c-Abl tyrosine kinase interacts with MAVS and regulates innate immune response

The tyrosine kinase, c-Abl, plays important roles in many aspects of cellular function. Previous reports showed that c-Abl is involved in NF-κB signaling. However, the functions of c-Abl in innate immunity are still unknown. Here we demonstrate that the mitochondrial antiviral signaling (MAVS) protein can be physically associated with c-Abl in vivo and in vitro. MAVS interacted with c-Abl through its Card and TM domain. A phosphotyrosine-specific antibody indicated that MAVS was phosphorylated by c-Abl. Functional impairment of c-Abl attenuated MAVS or VSV induced type-I IFN production. Importantly, c-Abl knockdown in MCF7 cells displayed impaired MAVS-mediated NF-κB and IRF3 activation. Taken together, our results suggest that c-Abl modulates innate immune response through MAVS.

Structured summary

MINT-7297498, MINT-7297511, MINT-7297557, MINT-7297574: MAVS (uniprotkb:Q7Z434) physically interacts (MI:0915) with c-Abl (uniprotkb:P00519) by anti tag coimmunoprecipitation (MI:0007)MINT-7297542: c-Abl (uniprotkb:P00519) physically interacts (MI:0915) with MAVS (uniprotkb:Q7Z434) by anti bait coimmunoprecipitation (MI:0006)MINT-7297526: c-Abl (uniprotkb:P00519) physically interacts (MI:0915) with MAVS (uniprotkb:Q7Z434) by far western blotting (MI:0047)     

Proliferating cell nuclear antigen destabilizes c-Abl tyrosine kinase and regulates cell apoptosis in response to DNA damage

The tyrosine kinase, c-Abl, plays important roles in many aspects of cellular function. The activity of c-Abl is tightly controlled, but the underlying mechanism is unclear. Recent studies suggest that c-Abl function is regulated by distinct lipids in different cell types. In the present study, we show that the DNA replication factor, proliferating cell nuclear antigen (PCNA), interacts with c-Abl and destabilizes c-Abl by promoting its polyubiquitination and degradation. Moreover, deletion of a domain in c-Abl, the PIP box, disrupts its interaction with PCNA, abolishes the PCNA-induced degradation of nuclear c-Abl, and substantially increases the nuclear c-Abl apoptotic function. These findings indicate that PCNA negatively regulates the stability of c-Abl and thereby inhibits apoptosis in the response to DNA damage. Xiang He, Congwen Wei, and Ting Song contribute equally to this work. Wei Shi and Hui Zhong are co-correspondence authors.     

Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.     

c-Abl tyrosine kinase regulates caspase-9 autocleavage in the apoptotic response to DNA damage

Activation of the initiator caspase-9 is essential for induction of apoptosis by developmental signals, oncogenic transformation, and genotoxic stress. The c-Abl tyrosine kinase is also involved in the apoptotic response to DNA damage. The present results demonstrate that c-Abl binds directly to caspase-9. We show that c-Abl phosphorylates caspase-9 on Tyr-153 in vitro and in cells treated with DNA damaging agents. Moreover, inhibition of c-Abl with STI571 blocked DNA damage-induced autoprocessing of caspase-9 to the p35 subunit and activation of caspase-3. Caspase-9(Y153F) also attenuated DNA damage-induced processing of caspase-9 to p35, activation of caspase-3, and apoptosis. These findings indicate that caspase-9 autoprocessing is regulated by c-Abl in the apoptotic response to genotoxic stress.     

Phosphorylation of Bruton's tyrosine kinase by c-Abl

Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.     

c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage

The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage. The mechanisms by which c-Abl signals the induction of apoptosis are not understood. Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein. The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9. c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents. The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-x(L) protein. The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis. These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.     

c-Abl tyrosine kinase binds and phosphorylates phospholipid scramblase 1

Phospholipid scramblase 1 (PLSCR1) is a plasma membrane protein that has been proposed to play a role in the transbilayer movement of plasma membrane phospholipids. PLSCR1 contains multiple proline-rich motifs resembling Src homology 3 (SH3) domain-binding sites. An initial screen against 13 different SH3 domains revealed a marked specificity of PLSCR1 for binding to the Abl SH3 domain. Binding between intracellular PLSCR1 and c-Abl was demonstrated by co-immunoprecipitation of both proteins from several cell lines. Deletion of the proline-rich segment in PLSCR1 (residues 1--118) abolished its binding to the Abl SH3 domain. PLSCR1 was Tyr-phosphorylated by c-Abl in vitro. Phosphorylation was abolished by mutation of Tyr residues Tyr(69)/Tyr(74) within the tandem repeat sequence (68)VYNQPVYNQP(77) of PLSCR1, implying that these residues are the likely sites of phosphorylation. Cellular PLSCR1 was found to be constitutively Tyr-phosphorylated in several cell lines. The Tyr phosphorylation of PLSCR1 was increased upon overexpression of c-Abl and significantly reduced either upon cell treatment with the Abl kinase inhibitor STI571, or in Abl-/- mouse fibroblasts, suggesting that cellular PLSCR1 is a normal substrate of c-Abl. Cell treatment with the DNA-damaging agent cisplatin activated c-Abl kinase and increased Tyr phosphorylation of PLSCR1. The cisplatin-induced phosphorylation of PLSCR1 was inhibited by STI571 and was not observed in Abl-/- fibroblasts. These findings indicate that c-Abl binds and phosphorylates PLSCR1, and raise the possibility that an interaction between c-Abl and plasma membrane PLSCR1 might contribute to the cellular response to genotoxic stress.     

Inhibition of c-Abl tyrosine kinase activity by filamentous actin

The catalytic activity of c-Abl tyrosine kinase is reduced in fibroblasts that are detached from the extracellular matrix. We report here that a deletion of the extreme C terminus of c-Abl (DeltaF-actin c-Abl) can partially restore kinase activity to c-Abl from detached cells. Because the extreme C terminus of c-Abl contains a consensus F-actin binding motif, we investigated the effect of F-actin on c-Abl tyrosine kinase activity. We found that F-actin can inhibit the kinase activity of purified c-Abl protein. Mutations of the extreme C-terminal region of c-Abl disrupted both the binding of c-Abl to F-actin and the inhibition of c-Abl by F-actin. Mutations of the SH3, SH2, and DNA binding domains did not abolish the inhibition of c-Abl kinase by F-actin. Catalytic domain substitutions that affect the regulation of c-Abl by the retinoblastoma protein or the ataxia telangiectasia-mutated kinase also did not abolish the inhibition of c-Abl by F-actin. Interestingly, among these c-Abl mutants, only the DeltaF-actin c-Abl retained kinase activity in detached cells. Taken together, the data suggest that F-actin is an inhibitor of the c-Abl tyrosine kinase and that this inhibition contributes in part to the reduced Abl kinase activity in detached cells.     

Structural basis for the autoinhibition of c-Abl tyrosine kinase

c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks a phosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571) to inhibit the catalytic activity of Abl, but not that of c-Src.     

T cell survival and function requires the c-Abl tyrosine kinase

C-Abl (Abl) regulates multiple cellular processes, including proliferation, survival, shape determination and motility, and participates in cellular responses to genotoxic and oxidative stress stimuli. Mice lacking Abl exhibit retarded growth, osteoporosis and defects in the immune system resulting in lymphopoenia and susceptibility to infections, leading to early death. To define the role of Abl in the regulation of adult T cells we ablated Abl exclusively in T cells by generating mice with floxed abl alleles and expressing an Lck-Cre transgene (Abl-T-/-). These mice exhibited thymic atrophy and abnormally reduced T cell numbers in the periphery. The thymic atrophy was caused by increased susceptibility of thymocytes to cell death. Importantly, Abl deficient T cells displayed abnormally reduced response to mitogenic stimulation in vitro. Consequently, Abl-T-/- mice exhibited impaired ability to reject syngeneic tumor, to induce T-mediated tumor cell killing, and to generate anti-tumor antibodies. These results demonstrate a cell-autonomous role for Abl in T cell function and survival.     

c-Abl kinase regulates the protein binding activity of c-Crk.

c-Crk is a proto-oncogene product composed largely of Src homology (SH) 2 and 3 domains. We have identified a kinase activity, which binds to the first Crk SH3 domain and phosphorylates c-Crk on tyrosine 221 (Y221), as c-Abl. c-Abl has a strong preference for c-Crk, when compared with common tyrosine kinase substrates. The phosphorylation of c-Crk Y221 creates a binding site for the Crk SH2 domain. Bacterially expressed c-Crk protein lacks phosphorylation on Y221 and can bind specifically to several proteins, while mammalian c-Crk, which is phosphorylated on tyrosine, remains uncomplexed. The protein binding activity of c-Crk is therefore likely regulated by a mechanism similar to that of the Src family kinases. v-Crk is truncated before c-Crk Y221 and forms constitutive complexes with c-Abl and other proteins. Our results suggest that c-Abl regulates c-Crk function and that it could be involved in v-Crk transformation.     

c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.     

Activation of the cytoplasmic c-Abl tyrosine kinase by reactive oxygen species

The ubiquitously expressed c-Abl protein tyrosine kinase localizes to both the nucleus and cytoplasm. The nuclear form of c-Abl is activated in the cellular response to genotoxic stress. Here we show that cytoplasmic c-Abl is activated by oxidative stress. The results also demonstrate that mitochondrial cytochrome c is released in the cellular response to H(2)O(2) and that this effect is mediated by a c-Abl-dependent mechanism. In concert with these results, we show that H(2)O(2)-induced apoptosis is attenuated in c-Abl-deficient cells. These findings demonstrate that cytoplasmic c-Abl is involved in the apoptotic response of cells to oxidative stress.     

Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl

We investigated the mechanism of H(2)O(2) activation of the Ca(2+)-regulated NADPH oxidase NOX5. H(2)O(2) induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H(2)O(2) is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H(2)O(2). H(2)O(2) NOX5 activation was inhibited by extracellular Ca(2+) chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP-c-Abl colocalized with vesicular sites of superoxide production in a Ca(2+)-dependent manner. In contrast to H(2)O(2), the Ca(2+) ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H(2)O(2) treatment. Furthermore, H(2)O(2)-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP-Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca(2+)-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.     

p38 MAP kinase regulates rapid matrix metalloproteinase-9 release from eosinophils

Eosinophils constitutively produce and store matrix metalloproteinase-9 (MMP-9), a protease implicated in tissue remodeling observed in asthma. In this study, we examined the rapid release of stored MMP-9 from eosinophils following stimulation with either tumor necrosis factor-alpha (TNF-alpha or the bacterial product fMLP. TNF-alpha induced rapid and robust pro-MMP-9 release from eosinophils. MMP-9 could be detected in the cell-free supernatant as early as 15min after stimulation. Rapid MMP-9 release was similarly induced by fMLP. TNF-alpha stimulation activated the mitogen-activated protein (MAP) kinases p38 MAP kinase and extracellular signal-regulated kinase-2 (Erk-2) at times and concentrations similar to that observed for MMP-9 release. Using pharmacological inhibitors, we found that TNF-alpha-stimulated MMP-9 release was mediated by p38 MAP kinase, but not Erk-1/2. Signaling through p38 MAP kinase may represent a universal mechanism for MMP-9 release from eosinophils, as fMLP-induced MMP-9 release was also regulated by p38 MAP kinase.