Louis B Justement
University Of Alabama At Birmingham
Project start date: 1998-01-01
Project end date: 2013-06-30
Sponsored Links Excellgen http://Excellgen.com
CD19 TYROSINE-MEDIATED SIGNAL TRANSDUCTION IN VIVO
Louis B Justement
University Of Alabama At Birmingham, 1530 3rd Avenue South, Birmingham, Al 35294
Grant 5R01AI042265-12 from National Institute Of Allergy And Infectious Diseases
Abstract: B lymphocytes are required for host defense against infectious pathogens but abnormal activation of B cells can lead to autoimmunity. Thus, B cells are important in a wide range of physiologic and pathophysiologic processes. Mature B cells in the spleen enter into one of two compartments, the marginal zone or the follicular. Marginal zone B cells are responsible for rapid responses to pathogens that have reached the circulation. Follicular B cells are recruited into germinal centers, where affinity maturation and the generation of memory B cells lead to heightened responses upon subsequent encounters with a pathogen, which is crucial to immunization by vaccines. Mice lacking CD19, a B cell surface receptor, fail to form marginal zone B cells or germinal centers and have a propensity to produce autoantibodies. Our new data demonstrate that lack of marginal zone B cells results in defects in other components of the marginal zone, including marginal zone macrophages and dendritic cells. However, the macrophages and dendritic cells reappear following reconstitution of the marginal zone B cells by adoptive transfer of wild type B cells. Aim 1 will dissect the mechanisms by which CD19 on B cells regulates the differentiation of MZ B cells, and thereby other constituents of the marginal zone. Additional new preliminary data suggest that that failure to form germinal centers is associated with a failure of activation of Follicular Dendritic Cells (FDC) in CD19-/- mice. Adoptive transfer of wild type B cells also reconstitutes the ability to activate FDC in CD19-/- recipient mice, which recover the ability to form germinal centers. In Aim 2, we will test three hypotheses as to how CD19- dependent mechanisms regulate FDC through activation of follicular B cells, through effects on the marginal zone that control delivery of antigen to FDC, and through formation of immune complexes. In Aim 3, we will determine how CD19 regulates autoimmunity by experiments to test the role of CD19 in central selection, selection in the periphery, and in a model of autoimmunity. These aims are proposed not just to understand the function of CD19, but to use CD19 to probe basic mechanisms that are fundamental to immunization and protection from pathogens, without autoimmunity. The marginal zone of the spleen is required for survival of infections that have reached the blood while germinal centers are of fundamental importance in vaccination and immunological memory. Despite their crucial roles in protection from infections, the marginal zone and the germinal center responses are poorly understood. This project will use CD19, a protein expressed on B lymphocytes that is required for both types of response, as a tool to investigate the functions of B cells in the marginal zone and in germinal centers in vivo
Keywords: ATGN; Adhesions; Adoptive Transfer; Affinity; Angiitis; Antibody Formation; Antibody Production; Antibody Response; Antigen-Antibody Complex; Antigens; Autoantibodies; Autoimmune Diseases; Autoimmune Status; Autoimmunity; B blood cells; B-Cell Activation; B-Cells; B-Lymphocytes; Blood; Blood Circulation; Bloodstream; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; CD19; CD19 gene; CD19 tyrosine; Cell Communication and Signaling; Cell Locomotion; Cell Migration; Cell Movement; Cell Signaling; Cell Surface Receptors; Cells; Cellular Migration; Circulation; Data; Defect; Dendritic Cells; Dendritic cell activation; FLR; Failure (biologic function); Follicular Dendritic Cells; Gamma Globulin, 19S; Generations; Germinal Center; Goals; Homing; Host Defense; Hour; Human; Human, General; IgM; Immune Complex; Immunity; Immunization; Immunoglobulin M; Immunologic Memory; Immunologic Stimulation; Immunological Memory; Immunological Stimulation; Immunostimulation; Infection; Integral Membrane Protein; Intracellular Communication and Signaling; Intrinsic Membrane Protein; Kidney Diseases; Lead; Link; Lupus; Mammals, Mice; Man (Taxonomy); Man, Modern; Mature B-Cell; Mature B-Lymphocyte; Mediating; Memory B Cell; Memory B-Lymphocyte; Mice; Modeling; Motility; Motility, Cellular; Murine; Mus; Nephropathy; Pathway interactions; Pb element; Physiologic; Physiological; Physiology; Process; Production; Proteins; Publishing; Recovery; Recruitment Activity; Regulation; Renal Disease; Reporting; Reticuloendothelial System, Blood; Reticuloendothelial System, Spleen; Role; SEQ-AN; SLEB1 gene; Scleroderma; Sensitization, Immunologic; Sensitization, Immunological; Sequence Analyses; Sequence Analysis; Signal Transduction; Signal Transduction Systems; Signaling; Site; Spleen; Structure of germinal center of lymph node; Testing; Transgenes; Transmembrane Protein; Vaccination; Vaccines; Vasculitis; Veiled Cells; anamnestic reaction; antibody biosynthesis; autoimmune antibody; autoimmune disorder; base; biological signal transduction; cell motility; dermatosclerosis; experiment; experimental research; experimental study; failure; gene product; heavy metal Pb; heavy metal lead; immunogen; immunoglobulin biosynthesis; in vivo; kidney disorder; macrophage; pathogen; pathway; public health relevance; reconstitute; reconstitution; recruit; renal disorder; research study; response; secondary immune response; self reactive antibody; self recognition (immune); social role; systemic lupus erythematosus susceptibility 1; tool; trafficking
Project start date: 1998-01-01
Project end date: 2013-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
5R01AI042265-12 (2010): $358875
5R01AI042265-11 (2009): $362500
Grants awarded to Louis B Justement
MOLECULAR MECHANISMS MEDIATING CD22 ACCESSORY FUNCTION
Louis B Justement, Professor
University Of Alabama At Birmingham 1530 3rd Avenue South Birmingham, Al 35294
Grant 5R01AI036401-10 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: The objective of this proposal is to determine the in vivo functional role of effector proteins that interact with the B cell co-receptor CD22 by generating transgenic mice that express wild type or altered CD22 on the CD22-/- background. CD22 is a B cell specific transmembrane glycoprotein that regulates the threshold of signaling via the B cell antigen receptor (BCR). Therefore, CD22 plays an important role in regulating the balance between tolerance and immunity in the B cell. The cytoplasmic domain of CD22 contains six tyrosine residues, one or more of which are phosphorylated in response to BCR cross-linking. The resultant phosphotyrosine motifs function as docking sites for the recruitment of SH2 domain-containing effector proteins. Studies performed in this laboratory have determined that CD22 recruits three "classes" of effector proteins including the inhibitory effector protein SHP-1 and the stimulatory effector proteins PLOgamma, PI 3-K, Grb2 and Syk. Additionally, studies performed in this laboratory have determined that CD22 physically interacts with AP50, the medium chain subunit of the AP-2 complex, via a tyrosine-containing motif. Thus it is likely that CD22 expression, and presumably its function, are regulated through its association with clathrin-coated pits. Although it is apparent the CD22 negatively regulates signal transduction via the BCR; questions remain regarding the functional role of stimulatory and inhibitory effector proteins that associate with it. Therefore, studies are proposed to determine the functional importance of effector proteins that are recruited to CD22. The specific aims of the proposal include the following 1) to reconstitute CD22-/- mice with wild type and altered forms of murine CD22 that no longer bind to selected effector proteins; 2) to utilize the CD22 transgenic mice to determine the physiologic importance of effector protein binding to the cytoplasmic domain of CD22; 3) to define the molecular and biochemical processes that regulate CD22 expression; and 4) to determine the physiologic importance of the interaction between CD22 and the AP-2 complex in vivo. The proposed studies will precisely determine the mechanism by which CD22 regulates BCR signal transduction in vivo. The results from these studies will provide information to further our understanding of the molecular processes that regulate the balance between tolerance and immunity. This information can then be used to facilitate the development of biotherapeutic agents that will modulate the immune system to control disease.
Keywords: B lymphocyte, CD22 molecule, biological signal transduction, leukocyte activation /transformation, B cell receptor, calcium flux, endocytosis, gene expression, gene mutation, phosphorylation, protein protein interaction, transcription factor, laboratory mouse, molecular cloning, nucleic acid sequence, transgenic animal
Project start date: 1995-03-15
Project end date: 2005-03-31
5R01AI036401-10 (2003): $234561
5R01AI036401-09 (2002): $227726
5R01AI036401-08 (2001): $217306
5R01AI036401-07 (2000): $202140
REGULATION OF B LYMPHOCYTE SURVIVAL AND DIFFERENTIATION BY HSH2
Louis B Justement
University Of Alabama At Birmingham, 1530 3rd Avenue South, Birmingham, Al 35294
Grant 5R01AI072647-04 from National Institute Of Allergy And Infectious Diseases
Abstract: The goal of this application is to elucidate the physiological role of the novel adaptor protein HSH2 (Hematopoietic SH2 protein) in regulation of B lymphocyte biology. Studies conducted in our laboratory have demonstrated that HSH2 is expressed at low basal levels in splenic B cells and that its expression is induced in response to stimuli that bind to distinct families of receptors that promote survival and differentiation, including CD40L, IL-4, LPS, CpG DMA and BLyS (BAFF). Up-regulation of HSH2 expression was shown to be dependent on activation of NF-?B and correlates with initiation of a survival response that includes up-.regulation of Bcl-XL. Retroviral-mediated expression of HSH2 in the WEHI-231 B cell line, which undergoes apoptosis in response to BCR ligation, was observed to enhance survival and mitochondrial stability. Similarly, enhanced survival of WEHI-231 cells in response to CD40-mediated signaling directly correlated with up-regulation of HSH2 expression. Although HSH2 does not significantly alter BCR-proximal signal transduction, it was observed to maintain mitochondrial stability and this correlated with its ability to block up-regulation of Bim in response to BCR signaling. Moreover, HSH2 was found to interact with the anti-apoptotic protein HAX-1, which possesses a membrane-spanning region that targets it to the outer mitochondrial membrane. Preliminary studies have shown that the interaction between HSH2 and HAX-1 is important for the anti-apoptotic activity of HSH2. Therefore, HSH2 and HAX-1 may function together to regulate mitochondrial integrity and cell survival. To further elucidate the physiological role of HSH2 in regulation of B lymphocyte survival/differentiation, three specific aims have been proposed that will 1) determine the physiological role of HSH2 in B lymphocyte development, homeostasis and immune function; 2) elucidate the role that HSH2 plays in regulating Bim expression in response to co-stimulation; and 3) determine the functional importance of the interaction between HSH2 and HAX-1 in regulating mitochondrial stability. Because HSH2 expression is induced in response to many of the key stimuli that are known to promote B cell survival and differentiation, this adaptor protein is likely to play an important role in regulating B cell homeostasis and immune function. Therefore, these studies will provide novel insight into the molecular mechanisms that maintain the balance between B lymphocyte survival and death leading to differentiation into humoral effector cells and will provide insight into the etiology and progression of diseases associated with aberrant B cell function, including cancer and autoimmunity
Keywords: Adaptor Protein; Adaptor Signaling Protein; Antigen Receptors; Apoptosis; Apoptosis Pathway; Apoptotic; Attenuated; Autoimmune Status; Autoimmunity; Autoregulation; B blood cells; B cell activating factor; B cell growth factor; B-Cell Development; B-Cell Differentiation Factor-1; B-Cell Growth Factor-1; B-Cell Growth Factor-I; B-Cell Proliferating Factor; B-Cell Stimulating Factor; B-Cell Stimulating Factor-1; B-Cell Stimulation Factor-1; B-Cell Stimulatory Factor-1; B-Cells; B-Lymphocytes; BAF; BCDF-1; BCGF; BCGF-1; BCSF 1; BSF-1; BSF1; BSF1 (B cell stimulating factor 1); Binding; Binding (Molecular Function); Binetrakin; Biochemical; Blotting, Western; Bone Marrow; Bp50; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; CD154; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; CD40; CD40L; CD40LG; CDW40; Cancers; Causality; Cell Communication and Signaling; Cell Death, Programmed; Cell Function; Cell Line; Cell Lines, Strains; Cell Process; Cell Signaling; Cell Survival; Cell Viability; Cell physiology; CellLine; Cells; Cells, CD4; Cellular Function; Cellular Physiology; Cellular Process; Cellular biology; Cessation of life; Closure by Ligation; Cytofluorometry, Flow; DNA; Death; Deoxyribonucleic Acid; Development; Disease Progression; Doctor of Philosophy; Effector Cell; Equilibrium; Etiology; Event; Exhibits; Family; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Gene Targeting; Goals; Hematopoietic; Homeostasis; Hour; IL-4; IL4; IL4 Protein; Immune Function, Cellular; Immunofluorescence; Immunofluorescence Immunologic; Immunologic, Immunofluorescence; Interleukin-4; Interleukin-4 Precursor; Intracellular Communication and Signaling; Investigators; Ionomycin; Laboratories; Lead; Life; Ligation; Link; Lymphocyte; Lymphocyte Biology; Lymphocyte Stimulatory Factor 1; Lymphocytic; MCGF-2; MGC9013; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Mast Cell Growth Factor-2; Mediating; Membrane; Mice; Microfluorometry, Flow; Mitochondria; Molecular; Molecular Interaction; Murine; Mus; Nature; Outer Mitochondrial Membrane; PERNUM; Pathway interactions; Pb element; Peripheral; Peritoneum; Ph.D.; PhD; Physiologic; Physiological; Physiological Homeostasis; Play; Population; Programs (PT); Programs [Publication Type]; Proteins; Receptor Protein; Receptors, Antigen; Regulation; Regulatory Pathway; Research Personnel; Researchers; Reticuloendothelial System, Bone Marrow; Reticuloendothelial System, Spleen; Reticuloendothelial System, Thymus; Role; SH2-containing protein; SHC; SHC1 (Src homology 2 domain-containing) protein; Shc protein; Signal Transduction; Signal Transduction Systems; Signaling; Spleen; Src homology 2 domain-containing protein; Src homology 2 domain-containing, transforming protein 1; Staining method; Stainings; Stains; Stains, Tissue; Stimulus; Structure; Subcellular Process; T-Cell Growth Factor 2; T-Cells; T-Lymphocyte; T4 Cells; T4 Lymphocytes; TNFRSF5; TNFRSF5 gene; TNFSF5; TNFSF5 gene; TRAP Gene; Targetings, Gene; Thymus; Thymus Gland; Thymus Proper; Thymus-Dependent Lymphocytes; Tissue Stains; Transgenes; Tumor Necrosis Factor Receptor Superfamily Member 5 Gene; Up-Regulation; Up-Regulation (Physiology); Upregulation; Western Blotting; Western Blottings; Western Immunoblotting; anti-IgM; balance; balance function; biological signal transduction; cell biology; computerized data processing; cultured cell line; data processing; disease causation; disease etiology; disease/disorder etiology; disorder etiology; experiment; experimental research; experimental study; flow cytophotometry; gene product; heavy metal Pb; heavy metal lead; helper T cell; immune function; insight; knock-down; loss of function; lymph cell; macrophage; malignancy; membrane structure; mitochondrial; neoplasm/cancer; novel; p50; pathway; population based; programs; protein blotting; protein expression; protein function; receptor; research study; response; retroviral-mediated; self recognition (immune); shRNA; short hairpin RNA; signal processing; small hairpin RNA; social role; thymocyte; thymus derived lymphocyte
Project start date: 2007-03-05
Project end date: 2012-02-29
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
5R01AI072647-04 (2010): $316851
1R01AI072647-01 (2007): $326250
CD19 TYROSINE-MEDIATED SIGNAL TRANSDUCTION IN VIVO
Louis B Justement
University Of Alabama At Birmingham, 1530 3rd Avenue South, Birmingham, Al 35294
Grant 3R01AI042265-12S1 from National Institute Of Allergy And Infectious Diseases
Abstract: B lymphocytes are required for host defense against infectious pathogens but abnormal activation of B cells can lead to autoimmunity. Thus, B cells are important in a wide range of physiologic and pathophysiologic processes. Mature B cells in the spleen enter into one of two compartments, the marginal zone or the follicular. Marginal zone B cells are responsible for rapid responses to pathogens that have reached the circulation. Follicular B cells are recruited into germinal centers, where affinity maturation and the generation of memory B cells lead to heightened responses upon subsequent encounters with a pathogen, which is crucial to immunization by vaccines. Mice lacking CD19, a B cell surface receptor, fail to form marginal zone B cells or germinal centers and have a propensity to produce autoantibodies. Our new data demonstrate that lack of marginal zone B cells results in defects in other components of the marginal zone, including marginal zone macrophages and dendritic cells. However, the macrophages and dendritic cells reappear following reconstitution of the marginal zone B cells by adoptive transfer of wild type B cells. Aim 1 will dissect the mechanisms by which CD19 on B cells regulates the differentiation of MZ B cells, and thereby other constituents of the marginal zone. Additional new preliminary data suggest that that failure to form germinal centers is associated with a failure of activation of Follicular Dendritic Cells (FDC) in CD19-/- mice. Adoptive transfer of wild type B cells also reconstitutes the ability to activate FDC in CD19-/- recipient mice, which recover the ability to form germinal centers. In Aim 2, we will test three hypotheses as to how CD19- dependent mechanisms regulate FDC through activation of follicular B cells, through effects on the marginal zone that control delivery of antigen to FDC, and through formation of immune complexes. In Aim 3, we will determine how CD19 regulates autoimmunity by experiments to test the role of CD19 in central selection, selection in the periphery, and in a model of autoimmunity. These aims are proposed not just to understand the function of CD19, but to use CD19 to probe basic mechanisms that are fundamental to immunization and protection from pathogens, without autoimmunity. The marginal zone of the spleen is required for survival of infections that have reached the blood while germinal centers are of fundamental importance in vaccination and immunological memory. Despite their crucial roles in protection from infections, the marginal zone and the germinal center responses are poorly understood. This project will use CD19, a protein expressed on B lymphocytes that is required for both types of response, as a tool to investigate the functions of B cells in the marginal zone and in germinal centers in vivo
Keywords: ATGN; Adhesions; Adoptive Transfer; Affinity; Angiitis; Antibody Formation; Antibody Production; Antibody Response; Antigen-Antibody Complex; Antigens; Autoantibodies; Autoimmune Diseases; Autoimmune Status; Autoimmunity; B blood cells; B-Cell Activation; B-Cells; B-Lymphocytes; Blood; Blood Circulation; Bloodstream; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; CD19; CD19 gene; CD19 tyrosine; Cell Communication and Signaling; Cell Locomotion; Cell Migration; Cell Movement; Cell Signaling; Cell Surface Receptors; Cells; Cellular Migration; Circulation; Data; Defect; Dendritic Cells; Dendritic cell activation; FLR; Failure (biologic function); Follicular Dendritic Cells; Gamma Globulin, 19S; Generations; Germinal Center; Goals; Homing; Host Defense; Hour; Human; Human, General; IgM; Immune Complex; Immunity; Immunization; Immunoglobulin M; Immunologic Memory; Immunologic Stimulation; Immunological Memory; Immunological Stimulation; Immunostimulation; Infection; Integral Membrane Protein; Intracellular Communication and Signaling; Intrinsic Membrane Protein; Kidney Diseases; Lead; Link; Lupus; Mammals, Mice; Man (Taxonomy); Man, Modern; Mature B-Cell; Mature B-Lymphocyte; Mediating; Memory B Cell; Memory B-Lymphocyte; Mice; Modeling; Motility; Motility, Cellular; Murine; Mus; Nephropathy; Pathway interactions; Pb element; Physiologic; Physiological; Physiology; Process; Production; Proteins; Publishing; Recovery; Recruitment Activity; Regulation; Renal Disease; Reporting; Reticuloendothelial System, Blood; Reticuloendothelial System, Spleen; Role; SEQ-AN; SLEB1 gene; Scleroderma; Sensitization, Immunologic; Sensitization, Immunological; Sequence Analyses; Sequence Analysis; Signal Transduction; Signal Transduction Systems; Signaling; Site; Spleen; Structure of germinal center of lymph node; Testing; Transgenes; Transmembrane Protein; Vaccination; Vaccines; Vasculitis; Veiled Cells; anamnestic reaction; antibody biosynthesis; autoimmune antibody; autoimmune disorder; base; biological signal transduction; cell motility; dermatosclerosis; experiment; experimental research; experimental study; failure; gene product; heavy metal Pb; heavy metal lead; immunogen; immunoglobulin biosynthesis; in vivo; kidney disorder; macrophage; pathogen; pathway; public health relevance; reconstitute; reconstitution; recruit; renal disorder; research study; response; secondary immune response; self reactive antibody; self recognition (immune); social role; systemic lupus erythematosus susceptibility 1; tool; trafficking
Project start date: 2010-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
3R01AI042265-12S1 (2010): $99870
MOLECULAR MECHANISMS MEDIATING CD22 ACCESSORY FUNCTION
Louis B Justement, Professor
University Of Alabama At Birmingham 1530 3rd Avenue South Birmingham, Al 35294
Grant 5R01AI036401-05 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: B cell activation is dependent on ligand binding and cross-liking of the antigen receptor resulting in activation of multiple protein tyrosine kinases. Tyrosine phosphorylation of antigen receptor-associated proteins is critical for entry into the cell cycle and proliferative expansion of the activated B cell. CD22 is a B lineage-restricted transmembrane glycoprotein that is rapidly phosphorylated on tyrosine in response to antigen receptor cross-linking. Recent studies have demonstrated that CD22 functions as an adhesion molecule and an accessory protein that regulates signal transduction via the B cell antigen receptor. Simultaneous cross- linking of CD22 and membrane immunoglobulin significantly decreases the threshold of activation required for entry of the B cell cycle. Moreover, the lack of CD22 expression has been shown to alter signal transduction processes initiated by ligand binding to the antigen receptor. Thus, it is apparent that membrane immunoglobulin and CD22 are both important for B cell activation by virtue of the fact that they act in concert to regulate the generation of signals that lead to increased gene transcription within the cell. We propose to examine the molecular mechanisms that regulate the accessory function of CD22 in the B cell. Studies will be performed to identify the regions of CD22 that are important for its interaction with the antigen receptor and the protein tyrosine phosphatase, CD45, both of which have been shown to regulate tyrosine phosphorylation of CD22. The specific antigen receptor-associated protein tyrosine kinase(s) that is responsible for phosphorylation of CD22 will be identified. Regulation of CD22 tyrosine phosphorylation by the relevant protein tyrosine kinase and the protein tyrosine phosphatase, CD45, will be examined in order to identify the specific target residues on CD22 that are substrates for these enzymes. Additionally, the physiological importance of CD22 phosphorylation will be examined with regard to the binding of SH2-containing proteins. Experiments will be performed to measure phosphotyrosine-dependent interactions between CD22 and SH2-containing signal transduction proteins as well as to define the specific tyrosine residues to which these proteins bind. Finally, studies will be performed to determine the physiological importance of accessory protein binding to CD22. CD22-negative cell lines will be reconstituted with mutated forms of CD22 that no longer associate with specific SH2-containing proteins. The ability of mutated CD22 molecules to reconstitute signal transduction via the antigen receptor will be assessed. These structure/function studies will contribute significantly to our understanding of the molecular basis by which CD22 regulates B cell activation. In view of the fact the CD22 appears to play an important role in regulating and/or potentiating entry of the B cell into the cell cycle, these studies may shed light on processes related to autoimmune disease and/or cancer that result in aberrant activation and proliferation of B cells.
Keywords: B lymphocyte, CD antigen, biological signal transduction, leukocyte activation /transformation, B cell receptor, calcium flux, chimeric protein, enzyme mechanism, phosphoprotein phosphatase, phosphorylation, protein tyrosine kinase, tissue /cell culture, transfection
Project start date: 1995-03-15
Project end date: 1999-02-28
5R01AI036401-05 (1998): $162001
5R01AI036401-04 (1997): $155771
Sponsored Links Excellgen http://Excellgen.com
REGULATION OF BCR MEDIATED SIGNAL TRANSDUCTION BY CD45
Louis B Justement, Professor
Microbiologyuniversity Of Alabama At Birmingham
1530 3rd Avenue South
birmingham, Al 35294
Grant 5R01GM046524-10 from National Institute Of General Medical Sciences IRG: ALY
Abstract: B cell activation and differentiation are dependent on ligand binding to the antigen receptor, membrane immunoglobulin (mIg), which transduces a signal across the plasma membrane that activates both the phosphoinositide pathway and a mIg-regulated protein tyrosine kinase (PTK). Activation of these biochemical pathways is required for subsequent gene transcription and entry into the cell cycle. In addition, we recently demonstrated that expression of a phosphotyrosine phosphatase (PTPase), CD45, is required for signaling following mIg ligation. Thus, based on the above observations it is apparent that mIg-mediated signal transduction is regulated by the dynamic interplay between a PTK and a PTPase (CD45). We propose to examine the physiologic role of CD45 in regulation of B cell activation following antigen receptor ligation. Studies will be carried out to gain a better understanding of the physical interaction that occurs between CD45 and the antigen receptor complex. Co-capping and co- modulation experiments as well as chemical crosslinking studies will be performed to document functional associations between these proteins in the plasma membrane. Further, we will determine whether CD45 expression is necessary for PTK activation. Identification of the specific PTK associated with the antigen receptor complex by coimmunoprecipitation with mIg or its associated proteins (MB-1 and B29) will enable us to determine how its activity is regulated by tyrosine phosphorylation, and what role CD45 plays in this process. Additional experiments will be carried out to determine whether CD45 regulates the tyrosine phosphorylation of specific residues on MB-1 and B29 following their isolation from CD45-variant cell lines. Differences in tyrosine phosphorylation will be correlated with altered function by examining the ability of these proteins to couple mIg to the PTK in CD45-variant cell lines. Finally, we will utilize site-directed mutagenesis of the catalytic domain of CD45 in order to gain a better understanding of the structure-function relationships which control the activity of this PTPase. Mutagenized forms of CD45 will be examined for their ability to reconstitute signal transduction in CD45-negative cells. These studies will contribute significantly to our understanding of the molecular basis of B cell activation and will provide the foundation for further studies concerning the regulation of cell function by tyrosine phosphorylation/dephosphorylation. Specifically, it has been suggested that PTPase expression is critical for the prevention of cellular transformation by counterbalancing the effects of PTK´s on cellular processes. Thus, the studies proposed above may provide information that enhances our understanding of the events which result in aberrant activation of the B cell
Keywords: B cell receptor, B lymphocyte, CD antigen, biological signal transduction, protein tyrosine phosphatase calcium flux, casein kinase, enzyme activity, guanine nucleotide binding protein, leukocyte activation /transformation, protein tyrosine kinase molecular cloning, site directed mutagenesis, tissue /cell culture
Project start date: 1991-08-01
Project end date: 2003-11-30
5R01GM046524-10 (2002): $223043
5R01GM046524-09 (2001): $216610
5R01GM046524-08 (2000): $210362
REGULATION OF MEMBRANE IMMUNOGLOBULIN-MEDIATED SIGNAL TR
Louis B Justement, Professor
University Of Texas Medical Br Galveston
301 University Blvd
galveston, Tx 77555
Grant 5R29GM046524-03 from National Institute Of General Medical Sciences IRG: ALY
Abstract: B cell activation and differentiation are dependent on ligand binding to the antigen receptor, membrane immunoglobulin (mIg), which transduces a signal across the plasma membrane that activates both the phosphoinositide pathway and a mIg-regulated protein tyrosine kinase (PTK). Activation of these biochemical pathways is required for subsequent gene transcription and entry into the cell cycle. In addition, we recently demonstrated that expression of a phosphotyrosine phosphatase (PTPase), CD45, is required for signaling following mIg ligation. Thus, based on the above observations it is apparent that mIg-mediated signal transduction is regulated by the dynamic interplay between a PTK and a PTPase (CD45). We propose to examine the physiologic role of CD45 in regulation of B cell activation following antigen receptor ligation. Studies will be carried out to gain a better understanding of the physical interaction that occurs between CD45 and the antigen receptor complex. Co-capping and co- modulation experiments as well as chemical crosslinking studies will be performed to document functional associations between these proteins in the plasma membrane. Further, we will determine whether CD45 expression is necessary for PTK activation. Identification of the specific PTK associated with the antigen receptor complex by coimmunoprecipitation with mIg or its associated proteins (MB-1 and B29) will enable us to determine how its activity is regulated by tyrosine phosphorylation, and what role CD45 plays in this process. Additional experiments will be carried out to determine whether CD45 regulates the tyrosine phosphorylation of specific residues on MB-1 and B29 following their isolation from CD45-variant cell lines. Differences in tyrosine phosphorylation will be correlated with altered function by examining the ability of these proteins to couple mIg to the PTK in CD45-variant cell lines. Finally, we will utilize site-directed mutagenesis of the catalytic domain of CD45 in order to gain a better understanding of the structure-function relationships which control the activity of this PTPase. Mutagenized forms of CD45 will be examined for their ability to reconstitute signal transduction in CD45-negative cells. These studies will contribute significantly to our understanding of the molecular basis of B cell activation and will provide the foundation for further studies concerning the regulation of cell function by tyrosine phosphorylation/dephosphorylation. Specifically, it has been suggested that PTPase expression is critical for the prevention of cellular transformation by counterbalancing the effects of PTK´s on cellular processes. Thus, the studies proposed above may provide information that enhances our understanding of the events which result in aberrant activation of the B cell
Keywords: B lymphocyte, biological signal transduction, immune complex, immunoglobulin, immunoregulation, membrane protein, surface antigen RNA splicing, antigen receptor, cell type, enzyme substrate, gene expression, leukocyte activation /transformation, nucleic acid sequence, phosphatidylinositol, phosphorylation, protein structure function, protein tyrosine kinase, synthetic peptide crosslink, laboratory mouse, laboratory rabbit, site directed mutagenesis, western blotting
Project start date: 1991-08-01
Project end date: 1996-07-31
5R29GM046524-03 (1993): $103565
5R29GM046524-02 (1992): $104719
MOLECULAR MECHANISMS MEDIATING CD22 ACCESSORY FUNCTION
Louis B Justement, Professor
University Of Alabama At Birmingham 1530 3rd Avenue South Birmingham, Al 35294
Grant 7R01AI036401-03 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Project start date: 1995-03-15
Project end date: 1999-02-28
7R01AI036401-03 (1996): $149780
5R01AI036401-02 (1996): $1
REGULATION OF MEMBRANE IMMUNOGLOBULIN-MEDIATED SIGNAL TR
Louis B Justement, Professor
University Of Texas Medical Br Galveston
301 University Blvd
galveston, Tx 77555
Grant 3R29GM046524-02S2 from National Institute Of General Medical Sciences IRG: ALY
Project start date: 1991-08-01
Project end date: 1996-07-31
3R29GM046524-02S2 (1993): $4629