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S Koyasu

Publications and source records attributed to S Koyasu.

At least 73 records · Page 4Linked to original sources
PubMed1991

CD3 eta and CD3 zeta are alternatively spliced products of a common genetic locus and are transcriptionally and/or post-transcriptionally regulated during T-cell development.

The CD3 eta subunit of the T-cell receptor is thought to subserve an important role in signal transduction and possibly T-cell development. Herein we characterize the organization of the mouse CD3 eta gene and show that it is part of one gene locus that also encodes CD3 zeta on chromosome 1. The NH2-terminal sequence of CD3 zeta and CD3 eta, which share the same leader peptide and are identical through amino acid 122 of each mature protein, is encoded by exons 1-7. However, exons 8 and 9 are differentially spliced to give rise to CD3 zeta and CD3 eta: exons 1-8 encode CD3 zeta and exons 1-7 plus 9 encode CD3 eta. RNase protection analysis with RNA from a variety of fetal, neonatal, and adult cell types indicates that expression of both gene products is T-lineage-restricted. Importantly, expression of CD3 zeta and CD3 eta mRNA appears before or on day 16 of fetal gestation. Expression is apparently coordinate since no cell types tested express CD3 zeta or CD3 eta alone. The steady-state level of CD3 zeta mRNA is greater than or equal to 40-60 times that of CD3 eta mRNA. In immature CD4+CD8+CD3low double-positive thymocytes and CD4+CD8-CD3high or CD4-CD8+CD3high single-positive thymocytes, the respective steady-state CD3 zeta and CD3 eta mRNA levels are equivalent, whereas the amount of receptor-associated CD3 zeta and CD3 eta proteins in double-positive thymocytes is approximately 10 times less than in single-positive thymocytes. Nevertheless, the CD3 zeta/CD3 eta protein ratio remains constant in all populations (40-60:1). Furthermore, discordance between mRNA and protein levels for CD3 zeta and CD3 eta is also observed in splenic T cells. Thus, posttranscriptional and/or transcriptional regulatory mechanisms control CD3 zeta and CD3 eta expression during T-cell development.

Amino Acid Sequence↗
PubMed1991

Differential signal transduction via T-cell receptor CD3 zeta 2, CD3 zeta-eta, and CD3 eta 2 isoforms.

The T-cell antigen receptor (TCR) consists of an antigen-binding heterodimer, termed Ti, which is noncovalently associated with the invariant CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3 zeta and -eta subunits form either homodimeric or heterodimeric structures in turn associated with the other components of the TCR complex. This feature increases the structural complexity of TCRs by creating "isoforms." Both CD3 zeta and -eta are thought to play an important role in signal transduction triggered by antigen/major histocompatibility complex. To compare signaling functions of TCR isoforms, MA5.8, a CD3 zeta-eta- variant of the cytochrome c-specific, I-Ek-restricted T-cell hybridoma 2B4.11, was stably transfected with cDNAs encoding CD3 zeta and/or CD3 eta, and resulting clones were characterized. The findings indicate that signals inducing Ca2+ mobilization, phosphatidylinositol turnover, and interleukin 2 production are each transmitted by the above TCR isoforms. In contrast, tyrosine phosphorylation of the CD3 zeta subunit but not the CD3 eta subunit follows TCR stimulation. Given the general importance of tyrosine phosphorylation for receptor signaling, it is likely that this difference between TCR isoforms plays a regulatory role in T-lineage function by qualitatively or quantitatively altering signaling events.

Animals↗
PubMed1991

T cell receptor-independent CD2 signal transduction in FcR+ cells.

CD2 subserves both adhesion and signal transduction functions in T cells, thymocytes, and natural killer (NK) cells. In mature T lymphocytes, CD2-mediated signaling function apparently requires surface expression of T cell receptors (TCRs). In contrast, in CD2+ CD3- NK cells and thymocytes, signal transduction through CD2 is TCR independent. To resolve this paradox and characterize TCR-independent triggering mechanisms, we transfected a human CD2 cDNA into a murine mast cell line, C1.MC/57 (Fc epsilon RI+, Fc gamma RII+, Fc gamma RIII+), which is known to produce interleukin 6 (IL-6) as well as release histamine in response to crosslinking of Fc epsilon RI. In the CD2 transfectant, a combination of anti-T11(2) + anti-T11(3) monoclonal antibodies (mAbs) induced a rise in intracellular free calcium [( Ca2+]i), IL-6 production, and histamine release. As expected, no activation was mediated by the same mAbs in C1.MC/57. F(ab)'s fragments of the activatory combination of anti-T11(2) + anti-T11(3) mAbs induced IL-6 in the CD2-transfected mast cells, demonstrating an Fc gamma receptor ectodomain-independent triggering mechanism. In addition, either intact anti-T11(2) or anti-T11(3) IgG alone, which failed to induce [Ca2+]i mobilization in the transfectant, was able to induce IL-6 production. A mAb directed against both Fc gamma RII (previously denoted as Fc gamma RIIb) and Fc gamma RIII (previously denoted as Fc gamma RIIa) inhibits this induction. These results indicate that: (a) Ca2+ mobilization is not essential for IL-6 production; and (b) crosslinking of CD2 and Fc gamma receptors via intact anti-CD2 IgG stimulates IL-6 production. Thus, CD2-mediated IL-6 production occurs by both Fc receptor ectodomain-independent as well as Fc receptor ectodomain-dependent mechanisms in these nonlymphoid cells. Northern blot analysis demonstrates that although the mast cells do not express CD3 zeta or CD3 eta mRNA, they express Fc epsilon RI gamma mRNA. The latter is a known component of Fc gamma RIII as well as Fc epsilon RI, has significant homology to CD3 zeta/eta, and is thought to have a signal transduction function. In these mast cells, CD2 signaling machinery does not require CD3 zeta/eta and may be linked to the Fc epsilon RI gamma subunit. We predict that this subunit or a related structure may confer a TCR-independent signal transduction pathway upon CD2 in CD3- NK cells, thymocytes, and certain B lymphocytes.

Animals↗
PubMed1991

The expression of IL-2R alpha-chain is enhanced by activation of adenylate cyclase in large granular lymphocytes and natural killer cells.

An increase in intracellular cAMP level induced the expression of IL-2R alpha-chain, the 55-kDa component of IL-2R complex, in a human NK-like cell line, YT. We show here that forskolin also induces the expression of IL-2R alpha-chain on mouse large granular lymphocytes (LGL) but not on T cells. In contrast, treatment with a combination of phorbol ester and calcium ionophore, which is a strong inducer of IL-2R alpha-chain on T cells, does not induce the expression of the alpha-chain on LGL cells. Forskolin was shown to activate the transcription of IL-2R alpha-chain gene in YT cells as revealed by the chloramphenicol acetyltransferase assay. Chemical cross-linking experiments using radio-iodinated IL-2 also supported the enhanced expression of IL-2R alpha-chain by treatment with forskolin. In contrast to the alpha-chain, IL-2R beta-chain was not induced by forskolin as revealed by flow cytofluorometry with a mAb against the beta-chain molecule. These results indicate that the activation of adenylate cyclase induces or/and enhance the expression of IL-2R alpha-chain at the transcriptional level in LGL/NK cells including mouse LGL and human YT cell, which leads to the enhanced expression of high affinity IL-2 receptors.

Adenylyl Cyclases↗
PubMed1991

Complementary roles for CD2 and LFA-1 adhesion pathways during T cell activation.

The influence of T cell receptor (TcR) triggering on T cell adhesion function has been systematically investigated in the present studies; we show that the adhesion function of LFA-1 is minimal in non-activated T cells but is augmented within minutes following TcR-mediated activation. In contrast, CD2 function is essentially optimal in non-activated T cells and undergoes no detectable modification within 12 h of TcR stimulation. Protein kinase C activation augments LFA-1 but not CD2 adhesion function and cyclic AMP reduces LFA-1 adhesion without affecting CD2-LFA-3 interactions. Up-regulation of the LFA-1 pathway occurs in the absence of any detectable surface redistribution of this molecule, suggesting an activation dependent modification leading to a high-affinity ICAM-1 binding state. The TcR independence of CD2 adhesion function implies a critical role of the CD2 pathway in initiating cell-cell interactions prior to TcR engagement and LFA-1-ICAM-1 binding and underscores the complementary nature of the CD2 and LFA-1 adhesion pathways during the immune response.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

T-cell receptor isoforms and signal transduction.

Recent cDNA and genomic cloning have identified CD3 eta as an alternatively spliced product of the same gene locus that encodes CD3 zeta. Three distinct T-cell receptor isoforms have now been identified. A current view of the signal transduction function of these isoforms in thymocytes and T cells is discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Characterization of thymus-derived lymphocytes expressing Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta antigen receptor isoforms: analysis by gene transfection.

To characterize the function of the CD3 eta subunit of the T cell receptor (TCR), we have used cDNAs encoding CD3 zeta, CD3 eta, or both to reconstitute a variant of a cytochrome c-specific, I-Ek-restricted murine T cell hybridoma, termed MA5.8, which lacks CD3 zeta and CD3 eta proteins. We provide direct evidence that assembly and surface expression of TCRs can be mediated by either of these subunits separately or together. However, the level of TCR expression on zeta transfectants is up to one order of magnitude greater than that on eta transfectants, implying that CD3 eta is weakly associated with the pentameric Ti alpha-beta CD3 gamma delta epsilon complex and/or inefficient at salvaging the incomplete TCR from lysosomal degradation. As a component of the TCR, the CD3 eta subunit preferentially forms a heterodimer with CD3 zeta, but is also able to form a CD3 eta-eta homodimer. Crosslinking of Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta, or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta TCR isotypes with anti-CD3 epsilon monoclonal antibody or a cytochrome c peptide epitope on I-Ek antigen-presenting cells mediates signal transduction resulting in reversible cell-cycle arrest of transfected clones. Given the potential for diversity of signals generated by these functional TCR isotypes and the expression of the CD3 eta gene product in the thymus, CD3 eta is likely to play a role in selection and/or activation of thymocytes during development.

Animals↗
PubMed1990

A fraction of CD3 epsilon subunits exists as disulfide-linked dimers in both human and murine T lymphocytes.

In a T cell antigen receptor complex (TCR), the clonotypic disulfide-linked Ti heterodimer is noncovalently associated with the invariant CD3 polypeptides. The latter are composed of three monomeric subunits (gamma, delta, epsilon) and either a disulfide-linked homodimer (zeta zeta) or a disulfide-linked heterodimer (zeta eta). The exact stoichiometry of the Ti-CD3 subunits in a given complex is still largely unknown. Here, we report the presence of a CD3 epsilon dimer in a fraction of the TCR. When TCRs from both human and murine T lymphocytes were immunoprecipitated with monoclonal antibodies against either CD3 epsilon or Ti, a 40-kDa disulfide-linked dimer was coprecipitated with the other TCR subunits from digitonin lysates. Amino acid sequence analysis of peptides obtained by in situ CNBr cleavage of the 20-kDa product blotted to polyvinyl difluoride membranes from reducing/nonreducing two-dimensional gels identified human CD3 epsilon. Assuming this CD3 epsilon to derive from a homodimer, then either some TCRs contain more than one CD3 epsilon chain or several TCRs are covalently associated with one another via their CD3 epsilon subunits. Although it has been suggested that a putative TCR association with CD2 exists under similar conditions to those utilized to detect CD3 epsilon dimers, the CD2 molecule was not coimmunoprecipitated with the TCR by any of a series of anti-CD3 epsilon monoclonal antibodies. In conjunction with the fact that CD2 and the TCR do not colocalize during conjugate formation between T cells and antigen-presenting cells (Koyasu, S., Lawton, T., Novick, D., Recny, M. A., Siliciano, R. F., Wallner, B. P., and Reinherz, E. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2603-2607), we conclude that CD2 and the TCR are not physically associated on the T cell surface.

Amino Acid Sequence↗
PubMed1990

Role of interaction of CD2 molecules with lymphocyte function-associated antigen 3 in T-cell recognition of nominal antigen.

The role of the interaction of CD2 molecules with lymphocyte function-associated antigen 3 (LFA-3) in facilitating nominal antigen recognition by T lymphocytes was studied by utilizing an HLA-DR4-restricted CD4+ cytotoxic human T-cell clone specific for human immunodeficiency virus envelope glycoprotein gp120 as a responder and murine fibroblasts transfected with human class II major histocompatibility complex (MHC) and/or human LFA-3 molecules as antigen-presenting cells (APC). Although expression of the DR4 restriction element in fibroblasts is sufficient for T-cell recognition of a gp120 peptide as judged by induction of proliferation coexpression of human LFA-3 on DR4+ APC decreases the molar requirement of nominal antigen by greater than one order of magnitude. Both LFA-3 and the relevant class II MHC molecules are necessary for antigen-independent conjugate formation, but the binding is further enhanced by specific nominal antigen. CD2-LFA-3 interaction is independent of T-cell receptor-MHC interaction and contributes directly to the stabilized conjugate between the T cell and LFA-3-bearing APC; soluble CD2 and monoclonal antibodies to LFA-3 and CD2 reduce T-cell-APC binding to the level mediated by nominal antigen and MHC. During conjugate formation, CD2 but not CD3 molecules are reorganized into the cell-cell interaction site in an antigen-independent manner. Thus, reorganization and/or coassociation of CD2 with CD3 molecules is not essential for T-cell activation.

Animals↗
PubMed1990

Molecular cloning of the CD3 eta subunit identifies a CD3 zeta-related product in thymus-derived cells.

The CD3 eta subunit of the T-cell antigen receptor forms a heterodimeric structure with the CD3 zeta subunit in thymus-derived lymphoid cells and is apparently involved in signal transduction through the receptor. Here we report the primary structure of murine CD3 eta as deduced from protein microsequencing and cDNA cloning. The mature protein is divided into three domains: a 9-amino acid extracellular segment, a 21-amino acid transmembrane segment including a negatively charged residue characteristic of CD3 subunits, and a 155-amino acid cytoplasmic tail. The NH2-terminal sequences of CD3 eta and CD3 zeta are identical through amino acid 122 of each mature protein but then diverge in the remainder of their respective COOH-terminal regions, consistent with alternatively spliced products of a common gene. The cytoplasmic domain of CD3 eta is 42 amino acids larger than that of CD3 zeta but lacks one of six potential tyrosine phosphorylation sites as well as a putative nucleotide binding site previously identified in CD3 zeta. These structural features presumably account for the difference between CD3 eta and CD3 zeta function and are consistent with the notion that CD3 eta may be an important component of a T-cell receptor isoform(s) during thymic development.

Amino Acid Sequence↗
PubMed1990

Identification of a cell surface 105 kd protein (Aic-2 antigen) which binds interleukin-3.

A mouse interleukin-3 (IL-3)-binding molecule that is an essential constituent of the mouse IL-3 receptor complex was identified as a cell surface protein of Mr 105 kd. A rat monoclonal antibody, anti-Aic-2 IgM, recognized and immunoprecipitated a cell surface 105 kd protein (Aic-2 antigen). The antigen (Aic-2) and IL-3 receptor were co-down-regulated upon incubation of IL-3-dependent mouse IC2 cells with either anti-Aic-2 IgM or IL-3 at 37 degrees C, whereas anti-Aic-2 did not inhibit the binding of IL-3 to IC2 cells at 15 degrees C. The Aic-2 antigen and IL-3 receptor were co-distributed on various cell lines and cell types. IL-3 was shown to bind specifically to the Aic-2 antigen (Mr 105 kd) in the immunoprecipitated complex with anti-Aic-2. Chemically cross-linking of IL-3 to surface molecules of IC2 cells produced three types of complexes with Mr 95, 140, and 200-340 kd in SDS-PAGE, and pre-incubation with anti-Aic-2 IgM at 37 degrees C reduced the intensity of the three bands to the same degree. Moreover, anti-Aic-2 immunoprecipitated these three IL-3-cross-linked complexes, whereas the same monoclonal antibody recognized a single 105 kd molecule. Anti-Aic-2 IgM enhanced IL-3-dependent growth of IC2 cells though it did not promote proliferation of IC2 cells.

Animals↗
PubMed1990

70-75 kd molecules expressed on LGL and T cells recognized by a mitogenic monoclonal antibody YTA-1; co-modulation and functional association with the interleukin 2 receptor p75.

We have raised a mAb YTA-1 which recognizes 75 kd antigen (Ag) on large granular lymphocytes (LGL) and T cells using a human natural killer (NK)/LGL cell line YT as the immunogen. YTA-1 mAb not only enhanced the growth of peripheral blood mononuclear cells (PBMC) but also down-regulated both high- and intermediate-affinity interleukin 2 receptors (IL-2R) on resting PBMC. When incubated with 5-50 micrograms/ml of YTA-1 mAb at 37 degrees C, up to 50% of high- and intermediate-affinity IL-2R of the Tac+ YT subclone (YTCl.1) were down-regulated within 120 min. YTA-1 mAb also down-regulated the intermediate-affinity IL-2R on YT2C2 cells, which express only IL-2Rp75. On the other hand, 5 nM recombinant IL-2 down-regulated the expression of YTA-1 Ag on YT cells after 120 min culture, indicating a close association between YTA-1 Ag and IL-2R. However, YTA-1 mAb did not affect the low-affinity IL-2R on an HTLV-1+ T cell line MT-1. Furthermore, interleukin 1 receptor (IL-1R) on YT cells was not affected by YTA-1 mAb. It appears that the down-regulation by YTA-1 mAb is selective for IL-2Rp75 of the IL-2R complex. Pre-incubation of YT cells with 20 micrograms/ml YTA-1 mAb accelerated the internalization of IL-2Rp75 but did not accelerate that of IL-2Rp55, indicating that YTA-1-induced down-regulation of IL-2R is associated with the internalization of IL-2Rp75.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1989

Protein kinase C-dependent and -independent pathways in the growth factor-induced cytoskeletal reorganization.

Human epidermoid carcinoma KB cells exhibit rapid induction of membrane ruffling in response to epidermal growth factor (EGF), insulin, and insulin-like growth factor-I (IGF-I) (Kadowaki, T., Koyasu, S., Nishida, E., Sakai, H., Takaku, F., Yahara, I., and Kasuga, M. (1986) J. Biol. Chem. 261, 16141-16147). We have analyzed the role of protein kinase C (PKC) in this response. Treatment of KB cells with 4 beta-phorbol 12,13-dibutyrate (PDBu) (100 ng/ml) for 30 min caused translocation of PKC to the membrane. This treatment completely inhibited the induction of membrane ruffling by EGF, insulin, and IGF-I. Prolonged treatment with PDBu (200 ng/ml for 15 h) induced complete depletion of the PKC activity in the cells. Under these conditions, EGF binding to cells and autophosphorylation of the EGF receptor occurred normally, while EGF could not induce membrane ruffling. In contrast, insulin- or IGF-I-induced membrane ruffling occurred normally in the PKC-depleted cells. Moreover, H-7 (PKC inhibitor) inhibited only EGF-induced membrane ruffling in a dose-dependent manner. We further found that EGF, but not insulin/IGF-I, caused transient translocation of PKC to the membrane. All these results suggest that PKC is required for the membrane ruffling induced by EGF but not for that induced by insulin/IGF-I. Therefore, there are PKC-dependent and independent pathways in the growth factor-induced membrane ruffling. Furthermore, we propose dual roles of PKC in the EGF signaling, a signal transmitting role and a negative feedback role.

Cell Membrane↗
PubMed1989

HSP100, a 100-kDa heat shock protein, is a Ca2+-calmodulin-regulated actin-binding protein.

The 100-kDa heat shock protein, HSP100, was purified from mouse lymphoma cells. Amino acid sequences of three peptide fragments which were obtained from the purified protein by lysylendopeptidase digestion were completely or nearly identical with those of a mouse endoplasmic reticulum protein, ERp99, of a hamster glucose-regulated protein, GRP94, and of a chicken heat shock protein, HSP108, all of which have been known to have strong homology with the 90-kDa heat shock protein, HSP90. HSP100 bound to actin filaments and an apparent Kd for the binding was determined to be 8 x 10(-7) M in 2 mM MgCl2 + 100 mM KCl. Calmodulin inhibited the binding in a Ca2+-dependent manner. Equilibrium gel filtration demonstrated that HSP100 has an ability to bind to calmodulin only in the presence of Ca2+. Moreover, HSP100 competed with HSP90 for binding to actin filaments. These results together with our previous findings that HSP90 and HSP100 have similar physicochemical properties (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8054-8058) and HSP90 is a calmodulin-regulated actin-binding protein (Nishida, E., Koyasu, S., Sakai, H., and Yahara, I. (1986) J. Biol. Chem. 261, 16033-16036), strongly suggest that HSP100 is structurally and functionally related to HSP90.

Actin Cytoskeleton↗
PubMed1989

Regulation by intracellular Ca2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis.

Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes [T. Kadowaki et al. (1986) J. Biol. Chem. 261, 16,141-16,147] and stimulate the fluid-phase endocytosis and exocytosis [Y. Miyata et al. (1988) Exp. Cell Res. 178, 73-83] in human epidermoid carcinoma KB cells. An increase in intracellular Ca2+ concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca2+ or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca2+ and cAMP. 125I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca2+- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.

Bucladesine↗