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Y Nojima

Publications and source records attributed to Y Nojima.

At least 73 records · Page 4Linked to original sources
PubMed1996

Tyrosine phosphorylation of p130Cas in cell adhesion and transformation.

Integrins comprise the major class of receptors used by cells to interact with the extracellular matrix. Integrin/matrix interactions play a critical role in a variety of biological processes, including embryonic development, wound healing, tumor metastasis, cell growth and differentiation. It is now evident that integrins can transduce biochemical signals across the plasma membrane to the cell interior. Protein tyrosine phosphorylation has attracted much attention as an important regulator for integrin-mediated signal transduction. Recently, we have identified a novel signaling molecule, p130Cas, which participates in integrin-mediated signal transduction. p130Cas was originally identified as a protein hyperphosphorylated in cells expressing transforming gene products p47v-crk (v-Crk) and p60v-crk (v-Src). In this brief review, we will discuss about a role of p130Cas in signal transduction triggered by cell adhesion and transformation.

Cell Adhesion↗
PubMed1995

Role of the VLA-4 molecule in T cell costimulation. Identification of the tyrosine phosphorylation pattern induced by the ligation of VLA-4.

The very late activated Ag (VLA) molecules not only mediate T cell adhesions, but also provide costimulation in a TCR/CD3-dependent manner. However, little is known about the signals mediated by the ligation of VLA molecules. Previous work from our laboratory identified a 105-kDa protein that is predominantly phosphorylated on tyrosine residue upon engagement of VLA-4 in a human T lymphoblastic cell line, H9, and in peripheral T cells. In the present study, we have shown that the A and B epitope of VLA-4 plays a key role in VLA-4-mediated T cell costimulation. Moreover, we have demonstrated that the solid phase cross-linking of VLA-4 using Ab (against A and B) or the CS-1 region of fibronectin, stimulated tyrosine phosphorylation of 140-, 120-, 80- to 70-, 60- to 55-, 50-, and 45-kDa proteins in addition to the 105-kDa protein. In contrast, Ab ligation of the C epitope of VLA-4 mainly induced tyrosine phosphorylation of pp105, weakly induced other protein tyrosine phosphorylation, and additionally induced only minimal T cell costimulation. Using immunoblotting, we have identified some of the tyrosine-phosphorylated proteins to be phospholipase C gamma (pp140), pp125 focal adhesion kinase (pp120), paxillin (pp70 and pp50), p59fyn/p56lck (pp60-55), and mitogen-activated protein kinase (pp45). Since solid phase cross-linking of VLA-4 by B2 epitope-specific Ab induced T cell costimulation most strongly via the CD3 pathway, our results suggested that the above tyrosine-phosphorylated proteins may play an important role in VLA-4-mediated T cell costimulatory signaling events.

Antibodies, Monoclonal↗
PubMed1995

Stretching mesangial cells stimulates tyrosine phosphorylation of focal adhesion kinase pp125FAK.

Mechanical loading of cultured mesangial cells has been shown to induce both increased production of matrix proteins and cell proliferation. This in vitro phenomenon has been implicated in the progression of glomerular sclerosis observed in a variety of glomerular diseases. However, it is not yet known how cells sense mechanical stress and transfer this sense into biochemical and biological events. In this study, we show that mesangial stretching rapidly stimulates tyrosine phosphorylation of non-receptor tyrosine kinase pp125FAK. Since pp125FAK is a major constituent of focal adhesions, our findings suggest that the focal adhesion may be a site where mechanical forces are translated into biochemical events, and that pp125FAK may play an important role in this signaling cascade.

Animals↗
PubMed1995

Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs.

p130Cas (Cas) has been recently identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues and is stably associated with p47v-crk (v-Crk) and p60v-src (v-Src) oncogene products in cells transformed by the respective genes. Cas is a novel signaling molecule having a single Src homology (SH) 3 domain and a cluster of multiple SH2-binding motifs. While the tight association of Cas with v-Crk and v-Src is strongly suggestive of a significant role in regulating cellular transformation, the function of Cas in normal untransformed cells is totally unknown. We report here that cell adhesion to fibronectin rapidly promotes tyrosine phosphorylation of Cas in human and rat fibroblast cell lines. The response was equally induced by cell adhesion to plates coated with vitronectin, laminin, and collagen but not by cell attachment to nonspecific substrate poly-L-lysine. The kinetic profile of Cas phosphorylation was almost identical with that of tyrosine phosphorylation of focal adhesion kinase pp125FAK (Fak), which is well known to be activated subsequent to integrin-mediated cell adhesion. Adhesion-dependent Cas phosphorylation was completely inhibited by treating cells with cytochalasin D, an agent that disrupts polymerization of actin stress fibers. These results suggest that tyrosine phosphorylation of Cas is stimulated by normal cell adhesion in close association with Fak phosphorylation and the formation of actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the tyrosine phosphorylation of Cas is markedly increased in an adhesion-independent manner that is insensitive to treatment with cytochalasin D. Thus, Cas plays a role in signaling pathways mediated by cell adhesion as well as by transformation. We propose that Cas may amplify and propagate integrin-mediated signals by interacting with SH2-containing molecule(s).

Animals↗
PubMed1995

Matrix/integrin interaction activates the mitogen-activated protein kinase, p44erk-1 and p42erk-2.

Cell adhesion to extracellular matrix proteins is a dynamic process leading to dramatic changes in the cell phenotype. Integrins are one of the major receptor families that mediate cell-matrix contact. Evidence that integrins can act as signal transducing molecules has accumulated over the past few years. We report here that p44erk-1 and p42erk-2 mitogen-activated protein (MAP) kinases are rapidly phosphorylated on tyrosine residues upon adhesion of human skin fibroblasts to fibronectin or upon cross-linking of beta 1 integrins with antibody. The tyrosine phosphorylation of both kinases is associated with increased enzymatic activity. Pretreatment of the cells with cytochalasin D, which selectively disrupts the network of the actin filaments, completely inhibits this adhesion-mediated MAP kinase activation. Thus, our findings indicate that ligation of beta 1 integrins induces an increase in both tyrosine phosphorylation and enzymatic activity of p44erk-1 and p42erk-2 MAP kinases, and that the integrity of the actin cytoskeleton is essential in this process. Since MAP kinase behaves as a convergence point for diverse receptor-initiated signaling events at the plasma membrane, this serine/threonine kinase plays a key role and helps to account for the diversity of integrin-dependent cell functions.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Focal adhesion kinase (pp125FAK) is tyrosine phosphorylated after engagement of alpha 4 beta 1 and alpha 5 beta 1 integrins on human T-lymphoblastic cells.

pp125FAK is a novel protein tyrosine kinase associated with focal adhesions. It has been shown that ligation of VLA beta 1 integrins on a number of cell types enhanced tyrosine phosphorylation and kinase activity of pp125FAK. Cellular transformation by retroviral oncogene products or mitogenic neuropeptides also result in the activation of this kinase. On the basis of these observations, pp125FAK has been proposed to be a key regulatory molecule connecting cell adhesion, transformation, and growth. We have previously shown that ligation of VLA beta 1 integrins induced CD3-dependent T cell proliferation and stimulated tyrosine phosphorylation of a molecular mass with a 105-kDa protein (pp105). Here we report that engagement of alpha 4 beta 1 and alpha 5 beta 1 integrins by adherence to their respective ligands stimulated tyrosine phosphorylation of 105- to 120-kDa proteins (pp105 and pp120, respectively) in human H9 T-lymphoblastic cells. At least one component of the 105- to 120-kDa proteins was found to be tyrosine-phosphorylated pp125FAK. While kinetics of adherence-dependent tyrosine phosphorylation of pp120/pp125FAK and pp105 are closely similar, pp105 appeared to be distinct from pp125FAK. Given T cell costimulation induced by VLA beta 1 integrins and the putative functional role of pp125FAK in cell growth, tyrosine phosphorylation of these two distinct proteins may be involved in T cell activation and proliferation.

Cell Adhesion↗
PubMed1995

Detection of antiribosomal P protein antibodies in patients with systemic sclerosis.

This study investigated the prevalence and clinical significance of anti-ribosomal P protein (anti-P) antibodies in patients with systemic sclerosis (SSc). Serum samples from 150 patients with SSc were examined by indirect immunofluorescence. ELISA and immunoblotting. Anti-P antibodies were detected in four (3%) patients with SSc. Three of the four patients showed SSc/SLE (systemic lupus erythematosus) overlap syndrome, but psychiatric disorders were not observed in these patients. By longitudinal immunoblotting analysis one patient, who was initially diagnosed with SSc, later developed anti-P antibodies along clinical manifestations of SLE. Our data suggest that anti-P antibodies are uncommon in SSc and that the presence of anti-P antibodies in patients with SSc indicates an overlap with SLE.

Adult↗
PubMed1995

[Cytotoxicity and mutagenicity studies of T-3761].

We investigated cytotoxicity and mutagenicity of T-3761. The mutagenicity was evaluated using reverse mutation test with bacteria, chromosome aberration test with cultured cells and micronucleus test with mice. The following results were obtained. 1. Cytotoxicity test: The cell growth was examined using Chinese hamster (V79) cells. The 50% inhibition doses of T-3761 for cell growth (ID50) were 490 micrograms/ml (cultured for 24 hours) and 220 micrograms/ml (cultured for 48 hours). The inhibitory effect of T-3761 was 2-4 times lower than those of ciprofloxacin or norfloxacin and approximately equal to cephalothin. 2. Reverse mutation test with bacteria: The preincubation method with Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, and the induced mutation frequency (IMF) test with Salmonella typhimurium TA100, TA98 were performed. The number of revertant colonies were not increased in any strains treated with T-3761 in the presence or absence of S9 mix. 3. Chromosome aberration test: V79 cells were treated with 50-200 micrograms/ml of T-3761 for 24 or 48 hours, and were treated with 400-3,200 micrograms/ml of T-3761 for 6 hours with S9 mix. The number of cells showing chromosomal aberrations were not increased in any conditions tested for T-3761. 4. Micronucleus test: The male ICR mice were given a single (500-5,000 mg/kg) or five consecutive (150-1,500 mg/kg) oral administration of T-3761. The number of polychromatic erythrocytes with micronuclei were not increased at any dosage groups of T-3761. From these results, it is concluded that T-3761 has low cytotoxicity, and has no mutagenicity.

Animals↗
PubMed1994

3H11, a unique cell surface molecule involved in the function of the CD45RA+ subset of CD4+ cells.

We have developed a mAb anti-3H11 by immunizing mice with a T cell line derived from the Callithrix jacchus (common marmoset). Anti-3H11 is reactive with approximately 48% of unfractionated T cells, 62% of CD4+ cells and 39% of CD8+ cells. Among CD4 cells, anti-3H11 preferentially reacts with the CD45RA+ T cell subset. The majority of helper activity for pokeweed mitogen (PWM)-driven B cell IgG synthesis and T cell response to recall antigen such as tetanus toxoid was found within the 3H11-CD4 cell population, whereas anti-3H11+CD4+ cells provided poor helper function for PWM-driven B cell IgG synthesis and were more responsive to concanavalin A and autologous mixed lymphocyte reaction. Biochemical characterization showed that anti-3H11 precipitated a single protein band with a relative molecular weight of 32,000 from 125I-surface labeled cell lysate. Biochemical, phenotypic and functional studies revealed that the 3H11 molecule appeared to be different from previously established molecules on the T cell surface. Interestingly, addition of anti-3H11 to the combination of CD4 and B cells in the presence of CD8 cells but not to the combination of CD4 and B cells resulted in enhancement of the suppression of PWM-driven B cell IgG synthesis. Moreover, anti-3H11 had a co-mitogenic effect on T cells via the CD2 and CD3 pathways, and this co-mitogenic activity is restricted to the CD45RA+ T cells. Taken together, our results show that the 3H11 molecule is a novel antigen which may play an important role in the activation and function of the CD45RA+ subset of T cells.

Animals↗
PubMed1994

Prolonged impairment of very late activating antigen-mediated T cell proliferation via the CD3 pathway after T cell-depleted allogeneic bone marrow transplantation.

One of the major obstacles in allogeneic bone marrow transplantation (allo-BMT) is prolonged T cell dysfunction resulting in a variety of infectious complications in the months to years after hematologic engraftment. We previously showed that immobilized extracellular matrix (ECM) proteins such as fibronectin (FN), the CS-1 domain of FN, or collagen (CO) acted synergistically with immobilized anti-CD3 to induce T cell proliferation. In addition, the comitogenic effect of ECMs could be mimicked by immobilized mAb reactive with a common beta 1 chain (CD29) of very late activating (VLA) antigens which include ECM receptors. Since the interaction of T cells with ECMs appears to play an important role in the process of T cell reconstitution following allo-BMT, we examined the expression of VLA antigens (alpha 1-alpha 6, beta 1) and their functional roles in CD3-mediated T cell proliferation at various times after T cell depleted allo-BMT. VLA beta 1 as well as VLA alpha 4, alpha 5, and alpha 6 expression was lower than normal controls during the first 3 mo after allo-BMT and auto-BMT, whereas these expressions returned to normal levels by 4 mo after allo-BMT and auto-BMT. Although alpha 1 and alpha 2 were not expressed on lymphocytes from normal controls, these antigens were expressed on lymphocytes at the detectable levels (5-15%) from patients after allo-BMT and auto-BMT. Both CD29 and CD3 were expressed at normal levels on lymphocytes from patients > 3 mo after allo-BMT, whereas T cell interaction with ECM through VLA proteins or crosslinking of VLA beta 1 expressed by T cells with anti-CD29 mAb results in poor induction of CD3-mediated T cell proliferation for a prolonged period (> 1 yr) after allo-BMT. In contrast, T cell proliferation induced by crosslinking of anti-CD2 or anti-CD26 with anti-CD3 was almost fully recovered by 1 yr post-allo-BMT. After autologous BMT, impaired VLA-mediated T cell proliferation via the CD3 pathway after auto-BMT returned to normal levels within 1 yr despite no significant difference in CD3 and CD29 expression following either allo- or auto-BMT. The adhesion of T cells from post-allo-BMT patients to FN-coated plate was normal or increased compared to that of normal controls. Moreover, the induction of the tyrosine phosphorylation of pp105 protein by the ligation of VLA molecules was not impaired in allo-BMT patients. These results suggest that there are some other defects in the process of VLA-mediated signal transduction in such patients. Our results imply that disturbance of VLA function could explain, at least in part, the persistent immunoincompetent state after allo-BMT and may be involved in susceptibility to opportunistic infections after allo-BMT.

Antigens, CD↗
PubMed1993

Direct association of adenosine deaminase with a T cell activation antigen, CD26.

CD26, the T cell activation molecule dipeptidyl peptidase IV (DPPIV), associates with a 43-kilodalton protein. Amino acid sequence analysis and immunoprecipitation studies demonstrated that this 43-kilodalton protein was adenosine deaminase (ADA). ADA was coexpressed with CD26 on the Jurkat T cell lines, and an in vitro binding assay showed that the binding was through the extracellular domain of CD26. ADA deficiency causes severe combined immunodeficiency disease (SCID) in humans. Thus, ADA and CD26 (DPPIV) interact on the T cell surface, and this interaction may provide a clue to the pathophysiology of SCID caused by ADA deficiency.

Adenosine Deaminase↗
PubMed1993

The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes.

CD22 is a B lineage-restricted member of the Ig superfamily that serves as an adhesion receptor expressed by mature B lymphocytes. In this study, the ability of different cell types to attach to COS cells transiently transfected with a full-length CD22 cDNA (COS-CD22) was examined to determine the cellular distribution of the ligand for CD22. T and B lymphocytes, monocytes, erythrocytes, and neutrophils formed specific rosettes with COS-CD22 cells at 4 degrees C. A panel of 33 new mAb directed against CD22 were developed to examine the regions of CD22 that mediate adhesion. Four of these mAb, HB22-7, -22, -23, and -33 (at 1 to 5 micrograms/ml) specifically blocked adhesion (75 to 95%) of all cell types to COS-CD22 cells. Each of these mAb cross-blocked each other's binding, suggesting that ligand binding occurs through a single region of CD22. These mAb also identify a region of CD22 distinct from those defined by previously described CD22 mAb. CD22-mediated adhesion of cell lines to COS-CD22 cells was independent of CD45RO and CDw75 expression, and it was not inhibited by mAb against known integrins. Although alpha-2,6-linked sialic acid expressed on the surface of COS cells did not serve as a ligand for CD22, the CD22 ligand may contain a critical sialic acid determinant, as neuraminidase treatment of all target cells eliminated CD22-mediated adhesion. CD22-mediated adhesion was Ca2+/Mg2+ independent, again suggesting that integrins were not involved. An inhibitory substance for CD22-mediated adhesion was found to be present in FCS and some ascites fluid. Analysis of CD22 mRNA and protein revealed that although multiple mRNA splice variants of CD22 mRNA can be detected, only a single protein isoform was detected on the cell surface. Therefore, although the identity of the CD22 ligands remains incompletely characterized, it is possible that a single major ligand is expressed by RBC and leukocytes, which binds to a single region of CD22.

Animals↗
PubMed1993

Stimulation of protein tyrosine phosphorylation in human B cells after ligation of the beta 1 integrin VLA-4.

B lymphocytes express several adhesion molecules that are involved in cell-cell and cell-extracellular matrix interactions. The alpha 4 beta 1 integrin VLA-4, expressed on pre-B and mature/activated B cells, mediates adhesion of these cells to its two ligands, VCAM-1 and fibronectin. Recent evidence suggests that VLA-4 is involved in T lymphocyte activation; however, relatively little is known of the role of VLA-4 in B cell differentiation. To begin to assess the potential involvement of VLA-4 in B cell activation, we have examined the effect of ligation of VLA-4 on protein tyrosine phosphorylation in B cells. We found that cross-linking of VLA-4 by either mAb or natural ligands (i.e., VCAM-1 and the FN-40 cleavage fragment of fibronectin) induced the tyrosine phosphorylation of a 110-kDa protein in a human pre-B cell line (Nalm-6), an EBV-transformed B cell line (SB), and normal tonsillar B cells. These findings suggest that VLA-4 can activate a tyrosine kinase in B cells and B cell lines. These signals may be involved in the subsequent differentiation of pre-B and mature B cells within specific microenvironments where VLA-4 mediated adhesion is operational.

Antigens, CD↗
PubMed1993

The 4F9 antigen is a member of the tetra spans transmembrane protein family and functions as an accessory molecule in T cell activation and adhesion.

In this report, we describe a 43- to 50-kDa protein, which may function as a costimulatory molecule for full activation of human T cells. This Ag, defined by a mouse monoclonal antibody (mAb) anti-4F9, is primarily distributed on "helper/inducer" or "memory" CD4+CD45RO+ subset. Like mAbs against many other accessory/costimulatory molecules, coimmobilization of anti-4F9 with anti-CD3 resulted in synergistic T cell proliferation. In addition, immobilized anti-4F9 on plastic plates induced T cell spreading characterized by the development of prominent dendritic processes. A cDNA encoding the 4F9 Ag was isolated from a cDNA library constructed from PHA/PMA-activated T cells using a COS cell expression system. The sequence of the cDNA and a homology search revealed that the 4F9 Ag was identical to R2, a molecule recently cloned by subtractive hybridization. The 4F9/R2 Ag belongs to a newly identified supergene family (tetra spans transmembrane protein family) characterized by four putative transmembrane domains which are highly conserved between the members of this family. Based upon the phenotypical and functional studies described here, we propose that the 4F9 Ag is an integral membrane protein which can transmit signals involved in T cell proliferation and adhesion. The preferential distribution of this molecule on the CD4+CD45RO+ subset of T cells may contribute to the distinct activation profile and functional repertoire of these cells.

Animals↗
PubMed1993

Changes in subset specificity of anti-T cell autoantibodies in systemic lupus erythematosus.

Relative reactivity of anti-lymphocyte autoantibodies (ALA) from patients with systemic lupus erythematosus (SLE) against CD4+ and CD8+ T cells was studied using C-dependent microcytotoxicity assay. Of 46 SLE sera screened for anti-T cell autoantibodies, 27 sera (59%) showed significant cytotoxic reactivity. Of these, positive correlation between the titer of anti-T cell antibody and CD4/CD8 killing ratio (p < 0.01) was demonstrated. In time course study of individual patients, the CD4/CD8 killing ratio increased and decreased as the disease flared and subsided and was accompanied by parallel changes in the titer of anti-T cell antibody titer. Moreover, as sera were serially diluted, the CD4/CD8 killing ratio decreased in 5 out of 10 sera. These results suggest that discrepancy among reports concerning the subset specificity of anti-T cell antibodies may be due, in part, to differences in the titer of ALA in the sera studied and to the dilution of serum used.

Animals↗
PubMed1993

Protein kinase A-mediated phosphorylation of the T-cell surface antigen CD27.

CD27 is a T-cell surface antigen expressed on the majority of peripheral T cells and belongs to a newly defined receptor family including the low-affinity nerve growth factor receptor, tumour necrosis factor (TNF) receptors, the B-cell activation antigen CD40, and the Fas antigen. Although the function of CD27 has not been defined, several experimental observations support the notion that this molecule plays an important role in the process of T-cell activation. In this paper, we have demonstrated that a rapid hyperphosphorylation of CD27 is induced by a cyclic AMP-inducing agent, forskolin, and a membrane-permeable cAMP analogue, 8-bromo-cAMP, as well as phorbol 12-myristate 13-acetate (PMA). In addition, increased phosphorylation of CD27 in T-cell activation either via CD2 or CD3 pathways was strongly suppressed by a cyclic nucleotide-dependent kinase inhibitor, H-8, but only slightly by a protein kinase C inhibitor, staurosporine. These results suggest that protein kinase A might be a key kinase responsible for CD27 phosphorylation in the process of T-cell activation. CD27 is the first T-cell surface antigen demonstrated to be phosphorylated by the cyclic AMP-protein kinase A-mediated pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Ligation of VLA-4 on T cells stimulates tyrosine phosphorylation of a 105-kD protein.

The VLA/integrins are a family of heterodimeric adhesion receptors shown to be involved in cell-to-cell and cell-to-extracellular matrix (ECM) interactions. Given recent evidence that VLA molecules can synergize with the CD3/T cell receptor (TCR) pathway to activate T cells, it is important to identify biochemical event(s) generated by these molecules. Here, we report that the engagement of VLA-4 on T cells with specific antibodies or its ligand activates protein-tyrosine kinase (PTK) activity as detected by antiphosphotyrosine immunoblotting. The crosslinking of VLA-beta 1 (CD29) with a specific monoclonal antibody (mAb) (anti-4B4) plus anti-mouse immunoglobulin resulted in the rapid tyrosine phosphorylation of a 105-kD protein (pp105) in the human T cell line H9, as well as in peripheral resting T cells. The increase in tyrosine phosphorylation of pp105 was specifically mediated by VLA-4, since mAbs against alpha 4, but not against other VLA alpha chains, could induce this phosphorylation. In addition, the binding of T cells with the CS1 alternatively spliced segment of fibronectin (the binding site recognized by VLA-4) induced pp105 tyrosine phosphorylation. Crosslinking the CD3 complex or VLA-4 molecules with mAbs demonstrated that each of these molecules stimulated the tyrosine phosphorylation of unique sets of proteins with different kinetics, suggesting that these two receptor systems are coupled to distinct PTKs. Since tyrosine phosphorylation of cellular proteins has been shown to be a crucial biochemical event in cell growth, our findings suggest that the induction of pp105 tyrosine phosphorylation via VLA-4 may play a role in the transduction of activation signals through this molecule.

Antigens, CD↗