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Biomedical subjects

Y Gotoh

Publications and source records attributed to Y Gotoh.

At least 73 records · Page 4Linked to original sources
PubMed1997

Activation and involvement of p38 mitogen-activated protein kinase in glutamate-induced apoptosis in rat cerebellar granule cells.

In the mammalian central nervous system glutamate is the major excitatory neurotransmitter and plays a crucial role in plasticity and toxicity of certain neural cells. We found that glutamate stimulated activation of p38 and stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase (JNK)), two subgroup members of the mitogen-activated protein kinase superfamily in matured cerebellar granule cells. The p38 activation was largely mediated by N-methyl-D-aspartate receptors. Furthermore, we have revealed a novel signaling pathway, that is, Ca2+-mediated activation of p38 in glutamate-treated granule cells. The glutamate concentration effective for inducing apoptosis correlated with that for inducing p38 activation. SB203580, a specific inhibitor for p38, inhibited glutamate-induced apoptosis. Thus p38 might be involved in glutamate-induced apoptosis in cerebellar granule cells.

Animals↗
PubMed1997

Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase.

The mitogen-activated protein kinase (MAPK) cascade consisting of MAPK and its direct activator, MAPK kinase (MAPKK), is essential for signaling of various extracellular stimuli to the nucleus. Upon stimulation, MAPK is translocated to the nucleus, whereas MAPKK stays in the cytoplasm. It has been shown recently that the cytoplasmic localization of MAPKK is determined by its nuclear export signal (NES) in the near N-terminal region (residues 33-44). However, the mechanism determining the subcellular distribution of MAPK has been poorly understood. Here, we show that introduction of v-Ras, active STE11 or constitutively active MAPKK can induce nuclear translocation of MAPK in mammalian cultured cells. Furthermore, we show evidence suggesting that MAPK is localized to the cytoplasm through its specific association with MAPKK and that nuclear accumulation of MAPK is accompanied by dissociation of a complex between MAPK and MAPKK following activation of the MAPK pathway. We have identified the MAPK-binding site of MAPKK as its N-terminal residues 1-32. Moreover, a peptide encompassing the MAPK-binding site and the NES sequence of MAPKK has been shown to be sufficient to retain MAPK to the cytoplasm. These findings reveal the molecular basis regulating subcellular distribution of MAPK, and identify a novel function of MAPKK as a cytoplasmic anchoring protein for MAPK.

Animals↗
PubMed1997

TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase.

Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes. It has recently been reported that ceramide activates stress-activated protein kinase (SAPK, also known as c-Jun NH2-terminal kinase JNK), a subfamily member of mitogen-activated protein kinase superfamily molecules and that the ceramide/SAPK/JNK signaling pathway is required for stress-induced apoptosis. However, the molecular mechanism by which ceramide induces SAPK/JNK activation is unknown. Here we show that TAK1, a member of the mitogen-activated protein kinase kinase kinase family, is activated by treatment of cells with agents and stresses that induce an increase in ceramide. Ceramide itself stimulated the kinase activity of TAK1. Expression of a constitutively active form of TAK1 resulted in activation of SAPK/JNK and SEK1/MKK4, a direct activator of SAPK/JNK. Furthermore, expression of a kinase-negative form of TAK1 interfered with the activation of SAPK/JNK induced by ceramide. These results indicate that TAK1 may function as a mediator of ceramide signaling to SAPK/JNK activation.

Animals↗
PubMed1997

MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts.

In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.

Animals↗
PubMed1997

Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.

Mitogen-activated protein (MAP) kinase cascades are activated in response to various extracellular stimuli, including growth factors and environmental stresses. A MAP kinase kinase kinase (MAPKKK), termed ASK1, was identified that activated two different subgroups of MAP kinase kinases (MAPKK), SEK1 (or MKK4) and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and p38 subgroups of MAP kinases, respectively. Overexpression of ASK1 induced apoptotic cell death, and ASK1 was activated in cells treated with tumor necrosis factor-alpha (TNF-alpha). Moreover, TNF-alpha-induced apoptosis was inhibited by a catalytically inactive form of ASK1. ASK1 may be a key element in the mechanism of stress- and cytokine-induced apoptosis.

Amino Acid Sequence↗
PubMed1997

Further observations on carbendazim-induced abnormalities of spermatid morphology in rats.

The effects of a microtubule poison, carbendazim, on rat spermiogenesis were examined for abnormalities of nuclei, acrosome and manchette in round and elongating spermatids in Stages VII-XII on days 7.5, 9.5, 10.0 and 10.5 post-treatment using routine electron microscopy. Spermatid nuclear abnormalities were observed in Stages IX-XI on day 9.5 and at greater post-treatment intervals. Nuclear abnormalities included nuclear distortions, various types of nuclear invaginations and abnormal positioning of the modified nuclear envelope. Acrosomal abnormalities were noted on day 7.5 and at greater intervals. Discontinuous, multiple granular and fragmentary acrosomes were observed in Stages VII-XI. In addition, spermatids with complete absence of acrosome (acrosome-deficient spermatids) were observed in Stages VII-X. Poorly-formed and absent ectoplasmic specializations were seen in the cytoplasm of Sertoli cells next to the acrosome-deficient spermatids. A major abnormality of the manchette was irregular positioning of the manchette microtubules in steps 9-11 spermatids on day 9.5 and at greater intervals, which resulted in nuclear invagination. The results indicate that carbendazim induces abnormalities in spermatid morphology that are common to those reported in testes treated with several chemical compounds and in testes of mutant animals.

Acrosome↗
PubMed1997

Synthesis of poly(ethylene glycol)-silk fibroin conjugates and surface interaction between L-929 cells and the conjugates.

Poly(ethylene glycol) (PEG)-silk fibroin (SF) conjugates (PEG2-SF) were prepared by the chemical modification of solubilized SF with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (actPEG2) in borate buffer at 37 degrees C. The IR spectra and DSC curves of PEG2-SF and SF suggested the introduction of PEG into SF by the modification and the beta-sheet structure of both SF and PEG2-SF induced by the treatment with methanol aqueous solutions. The content of the PEG component in PEG2-SF was evaluated to be 67% by weight from the melting enthalpy change of PEG observed on the DSC thermogram of PEG2-SF. Water content and contact angle measurements of SF before and after the modification indicated that the hydrophilicity of the PEG2-SF surface increased compared with that of SF. The attachment and growth of fibroblast cells (L-929) on the matrix of PEG2-SF were studied by a cell culture method. PEG2-SF exhibited very low cell attachment and growth, though SF exhibited high cell attachment and growth. The filopodium of the cells attached to PEG2-SF could not be found, and the cells aggregated to form masses in scanning electron microscopy images. These results could be explained in terms of the increased hydrophilicity of the PEG2-SF surface.

Animals↗
PubMed1997

The association of platelet-derived growth factor (PDGF) receptor tyrosine phosphorylation to mitogenic response of human osteoblastic cells in vitro.

OBJECTIVES: The purpose of this study was to make clear the relationship of human osteoblastic cell growth, induced by platelet-derived growth factor (PDGF), to PDGF receptor tyrosine phosphorylation. MATERIALS AND METHODS: Osteoblastic cells derived from human maxilla were cultured with human PDGF. The cell growth was evaluated by cell number and DNA synthesis. PDGF receptor tyrosine phosphorylation was detected by immunoblot analysis using anti-PDGF receptor alpha, beta subunits and anti-phosphotyrosine antibodies. Genistein, a tyrosine kinase inhibitor, was added to the culture to investigate the effect on osteoblastic cell growth and PDGF receptor tyrosine phosphorylation induced by PDGF. RESULTS AND CONCLUSIONS: PDGF stimulated the proliferation of human osteoblastic cells and this effect was synergetic with serum stimulation. DNA synthesis of osteoblastic cells was elevated by PDGF in a dose dependent manner at the minimum concentration of 1 ng ml-1. PDGF also induced PDGF receptor tyrosine phosphorylation within 1 min on osteoblastic cells, and tyrosine phosphorylation occurred on PDGF receptor subunits alpha and beta. Genistein inhibited cell growth and receptor tyrosine phosphorylation, which was induced by PDGF on these cells. In conclusion, human osteoblastic cell growth induced by PDGF is shown to relate to tyrosine kinase of PDGF receptors.

Becaplermin↗
PubMed1996

Suppression of nerve growth factor-induced neuronal differentiation of PC12 cells. N-acetylcysteine uncouples the signal transduction from ras to the mitogen-activated protein kinase cascade.

The cellular redox state is thought to play an important role in a wide variety cellular signaling pathways. Here, we investigated the involvement of redox regulation in the nerve growth factor (NGF) signaling pathway and neuronal differentiation in PC12 cells. N-acetyl-L-cysteine (NAC), which acts as a reductant in cells both by its direct reducing activity and by increasing the synthesis of the cellular antioxidant glutathione, inhibited neuronal differentiation induced by NGF or by the expression of oncogenic ras in PC12 cells. NAC suppressed NGF-induced c-fos gene expression and AP-1 activation. These results suggest that neuronal differentiation and NGF signaling are subject to regulation by the cellular redox state. NAC also suppressed the NGF-induced activation of mitogen-activated protein kinases (MAPKs) and decreased the amount of tyrosine phosphorylation of MAPKs. The suppression of MAPK by NAC was independent of glutathione synthesis. In parallel with the suppression of MAPK, the activation of MAPK kinase kinase activity was also suppressed in the presence of NAC. In contrast, NGF-induced activation of Ras was not inhibited by NAC. The inhibitory effect of NAC on the MAPK cascade was independent of transcription and translation. Thus, NAC suppresses NGF-induced neuronal differentiation by uncoupling the signal transduction from Ras to the MAP kinase cascade in PC12 cells.

Acetylcysteine↗
PubMed1996

Purification and identification of a major activator for p38 from osmotically shocked cells. Activation of mitogen-activated protein kinase kinase 6 by osmotic shock, tumor necrosis factor-alpha, and H2O2.

A stress-activated, serine/threonine kinase, p38 (also known as HOG1 or MPK2) belongs to a subgroup of mitogen-activated protein kinase (MAPK) superfamily molecules. An activity to activate p38 (p38 activator activity) as well as p38 activity itself were greatly stimulated by hyperosmolar media in mouse lymphoma L5178Y cells. The activator activity has been purified by sequential chromatography. A 36-kDa polypeptide that was coeluted with the activity in the final chromatography step was identified as MAPK kinase 6 (MAPKK6) by protein microsequencing analysis. Monoclonal and polyclonal antibodies raised against recombinant MAPKK6 recognized specifically the 36-kDa MAPKK6 protein but did not cross-react with MKK3 proteins. The use of these anti-MAPKK6 antibodies revealed that two major peaks of the p38 activator activity in the first chromatography step reside in the activated MAPKK6. Using a genetic screen in yeast, we isolated MKK3b, an alternatively spliced form of MKK3. Like MKK3 and MAPKK6, MKK3b was shown to be a specific activator for p38 and was activated by osmotic shock when expressed in COS7 cells. Immunoblotting analysis revealed that MAPKK6 is expressed highly in HeLa and KB cells and scarcely in PC12 cells, whereas MKK3 and MKK3b are expressed in all cells examined. Immunodepletion of MAPKK6 from the extracts obtained from L5178Y cells and KB cells exposed to hyperosmolar media depleted them of almost all of the p38 activator activity, indicating that MAPKK6 is a major activator for p38 in an osmosensing pathway in these cells. In addition, MAPKK6 was activated strongly by tumor necrosis factor-alpha, H2O2, and okadaic acid and moderately by cycloheximide in KB cells. Thus, there are at least three members of p38 activator, MKK3, MKK3b, and MAPKK6, and MAPKK6 may function as a major activator for p38 when expressed.

Amino Acid Sequence↗
PubMed1996

Ras-dependent and Ras-independent activation pathways for the stress-activated-protein-kinase cascade.

We have previously shown that osmotic stress activates both the mitogen-activated protein kinase (MAPK) cascade and the stress-activated protein kinase (SAPK, also known as JNK) cascade in rat fibroblastic 3Y1 cells and rat PC12 cells. Here, we show that treatment of these cells with sodium arsenite, a chemical compound that mimics the effects of heat shock, or anisomycin, a protein synthesis inhibitor, induces activation of SAPKs potently. These chemical compounds also stimulated the activity of SEK1/MKK4/JNKK, SAPK activator, and the activity of MEKK, SEK1 activator. Expression of a dominant negative mutant of Ras blocked the anisomycin-induced activation of SAPK and SEK1, but did not affect markedly the arsenite-induced or heat shock-induced activation in PC12 cells. The osmotic-stress-induced activation of SAPK was insensitive to the expression of a dominant negative Ras, but was partly sensitive to down-regulation of protein kinase C. These results suggest the existence of Ras-dependent and Ras-independent activation pathways for the SAPK cascade triggered by environmental stresses including chemical stress in PC12 cells. Cell staining with a specific anti-SAPK serum showed that SAPKs were present in both the cytoplasm and the nucleus under normal conditions, and became located mainly in the nucleus after osmotic stress or ultraviolet treatment, suggesting the nuclear translocation of SAPKs.

Animals↗
PubMed1996

Isolation and characterization of neutralizing single-chain antibodies against Xenopus mitogen-activated protein kinase kinase from phage display libraries.

MAP kinase kinase (MAPKK) is a dual specificity protein kinase that phosphorylates and activates MAP kinase in vivo. In this study, four mouse monoclonal single-chain Fv (scFv) antibodies (Y1-6, Y1-7, Y3-6, and Y3-11) that can specifically bind to Xenopus MAPKK were isolated from combinatorial scFv-displaying phage libraries. Three scFv clones (Y1-6, Y1-7, and Y3-6) were shown to efficiently inhibit MAPKK activity in vitro. Point mutation (D98K) at VH-CDR3 of one (Y1-6) of these three clones markedly reduced its neutralizing activity. The wild-type scFv (Y1-6) inhibited the Mos-induced MAP kinase activation and germinal vesicle breakdown when injected into immature Xenopus oocytes, whereas the mutant scFv, Y1-6 (D98K), did not. The three neutralizing scFv clones (Y1-6, Y1-7, and Y3-6) were shown to bind to NH2-terminal residues 1-23 of Xenopus MAPKK, whereas the epitope of a Y3-11 clone with no neutralizing activity was shown to lie between residues 33 and 67 of MAPKK. Furthermore, a synthetic peptide (the N16 peptide) corresponding to residues 2-17 of MAPKK suppressed the neutralizing activity of the wild-type Y1-6, and a rabbit polyclonal antibody against the N16 peptide was found to possess a strong neutralizing activity against MAPKK. These results demonstrate that the neutralizing antibodies characterized here inhibit the kinase activity of MAPKK by binding to the NH2-terminal segment of MAPKK.

Amino Acid Sequence↗
PubMed1996

Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2-terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal.

Mitogen-activated protein kinase (MAPK) is activated in cytoplasm in response to extracellular signals and then is translocated to nucleus. A directed activator for MAPK, MAPK kinase (MAPKK), stays in cytoplasm to transmit the signal from the plasma membrane to MAPK. Here we show that MAPKK contains a short amino acid sequence in the N-terminal region (residues 32-44), which acts as a nuclear export signal (NES) and thus is required for cytoplasmic localization of MAPKK. This NES sequence of MAPKK, like that of protein kinase inhibitor of cAMP-dependent protein kinase or Rev, is rich in leucine residues, which are crucial for the NES activity. Furthermore, the NES peptide of protein kinase inhibitor, as well as the NES peptide of MAPKK, inhibited the nuclear export of ovalbumin conjugated to the NES peptide of MAPKK. These results may suggest a common mechanism of nuclear export using a general leucine-rich NES.

Amino Acid Sequence↗
PubMed1996

A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3.

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 was shown to phosphorylate and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 was also shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 were strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells resulted in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.

Amino Acid Sequence↗
PubMed1996

TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures.

As part of ongoing studies aimed at clarifying the early events of bone matrix deposition and mineralization, we have characterized primary osteoblast cultures using ultrastructural and immunocytochemical methods. Osteogenic cells were isolated by sequential enzymatic digestion of newborn rat (2-4-day-old) calvariae and grown for periods of 7 to 28 days on polystyrene, Thermanox plastic, or sputtered titanium. Bone-like nodules, showing a stratified organization of cells and collagen, were examined by scanning and transmission electron microscopy, and further characterized for mineral by backscattered electron imaging and X-ray microanalysis. Colloidal gold immunocytochemistry was used to examine the distribution of osteopontin in these nodules. Cells at the surface of the nodules were rounded, while those within the nodules generally appeared more flattened. Both cell types, particularly at early culture intervals, exhibited well-developed protein synthetic organelles. Collagen fibrils were present between the cell layers and some individual fibrils appeared mineralized. Aggregates of needle-shaped crystallites were sometimes apposed to the cell surface, frequently within invaginated regions of the cell membrane, while other mineralized masses of various sizes were present within the collagenous scaffolding. The periphery of the mineralized masses was often delimited by an electron-dense, lamina limitans-like layer. Focal accumulations and/or a more complete layer of afibrillar, mineralized organic matrix were sometimes observed at the interface between the cells and the surface of the culture dish. Osteopontin was immunodetected over the afibrillar and collagenous mineralized matrix throughout the cultures and, in some cases, labeling was concentrated over the peripheral, electron-dense material delimiting the mineralized masses. In conclusion, these data indicate that calvaria-derived osteoblasts produce an extracellular matrix with structural and compositional similarities to bone. Although not a regular observation, the accumulation of osteopontin on the surface of the culture substrate and at the periphery of masses of mineralized matrix may be analogous to what takes place in vivo at naturally occurring bone interfaces.

Animals↗