Coreceptors and adapter proteins in T-cell signaling.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
S J Burakoff
Publications and source records attributed to S J Burakoff.
Interaction of Shc with Grb2 regulates the Grb2 association with mSOS.
Explore the source record for details and available documents.
Adaptor Proteins, Signal Transducing↗
Solution structure of the Shc SH2 domain complexed with a tyrosine-phosphorylated peptide from the T-cell receptor.
She is a widely expressed adapter protein that plays an important role in signaling via a variety of cell surface receptors and has been implicated in coupling the stimulation of growth factor, cytokine, and antigen receptors to the Ras signaling pathway. She interacts with several tyrosine-phosphorylated receptors through its C-terminal SH2 domain, and one of the mechanisms of T-cell receptor-mediated Ras activation involves the interaction of the Shc SH2 domain with the tyrosine-phosphorylated zeta chain of the T-cell receptor. Here we describe a high-resolution NMR structure of the Shc SH2 domain complexed to a phosphopeptide (GHDGLpYQGLSTATK) corresponding to a portion of the zeta chain of the T-cell receptor. Although the overall architecture of the protein is similar to other SH2 domains, distinct structural differences were observed in the smaller beta-sheet, BG loop, (pY + 3) phosphopeptide-binding site, and relative position of the bound phosphopeptide.
Adaptor Proteins, Signal Transducing↗
CD8 beta chain influences CD8 alpha chain-associated Lck kinase activity.
The CD8 molecule plays an important role in the differentiation of CD8+ T cells in the thymus and in their normal function in the periphery. CD8 exists on the cell surface in two forms, the alpha alpha homodimer and the alpha beta heterodimer. Recent studies indicate an important role for the CD8 beta chain in thymic development of CD8+ T cells and suggest that signaling via CD8 alpha beta may be distinct from CD8 alpha alpha. To better understand these differences, we introduced the CD8 beta gene into a T cell hybridoma which only expressed the CD8 alpha alpha homodimer. In the parent hybridoma, cross-linking of the CD8 alpha chain led to minimal enhancement of CD8-associated Lck tyrosine kinase activity. In the CD8 beta+ transfectants, several observations suggested that CD8 beta modifies CD8 alpha-associated Lck tyrosine kinase activity: (a) in in vitro kinase assays, antibody-mediated crosslinking of CD8 alone, or CD8 cross-linking with the TCR, resulted in 10-fold greater activation of Lck kinase activity, compared to cells expressing CD8 alpha alpha alone; (b) in vivo, markedly enhanced tyrosine phosphorylation of several intracellular proteins was observed upon CD8 cross-linking with the TCR in CD8 alpha beta-expressing cells, compared to cells expressing CD8 alpha alpha alone; and (c) Lck association with CD8 alpha was stabilized by the coexpression of CD8 beta. Thus, the differential Lck kinase activation and tyrosine phosphorylation seen with CD8 alpha alpha vs. CD8 alpha beta may reflect the unique signaling capabilities of the CD8 beta molecule. These differences in signaling may, in part, account for the diminished ability to generate CD8 single positive thymocytes in mice bearing a homozygous disruption of the CD8 beta gene.
Animals↗
Expression and induction of nuclear factor-kappa B-related proteins in thymocytes.
Thymocytes mature in response to the cues from the thymic micro-environment, which regulate stage-specific gene expression during development. We find that several proteins that bind the kappa B sequence in vitro are constitutively activated in freshly isolated thymocytes. These include the rel-related p50 homodimers, p50/p65 heterodimers, low levels of c-rel, and two other factors that may be thymus specific. Disruption of the thymic micro-environment resulted in loss of DNA-binding, suggesting that lymphocyte-stromal cell interactions induce and maintain these proteins in a DNA-binding form. Phorbol ester and ionomycin treatment induced p50, p65, and p68 c-rel kappa B DNA-binding activity. Expression of p68 c-rel protein, but not p50 or p65, was suppressed by the immunosuppressive drug FK506. Because FK506 specifically inhibits the appearance of mature single-positive thymocytes, gene expression regulated by p68 c-rel may play a role in selection and maturational signals involved in the double-positive to single-positive transition.
Animals↗
FK506 inhibits antigen receptor-mediated induction of c-rel in B and T lymphoid cells.
Stimulation of B and T cells via the antigen receptor, by phorbol ester or by phorbol ester and ionomycin, leads to nuclear translocation of the inducible transcription factor NF-kappa B, comprising the p50 and p65 rel-related polypeptides. In this report we show that c-rel is a component of the antigen receptor-induced kappa B binding proteins in both B and T cells. Whereas NF-kappa B can be induced by phorbol ester alone, optimal induction of c-rel requires stimulation by both phorbol ester and ionomycin, the dual signal that is necessary for proliferation of untransformed lymphocytes. Furthermore, c-rel induction is blocked by the immunosuppressive drug FK506 that is known to inhibit B and T cell activation. c-rel-dependent transactivation of the interleukin-2 receptor alpha chain (IL-2R alpha) promoter is augmented by coexpression of calcineurin, suggesting the involvement of a calcineurin-dependent intracellular pathway. Our results identify c-rel as a target of immunosuppressive agents and illustrate the similarity of activation pathways in both B and T cells.
Amino Acid Sequence↗
Crk interacts with tyrosine-phosphorylated p116 upon T cell activation.
Products of the crk oncogene are expressed in all tissues. Crk proteins are composed exclusively of Src homology 2 (SH2) and Src homology 3 (SH3) domains, and they have been implicated in intracellular signaling. For example, they participate as mediators of Ras activation during nerve growth factor stimulation of PC12 pheochromocytoma cells. We examined the role of Crk proteins during T cell receptor-mediated signaling and observed that Crk proteins specifically interact, via their SH2 domains, with a tyrosine-phosphorylated 116-kDa protein upon T cell activation. p116 may be related to the recently cloned fibroblast p130cas and/or p120-Cbl. In addition, we observed that GST-Crk fusion proteins and Crk-L bind, most likely via their SH3 domain, to C3G, a Ras guanine nucleotide exchange factor. Thus, the interaction of Crk with p116 and C3G strongly implicates Crk as a mediator of T cell receptor signaling, possibly involved in Ras activation.
Adrenal Gland Neoplasms↗
The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells.
FK506 and cyclosporin A (CsA) are immunosuppressive agents that inhibit IL-2 production by activated T cells, but only CsA inhibits IgE activation-induced cytokine transcripts in mouse IL-3-dependent, bone marrow-derived mast cells (BMMC). We previously associated the resistance of BMMC to FK506 with a deficiency in the expression of FK506 binding protein (FKBP) 12, a molecule that forms a complex with FK506 capable of inhibiting calcineurin phosphatase activity in vitro. In this report, we establish that FKBP12 mediates FK506 inhibition of both calcineurin phosphatase activity and IgE activation-induced cytokine transcripts in a Kirsten murine sarcoma virus-immortalized mast cell line that is FKBP12 deficient. Overexpression of FKBP12 by transfection enhanced the ability of FK506 to inhibit calcineurin phosphatase activity (IC50 = 2 nM), compared with cells transfected with the expression vector alone (IC50 > 30 nM). The IC50 value for FK506 inhibition of IgE activation-induced transcripts for TNF-alpha decreased from 40 nM in vector control cells to 10 nM in FKBP12 transfectants. Similarly, the IC50 value for inhibition of IL-6 transcripts decreased from > 1000 nM in vector control cells to 35 nM in FKBP12 transfectants. In contrast, activation-elicited release of the secretory granule mediator beta-hexosaminidase was only partially inhibited by FK506 at 1000 nM, regardless of the levels of FKBP12 expressed by the cells. Thus, FKBP12 is the dominant cytosolic protein that mediates FK506 inhibition of TNF-alpha and IL-6 transcripts.
Animals↗
Developmental control point in induction of thymic cortex regulated by a subpopulation of prothymocytes.
T lymphocytes of the alpha/beta T-cell receptor (TCR) lineage mature in the thymus, where they undergo a series of differentiation, expansion and selection events. For normal T-cell ontogeny to occur, thymocytes must interact physically with cortical and medullary thymic stroma cells. In parallel, interactions of the thymic stromal cells with TCR-positive thymocytes are necessary for the development of the thymic medulla. Comparable requirements for the differentiation of the cortex have not been defined, however. Here we analyse mutant mouse strains to assess the function of early prothymocytes in the induction of the thymic cortex. We find that animals with a developmental block at the earliest stage of T-lineage commitment lack a functional thymic cortex. This abnormality could be corrected in fetal but not adult animals by transplantation of either fetal or adult wild-type haematopoietic stem cells. Thus a developmentally restricted interaction of fetal stromal cells with early prothymocytes is required for the induction of a cortical microenvironment. In addition, a normal thymic architecture is necessary for sustained T-cell ontogeny.
Aging↗
FK506 binding protein 12 mediates sensitivity to both FK506 and rapamycin in murine mast cells.
The immunosuppressive drugs FK506 and rapamycin bind to a family of intracellular proteins termed FK506-binding proteins (FKBP). FK506 and rapamycin inhibit lymphocyte-activation pathways by forming complexes with an FKBP; subsequently, the drug/FKBP complexes interact with target molecules involved in signal transduction. A key target of FK506/FKBP12 complexes is calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase. In mammalian cells, rapamycin treatment is associated with inhibition of the activity of several cellular serine/threonine kinases, including p70 S6 kinase. These kinases may function in signaling pathways involving TOR gene producs, which have been shown to interact with rapamycin/FKBP12 complexes in vitro. To determine if FKBP12 mediates the effects of both FK506 and rapamycin in mammalian cells, we overexpressed FKBP12 in a murine mast cell line. Increased expression of FKBP12 resulted in increased sensitivity to FK506 and rapamycin, as measured by inhibition of calcineurin activity and p70 S6 kinase activity, respectively. In contrast, overexpression of FKBP25 had no effect on sensitivity to either drug. Two distinct point mutations in FKBP12, one altering a hydrophobic residue within the drug-binding pocket and the other changing a charged surface residue of FKBP12, abrogated its ability to mediate sensitivity to FK506 and rapamycin. These results establish that FKBP12 can mediate sensitivity to both FK506 and rapamycin in mammalian cells.
Animals↗
Severe colitis in mice with aberrant thymic selection.
Tg epsilon 26 mice display an arrest very early in T cell development that has a profound effect on the architecture of thymic stromal cells. We have recently demonstrated that transplantation of wild-type bone marrow cells restores the thymic microenvironment of fetal but not adult Tg epsilon 26 mice. Here, we report that T cell-reconstituted adult Tg epsilon 26 mice develop a spontaneous wasting syndrome characterized by extensive inflammation of the colon, resembling human ulcerative colitis. Colitis in these animals was marked by substantial infiltration of the colon by activated thymus-derived CD4+ T cells. Importantly, bone marrow-transplanted Tg epsilon 26 mice previously engrafted with a fetal Tg epsilon 26 thymus did not develop colitis. These results suggest that T cells selected in an aberrant thymic microenvironment contain a population of cells able to induce severe colitis that can be prevented by T cells that have undergone normal thymic development.
Animals↗
Interaction of Shc with Grb2 regulates association of Grb2 with mSOS.
The adapter protein Shc has been implicated in Ras signaling via many receptors, including the T-cell antigen receptor (TCR), B-cell antigen receptor, interleukin-2 receptor, interleukin-3 receptor, erythropoietin receptor, and insulin receptor. Moreover, transformation via polyomavirus middle T antigen is dependent on its interaction with Shc and Shc tyrosine phosphorylation. One of the mechanisms of TCR-mediated, tyrosine kinase-dependent Ras activation involves the simultaneous interaction of phosphorylated Shc with the TCR zeta chain and with a second adapter protein, Grb2. Grb2, in turn, interacts with the Ras guanine nucleotide exchange factor mSOS, thereby leading to Ras activation. Although it has been reported that in fibroblasts Grb2 and mSOS constitutively associate with each other and that growth factor stimulation does not alter the levels of Grb2:mSOS association, we show here that TCR stimulation leads to a significant increase in the levels of Grb2 associated with mSOS. This enhanced Grb2:mSOS association, which occurs through an SH3-proline-rich sequence interaction, is regulated through the SH2 domain of Grb2. The following observations support a role for Shc in regulating the Grb2:mSOS association: (i) a phosphopeptide corresponding to the sequence surrounding Tyr-317 of Shc, which displaces Shc from Grb2, abolished the enhanced association between Grb2 and mSOS; and (ii) addition of phosphorylated Shc to unactivated T cell lysates was sufficient to enhance the interaction of Grb2 with mSOS. Furthermore, using fusion proteins encoding different domains of Shc, we show that the collagen homology domain of Shc (which includes the Tyr-317 site) can mediate this effect. Thus, the Shc-mediated regulation of Grb2:mSOS association may provide a means for controlling the extent of Ras activation following receptor stimulation.
Adaptor Proteins, Signal Transducing↗
Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.
The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.
Amino Acid Sequence↗
Disruption of T cell development and repertoire selection by calcineurin inhibition in vivo.
The microbial products FK506 and CsA are potent immunosuppressive agents that prevent early transcriptional events in TcR-mediated activation. Their mode of action is dependent upon the inhibition of calcineurin, a serine/threonine phosphatase positioned within the calcium-dependent signaling pathway. TcR-mediated activation of thymocytes constitutes an important prerequisite for their development and selection to mature T cells. Disruption of the cross-talk between thymic APC and thymocytes results in the loss of normal T cell ontogeny. To study the role of calcineurin in T cell maturation and repertoire selection in vivo, mice were treated with either FK506 or CsA. Administration of either drug inhibited the progression of CD4+CD8+ positive thymocytes to mature single positive T cells. Furthermore, both drugs disrupted the process of negative thymic selection, causing an increased frequency of self-reactive cells among the few positively selected T cells. These effects correlated directly with the degree of inhibition of in vivo calcineurin enzyme activity. Blocking calcineurin activity appears to disrupt positive thymic selection and to prevent the deletion of self-reactive thymocytes.
Animals↗
Immunophilins in protein folding and immunosuppression.
Lymphocyte activation requires the transmission of signals from molecules at the plasma membrane to nuclear signals that regulate gene expression. In recent years, several immunosuppressive compounds have been used as probes to identify important and potentially novel molecules involved in lymphocyte signal transduction processes. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin have been studied in particular detail. Two distinct classes of immunosuppressant binding proteins have been identified, and collectively termed immunophilins. The cyclophilin family of immunophilins binds CsA, whereas the FK506-binding protein (FKBP) family binds FK506 and rapamycin. This review will discuss both the endogenous functions of immunophilins as well as their roles in mediating immunosuppression.
Amino Acid Isomerases↗
Loss of normal thymic repertoire selection and persistence of autoreactive T cells in graft vs host disease.
To assess the influence of graft vs host disease (GVHD) on T cell development and thymic repertoire selection, a murine transplantation model was chosen, in which donor (B10.BR: Mls-1b, Mls-2b) and recipient (CBA/J: Mls-1a, Mls-2a) mice differ in their minor lymphocyte-stimulating Ag. Mature splenic T cells of donor origin were added to the T cell-depleted bone marrow cells to induce moderate GVHD. When analyzed 5 and 10 wk after transplantation, animals with GVHD, but not disease-free controls, demonstrated aberrant thymic maturation with an increase in single positive, TCRhigh+ thymocytes. Phenotypic analysis of TCR V beta expression in mature thymocytes and peripheral T cells revealed a disruption of negative thymic selection of Mls-reactive T cells with the subsequent emergence of V beta 3+ and V beta 6+ T cells in mice with GVHD but not in controls. Using retroviral-mediated gene transfer to tag mature T cells, these V beta 3+ and V beta 6+ T cells could be shown to be derived from donor bone marrow precursors. Thymocytes expressing V beta 3 and V beta 6 were efficiently activated upon cross-linking of their TCRs and peripheral T cells from mice with GVHD proliferated to host-derived splenocytes in a MLR. When transferred to sublethally irradiated CBA/J recipients, only T cells from mice with GVHD expanded dramatically. These data suggest that the lack of proper thymic selection after bone marrow transplantation results in the survival and persistence of self-reactive T cells, which might be responsible for the autoimmune manifestations of chronic GVHD.
Animals↗
Adhesion receptors in lymphocyte activation.
The past several years have seen significant progress in understanding the role of T lymphocyte coreceptors in adhesion and activation. New insights have been gained in several areas: the avidity regulation of beta 1 and beta 2 integrins and their role in signal transduction; the regulation of CD8 avidity; the role of Lck in CD4 coreceptor activity; and the novel role for CD2 adhesion in the T cell antigen response.
Animals↗
Tyrosine kinase activity of CD4-associated p56lck may not be required for CD4-dependent T-cell activation.
The lymphoid-specific tyrosine kinase p56lck (Lck) is critical for the development and activation of T lymphocytes, and Lck kinase activity has been implicated in both T-cell antigen receptor/CD3- and CD4-mediated signaling. CD4-dependent T-cell activation has been demonstrated to be dependent upon the association of CD4 with Lck. To examine the role of the kinase activity of Lck in CD4-dependent T-cell activation, we have generated several kinase-deficient mutants of Lck. When transfected into CD4+ murine T-cell hybridoma cells, these mutants cause approximately 90% diminution in CD4-associated Lck kinase activity. Specifically, upon CD4 crosslinking there is decreased Lck autophosphorylation and decreased phosphorylation of an exogenous substrate. When CD4 is crosslinked to the T-cell antigen receptor-CD3 complex, decreased phosphorylation of associated substrates is also observed. In spite of this striking inhibition of Lck kinase function, cells expressing the kinase-deficient mutants demonstrate normal or enhanced CD4-dependent antigen responsiveness. These data demonstrate that the level of Lck kinase activity does not correlate with its CD4-associated function and suggest that the kinase activity of Lck may not be required for CD4-mediated signaling.
Animals↗