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Biomedical subjects

T Toyofuku

Publications and source records attributed to T Toyofuku.

At least 19 recordsLinked to original sources
PubMed2001

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.

Amino Acid Sequence↗
PubMed2000

c-Src regulates the interaction between connexin-43 and ZO-1 in cardiac myocytes.

Connexin-43 is known to interact directly with ZO-1 in cardiac myocytes, but little is known about the role of ZO-1 in connexin-43 function. In cardiac myocytes, constitutively active c-Src inhibited endogenous interaction between connexin-43 and ZO-1 by binding to connexin-43. In HEK293 cells, by contrast, a connexin-43 mutant lacking the Src phosphorylation site (Tyr265) interacted with ZO-1 despite cotransfection of a constitutively active c-Src. Moreover, in vitro binding assays using recombinant proteins synthesized from regions of connexin-43 and ZO-1 showed that the tyrosine-phosphorylated C terminus of connexin-43 interacts with the c-Src SH2 domain in parallel with the loss of its interaction with ZO-1. Cell surface biotinylation revealed that, by phosphorylating Tyr265, constitutively active c-Src reduces total and cell surface connexin-43 down to the levels seen in cells expressing a mutant connexin-43 lacking the ZO-1 binding domain. Finally, electrophysiological analysis showed that both the tyrosine phosphorylation site and the ZO-1-binding domain of connexin-43 were involved in the regulation of gap junctional function. We therefore conclude that c-Src regulates the interaction between connexin-43 and ZO-1 through tyrosine phosphorylation and through the binding of its SH2 domain to connexin-43.

Cell Line↗
PubMed2000

Wnt/frizzled-2 signaling induces aggregation and adhesion among cardiac myocytes by increased cadherin-beta-catenin complex.

Wingless is known to be required for induction of cardiac mesoderm in Drosophila, but the function of Wnt family proteins, vertebrate homologues of wingless, in cardiac myocytes remains unknown. When medium conditioned by HEK293 cells overexpressing Wnt-3a or -5a was applied to cultured neonatal cardiac myocytes, Wnt proteins induced myocyte aggregation in the presence of fibroblasts, concomitant with increases in beta-catenin and N-cadherin in the myocytes and with E- and M-cadherins in the fibroblasts. The aggregation was inhibited by anti-N-cadherin antibody and induced by constitutively active beta-catenin, but was unaffected by dominant negative and dominant positive T cell factor (TCF) mutants. Thus, increased stabilization of complexed cadherin-beta-catenin in both cell types appears crucial for the morphological effect of Wnt on cardiac myocytes. Furthermore, myocytes overexpressing a dominant negative frizzled-2, but not a dominant negative frizzled-4, failed to aggregate in response to Wnt, indicating frizzled-2 to be the predominant receptor mediating aggregation. By contrast, analysis of bromodeoxyuridine incorporation and transcription of various cardiogenetic markers showed Wnt to have little or no impact on cell proliferation or differentiation. These findings suggest that a Wnt-frizzled-2 signaling pathway is centrally involved in the morphological arrangement of cardiac myocytes in neonatal heart through stabilization of complexed cadherin- beta-catenin.

Animals↗
PubMed2000

Single-strand conformation polymorphism analysis on the delta-sarcoglycan gene in Japanese patients with hypertrophic cardiomyopathy.

To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the delta-sarcoglycan gene (SG), which is reported to be the causal gene for HC in the Syrian hamster BIO14.6. We performed polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses on the delta-SG in 102 patients with HC. SSCP was detected in exon 2 of the gene, but not in the other exons. The direct sequencing analysis of exon 2 revealed a C-->T substitution at nucleotide residue 84 (TAC-->TAT) with no amino acid alteration (Tyr-->Tyr). There were no significant differences in allele frequencies of C/T between the patients with HC and the control group. Patients with HC were classified into 4 subgroups: obstructive HC, nonobstructive HC, apical HC, and familial HC. The allele frequency of C/T polymorphism in each of these groups was compared with that of the control group. The obstructive HC group showed a significantly greater frequency of the allele T than in the control group (31.6% vs 15.1%, RR = 2.6, p = 0.023). No other significant differences were observed. Thus, amino acid alteration in delta-SG may not be a common cause of HC in Japanese patients.

Adult↗
PubMed1999

Functional role of c-Src in gap junctions of the cardiomyopathic heart.

Given the essential role played by gap junctions in the coordination of cardiac muscle contraction, it is plausible that down-regulation of gap junctional conduction is in part responsible for the contractile dysfunction observed in hypertrophied and failing hearts. In the present study, we analyzed the expression and function of the gap junction protein, connexin43, in the ventricular myocardium of hereditary cardiomyopathic, Syrian BIO 14.6 hamsters. Immunoprecipitation and immunoblot analyses revealed that levels of tyrosine phosphorylated connexin43 were increased in BIO 14.6 hamsters at the late stage of congestive heart failure. Furthermore, the increased tyrosine phosphorylation was correlated with increased c-Src activity. The functional consequences of tyrosine phosphorylation of connexin43 in gap junction were assessed using transfected cells expressing constitutively active c-Src. It was found that constitutively active c-Src diminished propagation of Ca(2+) waves in HEK293 cells and reduced gap junctional conductance between pairs of cardiac myocytes. We, therefore, conclude that during the progression of cardiac dysfunction in the cardiomyopathic heart, gap junctional communication is reduced via c-Src-mediated tyrosine phosphorylation of connexin43.

Animals↗
PubMed1998

Involvement of NF-Y in transcriptional regulation of the phospholamban gene.

To understand the transcriptional regulation of the phospholamban gene, we analyzed a 5'-upstream region of the gene. Using a series of deletion constructs, we demonstrated that the region from -96 bp to -78 bp, containing the CCAAT sequence, is essential for transcription of this gene. This region specifically bound to nuclear proteins extracted from rat hearts, and gel-shift assays using competitive oligonucleotides, antibodies and recombinant proteins showed that this region binds to the NF-YA and NF-YB, members of the CCAAT-binding nuclear protein family. This region-dependent transcription in cardiac myocytes transfected with antisense cDNAs encoding NF-YA and NF-YB was decreased to approximately 50% of that seen in cells transfected with the same sense cDNAs. We, therefore, conclude that the region from -96 bp to -78 bp plays a critical role in expression of the phospholamban gene, which is regulated by binding of the nuclear protein NF-Y.

Animals↗
PubMed1998

Sites of regulatory interaction between calcium ATPases and phospholamban.

Phospholamban (PLN) is a 52-amino acid, integral membrane protein that interacts with and reversibly inhibits the activity of the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a). We have used site-directed mutagenesis to analyze the sites of interaction between PLN and SERCA2a. First, we used chimera formation between SERCA2a and SERCA3 (which is weakly inhibited by PLN) to determine the interacting residues in cytoplasmic sequences of SERCA2 and PLN. Then, we expressed SERCA2a with the transmembrane sequence of PLN and demonstrated that the sites of inhibitory interaction are located in transmembrane sequences of the two proteins. We proposed that a four-base circuit involving noninhibitory cytoplasmic and inhibitory transmembrane sites in PLN and SERCA2a best describes the interaction. Recently, we have used alanine-scanning mutagenesis to show an asymmetric distribution of function in the transmembrane domain of PLN--one helical face interacts with PLN molecules in a pentamer, and the other interacts with SERCA2a. Gain of function by mutation of PLN-interacting residues indicates that the inhibitory species of PLN is a monomer. Thus regulatory steps include PLN dissociation, PLN/SERCA2a inhibitory association, and PLN/SERCA2a dissociation induced by phosphorylation of PLN (in the noninhibitory cytoplasmic domain) or by binding of Ca2+ by SERCA2a (in the inhibitory transmembrane domain).

Amino Acid Sequence↗
PubMed1998

Molecular regulation of phospholamban function and gene expression.

Ca-ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic reticulum. Phospholamban (PLN) functions as an inhibitory cofactor for cardiac Ca-ATPase (SERCA2). To define the molecular mode of interaction between two proteins, interaction sites have been identified. Studies using photoactivated cross-linker and chimeric Ca-ATPase between SERCA2 and nonmuscle Ca-ATPase (SERCA3) indicated that potential binding residues are located just downstream of the active ATPase site (Asp351) of SERCA2. Site-directed mutagenesis study of this region showed that six residues, Lys-Asp-Asp-Lys-Pro-Val402, of SERCA2 are functionally important for the interaction. Further, mutagenesis study of PLN showed that the cytoplasmic region of PLN contains a potential binding site with SERCA2. The unique expression of PLN in cardiac cells has been analyzed by the transcriptional level of its gene using luciferase activity and Gel shift assays. CCAAT-box in the 5'-upstream region was found to be essential for its expression by associating with Y-box binding transcriptional factors.

Amino Acid Sequence↗
PubMed1998

Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes.

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and alpha-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with alpha-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the N-terminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and alpha-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.

Amino Acid Sequence↗
PubMed1998

Intercellular calcium signaling via gap junction in connexin-43-transfected cells.

In excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+ propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187 and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.

Calcium↗
PubMed1998

Molecular regulation of phospholamban function and expression.

Intracellular levels of cAMP regulated by the beta-adrenergic actions of catecholamines play a key in the metabolic, electrical, and mechanical performance of the cardiac muscles. Among a number of biological actions of cAMP, the excitation-contraction coupling process in cardiac myocytes is markedly affected by cAMP through its stimulatory effect on cAMP-dependent protein kinase. Phospholamban, which is expressed in the sarcoplasmic reticulum of cardiac, slow-twitch skeletal, and smooth muscles, is one of the substrates for cAMP-dependent protein kinase. Phospholamban regulates the activity of Ca ATPase in the sarcoplasmic reticulum membranes in a manner dependent on the phosphorylation state of cAMP-dependent protein kinase, thereby changing the mechanical performance of the cardiac muscles. This Ca regulatory mechanism of phospholamban-Ca ATPase system is mediated by a direct protein-protein interaction between two proteins. This review focuses on recent advances in understanding the role of phospholamban molecule in the regulation of Ca transport by cardiac muscle sarcoplasmic reticulum.

Journal Article↗
PubMed1997

Sites of regulatory interaction between calcium ATPases and phospholamban.

In an effort to define the amino acids that are involved in functional interactions between phospholamban (PLN) and the Ca2+ ATPase of cardiac sarcoplasmic reticulum (SERCA2), we have co-expressed wild type and mutant forms of phospholamban with wild type and mutant forms of SERCA2, isolated microsomal fractions and measured Ca2+ dependence of Ca2+ transport. We have found that both charged and hydrophobic residues in the cytoplasmic domains of both PLN and SERCA2 make up the cytoplasmic interaction site. In SERCA2, this site is the linear sequence Lys-Asp-Asp-Lys-Pro-Val402: In PLN, the site is more diffuse and complex. Function was retained if the net charge over the first 20 amino acids was +1 or +2, but function was lost if the net charge was -3, -2, 0 or +3. Function was also lost if the long alkyl side chains of Val4, Leu7 or Ile12 were replaced with the methyl group of Ala. We have also obtained evidence that a site of functional interaction is present in the transmembrane domains of PLN and SERCA2.

Animals↗
PubMed1996

SR Ca(2+)-ATPase/phospholamban in cardiomyocyte function.

Ca ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic and endoplasmic reticulum (SER). Phospholamban was first identified as a phosphoprotein in cardiac myocytes. Functional properties of phospholamban by steady-state and presteady-state kinetic studies of Ca pump ATPase suggest that phospholamban functions as an inhibitory co-factor for cardiac Ca ATPase (SERCA 2). Protein kinase A-catalyzed phosphorylation of phospholamban results in the dissociation of phospholamban from the Ca ATPase, thus augmenting the ATPase activity. Phospholamban is found as a homo-pentamer, formed from subunits of 6080 Da in size. PKA-catalyzed and CAM kinase- catalyzed phosphorylation residues (Ser 16 and Thr 17) are located in the N-terminal cytoplasmic domain, whereas the C-terminal 22 residues are extremely hydrophobic and are considered to be embedded in the SR membrane. At least three kinds of Ca ATPase have been found. SERCA 1 is expressed in fast-twitch skeletal muscle, while the SERCA 2 gene encodes two alternatively spliced products, SERCA 2a and 2b. SERCA 2a is expressed in cardiac and slow-twitch skeletal muscles; SERCA 2b in smooth muscle and non-muscle tissues. SERCA 3 is expressed in a broad variety of muscle and non-muscle tissues. In vitro expression systems revealed that the functional properties of Ca transport of SERCA 2 are identical to SERCA 1, but not SERCA 3. In particular, the Ca affinity for Ca transport of SERCA 1 or 2 is lowered by co-expression with phospholamban, whereas that of SERCA 3 is not. Identification of the interaction sites of phospholamban and SERCA 2 helps defining the molecular mode of interaction between the two proteins. Photoactivated cross-linking studies indicated that potential binding residues are located just downstream of the active ATPase site (Asp 351) of SERCA 2, but SERCA 3 is devoid of this sequence. If a chimeric Ca ATPase (CH2) is made from SERCA 2 and 3, in which the SERCA 3 region corresponding to the phospholamban-binding sequence of SERCA 2 is introduced into the remainder of the SERCA 2 molecule, then the interaction with phospholamban is lost. These results suggest that this region of SERCA 2 contains amino acids which are involved in the interaction with phospholamban. By site-directed mutagenesis of amino acids of this region, we were able to show that 6 residues, Lys-Asp-Asp-Lys-Pro-Val402, of SERCA 2 are functionally important for the interaction. When the chimera CH2 was mutated back to SERCA 2 type, mutated CH2 containing these 6 residues of SERCA 2 restored the interaction with phospholamban. Altogether, these 6 residues of SERCA 2 represent the interaction sites for phospholamban. Mutagenesis studies of phospholamban also demonstrated that the hydrophilic, cytoplasmic region of phospholamban contains a potential binding site for SERCA 2. We therefore conclude that the functional interaction between the two proteins occurs in the cytoplasmic region.

Adenosine Triphosphatases↗
PubMed1994

Identification of Ser38 as the site in cardiac sarcoplasmic reticulum Ca(2+)-ATPase that is phosphorylated by Ca2+/calmodulin-dependent protein kinase.

In previous studies (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397), the Ca(2+)-ATPase of cardiac muscle sarcoplasmic reticulum (SERCA2) was shown to be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase) on a serine residue, likely to be either Ser38, Ser167, or Ser531. SERCA2 and SERCA2 mutants S38A, S167A, and S531A were expressed in HEK-293 cells and tested for phosphorylation with CaM kinase. Mutant S38A was not phosphorylated, while mutants S167A and S531A were phosphorylated, suggesting that Ser38 is the site of CaM kinase phosphorylation in SERCA2. This conclusion was supported by the observation that phosphorylation of SERCA2 and mutants S167A and S531A by CaM kinase increased the Vmax for Ca2+ transport, while the Vmax for Ca2+ transport by mutant S38A was unaffected by exposure to a phosphorylation reaction mix. SERCA1, containing a potential CaM kinase phosphorylation site at Ser167 and two SERCA1 mutants, K35R plus H38S and T532S, in which potential CaM kinase sites were created, were not phosphorylated by CaM kinase, and Vmax for Ca2+ transport was unaffected by exposure to a phosphorylation reaction mix. Thus phosphorylation of Ser38 in SERCA2 results in a unique activation of Vmax for Ca2+ transport, providing a potential regulatory mechanism for Ca2+ removal from cardiac and other tissues in which SERCA2 is expressed.

Animals↗
PubMed1994

Amino acids Lys-Asp-Asp-Lys-Pro-Val402 in the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum are critical for functional association with phospholamban.

Phospholamban interacts with SERCA2 (sarco/endoplasmic reticulum calcium ATPase isoform 2) but not SERCA3. The use of chimeric SERCA2/SERCA3 molecules has revealed that amino acids 336-412 in the phosphorylation domain of SERCA2 are essential for functional association with phospholamban (Toyofuku, T., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1993) J. Biol. Chem. 268, 2809-2815). When mutations were made in SERCA2 between amino acids 336 and 412 and the mutants were coexpressed with phospholamban, only mutation of amino acids Lys397 to Val402 affected phospholamban association with the Ca(2+)-ATPase. A chimeric Ca(2+)-ATPase, CH2, was created in which the phosphorylation domain of SERCA2 was replaced with that of SERCA3, disrupting functional interaction with phospholamban. The SERCA3 sequence QGEQLV402 was then mutated to the corresponding SERCA2 sequence, KDD-KPV402, and to the sequence KGEKPV402, resulting in restoration of functional interaction with phospholamban. Mutation to KGNKPV402 or to QGEQPV402 did not restore functional interaction with phospholamban. These results demonstrate that amino acids Lys397-Val402 comprise the interaction site with phospholamban in SERCA2, and probably also in SERCA1, and that the appropriate balance of charged and hydrophobic residues is an important feature of the interaction.

Amino Acid Sequence↗