Journal articles on the topic 'AHL Receptor Binding'
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1
Cui, Yaya, Asita Chatterjee, Hiroaki Hasegawa, and Arun K. Chatterjee. "Erwinia carotovora Subspecies Produce Duplicate Variants of ExpR, LuxR Homologs That Activate rsmA Transcription but Differ in Their Interactions with N-Acylhomoserine Lactone Signals." Journal of Bacteriology 188, no. 13 (July 1, 2006): 4715–26. http://dx.doi.org/10.1128/jb.00351-06.
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ABSTRACT The N-acylhomoserine lactone (AHL) signaling system comprises a producing system that includes acylhomoserine synthase (AhlI, a LuxI homolog) and a receptor, generally a LuxR homolog. AHL controls exoprotein production in Erwinia carotovora and consequently the virulence for plants. In previous studies we showed that ExpR, a LuxR homolog, is an AHL receptor and that it activates transcription of rsmA, the gene encoding an RNA binding protein which is a global negative regulator of exoproteins and secondary metabolites. An unusual finding was that the transcriptional activity of ExpR was neutralized by AHL. We subsequently determined that the genomes of most strains of E. carotovora subspecies tested possess two copies of the expR gene: expR1, which was previously studied, and expR2, which was the focus of this study. Comparative analysis of the two ExpR variants of E. carotovora subsp. carotovora showed that while both variants activated rsmA transcription, there were significant differences in the patterns of their AHL interactions, the rsmA sequences to which they bound, and their relative efficiencies of activation of rsmA transcription. An ExpR2− mutant produced high levels of exoproteins and reduced levels of RsmA in the absence of AHL. This contrasts with the almost complete inhibition of exoprotein production and the high levels of RsmA production in an AhlI− mutant that was ExpR1−. Our results suggest that ExpR2 activity is responsible for regulating exoprotein production primarily by modulating the levels of an RNA binding protein.
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Rajamani, Sathish, Wolfgang D. Bauer, Jayne B. Robinson, John M. Farrow, Everett C. Pesci, Max Teplitski, Mengsheng Gao, Richard T. Sayre, and Donald A. Phillips. "The Vitamin Riboflavin and Its Derivative Lumichrome Activate the LasR Bacterial Quorum-Sensing Receptor." Molecular Plant-Microbe Interactions® 21, no. 9 (September 2008): 1184–92. http://dx.doi.org/10.1094/mpmi-21-9-1184.
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Many bacteria use quorum sensing (QS) as an intercellular signaling mechanism to regulate gene expression in local populations. Plant and algal hosts, in turn, secrete compounds that mimic bacterial QS signals, allowing these hosts to manipulate QS-regulated gene expression in bacteria. Lumichrome, a derivative of the vitamin riboflavin, was purified and chemically identified from culture filtrates of the alga Chlamydomonas as a QS signal-mimic compound capable of stimulating the Pseudomonas aeruginosa LasR QS receptor. LasR normally recognizes the N-acyl homoserine lactone (AHL) signal, N-3-oxo-dodecanoyl homoserine lactone. Authentic lumichrome and riboflavin stimulated the LasR receptor in bioassays and lumichrome activated LasR in gel shift experiments. Amino acid substitutions in LasR residues required for AHL binding altered responses to both AHLs and lumichrome or riboflavin. These results and docking studies indicate that the AHL binding pocket of LasR recognizes both AHLs and the structurally dissimilar lumichrome or riboflavin. Bacteria, plants, and algae commonly secrete riboflavin or lumichrome, raising the possibility that these compounds could serve as either QS signals or as interkingdom signal mimics capable of manipulating QS in bacteria with a LasR-like receptor.
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He, Qing, Kang Wang, Tiantian Su, Feng Wang, Lichuan Gu, and Sujuan Xu. "Crystal structure of the N-terminal domain of VqsR fromPseudomonas aeruginosaat 2.1 Å resolution." Acta Crystallographica Section F Structural Biology Communications 73, no. 7 (June 28, 2017): 431–36. http://dx.doi.org/10.1107/s2053230x17009025.
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VqsR is a quorum-sensing (QS) transcriptional regulator which controls QS systems (las,rhlandpqs) by directly downregulating the expression ofqscRinPseudomonas aeruginosa. As a member of the LuxR family of proteins, VqsR shares the common motif of a helix–turn–helix (HTH)-type DNA-binding domain at the C-terminus, while the function of its N-terminal domain remains obscure. Here, the crystal structure of the N-terminal domain of VqsR (VqsR-N; residues 1–193) was determined at a resolution of 2.1 Å. The structure is folded into a regular α–β–α sandwich topology, which is similar to the ligand-binding domain (LBD) of the LuxR-type QS receptors. Although their sequence similarity is very low, structural comparison reveals that VqsR-N has a conserved enclosed cavity which could recognize acyl-homoserine lactones (AHLs) as in other LuxR-type AHL receptors. The structure suggests that VqsR could be a potential AHL receptor.
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Chatterjee, Asita, Yaya Cui, Hiroaki Hasegawa, Nathan Leigh, Vaishali Dixit, and Arun K. Chatterjee. "Comparative Analysis of Two Classes of Quorum-Sensing Signaling Systems That Control Production of Extracellular Proteins and Secondary Metabolites in Erwinia carotovora Subspecies." Journal of Bacteriology 187, no. 23 (December 1, 2005): 8026–38. http://dx.doi.org/10.1128/jb.187.23.8026-8038.2005.
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ABSTRACT In Erwinia carotovora subspecies, N-acyl homoserine lactone (AHL) controls the expression of various traits, including extracellular enzyme/protein production and pathogenicity. We report here that E. carotovora subspecies possess two classes of quorum-sensing signaling systems defined by the nature of the major AHL analog produced as well as structural and functional characteristics of AHL synthase (AhlI) and AHL receptor (ExpR). Class I strains represented by E. carotovora subsp. atroseptica strain Eca12 and E. carotovora subsp. carotovora strains EC153 and SCC3193 produce 3-oxo-C8-HL (N-3-oxooctanoyl-l-homoserine lactone) as the major AHL analog as well as low but detectable levels of 3-oxo-C6-HL (N-3-oxohexanoyl-l-homoserine lactone). In contrast, the members of class II (i.e., E. carotovora subsp. betavasculorum strain Ecb168 and E. carotovora subsp. carotovora strains Ecc71 and SCRI193) produce 3-oxo-C6-HL as the major analog. ExpR species of both classes activate rsmA (Rsm, repressor of secondary metabolites) transcription and bind rsmA DNA. Gel mobility shift assays with maltose-binding protein (MBP)-ExpR71 and MBP-ExpR153 fusion proteins show that both bind a 20-mer sequence present in rsmA. The two ExpR functions (i.e., expR-mediated activation of rsmA expression and ExpR binding with rsmA DNA) are inhibited by AHL. The AHL effects are remarkably specific in that expR effect of EC153, a strain belonging to class I, is counteracted by 3-oxo-C8-HL but not by 3-oxo-C6-HL. Conversely, the expR effect of Ecc71, a strain belonging to class II, is neutralized by 3-oxo-C6-HL but not by 3-oxo-C8-HL. The AHL responses correlated with expR-mediated inhibition of exoprotein and secondary metabolite production.
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Cui, Yaya, Asita Chatterjee, Hiroaki Hasegawa, Vaishali Dixit, Nathan Leigh, and Arun K. Chatterjee. "ExpR, a LuxR Homolog of Erwinia carotovora subsp. carotovora, Activates Transcription of rsmA, Which Specifies a Global Regulatory RNA-Binding Protein." Journal of Bacteriology 187, no. 14 (July 2005): 4792–803. http://dx.doi.org/10.1128/jb.187.14.4792-4803.2005.
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ABSTRACT N-acyl homoserine lactone (AHL) is required by Erwinia carotovora subspecies for the expression of various traits, including extracellular enzyme and protein production and pathogenicity. Previous studies with E. carotovora subsp. carotovora have shown that AHL deficiency causes the production of high levels of RsmA, an RNA binding protein that functions as a global negative regulator of extracellular enzymes and proteins and secondary metabolites (Rsm, regulator of secondary metabolites). We document here that ExpR, a putative AHL receptor belonging to the LuxR family of regulators, activates RsmA production. In the absence of AHL, an ExpR+ E. carotovora subsp. carotovora strain compared to its ExpR− mutant, produces higher levels of rsmA RNA and better expresses an rsmA-lacZ transcriptional fusion. Moreover, the expression of the rsmA-lacZ fusion in Escherichia coli is much higher in the presence of expR71 (the expR gene of E. carotovora subsp. carotovora strain Ecc71) than in its absence. We also show that purified preparation of MBP-ExpR71 binds (MBP, maltose binding protein) rsmA DNA. By contrast, MBP-ExpR71 does not bind ahlI (gene for AHL synthase), pel-1 (gene for pectate lyase), or rsmB (gene for regulatory RNA that binds RsmA), nor does ExpR71 activate expression of these genes. These observations strongly suggest transcriptional activation of rsmA resulting from a direct and specific interaction between ExpR71 and the rsmA promoter. Several lines of evidence establish that N-3-oxohexanoyl-l-homoserine lactone (3-oxo-C6-HL), the major AHL analog produced by E. carotovora subsp. carotovora strain Ecc71, inhibits ExpR71-mediated activation of rsmA expression. These findings for the first time establish that the expR effect in E. carotovora subsp. carotovora is channeled via RsmA, a posttranscriptional regulator of E. carotovora subspecies, and AHL neutralizes this ExpR effect.
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Pérez, Daniela, Maicol Ahumedo, Eileen Herrera, Catalina Vivas-Gomez, and Ricardo Vivas-Reyes. "Computational study of the interactions among structural analogues of acyl homoserine lactones (AHLs) and the Agrobacterium tumefaciens TraR binding site." F1000Research 8 (December 5, 2019): 2062. http://dx.doi.org/10.12688/f1000research.20793.1.
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Background: In the present investigation, relationships between a set of 34 analogues of N-acyl-L-homoserine lactones (AHL) and the TraR receptor were studied. The aim was to use molecular modeling as a strategy for elucidating important aspects of the mechanism of chemical signaling in the Gram-negative bacteria Agrobacterium tumefaciens, with the idea of identifying some of analogues’ structural characteristics and molecular interactions with the active site of the TraR receptor. Methods: For this purpose, we combine two molecular modeling strategies: molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR). First, the molecular docking methodology was applied to a series of 34 analogues of AHL on the TraR transcriptional receptor to simulate the binding of analogues at the active TraR site. Secondly, 3D-QSAR models were generated to describe the correlation with the experimental biological activity using partial least squares (PLS) calculations and steric and electrostatic properties, which theoretically predict the activity of the 34 AHL analogues through statistical parameters and evaluate the prediction of the models obtained. Two alignment models were constructed; one using the optimized structures of the 34 analogues (ligand-based model) and another using the conformations of the best poses generated in the docking with TraR (receptor-based model). Results: The outcomes obtained for each protein-ligand complex showed that the Aspartic acid 70 and Threonine 129 residues are residues that participate in the formation of hydrogen bonds, while residues Alanine 38, Leucine, 40, Tyrosine 53, Glutamine 58, Tyrosine 61, Phenylalanine 62 and Valine 72 form hydrophobic interactions. These interactions are important in determining the antagonistic activity of the analogues under study against TraR. Conclusions: The ligand-based model produces better statistical results expressed in terms of several rigorous evaluation criteria, such as Q2 and R2 for the data sets than those of the receptor-based model.
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Herrera-Arizmendi, José Luis, Everardo Curiel-Quesada, José Correa-Basurto, Martiniano Bello, and Alicia Reyes-Arellano. "Effect of New Analogs of Hexyloxy Phenyl Imidazoline on Quorum Sensing in Chromobacterium violaceum and In Silico Analysis of Ligand-Receptor Interactions." Journal of Chemistry 2020 (February 25, 2020): 1–18. http://dx.doi.org/10.1155/2020/8735190.
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The increasing common occurrence of antibiotic-resistant bacteria has become an urgent public health issue. There are currently some infections without any effective treatment, which require new therapeutic strategies. An attractive alternative is the design of compounds capable of disrupting bacterial communication known as quorum sensing (QS). In Gram-negative bacteria, such communication is regulated by acyl-homoserine lactones (AHLs). Triggering of QS after bacteria have reached a high cell density allows them to proliferate before expressing virulence factors. Our group previously reported that hexyloxy phenylimidazoline (9) demonstrated 71% inhibitory activity of QS at 100 μM (IC50 = 90.9 μM) in Chromobacterium violaceum, a Gram-negative bacterium. The aim of the present study was to take 9 as a lead compound to design and synthesize three 2-imidazolines (13–15) and three 2-oxazolines (16–18), to be evaluated as quorum-sensing inhibitors on C. violaceum CV026. We were looking for compounds with a higher affinity towards the Cvi receptor of this bacterium and the ability to inhibit QS. The binding mode of the test compounds on the Cvi receptor was explored with docking studies and molecular dynamics. It was found that 8-pentyloxyphenyl-2-imidazoline (13) reduced the production of violacein (IC50 = 56.38 μM) without affecting bacterial growth, suggesting inhibition of quorum sensing. Indeed, compound 13 is apparently one of the best QS inhibitors known to date. Molecular docking revealed the affinity of compound 13 for the orthosteric site of N-hexanoyl homoserine lactone (C6-AHL) on the CviR protein. Ten amino acid residues in the active binding site of C6-AHL in the Cvi receptor interacted with 13, and 7 of these are the same as those interacting with AHL. Contrarily, 8-octyloxyphenyl-2-imidazoline (14), 8-decyloxyphenyl-2-imidazoline (15), and 9-decyloxyphenyl-2-oxazoline (18) bound only to an allosteric site and thus did not compete with C6-AHL for the orthosteric site.
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Deryabin, Dmitry, Anna Galadzhieva, Dianna Kosyan, and Galimjan Duskaev. "Plant-Derived Inhibitors of AHL-Mediated Quorum Sensing in Bacteria: Modes of Action." International Journal of Molecular Sciences 20, no. 22 (November 8, 2019): 5588. http://dx.doi.org/10.3390/ijms20225588.
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Numerous gram-negative phytopathogenic and zoopathogenic bacteria utilise acylated homoserine lactone (AHL) in communication systems, referred to as quorum sensing (QS), for induction of virulence factors and biofilm development. This phenomenon positions AHL-mediated QS as an attractive target for anti-infective therapy. This review focused on the most significant groups of plant-derived QS inhibitors and well-studied individual compounds for which in silico, in vitro and in vivo studies provide substantial knowledge about their modes of anti-QS activity. The current data about sulfur-containing compounds, monoterpenes and monoterpenoids, phenylpropanoids, benzoic acid derivatives, diarylheptanoids, coumarins, flavonoids and tannins were summarized; their plant sources, anti-QS effects and bioactivity mechanisms have also been summarized and discussed. Three variants of plant-derived molecules anti-QS strategies are proposed: (i) specific, via binding with LuxI-type AHL synthases and/or LuxR-type AHL receptor proteins, which have been shown for terpenes (carvacrol and l-carvone), phenylpropanoids (cinnamaldehyde and eugenol), flavonoid quercetin and ellagitannins; (ii) non-specific, by affecting the QS-related intracellular regulatory pathways by lowering regulatory small RNA expression (sulphur-containing compounds ajoene and iberin) or c-di-GMP metabolism reduction (coumarin); and (iii) indirect, via alteration of metabolic pathways involved in QS-dependent processes (vanillic acid and curcumin).
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Gamage, Akshamal Mihiranga, Guanghou Shui, Markus R. Wenk, and Kim Lee Chua. "N-Octanoylhomoserine lactone signalling mediated by the BpsI–BpsR quorum sensing system plays a major role in biofilm formation of Burkholderia pseudomallei." Microbiology 157, no. 4 (April 1, 2011): 1176–86. http://dx.doi.org/10.1099/mic.0.046540-0.
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The genome of Burkholderia pseudomallei encodes three acylhomoserine lactone (AHL) quorum sensing systems, each comprising an AHL synthase and a signal receptor/regulator. The BpsI–BpsR system produces N-octanoylhomoserine lactone (C8HL) and is positively auto-regulated by its AHL product. The products of the remaining two systems have not been identified. In this study, tandem MS was used to identify and quantify the AHL species produced by three clinical B. pseudomallei isolates – KHW, K96243 and H11 – three isogenic KHW mutants that each contain a null mutation in an AHL synthase gene, and recombinant Escherichia coli heterologously expressing each of the three B. pseudomallei AHL synthase genes. BpsI synthesized predominantly C8HL, which accounted for more than 95 % of the extracellular AHLs produced in stationary-phase KHW cultures. The major products of BpsI2 and BpsI3 were N-(3-hydroxy-octanoyl)homoserine lactone (OHC8HL) and N-(3-hydroxy-decanoyl)homoserine lactone, respectively, and their corresponding transcriptional regulators, BpsR2 and BpsR3, were capable of driving reporter gene expression in the presence of these cognate lactones. Formation of biofilm by B. pseudomallei KHW was severely impaired in mutants lacking either BpsI or BpsR but could be restored to near wild-type levels by exogenous C8HL. BpsI2 was not required, and BpsI3 was partially required for biofilm formation. Unlike the bpsI mutant, biofilm formation in the bpsI3 mutant could not be restored to wild-type levels in the presence of OHC8HL, the product of BpsI3. C8HL and OHC8HL had opposite effects on biofilm formation; exogenous C8HL enhanced biofilm formation in both the bpsI3 mutant and wild-type KHW while exogenous OHC8HL suppressed the formation of biofilm in the same strains. We propose that exogenous OHC8HL antagonizes biofilm formation in B. pseudomallei, possibly by competing with endogenous C8HL for binding to BpsR.
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Koch, B., T. Liljefors, T. Persson, J. Nielsen, S. Kjelleberg, and M. Givskov. "The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors." Microbiology 151, no. 11 (November 1, 2005): 3589–602. http://dx.doi.org/10.1099/mic.0.27954-0.
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The function of LuxR homologues as quorum sensors is mediated by the binding of N-acyl-l-homoserine lactone (AHL) signal molecules to the N-terminal receptor site of the proteins. In this study, site-directed mutagenesis was carried out of the amino acid residues comprising the receptor site of LuxR from Vibrio fischeri, and the ability of the L42A, L42S, Y62F, W66F, D79N, W94D, V109D, V109T and M135A LuxR mutant proteins to activate green fluorescent protein expression from a PluxI promoter was measured. X-ray crystallographic studies of the LuxR homologue TraR indicated that residues Y53 and W57 form hydrogen bonds to the 1-carbonyl group and the ring carbonyl group, respectively, of the cognate AHL signal. Based on the activity and signal specificity of the LuxR mutant proteins, and on molecular modelling, a model is suggested in which Y62 (corresponding to Y53 in TraR) forms a hydrogen bond with the ring carbonyl group rather than the 1-carbonyl group, while W66 (corresponding to W57 in TraR) forms a hydrogen bond to the 1-carbonyl group. This flips the position of the acyl side chain in the LuxR/signal molecule complex compared to the TraR/signal molecule complex. Halogenated furanones from the marine alga Delisea pulchra and the synthetic signal analogue N-(sulfanylacetyl)-l-homoserine lactone can block quorum sensing. The LuxR mutant proteins were insensitive to inhibition by N-(propylsulfanylacetyl)-l-homoserine lactone. In contrast, the mutations had only a minor effect on the sensitivity of the proteins to halogenated furanones, and the data strongly suggest that these compounds do not compete in a ‘classic’ way with N-3-oxohexanoyl-l-homoserine lactone for the binding site. Based on modelling and experimental data it is suggested that these compounds bind in a non-agonist fashion.
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Markus, Victor, Orr Share, Marilou Shagan, Barak Halpern, Tal Bar, Esti Kramarsky-Winter, Kerem Teralı, et al. "Inhibitory Effects of Artificial Sweeteners on Bacterial Quorum Sensing." International Journal of Molecular Sciences 22, no. 18 (September 13, 2021): 9863. http://dx.doi.org/10.3390/ijms22189863.
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Despite having been tagged as safe and beneficial, recent evidence remains inconclusive regarding the status of artificial sweeteners and their putative effects on gut microbiota. Gut microorganisms are essential for the normal metabolic functions of their host. These microorganisms communicate within their community and regulate group behaviors via a molecular system termed quorum sensing (QS). In the present study, we aimed to study the effects of artificial sweeteners on this bacterial communication system. Using biosensor assays, biophysical protein characterization methods, microscale thermophoresis, swarming motility assays, growth assays, as well as molecular docking, we show that aspartame, sucralose, and saccharin have significant inhibitory actions on the Gram-negative bacteria N-acyl homoserine lactone-based (AHL) communication system. Our studies indicate that these three artificial sweeteners are not bactericidal. Protein-ligand docking and interaction profiling, using LasR as a representative participating receptor for AHL, suggest that the artificial sweeteners bind to the ligand-binding pocket of the protein, possibly interfering with the proper housing of the native ligand and thus impeding protein folding. Our findings suggest that these artificial sweeteners may affect the balance of the gut microbial community via QS-inhibition. We, therefore, infer an effect of these artificial sweeteners on numerous molecular events that are at the core of intestinal microbial function, and by extension on the host metabolism.
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Qazi, Saara, Barry Middleton, Siti Hanna Muharram, Alan Cockayne, Philip Hill, Paul O'Shea, Siri Ram Chhabra, Miguel Cámara, and Paul Williams. "N-Acylhomoserine Lactones Antagonize Virulence Gene Expression and Quorum Sensing in Staphylococcus aureus." Infection and Immunity 74, no. 2 (February 2006): 910–19. http://dx.doi.org/10.1128/iai.74.2.910-919.2006.
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ABSTRACT Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 μM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.
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Okuda, K., A. D’Andrea, R. A. Van Etten, and J. D. Griffin. "The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein." Blood 92, no. 10 (November 15, 1998): 3848–56. http://dx.doi.org/10.1182/blood.v92.10.3848.
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Abstract Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.
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Okuda, K., A. D’Andrea, R. A. Van Etten, and J. D. Griffin. "The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein." Blood 92, no. 10 (November 15, 1998): 3848–56. http://dx.doi.org/10.1182/blood.v92.10.3848.422k44_3848_3856.
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Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.
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Baggen, Jim, Daniel L. Hurdiss, Georg Zocher, Nitesh Mistry, Richard W. Roberts, Jasper J. Slager, Hongbo Guo, et al. "Role of enhanced receptor engagement in the evolution of a pandemic acute hemorrhagic conjunctivitis virus." Proceedings of the National Academy of Sciences 115, no. 2 (December 28, 2017): 397–402. http://dx.doi.org/10.1073/pnas.1713284115.
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Acute hemorrhagic conjunctivitis (AHC) is a painful, contagious eye disease, with millions of cases in the last decades. Coxsackievirus A24 (CV-A24) was not originally associated with human disease, but in 1970 a pathogenic “variant” (CV-A24v) emerged, which is now the main cause of AHC. Initially, this variant circulated only in Southeast Asia, but it later spread worldwide, accounting for numerous AHC outbreaks and two pandemics. While both CV-A24 variant and nonvariant strains still circulate in humans, only variant strains cause AHC for reasons that are yet unknown. Since receptors are important determinants of viral tropism, we set out to map the CV-A24 receptor repertoire and establish whether changes in receptor preference have led to the increased pathogenicity and rapid spread of CV-A24v. Here, we identify ICAM-1 as an essential receptor for both AHC-causing and non-AHC strains. We provide a high-resolution cryo-EM structure of a virus–ICAM-1 complex, which revealed critical ICAM-1–binding residues. These data could help identify a possible conserved mode of receptor engagement among ICAM-1–binding enteroviruses and rhinoviruses. Moreover, we identify a single capsid substitution that has been adopted by all pandemic CV-A24v strains and we reveal that this adaptation enhances the capacity of CV-A24v to bind sialic acid. Our data elucidate the CV-A24v receptor repertoire and point to a role of enhanced receptor engagement in the adaptation to the eye, possibly enabling pandemic spread.
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Brunetti, Lorenzo, Giovanna Abate, Marisa Gorrese, Maddalena Raia, Caterina Pascariello, Giulia Scalia, Bruno Rotoli, and Luigi Del Vecchio. "CD200: a New Target for Immunotherapy in Hematologic Malignancies." Blood 112, no. 11 (November 16, 2008): 1598. http://dx.doi.org/10.1182/blood.v112.11.1598.1598.
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Abstract CD200 is a transmembrane glycoprotein expressed on several tissues in rats and humans. It plays an immunoregulatory function by switching cytokine production from a TH1 to TH2 pattern, thus reducing cytotoxic response while indirectly enabling tumor escape and growth. Interestingly, CD200 is also a target for a novel humanized monoclonal antibody (Anti-CD200 MoAb, Alexion Pharmaceuticals, Cheshire, CT, USA). Anti-CD200 inhibits CD200 binding to its receptor, so modifying cytokine production and improving T-cell mediated cytotoxic response. Expression of CD200 has been already described in chronic lymphocytic leukemia/small lymphocyte lymphoma (CLL/SLL), multiple myeloma (MM) and acute myeloid leukemia (AML). Moreover, CD200 expression is considered an unfavorable prognostic factor in MM and AML. The goal of this work was to study, using flow cytometry, CD200 expression on a large number of onco-hematological samples, with the aim to exactly describe conditions in which anti-CD200 MoAb could be a therapeutic option. Analysis was conduced using a six-color FACSCanto II cytometer (Becton Dickinson, BD, San Jose, CA, USA) equipped with the FACSDiva software (BD). CD200 expression was evaluated by using CD200-PE-conjugated antibody (BD/Pharmingen). During the last year we analyzed CD200 expression in 184 samples: 131 bone marrow aspirates (BM), 33 peripheral blood specimens (PB) and 20 fine needle aspiration cytology samples (FNAC). One hundred and four were lymphoproliferative disorders, 12 MM, 16 myelodysplastic syndromes (MDS) and 52 acute leukemias. CD200 positivity was assigned to every single case when CD200 mean fluorescence intensity (MFI) was higher then 256 arbitrary units, a channel close to the cut-off point between positive and negative cells, in our experience. All results concerning our analysis are showed in the table. Three classes of hematologic neoplasms displayed a constant positivity for CD200 with a high level of MFI: CLL/SLL, hairy cell leukemia (HCL) and B-cell acute lymphoblastic leukemia (B-ALL). Lymphoplasmacytic lymphoma (LPL) was positive in 100% of cases but with lower MFI as compared to CLL/SLL, HCL and B-ALL (p<0.001). MM/MGUS plasma cells showed CD200 positivity in 80% of cases. CD200 was also expressed in cases of marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), T-non Hodgkin lymphoma (T-NHL), AML, acute hybrid leukemia (AHL) and T-cell acute lymphoblastic leukemia (T-ALL). Follicular Lymphoma (FL) samples were always negative but one, which expressed the antigen with a very low MFI. In AML we also compared CD200 expression with that of a large number of antigens (n=40), finding a statistically significant inverse correlation with myeloperoxidase (MPO-7) (Spearman’s r= −0.54; p<0.001). Difference in MFI between AML and B-ALL was statistically significant (p=0.003). No case of acute promyelocytic leukemia (APL) was positive for CD200. In conclusion, CD200 is candidate as a new specific target for immunotherapy with anti-CD200 in all cases of CLL/SLL, B-ALL and HCL as well as in selected cases of MZL, DLBCL, MCL, LPL, MM, T-NHL, AML, AHL and T-ALL. Disease N Positive N (%) PPC (mean) PPC (25°–75° percentile) MFI (median) MFI (25°–75° percentile) PPC= percent positive cells MFI= mean fluorescence intensity AML 38 24 (63) 32.9 8–57 294 138–695 APL 2 0 (0) 2.5 - 33 - MDS 16 12 (75) 34.2 15–57 516 263–1122 AHL 5 4 (80) 60.2 27–87 420 238–1194 ALL 7 6 (86) 63.6 57–85 1099 288–1444 CLL/SLL 47 47 (100) 98.4 98–99 3410 2300–4733 LPL 5 5 (100) 54.8 41–65 492 377–715 MCL 11 8 (72) 43 3–71 665 20–835 FL 10 1 (10) 4.7 2–8 46 30–83 DLCL 12 6 (50) 29.8 3–71 204 66–754 MZL 9 6 (66) 39.2 4–85 352 47–864 HCL 6 6 (100) 87 72–98 3841 1271–7527 MM 12 11 (92) 52.7 18–79 2956 979–5937 T-NHL 4 2 (50) 23.5 2–62 287 23–2477
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Brameyer, Sophie, and Ralf Heermann. "Specificity of Signal-Binding via Non-AHL LuxR-Type Receptors." PLOS ONE 10, no. 4 (April 29, 2015): e0124093. http://dx.doi.org/10.1371/journal.pone.0124093.
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Schmoker, Anna M., Jaye L. Weinert, Kyle J. Kellett, Hannah E. Johnson, Ryan M. Joy, Marion E. Weir, Alicia M. Ebert, and Bryan A. Ballif. "Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding receptors DCBLD1 and DCBLD2." Biochemical Journal 474, no. 23 (November 21, 2017): 3963–84. http://dx.doi.org/10.1042/bcj20170615.
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Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL–SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL–SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL–SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.
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TUCKER, Amy L., LiGuo JIA, Diane HOLETON, Allen J. TAYLOR, and Joel LINDEN. "Dominance of Gs in doubly Gs/Gi-coupled chimaeric A1/A2A adenosine receptors in HEK-293 cells." Biochemical Journal 352, no. 1 (November 7, 2000): 203–10. http://dx.doi.org/10.1042/bj3520203.
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A1 adenosine receptors inhibit adenylate cyclase by activating Gi/Go, whereas A2A receptors activate Gs. We examined how regions of A1 and A2A receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to Gi/Go and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A1/A2A 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A1 3ICL sequence (A2A/A1L or A2A/A1LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to Gi/Go. These chimaeras have basal cAMP levels lower than those of wild-type A2A receptors, similar to wild-type A1 receptors. The A1 C-terminus modulates the coupling of receptors with A1 3ICL such that A2A/A1LT is better coupled to Gi/Go than A2A/A1L. The C-terminus has little impact on coupling to receptors containing A2A 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to Gi/Go mediated by A1 3ICL and not by other intracellular domains that favour Gi coupling. The C-terminus sequence has little or no effect on coupling to Gs. For doubly Gs/Gi-coupled adenosine receptors in HEK-293 cells, Gs-mediated stimulation predominates over Gi/Go-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.
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Yen, Ryan, Lambert Yue, Steven Pelech, and Xiaoyan Jiang. "The AHI-1-BCR-ABL-DNM2 Complex Mediates Mitochondrial Dynamics in Drug-Resistant BCR-ABL+ Cells." Blood 134, Supplement_1 (November 13, 2019): 189. http://dx.doi.org/10.1182/blood-2019-128712.
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Chronic myeloid leukemia (CML) is driven by the BCR-ABL1 oncoprotein with constitutively active protein-tyrosine kinase activity, perturbing multiple signaling pathways. Although therapies with tyrosine kinase inhibitors (TKIs) can effectively treat early phase CML, relapses and emergence of TKI resistance are problematic, due to BCR-ABL kinase domain mutations and TKI unresponsive quiescent leukemic stem cells (LSCs). These observations point towards a need for alternate treatment strategies to prevent the development of resistant LSCs. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs and that its WD40-repeat domain physically interacts with BCR-ABL, enhancing leukemia-initiating activity. AHI-1 also contains an SH3 domain, which mediates TKI resistance in LSCs. This domain interacts with dynamin-2 (DNM2) and forms a complex with BCR-ABL, to enhance the phosphorylation and activity of DNM2. The AHI-1-BCR-ABL-DNM2 complex is shown to regulate leukemic properties in patient LSCs, including increased ROS production, endocytosis and autophagy. Interestingly, deletion of the Ahi-1 SH3 domain (Ahi-1 SH3Δ) results in a defect in Ahi-1 localization, with most being present in the nucleus. To test whether Ahi-1 SH3 domain activity directly affects cytoplasmic anchoring and localization, we have generated two Ahi-1 mutants, using site-directed mutagenesis: a mutation in the key tryptophan residue (W939A) involved in SH3 domain binding and in a non-conserved surface residue (M906A), as a negative control, based on the crystal structure of the AHI-1 SH3 domain. Interestingly, the cytoplasm-to-nucleus signal ratio of Ahi-1 W939A was significantly reduced compared to the negative control or wildtype Ahi-1, as assessed by immunofluorescence and confocal microscopy (70% reduction, p<0.0001), indicating that changes in localization of Ahi-1 SH3Δ may result in disruption of the complex and allow for new interactions with nuclear proteins. Investigating changes in the proteome may help uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance. Advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL+ cells co-transduced with wildtype Ahi-1 or Ahi-1 SH3Δ. This system quantifies the differences in expression and phosphorylation states of key signaling proteins simultaneously, using 878 antibodies in duplicate. Twenty leads were identified by the following criteria: a large signal difference of at least 1.5-fold change, high signal strength for high expression, and low error between duplicates. These leads were validated by Western blot analysis and several of them were confirmed. Particularly, phosphorylation of cyclin-dependent kinase 1 (CDK1), a key player in cell cycle control and mitochondrial dynamics, was greatly reduced in cells expressing wildtype Ahi-1 compared to Ahi-1 SH3Δ, indicating that AHI-1-mediated phosphorylation changes in CDK1 may contribute to regulation of mitochondrial functions. Indeed, BCR-ABL-transduced cells co-expressing wildtype Ahi-1 showed increased mitochondria potential in response to TKI treatment or serum starvation, in MitoTracker analysis (p<0.05). However, this was not observed in BCR-ABL-transduced cells co-expressing the Ahi-1 SH3Δ mutant. A similar trend was also observed in immunofluorescence confocal microscopy analysis of the mitochondrial importer receptor, TOM20. To further study the role of DNM2 in mediating mitochondrial dynamics associated with AHI-1 and BCR-ABL, CRISPR-Cas9 mediated DNM2 knockdown was performed in TKI-resistant cells, using two different DNM2-targeting guide RNAs; these resulted in significant reduction in DNM2 (78% & 75%) in Western blot analysis. The knockdown cells showed a reduction in viability (60% reduction) and increased sensitivity to TKI treatment compared to the control (90% vs. 30% reduction) after 48 hours and changes in mitochondrial activity were also observed in these cells. These results support a role for mitochondrial dynamics in the AHI-1-BCR-ABL-DNM2 complex-mediated TKI response and that targeting key biological processes regulated by the AHI-1-BCR-ABL-DNM2 complex and its pathways may lead to new therapeutic strategies to overcome TKI resistance in CML. Disclosures Pelech: Kinexus Bioinformatics Corporation: Equity Ownership.
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Ito, M., R. Yu, and J. L. Jameson. "DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita." Molecular and Cellular Biology 17, no. 3 (March 1997): 1476–83. http://dx.doi.org/10.1128/mcb.17.3.1476.
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X-linked adrenal hypoplasia congenita (AHC) with hypogonadotropic hypogonadism was recently shown to be caused by mutations in a gene referred to as DAX-1, which encodes a novel member of the orphan nuclear receptor family. DAX-1 is homologous to other nuclear receptors in its carboxy-terminal region, but it lacks the characteristic zinc finger DNA-binding domain. The tissue distribution of DAX-1 (adrenal cortex, gonads, hypothalamus, and pituitary) is the same as that of another orphan nuclear receptor, steroidogenic factor 1 (SF-1), that is required for development of the adrenal glands and gonads. We examined whether DAX-1 and SF-1 might interact in the regulation of SF-1-responsive target genes. Coexpression of DAX-1 and SF-1 inhibited SF-1-mediated transactivation. DAX-1 was shown to interact directly with SF-1 in in vitro protein binding studies; however, it did not interfere with SF-1 binding to DNA in gel mobility shift assays. Transactivation by GAL4-SF-1 constructs was inhibited by DAX-1, indicating that neither the SF-1 DNA-binding domain nor the SF-1 binding sites are required for inhibition by DAX-1. A series of DAX-1 deletion mutants localized the inhibitory domain to the carboxy-terminal region of the protein. Deletion of this domain also reduced basal transcriptional silencing by GAL4-DAX-1. This inhibitory domain has been deleted in all naturally occurring AHC deletion mutants described to date. In addition, two naturally occurring point mutations in DAX-1 exhibited impaired inhibition of SF-1. We conclude that DAX-1 can inhibit SF-1 transcriptional activity and suggest that the loss of this inhibitory property in DAX-1 may account in part for the phenotype of AHC.
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Ghaffari, Saghi, Claire Kitidis, Mark D. Fleming, Hans Neubauer, Klaus Pfeffer, and Harvey F. Lodish. "Erythropoiesis in the absence of janus-kinase 2: BCR-ABL induces red cell formation in JAK2−/− hematopoietic progenitors." Blood 98, no. 10 (November 15, 2001): 2948–57. http://dx.doi.org/10.1182/blood.v98.10.2948.
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Abstract The receptor-associated protein tyrosine kinase janus-kinase 2 (JAK2) is essential for normal red cell development and for erythropoietin receptor (EpoR) signaling. JAK2−/− embryos are severely deficient in erythropoiesis and die at an early stage of development from fetal anemia. The binding of erythropoietin (Epo) to the EpoR triggers the activation of JAK2, the phosphorylation of the EpoR, and the initiation of the EpoR signaling cascade. In addition to Epo binding to its receptor, signaling pathways downstream of the EpoR can also be stimulated by the BCR-ABL oncoprotein. This study explored whether JAK2 is required for BCR-ABL–mediated stimulation of erythropoiesis. Here, it is shown that JAK2 is constitutively tyrosine phosphorylated in cultured and primary erythroid cells expressing BCR-ABL. However, BCR-ABL effectively supports normal erythroid proliferation, differentiation, and maturation in JAK2-deficient fetal liver cells. Using mutants of BCR-ABL, this study shows that certain signaling pathways activated by BCR-ABL segments distinct from its tyrosine kinase domain are essential for rescue of erythropoiesis in JAK2−/− progenitors. The consequences of these multiple signaling pathways for normal erythroid development are discussed.
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Barilà, Daniela, Alessandra Rufini, Ivano Condò, Natascia Ventura, Karel Dorey, Giulio Superti-Furga, and Roberto Testi. "Caspase-Dependent Cleavage of c-Abl Contributes to Apoptosis." Molecular and Cellular Biology 23, no. 8 (April 15, 2003): 2790–99. http://dx.doi.org/10.1128/mcb.23.8.2790-2799.2003.
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ABSTRACT The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program.
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Savli, M., A. Bauer, D. Häusler, T. Kroll, A. Hahn, F. Rattay, M. Mitterhauser, W. Wadsak, S. Kasper, and R. Lanzenberger. "In vivo molecular imaging reveals distinct distributions of the serotonin transporter, the major inhibitory and excitatory serotonin receptors." European Psychiatry 26, S2 (March 2011): 953. http://dx.doi.org/10.1016/s0924-9338(11)72658-x.
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IntroductionBased on evidences in molecular neuroimaging, postmortem and genetic studies, impaired serotonergic neurotransmission has been implicated with affective disorders. Moreover, a growing number of evidences showed strong interrelations within the serotonergic system suggesting a common mechanism in the modulation of receptor and transporter densities.ObjectiveHere we directly investigated the regional expression of the 5-HT1A, 5-HT2A and 5-HTT using PET and the three highly selective and specific radioligands [carbonyl-11C]WAY-100635, [18F]Altanserin and [11C]DASB in healthy subjects.MethodsA total of 55 healthy subjects (5-HT1A: 36 subjects, 18 males, age = 26.0 ± 4.9; 5-HT2A: 19 subjects, 11 males, age = 28.2 ± 5.9; 5-HTT: 8 males, age = 28.12 ± 3.6) were included in this study. Binding potential (BPND) values were quantified according to the AAL parcellation scheme.ResultsBPND values averaged over both hemispheres ranged from 0.40–6.35 for the 5-HT1A receptor; 0.01–2.01 for the 5-HT2A receptor and 0.09–2.05 for the 5-HTT, respectively. There was a specific topological pattern according to the ratio between the 5-HT1A, 5-HT2A receptors and 5-HTT (“fingerprints”).ConclusionsSuch information can be essential for detecting potential local alterations in the ratio between different binding proteins on a network level in pathological conditions.Moreover, these data might provide further insight in area-specific effects of frequently prescribed selective serotonin re-uptake inhibitors (SSRI): 1)due to the distinct local receptor and transporter availability;2)SSRI application alters the postsynaptic receptor expression and thus;3)leads to a modified interaction of inhibitory and exhibitory receptors.
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Schmoker, Anna M., Jaye L. Weinert, Jacob M. Markwood, Kathryn S. Albretsen, Michelle L. Lunde, Marion E. Weir, Alicia M. Ebert, Karen L. Hinkle, and Bryan A. Ballif. "FYN and ABL Regulate the Interaction Networks of the DCBLD Receptor Family." Molecular & Cellular Proteomics 19, no. 10 (June 30, 2020): 1586–601. http://dx.doi.org/10.1074/mcp.ra120.002163.
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The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.
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Chatterjee, Asita, Yaya Cui, Pranjib Chakrabarty, and Arun K. Chatterjee. "Regulation of Motility in Erwinia carotovora subsp. carotovora: Quorum-Sensing Signal Controls FlhDC, the Global Regulator of Flagellar and Exoprotein Genes, by Modulating the Production of RsmA, an RNA-Binding Protein." Molecular Plant-Microbe Interactions® 23, no. 10 (October 2010): 1316–23. http://dx.doi.org/10.1094/mpmi-01-10-0017.
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Erwinia carotovora subsp. carotovora causes soft-rotting (tissue-macerating) disease in many plants and plant organs. Although pectinases are the primary determinants of virulence, several ancillary factors that augment bacterial virulence have also been identified. One such factor is bacterial motility. Flagellum formation and bacterial movement are regulated in many enterobacteria, including E. carotovora subsp. carotovora, by FlhDC, the master regulator of flagellar genes and FliA, a flagellum-specific σ factor. We document here that motility of E. carotovora subsp. carotovora is positively regulated by the quorum-sensing signal, N-acylhomoserine lactone (AHL), and negatively regulated by RsmA, a post-transcriptional regulator. RsmA, an RNA-binding protein, causes translational repression and promotes RNA decay. Our data show that RsmA negatively regulates flhDC and fliA expression. Moreover, the chemical stabilities of transcripts of these genes are greater in an RsmA– mutant than in RsmA+ bacteria. These observations contrast with positive regulation of flhDC and motility by CsrA (= RsmA) in Escherichia coli. In the absence of AHL, the AHL receptors ExpR1/ExpR2 (= AhlR) in Erwinia carotovora subsp. carotovora negatively regulate motility and expression of flhDC and fliA by activating RsmA production. In the presence of AHL, regulatory effects of ExpR1/ExpR2 are neutralized, resulting in reduced levels of rsmA expression and enhanced motility.
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Carlesso, N., D. A. Frank, and J. D. Griffin. "Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl." Journal of Experimental Medicine 183, no. 3 (March 1, 1996): 811–20. http://dx.doi.org/10.1084/jem.183.3.811.
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Bcr/Abl is a chimeric oncogene that can cause both acute and chronic human leukemias. Bcr/Abl-encoded proteins exhibit elevated kinase activity compared to c-Abl, but the mechanisms of transformation are largely unknown. Some of the biological effects of Bcr/Abl overlap with those of hematopoietic cytokines, particularly interleukin 3 (IL-3). Such effects include mitogenesis, enhanced survival, and enhanced basophilic differentiation. Therefore, it has been suggested that p210Bcr/Abl and the IL-3 receptor may activate some common signal transduction pathways. An important pathway for IL-3 signaling involves activation of the Janus family kinases (JAKs) and subsequent tyrosyl phosphorylation of STAT proteins (signal transducers and activators of transcription). This pathway directly links growth factor receptors to gene transcription. We analyzed JAK activation, STAT protein phosphorylation, and the formation of specific DNA-binding complexes containing STAT proteins, in a series of leukemia cell lines transformed by Bcr/Abl or other oncogenes. We also examined these events in cell lines transformed by a temperature sensitive (ts) mutant of Bcr/Abl, where the kinase activity of Abl could be regulated. STAT1 and STAT5 were found to be constitutively phosphorylated in 32D, Ba/F3, and TF-1 cells transformed by Bcr/Abl, but not in the untransformed parental cell lines in the absence of IL-3. Phosphorylation of STAT1 and STAT5 was also observed in the human leukemia cell lines K562 and BV173, which express the Bcr/Abl oncogene, but not in several Bcr/Abl-negative leukemia cell lines. Phosphorylation of STAT1 and STAT5 was directly due to the tyrosine kinase activity of Bcr/Abl since it could be activated or deactivated by temperature shifting of cells expressing the Bcr/Abl ts mutant. DNA-STAT complexes were detected in all Bcr/Abl-transformed cell lines and they were supershifted by antibodies against STAT1 and STAT5. DNA-STAT complexes in 32Dp210Bcr/Abl cells were similar, but not identical, to those formed after IL-3 stimulation. It is interesting to note that JAK kinases (JAK1, JAK2, JAK3, and Tyk2) were not consistently activated in Bcr/Abl-positive cells. These data suggest that STATs can be activated directly by Bcr/Abl, possibly bypassing JAK family kinase activation. Overall, our results suggest a novel mechanism that could contribute to some of the major biological effects of Bcr/Abl transformation.
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Swimm, Alyson I., William Bornmann, Mengxi Jiang, Michael J. Imperiale, Aron E. Lukacher, and Daniel Kalman. "Abl Family Tyrosine Kinases Regulate Sialylated Ganglioside Receptors for Polyomavirus." Journal of Virology 84, no. 9 (February 24, 2010): 4243–51. http://dx.doi.org/10.1128/jvi.00129-10.
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ABSTRACT Sialylated lipids serve as cellular receptors for polyomaviruses. Using pharmacological inhibitors and cell lines derived from knockout mice, we demonstrate that Abl family tyrosine kinases are required for replication of mouse polyomavirus and BK virus, a human polyomavirus associated with allograft failure following kidney transplantation. We show that decreasing Abl family kinase activity results in low levels of cell surface ganglioside receptors for mouse polyomavirus and that inhibition of sialidase activity promotes virion binding in the absence of Abl family kinase activity. These data provide evidence that Abl family kinases reduce ganglioside turnover in the plasma membrane by inhibiting host cell sialidase activity. Thus, Abl family kinases regulate the susceptibility of cells to polyomavirus infection by modulating gangliosides required for viral attachment.
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Hattersley, Gary, Thomas Dean, Braden A. Corbin, Hila Bahar, and Thomas J. Gardella. "Binding Selectivity of Abaloparatide for PTH-Type-1-Receptor Conformations and Effects on Downstream Signaling." Endocrinology 157, no. 1 (January 1, 2016): 141–49. http://dx.doi.org/10.1210/en.2015-1726.
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Abstract The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1–34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1–34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1–34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R0 and RG, and that efficient binding to R0 results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R0 PTHR1 conformation than does PTH (1–34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1–34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone.
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Manley, Paul W., Sandra W. Cowan-Jacob, Robert Cozens, Gabriele Fendrich, Wolfgang Jahnke, Johannes L. Roesel, and Doriano Fabbro. "Fingolimod (FTY720) Inhibits BCR-ABL Signaling Allosterically by Binding to the Myristate Binding Site." Blood 118, no. 21 (November 18, 2011): 2746. http://dx.doi.org/10.1182/blood.v118.21.2746.2746.
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Abstract Abstract 2746 Background: The tyrosine kinase (TK) activity of the ABL protein is normally tightly regulated, with the N-terminal cap (NCap) region (≈ 80 amino-acid residues) of the SH3 domain playing an important role. One regulatory mechanism involves the NCap Gly-2 residue being myristoylated and then interacting with a myristate binding site within the SH1 catalytic domain. Chronic myeloid leukemia (CML) results from a chromosome translocation leading to expression of the chimeric BCR-ABL oncoprotein, which lacks the NCap and has constitutively active TK activity. Although drugs that inhibit the TK activity of the BCR-ABL oncoprotein via an ATP-competitive mechanism are effective in the treatment of chronic myeloid leukemia (CML), some patients relapse due to the emergence of drug-resistant clones, in which mutations in the SH1 domain compromise inhibitor binding. Recently, we have shown that compounds that bind to the SH1 myristate pocket can also inhibit the TK activity of BCR-ABL, and that these maintain activity against mutant enzymes (Nature 2010;263:501). FTY720 is a drug approved for the treatment of multiple sclerosis which, upon phosphorylation, targets sphingosine-1-phosphate receptors leading to immunomodulatory activity. FTY720 also activates the tumour suppressor protein phosphatase 2A, that is inactivated in CML and upon reactivation impairs BCR-ABL expression and function, leading to growth suppression, enhanced apoptosis, impaired clonogenicity, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR-ABL expressing cell lines and primary myeloid BP-CML cells (Br J Cancer 2006;95:775). Based upon structural insight it was postulated that FTY720 might bind to the ABL myristate site, and here we report on both the binding and allosteric inhibition of ABL TK activity. Methods and Results: In order to evaluate whether FTY720 could interact with ABL, nuclear magnetic resonance (NMR) studies were employed: In a conformational assay (JACS 2010;132:7043), FTY720 induced bending of the SH1 C-terminal helix I, that is a prerequisite for inhibitory activity of allosteric BCR-ABL ligands, and showed a binding affinity, Kd of 3 μM. The effects of FTY720 and it's phosphorylated analogue on the kinase activity of ABL64–515 (containing SH3SH2SH1 domains, lacking the C-terminal domain encoded by exon 11) and ABL229–515 (SH1 domain only) were determined using radiometic filter-binding assays using Poly-(AlaGluLysTyr) as protein substrate (Biochim Biophys Acta 2010;1804:454). FTY720 dose-dependently inhibited the transphosphorylation activity of ABL64–515 with an IC50 value of 1.4 ± 1.1 μM, but did not effect the catalytic activity of ABL229–515 at concentrations < 25 μM; furthermore phosphorylated-FTY720 was inactive. However, in pharmacological assays FTY720 only weakly inhibited the BCR-ABL dependent proliferation of either murine 32D or Ba/F3 cells (GI50 > 3 μM) and had, in comparison to nilotinib, modest dose-dependent effects in a murine leukemia model (52% reduction in growth compared to vehicle-treated controls at 10 mg/kg p.o. q24h; nilotinib gave 89% reduction at 20 mg/kg q24h). Discussion: Biochemical and biophysical data confirm that FTY720 is an allosteric inhibitor of the TK activity of ABL. Although the extent of this effect is insufficient to produce substantial efficacy in BCR-ABL transfected cells, the findings are of mechanistic interest since they probably contribute to the previously published effects of FTY720 in CML models and provide a new structural lead for allosteric inhibitors of ABL. Disclosures: Manley: Novartis Pharma AG: Employment. Cowan-Jacob:Novartis Pharma AG: Employment. Cozens:Novartis Pharma AG: Employment. Fendrich:Novartis Pharma AG: Employment. Jahnke:Novartis Pharma AG: Employment. Roesel:Novartis Pharma AG: Employment. Fabbro:Novartis Pharma AG: Employment.
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Forster-Gibson, Cynthia J., Yu Xiao Fei, and Michael J. Dufresne. "The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells." Biochemistry and Cell Biology 66, no. 12 (December 1, 1988): 1278–86. http://dx.doi.org/10.1139/o88-148.
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Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p < 0.005), two had approximately the same, and two had significantly higher (p < 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 ± 0.09 for clone 1 (Hs-1) and 0.94 ± 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p < 0.005), two had approximately the same, and one had significantly higher levels (p < 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).
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Chen, Ru, Gwen Mahon, and Ian P. Whitehead. "Ubiquitin-Mediated Binding and Stabilization of β-Catenin by p210 BCR/ABL." Blood 114, no. 22 (November 20, 2009): 3278. http://dx.doi.org/10.1182/blood.v114.22.3278.3278.
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Abstract Abstract 3278 Poster Board III-1 Chronic myelogenous leukemia (CML) is a malignant hematopoietic stem cell disorder that is invariably associated with a balanced reciprocal translocation between chromosomes 22 and 9. The major product of the rearrangement is a 210 kD in-frame fusion protein (p210 BCR/ABL) that contains amino-terminal sequences from BCR, and carboxy-terminal sequences from the non-receptor tyrosine kinase ABL. This fusion event leads to the constitutive activation of the ABL-encoded kinase activity, which is the principal driving force behind CML. Thus, inhibitors that target the kinase activity produce durable clinical remissions. Although the ABL-encoded kinase activity is essential for disease progression, several studies have shown that BCR encoded sequences are also necessary for BCR/ABL-mediated leukemogenesis. In this current study we have identified ubiquitin as a binding partner for BCR, and demonstrated that binding is retained in p210 BCR/ABL. We have localized the docking site for ubiquitin to residues 180-191 which is present in all fusion variants of BCR/ABL (p190, p210 and p230). This putative ubiquitin binding domain (UBD) does not conform to any known consensus sequence, and is unique to BCR. Deletion of the UBD does not impair the auto-or trans-kinase activity of p210 BCR/ABL, nor does it impair the interaction between p210 BCR/ABL and GRB2, or the ability of p210 BCR/ABL to activate ERK1/2. A mutation at residue tyr-177 of p210 BCR/ABL also does not impair the interaction with ubiquitin suggesting that the GRB2 and ubiquitin binding sites are adjacent, but separable. β-catenin has been identified as a binding partner for BCR and BCR/ABL, and the docking site has been mapped to a region of BCR/ABL that contains the UBD. Over-expression of p210 BCR/ABL, but not the ubiquitin binding mutant, leads to an accumulation of β-catenin that is phosphorylated on the serine residues that normally trigger ubiquitin-mediated turnover. This difference cannot be attributed to a difference in the activation status of GSK-3β or Pin-1. These results suggest that either β-catenin and ubiquitin share a docking site on p210 BCR/ABL, or that the interaction between β-catenin and p210 BCR/ABL is ubiquitination dependent. The latter possibility is consistent with a previous study which showed that glycine residue in β-catenin is required for Bcr interaction. We propose a model in which p210 BCR/ABL may influence the Wnt signaling pathway by binding ubiquitinated β-catenin, and stabilizing it against degradation. Disclosures: No relevant conflicts of interest to declare.
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Lalli, Enzo, Kenji Ohe, Colette Hindelang, and Paolo Sassone-Corsi. "Orphan Receptor DAX-1 Is a Shuttling RNA Binding Protein Associated with Polyribosomes via mRNA." Molecular and Cellular Biology 20, no. 13 (July 1, 2000): 4910–21. http://dx.doi.org/10.1128/mcb.20.13.4910-4921.2000.
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ABSTRACT The DAX-1 (NR0B1) gene encodes an unusual member of the nuclear hormone receptor superfamily which acts as a transcriptional repressor. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita (AHC) associated with hypogonadotropic hypogonadism (HHG). We have studied the intracellular localization of the DAX-1 protein in human adrenal cortex and mouse Leydig tumor cells and found it to be both nuclear and cytoplasmic. A significant proportion of DAX-1 is associated with polyribosomes and is found complexed with polyadenylated RNA. DAX-1 directly binds to RNA, two domains within the protein being responsible for cooperative binding activity and specificity. Mutations in DAX-1 found in AHC-HHG patients significantly impair RNA binding. These findings reveal that DAX-1 plays multiple regulatory roles at the transcriptional and posttranscriptional levels.
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Lambert, Que T., Anuradha Pradhan, J. Devon Roll, and Gary W. Reuther. "Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity." Biochemical Journal 438, no. 1 (July 27, 2011): 155–64. http://dx.doi.org/10.1042/bj20110351.
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Cytokines and their receptors regulate haemopoiesis by controlling cellular growth, survival and differentiation. Thus it is not surprising that mutations of cytokine receptors contribute to the formation of haemopoietic disorders, including cancer. We recently identified transforming properties of IL27R, the ligand-binding component of the receptor for interleukin-27. Although wild-type IL27R exhibits transforming properties in haemopoietic cells, in the present study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations identified involve deletions of amino acids in the cytoplasmic juxtamembrane region of the receptor. Expression of each of these mutant IL27R proteins led to rapid cytokine-independent transformation in haemopoietic cells. Moreover, the rate of transformation induced by these mutants was significantly greater than that induced by wild-type IL27R. Expression of these IL27R mutants also induced enhanced activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling compared with wild-type. An activating deletion mutation of IL27R enhanced homodimerization of the receptor by a mechanism that may involve disulfide bonding. These transforming IL27R mutants displayed equal or greater transforming activity than bona fide haemopoietic oncogenes such as BCR–ABL (breakpoint cluster region–Abelson murine leukaemia viral oncogene homologue) and JAK2-V617F. Since IL27R is expressed on haemopoietic stem cells, lymphoid cells and myeloid cells, including acute myeloid leukaemia blast cells, mutation of this receptor has the potential to contribute to a variety of haemopoietic neoplasms.
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Trampont, Paul C., Li Zhang, Amber J. Giles, Scott F. Walk, Jing J. Gu, Ann Marie Pendergast, and Kodi S. Ravichandran. "ShcA Regulates Thymocyte Proliferation through Specific Transcription Factors and a c-Abl-Dependent Signaling Axis." Molecular and Cellular Biology 35, no. 8 (February 17, 2015): 1462–76. http://dx.doi.org/10.1128/mcb.01084-14.
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Signaling via the pre-T-cell receptor (pre-TCR), along with associated signals from Notch and chemokine receptors, regulates the β-selection checkpoint that operates on CD4−CD8−doubly negative (DN) thymocytes. Since many hematopoietic malignancies arise at the immature developmental stages of lymphocytes, understanding the signal integration and how specific signaling molecules and distal transcription factors regulate cellular outcomes is of importance. Here, a series of molecular and genetic approaches revealed that the ShcA adapter protein critically influences proliferation and differentiation during β-selection. We found that ShcA functions downstream of the pre-TCR and p56Lckand show that ShcA is important for extracellular signal-regulated kinase (ERK)-dependent upregulation of transcription factors early growth factor 1 (Egr1) and Egr3 in immature thymocytes and, in turn, of the expression and function of the Id3 and E2A helix-loop-helix (HLH) proteins. ShcA also contributes to pre-TCR-mediated induction of c-Myc and additional cell cycle regulators. Moreover, using an unbiasedSaccharomyces cerevisiae(yeast) screen, we identified c-Abl as a binding partner of phosphorylated ShcA and demonstrated the relevance of the ShcA–c-Abl interaction in immature thymocytes. Collectively, these data identify multiple modes by which ShcA can fine-tune the development of early thymocytes, including a previously unappreciated ShcA–c-Abl axis that regulates thymocyte proliferation.
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Gely-Pernot, Aurore, Mathilde Raverdeau, Catherine Célébi, Christine Dennefeld, Betty Feret, Muriel Klopfenstein, Shosei Yoshida, Norbert B. Ghyselinck, and Manuel Mark. "Spermatogonia Differentiation Requires Retinoic Acid Receptor γ." Endocrinology 153, no. 1 (January 1, 2012): 438–49. http://dx.doi.org/10.1210/en.2011-1102.
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Vitamin A is instrumental to mammalian reproduction. Its metabolite, retinoic acid (RA), acts in a hormone-like manner through binding to and activating three nuclear receptor isotypes, RA receptor (RAR)α (RARA), RARβ, and RARγ (RARG). Here, we show that 1) RARG is expressed by A aligned (Aal) spermatogonia, as well as during the transition from Aal to A1 spermatogonia, which is known to require RA; and 2) ablation of Rarg, either in the whole mouse or specifically in spermatogonia, does not affect meiosis and spermiogenesis but impairs the Aal to A1 transition in the course of some of the seminiferous epithelium cycles. Upon ageing, this phenomenon yields seminiferous tubules containing only spermatogonia and Sertoli cells. Altogether, our findings indicate that RARG cell-autonomously transduces, in undifferentiated spermatogonia of adult testes, a RA signal critical for spermatogenesis. During the prepubertal spermatogenic wave, the loss of RARG function can however be compensated by RARA, as indicated by the normal timing of appearance of meiotic cells in Rarg-null testes. Accordingly, RARG- and RARA-selective agonists are both able to stimulate Stra8 expression in wild-type prepubertal testes. Interestingly, inactivation of Rarg does not impair expression of the spermatogonia differentiation markers Kit and Stra8, contrary to vitamin A deficiency. This latter observation supports the notion that the RA-signaling pathway previously shown to operate in Sertoli cells also participates in spermatogonia differentiation.
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Ptasznik, Andrzej, Elzbieta Urbanowska, Suneetha Chinta, Melinda A. Costa, Benjamin A. Katz, Marisha A. Stanislaus, Gokhan Demir, Diana Linnekin, Zhixing K. Pan, and Alan M. Gewirtz. "Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells." Journal of Experimental Medicine 196, no. 5 (September 2, 2002): 667–78. http://dx.doi.org/10.1084/jem.20020519.
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Stromal-derived factor (SDF)-1 and its G protein–coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein–coupled receptor signaling and function of mammalian precursors.
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Piskorska-Pliszczynska, J., B. Keys, S. Safe, and M. S. Newman. "The cytosolic receptor binding affinities and ahh induction potencies of 29 polynuclear aromatic hydrocarbons." Toxicology Letters 34, no. 1 (November 1986): 67–74. http://dx.doi.org/10.1016/0378-4274(86)90146-3.
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Dewar, Andrea L., Antony C. Cambareri, Andrew C. W. Zannettino, Bernadette L. Miller, Kathleen V. Doherty, Timothy P. Hughes, and A. Bruce Lyons. "Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib." Blood 105, no. 8 (April 15, 2005): 3127–32. http://dx.doi.org/10.1182/blood-2004-10-3967.
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AbstractImatinib is a tyrosine kinase inhibitor that suppresses the growth of bcr-abl–expressing chronic myeloid leukemia (CML) progenitor cells by blockade of the adenosine triphosphate (ATP)–binding site of the kinase domain of bcr-abl. Imatinib also inhibits the c-abl, platelet-derived growth factor (PDGF) receptor, abl-related gene (ARG) and stem-cell factor (SCF) receptor tyrosine kinases, and has been used clinically to inhibit the growth of malignant cells in patients with CML and gastrointestinal stromal tumors (GISTs). Although initially considered to have minimal effects of normal hematopoiesis, recent studies show that imatinib also inhibits the growth of some nonmalignant hematopoietic cells, including monocyte/macrophages. This inhibition could not be attributed to the known activity profile of imatinib. Here, we demonstrate for the first time that imatinib targets the macrophage colony-stimulating factor (M-CSF) receptor c-fms. Phosphorylation of c-fms was inhibited by therapeutic concentrations of imatinib, and this was not due to down-regulation in c-fms expression. Imatinib was also found to inhibit M-CSF–induced proliferation of a cytokine–dependent cell line, further supporting the hypothesis that imatinib affects the growth and development of monocyte and/or macrophages through inhibition of c-fms signaling. Importantly, these results identify an additional biologic target to those already defined for imatinib. Imatinib should now be assessed for activity in diseases where c-fms activation is implicated, including breast and ovarian cancer and inflammatory conditions.
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Miñambres, Inka, Rosa Corcoy, Anthony P. Weetman, and E. Helen Kemp. "Autoimmune Hypercalcemia Due to Autoantibodies Against the Calcium-sensing Receptor." Journal of Clinical Endocrinology & Metabolism 105, no. 7 (April 20, 2020): 2229–36. http://dx.doi.org/10.1210/clinem/dgaa219.
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Abstract Context Autoimmune hypocalciuric hypercalcemia (AHH) is an acquired disorder caused by the presence of blocking autoantibodies against the calcium-sensing receptor (CaSR). Few cases of this condition have been described to date in the literature. Objective The objectives of this study were to describe 2 patients in whom the presence of AHH was suspected and to assess the patients for the presence of CaSR antibodies. Methods CaSR antibodies were detected and characterised by immunoprecipitation assays, CaSR peptide ELISAs, and functional assays based on the calcium-stimulated accumulation of inositol-1-phosphate in a mammalian cell line expressing the CaSR. Results Both patients presented with an acquired form of hypocalciuric hypercalcemia. Mutational analyses of CASR, GNA11, and AP2S1 for familial hypocalciuric hypercalcemia were negative. According to the presence of Hashimoto’s disease in 1 patient and latent autoimmune diabetes of adulthood and thyroid autoimmunity in the other, AHH was suspected. Immunoprecipitation assays detected CaSR antibodies in both patients. Analysis of the antibody binding sites revealed 2 main epitopes at amino acids 41–69 and 114–126. Preincubation with purified CaSR antibodies against epitope 114–126 resulted in a significant decrease in inositol-1-phophate accumulation upon calcium-stimulation of mammalian cells expressing the CaSR, suggesting that the antibodies had receptor-blocking activity. Conclusions AHH is to be suspected in patients with an acquired biochemical pattern of PTH-dependant hypocalciuric hypercalcemia, especially in those with other concomitant autoimmune diseases. Diagnosis by means of detecting CaSR antibodies may help to better characterise this probably under-reported condition.
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Ring, Colleen, Mark H. Ginsberg, Jacob Haling, and Ann Marie Pendergast. "Abl-interactor-1 (Abi1) has a role in cardiovascular and placental development and is a binding partner of the α4 integrin." Proceedings of the National Academy of Sciences 108, no. 1 (December 20, 2010): 149–54. http://dx.doi.org/10.1073/pnas.1012316108.
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Dynamic signals linking the actin cytoskeleton and cell adhesion receptors are essential for morphogenesis during development and normal tissue homeostasis. Abi1 is a central regulator of actin polymerization through interactions with multiple protein complexes. However, the in vivo role of Abi1 remains to be defined. The α4 integrin adhesion receptor is associated with enhanced protrusive activity and regulation of directional cell migration. Among integrin subunits, α4 exhibits unique properties in that it predominantly accumulates at the leading edge of migrating cells; however, the pathways that link the actin-regulatory machinery to α4 at the leading edge have remained elusive. We generated Abi1 KO mice and found that loss of Abi1 phenocopies KO of α4. Mice lacking Abi1 or α4 exhibit midgestational lethality with abnormalities in placental and cardiovascular development. Notably, purified Abi1 protein binds directly to the α4 cytoplasmic tail and endogenous Abi1 colocalizes with phosphorylated α4 at the leading edge of spreading cells. Moreover, Abi1-deficient cells expressing α4 have impaired cell spreading, which is rescued by WT Abi1 but not an Abi1 mutant lacking the α4-binding site. These data reveal a direct link between the α4 integrin and actin polymerization and uncover a role for Abi1 in the regulation of morphogenesis in vivo. The Abi1–α4 interaction establishes a mechanistic paradigm for signaling between adhesion events and enhanced actin polymerization at the earliest stages of protrusion.
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Huang, Ying, Allan B. Okey, and Patricia A. Harper. "Aromatic hydrocarbon receptor in cultured fetal cells from C57BL/6J and DBA/2J mice: similarity in molecular mass to receptors in adult livers." Canadian Journal of Physiology and Pharmacology 73, no. 1 (January 1, 1995): 18–26. http://dx.doi.org/10.1139/y95-003.
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In liver of adult responsive C57BL/6J (B6) mice the aromatic hydrocarbon receptor (AHR) has high affinity for specific halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as nonhalogenated aromatic hydrocarbons (PAHs), such as benz[a]anthracene (BA) or 3-methylcholanthrene (MC). In livers of adult nonresponsive DBA/2J (D2) mice TCDD binds to a low-affinity variant form of AHR. Both TCDD and MC induce aryl hydrocarbon hydroxylase (AHH) in adult B6 mice, whereas adult D2 mouse liver is nonresponsive to MC. In fetal cell cultures derived from D2 mice AHH is induced by PAHs such as MC or BA, and these PAHs bind to cytosolic AHR (P.A. Harper, C.L. Golas, and A.B. Okey. Mol. Pharmacol. 40: 818–826, 1991). We compared AHR from fetal cell cultures with AHR from adult livers to determine whether there was some structural difference in receptors expressed in fetal cell culture that might permit cells from "nonresponsive" mice to respond to PAHs. The apparent molecular mass of AHR from cells cultured from 18-day fetuses is identical with that from adult liver within each strain of inbred mice tested (Mr ~ 95 kDa in B6 and ~ 105 kDa in D2 mice). The AHR in D2 fetal cells was able to activate a transfected chloramphenicol acetyltransferase linked to a dioxin-responsive element nucleotide sequence (DRE–CAT) when the cells were treated with TCDD or MC. The potency of CAT expression in D2 fetal cells was similar to that in B6 fetal cells. Our data suggest that the responsiveness of fetal cells from "nonresponsive" mice is likely mediated by AHR in these cells but is not due to expression of a different allelic form of AHR ligand-binding subunit in fetal cells versus adult liver.Key words: aromatic hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin, cultured fetal cells, C57BL/6J mice, DBA/2J mice.
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Fantin, Alessandro, Anastasia Lampropoulou, Valentina Senatore, James T. Brash, Claudia Prahst, Clemens A. Lange, Sidath E. Liyanage, et al. "VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation." Journal of Experimental Medicine 214, no. 4 (March 13, 2017): 1049–64. http://dx.doi.org/10.1084/jem.20160311.
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The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth.
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Danial, Nika N., Julie A. Losman, Tianhong Lu, Natalie Yip, Kartik Krishnan, John Krolewski, Stephen P. Goff, Jean Y. J. Wang, and Paul B. Rothman. "Direct Interaction of Jak1 and v-Abl Is Required for v-Abl-Induced Activation of STATs and Proliferation." Molecular and Cellular Biology 18, no. 11 (November 1, 1998): 6795–804. http://dx.doi.org/10.1128/mcb.18.11.6795.
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ABSTRACT In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl–Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abloncogene.
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Safe, Stephen, Stelvio Bandiera, Tom Sawyer, Bozena Zmudzka, Grant Mason, Marjorie Romkes, Mary Anne Denomme, Judy Sparling, Allan B. Okey, and Toshio Fujita. "Effects of Structure on Binding to the 2,3,7,8-TCDD Receptor Protein and AHH Induction. Halogenated Biphenyls." Environmental Health Perspectives 61 (September 1985): 21. http://dx.doi.org/10.2307/3430059.
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Safe, S., S. Bandiera, T. Sawyer, B. Zmudzka, G. Mason, M. Romkes, M. A. Denomme, J. Sparling, A. B. Okey, and T. Fujita. "Effects of structure on binding to the 2,3,7,8-TCDD receptor protein and AHH induction--halogenated biphenyls." Environmental Health Perspectives 61 (September 1985): 21–33. http://dx.doi.org/10.1289/ehp.856121.
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Tauchi, T., H. S. Boswell, D. Leibowitz, and H. E. Broxmeyer. "Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway." Journal of Experimental Medicine 179, no. 1 (January 1, 1994): 167–75. http://dx.doi.org/10.1084/jem.179.1.167.
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Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.
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Neumeister, E. N., Y. Zhu, S. Richard, C. Terhorst, A. C. Chan, and A. S. Shaw. "Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins." Molecular and Cellular Biology 15, no. 6 (June 1995): 3171–78. http://dx.doi.org/10.1128/mcb.15.6.3171.
Full textAbstract:
ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.
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Tamura, Tomohiko, Hee Jeong Kong, Chainarong Tunyaplin, Hideki Tsujimura, Kathryn Calame, and Keiko Ozato. "ICSBP/IRF-8 inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid progenitor cells." Blood 102, no. 13 (December 15, 2003): 4547–54. http://dx.doi.org/10.1182/blood-2003-01-0291.
Full textAbstract:
AbstractInterferon consensus sequence binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) is a transcription factor that controls myeloid cell development. ICSBP-/- mice develop a chronic myelogenous leukemia (CML)-like syndrome. Several observations on patients and mouse models have implicated ICSBP in the pathogenesis of CML. In this paper, we investigated whether ICSBP modulates the growth-promoting activity of Bcr/Abl, the causal oncoprotein for CML. When transformed with p210 Bcr/Abl, ICSBP-/- myeloid progenitor cells lost growth factor dependence and grew in the absence of granulocyte-macrophage colony-stimulating factor. When ICSBP was ectopically expressed, Bcr/Abl-transformed cells underwent complete growth arrest and differentiated into mature, functional macrophages without inhibiting the kinase activity of Bcr/Abl. Providing a mechanistic basis for the growth arrest, ICSBP markedly repressed c-Myc messenger RNA (mRNA)-expression, a downstream target of Bcr/Abl. A further analysis with the ICSBP/estrogen receptor chimera showed that ICSBP repression of c-Myc is indirect and is mediated by another gene(s). We identified Blimp-1 and METS/PE1, potent c-Myc repressors, as direct targets of ICSBP activated in these cells. Consistent with this, ectopic Blimp-1 repressed c-Myc expression and inhibited cell growth. These results indicate that ICSBP inhibits growth of Bcr/Abl-transformed myeloid progenitor cells by activating several genes that interfere with the c-Myc pathway. (Blood. 2003;102:4547-4554)
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Aichberger, Karl J., Matthias Mayerhofer, Anja Vales, Maria-Theresa Krauth, Karoline V. Gleixner, Martin Bilban, Harald Esterbauer, et al. "The CML-related oncoprotein BCR/ABL induces expression of histidine decarboxylase (HDC) and the synthesis of histamine in leukemic cells." Blood 108, no. 10 (November 15, 2006): 3538–47. http://dx.doi.org/10.1182/blood-2005-12-028456.
Full textAbstract:
Abstract Basophil numbers are typically elevated in chronic myeloid leukemia (CML) and increase during disease progression. Histamine is an essential mediator and marker of basophils and is highly up-regulated in CML. We examined the biochemical basis of histamine synthesis in CML cells. The CML-specific oncoprotein BCR/ABL was found to promote expression of histidine decarboxylase (HDC) and synthesis of histamine in Ba/F3 cells. Moreover, the BCR/ABL tyrosine kinase inhibitors imatinib (STI571) and nilotinib (AMN107) decreased histamine levels and HDC mRNA expression in BCR/ABL-transformed Ba/F3 cells, in the CML-derived basophil cell line KU812, and in primary CML cells. Synthesis of histamine was found to be restricted to the basophil compartment of the CML clone and to depend on signaling through the PI3-kinase pathway. CML cells also expressed histamine receptors (HRs), including HR-1, HR-2, HR-4, and histamine-binding CYP450 isoenzymes which also serve as targets of HR antagonists. The HR-1 antagonists loratadine and terfenadine, which bind to CYP450, were found to counteract proliferation of CML cells, whereas no growth inhibition was observed with the HR-1 antagonist fexofenadine which is not targeted or metabolized by CYP450. Moreover, DPPE, an inhibitor of histamine-binding CYP450 isoenzymes, produced growth inhibition in CML cells. Together, these data show that BCR/ABL promotes histamine production in CML cells and that certain HR-targeting drugs exert antileukemic effects on CML cells.