T. Kawakami - Academia.edu (original) (raw)

Papers by T. Kawakami

Blood, 2007

IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and... more IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and other allergic reactions. The Src family tyrosine kinase (SFK) Lyn is activated by the cross-linking of high-affinity IgE receptors (FcϵRI). Activated Lyn phosphorylates the FcϵRI subunits, β and γ, leading to subsequent activation of various signaling pathways. Lyn also plays a negative regulatory function by activating negative regulatory molecules. Another SFK, Fyn, also contributes to mast cell degranulation by inducing Gab2-dependent microtubule formation. Here we show that a third SFK, Hck, plays a critical role in mast cell activation. Degranulation and cytokine production are reduced in FcϵRI-stimulated hck−/− mast cells. The reduced degranulation can be accounted for by defects in Gab2 phosphorylation and microtubule formation. Importantly, Lyn activity is elevated in hck−/− cells, leading to increased phosphorylation of several negative regulators. However, positive regulatory...

Immunity, 2009

Crosslinking of IgE-bound Fc3RI triggers mast cell degranulation. Previous fluorescence recovery ... more Crosslinking of IgE-bound Fc3RI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that Fc3RI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-Fc3RI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. Fc3RI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

Gene, 1993

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which ... more The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or cr-ketobutyrate are converted into cl-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes (ZLV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (CM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ZLV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILL' genes have been cloned, thus allowing direct molecular analyses of their regulation.

Gene, 1993

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which ... more The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or cr-ketobutyrate are converted into cl-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes (ZLV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (CM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ZLV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILL' genes have been cloned, thus allowing direct molecular analyses of their regulation.

Proceedings of the National Academy of Sciences, 2000

Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the prot... more Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the protein kinase C (PKC) subfamily of serine/threonine kinases play crucial roles in signal transduction through antigen receptors in B lymphocytes and high-affinity IgE receptors (FcɛRI) in mast cells. The present study provides genetic, biochemical, and pharmacological evidence that, on FcɛRI stimulation, Syk regulates Btk, and Btk selectively regulates the membrane translocation and enzymatic activity of PKCβI among the conventional PKC isoforms (α, βI, and βII) expressed in mast cells. Syk/Btk-mediated PKCβI regulation is involved in transcriptional activation of the IL-2 and tumor necrosis factor α genes through the JNK pathway induced by FcɛRI stimulation. Accordingly, FcɛRI-induced production of these cytokines is inhibited by specific inhibitors of Btk and Syk, as well as broad-specificity inhibitors of PKC and a selective inhibitor of PKCβ. Specific regulation of PKCβI by Btk is consi...

The Open Allergy Journal, 2012

Histamine-releasing factor (HRF), also termed translationally controlled tumor protein (TCTP) and... more Histamine-releasing factor (HRF), also termed translationally controlled tumor protein (TCTP) and fortilin, is a highly conserved, multi-functional protein. This protein within a cell plays a critical role in the fundamental processes of cell-cycle progression, proliferation, survival, and malignant transformation. The same protein, despite the lack of signal sequence, is secreted through a nonclassical secretory pathway. The secreted protein usually termed HRF can activate IgE-primed basophils and mast cells, and works as a B cell growth factor and a chemoattractant for eosinophils. This structurally well-characterized protein interacts with many proteins to perform its intracellular and extracellular functions. This review summarizes recent studies of HRF/TCTP-interacting proteins as a major driving force to decipher its functions.

The Journal of Immunology

Cross-linking of the high affinity receptor for IgE (Fc epsilon RI) initiates various biochemical... more Cross-linking of the high affinity receptor for IgE (Fc epsilon RI) initiates various biochemical and morphologic changes leading to degranulation and synthesis and release of cytokines and lipid mediators. Tyrosine phosphorylation of several cellular proteins was previously reported as the earliest signaling event for the Fc epsilon RI signal transduction pathway. By amino acid sequence determination and cDNA cloning analysis, a 75-kDa protein, termed SPY75, was identified as a major tyrosine-phosphorylated protein in activated mouse mast cells. SPY75, barely tyrosine phosphorylated in resting cells, was rapidly and transiently tyrosine phosphorylated on Fc epsilon RI cross-linking in an Ag concentration-dependent manner. Similar SPY75 tyrosine phosphorylation was observed when Ag receptors on B and T lymphocytes were cross-linked by appropriate antibodies. However, IL-3, granulocyte macrophage-CSF, or stem cell factor did not induce tyrosine phosphorylation of SPY75 in PT-18 mast ...

Virology, 1987

The nucleotide sequence of the integrated form of the genome of the equine infectious anemia viru... more The nucleotide sequence of the integrated form of the genome of the equine infectious anemia virus was determined. By comparison with LTR sequences of other retroviruses, signals for the control of viral gene transcription and translation could be identified in the EIAV LTR. Open reading frames for gag and pol genes were identified and their sequences matched very closely to those determined previously by others. However, in the present study, the pol gene reading frame was open throughout its entire length. The open reading frame for the env gene product was constructed from the sequences of two independent EIAV clones. Thus, a noninfectious genomic-length clone was shown to contain a frameshift mutation approximately in the middle of the presumed env gene coding sequence, whereas the sequence of another clone was open in this region. The deduced amino acid sequences of the EIAV gag and pol products showed closer evolutionary relationships to those of known lentiviruses than to other retroviruses. There was also partial sequence homology between predicted env gene products of EIAV, visna virus, and HTLV-III/LAV. Sequences analogous to the sor region of other lentiviruses could not be identified in our EIAV clone. A short open reading frame at the 3' end of the genome that overlapped env but not the 3' LTR was present but lacked significant sequence similarity to the 3' open reading frames of other lentiviruses. Thus, the sequence and general structure of EIAV most closely resemble those of known lentiviruses.

The American Journal of Pathology, 2004

Blood, 2003

The Syk tyrosine kinase is essential for immunoreceptor and multiple integrin functions as well a... more The Syk tyrosine kinase is essential for immunoreceptor and multiple integrin functions as well as being implicated in signaling from G-protein–coupled receptors (GPCR) in cell lines, transfection systems, and pharmacologic studies. In contrast, using Syk-deficient primary cells, we show here that Syk does not play a major functional role in chemoattractant/chemokine signaling in neutrophils and mast cells. syk−/− neutrophils showed normal respiratory burst and degranulation in response to the bacterial peptide formyl-Met-Leu-Phe (fMLP). The migration of neutrophils toward fMLP was similarly not affected by the syk−/−mutation. fMLP initiated normal Ca2+-signal, activation of the extracellular signal-related kinase (ERK) and p38 mitogen–activated protein (MAP) kinase cascades, and polymerization of cellular actin in the absence of Syk.syk−/− and wild-type neutrophils also responded similarly to LTB4, C5a, and the chemokines macrophage inflammatory protein-1 (MIP-1)α or MIP-2, both in...

Proceedings of the National Academy of Sciences, 1994

T lymphocytes require two signals to be activated. The antigen-specific T-cell receptor can deliv... more T lymphocytes require two signals to be activated. The antigen-specific T-cell receptor can deliver the first signal, while ligation of the T-cell surface molecule CD28 by antibodies or its cognate ligands B7-1 (CD80) or B7-2 has been demonstrated to be sufficient for the delivery of the second signal. Signaling via CD28 and the T-cell receptor results (i) in their costimulation of T cells to produce numerous lymphokines including interleukin 2 and (ii) in the prevention of anergy induction. Little is known about the pathway by which CD28 mediates its signals except that protein-tyrosine phosphorylation is involved. We show here in human Jurkat cells that the Tec-family protein-tyrosine kinase ITK/EMT (p72ITK/EMT) is associated with CD28 and becomes tyrosine-phosphorylated and activated within seconds of CD28 ligation. This tyrosine phosphorylation of p72ITK/EMT is rapid (within 30 sec), occurs in the absence of LCK activation, and precedes tyrosine phosphorylation of the guanine nu...

Summary We investigated the role of Bruton's tyrosine kinase (Btk) in Fc e RI-dependent acti... more Summary We investigated the role of Bruton's tyrosine kinase (Btk) in Fc e RI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell de- velopment is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in Fc e RI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon Fc e RI cross-linking. Moreover, the transcriptional ac- tivities of these cytokine genes were severely reduced in Fc e RI-stimulated btk mutant mast cells. The specificity of these effects of btk muta...

Molecular and Cellular Biology, 1994

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events ind... more Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-...

Journal of Experimental Medicine, 1998

We investigated the role of Bruton's tyrosine kinase (Btk) in FcεRI-dependent activation of m... more We investigated the role of Bruton's tyrosine kinase (Btk) in FcεRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcεRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcεRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcεRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed b...

Proceedings of the National Academy of Sciences, 1999

Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell d... more Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of...

The Journal of …, 2006

Galectin-3 is a member of the -galactoside-binding animal lectin family expressed in various cell... more Galectin-3 is a member of the -galactoside-binding animal lectin family expressed in various cell types, including mast cells. To determine the role of galectin-3 in the function of mast cells, we studied bone marrow-derived mast cells (BMMC) from wild-type (gal3 +/+ ) and ...

Allergology International, 2007

Background: Atopic dermatitis is a chronic or chronically relapsing, pruritic inflammatory skin d... more Background: Atopic dermatitis is a chronic or chronically relapsing, pruritic inflammatory skin disease. The incidence of atopic dermatitis has dramatically increased during the past three decades in industrialized countries. We attempted to develop an improved method to induce an animal model of atopic dermatitis and to use it to evaluate the efficacy of a Tec family kinase inhibitor. Methods: We treated dermatitis-prone inbred mice, NC! Nga, by repetitive epicutaneous applications of a house dust mite allergen and staphylococcal enterotoxin B to induce atopic dermatitis-like skin lesions. Results: We established a highly efficient protocol to induce skin lesions in NC! Nga mice, which were histologically and immunologically similar to human atopic dermatitis. Similar to human patients, serum IgE levels were increased in dermatitis-induced mice. Consistent with the proposed roles of infiltrated immune cells in the pathogenesis of human atopic dermatitis, skin lesions were treatable with terreic acid, an inhibitor of Tec family kinases, as well as dexamethasone. Conclusions: We established a highly efficient, highly reproducible protocol to induce skin lesions in NC! Nga mice and successfully applied it to show the efficacy of terreic acid in treating skin lesions. This mouse model of atopic dermatitis will be useful to study the pathogenetic processes of atopic dermatitis and to evaluate the efficacy of drug candidates.

The Journal of …, 2005

Aggregation of the high affinity receptor for IgE (Fc⑀RI) induces activation of mast cells. In th... more Aggregation of the high affinity receptor for IgE (Fc⑀RI) induces activation of mast cells. In this study we show that upon low intensity stimulation of Fc⑀RI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with Fc⑀RI ␤ subunit, whereas high intensity stimulation enhanced Lyn activity and its association with Fc⑀RI ␤. The latter induced much higher levels of Fc⑀RI ␤ phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with Fc⑀RI ␤, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant Fc⑀RI ␤ showed that Fc⑀RI ␤ subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of Fc⑀RI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the Fc⑀RI ␤-ITAM.

The EMBO journal, Jan 15, 2001

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in hu... more Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates ...

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997

Protein tyrosine phosphorylation and other biochemical events have been shown to occur after cros... more Protein tyrosine phosphorylation and other biochemical events have been shown to occur after cross-linking of Fc epsilonRI in rodent mast cells. To investigate the mechanism of Fc epsilonRI signal transduction in human mast cells, we used human cultured mast cells (HCMC) generated from cord blood cells in the presence of recombinant human stem cell factor and IL-6. We found that on cross-linking of Fc epsilonRI: 1) HCMC released histamine; 2) rapid tyrosine phosphorylation of multiple cellular substrates, including Syk, HS1, c-Cbl, ERK-1, and ERK-2, was observed; 3) intracellular Ca2+ and inositol phosphate production were increased within the first minute after Fc epsilonRI cross-linking; and 4) genistein, a tyrosine kinase inhibitor, inhibited both protein tyrosine phosphorylation and histamine release in a dose-dependent manner. These results were consistent with previous studies in rodent mast cells. In contrast, no tyrosine phosphorylation of phospholipase C gamma1 and Btk (Bru...

Blood, 2007

IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and... more IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and other allergic reactions. The Src family tyrosine kinase (SFK) Lyn is activated by the cross-linking of high-affinity IgE receptors (FcϵRI). Activated Lyn phosphorylates the FcϵRI subunits, β and γ, leading to subsequent activation of various signaling pathways. Lyn also plays a negative regulatory function by activating negative regulatory molecules. Another SFK, Fyn, also contributes to mast cell degranulation by inducing Gab2-dependent microtubule formation. Here we show that a third SFK, Hck, plays a critical role in mast cell activation. Degranulation and cytokine production are reduced in FcϵRI-stimulated hck−/− mast cells. The reduced degranulation can be accounted for by defects in Gab2 phosphorylation and microtubule formation. Importantly, Lyn activity is elevated in hck−/− cells, leading to increased phosphorylation of several negative regulators. However, positive regulatory...

Immunity, 2009

Crosslinking of IgE-bound Fc3RI triggers mast cell degranulation. Previous fluorescence recovery ... more Crosslinking of IgE-bound Fc3RI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that Fc3RI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-Fc3RI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. Fc3RI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

Gene, 1993

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which ... more The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or cr-ketobutyrate are converted into cl-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes (ZLV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (CM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ZLV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILL' genes have been cloned, thus allowing direct molecular analyses of their regulation.

Gene, 1993

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which ... more The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or cr-ketobutyrate are converted into cl-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes (ZLV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (CM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ZLV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILL' genes have been cloned, thus allowing direct molecular analyses of their regulation.

Proceedings of the National Academy of Sciences, 2000

Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the prot... more Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the protein kinase C (PKC) subfamily of serine/threonine kinases play crucial roles in signal transduction through antigen receptors in B lymphocytes and high-affinity IgE receptors (FcɛRI) in mast cells. The present study provides genetic, biochemical, and pharmacological evidence that, on FcɛRI stimulation, Syk regulates Btk, and Btk selectively regulates the membrane translocation and enzymatic activity of PKCβI among the conventional PKC isoforms (α, βI, and βII) expressed in mast cells. Syk/Btk-mediated PKCβI regulation is involved in transcriptional activation of the IL-2 and tumor necrosis factor α genes through the JNK pathway induced by FcɛRI stimulation. Accordingly, FcɛRI-induced production of these cytokines is inhibited by specific inhibitors of Btk and Syk, as well as broad-specificity inhibitors of PKC and a selective inhibitor of PKCβ. Specific regulation of PKCβI by Btk is consi...

The Open Allergy Journal, 2012

Histamine-releasing factor (HRF), also termed translationally controlled tumor protein (TCTP) and... more Histamine-releasing factor (HRF), also termed translationally controlled tumor protein (TCTP) and fortilin, is a highly conserved, multi-functional protein. This protein within a cell plays a critical role in the fundamental processes of cell-cycle progression, proliferation, survival, and malignant transformation. The same protein, despite the lack of signal sequence, is secreted through a nonclassical secretory pathway. The secreted protein usually termed HRF can activate IgE-primed basophils and mast cells, and works as a B cell growth factor and a chemoattractant for eosinophils. This structurally well-characterized protein interacts with many proteins to perform its intracellular and extracellular functions. This review summarizes recent studies of HRF/TCTP-interacting proteins as a major driving force to decipher its functions.

The Journal of Immunology

Cross-linking of the high affinity receptor for IgE (Fc epsilon RI) initiates various biochemical... more Cross-linking of the high affinity receptor for IgE (Fc epsilon RI) initiates various biochemical and morphologic changes leading to degranulation and synthesis and release of cytokines and lipid mediators. Tyrosine phosphorylation of several cellular proteins was previously reported as the earliest signaling event for the Fc epsilon RI signal transduction pathway. By amino acid sequence determination and cDNA cloning analysis, a 75-kDa protein, termed SPY75, was identified as a major tyrosine-phosphorylated protein in activated mouse mast cells. SPY75, barely tyrosine phosphorylated in resting cells, was rapidly and transiently tyrosine phosphorylated on Fc epsilon RI cross-linking in an Ag concentration-dependent manner. Similar SPY75 tyrosine phosphorylation was observed when Ag receptors on B and T lymphocytes were cross-linked by appropriate antibodies. However, IL-3, granulocyte macrophage-CSF, or stem cell factor did not induce tyrosine phosphorylation of SPY75 in PT-18 mast ...

Virology, 1987

The nucleotide sequence of the integrated form of the genome of the equine infectious anemia viru... more The nucleotide sequence of the integrated form of the genome of the equine infectious anemia virus was determined. By comparison with LTR sequences of other retroviruses, signals for the control of viral gene transcription and translation could be identified in the EIAV LTR. Open reading frames for gag and pol genes were identified and their sequences matched very closely to those determined previously by others. However, in the present study, the pol gene reading frame was open throughout its entire length. The open reading frame for the env gene product was constructed from the sequences of two independent EIAV clones. Thus, a noninfectious genomic-length clone was shown to contain a frameshift mutation approximately in the middle of the presumed env gene coding sequence, whereas the sequence of another clone was open in this region. The deduced amino acid sequences of the EIAV gag and pol products showed closer evolutionary relationships to those of known lentiviruses than to other retroviruses. There was also partial sequence homology between predicted env gene products of EIAV, visna virus, and HTLV-III/LAV. Sequences analogous to the sor region of other lentiviruses could not be identified in our EIAV clone. A short open reading frame at the 3' end of the genome that overlapped env but not the 3' LTR was present but lacked significant sequence similarity to the 3' open reading frames of other lentiviruses. Thus, the sequence and general structure of EIAV most closely resemble those of known lentiviruses.

The American Journal of Pathology, 2004

Blood, 2003

The Syk tyrosine kinase is essential for immunoreceptor and multiple integrin functions as well a... more The Syk tyrosine kinase is essential for immunoreceptor and multiple integrin functions as well as being implicated in signaling from G-protein–coupled receptors (GPCR) in cell lines, transfection systems, and pharmacologic studies. In contrast, using Syk-deficient primary cells, we show here that Syk does not play a major functional role in chemoattractant/chemokine signaling in neutrophils and mast cells. syk−/− neutrophils showed normal respiratory burst and degranulation in response to the bacterial peptide formyl-Met-Leu-Phe (fMLP). The migration of neutrophils toward fMLP was similarly not affected by the syk−/−mutation. fMLP initiated normal Ca2+-signal, activation of the extracellular signal-related kinase (ERK) and p38 mitogen–activated protein (MAP) kinase cascades, and polymerization of cellular actin in the absence of Syk.syk−/− and wild-type neutrophils also responded similarly to LTB4, C5a, and the chemokines macrophage inflammatory protein-1 (MIP-1)α or MIP-2, both in...

Proceedings of the National Academy of Sciences, 1994

T lymphocytes require two signals to be activated. The antigen-specific T-cell receptor can deliv... more T lymphocytes require two signals to be activated. The antigen-specific T-cell receptor can deliver the first signal, while ligation of the T-cell surface molecule CD28 by antibodies or its cognate ligands B7-1 (CD80) or B7-2 has been demonstrated to be sufficient for the delivery of the second signal. Signaling via CD28 and the T-cell receptor results (i) in their costimulation of T cells to produce numerous lymphokines including interleukin 2 and (ii) in the prevention of anergy induction. Little is known about the pathway by which CD28 mediates its signals except that protein-tyrosine phosphorylation is involved. We show here in human Jurkat cells that the Tec-family protein-tyrosine kinase ITK/EMT (p72ITK/EMT) is associated with CD28 and becomes tyrosine-phosphorylated and activated within seconds of CD28 ligation. This tyrosine phosphorylation of p72ITK/EMT is rapid (within 30 sec), occurs in the absence of LCK activation, and precedes tyrosine phosphorylation of the guanine nu...

Summary We investigated the role of Bruton's tyrosine kinase (Btk) in Fc e RI-dependent acti... more Summary We investigated the role of Bruton's tyrosine kinase (Btk) in Fc e RI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell de- velopment is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in Fc e RI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon Fc e RI cross-linking. Moreover, the transcriptional ac- tivities of these cytokine genes were severely reduced in Fc e RI-stimulated btk mutant mast cells. The specificity of these effects of btk muta...

Molecular and Cellular Biology, 1994

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events ind... more Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-...

Journal of Experimental Medicine, 1998

We investigated the role of Bruton's tyrosine kinase (Btk) in FcεRI-dependent activation of m... more We investigated the role of Bruton's tyrosine kinase (Btk) in FcεRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcεRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcεRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcεRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed b...

Proceedings of the National Academy of Sciences, 1999

Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell d... more Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of...

The Journal of …, 2006

Galectin-3 is a member of the -galactoside-binding animal lectin family expressed in various cell... more Galectin-3 is a member of the -galactoside-binding animal lectin family expressed in various cell types, including mast cells. To determine the role of galectin-3 in the function of mast cells, we studied bone marrow-derived mast cells (BMMC) from wild-type (gal3 +/+ ) and ...

Allergology International, 2007

Background: Atopic dermatitis is a chronic or chronically relapsing, pruritic inflammatory skin d... more Background: Atopic dermatitis is a chronic or chronically relapsing, pruritic inflammatory skin disease. The incidence of atopic dermatitis has dramatically increased during the past three decades in industrialized countries. We attempted to develop an improved method to induce an animal model of atopic dermatitis and to use it to evaluate the efficacy of a Tec family kinase inhibitor. Methods: We treated dermatitis-prone inbred mice, NC! Nga, by repetitive epicutaneous applications of a house dust mite allergen and staphylococcal enterotoxin B to induce atopic dermatitis-like skin lesions. Results: We established a highly efficient protocol to induce skin lesions in NC! Nga mice, which were histologically and immunologically similar to human atopic dermatitis. Similar to human patients, serum IgE levels were increased in dermatitis-induced mice. Consistent with the proposed roles of infiltrated immune cells in the pathogenesis of human atopic dermatitis, skin lesions were treatable with terreic acid, an inhibitor of Tec family kinases, as well as dexamethasone. Conclusions: We established a highly efficient, highly reproducible protocol to induce skin lesions in NC! Nga mice and successfully applied it to show the efficacy of terreic acid in treating skin lesions. This mouse model of atopic dermatitis will be useful to study the pathogenetic processes of atopic dermatitis and to evaluate the efficacy of drug candidates.

The Journal of …, 2005

Aggregation of the high affinity receptor for IgE (Fc⑀RI) induces activation of mast cells. In th... more Aggregation of the high affinity receptor for IgE (Fc⑀RI) induces activation of mast cells. In this study we show that upon low intensity stimulation of Fc⑀RI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with Fc⑀RI ␤ subunit, whereas high intensity stimulation enhanced Lyn activity and its association with Fc⑀RI ␤. The latter induced much higher levels of Fc⑀RI ␤ phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with Fc⑀RI ␤, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant Fc⑀RI ␤ showed that Fc⑀RI ␤ subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of Fc⑀RI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the Fc⑀RI ␤-ITAM.

The EMBO journal, Jan 15, 2001

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in hu... more Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates ...

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997

Protein tyrosine phosphorylation and other biochemical events have been shown to occur after cros... more Protein tyrosine phosphorylation and other biochemical events have been shown to occur after cross-linking of Fc epsilonRI in rodent mast cells. To investigate the mechanism of Fc epsilonRI signal transduction in human mast cells, we used human cultured mast cells (HCMC) generated from cord blood cells in the presence of recombinant human stem cell factor and IL-6. We found that on cross-linking of Fc epsilonRI: 1) HCMC released histamine; 2) rapid tyrosine phosphorylation of multiple cellular substrates, including Syk, HS1, c-Cbl, ERK-1, and ERK-2, was observed; 3) intracellular Ca2+ and inositol phosphate production were increased within the first minute after Fc epsilonRI cross-linking; and 4) genistein, a tyrosine kinase inhibitor, inhibited both protein tyrosine phosphorylation and histamine release in a dose-dependent manner. These results were consistent with previous studies in rodent mast cells. In contrast, no tyrosine phosphorylation of phospholipase C gamma1 and Btk (Bru...