Abl kinases are required for invadopodia formation and chemokine-induced invasion

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1α, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.     

Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering,Tubulogenesis and Motility

Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling.     

Accelerated clearance of Escherichia coli in experimental peritonitis of histamine-deficient mice

IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5Ralpha is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5Ralpha in eosinophils. To identify IL-5Ralpha-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5Ralpha under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5Ralpha. Lyn kinase phosphorylates both IL-5Ralpha and betacR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn(-/-) mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5Ralpha signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.     

Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.     

Structure-Function Analysis of Lyn Kinase Association with Lipid Rafts and Initiation of Early Signaling Events after Fcɛ Receptor I Aggregation

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.     

How do Abl family kinases regulate cell shape and movement?

Genetic analysis and studies of normal and leukemia cells in culture have shown that Abl family nonreceptor tyrosine kinases regulate cell morphogenesis and motility. Abl family kinases, which include Drosophila (D-) Abl and the vertebrate Abl and Arg proteins, relay signals from cell surface growth-factor and adhesion receptors to promote cytoskeletal rearrangements. Recent biochemical and crystallographic analyses have clarified the mechanisms by which growth-factor and adhesion receptors might regulate the activity of Abl family kinases. When activated, Abl family kinases can regulate cytoskeletal dynamics by phosphorylating several known cytoskeletal regulatory proteins. In addition, the C-terminal half of Abl family kinases has several domains that bind to cytoskeletal components. Emerging evidence suggests that Abl family kinases can use these domains to directly organize cytoskeletal structure in vivo.     

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 Family Kinases Regulate Endothelial Barrier Function In Vitro and in Mice

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 Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. 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.     

Activation of Lyn Tyrosine Kinase through Decreased Membrane Cholesterol Levels during a Change in Its Membrane Distribution upon Cell Detachment

Cellular membranes, which can serve as scaffolds for signal transduction, dynamically change their characteristics upon cell detachment. Src family kinases undergo post-translational lipid modification and are involved in a wide range of signaling events at the plasma membrane, such as cell proliferation, cell adhesion, and survival. Previously, we showed the differential membrane distributions among the members of Src family kinases by sucrose density gradient fractionation. However, little is known about the regulation of the membrane distribution of Src family kinases upon cell detachment. Here, we show that cell detachment shifts the main peak of the membrane distribution of Lyn, a member of Src family kinase, from the low density to the high density membrane fractions and enhances the kinase activity of Lyn. The change in Lyn distribution upon cell detachment involves both dynamin activity and a decrease in membrane cholesterol. Cell detachment activates Lyn through decreased membrane cholesterol levels during a change in its membrane distribution. Furthermore, cholesterol incorporation decreases Lyn activity and reduces the viability of suspension cells. These results suggest that cell detachment-induced Lyn activation through the change in the membrane distribution of Lyn plays an important role in survival of suspension cells.     

Reciprocal regulation of Abl and receptor tyrosine kinases

Previously, we showed that Abl kinases (c-Abl, Arg) are activated downstream of PDGF in a manner dependent on Src kinases and PLC-γ1, and promote PDGF-mediated proliferation and migration of fibroblasts. We additionally demonstrated that Abl kinases bind directly to PDGFR-β via their SH2 domains. In this study, we extend these findings by demonstrating that Abl kinases also are activated downstream of a PDGF autocrine growth loop in glioblastoma cells, indicating that the PDGFR-Abl signaling pathway also is likely to be important in glioblastoma development and/or progression. We recently showed that Abl kinases are highly active in many breast cancer cell lines, and the Her-2 receptor tyrosine kinase contributes to c-Abl and Arg kinase activation. In this study, we show that Abl kinase SH2 domains bind directly to Her-2, and like PDGFR-β, Her-2 directly phosphorylates c-Abl. Previously, we demonstrated that PDGFR-β directly phosphorylates Abl kinases in vitro, and Abl kinases reciprocally phosphorylate PDGFR-β. Here, we show that PDGFR-β-phosphorylation of Abl kinases has functional consequences as PDGFR-β phosphorylates Abl kinases on Y245 and Y412, sites known to be required for activation of Abl kinases. Moreover, PDGFR-β phosphorylates Arg on two additional unique sites whose function is unknown. Importantly, we also show that Abl-dependent phosphorylation of PDGFR-β has functional and biological significances. c-Abl phosphorylates three tyrosine residues on PDGFR-β (Y686, Y934, Y970), while Arg only phosphorylates Y686. Y686 and Y934 reside in PDGFR-β catalytic domains, while Y970 is in the C-terminal tail. Using site-directed mutagenesis, we show that Abl-dependent phosphorylation of PDGFR-β activates PDGFR-β activity, in vitro, but serves to downregulate PDGFR-mediated chemotaxis. These data are exciting as they indicate that Abl kinases not only are activated by PDGFR and promote PDGFR-mediated proliferation and migration, but also act in an intricate negative feedback loop to turn-off PDGFR-mediated chemotaxis.     

Abl kinases regulate autophagy by promoting the trafficking and function of lysosomal components

Autophagy is a lysosome-dependent degradative pathway that regulates the turnover of intracellular organelles, parasites, and long-lived proteins. Deregulation of autophagy results in a variety of pathological conditions, but little is known regarding the mechanisms that link normal cellular and pathological signals to the regulation of distinct stages in the autophagy pathway. Here we uncover a novel role for the Abl family kinases in the regulation of the late stages of autophagy. Inhibition, depletion, or knockout of the Abl family kinases, Abl and Arg, resulted in a dramatic reduction in the intracellular activities of the lysosomal glycosidases alpha-galactosidase, alpha-mannosidase and neuraminidase. Inhibition of Abl kinases also reduced the processing of the precursor forms of cathepsin D and cathepsin L to their mature, lysosomal forms, which coincided with the impaired turnover of long-lived cytosolic proteins and accumulation of autophagosomes. Furthermore, defective lysosomal degradation of long-lived proteins in the absence of Abl kinase signaling was accompanied by a perinuclear redistribution of lysosomes and increased glycosylation and stability of lysosome-associated membrane proteins, which are known to be substrates for lysosomal enzymes and play a role in regulating lysosome mobility. Our findings reveal a role for Abl kinases in the regulation of late-stage autophagy and have important implications for therapies that employ pharmacological inhibitors of the Abl kinases.     

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.     

Two distinct phosphorylation pathways have additive effects on Abl family kinase activation

The activities of the related Abl and Arg nonreceptor tyrosine kinases are kept under tight control in cells, but exposure to several different stimuli results in a two- to fivefold stimulation of kinase activity. Following the breakdown of inhibitory intramolecular interactions, Abl activation requires phosphorylation on several tyrosine residues, including a tyrosine in its activation loop. These activating phosphorylations have been proposed to occur either through autophosphorylation by Abl in trans or through phosphorylation of Abl by the Src nonreceptor tyrosine kinase. We show here that these two pathways mediate phosphorylation at distinct sites in Abl and Arg and have additive effects on Abl and Arg kinase activation. Abl and Arg autophosphorylate at several sites outside the activation loop, leading to 5.2- and 6.2-fold increases in kinase activity, respectively. We also find that the Src family kinase Hck phosphorylates the Abl and Arg activation loops, leading to an additional twofold stimulation of kinase activity. The autoactivation pathway may allow Abl family kinases to integrate or amplify cues relayed by Src family kinases from cell surface receptors.     

Crystal structures of the Lyn protein tyrosine kinase domain in its Apo- and inhibitor-bound state

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its "active" conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.     

Involvement of Golgi-associated Lyn tyrosine kinase in the translocation of annexin II to the endoplasmic reticulum under oxidative stress

Src-family tyrosine kinases, known to participate in signaling pathways of a variety of receptors at the plasma membrane, are found in cellular endomembranes such as the Golgi apparatus and endosomes. Recently, we showed that Lyn, a member of the Src kinases, accumulates on the Golgi apparatus and then traffics to the plasma membrane. We show here that a majority of endogenous Lyn but not c-Src is accumulated in Golgi-enriched heavy-membrane fractions on a sucrose-density gradient, whereas a small amount of endogenous Lyn is present in light-membrane fractions containing the plasma membrane. Inducible expression of kinase-active Lyn, which biosynthetically reaches the Golgi apparatus, triggers tyrosine phosphorylation of proteins including annexin II. Coimmunoprecipitation analyses reveal that Lyn physically associates with annexin II, and an in vitro kinase assay shows that Lyn phosphorylates annexin II directly. Furthermore, stimulation of cells with H2O2 induces tyrosine phosphorylation of annexin II on the Golgi apparatus in a manner that is dependent on the kinase activity of Src kinases, leading to the translocation of annexin II from the Golgi apparatus to the endoplasmic reticulum. Thus, these results suggest that endomembranes containing the Golgi apparatus where Lyn is anchored can serve as a signaling platform under oxidative stress.     

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.     

Src和Abl酪氨酸蛋白激酶家族参与病原微生物感染的研究进展

Src和Abl家族激酶属于非受体型酪氨酸激酶(Nonreceptor tyrosine kinase,NRTK)家族重要成员,广泛存在于各种细胞中,参与细胞内信号传递并调节细胞生理过程,它们在维持细胞、组织和器官稳态功能中发挥着至关重要的作用。研究表明,Src和Abl家族激酶通过多种机制参与病原微生物的感染(如与病原微生物的脯氨酸基序-PXXP互作)。因此,从Src和Abl家族激酶角度出发探究病原微生物感染机制逐渐成为一个热点。本文就Src和Abl家族激酶的结构特点以及参与病原微生物感染的研究报道进行综述,以期为病原微生物感染的致病机制、防控和药物研发提供参考。