascular leakage of Evans blue dye in mice following intradermal injection of VEGF (100ng, 15 minutes) with or without concomitant treatment with imatinib or GNF-2 (15μM). Dye extravasation was normalized to tissue weight. Values are presented as means +/- SD (n=12). (<b>B</b>) Quantification of VEGF-induced dermal vascular leakage of Evans blue dye in <i>Abl</i>^<i>ECKO</i><i>; Arg</i>^<i>+/-</i> (Abl^flox/flox; Arg^+/-; Tie2-Cre^+/-) and age/sex-matched Arg^+/- control mice (Abl^flox/flox; Arg^+/-; Tie2-Cre^-/-). Dye extravasation was normalized to tissue weight. Values are presented as means +/- SD (Arg^+/- controls, n=8; <i>Abl</i>^<i>ECKO</i><i>; Arg</i>^<i>+/-</i>, n=6). *P<0.05; **P<0.01.</p