Genomes and Genes
Signaling by Cytoplasmic Tyrosine Kinases in Leukocytes
Principal Investigator: Clifford A Lowell
Abstract: DESCRIPTION (provided by applicant): The Src-family tyrosine kinases play pivotal roles in initiating intracellular signaling from a diverse repertoire of receptors on innate immune leukocytes. The Src-family kinases initiate intracellular signaling from these receptors by phosphorylating specific tyrosine residues within ITAM (Immunoreceptor Tyrosine-based Activation Motifs) domains that are either part of the cytoplasmic domain of the receptor or are located on receptor-associated signaling adapters, such as the FcR? or DAP-12 molecules. Phosphorylation of these ITAM domains leads to recruitment of Syk/Zap70 kinases which in turn initiate downstream signaling, leading to cellular activation. Src-family kinases also phosphorylate inhibitory receptors on specific tyrosines within ITIM (Immunoreceptor Tyrosine-based Inhibitory Motifs) domains that are located within the cytoplasmic regions of each receptor. ITIM phosphorylation leads to recruitment of the tyrosine phosphatase SHP-1, which in turn dephosphorylates both the Src kinases as well as downstream substrates in the signaling cascade. The overall balance between activating and inhibitory signaling determines the cellular response. As a result, the functions of the Src-family kinases are opposed in large part by SHP-1, which serves as a major brake to intracellular signaling in innate cells. Mutation of the SHP-1 gene (Ptpn6) results in severely hyperactive lymphocytes, myeloid cells and platelets. As a result, SHP-1 deficient animals (motheaten (me/me) or motheaten viable (mev/mev)) develop dramatic autoimmunity, inflammation and early mortality due to lung inflammation (~4 weeks for complete SHP-1 loss in me/me mice and ~8-10 weeks in the hypomorphic mev/mev mice). Though these SHP-1 deficient animals have been available for years, the complex interactions and indirect effects between the many hyperactive leukocyte subsets has limited their utility. Thus it remains unclear which specific SHP-1 regulated signaling pathways in each of the different hyperactive leukocyte types is responsible for the different aspects of the autoimmune/inflammatory disease. To address this problem, we have bred the Ptpn6flx/flx (SHP-1flx) mice to a series of Cre expressing animals to achieve lineage-specific deletion of SHP-1. Deletion of SHP-1 specifically in neutrophils (using MRP8-Cre) recapitulates much of the inflammatory disease in present in mev/mev mice but the animals don't get autoimmunity. Deletion of SHP-1 in dendritic cells (using CD11c-Cre) results in autoimmunity, but no inflammation. Deletion of SHP-1 in both neutrophils and macrophages (using LysM-Cre) results in no phenotype at all (despite the presence of activated neutrophils). Finally, deletion of SHP-1 in platelets (using PF4-Cre) results in early mortality due to lung inflammation, without evidence of other disease. By subdividing the overall disease phenotype caused by global SHP-1 deficiency, this set of lineage-specific mutant mice will allow us to characterize the in vivo physiologic functions of SHP-1 regulated signaling in defined leukocytes which has been impossible to do with previous animal models.. We hypothesize that dysregulation of integrin and Toll-like receptor (TLR) signaling are the major drivers to the inflammatory versus autoimmune phenotypes in these different conditional SHP-1 mutant mice. We will examine this using both biochemical and genetic approaches, the latter of which will depend on the generation of double floxed mice that delete specific signaling pathways in each conditional murine line (for example SHP-1flxMyD88flxCD11c-Cre). We will also carry out unbiased screens of signaling pathways in the cells from the various conditional mutant mice. Finally, we will carry out platelet studies and use a novel two-photon imaging method to examine platelet/neutrophil interactions in the inflamed lungs of SHP-1flxPF4-Cre mice, to determine the mechanisms by which dysregulation of platelet function can lead to lethal pulmonary inflammation. Overall, these studies will help define how hyperactivation of specific tyrosine kinase based signaling pathways leads to inflammation and autoimmunity. Since it is well appreciated that alterations in these signaling pathways play a major role in human autoimmune, inflammatory and malignant disease, a more complete in vivo dissection of SHP-1 regulated signaling will help guide therapeutic development. PUBLIC HEALTH RELEVANCE: Immune cells sense pathogens such as bacteria, fungi and viruses through a complex array of cellular receptors. Binding of the pathogen molecules to these receptors activates intracellular enzymes that initiate intracellular signaling pathways that lead to immune cell activation to eliminate the pathogen. Appropriate control of these intracellular signaling pathways is critical to prevent immune cell hyperactivation that can cause damage to host tissues during responses to pathogen invasion. One of the major molecules that downregulates immune cells are the tyrosine phosphatase SHP-1. We have generated a series of mutant mice that lack the SHP-1 protein in defined immune cell types. These conditional SHP-1 mutant animals develop a spectrum of diseases, including severe skin inflammation, autoimmunity or lethal pulmonary inflammation. By studying the specific intracellular signaling pathways that are affected by these different SHP-1 conditional mutations, we will gain a new insight into the regulation of immune cell reactivity. This will lead to better design of therapeutcs to target immune diseases, as well as leukemia and lymphoma.
Funding Period: 2000-08-01 - 2017-01-31
more information: NIH RePORT
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Department of Pathology, Center for Excellence in Vascular Biology, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
Immunity 25:271-83. 2006..These data suggest that Mac-1 engagement of complement activates tyrosine kinases to promote elastase-dependent blood vessel injury in vivo...
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Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
Dev Cell 24:372-83. 2013..By coupling to FcRγ, CD36 is able to engage Src-family kinases and Syk, which in turn drives the internalization of CD36 and its bound ligands...
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Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
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Division of Immunology, Cedars Sinai Medical Center, Los Angeles, California 90048, USA
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Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
J Thromb Haemost 10:1631-45. 2012..The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbβ3, and in vivo...
- Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injuryHelena Block
Department of Anesthesiology and Critical Care Medicine, University of Munster, 48149 Munster, Germany
J Exp Med 209:407-21. 2012..Thus, SLP-76 and ADAP are involved in E-selectin-mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion-induced AKI in humans...
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Immunology Program, Benaroya Research Institute, Seattle, WA 98101, USA
Proc Natl Acad Sci U S A 109:267-72. 2012..Therefore, BCAP is an essential activator of the PI3K pathway downstream of TLR signaling, providing a brake to limit potentially pathogenic excessive TLR responses...
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Department of Medicine, Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis, University of California, San Francisco, San Francisco, CA 94143, USA
Immunity 35:757-69. 2011..Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways...
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Section of General Pathology, Department of Pathology and Diagnostics, University of Verona, Verona 37134, Italy
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Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco 94143, USA
Immunity 38:489-501. 2013..Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease...
- STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defenseHong Zhang
Department of Laboratory Medicine
Blood 123:2238-49. 2014..These results demonstrate the critical role of STIM1-mediated SOCE and define major protein targets of calcium signaling in neutrophil activation during inflammatory disease. ..
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Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
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Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143
J Immunol 192:919-28. 2014..Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease. ..
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Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, United States of America
PLoS ONE 8:e77542. 2013..These data indicate that expression of TEL-Syk in fetal liver hematopoietic cells results in JAK-independent STAT5 phosphorylation ultimately leading to a uniquely aggressive and lethal form of myelofibrosis. ..
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Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women s Hospital and Harvard Medical School, Boston, Massachusetts 20115 email
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- Neutrophils mediate antibody-induced antitumor effects in miceMarcello Albanesi
Departement d Immunologie, Laboratoire Anticorps en Thérapie et Pathologie, Institut Pasteur, Paris, France
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- Hyperactivated MyD88 signaling in dendritic cells, through specific deletion of Lyn kinase, causes severe autoimmunity and inflammationChrystelle Lamagna
Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA
Proc Natl Acad Sci U S A 110:E3311-20. 2013..Thus, we demonstrate that hyperactivation of MyD88-dependent signaling in DCs is sufficient to drive pathogenesis of lupus-like disease, illuminating the fact that dysregulation in innate immune cells alone can lead to autoimmunity. ..
- Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursorsChristel Verollet
Centre National de la Recherche Scientifique CNRS, Unité Mixte de Recherche UMR 5089, Institut de Pharmacologie et de Biologie Structurale IPBS, Toulouse, France
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- Strain-dependent induction of neutrophil histamine production and cell death by Pseudomonas aeruginosaXiang Xu
Department of Medicine, University of California at San Francisco, San Francisco, California, USA
J Leukoc Biol 91:275-84. 2012..These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine...
- Deletion of Syk in neutrophils prevents immune complex arthritisEmily R Elliott
Biomedical Sciences Program, University of California, San Francisco, San Francisco, CA 94143, USA
J Immunol 187:4319-30. 2011..Basophil-deficient mice also responded normally to K/BxN serum transfer. These results demonstrate that Syk-dependent signaling in neutrophils alone is critically required for arthritis development in the serum transfer model...
- Generation of a novel system for studying spleen tyrosine kinase function in macrophages and B cellsAllison L Miller
Department of Laboratory Medicine, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA
J Immunol 182:988-98. 2009....
- Transmigration of neutrophils across inflamed endothelium is signaled through LFA-1 and Src family kinaseMelissa R Sarantos
Department of Biomedical Engineering, University of California, Davis, CA 95616, USA
J Immunol 181:8660-9. 2008..We conclude that dimeric bond clusters of LFA-1/ICAM-1 provide a key outside-in signal for orienting cytoskeletal dynamics that direct PMN extravasation at sites of inflammation...
- Myeloid Src kinases regulate phagocytosis and oxidative burst in pneumococcal meningitis by activating NADPH oxidaseRobert Paul
Department of Neurology, Klinikum Grosshadern, Ludwig Maximilians University, Marchioninistr 15, D 81377 Munich, Germany
J Leukoc Biol 84:1141-50. 2008..These data support the role of SFKs as critical mediators of CR3 signal transduction in host defense...
- The diverse functions of Src family kinases in macrophagesClare L Abram
Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA
Front Biosci 13:4426-50. 2008..In general, SFKs may function more like rheostats, influencing the amplitude of many pathways...
- Increased TLR responses in dendritic cells lacking the ITAM-containing adapters DAP12 and FcRgammaChing Liang Chu
Immunology Research Center, National Health Research Institutes, Taiwan
Eur J Immunol 38:166-73. 2008....
- Immunoreceptor-like signaling by beta 2 and beta 3 integrinsZoltan Jakus
Department of Physiology, Semmelweis University School of Medicine, 1088 Budapest, Hungary
Trends Cell Biol 17:493-501. 2007..These and other reports reveal an unexpected similarity between the signal-transduction mechanisms used by integrins and immune recognition receptors...
- Convergence of immunoreceptor and integrin signalingClare L Abram
Department of Laboratory Medicine, University of California, San Francisco, CA 94143 0451, USA
Immunol Rev 218:29-44. 2007..In this review, we discuss the convergence of immunoreceptor and integrin signaling, focusing on how these pathways modulate leukocyte activation...
- Neutrophil histamine contributes to inflammation in mycoplasma pneumoniaXiang Xu
Cardiovascular Research Institute, Department of Microbiology Immunology, University of California at San Francisco, and Veterans Affairs Medical Center, San Francisco 94121, USA
J Exp Med 203:2907-17. 2006..These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation...
- Multiple roles of Lyn kinase in myeloid cell signaling and functionPatrizia Scapini
Department of Laboratory Medicine, University of California, San Francisco, CA 94143 0451, USA
Immunol Rev 228:23-40. 2009..In this review, we discuss the current knowledge of the duplicitous nature of Lyn in the modulation of myeloid cell signaling and function...
- The ins and outs of leukocyte integrin signalingClare L Abram
Program in Immunology, Department of Laboratory Medicine, University of California, San Francisco, California 94143 0451, USA
Annu Rev Immunol 27:339-62. 2009....
- B cell-derived IL-10 suppresses inflammatory disease in Lyn-deficient micePatrizia Scapini
Department of Laboratory Medicine and Microbiology Immunology, University of California, San Francisco, CA 94143, USA
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- Overview: studying integrins in vivoClifford A Lowell
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA
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- Neutrophils give us a shockClifford A Lowell
Department of Laboratory Medicine, UCSF, San Francisco, California 94143 0451, USA
J Clin Invest 121:1260-3. 2011..These exciting results suggest that we have to reevaluate our models for anaphylaxis in humans, which will have a direct impact on our therapeutic approaches for prevention of this potential deadly hypersensitivity reaction...
- Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infectionLynn A Kamen
Program in Immunology, Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
J Immunol 186:1656-65. 2011..These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents...
- Myeloid cells, BAFF, and IFN-gamma establish an inflammatory loop that exacerbates autoimmunity in Lyn-deficient micePatrizia Scapini
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
J Exp Med 207:1757-73. 2010..Our findings uncover an important pathological role of BAFF in autoimmune disorders...
- The expanding roles of ITAM adapters FcRgamma and DAP12 in myeloid cellsJessica A Hamerman
Immunology Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA
Immunol Rev 232:42-58. 2009..In this review, we discuss the newly described receptors that utilize DAP12 and/or FcRgamma adapters to modulate innate immune responses...
- Neutrophil-specific deletion of Syk kinase results in reduced host defense to bacterial infectionJessica A Van Ziffle
Immunology Program and the Department of Laboratory Medicine, University of California San Francisco, 513 Parnassus Ave, San Francisco, CA 94143 0451, USA
Blood 114:4871-82. 2009..These results indicate that loss of Syk kinase-mediated integrin signaling impairs leukocyte activation, leading to reduced host defense responses...
- Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter miceClare L Abram
Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco, CA 94143, USA
J Immunol Methods 408:89-100. 2014..By focusing on myeloid subsets, we directly compare the relative efficiency and specificity of myeloid deletion in these strains under steady-state conditions. ..
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