ABSTRACT
SLAP-130/Fyb (SLP-76-associated phosphoprotein or Fyn-binding protein; also known as Fyb/Slap) is a hematopoietic-specific adapter, which associates with and modulates function of SH2-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). T cells from mice lacking SLAP-130/Fyb show markedly impaired proliferation following CD3 engagement. In addition, the T cell receptor (TCR) in SLAP-130/Fyb mutant cells fails to enhance integrin-dependent adhesion. Although TCR-induced actin polymerization is normal, TCR-stimulated clustering of the integrin LFA-1 is defective in SLAP-130/Fyb-deficient cells. These data indicate that SLAP-130/Fyb is important for coupling TCR-mediated actin cytoskeletal rearrangement with activation of integrin function, and for T cells to respond fully to activating signals.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Lymphocyte Function-Associated Antigen-1/physiology , Phosphoproteins/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/physiology , Actins/metabolism , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , CD3 Complex/immunology , Carrier Proteins/genetics , Cell Adhesion , Cell Membrane/metabolism , Immunologic Capping , Intercellular Adhesion Molecule-1/metabolism , Interleukin-2/biosynthesis , Interleukin-2/pharmacology , Lectins, C-Type , Lymphocyte Activation , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/genetics , Protein-Tyrosine Kinases/metabolism , Receptors, Interleukin-2/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Membrane Proteins/metabolism , Nuclear Proteins , Receptors, Antigen, T-Cell/metabolism , Receptors, Cell Surface , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , CD4-Positive T-Lymphocytes/immunology , CD5 Antigens/metabolism , CD8-Positive T-Lymphocytes/immunology , DNA-Binding Proteins/metabolism , Flow Cytometry , Gene Expression Regulation , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Humans , Jurkat Cells , Lectins, C-Type , Liver/cytology , Liver/embryology , Membrane Proteins/genetics , Mice , Mice, Transgenic , NFATC Transcription Factors , Promoter Regions, Genetic/genetics , Receptor, Notch1 , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Response Elements/genetics , Signal Transduction , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism , Transcription Factor AP-1/metabolism , Transcription Factors/metabolismABSTRACT
The hematopoietic cell-specific adaptor protein, SLP-76, is critical for T cell development and mature T cell receptor (TCR) signaling; however, the structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. In this study, transgenic mice were generated to examine the requirements for specific domains of SLP-76 in thymocytes and peripheral T cells in vivo. Examination of mice expressing various mutants of SLP-76 on the null background demonstrates a differential requirement for specific domains of SLP-76 in thymocytes and T cells and provides new insight into the molecular mechanisms underlying SLP-76 function.
Subject(s)
Adaptor Proteins, Signal Transducing , Membrane Proteins , Phosphoproteins/physiology , T-Lymphocytes/cytology , Amino Acid Motifs , Amino Acid Substitution , Animals , Binding Sites , CD3 Complex/immunology , Calcium Signaling , Carrier Proteins/physiology , Cell Differentiation , Clonal Deletion/physiology , Immunophenotyping , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mutation, Missense , Phosphoproteins/chemistry , Phosphoproteins/deficiency , Phosphoproteins/genetics , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/immunology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/physiology , Sequence Deletion , Signal Transduction/physiology , Spleen/immunology , Structure-Activity Relationship , T-Lymphocytes/immunology , Thymus Gland/immunology , src Homology DomainsABSTRACT
Adaptor proteins, molecules that mediate intermolecular interactions, are now known to be as crucial for lymphocyte activation as are receptors and effectors. Extensive work from numerous laboratories has identified and characterized many of these adaptors, demonstrating their roles as both positive and negative regulators. Studies into the molecular basis for the actions of these molecules shows that they function in various ways, including: recruitment of positive or negative regulators into signalling networks, modulation of effector function by allosteric regulation of enzymatic activity, and by targeting other proteins for degradation. This review will focus on a number of adaptors that are important for lymphocyte function and emphasize the various ways in which these proteins carry out their essential roles.
Subject(s)
Adaptor Proteins, Signal Transducing , Lymphocyte Activation/immunology , Membrane Proteins , Proteins/immunology , T-Lymphocytes/immunology , Animals , Carrier Proteins/immunology , Cell Movement , Gene Targeting , Humans , Models, Immunological , Phosphoproteins/immunology , Proteins/genetics , Receptors, Antigen, T-Cell/immunology , Signal Transduction , T-Lymphocytes/cytologyABSTRACT
Mice deficient in the hematopoietic cell-specific adapter protein SLP-76 demonstrate a failure of T cell development and fetal hemorrhage. Although SLP-76-deficient platelets manifest defective collagen receptor signaling, this alone may not explain the observed bleeding diathesis. Because alpha IIb beta 3, the platelet fibrinogen receptor, is required for normal hemostasis, we explored a potential role for SLP-76 in alpha IIb beta 3 signaling. Interaction of soluble or immobilized fibrinogen with normal human or murine platelets triggers rapid tyrosine phosphorylation of SLP-76. Moreover, platelet adhesion to fibrinogen stimulates actin rearrangements, filopodial and lamellipodial extension, and localization of tyrosine phosphorylated proteins to the cell periphery. In contrast, SLP-76-deficient murine platelets bind fibrinogen normally, but spread poorly and exhibit reduced levels of phosphotyrosine. The in vivo bleeding diathesis as well as the defects in platelet responses to fibrinogen and collagen are reversed by retroviral transduction of SLP-76 into bone marrow derived from SLP-76-deficient mice. These studies establish that SLP-76 functions downstream of alpha IIb beta 3 and collagen receptors in platelets. Furthermore, expression of SLP-76 in hematopoietic cells, including platelets, plays a necessary role in hemostasis.
Subject(s)
Blood Platelets/physiology , Hematopoiesis , Hemostasis/physiology , Integrins/physiology , Phosphoproteins/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Signal Transduction/physiology , Adaptor Proteins, Signal Transducing , Animals , Fibrinogen/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Phosphorylation , Protein Binding , Receptors, Collagen , Tyrosine/metabolismABSTRACT
Expression of SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), a hematopoietic cell-specific adapter protein, is required to couple Syk family tyrosine kinase activation to downstream mediators such as phospholipase C (PLC)-gamma following TCR, platelet collagen receptor and mast cell Fc epsilon R stimulation. In addition to T cells, mast cells and platelets, SLP-76 is expressed in monocytes and macrophages. To determine the role of SLP-76 in Fc gamma R-stimulated signaling pathways in macrophages, we examined cultured bone marrow-derived macrophages (BMM) from SLP-76(-/-) and wild-type mice. In this study, we show that Fc gamma R cross-linking rapidly induces tyrosine phosphorylation of SLP-76 in wild-type BMM. Surprisingly, however, BMM from SLP-76(-/-) mice activate ERK2 and phosphorylate PLC-gamma 2 following Fc gamma R ligation. Furthermore, SLP-76(-/-) BMM display normal Fc gamma R-dependent phagocytic function and reactive oxygen intermediate production. SLP-76(-/-) and SLP-76(+/+) BMM secrete comparable levels of IL-12 in response to lipopolysaccharide and IFN-gamma. To examine macrophage function in vivo, SLP-76(-/-) mice were challenged i.v. with Listeria monocytogenes. SLP-76(-/-) mice survive and efficiently contain the acute phase of infection similar to wild-type mice but exhibit a stable chronic infection attributed to the lack of mature T cells. These data show that, although SLP-76 is required to couple Syk family PTK activity to downstream mediators and effector functions in Fc gamma R-induced pathways in some cell types, activation of Fc gamma R-dependent pathways occurs independently of SLP-76 in BM
Subject(s)
Macrophages/physiology , Adaptor Proteins, Signal Transducing , Animals , Interleukin-12/biosynthesis , Listeriosis/mortality , Mice , Mitogen-Activated Protein Kinase 1/physiology , Phagocytosis , Phosphoproteins/physiology , Phosphorylation , Reactive Oxygen Species , Receptors, IgG/physiology , Sheep , Type C Phospholipases/physiology , Tyrosine/metabolismABSTRACT
Adapter molecules contain discrete modular domains that direct specific intermolecular interactions to orchestrate assembly of signaling complexes. A number of adapter proteins play critical roles in both positive and negative regulation of antigen-receptor signaling, influencing lymphocyte development and function.
Subject(s)
Lymphocytes/immunology , Proteins/metabolism , Receptors, Antigen/metabolism , Signal Transduction , Models, Immunological , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/metabolismABSTRACT
Challenge of macrophages with DNA containing an internal CpG motif results in IL-12 p40 secretion. In the presence of IFN-gamma, CpG DNA induces more p40 secretion than does LPS. In the RAW 264 macrophage cell line, both CpG DNA and LPS activate a p40 promoter-reporter construct, and the promoter response to either agent is augmented 2- to 5-fold by IFN-gamma. While either LPS or CpG DNA induces p40 promoter activity, only CpG DNA induces an increase in p40 mRNA or protein secretion. Even though IFN-gamma augmented LPS-driven p40 promoter activity in RAW 264 cells, the combination of IFN-gamma and LPS induced less p40 mRNA or protein secretion than the combination of IFN-gamma and CpG DNA. The ability of IFN-gamma to augment LPS or CpG DNA-induced p40 promoter activation was observed with truncation mutants of the IL-12 promoter containing as few as 250 bp 5' of the TATA box. Although LPS alone is a poor inducer of p40 transcription, both LPS and CpG DNA induce similar nuclear translocation of NF-kappaB. This binding is not augmented by costimulation with IFN-gamma. Thus, CpG DNA induces p40 transcription by a mechanism that includes NF-kappaB translocation; however, CpG DNA appears to induce other factor(s) necessary for p40 transcription. These results illustrate fundamental differences between CpG DNA and LPS with respect to activation of IL-12 p40 secretion.
Subject(s)
CpG Islands/immunology , DNA/pharmacology , Gene Expression Regulation/immunology , Interleukin-12/genetics , Interleukin-12/metabolism , Lipopolysaccharides/pharmacology , Promoter Regions, Genetic/immunology , Adjuvants, Immunologic/pharmacology , Animals , Binding Sites/genetics , Binding Sites/immunology , Cell Line , Interferon-gamma/pharmacology , Mice , Mice, Inbred BALB C , NF-kappa B/genetics , NF-kappa B/metabolism , Oligonucleotides/pharmacology , Response Elements/immunology , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.