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Biomedical subjects

K Fukunaga

Publications and source records attributed to K Fukunaga.

At least 19 recordsLinked to original sources
PubMed1999

Tyrosine phosphorylation of p62(Dok) induced by cell adhesion and insulin: possible role in cell migration.

Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokDeltaPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Differential effects of a calcineurin inhibitor on glutamate-induced phosphorylation of Ca2+/calmodulin-dependent protein kinases in cultured rat hippocampal neurons.

Calcium/calmodulin-dependent protein kinases (CaM kinases) are major multifunctional enzymes that play important roles in calcium-mediated signal transduction. To characterize their regulatory mechanisms in neurons, we compared glutamate-induced phosphorylation of CaM kinase IV and CaM kinase II in cultured rat hippocampal neurons. We observed that dephosphorylation of these kinases followed different time courses, suggesting different regulatory mechanisms for each kinase. Okadaic acid, an inhibitor of protein phosphatase (PP) 1 and PP2A, increased the phosphorylation of both kinases. In contrast, cyclosporin A, an inhibitor of calcineurin, showed different effects: the phosphorylation and activity of CaM kinase IV were significantly increased with this inhibitor, but those of CaM kinase II were not significantly increased. Cyclosporin A treatment of neurons increased phosphorylation of Thr196 of CaM kinase IV, the activated form with CaM kinase kinase, which was recognized with an anti-phospho-Thr196 antibody. Moreover, recombinant CaM kinase IV was dephosphorylated and inactivated with calcineurin as well as with PP1, PP2A, and PP2C in vitro. These results suggest that CaM kinase IV, but not CaM kinase II, is directly regulated with calcineurin.

Animals↗
PubMed1999

Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites.

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

Amino Acid Sequence↗
PubMed1999

Endothelin antagonist treatment for successful liver transplantation from non-heart-beating donors.

BACKGROUND: With the shortage of cadaveric donors, non-heart-beating donors (NHBDs) are a potential source of liver allografts. However, warm ischemic injury in NHBDs seriously affects the viability of graft liver. Endothelin (ET)-1 has been reported to be involved in the hepatic microcirculatory disturbances after ischemia-reperfusion. METHODS: In a porcine orthotopic liver transplantation model, changes in the serum and liver tissue ET-1 concentration were measured and the effects of an ET receptor antagonist, TAK-044, were evaluated. After cardiac arrest of the donors, liver allografts were subjected to 90 min of warm ischemia, flushed, and preserved for 4 hr at 4 degrees C. The pigs were divided into two groups: a control group (no drug treatment) and a drug-treated group, in which donors and recipients were treated with TAK-044 (10 mg/kg body, drip intravenous injection). Both groups had six donor/recipient pairs. RESULTS: -The ET-1 concentration in the hepatic venous blood increased after reperfusion of the graft in the control group recipients. ET-1 in the graft liver significantly increased during the cold preservation period. TAK-044 treatment significantly increased recipient 7-day survival rate. After reperfusion of the graft, the concentrations of serum liver enzymes and arterial lactate in the drug-treated group were significantly lower than in the control group. The postoperative increase in portal venous pressure was significantly reduced in the drug-treated group. Measurements of liver enzymes in the washed-out preservation fluid at the time of graft rinsing indicated that TAK-044 treatment of the donors significantly suppressed liver enzyme release during ischemia. CONCLUSIONS: These findings indicate TAK-044 treatment has protective effects on postoperative function of hepatic allografts procured from NHBDs.

Alanine Transaminase↗
PubMed1999

Cloning from insulinoma cells of synapsin I associated with insulin secretory granules.

Synapsin I is a synaptic vesicle-associated protein involved in neurotransmitter release. The functions of this protein are apparently regulated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). We reported evidence for CaM kinase II and a synapsin I-like protein present in mouse insulinoma MIN6 cells (Matsumoto, K., Fukunaga, K., Miyazaki, J., Shichiri, M., and Miyamoto, E. (1995) Endocrinology 136, 3784-3793). Phosphorylation of the synapsin I-like protein in these cells correlated with the activation of CaM kinase II and insulin secretion. In the present study, we screened the MIN6 cDNA library with the full-length cDNA probe of rat brain synapsin Ia and obtained seven positive clones; the largest one was then sequenced. The largest open reading frame deduced from the cDNA sequence of 3695 base pairs encoded a polypeptide of 670 amino acids, which exhibited significant sequence similarity to rat synapsin Ib. The cDNA contained the same sequence as the first exon of the mouse synapsin I gene. These results indicate that synapsin Ib is present in MIN6 cells. Synapsin I was expressed in normal rat islets, as determined by reverse transcriptase-polymerase chain reaction analysis. Immunoblot analysis after subcellular fractionation of MIN6 cells demonstrated that synapsin Ib and delta subunit of CaM kinase II co-localized with insulin secretory granules. By analogy concerning regulation of neurotransmitter release, our results suggest that phosphorylation of synapsin I by CaM kinase II may induce the release of insulin from islet cells.

Amino Acid Sequence↗
PubMed1999

PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor.

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS-dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS-independent manner in CHO cells that overexpress a dominant-negative mutant of the guanine nucleotide exchange protein SOS (CHO-DeltaSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO-DeltaSOS cells. The RAS-independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by overexpression of a dominant-negative mutant of the gamma isoform of PI3K. Furthermore, LPA induced the activation of the atypical zeta isoform of protein kinase C (PKC-zeta) in CHO-DeltaSOS cells in a manner that was sensitive to wortmannin or to the dominant-negative mutant of PI3Kgamma, and overexpression of a dominant-negative mutant of PKC-zeta inhibited LPA-induced activation of MAP kinase. These observations indicate that Gi protein-coupled receptors induce activation of MEK and MAP kinase through a RAS-independent pathway that involves PI3Kgamma-dependent activation of atypical PKC-zeta.

Animals↗
PubMed1999

Phosphorylation of myristoylated alanine-rich protein kinase C substrate by mitogen-activated protein kinase in cultured rat hippocampal neurons following stimulation of glutamate receptors.

Glutamate-induced phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) was investigated in cultured rat hippocampal neurons. In 32P-labeled hippocampal neurons, exposure to 10 microM glutamate induced a long lasting increase in phosphorylation of MARCKS. The long lasting increase in MARCKS phosphorylation mainly required activation of the N-methyl-D-aspartate receptor. Unexpectatively, the MARCKS phosphorylation after the 10-min incubation with glutamate was not inhibited by treatment with calphostin C, a potent inhibitor for protein kinase C (PKC), or down-regulation of PKC but was largely prevented by PD098059, a selective inhibitor for mitogen-activated protein (MAP) kinase kinase. In contrast, the phosphorylation following the short exposure to glutamate was prevented by a combination of PD098059 and calphostin C. The phosphopeptide mapping and immunoblotting analyses confirmed that PKC-dependent phosphorylation of MARCKS was transient and the MAP kinase-dependent phosphorylation was relatively persistent. Investigations of the functional properties also showed that the MARCKS phosphorylation by MAP kinase regulates its calmodulin-binding ability and its interaction with F-actin as seen in the PKC-dependent phosphorylation. These results suggest that glutamate causes a long lasting increase in MARCKS phosphorylation through activation of the N-methyl-D-aspartate receptor and subsequent activation of MAP kinase in the hippocampal neurons.

Animals↗
PubMed1999

In vitro comparison study of CD63 and CD62P expression after contacting leukocyte filters.

CD63 and CD62P have been recognized as platelet activation markers. This study investigated the secretion of these antigens to compare the platelet activation between a newly developed stainless steel leukocyte filter (SSLF) and 7 polyester or polyurethane commercially available leukocyte filters. Flow cytometry demonstrated that the SSLF initiated significantly smaller effects in terms of mean fluorescence intensity of CD63 (p<0.03) and of the amount of CD62P expressing platelets (p<0.002) compared to the polyurethane filters. However, there was no statistical difference between the SSLF and polyester filters. The result of this study suggests that the SSLF caused less platelet activation than the polyurethane filters and has biocompatible characteristics comparable to the currently available polyester filters. Stainless steel was selected because of its physicochemical conductivity. With these results, further evaluation of the SSLF will be continued in an attempt to develop an active immunomodulator using this unique characteristic.

Adult↗
PubMed1999

Preliminary evaluation study of a prototype hollow fiber membrane for the continuous membrane autotransfusion system.

A totally new autotransfusion system has been developed utilizing a hollow fiber membrane, based upon plasmapheresis technology. Prior to fabricating the system, it was essential to evaluate the basic performance characteristics of the filter, which was designed particularly for the new system. The objective of this study was to prove or disprove that such a system would be available using this filter. An in vitro study was conducted on the filter using bovine blood. The result of the study showed that the filter could process 2-20% of hematocrit blood at a flow rate greater than 250 ml/min of inlet blood continuously. Moreover, it could concentrate 5-20% hematocrit blood to hematocrit percentages greater than 40% by a single passage through the filter. These results seemed to prove that a rapid, continuous, and compact autotransfusion system could be developed using this filter.

Blood Transfusion, Autologous↗
PubMed1999

Plasma platelet-activating factor acetylhydrolase deficiency in Japanese patients with asthma.

Platelet-activating factor (PAF), a phospholipid with a wide range of proinflammatory actions, is immediately degraded and inactivated in vivo by PAF acetylhydrolase (PAF-AH). Surprisingly, 4% of the Japanese population lacks the extracellular isoform of this enzyme, plasma PAF-AH, due to a genetic missense (V279F) mutation. We studied the association of this mutation with asthma prevalence and phenotypes in the Japanese adult population. The allele frequency of V279F mutation was 18.6% in 279 patients with asthma (28.7% heterozygotes and 4.3% homozygotes) and 21.7% in 217 healthy subjects (32.3% heterozygotes and 5.5% homozygotes). V279F mutant allele prevalence was consistent regardless of asthma type (16.3% in atopic [n = 156] and 21.6% in nonatopic [n = 123]), or the severity of disease (21.7% in patients with mild [n = 97], 17.5% in those with moderate [n = 131], and 15.8% in those with severe [n = 51] asthma). Plasma PAF-AH activity was inversely proportional to the number of mutant alleles, and did not correlate with asthma prevalence, type, or severity. We concluded that plasma PAF-AH deficiency due to V279F mutation is not essential to the pathophysiology of asthma in the Japanese adult population.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1999

Current studies on a working model of CaM kinase II in hippocampal long-term potentiation and memory.

There have been recent advances in understanding the molecular basis of the long-term potentiation (LTP) of synaptic transmission in the hippocampus. This review documents current views on mechanisms underlying LTP induction, from activation of the NMDA receptor to stimulation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). We will focus in particular on recent findings of how CaM kinase II encodes the frequency of synaptic usage and serves as a molecular memory switch at the synapse. Furthermore, a role for CaM kinase II in spatial learning and memory is demonstrated by recent studies using transgenic mice.

Animals↗
PubMed1998

Serine/threonine phosphatase activity of calcineurin is inhibited by sodium orthovanadate and dithiothreitol reverses the inhibitory effect.

Orthovanadate is known to be an inhibitor of protein tyrosine phosphatases. However, we found that it inhibited calcineurin which has the activity of a serine/threonine protein phosphatase, using casein phosphorylated by cyclic AMP-dependent protein kinase as a substrate. Orthovanadate inhibits the Mn2+-activated activity of purified calcineurin to 20%; this is not the case without Mn2+. Furthermore, 10 mM dithiothreitol (DTT) reversed the inhibitory effects of orthovanadate. Orthovanadate showed the same inhibitory effect for calcineurin activity in homogenates as for the purified enzyme; the inhibitory effect was reversed by DTT. These results indicate that orthovanadate inhibits not only protein tyrosine phosphatases as reported, but also serine/threonine phosphatase activity of calcineurin.

Animals↗
PubMed1998

Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family kinase.

SHPS-1 is an approximately 120 kDa glycosylated receptor like protein that contains three immunoglobulin-like domains in its extracellular region as well as four potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites in its cytoplasmic region. Lysophosphatidic acid (LPA) stimulated the rapid tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, a protein tyrosine phosphatase containing SH2 domains in Rat-1 fibroblasts. LAP-induced tyrosine phosphorylation of SHPS-1 was inhibited by Clostridium botulinum C3 exoenzyme (which inactivates RHO) but not by pertussis toxin. The protein kinase C activator phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulated tyrosine phosphorylation of SHPS-1; however, down-regulation of protein kinase C by prolonged exposure of cells to TPA did not affect LAP-induced tyrosine phosphorylation of SHPS-1. LPA-induced tyrosine phosphorylation of SHPS-1 was markedly reduced in either focal adhesion kinase (FAK)-deficient mouse cells or CHO cells overexpressing the tyrosine kinase CSK. Overexpression of a catalytically inactivate SHP-2 markedly inhibited MAP kinase activation in response to low concentrations of LPA in CHO cells, whereas overexpression of a wild-type SHPS-1 did enhance this effect of LPA. Furthermore, MAP kinase activation in response to a low concentration of LPA was inhibited by botulinum C3 exoenzyme. These results indicate that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 may be mediated by a RHO-dependent pathway that includes FAK and a SRC family kinase. Thus, in addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, the formation of a complex between SHPS-1 and SHP-2 may, in part, play an important role in the activation of MAP kinase in response to low concentrations of LPA.

ADP Ribose Transferases↗
PubMed1998

Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases.

SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, an Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins such as fibronectin and laminin also induced the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses were markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase did not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibited activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 did not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix.

Animals↗
PubMed1998

Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation.

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.

Amino Acid Sequence↗
PubMed1998

Role of MAP kinase in neurons.

Extracellular stimuli such as neurotransmitters, neurotrophins, and growth factors in the brain regulate critical cellular events, including synaptic transmission, neuronal plasticity, morphological differentiation and survival. Although many such stimuli trigger Ser/Thr-kinase and tyrosine-kinase cascades, the extracellular signal-regulated kinases, ERK1 and ERK2, prototypic members of the mitogen-activated protein (MAP) kinase family, are most attractive candidates among protein kinases that mediate morphological differentiation and promote survival in neurons. ERK1 and ERK2 are abundant in the central nervous system (CNS) and are activated during various physiological and pathological events such as brain ischemia and epilepsy. In cultured hippocampal neurons, simulation of glutamate receptors can activate ERK signaling, for which elevation of intracellular Ca2+ is required. In addition, brain-derived neurotrophic factor and growth factors also induce the ERK signaling and here, receptor-coupled tyrosine kinase activation has an association. We describe herein intracellular cascades of ERK signaling through neurotransmitters and neurotrophic factors. Putative functional implications of ERK and other MAP-kinase family members in the central nervous system are give attention.

Animals↗