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R Fukunaga

Publications and source records attributed to R Fukunaga.

At least 19 recordsLinked to original sources
PubMed2001

The fused protein kinase regulates Hedgehog-stimulated transcriptional activation in Drosophila Schneider 2 cells.

The Drosophila segment polarity gene fused encodes a putative protein-serine/threonine kinase, and plays a critical role in the signal transduction for Hedgehog (Hh)-dependent gene expression. We show that the Drosophila Schneider 2 (S2) cell line has the potential to transduce the Hh-triggered intracellular signals, leading to the activation of target gene expression, when a transcription factor, Cubitus interruptus (Ci), is provided exogenously. Using S2 cells transfected with the Ci-expressing plasmid and a patched promoter reporter construct, we demonstrate that the forced expression of Fused (Fu) stimulates Hh-triggered and Ci-dependent transcriptional activation. The N-terminal kinase domain of Fu is required for this activity, but the C-terminal domain is not. Two kinase-inactive Fu mutants fail to enhance the reporter activation, indicating that the kinase catalytic activity is essential for this function. Negative components of the Hh-signaling pathway, Costal-2 and Suppressor of Fused, strongly antagonize the Fu activity, irrespective of the presence or absence of the Fu C-terminal domain, suggesting an indirect mechanism for the inhibition of Fu by these proteins. Furthermore, mutational analyses of threonine 158 and serine 159, in the activation segment of the Fu protein kinase, indicate that threonine 158 is essential for Fu activity and that phosphorylation of this threonine residue may be involved in the activation of the kinase catalytic activity upon Hh stimulation.

Animals↗
PubMed2000

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.

Animals↗
PubMed1999

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E-binding proteins (4E-BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK-activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C-terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G-binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C-terminal region of the translational inhibitor p97, an eIF4G-related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.

Animals↗
PubMed1998

PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis.

We have identified a novel human protein, PRC1, that is involved in cytokinesis. PRC1 is a good substrate for several CDKs in vitro and is phosphorylated in vivo at sites that are phosphorylated by CDK in vitro, strongly suggesting that PRC1 is an in vivo CDK substrate. PRC1 has sequence homology to the budding yeast anaphase spindle elongation factor Ase1p. Like Ase1p, PRC1 protein levels are high during S and G2/M and drop dramatically after cells exit mitosis and enter G1. PRC1 is a nuclear protein in interphase, becomes associated with mitotic spindles in a highly dynamic manner during mitosis, and localizes to the cell mid-body during cytokinesis. Microinjection of anti-PRC1 antibodies into HeLa cells blocked cellular cleavage, but not nuclear division, indicating a functional role for PRC1 in the process of cytokinesis.

Amino Acid Sequence↗
PubMed1997

MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates.

We have developed a novel expression screening method for identifying protein kinase substrates. In this method, a lambda phage cDNA expression library is screened by in situ, solid-phase phosphorylation using purified protein kinase and [gamma-32P]ATP. Screening a HeLa cDNA library with ERK1 MAP kinase yielded cDNAs of previously characterized ERK substrates, c-Myc and p90RSK, demonstrating the utility of this method for identifying physiological protein kinase substrates. A novel clone isolated in this screen, designated MNK1, encodes a protein-serine/threonine kinase, which is most similar to MAP kinase-activated protein kinase 2 (MAPKAP-K2), 3pK/MAPKAP-K3 and p90RSK. Bacterially expressed MNK1 was phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 was activated upon stimulation of HeLa cells with 12-O-tetradecanoylphorbol-13-acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor-alpha, interleukin-1beta, or osmotic shock, and the activation by these stimuli was differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways.

Amino Acid Sequence↗
PubMed1997

In vivo hypoxia-induced neuronal damage in dentate gyrus of rat hippocampus: changes in NMDA receptors and the effect of MK-801.

Hypoxia is a major cause of ischaemia-induced neuronal damage. In the present study, we examined the effects of in vivo hypoxia on N-methyl-D-aspartate receptors (NMDAR) in the rat hippocampus. This model of in vivo hypoxia involved placing rats in a hypoxic chamber containing 5% O2 and 95% N2 for 30 min. In the hippocampus, neuronal cells in the CA3, the hilus of the dentate gyrus and the dentate gyrus (DG) were damaged. In the CA1, which is known to be vulnerable to ischaemic damage, neuronal cells did not show hypoxia-induced damage. In vivo hypoxia-induced damage caused morphological changes in neuronal cells, such as shrunken, spindle or triangular shapes accompanied by pyknotic nuclei, but did not induce the loss of neuronal cells. On the other hand, the number of binding sites for [3H]-1-[1-(2-thienyl)cyclohexyl]-3,4-piperidine hydrochloride (TCP) gradually decreased on and after 7 days, and then maximally decreased by 25% at 21 days after hypoxia. The number of NMDAR1-immunopositive cells was decreased by 22% in the DG, but was unchanged in the CA3. Furthermore, we examined the effect of a non-competitive NMDA antagonist, (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,b] cyclohepten-5,10-imine hydrogen maleate (MK-801), on against in vivo hypoxia. The administration of MK-801 (3 mg/kg, i.p.), 30 min before hypoxia treatment, partly protected against neuronal damage in the DG, but not in the CA3. These results suggest that hypoxia-induced neuronal damage in the DG involves, in part, the activation of NMDAR.

Animals↗
PubMed1994

Ligand binding domain of granulocyte colony-stimulating factor receptor.

The amino-terminal domain of the cytokine receptor homologous region (BN domain; roughly 100 amino acid residues) in the receptor for murine granulocyte colony-stimulating factor (G-CSF) was secreted as a maltose-binding protein fusion into the Escherichia coli periplasm. The murine BN domain (mBN) was prepared from the fusion protein by restriction protease Factor Xa digestion and purified to homogeneity. The purified BN domain specifically and stoichiometrically bound G-CSF, with an apparent dissociation constant (Kd) of 3-8 x 10(-8) M. The CD spectrum of the mBN domain was similar to that of the extracellular region of the human growth hormone (GH) receptor, which is composed of turns and beta-sheets held together by disulfide bonds. Tertiary folding and the beta-sheet of this small domain was confirmed by NMR spectroscopy. Disulfide bonds determined by peptide mapping were in the following locations: Cys107-Cys118, Cys153-Cys162, and Cys143-Cys194. Among them, the first and the second produce small loops (roughly 10 amino acid residues) as found in the human GH receptor. These results suggested that the mBN domain of the G-CSF receptor expressed by E. coli has a GH receptor-like structure. However, the third disulfide bond varied considerably between the G-CSF and GH receptors. Disruption of these disulfide bonds in the BN domain of the G-CSF receptor suggested that all of them are critical for maintaining a stably folded protein. Our results will facilitate understanding of the biophysical and structural properties of this receptor.

Amino Acid Sequence↗
PubMed1994

Delayed expression of c-fos protein in rat hippocampus and cerebral cortex following transient in vivo exposure to hypoxia.

The time course of c-fos protein expression after hypoxia was examined in rat hippocampus and cerebral cortex using an immunohistochemical method. The rats were exposed to in vivo hypoxia for 30 min in a chamber containing 5% O2 and 95% N2. Immediately after the treatment, c-fos protein-like immunoreactivity was observed in the granule cell layer of the dentate gyrus. The change was transient, and the density of immunoreactive cells returned quickly to a control level 3 h after the exposure. However, the density of positive cells was again increased 1 day after hypoxia and reached the maximum 7 days after. In the cerebral cortex, on the other hand, no change was detected in the pattern of staining at any time, with an exception on 21 days after hypoxia. At this period, positively stained neurons were significantly increased in both density and intensity throughout the entire extent of the cerebral cortex including the cingulate gyrus. These results clearly indicate that hypoxia induces different patterns of c-fos protein expression among various regions of the brain. The biphasic pattern seen in the dentate gyrus as well as the delayed expression in the cerebral cortex may be related to delayed neuronal damages induced by hypoxia.

Animals↗
PubMed1994

Structural analysis of the functional gene and pseudogene encoding the murine granulocyte colony-stimulating-factor receptor.

Granulocyte colony-stimulating factor is a cytokine which specifically regulates the production of neutrophilic granulocytes. The granulocyte colony-stimulating-factor receptor (GCSFR) is mainly expressed in neutrophils and their precursor cells. In this study, we isolated the chromosomal gene for murine GCSFR and determined its structure. Like the human GCSFR gene homolog, it consists of 17 exons. The exon-intron organization of the murine and human GCSFR-encoding genes are very similar, except that exon 14 and exon 15 in the murine gene are interrupted by a larger intron (greater than 10 kbp) than that found in the human gene (128 bp). This GCSFR-encoding functional gene (Csfgr) was localized to the distal region of murine chromosome 4 by interspecific backcross mapping. A comparison of the 5' flanking sequence of murine and human Csfgr revealed that a sequence of approximately 300 bp upstream from the cap site is highly conserved. Within this region, an 18-nucleotide element conserved in the promoter of the genes for neutrophil-specific enzymes, was found approximately 140 bp upstream from the cap site, suggesting an involvement of this element in the specific expression of GCSFR in neutrophilic granulocytes. In addition to the functional GCSFR-encoding gene, we isolated a pseudogene for GCSFR, which is flanked by a 15-bp direct repeat at the 5' and 3' ends, and lacks all introns, exons 1-3 and exons 7-8 of the functional gene. The processed pseudogene has, in its most 5' region, a sequence of approximately 200 bp that is highly related to the DNA sequence approximately 1.2 kbp upstream of the cap site of the functional gene.

Animals↗
PubMed1994

Evaluation of cerebral vasoreactivity by three-dimensional time-of-flight magnetic resonance angiography.

BACKGROUND AND PURPOSE: Cerebral vasoreactivity is an important indicator of the reserve capacity of the cerebral circulation. To make a quantitative analysis of cerebral vasoreactivity in individual major arterial territories, we evaluated the response to acetazolamide using three-dimensional time-of-flight magnetic resonance angiography. METHODS: We examined 10 healthy volunteers and 6 patients with unilateral stenosis of the middle cerebral artery by a 1.5-T superconducting magnetic resonance imaging system. After a baseline vascular image was obtained, each subject received 17 mg/kg IV of acetazolamide; a second scan was performed 20 minutes later. Using a generally available personal computer and image analysis software, we measured the areas of the individual major arteries on collapsed axial vascular images and then calculated the vasoreactivity. RESULTS: The average vasoreactivity of individual major cerebral arterial territories in the healthy volunteers was as follows: anterior cerebral artery complex, 33%; right middle cerebral artery, 71%; left middle cerebral artery, 74%; right posterior cerebral artery, 68%; and left posterior cerebral artery, 68%. In the patient group, the vasoreactivity of the stenotic middle cerebral arteries was significantly smaller than that of the nonstenotic arteries (P < .05). In addition, the nonstenotic middle cerebral arteries showed significantly less vasoreactivity than the right arteries of the healthy volunteers (P < .01). CONCLUSIONS: Three-dimensional time-of-flight magnetic resonance angiography can be used to quantitatively evaluate acetazolamide-induced vasoreactivity in individual major cerebral arterial territories.

Acetazolamide↗
PubMed1994

Expression of granulocyte colony-stimulating factor and its receptor at the fetomaternal interface in murine and human pregnancy.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine which regulates proliferation and differentiation of neutrophilic granulocytes, and its receptor (G-CSF-R) is a member of the hemopoietic growth factor receptor family. We studied the expression of G-CSF and G-CSF-R at the fetomaternal interface in murine and human pregnancy. Immunohistochemical analysis and in situ hybridization indicated that both G-CSF and G-CSF-R are expressed in mouse spongiotrophoblasts and placental labyrinths, and human placental cytotrophoblasts and syncytiotrophoblasts. They were also detected in mouse decidual basalis cells and endometrial epithelial cells, and human decidual stromal cells and endometrial gland cells. These results suggest that G-CSF plays a role in decidual and placental functions by autocrine and paracrine mechanisms.

Abortion, Induced↗
PubMed1994

Acetazolamide challenge for three-dimensional time-of-flight MR angiography of the brain.

PURPOSE: We compared three-dimensional time-of-flight MR angiograms obtained before and after acetazolamide administration to evaluate whether use of this drug could improve visualization of small peripheral intracranial arteries and atherosclerotic stenosis. METHODS: For evaluation of small peripheral arteries, 10 patients with clinical diagnosis of ischemic cerebrovascular disease and 10 healthy volunteers were investigated, and for evaluation of stenosis, another 6 patients were investigated. Vascular images were obtained by three-dimensional time-of-flight MR angiography. After a baseline scan, 17 mg/kg acetazolamide was injected intravenously and the second scan was performed 20 minutes later. RESULTS: Several small peripheral arteries that had not been seen on the baseline images were visible on the acetazolamide images without any augmentation of the background signals. Stenotic lesions in the main trunks of the major cerebral arteries were detected more clearly on acetazolamide images. CONCLUSIONS: Acetazolamide improves visualization of small peripheral intracranial arteries and sensitivity in detecting atherosclerotic stenosis in the main trunk of major cerebral artery by three-dimensional time-of-flight MR angiography without changing MR apparatus and software.

Acetazolamide↗
PubMed1993

Unidirectional cross-phosphorylation between the granulocyte colony-stimulating factor and interleukin 3 receptors.

The mouse interleukin-3 (IL-3)-dependent hemopoietic precursor cell line 32DC13 responds to granulocyte colony-stimulating factor (G-CSF) for proliferation and differentiation. We established a subline (32D-FH) of the 32DC13 cells which has lost the ability to respond to G-CSF. When murine G-CSF receptor cDNA was introduced into the 32D-FH cell line, the transformants responded to G-CSF as well as to IL-3 for proliferation. Adding G-CSF to the transformants rapidly induced tyrosine phosphorylation not only of the G-CSF receptor but also of both subtypes of the IL-3 receptor beta-chain (AIC2A and AIC2B molecules). On the other hand, stimulation of the transformants with IL-3 induced tyrosine phosphorylation of the AIC2A and AIC2B but not of the G-CSF receptor. These results indicate that the tyrosine kinase activated through the G-CSF receptor interacts with the IL-3 receptor beta-chains but not vice versa. This unidirectional cross-phosphorylation between the G-CSF and IL-3 receptors may play a role in granulopoiesis induced by G-CSF.

Amino Acids↗
PubMed1993

In vivo and in vitro suppression of primary B lymphocytopoiesis by tumor-derived and recombinant granulocyte colony-stimulating factor.

Transplantation of a granulocytosis-inducing murine CE mammary carcinoma into mice suppresses primary B lymphopoiesis in the marrow. The mechanisms of this tumor-induced B-cell suppression were investigated using Whitlock-Witte-type lymphoid cultures. When seeded with normal marrow progenitors, stromal cells of tumor-bearing mice supported the production of B220+ cells as well as did either stomal cells derived from control mice or the stromal cell line S17. Cultured over normal stroma, marrow cells of tumor-bearing mice depleted of adherent cells and B220+ cells generated B220+ cells as effectively as a similar cell population from control mice. However, interleukin-7-responsive progenitors, were completely depleted from the marrow of tumor-bearing mice. When conditioned medium (CM) of cloned CE tumor cells known to produce granulocyte colony-stimulating factor (G-CSF) and macrophage-CSF, or recombinant murine G-CSF was added to the cultures established with S17 cells, B220+ cell production was significantly diminished. Antiserum to murine G-CSF blocked these effects. These in vitro observations were corroborated by the elimination of marrow B220+ cells in mice injected with G-CSF. These in vitro and in vivo studies suggest that G-CSF plays an inhibitory role in primary B lymphopoiesis by blocking stromal cell-mediated differentiation of early B-cell progenitors into phenotypically recognizable B220+ pre-B cells.

Animals↗
PubMed1993

Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that regulates the proliferation and differentiation of neutrophils. The G-CSF receptor (G-CSFR) is a member of the hemopoietic growth factor receptor family. A G-CSFR expression plasmid was introduced into interleukin-3 (IL-3)-dependent mouse myeloid precursor FDC-P1 cells that normally do not respond to G-CSF. G-CSF stimulated proliferation of the transformants, down-regulated Thy-1 and F4/80 antigens on the cell surface, and induced expression of neutrophil-specific genes such as myeloperoxidase (MPO) and leukocyte elastase. On the other hand, neither granulocyte/macrophage colony-stimulating factor (GM-CSF) nor IL-3 induced MPO gene expression, but they inhibited G-CSFR-mediated MPO gene expression. These results suggested that the G-CSFR, but not the IL-3/GM-CSF receptors, transduced the neutrophilic differentiation signal into cells. Mutational analysis of the G-CSFR indicated that the N-terminal region of its cytoplasmic domain is sufficient to transduce the proliferation signal into cells, while the C-terminal region plays an essential role in transducing the differentiation signal.

Animals↗
PubMed1993

Changes in protein kinase C isozymes in the rat hippocampus following transient hypoxia.

The effects of hypoxia on protein kinase C (PKC) isozymes (alpha, beta I, beta II, and gamma) were examined in the hippocampus from rats subjected to hypoxic conditions (5% O2 in 95% N2) for 30 min in a chamber. Western blot analysis revealed that the total amounts of PKC-alpha (-26.0% of control) and -gamma (-32.7% of control) were decreased significantly at the end of hypoxia, which was followed by the reduction of that of PKC-beta II (-23.7% of control at 7 days after hypoxia). Whereas, the PKC activities, which were measured by the incorporation of [gamma-32P] into a specific PKC substrate peptide, in both the cytosolic and the particulate fractions did not change. The reductions of PKC-gamma and -alpha at the end of hypoxia may be related to the following neuronal degeneration.

Animals↗
PubMed1993

Signal transduction mediated by growth hormone receptor and its chimeric molecules with the granulocyte colony-stimulating factor receptor.

The granulocyte colony-stimulating factor receptor (G-CSF-R) and growth hormone receptor (GH-R) belong to the cytokine receptor family and have some similarity in the cytokine receptor-homologous (CRH) domain of the extracellular region. Among members of this family, the G-CSF-R and GH-R seem to function as homodimers. Previously, we showed that mouse myeloid precursor FDC-P1 cells expressing the G-CSF-R can respond to G-CSF for growth. Here we show that the GH-R can also transduce the growth signal in FDC-P1 cells in the range 10 pM-100 nM GH. At a higher concentration of GH, GH did not promote the growth of the transformant cells. A series of chimeric receptor cDNAs between the G-CSF-R and GH-R cDNAs was constructed by exon swapping and was expressed in FDC-P1 cells. A ligand-binding assay with transformants expressing chimeric receptors indicated that the entire CRH domain is necessary for specific binding of the ligand. Although the transmembrane and cytoplasmic regions of the G-CSF-R and GH-R have no apparent similarity, these regions were interchangeable, resulting in growth-signal transduction in FDC-P1 cells.

Amino Acid Sequence↗