ABSTRACT
Essentials Endothelial injury is thought to be a key event in thrombotic thrombocytopenic purpura (TTP). Endothelial and cardiac damages were assessed in a model of TTP using ADAMTS-13 knockout mice. Damages of cardiac perfusion and function were associated with nitric oxide pathway alteration. Endothelial dysfunction constitutes a critical event in TTP development and cardiac injury. SUMMARY: Background Cardiac alterations represent a major cause of mortality in patients with thrombotic thrombocytopenic purpura (TTP). Endothelial injury remains poorly defined, but seems to be a key initiating event leading to the formation of platelet-rich thrombi in TTP patients. Objectives To assess the changes in endothelial function and the induced cardiac damage in a mouse model of TTP. Patients/methods We used an animal model in which TTP-like symptoms are triggered by injection of 2000 units kg-1 of recombinant von Willebrand factor in ADAMTS-13 knockout mice. Results These mice developed TTP-like symptoms, i.e. severe thrombocytopenia, schistocytosis, and anemia. On day 2, magnetic resonance imaging demonstrated a decrease in left ventricular perfusion associated with alteration of left ventricular ejection fraction, fractional shortening, and cardiac output, suggesting early systolic dysfunction. This was associated with decrease in endothelium-mediated relaxation responses to acetylcholine in mesenteric and coronary arteries, demonstrating severe early endothelial dysfunction. In parallel, we showed decreased cardiac expression of endothelial nitric oxide (NO) synthase and increased expression of antioxidant enzymes, suggesting alteration of the NO pathway. At this time, cardiac immunohistochemistry revealed an increase in the expression of VCAM-1 and E-selectin. Conclusion This study provides evidence that the heart is a sensitive target organ in TTP, and shows, for the first time, strong mesenteric and coronary endothelial dysfunction in an induced-TTP model. The mechanisms incriminated are the occurrence of a pro-oxidant state, and proadhesive and proinflammatory phenotypes. This previously largely unrecognized vascular dysfunction may represent an important contributor to the systemic organ failure occurring in TTP.
Subject(s)
ADAMTS13 Protein/genetics , Endothelium, Vascular/pathology , Purpura, Thrombotic Thrombocytopenic/diagnosis , Animals , Antioxidants/metabolism , Disease Models, Animal , E-Selectin/metabolism , Female , Heart Ventricles/pathology , Humans , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/chemistry , Nitric Oxide Synthase Type III/metabolism , Oxidants/metabolism , Perfusion , Phenotype , Purpura, Thrombotic Thrombocytopenic/pathology , Reactive Oxygen Species/metabolism , Recombinant Proteins/pharmacology , Thrombosis/pathology , Vascular Cell Adhesion Molecule-1/metabolism , Ventricular Function, Left , von Willebrand Factor/pharmacologyABSTRACT
BACKGROUND: ADAMTS13 cleaves hyperactive ultra-large von Willebrand factor (ULVWF) multimers into smaller and less active forms. It remains unknown whether VWF-mediated inflammatory processes play a role in the enhanced brain injury due to ADAMTS13 deficiency. OBJECTIVE: We tested the hypothesis that the deleterious effect of ADAMTS13 deficiency on ischemic brain injury is mediated through VWF-dependent enhanced vascular inflammation. METHODS: Transient focal cerebral ischemia was induced by 60 min of occlusion of the right middle cerebral artery. Myeloperoxidase (MPO) activity and inflammatory cytokines in the infarcted region were evaluated 23 h after reperfusion injury. Neutrophil infiltration within the infarct and surrounding areas was quantitated by immunohistochemistry. RESULTS: We report that ADAMTS13-deficient mice exhibited significantly enlarged infarct size, concordant with increased myeloperoxidase (MPO) activity, neutrophil infiltration and expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In contrast, VWF-deficient mice exhibited significantly reduced MPO activity, neutrophil infiltration and inflammatory cytokine induction, demonstrating a role of VWF in these inflammatory processes. Mice deficient for both ADAMTS13 and VWF exhibited an identical reduction of the same inflammatory parameters, demonstrating that the increased inflammation observed in ADAMTS13-deficient mice is VWF dependent. Finally, the increased infarct size observed in ADAMTS13-deficient mice was completely abrogated by prior immunodepletion of neutrophils, demonstrating a causal role for acute inflammation in the enhanced brain injury that occurs in the setting of ADAMTS13 deficiency. CONCLUSION: These findings provide new evidence for ADAMTS13 in reducing VWF-mediated acute cerebral inflammation following ischemic stroke.
Subject(s)
Brain/enzymology , Infarction, Middle Cerebral Artery/enzymology , Metalloendopeptidases/metabolism , Reperfusion Injury/enzymology , Vasculitis, Central Nervous System/prevention & control , von Willebrand Factor/metabolism , ADAMTS13 Protein , Acute Disease , Animals , Brain/blood supply , Brain/immunology , Brain/pathology , Disease Models, Animal , Immunohistochemistry , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/immunology , Infarction, Middle Cerebral Artery/pathology , Inflammation Mediators/metabolism , Interleukin-6/metabolism , Male , Metalloendopeptidases/deficiency , Metalloendopeptidases/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Infiltration , Peroxidase/metabolism , Reperfusion Injury/genetics , Reperfusion Injury/immunology , Reperfusion Injury/pathology , Time Factors , Tumor Necrosis Factor-alpha/metabolism , Vasculitis, Central Nervous System/enzymology , Vasculitis, Central Nervous System/genetics , Vasculitis, Central Nervous System/immunology , Vasculitis, Central Nervous System/pathology , von Willebrand Factor/geneticsABSTRACT
SUMMARY BACKGROUND: During sepsis, von Willebrand factor (VWF) is abundantly secreted; the main mechanism regulating its size involves specific proteolysis by the metalloprotease ADAMTS-13. OBJECTIVES: To determine whether ADAMTS-13 consumption due to its binding to, and/or cleavage, of VWF contributes to its decrease during sepsis and whether abrogating or enhancing ADAMTS-13 activity influences sepsis outcome. METHODS: ADAMTS-13 activity was evaluated in a model of sepsis induced by cecum ligature and puncture (CLP) in wild-type and Vwf(-/-) mice. Sepsis outcome was studied in those mice and in Adamts-13(-/-) mice. Finally, survival was studied in wild-type mice injected hydrodynamically with the human ADAMTS-13 gene. RESULTS: In wild-type mice, CLP-induced sepsis elicited a significant ADAMTS-13 decrease, and a strong negative correlation existed between VWF and ADAMTS-13. In Vwf(-/-) mice, CLP also induced severe sepsis, but ADAMTS-13 was not significantly diminished. Notably, Vwf(-/-) mice lived significantly longer than wild-type mice. In contrast, Adamts-13(-/-) mice and wild-type mice were comparable with regard to thrombocytopenia, VWF concentrations, absence of thrombi, and survival. Hydrodynamic hADAMTS-13 gene transfer with the pLIVE expression vector resulted in high and stable ADAMTS13 activity in CLP mice; however, no impact on survival was observed. CONCLUSIONS: VWF secretion is a major determinant of ADAMTS-13 decrease in the CLP model, and plays an important role in sepsis-induced mortality, but the complete absence of its regulating protease, ADAMTS-13, had no detectable impact in this sepsis model. Furthermore, increasing ADAMTS-13 activity had no impact on survival.
Subject(s)
Cecum/pathology , Metalloendopeptidases/metabolism , Sepsis/metabolism , von Willebrand Factor/physiology , ADAMTS13 Protein , Animals , Disease Models, Animal , Kidney/pathology , Liver/pathology , Male , Mice , Mice, Knockout , von Willebrand Factor/geneticsSubject(s)
Carcinoma, Lewis Lung/pathology , Lung Neoplasms/prevention & control , Melanoma, Experimental/pathology , von Willebrand Factor/pharmacology , Animals , Blood Coagulation Factors/genetics , Blood Coagulation Factors/pharmacology , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Mice , Mice, Knockout , Neoplasm Transplantation , von Willebrand Factor/geneticsABSTRACT
Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.
Subject(s)
Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , ADAM Proteins , ADAMTS13 Protein , Amino Acid Sequence , Amino Acids/chemistry , Animals , Blotting, Northern , Blotting, Western , Cell Line , Cloning, Molecular , DNA Primers/chemistry , DNA, Complementary/metabolism , Databases, Genetic , Dose-Response Relationship, Drug , Edetic Acid/pharmacology , Humans , Metalloendopeptidases/chemistry , Mice , Mice, Inbred BALB C , Models, Genetic , Molecular Sequence Data , Phylogeny , Poly A/metabolism , Polymerase Chain Reaction , Protein Binding , Protein Structure, Tertiary , Purpura, Thrombotic Thrombocytopenic/blood , RNA/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Tissue Distribution , Transfection , von Willebrand Factor/metabolismABSTRACT
Drosophila melanogaster has proven to be a good model for understanding the physiology of ion channels. We identified two novel Drosophila DEG/ ENaC proteins, Pickpocket (PPK) and Ripped Pocket (RPK). Both appear to be ion channel subunits. Expression of RPK generated multimeric Na+ channels that were dominantly activated by a mutation associated with neurodegeneration. Amiloride and gadolinium, which block mechanosensation in vivo, inhibited RPK channels. Although PPK did not form channels on its own, it associated with and reduced the current generated by a related human brain Na+ channel. RPK transcripts were abundant in early stage embryos, suggesting a role in development. In contrast, PPK was found in sensory dendrites of a subset of peripheral neurons in late stage embryos and early larvae. In insects, such multiple dendritic neurons play key roles in touch sensation and proprioception and their morphology resembles human mechanosensory free nerve endings. These results suggest that PPK may be a channel subunit involved in mechanosensation.
Subject(s)
Gene Expression Regulation, Developmental , Neurons, Afferent/physiology , Sodium Channels/biosynthesis , Amiloride/pharmacology , Amino Acid Sequence , Animals , Brain/physiology , Cloning, Molecular , Drosophila melanogaster/embryology , Embryo, Nonmammalian/physiology , Gadolinium/pharmacology , Humans , Molecular Sequence Data , Multigene Family , Neurons, Afferent/drug effects , Phylogeny , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Sodium Channels/chemistry , Sodium Channels/physiologyABSTRACT
We and others have shown that overexpression of SLP-76 augments TCR-stimulated IL-2 promoter activity in the Jurkat T cell line. In this report we investigate the signaling mechanisms through which SLP-76 mediates its effect on T cell activation. We show that overexpressed SLP-76 acts downstream of TCR-stimulated protein tyrosine kinases, but does not affect calcium signaling. Overexpression of SLP-76 does, however, augment TCR stimulation of both ERK (extracellular signal-regulated kinase) activity and a reporter construct driven by activating protein-1 binding sites. Structure/function analysis reveals that three distinct regions of SLP-76, each important for protein associations, are required for augmentation of TCR-induced nuclear factor-AT activity. These data suggest that SLP-76 functions as an adapter molecule that requires three unique domains to link proximal TCR signals in T cells.
Subject(s)
Nuclear Proteins , Phosphoproteins/physiology , Adaptor Proteins, Signal Transducing , Calcium/metabolism , DNA-Binding Proteins/physiology , Humans , Jurkat Cells , Mitogen-Activated Protein Kinases/metabolism , NFATC Transcription Factors , Nerve Tissue Proteins/metabolism , Phosphoproteins/chemistry , Receptors, Antigen, T-Cell/physiology , Transcription Factor AP-1/metabolism , Transcription Factors/physiologyABSTRACT
Previous work has demonstrated that SLP-76, a Grb2-associated tyrosine-phosphorylated protein, augments Interleukin-2 promoter activity when overexpressed in the Jurkat T cell line. This activity requires regions of SLP-76 that mediate protein-protein interactions with other molecules in T cells, suggesting that SLP-76-associated proteins also function to regulate signal transduction. Here we describe the molecular cloning of SLAP-130, a SLP-76-associated phosphoprotein of 130 kDa. We demonstrate that SLAP-130 is hematopoietic cell-specific and associates with the SH2 domain of SLP-76. Additionally, we show that SLAP-130 is a substrate of the T cell antigen receptor-induced protein tyrosine kinases. Interestingly, we find that in contrast to SLP-76, overexpression of SLAP-130 diminishes T cell antigen receptor-induced activation of the interleukin-2 promoter in Jurkat T cells and interferes with the augmentation of interleukin-2 promoter activity seen when SLP-76 is overexpressed in these cells. These data suggest that SLP-76 recruits a negative regulator, SLAP-130, as well as positive regulators of signal transduction in T cells.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Nuclear Proteins , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, T-Cell/metabolism , Amino Acid Sequence , Base Sequence , Carrier Proteins/biosynthesis , Carrier Proteins/chemistry , Cloning, Molecular , DNA-Binding Proteins/metabolism , Female , Humans , Jurkat Cells , Male , Molecular Sequence Data , Molecular Weight , NFATC Transcription Factors , Organ Specificity , Phosphoproteins/biosynthesis , Phosphoproteins/chemistry , Polymerase Chain Reaction , RNA, Messenger/analysis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , T-Lymphocytes , Transcription Factors/metabolism , Transcriptional Activation , TransfectionABSTRACT
Stimulation of the IgE high affinity receptor on rat basophilic leukemia RBL-2H3 cells results in activation of protein tyrosine kinases and rapid tyrosine phosphorylation of several substrates, many of which remain unidentified. In this report, we demonstrate that the Grb2 adapter protein, when expressed as a glutathione S-transferase fusion protein, associates with four tyrosine-phosphorylated molecules (116, 76, 36, and 31 kDa) from lysates of stimulated RBL-2H3 cells. We show further that the 76-kDa protein is SLP-76, a hematopoietic cell-specific protein first identified as a Grb2-binding protein in T cells. Upon stimulation of the high affinity receptor for IgE, SLP-76 undergoes rapid tyrosine phosphorylation and associates with two additional tyrosine phosphoproteins of 62 and 130 kDa via the SH2 domain of SLP-76. Additional studies demonstrate that the SLP-76 SH2 domain also binds a protein kinase from stimulated RBL-2H3 cell lysates. Furthermore, the phosphorylation of SLP-76 requires Syk activity but is not dependent on Ca+2 mobilization. These data, together with our previous work documenting its role in T-cell activation, suggest that SLP-76 and the proteins with which it associates may play a fundamental role in coupling signaling events in multiple cell types in the immune system.
Subject(s)
Adaptor Proteins, Signal Transducing , Leukemia, Basophilic, Acute/metabolism , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, IgE/metabolism , Animals , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enzyme Precursors/metabolism , GRB2 Adaptor Protein , Intracellular Signaling Peptides and Proteins , Proteins/metabolism , Rats , Syk Kinase , src Homology DomainsABSTRACT
SLP-76 (SH2 domain leukocyte protein of 76 kDa) is a recently identified substrate of the TCR-stimulated protein tyrosine kinases that functions in the signal transduction cascade linking the TCR with IL-2 gene expression. In this report, we demonstrate that engagement of the TCR results in tyrosine phosphorylation of SLP-76 in its amino-terminal acidic region. Two tyrosines (Y113 and Y128) fall within an identical five amino-acid motif and are shown to be phosphorylated upon TCR ligation. Although mutation of either Y113 and Y128 has a minimal effect on SLP-76 function, mutation of both residues decreases significantly the ability of SLP-76 to promote T cell activation. A third tyrosine within the amino-terminal region (Y145) appears to be the most important for optimal SLP-76 function, as altering it alone to phenylalanine has a potent impact on SLP-76 augmentation of NFAT promoter activity.
Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins , Phosphoproteins/chemistry , Promoter Regions, Genetic , Protein Processing, Post-Translational , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, T-Cell/physiology , Signal Transduction/physiology , Transcription Factors/metabolism , Tyrosine/physiology , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Gene Expression Regulation, Leukemic , Humans , Interleukin-2/biosynthesis , Interleukin-2/genetics , Leukemia-Lymphoma, Adult T-Cell/pathology , Molecular Sequence Data , NFATC Transcription Factors , Neoplasm Proteins/physiology , Phosphoproteins/pharmacology , Phosphorylation , Structure-Activity Relationship , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Cells, CulturedABSTRACT
The CD45 family of transmembrane protein-tyrosine phosphatases plays a critical role in T cell activation signaling by regulating the tyrosine phosphorylation of protein-tyrosine kinases and their substrates. Multiple alternatively spliced CD45 isoforms, differing only in their extracellular domains, are differentially expressed by subsets of T cells with distinct functional repertoires. However, the physiological function of the various isoforms remains elusive. Using a novel panel of Jurkat T cell clones that uniquely express either the smallest (CD45(0)) or the largest (CD45(ABC)) isoform, we previously demonstrated CD45 isoform-specific differences in interleukin-2 secretion and tyrosine phosphorylation of Vav. We now demonstrate differential activation-induced tyrosine phosphorylation of a 76-kDa Vav-associated protein (pp76) by cells expressing distinct CD45 isoforms. The tyrosine phosphorylation of Vav and associated pp76 follow parallel kinetics. pp76 interacts with the SH2 and SH3 domains of Vav. We have identified pp76 as SLP-76, a recently cloned Grb2-binding protein. After activation with anti-CD3, CD45(ABC) transfectants demonstrate increased tyrosine phosphorylation and physical association of SLP-76 with Vav compared to transfectants expressing CD45(0). These results establish a novel physical link between Vav and SLP-76 that is differentially regulated by CD45 isoform expression.
Subject(s)
Isoenzymes/metabolism , Oncogene Proteins/metabolism , Phosphoproteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Tyrosine/metabolism , Adaptor Proteins, Signal Transducing , Alternative Splicing , Blotting, Western , Cell Line , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Humans , Phosphorylation , Proto-Oncogene Proteins c-vav , Receptor-CD3 Complex, Antigen, T-Cell/metabolismSubject(s)
Chromosomes, Human, Pair 5/genetics , Phosphoproteins/genetics , Polymorphism, Single-Stranded Conformational , Adaptor Proteins, Signal Transducing , Alleles , Base Sequence , Chromosome Mapping , Female , Hematopoietic Stem Cells/metabolism , Humans , Lod Score , Male , Molecular Sequence DataABSTRACT
Ligation of the T-cell antigen receptor (TCR) results in the rapid activation of several protein tyrosine kinases, with the subsequent phosphorylation of numerous cellular proteins. We investigated the requirement for tyrosine phosphorylation of proteins which bind the Grb2 SH2 domain in TCR-mediated signal transduction by transfecting the Jurkat T-cell line with a cDNA encoding a chimeric protein designed to dephosphorylate these molecules. Stimulation of the TCR on cells expressing this engineered enzyme fails to result in sustained tyrosine phosphorylation of a 36-kDa protein likely to be the recently cloned pp36/Lnk. Interestingly, TCR ligation of the transfected cells also fails to induce soluble inositol phosphate production and intracellular calcium mobilization, although receptor-mediated tyrosine phosphorylation of phospholipase C gamma 1 still occurs. TCR-mediated Ras and mitogen-activated protein kinase activation remain intact in cells expressing the engineered phosphatase. These data demonstrate that tyrosine phosphorylation of a protein(s) which binds the SH2 domain of Grb2 correlates with phospholipase C gamma 1 activation and suggest that such a phosphoprotein(s) plays a critical role in coupling the TCR with the phosphatidylinositol second-messenger pathway.
Subject(s)
Adaptor Proteins, Signal Transducing , Isoenzymes/metabolism , Proteins/metabolism , T-Lymphocytes/metabolism , Type C Phospholipases/metabolism , Tyrosine/metabolism , Base Sequence , Cell Line , DNA Primers/genetics , Enzyme Activation , GRB2 Adaptor Protein , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Phosphatidylinositols/metabolism , Phospholipase C gamma , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Binding , Proteins/chemistry , Proteins/genetics , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Transfection , ras Proteins/metabolism , src Homology DomainsABSTRACT
T cell antigen receptor (TCR) stimulation induces tyrosine phosphorylation of many intracellular proteins, including the proto-oncogene Vav, which is expressed exclusively in hematopoietic and trophoblast cells. Vav is critical for lymphocyte development and activation. Overexpression of Vav in Jurkat T cells leads to potentiation of TCR-mediated IL-2 gene activation. However, the biochemical function of Vav is unknown. Here, we demonstrate that the major induced tyrosine phosphoprotein associated with Vav is the hematopoietic cell-specific SLP-76. The Vav SH2 domain is required for this interaction and for TCR-mediated Vav tyrosine phosphorylation. Similar to Vav, overexpression of SLP-76 markedly potentiates TCR-mediated NF-AT and IL-2 gene activation. Furthermore, overexpression of both Vav and SLP-76 synergistically induces basal and TCR-stimulated NF-AT activation. These results suggest that a signaling complex containing Vav and SLP-76 plays an important role in lymphocyte activation.
Subject(s)
Gene Expression Regulation/immunology , Interleukin-2/genetics , Oncogene Proteins/metabolism , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing , Gene Expression Regulation, Neoplastic/immunology , Humans , Leukemia, T-Cell , Oncogene Proteins/physiology , Phosphoproteins/physiology , Phosphorylation , Protein-Tyrosine Kinases/immunology , Proto-Oncogene Mas , Proto-Oncogene Proteins c-vav , Signal Transduction/genetics , Signal Transduction/immunology , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
Insulin stimulates the Ras/Raf/MEK/ERK pathway leading to feedback phosphorylation of the Ras guanylnucleotide exchange protein SOS and dissociation of Grb2 from SOS. Even though epidermal growth factor (EGF) also stimulates ERK activity and phosphorylation of SOS similar to insulin, EGF induces a dissociation of the Grb2-SOS complex from Shc. To determine the molecular basis for this difference, we examined the signaling properties of a mutant EGF receptor lacking the five major autophosphorylation sites. Although EGF stimulation of the mutant EGF receptor activates ERK and phosphorylation of both Shc and SOS, it fails to directly associate with either Shc or Grb2. However, under these conditions EGF induces a dissociation of the Grb2-SOS complex suggesting a role for receptor and/or plasma membrane targeting in the stabilization of Grb2-SOS interaction. Consistent with this hypothesis, expression of an SH2 domain Grb2 mutant which is unable to mediate plasma membrane targeting of the Grb2-SOS complex results in both insulin- and EGF-stimulated uncoupling of Grb2 from SOS. Furthermore, a plasma membrane-bound Grb2 fusion protein remains constitutively associated with SOS. Together, these data demonstrate that EGF stimulation prevents the feedback uncoupling of Grb2 from SOS by inducing a persistent plasma membrane receptor targeting of the Grb2-SOS complex.
Subject(s)
Adaptor Proteins, Signal Transducing , ErbB Receptors/physiology , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinases , Proteins/metabolism , Animals , CHO Cells , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cricetinae , Epidermal Growth Factor/pharmacology , GRB2 Adaptor Protein , Humans , Insulin/physiology , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Phosphorylation , Phosphotyrosine/metabolism , Protein Binding , Receptor, Insulin/metabolism , Recombinant Proteins , Signal Transduction , Son of Sevenless Proteins , src Homology DomainsABSTRACT
Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.
Subject(s)
Adaptor Proteins, Signal Transducing , Interleukin-2/biosynthesis , Nuclear Proteins , Phosphoproteins/metabolism , Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , Amino Acid Sequence , Binding Sites , Cells, Cultured , DNA-Binding Proteins/metabolism , GRB2 Adaptor Protein , Gene Expression Regulation , Genes, Reporter , Humans , Interleukin-2/genetics , Lymphocyte Activation , Molecular Sequence Data , NFATC Transcription Factors , Phosphoproteins/genetics , Phosphorylation , Promoter Regions, Genetic , Protein Binding , T-Lymphocytes , Transcription Factors/metabolism , Transfection , src Homology DomainsABSTRACT
The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-gamma 1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.
Subject(s)
Adaptor Proteins, Signal Transducing , Phosphoproteins/blood , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Tyrosine , Amino Acid Sequence , Animals , B-Lymphocytes/metabolism , Base Sequence , Cell Line , Cloning, Molecular , ErbB Receptors/metabolism , GRB2 Adaptor Protein , Gene Expression , HeLa Cells , Humans , Leukocytes/metabolism , Mice , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Organ Specificity , Phosphoproteins/biosynthesis , Phosphoproteins/isolation & purification , Proteins/isolation & purification , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Sequence Homology, Amino Acid , Spleen/metabolism , T-Lymphocytes/metabolism , Thymus Gland/metabolism , Tumor Cells, CulturedABSTRACT
Expression of CD45, the major transmembrane protein tyrosine phosphatase expressed on lymphoid cells, is required for optimal T cell receptor (TCR) signal transduction. We and others recently have demonstrated that surface expression of the cytoplasmic domain of CD45 in the absence of its extracellular and transmembrane domains is sufficient to restore TCR-mediated signaling events in CD45-deficient cell lines. Here we demonstrate that a single domain nonreceptor tyrosine phosphatase from yeast expressed as a chimeric protein with the extracellular and transmembrane domains of a major histocompatibility complex class I molecule also is able to restore proximal and distal TCR-mediated signal transduction events in the CD45-deficient T cell line J45.01. Ligation of the TCR on the cell line expressing the yeast phosphatase chimera results in the induction of protein tyrosine kinase activity, soluble inositol phosphate generation, and expression of the CD69 activation antigen. Furthermore, a phosphatase-inactive version of this molecule is unable to restore signal transduction, providing the first formal evidence that plasma membrane associated tyrosine phosphatase activity is required for TCR-mediated signaling.
Subject(s)
Leukocyte Common Antigens/analysis , Protein Tyrosine Phosphatases/deficiency , T-Lymphocytes/metabolism , Base Sequence , Cells, Cultured , Clone Cells , Humans , Leukocyte Common Antigens/biosynthesis , Molecular Sequence Data , Protein Tyrosine Phosphatases/biosynthesis , Receptors, Antigen, T-Cell/physiology , Recombinant Fusion Proteins/biosynthesis , Signal Transduction , Type C Phospholipases/metabolismABSTRACT
Numerous recent studies have implicated the src homology 2 and 3 domain-containing protein, Grb2, in coupling protein tyrosine kinase signaling pathways with the Ras signaling pathway. Ligation of the T cell antigen receptor results in the activation of both a PTK, and Ras; therefore, we investigated whether Grb2 may serve a similar function in T cells. Here we report that a GST/Grb2 fusion protein associates with several tyrosine phosphoproteins from lysates of T cell antigen receptor-stimulated Jurkat T cells. Two of these proteins, pp36 and pp116, bind to the Grb2 fusion protein with high affinity. Through the use of mutated Grb2 fusion proteins, we demonstrate that pp116 binds the amino-terminal src homology 3 domain of Grb2, the same domain of Grb2 thought to be primarily responsible for its interaction with SOS. We demonstrate further that pp116 associates with Grb2 in vivo, and we provide evidence that in the Jurkat T cell line Grb2 may exist complexed with either pp116 or with SOS.
Subject(s)
Adaptor Proteins, Signal Transducing , Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/metabolism , Base Sequence , Enzyme Activation , GRB2 Adaptor Protein , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Molecular Sequence Data , Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Surface expression of the CD45 tyrosine phosphatase is essential for the T cell antigen receptor (TCR) to couple optimally with its second messenger pathways. CD45 may be required to dephosphorylate a TCR-activated protein tyrosine kinase, which then transduces an activation signal from the TCR. A chimeric molecule that contained extracellular and transmembrane sequences from an allele of a major histocompatibility class I molecule and cytoplasmic sequences of CD45 restored TCR signaling in a CD45-deficient mutant T cell line. Thus, expression of the complex extracellular domain of CD45 is not required for the TCR to couple to its signaling machinery.