Abelson Tyrosine Kinase Research Articles

E. coli sabotages the in vivo production of O-linked β-N-acetylglucosamine-modified proteins

The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.

Distinct functional domains of the Abelson tyrosine kinase control axon guidance responses to Netrin and Slit to regulate the assembly of neural circuits

To develop a functional nervous system, axons must initially navigate through a complex environment, directed by guidance ligands and receptors. These receptors must link to intracellular signaling cascades to direct axon pathfinding decisions. The Abelson tyrosine kinase (Abl) plays a crucial role in multiple Drosophila axon guidance pathways during development, though the mechanism by which Abl elicits a diverse set of guidance outputs is currently unknown. We identified Abl in a genetic screen for genes that contribute to Netrin-dependent axon guidance in midline-crossing (commissural) neurons. We find that Abl interacts both physically and genetically with the Netrin receptor Frazzled, and that disrupting this interaction prevents Abl from promoting midline axon crossing. Moreover, we find that Abl exerts its diverse activities through at least two different mechanisms: (1) a partly kinase-independent, structural function in midline attraction through its C-terminal F-actin binding domain (FABD) and (2) a kinase-dependent inhibition of repulsive guidance pathways that does not require the Abl C terminus. Abl also regulates motor axon pathfinding through a non-overlapping set of functional domains. These results highlight how a multifunctional kinase can trigger diverse axon guidance outcomes through the use of distinct structural motifs.

Electrochemical investigations into kinase-catalyzed transformations of tau protein.

The formation of neurofibrillary tangles by hyperphosphorylated tau is a well-recognized hallmark of Alzheimer's disease. Resulting from malfunctioning protein kinases, hyperphosphorylated tau is unable to bind microtubules properly, causing it to self-associate and aggregate. The effects of tau phosphorylation on tau conformation and aggregation are still largely unexplored. The conformational analysis of tau and its hyperphosphorylated forms is usually performed by a variety of spectroscopic techniques, all of which require ample sample concentrations and/or volumes. Here we report on the use of surface based electrochemical techniques that allow for detection of conformational changes and orientation of tau protein as a function of tau phosphorylation by tyrosine and serine/threonine kinases. The electrochemical methods utilize 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative as a cosubstrate and tau immobilized on gold surface to probe the role of the following protein kinases: Sarcoma related kinase (Src), Abelson tyrosine kinase (Abl), tau-tubulin kinase (TTBK), proto-oncogene tyrosine protein kinase Fyn (Fyn), and glycogen synthase kinase 3-β (Gsk-3β). The single kinase and sequential kinase-catalyzed Fc-phosphorylations modulate the electrochemical signal, pointing to the dramatic changes around the Fc group in the Fc-phosphorylated tau films. The location and orientation of the Fc-group in Fc-tau film was investigated by the surface plasmon resonance based on antiferrocene antibodies. Additional surface characterization of the Fc-tau films by time-of-flight secondary ion-mass spectrometry and X-ray photoelectron spectroscopy revealed that Fc-phosphorylations influence the tau orientation and conformation on surfaces. When Fc-phosphorylations were performed in solution, the subsequently immobilized Fc-tau exhibited similar trends. This study illustrates the validity and the utility of the labeled electrochemical approach for probing the changes in protein film properties, conformation, and orientation as a function of the enzymatically catalyzed modifications.

Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries

Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library. Cleavage of the substrate fusion protein by the protease eliminates the ER retention sequence, facilitating transport to the yeast surface. Yeast cells that display Aga2 fusions in which only the selection substrate is cleaved are isolated by multicolor FACS with fluorescently labeled antiepitope tag antibodies. Using this system, the Tobacco Etch Virus protease (TEV-P), which strongly prefers Gln at P1 of its canonical ENLYFQ↓S substrate, was engineered to recognize selectively Glu or His at P1. Kinetic analysis indicated an overall 5,000-fold and 1,100-fold change in selectivity, respectively, for the Glu- and His-specific TEV variants, both of which retained high catalytic turnover. Human granzyme K and the hepatitis C virus protease were also shown to be amenable to this unique approach. Further, by adjusting the signaling strategy to identify phosphorylated as opposed to cleaved sequences, this unique system was shown to be compatible with the human Abelson tyrosine kinase.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

The Abelson tyrosine kinase regulates Notch endocytosis and signaling to maintain neuronal cell fate inDrosophilaphotoreceptors

The development of a functional organ requires coordinated programs of cell fate specification, terminal differentiation and morphogenesis. Whereas signaling mechanisms that specify individual cell fates are well documented, little is known about the pathways and molecules that maintain these fates stably as normal development proceeds or how their dysregulation may contribute to altered cell states in diseases such as cancer. In Drosophila, the tyrosine kinase Abelson (Abl) interfaces with multiple signaling pathways to direct epithelial and neuronal morphogenesis during embryonic and retinal development. Here we show that Abl is required for photoreceptor cell fate maintenance, as Abl mutant photoreceptors lose neuronal markers during late pupal stages but do not re-enter a proliferative state or undergo apoptosis. Failure to maintain the differentiated state correlates with impaired trafficking of the Notch receptor and ectopic Notch signaling, and can be suppressed by reducing the genetic dose of Notch or of its downstream transcriptional effector Suppressor of Hairless. Together, these data reveal a novel mechanism for maintaining the terminally differentiated state of Drosophila photoreceptors and suggest that neuronal fates in the fly retina retain plasticity late into development. Given the general evolutionary conservation of developmental signaling mechanisms, Abl-mediated regulation of Notch could be broadly relevant to cell fate maintenance and reprogramming during normal development, regeneration and oncogenic transformation.

Raf-1, Actin Dynamics, and Abelson Tyrosine Kinase in Human Airway Smooth Muscle Cells

Raf-1 is a serine/threonine protein kinase that has an essential role in cell proliferation. The mechanisms that regulate Raf-1 in airway smooth muscle are not well understood. In this study, treatment with platelet-derived growth factor (PDGF) induced spatial redistribution of Raf-1 from the cytoplasm to the periphery of human airway smooth muscle cells. Moreover, a pool of Raf-1 was found in F-actin of human airway smooth muscle cells. Activation with PDGF led to an increase in the association of Raf-1 with cytoskeletal actin. Treatment of cells with the actin polymerization inhibitor latrunculin A (LAT-A), but not the microtubule depolymerizer nocodazole, inhibited the interaction of Raf-1 with actin in response to PDGF activation. Because abelson tyrosine kinase (Abl) is known to specifically regulate actin dynamics in smooth muscle, the role of Abl in modulating the coupling of Raf-1 with actin was also evaluated. Abl knockdown by RNA interference attenuated the association of Raf-1 with actin, which is recovered by Abl rescue. Treatment with LAT-A, but not nocodazole, inhibited the spatial redistribution of Raf-1 during PDGF activation. However, treatment with both LAT-A and nocodazole attenuated smooth muscle cell proliferation. Finally, Abl knockdown attenuated the redistribution of Raf-1 and cell proliferation, which were restored by Abl reexpression. The results suggest a novel mechanism that the interaction of Raf-1 with cytoskeletal actin is critical for Raf-1 redistribution and airway smooth muscle cell proliferation during activation with the growth factor.

Abelson Tyrosine Kinase Controls Phagosomal Acidification Required for Killing of Mycobacterium tuberculosis in Human Macrophages

The mechanisms that regulate the acidification of intracellular compartments are key to host defense against pathogens. In this paper, we demonstrate that Abl tyrosine kinase, a master switch for cell growth and trafficking of intracellular organelles, controls the acidification of lysosomes in human macrophages. Pharmacological inhibition by imatinib and gene silencing of Abelson (Abl) tyrosine kinase reduced the lysosomal pH in human macrophages by increasing the transcription and expression of the proton pumping enzyme vacuolar-type H(+)-adenosine triphosphatase. Because lysosomal acidification is required for antimicrobial activity against intracellular bacteria, we determined the effect of imatinib on the growth of the major human pathogen Mycobacterium tuberculosis. Imatinib limited the multiplication of M. tuberculosis, and growth restriction was dependent on acidification of the mycobacterial compartment. The effects of imatinib were also active in vivo because circulating monocytes from imatinib-treated leukemia patients were more acidic than monocytes from control donors. Importantly, sera from imatinib-treated patients triggered acidification and growth restriction of M. tuberculosis in macrophages. In summary, our results identify the control of phagosomal acidification as a novel function of Abl tyrosine kinase and provide evidence that the regulation occurs on the level of the vacuolar-type H(+)-adenosine triphosphatase. Given the efficacy of imatinib in a mouse model of tuberculosis and our finding that orally administered imatinib increased the ability of human serum to trigger growth reduction of intracellular M. tuberculosis, clinical evaluation of imatinib as a complementary therapy of tuberculosis, in particular multidrug or extremely drug-resistant disease, is warranted.

Discovery of Tyrosine Kinase Inhibitors by Docking into an Inactive Kinase Conformation Generated by Molecular Dynamics

Several small molecules that bind to the inactive DFG-out conformation of tyrosine kinases (called type II inhibitors) have shown a good selectivity profile over other kinase targets. To obtain a set of DFG-out structures, we performed an explicit solvent molecular dynamics (MD) simulation of the complex of the catalytic domain of a tyrosine kinase receptor, ephrin type-A receptor 3 (EphA3), and a manually docked type II inhibitor. Automatic docking of four previously reported type II inhibitors was used to select a single snapshot from the MD trajectory for virtual screening. High-throughput docking of a pharmacophore-tailored library of 175,000 molecules resulted in about 4 million poses, which were further filtered by van der Waals efficiency and ranked according to a force-field-based energy function. Notably, around 20 % of the compounds with predicted binding energy smaller than -10 kcal mol(-1) are known type II inhibitors. Moreover, a series of 5-(piperazine-1-yl)isoquinoline derivatives was identified as a novel class of low-micromolar inhibitors of EphA3 and unphosphorylated Abelson tyrosine kinase (Abl1). The in silico predicted binding mode of the new inhibitors suggested a similar affinity to the gatekeeper mutant T315I of Abl1, which was verified in vitro by using a competition binding assay. Additional evidence for the type II binding mode was obtained by two 300 ns MD simulations of the complex between N-(3-chloro-4-(difluoromethoxy)phenyl)-2-(4-(8-nitroisoquinolin-5-yl)piperazin-1-yl)acetamide and EphA3.

Direct Binding Assay for the Detection of Type IV Allosteric Inhibitors of Abl

Abelson (Abl) tyrosine kinase is an important cellular enzyme that is rendered constitutively active in the breakpoint cluster region (BCR)-Abl fusion protein, contributing to several forms of leukemia. Although inhibiting BCR-Abl activity with imatinib shows great clinical success, many patients acquire secondary mutations that result in resistance to imatinib. Second-generation inhibitors such as dasatinib and nilotinib can overcome the majority of these mutations but fail to treat patients with an especially prevalent T315I mutation at the gatekeeper position of the kinase domain. However, a combination of nilotinib with an allosteric type IV inhibitor was recently shown to overcome this clinically relevant point mutation. In this study, we present the development of a direct binding assay that enables the straightforward detection of allosteric inhibitors which bind within the myristate pocket of Abl. The assay is amenable to high-throughput screening and exclusively detects the binding of ligands to this unique allosteric site.

Preclinical and clinical studies of estrogen deprivation support the PDGF/Abl pathway as a novel therapeutic target for overcoming endocrine resistance in breast cancer

IntroductionThe majority of breast tumors at primary diagnosis are estrogen receptor positive (ER+). Estrogen (E) mediates its effects by binding to the ER. Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ breast cancer. However, resistance remains a major clinical problem.MethodsTo identify molecular mechanisms associated with resistance to E-deprivation, we assessed the temporal changes in global gene expression during adaptation to long-term culture of MCF7 human breast cancer cells in the absence of estradiol (E2), long term estrogen deprived (LTED), that leads to recovery of proliferative status and models resistance to an aromatase inhibitor (AI). The expression levels of proteins were determined by western blotting. Proliferation assays were carried out using the dual platelet derived growth factor receptor (PDGFR)/Abelson tyrosine kinase (Abl) inhibitor nilotinib. Luciferase reporter assays were used to determine effects on ER-mediated transactivation. Changes in recruitment of cofactors to the gene regulated by estrogen in breast cancer 1 (GREB1) promoter were determined by chromatin immunoprecipitation (ChIP). Gene expression data were derived from 81 postmenopausal women with ER+ BC pre-treatment and at two-weeks post-treatment with single agent anastrozole in a neoadjuvant trial.ResultsThe PDGF/Abl canonical pathway was significantly elevated as early as one week post E-deprivation (P = 1.94 E-04) and this became the top adaptive pathway at the point of proliferative recovery (P = 1.15 E-07). Both PDGFRβ and Abl protein levels were elevated in the LTED cells compared to wild type (wt)-MCF7 cells. The PDGF/Abl tyrosine kinase inhibitor nilotinib, suppressed proliferation in LTED cells in the presence or absence of E. Nilotinib also suppressed ER-mediated transcription by destabilizing the ER and reducing recruitment of amplified in breast cancer-1 (AIB1) and the CREB binding protein (CBP) to the promoter of the E-responsive gene GREB1. High PDGFRβ in primary ER+ breast cancer of 81 patients prior to neoadjuvant treatment with an AI was associated with poorer antiproliferative response. Additionally PDGFRβ expression increased after two weeks of AI therapy (1.25 fold, P = 0.003).ConclusionsThese preclinical and clinical data indicate that the PDGF/Abl signaling pathway merits clinical evaluation as a therapeutic target with endocrine therapy in ER+ breast cancer.

Philadelphia chromosome-positive leukemia stem cells in acute lymphoblastic leukemia and tyrosine kinase inhibitor therapy

Leukemia stem cells (LSCs), which constitute a minority of the tumor bulk, are functionally defined on the basis of their ability to transfer leukemia into an immunodeficient recipient animal. The presence of LSCs has been demonstrated in acute lymphoblastic leukemia (ALL), of which ALL with Philadelphia chromosome-positive (Ph(+)). The use of imatinib, a tyrosine kinase inhibitor (TKI), as part of front-line treatment and in combination with cytotoxic agents, has greatly improved the proportions of complete response and molecular remission and the overall outcome in adults with newly diagnosed Ph(+) ALL. New challenges have emerged with respect to induction of resistance to imatinib via Abelson tyrosine kinase mutations. An important recent addition to the arsenal against Ph(+) leukemias in general was the development of novel TKIs, such as nilotinib and dasatinib. However, in vitro experiments have suggested that TKIs have an antiproliferative but not an antiapoptotic or cytotoxic effect on the most primitive ALL stem cells. None of the TKIs in clinical use target the LSC. Second generation TKI dasatinib has been shown to have a more profound effect on the stem cell compartment but the drug was still unable to kill the most primitive LSCs. Allogeneic stem cell transplantation (SCT) remains the only curative treatment available for these patients. Several mechanisms were proposed to explain the resistance of LSCs to TKIs in addition to mutations. Hence, TKIs may be used as a bridge to SCT rather than monotherapy or combination with standard chemotherapy. Better understanding the biology of Ph(+) ALL will open new avenues for effective management. In this review, we highlight recent findings relating to the question of LSCs in Ph(+) ALL.

Nilotinib, in Comparison to Both Dasatinib and Imatinib, Possesses a Greatly Prolonged Residence Time When Bound to the BCR-ABL Kinase SH1 Domain

Abstract 1674 Background:Imatinib, nilotinib and dasatinib are effective therapies for chronic myeloid leukemia, as a consequence of their inhibiting the Abelson tyrosine kinase activity of the BCR-ABL oncoprotein. Their rank-order of potency in assays assessing either effects on BCR-ABL autophosphorylation or cell proliferation is dasatinib > nilotinib > imatinib. However, such quantitative in vitro differences do not directly translate into pharmacological differences due to other factors (Laneuville et al. JCO 2010;28: e169), such as pharmacokinetics (dasatinib, nilotinib and imatinib have plasma half-lives in CML patients of 3–5, 19±6 and ≈20 hours respectively) and pharmacodynamics, including cell penetration and protein binding. Here we report the ABL binding kinetics for these drugs and discuss how the data can be related to target occupancy and the pharmacodynamic properties of the drugs. Methods:Active phosphorylated ABL (residues 118–535 of SH1 domain) was purified from insect cells and was treated with shrimp alkaline phosphatase to afford the dephosphorylated form. Drug binding kinetics were studied using the Proteros reporter displacement assay. This utilises reporter probes that bind to the target protein, with the proximity between reporter and protein resulting in the emission of an optical signal, so that compounds which bind to the same site as the reporter probe displace the probe, causing signal diminution. The rate of reporter displacement is measured over time after addition of compounds at various concentrations. Results and Conclusions:The kinetic parameters for the interaction between the drugs and either phosphorylated or dephosporylated ABL are tabulated below. The binding affinity (KD) is related to the association rate constant (kon) and the dissociation rate constant (koff) by KD = koff / kon. The kon rates (dasatinib > imatinib > nilotinib) reflect that dasatinib recognises the phosphorylated, catalytically-active resting-state conformation of ABL (DGF-in; Tokarski et al. Cancer Res 2006;66: 5790 and Vajpai et al. JBC 2008;283: 18292), whereas the other drugs bind to an inactive conformation (DFG-out) and this leads to dasatinib having the highest KD of the three drugs. However, in terms of their Koff rates, the rank order of activity is reversed, reflecting the strong binding of nilotinib to the DFG-out conformation and the thermodynamic stability of the ABL-nilotinib complex. The stability of the complexes between the drugs and ABL protein is quantifiable through the half-life of the complexes (residence times) and for nilotinib is over 3 hours.It is generally recognised that prolonged residence-times for drugs bound to their pharmacologically-relevant protein target, can uncouple pharmacodynamic target inhibition from tissue level pharmacokinetics. Furthermore, high off-rate selectivity can minimise the side-effects of drugs caused by off-target binding, which is reduced in the case of drugs with high target residence times. Thus this kinetic analysis can provide an explanation for the high efficacy of nilotinib in CML patients, at doses that cause relatively few side-effects. [Display omitted] Disclosures:Manley:Novartis Pharma AG: Employment. Cowan-Jacob:Novartis Pharma AG: Employment. Fendrich:Novartis Pharma AG: Employment. Jahnke:Novartis Pharma AG: Employment. Fabbro:Novartis Pharma AG: Employment.

The Drosophila Hem/Kette/Nap1 protein regulates asymmetric division of neural precursor cells by regulating localization of Inscuteable and Numb

The Hem/Kette/Nap1 protein is involved in many biological processes. We have recently reported that Hem is required for the normal migration of neurons in the Drosophila embryo. In this paper, we report that Hem regulates the asymmetric division of neural precursor cells. We find that a well-studied Hem/Kette mutant allele produces at least two main, but possibly more, phenotypic classes of mutant embryos, and these phenotypes correlate with variable levels of maternal wild type Hem protein in the developing embryo. While the weaker class exhibits weak axon guidance defect and the mis-migration of neurons, the stronger class causes severe axon guidance defects, mis-migration of neurons and symmetric division of ganglion mother cells (GMC) of the RP2/sib lineage. We also show that the basis for the loss of asymmetric division is due to non-localization of Inscuteable and Numb in GMC-1. A non-asymmetric Numb segregates to both daughter cells of GMC-1, which then prevents Notch signaling from specifying a sib fate. This causes both cells to adopt an RP2 fate. Furthermore, loss of function for Abelson tyrosine kinase also causes loss of asymmetric localization of Inscuteable and Numb and symmetric division of GMC-1, the loss of function for WAVE has a very weakly penetrant loss of asymmetry defect. These results define another role for Hem/Kette/Nap1 in a neural precursor cell during neurogenesis.

The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase

In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. We have previously shown that RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. Here, we show that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at al and fail to send out its axon projection. We report that Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, our data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. We also find that Abelson tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. Our results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl.

Abelson tyrosine kinase is required for Drosophila photoreceptor morphogenesis and retinal epithelial patterning

Coordinated differentiation and morphogenesis transform the Drosophila retina from a layer of epithelial cells into a complex three-dimensional organ. In this study we show that the Abelson (Abl) tyrosine kinase localizes to the dynamically remodeling apical-junctional membrane domains of the developing photoreceptor cells. Analyses of abl mutant clone phenotypes demonstrate that abl is required for enriched localization of adherens junction and apical polarity complex proteins at photoreceptor-photoreceptor cell junctions and apical membrane domains, respectively, for rhabdomere generation and for spatial organization of ommatidial cells along the apical-basal axis of the epithelium. Loss of abl does not alter expression or localization of Enabled (Ena) nor does heterozygosity for ena dominantly suppress the abl phenotypes, suggesting the downstream effector mechanisms used by Abl in the eye may differ from those used in the embryo. Together our results reveal a prominent role for Abl in coordinating multiple aspects of photoreceptor morphogenesis.

Neuronal c-Abl Overexpression Leads to Neuronal Loss and Neuroinflammation in the Mouse Forebrain

Several immunocytochemical studies have revealed that Abelson tyrosine kinase (c-Abl) is associated with both neuritic plaques and neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). Additionally, c-Abl has been shown to phosphorylate tau on tyrosine 394. The activity of c-Abl is also involved in the control of the cell cycle and apoptosis. To examine the consequences of c-Abl activation in the adult brain, we have constructed two lines of transgenic mice expressing either a constitutively active form of c-Abl (AblPP/tTA mice) or its sister protein, Arg (ArgPP/tTA mice), with a neuron-specific promoter (CamKIIα) regulated by doxycycline (Tet-Off). Expression of active c-Abl in adult mouse forebrain neurons results in severe neurodegeneration, particularly in the CA1 region of the hippocampus. Neuronal loss was preceded and accompanied by substantial microgliosis and astrocyctosis. Despite careful examination, no c-Abl expression is found in glial cells, indicating that neuronal c-Abl expression is responsible for the gliosis. In contrast, ArgPP/tTA mice have no evidence of neuronal loss or gliosis, even though protein expression and kinase activity levels are similar to those in the AblPP/tTA mice. Given the evidence of c-Abl activation in the human AD brain combined with the pathological phenotype of AblPP/tTA mice, it is likely that aberrant c-Abl activity may play a role in neurodegenerative disease.

Abstract 2479: Detecting Abelson tyrosine kinase activation by ATM and DNA-PK after exposure to ionizing radiation using a peptide biosensor

Abstract Abelson (Abl) kinase is a non-receptor tyrosine kinase that is involved in many cellular processes, including survival, differentiation and apoptosis. Abl is activated in the response to DNA damage, and is involved in the decision between DNA repair versus apoptosis. It is widely accepted that Abl is regulated in response to DNA double-strand breaks (DSBs) by ataxia telangiectasia mutated protein (ATM) via phosphorylation of Abl at S465. However, other aspects of this complex signaling response have been unclear, including the role of other kinases such as DNA-dependent protein kinase (DNA-PK), because of the difficulties of detecting subtle changes in Abl activation through endogenous substrates. Here, we show Abl is activated in a mutant cell line that cannot be phosphorylated by ATM at S465 (Abl-S465A-EGFP) after exposure to ionizing radiation (IR) using a peptide biosensor we developed for Abl kinase. This biosensor contains the following functional modules: a highly specific Abl kinase substrate, an Abl SH3 binding ligand, a photocleavable linker, a biotin tag and a cell penetrating peptide derived from Hiv-TAT to aid intracellular delivery of cargo across the plasma membrane. Using a combination of chemical biology approaches, Western blot for the phosphorylation of the biosensor peptide and high-resolution MS-based phosphoproteomics, we identified a potential role for DNA-PK in activating Abl kinase after IR, independent of ATM and S465. This information about the importance of DNA-PK in this signaling pathway may lead to a better understanding of c-Abl's function in the response to IR, and could be useful to develop strategies to combat radioresistance or improve radiosensitivity in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2011-2479

The abelson tyrosine kinase (c-Abl) expression on the mouse uterus and placenta during gestational period

c-Abl is a protein tyrosine kinase which has very important roles in signal transduction, control of the cell cycle, cell motility, proliferation, and inhibition of apoptosis. We hypothesized that c-Abl may play an important role on uterine remodeling during pre-receptive, receptive and non-receptive endometrium. Our aim is to investigate the expression of c-Abl protein tyrosine kinase in uterine remodeling and placental development in mouse gestational stage. We performed c-Abl immunohistochemistry on mouse uterine tissue sections on days 1-9, 11, 13, and 15 of pregnancy. c-Abl was highly upregulated in the uterine luminal epithelium and other endometrial structures including glands and blood vessels in pre-receptive and receptive endometrium. Therefore these results demonstrate a role for c-Abl in uterine remodeling during decidualization, implantation, and placentation throughout gestation.

A spatially propagating biochemical reaction.

A spatially propagating biochemical reaction.