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Z Honda

Publications and source records attributed to Z Honda.

At least 19 recordsLinked to original sources
PubMed1998

Essential roles of Lyn in fibronectin-mediated filamentous actin assembly and cell motility in mast cells.

Although the requirement for c-Src in extracellular matrix (ECM)-mediated fibroblast motility has been well established, the roles of hemopoietic Src family protein tyrosine kinases in leukocyte migration have not been fully elucidated. To address the issue, we analyzed fibronectin (Fn)-mediated adhesion signaling in rat basophilic leukemia (RBL) 2H3 cells overexpressing 1) Csk, 2) a membrane-anchored, gain-of-function Csk (mCsk), and 3) a kinase-defective mCsk (mCsk(-)). Parent RBL2H3 cells, expressing autoactivated c-kit, readily adhered to Fn-coated surface, developed typical leukocyte adhesion machinery (podosome), and migrated toward Fn without cytokine priming, thus provided a simple experimental system to analyze Fn-mediated outside-in signaling. While overexpression of Csk or the Csk mutants did not significantly affect cell adhesion to the Fn surface or alpha5 integrin recruitment to the attachment sites, Csk suppressed and mCsk almost abolished Fn-mediated tyrosine phosphorylation of paxillin, filamentous actin assembly to podosomes, and cell migration, but mCsk(-) did not. Coexpression of LynA devoid of C-terminal negative regulatory tyrosine in mCsk cells successfully restored Fn-mediated podosome formation and cell migration. Coexpression of c-Src lacking the C-terminal tyrosine reconstructed podosomes, but could not restore the cell migration regardless of its expression level. Collectively, these observations provide evidence that Src family protein tyrosine kinases are required, and that Lyn could transmit sufficient signal for Fn-mediated cytoskeletal changes leading to cell locomotion in RBL2H3 cells, and they suggest that Lyn and c-Src are differentially involved in cell motility.

Actins↗
PubMed1998

Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Actin Cytoskeleton↗
PubMed1996

Mutant of insulin receptor substrate-1 incapable of activating phosphatidylinositol 3-kinase did not mediate insulin-stimulated maturation of Xenopus laevis oocytes.

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by lipopolysaccharides.

We designed a microplate-based assay method for mitogen-activated protein (MAP) kinase. Using anion-exchanger resin, MAP kinases from murine macrophages were partially purified in 96-well plates. The activities of these purified enzymes correlated well with those detected in heretofore used assays. The micro-trap phosphorylation assay has advantages over conventional methods (immunoprecipitation, Western blotting for the detection of mobility shift, or kinase detection assay in myelin basic protein (MBP)-containing gel), in terms of sensitivity, economy and rapid execution for hundreds of samples. Using micro-trap phosphorylation assay, it was demonstrated that MAP kinase activities in macrophages were persistently increased by lipopolysaccharide (LPS) stimulation, and this activation was inhibited by polymyxin B or tyrosine kinase inhibitors. This method is expected to give a wide range of application, such as determining effects of drug inhibitors or antisense oligonucleotides on MAP kinases, or measuring the various protein kinases after specificity controls were done.

Animals↗
PubMed1995

Amino-acid sequence and tissue distribution of guinea-pig leukotriene A4 hydrolase.

The guinea-pig leukotriene A4 hydrolase (LTA4H)-encoding cDNA was isolated from a guinea-pig lung cDNA library by cross-hybridization using a human probe. The deduced amino acid (aa) sequence consists of 611 aa (68 756 Da) and contains all twelve internal peptide and N-terminal sequences determined from the purified enzyme from guinea-pig intestine. The aa identity of the guinea-pig enzyme with its human, mouse and rat counterparts was 92.9, 90.5 and 90.4%, respectively. The previously characterized zinc-binding motif and a putative active site were highly conserved, supporting the aminopeptidase activity described for this enzyme. RNA blot analysis demonstrated ubiquitous expression of the LTA4H mRNA.

Amino Acid Sequence↗
PubMed1995

Prostaglandin F2 alpha stimulates formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-protein-coupled pathway.

Prostaglandin (PG) F2 alpha activated mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3 cells by a mechanism that was completely inhibited by protein kinase inhibitors, staurosporine (20 nM) or H-7 (20 microM), but was insensitive to pretreatment with islet-activating protein (100 ng/ml; 24 h) or 12-O-tetradecanoylphorbol 13-acetate (2.5 microM; 24 h). PGF2 alpha stimulation also led to a significant increase in Ras.GTP complex. Transfection of a cDNA encoding a constitutively active mutant of Gq alpha-subunit (Q209L) mimicked PGF2 alpha-induced MAP kinase activation, increase in Ras.GTP complex, and DNA synthesis in these cells, suggesting that activation of Gq mediates the PGF2 alpha-activation of Ras-MAP kinase pathway and mitogenesis in NIH-3T3 cells. These data provide a new insight into regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric G-protein-mediated pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Gq pathway desensitizes chemotactic receptor-induced calcium signaling via inositol trisphosphate receptor down-regulation.

Desensitization of a chemotactic receptor is an adaptive process that terminates inflammation. Although homologous desensitization can be well explained by the action of specific receptor kinases, the mechanisms of heterologous desensitization remain elusive. As an approach to evaluate the roles of Gq pathway in desensitization of calcium signaling, we expressed a constitutively active Gq alpha mutant (Gq alpha Q-L) together with platelet-activating factor (PAF) receptor in Xenopus laevis oocytes. Gq alpha Q-L expression completely attenuated the calcium-sensitive chloride current and the 45Ca release elicited by PAF. The Gq-mediated desensitization could not be ascribed to G protein/receptor uncoupling via receptor phosphorylation, because (i) PAF-induced inositol 1,4,5-trisphosphate (IP3) synthesis was only partially suppressed and (ii) a mutated PAF receptor devoid of all Ser and Thr in the third cytoplasmic loop and in the C-terminal tail was also completely desensitized by Gq alpha Q-L. In Gq alpha Q-L expressing oocytes, microinjection of IP3 failed to elicit the calcium response, and the IP3 receptor, detected by a specific antibody, disappeared. Thus, the Gq-mediated desensitization can be most likely explained by IP3 receptor down-regulation. These novel mechanisms may explain in part heterologous desensitization in chemotactic factor-stimulated inflammatory cells.

Animals↗
PubMed1994

Activation of mitogen-activated protein kinase and arachidonate release via two G protein-coupled receptors expressed in the rat hippocampus.

Platelet-activating factor and somatostatin receptors, two G protein-coupled receptors expressed in the rat hippocampus, were analyzed for the downstream signaling pathways in Chinese hamster ovary cells stably expressing each receptor. Ligand stimulation to each CHO cell line induced (1) inhibition of forskolin-induced accumulation of cAMP, (2) arachidonate release, and (3) activation of mitogen-activated protein kinase and MAP kinase kinase. In contrast, inositol phosphate breakdown was seen only in the PAF-stimulated CHO cells. The induction of these signals accompanied no detectable Ras activation. Suppression of the signals by pertussis toxin was almost complete for the somatostatin receptor but partial for the PAF receptor, suggesting that the somatostatin receptor couples only with PTX-sensitive G protein, while the PAF receptor couples with both PTX-sensitive and -insensitive G proteins. A model of G protein-mediated signaling pathways was proposed in which the signals from Gi and those from Gq converge at MAP kinase kinase and lead to arachidonate release. The present system using CHO cells is useful for analyzing signaling pathways from G proteins to MAP kinase kinase and will thereby provide clues for understanding the mechanisms underlying the physiological and pathological events mediated by PAF, somatostatin, and other G protein-coupled receptors in the central nervous system and other tissues.

Amino Acid Sequence↗
PubMed1994

Role of cytoplasmic tail phosphorylation sites of platelet-activating factor receptor in agonist-induced desensitization.

The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.

Amino Acid Sequence↗
PubMed1994

Functional coupling of SSTR4, a major hippocampal somatostatin receptor, to adenylate cyclase inhibition, arachidonate release and activation of the mitogen-activated protein kinase cascade.

Somatostatin has a modulatory role in regulating the membrane conductance in hippocampal neurons. To examine the signal transducing molecules involved in this process, we isolated the cDNA encoding the dominant rat hippocampal somatostatin receptor, SSTR4. Distribution of SSTR4 in the adult central nervous system was restricted to the hippocampus, cerebral cortex, striatum, hypothalamus, and thalamus, as determined by Northern blot analysis and in situ hybridization. In SSTR4-expressing Chinese hamster ovary cells, SSTR4 was functionally coupled not only to inhibition of adenylate cyclase, but also to activation of both arachidonate release and mitogen-activated protein (MAP) kinase cascade, with similar ED50 values. All of these pathways, including both MAP kinase kinase and MAP kinase activation, were completely blocked by pretreatment with pertussis toxin. On the other hand, neither inositol 1,4,5-trisphosphate synthesis nor intracellular Ca2+ mobilization was induced upon SSTR4 stimulation. These data indicate that the hippocampal functions of somatostatin might be mediated through diverse but selective second messenger systems activated via SSTR4 and reveal an unsuspected coupling of a neuronal SSTR subtype to a mitogenic signaling pathway. SSTR4, in addition, provides a useful system to study the Ca(2+)-independent, Gi-dependent (pertussis toxin-sensitive) pathway of MAP kinase activation.

Adenylate Cyclase Toxin↗
PubMed1994

Cloning, expression and tissue distribution of rat platelet-activating-factor-receptor cDNA.

The biological functions of platelet-activating factor (PAF) have been extensively studied in the rat. However, the precise structure and distribution of rat PAF receptor has not been reported. To address this question, we isolated a rat PAF-receptor cDNA from a size-fractionated rat spleen cDNA library. The deduced amino acid sequence of the rat PAF receptor showed 80% and 79% identity with guinea pig and human PAF receptors, respectively. Pharmacological properties (ED50, inhibition by WEB2086) of rat PAF receptors expressed in Xenopus oocytes were similar to those for PAF receptors expressed from guinea pig or human cDNAs. Northern blot analysis showed a widespread distribution of PAF-receptor mRNA in almost all organs including spleen, small intestine, kidney, lung, liver and brain. Considerable difference in the PAF-receptor distribution detected among species suggests the existence of a species-specific and tissue-specific regulatory mechanism for PAF-receptor-mRNA expression. Isolation of rat PAF-receptor cDNA should facilitate further analysis of PAF-receptor function and pharmacology in diverse pathophysiological processes.

Amino Acid Sequence↗
PubMed1994

Transfected platelet-activating factor receptor activates mitogen-activated protein (MAP) kinase and MAP kinase kinase in Chinese hamster ovary cells.

The platelet-activating factor (PAF) was seen to potently activate mitogen-activated protein (MAP) kinase and MAP kinase kinase through the cloned guinea pig PAF receptor stably expressed in Chinese hamster ovary (CHO) cells. Both 42- and 44-kDa MAP kinases were activated and tyrosine-phosphorylated in response to PAF. The PAF receptor also triggered the production of inositol phosphates and the release of arachidonic acid and inhibited cyclic AMP accumulation. Differential inhibitory effects of pertussis toxin (PTX) on these signals suggested that the PAF receptor couples to both PTX-sensitive and -insensitive G proteins in CHO cells. MAP kinase and MAP kinase activations were partially regulated by PTX-sensitive G proteins. The PAF receptor did not trigger any detectable increase in the GTP form of Ras under the conditions in which the human insulin receptor expressed in the same parent CHO cells potently increased the level. Since these agonists induced comparable MAP kinase activations through cognate receptors, Ras seems to play different roles in MAP kinase activation by the two different classes of receptors. The activation of MAP kinase by the cloned PAF receptor may explain part of the mechanisms underlying PAF-induced differentiation and proliferation in non-inflammatory cells.

Animals↗
PubMed1993

Two distinct signal transduction pathways for the activation of guinea-pig macrophages and neutrophils by endotoxin.

Lipopolysaccharide (LPS, endotoxin) is a major component of the outer membrane of gram-negative bacteria. Although it interacts with many types of cells and is linked to numerous events associated with sepsis and endotoxic shock, the mechanisms underlying these actions are poorly understood. We found that Ca-signaling induced by endotoxin in guinea-pig neutrophils and macrophages is caused by cross-recognition of LPS with platelet activating factor (PAF) receptors. However, the synthesis of tumor necrosis factor-alpha or the priming effect of O2- production was not affected by PAF antagonists. Thus, at least two distinct pathways are involved in the actions of LPS, one via the PAF receptor, while the other is independent of a PAF receptor and Ca-signaling.

Animals↗
PubMed1993

Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor.

The human platelet-activating factor (PAF) receptor gene exists as a single copy on chromosome 1. We identified two 5'-noncoding exons, each of which has distinct transcriptional initiation sites. These exons are alternatively spliced to a common splice acceptor site on a third exon that contains the total open reading frame to yield two different species of functional mRNA (Transcript 1 and 2). Transcript 1 has consensus sequences for transcription factor NF-kappa B and Sp-1, and the Initiator (Inr) sequence homologous to the murine terminal deoxynucleotidyltransferase gene. Transcript 2 also contains consensus sequences for transcription factor AP-1, AP-2, and Sp-1. Transcripts 1 and 2 were both detected in heart, lung, spleen, and kidney, whereas only Transcript 1 was found in peripheral leukocytes, a differentiated human eosinophilic cell line (EoL-1 cells), and brain. Existence of distinct promoters was thus suggested to play a role in the regulatory control of PAF receptor gene expression in different human tissues and cells.

Alternative Splicing↗
PubMed1993

Characterization of prostaglandin F2 alpha receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes.

Prostaglandin (PG) F2 alpha increased [3H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with ED50 values of 2.0 x 10(-8) M, 4.6 x 10(-8) M, and 7.5 x 10(-8) M, respectively. The increase in [3H]thymidine incorporation with PGF2 alpha was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca2+]i increase with PGF2 alpha was still obvious in the absence of extracellular Ca2+ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF2 alpha, which was sensitive to GTP gamma S but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF2 alpha-specific Cl- current when examined by voltage clamp. This Cl- current was also insensitive to IAP pretreatment and not affected by extracellular Ca2+ concentration ([Ca2+]o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF2 alpha receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca2+ via an IAP-insensitive G-protein(s), 2) that this PGF2 alpha receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF2 alpha receptor can be expressed in the oocyte system.

3T3 Cells↗