ABSTRACT
PTP-PEST (encoded by Ptpn12) is an intracellular protein tyrosine phosphatase belonging to the same family as LYP. LYP inhibits secondary T cell responses by suppressing Src family protein tyrosine kinases and is implicated in human autoimmunity. To determine the function of PTP-PEST in T cells, we generated mice with a conditionally deleted allele of Ptpn12. By removing PTP-PEST in T cells, we determined that PTP-PEST was not necessary for T cell development or primary responses. However, PTP-PEST was required for secondary T cell responses, anergy prevention, and autoimmunity induction. PTP-PEST specifically regulated the phosphorylation of Pyk2, a substrate of the Src family kinase Fyn. It also promoted the formation of T cell homoaggregates, which are known to enhance T cell activation. Thus, PTP-PEST controls Pyk2 activity and is a positive regulator of secondary T cell activation. These data illustrate the critical role of protein tyrosine phosphatases in T cell regulation.
Subject(s)
Focal Adhesion Kinase 2/immunology , Focal Adhesion Kinase 2/metabolism , Lymphocyte Activation , Protein Tyrosine Phosphatase, Non-Receptor Type 12/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 12/metabolism , T-Lymphocytes/immunology , Animals , Cell Aggregation , Cell Proliferation , Encephalomyelitis, Autoimmune, Experimental/enzymology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuropeptides/metabolism , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 12/deficiency , T-Lymphocytes/cytology , T-Lymphocytes/enzymology , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding ProteinABSTRACT
CRACC is a self-associating member of the signaling lymphocytic activation molecule family that is expressed on cells of the immune system, including natural killer cells and activated T cells. Here we examine the function and mechanism of action of CRACC using several complementary approaches, including the generation of a CRACC-deficient mouse. Our results demonstrate that CRACC positively regulated natural killer cell functions by a mechanism dependent on the adaptor EAT-2 but not the related adaptor SAP. However, in the absence of EAT-2, CRACC potently inhibited natural killer cell function. CRACC was also inhibitory in T cells, which are typically devoid of EAT-2. Thus, CRACC can exert activating or inhibitory influences on cells of the immune system depending on cellular context and the availability of effector proteins.
Subject(s)
Killer Cells, Natural/immunology , Receptors, Immunologic/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing , Animals , Gene Expression Profiling , Killer Cells, Natural/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Receptors, Immunologic/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transcription Factors/immunologyABSTRACT
SAP (also named SH2D1A) is an intracellular adaptor molecule expressed in T cells, natural killer (NK) cells, and some B cells. The SAP gene is mutated in X-linked lymphoproliferative (XLP) disease, a human immunodeficiency characterized by a faulty immune response to Epstein-Barr virus infection. Previous reports documented severe defects in antibody production and germinal center (GC) formation in SAP-deficient humans and mice genetically engineered to lack SAP expression. However, in vitro studies and adoptive transfer experiments provided conflicting data as to whether this phenotype is caused by a functional defect resulting from SAP deficiency in T cells, B cells, or both. Here, we ascertained which cell types are responsible for this humoral immunity defect by using a conditional gene targeting approach. We also thoroughly examined the expression pattern of SAP in normal immune cells by using intracellular flow cytometry. The results showed that expression of SAP in T cells, but not in B cells or NK cells, is required and sufficient for SAP-dependent antibody production and GC formation. These data provide a critical insight into the mechanism by which SAP regulates humoral immunity. They also help elucidate the basis of a severe human immunodeficiency.
Subject(s)
Antibody Formation , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Intracellular Signaling Peptides and Proteins/physiology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Antibody Formation/genetics , B-Lymphocytes/pathology , Chickens , Female , Gene Expression Regulation/immunology , Gene Targeting , Haptens/immunology , Haptens/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Signaling Lymphocytic Activation Molecule Associated Protein , T-Lymphocytes/pathology , Trinitrobenzenes/immunology , Trinitrobenzenes/metabolism , gamma-Globulins/immunologyABSTRACT
SAP is an intracellular adaptor molecule composed almost exclusively of an SH2 domain. It is mutated in patients with X-linked lymphoproliferative disease, a human immunodeficiency. Several immune abnormalities were also identified in SAP-deficient mice. By way of its SH2 domain, SAP interacts with tyrosine-based motifs in the cytoplasmic domain of SLAM family receptors. SAP promotes SLAM family receptor-induced protein tyrosine phosphorylation, due to its capacity to recruit the Src-related kinase FynT. This unusual property relies on the existence of a second binding surface in the SAP SH2 domain, centered on arginine 78 of SAP, that binds directly to the FynT SH3 domain. Herein, we wanted to further understand the mechanisms controlling the interaction between SLAM-SAP and FynT. Our experiments showed that, unlike conventional associations mediated by SH3 domains, the interaction of the FynT SH3 domain with SLAM-SAP was strictly inducible. It was absolutely dependent on engagement of SLAM by extracellular ligands. We obtained evidence that this inducibility was not due to increased binding of SLAM to SAP following SLAM engagement. Furthermore, it could occur independently of any appreciable SLAM-dependent biochemical signal. In fact, our data indicated that the induced association of the FynT SH3 domain with SLAM-SAP was triggered by a change in the conformation of SLAM-associated SAP caused by SLAM engagement. Together, these data elucidate further the events initiating SLAM-SAP signaling in immune cells. Moreover, they identify a strictly inducible interaction mediated by an SH3 domain.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Glycoproteins/metabolism , Immunoglobulins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Proto-Oncogene Proteins c-fyn/metabolism , src Homology Domains , Adaptor Proteins, Signal Transducing/genetics , Animals , Antigens, CD , Cell Line , Glycoproteins/genetics , Humans , Immunoglobulins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Multiprotein Complexes , Protein Binding , Proto-Oncogene Proteins c-fyn/genetics , Receptors, Cell Surface , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction/physiology , Signaling Lymphocytic Activation Molecule Family Member 1 , Tyrosine/metabolismABSTRACT
EAT-2 is an adaptor expressed in innate immune cells, including natural killer (NK) cells. It is closely related to the adaptor SAP, which regulates signaling lymphocyte activation molecule (SLAM)-related receptors by recruiting the kinase FynT to the receptors. Here we have studied the function of EAT-2 in NK cells by creating mice lacking or overexpressing EAT-2. Like SAP, EAT-2 was associated with the SLAM-related receptor 2B4 in NK cells. However, unlike SAP, EAT-2 was an inhibitor of NK cell function. EAT-2 repressed natural cytotoxicity and interferon-gamma secretion by a mechanism involving tyrosine phosphorylation of its C terminus. We have demonstrated a similar function for the adaptor ERT, a newly identified SAP family member expressed in mouse NK cells. These data identify a previously unknown mechanism of NK cell inhibition. Moreover, they indicate that EAT-2 and SAP have distinct and at times opposing functions in natural immunity.
Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Killer Cells, Natural/immunology , Transcription Factors/physiology , src Homology Domains/physiology , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Animals, Genetically Modified , Antigens, CD/genetics , Antigens, CD/physiology , CHO Cells , Cell Line , Cricetinae , Cytotoxicity, Immunologic , Down-Regulation , Interferon-gamma/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Killer Cells, Natural/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Mice , Mice, Inbred BALB C , Mice, Knockout , Molecular Sequence Data , Phosphorylation , Receptors, Immunologic/genetics , Receptors, Immunologic/physiology , Sequence Alignment , Signaling Lymphocytic Activation Molecule Associated Protein , Signaling Lymphocytic Activation Molecule Family , Transcription Factors/deficiency , Transcription Factors/genetics , Tyrosine , src Homology Domains/geneticsABSTRACT
SAP is an adaptor mutated in X-linked lymphoproliferative disease. It plays a critical role in T helper 2 (T(H)2) cytokine production. This function was suggested to reflect the capacity of SAP to associate with SLAM family receptors and enable tyrosine phosphorylation signaling by these receptors through SAP-mediated recruitment of Src-related kinase FynT. Here, we addressed by genetic means the importance of the SAP-FynT interaction in normal T cell functions. By creating a mouse in which the FynT binding site of SAP was inactivated in the germ line (sap(R78A) mouse) and by analyzing mice lacking SAP, FynT or SLAM, evidence was obtained that the SAP-FynT cascade is indeed crucial for normal T(H)2 functions in vitro and in vivo. These data imply that SAP is necessary for T(H)2 cytokine regulation primarily as a result of its capacity to recruit FynT. They also establish a previously unappreciated role for FynT in SAP-dependent T(H)2 cytokine regulation.
Subject(s)
Cytokines/biosynthesis , Intracellular Signaling Peptides and Proteins/physiology , Proto-Oncogene Proteins/physiology , Th2 Cells/immunology , Animals , Antigens, CD , DNA-Binding Proteins/genetics , GATA3 Transcription Factor , Glycoproteins/physiology , Immunoglobulin E/biosynthesis , Immunoglobulins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-fyn , Receptors, Antigen, T-Cell/physiology , Receptors, Cell Surface , Signaling Lymphocytic Activation Molecule Associated Protein , Signaling Lymphocytic Activation Molecule Family Member 1 , Trans-Activators/geneticsABSTRACT
SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAP-mediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function.
