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Eisuke Nishida

Publications and source records attributed to Eisuke Nishida.

At least 37 records · Page 2Linked to original sources
PubMed2004

Negative feedback loop in T-cell activation through MAPK-catalyzed threonine phosphorylation of LAT.

Mitogen-activated protein kinase (MAPK) cascades are involved in a variety of cellular responses including proliferation, differentiation, and apoptosis. We have developed an expression screening method to detect in vivo substrates of MAPKs in mammalian cells, and identified a membrane protein, linker for activation of T cells (LAT), as an MAPK target. LAT, an adapter protein essential for T-cell signaling, is phosphorylated at its Thr 155 by ERK in response to T-cell receptor stimulation. Thr 155 phosphorylation reduces the ability of LAT to recruit PLCgamma1 and SLP76, leading to attenuation of subsequent downstream events such as [Ca2+]i mobilization and activation of the ERK pathway. Our data reveal a new role for MAPKs in a negative feedback loop in T-cell activation via threonine phosphorylation of LAT.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The Caenorhabditis elegans MAPK phosphatase VHP-1 mediates a novel JNK-like signaling pathway in stress response.

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP-regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp-1 gene, encoding an MKP that acts preferentially on the c-Jun N-terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp-1 are arrested during larval development. This vhp-1 defect is suppressed by loss-of-function mutations in the kgb-1, mek-1, and mlk-1 genes encoding a JNK-like MAPK, an MKK7-type MAPKK, and an MLK-type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP-1 in the KGB-1 pathway. Loss-of-function mutations in each component in the KGB-1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP-1 plays a pivotal role in the integration and fine-tuning of the stress response regulated by the KGB-1 MAPK pathway.

Amino Acid Motifs↗
PubMed2004

ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing.

In epithelial cell movements, which occur during wound healing or embryonic morphogenesis, sheets of cells move together as a unit. Molecular mechanisms that regulate this sheet movement have been largely unknown, although cell locomotion or movement mechanisms for individual cells, such as for fibroblastic cells, have been extensively studied. Here, we show that, during wound healing, sheets of MDCK epithelial cells migrate coordinately as a unit, and wound-induced activation of ERK MAP kinase (ERK1/2) propagates in cell sheets in accordance with the cell sheet movement. Inhibition of ERK1/2 activation by specific MEK inhibitors or by expressing dominant-negative ERK2 results in marked inhibition of the sheet movement during wound healing, and inhibition of the cell sheet movement by disrupting actin cytoskeleton suppresses propagation of ERK1/2 activation. These results indicate that cell movement and ERK1/2 activation form a positive feedback loop, which facilitates cell sheet migration. Moreover, we find that Src family kinase inhibitors suppress both cell migration and propagation of ERK1/2 activation, suggesting that Src family kinase may participate in this feedback loop. Interestingly, neither cell sheet migration as a unit nor migration-dependent propagation of ERK1/2 activation occurs during wound healing in fibroblastic 3Y1 cells. Thus, our results identify specific requirements of ERK1/2 MAP kinase for epithelial cell sheet movement.

Animals↗
PubMed2004

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences.

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.

3T3 Cells↗
PubMed2004

Shp2, an SH2-containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty.

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, we demonstrate that Sprouty (Spry) is a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 was able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry resulted in dissociation of Spry from Grb2. Furthermore, Shp2 could reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry.

Animals↗
PubMed2004

Sef is a spatial regulator for Ras/MAP kinase signaling.

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is among the key mechanisms for regulating a wide variety of cellular processes. In this study, we report that human Sef (hSef), a recently identified inhibitor whose action mechanism has not been fully defined, acts as a molecular switch for ERK signaling by specifically blocking ERK nuclear translocation without inhibiting its activity in the cytoplasm. Thus, hSef binds to activated forms of MEK, inhibits the dissociation of the MEK-ERK complex, and blocks nuclear translocation of activated ERK. Consequently, hSef inhibits phosphorylation and activation of the nuclear ERK substrate Elk-1, while it does not affect phosphorylation of the cytoplasmic ERK substrate RSK2. Downregulation of endogenous hSef by hSef siRNA enhances the stimulus-induced ERK nuclear translocation and the activity of Elk-1. These results thus demonstrate that hSef acts as a spatial regulator for ERK signaling by targeting ERK to the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed2004

Regulatory mechanisms and function of ERK MAP kinases.

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is a key factor for determining the specificity of cellular responses including cell proliferation, cell differentiation and cell survival. The fidelity of this signaling is regulated by docking interactions as well as scaffolding. Subcellular localization of ERK is controlled by cytoplasmic ERK anchoring proteins that have a nuclear export signal (NES), such as MEK. In quiescent cells, ERK and MEK localize to the cytoplasm. In response to stimulation, dissociation of the MEK-ERK complex is induced and activated ERK translocates to the nucleus. Recently, several negative regulators for Ras/ERK signaling have been identified and their detailed molecular mechanisms have been analyzed. Among them, Sprouty and Sef act as a temporal and a spatial regulator, respectively, for Ras/ERK signaling. Thus, multiple factors are involved in control of Ras/ERK signaling.

Animals↗
PubMed2004

An evolutionarily conserved gene required for proper microtubule architecture in Caenorhabditis elegans.

Microtubules are involved in many cellular events during the cell cycle and also in a variety of early embryonic developmental processes. Their architecture and properties change dramatically during the cell cycle and are properly regulated. However, these regulatory mechanisms have not been fully elucidated. C05D11.3 gene of Caenorhabditis elegans encodes a low molecular weight protein that is evolutionarily conserved from yeasts to mammals. A mouse homolog of the C05D11.3 product, APACD (ATP binding protein associated with cell differentiation), contains a thioredoxin-like domain and P-loop, and is present in both the nucleus and the cytoplasm, showing often localization to centrosomes and midbody. In C. elegans, C05D11.3 is expressed throughout development with higher levels of expression in most cells of the nervous system and in vulva. C05D11.3 RNAi-treated embryos show apparent defects in pronuclear migration or nuclear-centrosome rotation, and exhibit little astral microtubules and defective small spindles. These results indicate that C05D11.3, an evolutionarily conserved gene, is essential for proper microtubule organization and function in C. elegans. This gene family may be a conserved regulator of microtubule dynamics and function.

Amino Acid Sequence↗
PubMed2004

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37.

Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.

Amino Acid Sequence↗
PubMed2004

Supervision of multiple signaling protein kinases by the CK2-Cdc37 couple, a possible novel cancer therapeutic target.

Overexpression of a pleiotropic Ser/Thr kinase CK2 (casein kinase II), or of a kinase-targeting molecular chaperone Cdc37, induces neoplastic cell growth in animals. Recent genetic and biochemical evidence from several laboratories has revealed an unexpected direct link between CK2 and Cdc37. In this short review, we describe the basic characteristics of CK2 and Cdc37 and introduce recent findings on the interaction between CK2 and Cdc37. Cdc37 was identified as a multicopy suppressor of a temperature-sensitive allele of CK2 in Saccharomyces cerevisiae. CK2 phosphorylates a conserved serine residue in the N-terminal extremity of Cdc37 in vitro and in yeast as well as mammalian cells, and this is the unique phosphorylation site of Cdc37 under normal conditions. Mutations in the CK2-mediated phosphorylation site abolish the association of Cdc37 with various protein kinases. The same mutations in yeast cause severe growth and morphological defects. Specific inhibition of CK2 activity decreases intracellular levels of Cdc37-dependent protein kinases. Altogether, this evidence clearly indicates that the CK2-dependent phosphorylation is essential for the proper function of Cdc37 to bind and stabilize signaling protein kinases. In contrast, CK2 activity is enhanced by Cdc37 both in vitro and in vivo; thus, the CK2-Cdc37 couple seems to constitute a positive feedback control mechanism that may govern the activity of multiple protein kinases. Cdc37-dependent protein kinases include important signaling molecules whose disregulations are intimately related to neoplastic cell growth; hence, inhibition of the CK2-Cdc37 system may simultaneously suppress various cancer-promoting signal cascades. We propose that the CK2-Cdc37 couple can be a novel and efficient pharmacological target for cancer chemotherapy.

Amino Acid Sequence↗
PubMed2003

Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways.

We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.

Amino Acid Sequence↗
PubMed2003

Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate.

Plk1 (Polo-like kinase 1), an evolutionarily conserved serine/threonine kinase, is crucially involved in multiple events during the M phase. Here we have identified a consensus phosphorylation sequence for Plk1, by testing the ability of systematically mutated peptides derived from human Cdc25C to serve as a substrate for Plk1. The obtained results show that a hydrophobic amino acid at position +1 carboxyl-terminal of phosphorylated Ser/Thr and an acidic amino acid at position -2 are important for optimal phosphorylation by Plk1. We have then found that Myt1, an inhibitory kinase for MPF, has a number of putative phosphorylation sites for Plk1 in its COOH-terminal portion. While wild-type Myt1 (Myt1-WT) served as a good substrate for Plk1 in vitro, a mutant Myt1 (Myt1-4A), in which the four putative phosphorylation sites are replaced by alanines, did not. In nocodazole-treated cells, Myt1-WT, but not Myt1-4A, displayed its mobility shift in gel electrophoresis, due to phosphorylation. These results suggest that Plk1 phosphorylates Myt1 during M phase. Thus, this study identifies a novel substrate for Plk1 by determining a consensus phosphorylation sequence by Plk1.

Amino Acid Motifs↗
PubMed2003

Identification of a DAF-16 transcriptional target gene, scl-1, that regulates longevity and stress resistance in Caenorhabditis elegans.

In Caenorhabditis elegans, an insulin-like signaling pathway, which includes the daf-2 and age-1 genes, controls longevity and stress resistance. Downregulation of this pathway activates the forkhead transcription factor DAF-16, whose transcriptional targets are suggested to play an essential role in controlling the phenotypes governed by this pathway. We have surveyed the genes that have the DAF-16 consensus binding element (DBE) within putative regulatory regions. Here, we show that one such gene, termed scl-1, is a positive regulator of longevity and stress resistance. Expression of scl-1 is upregulated in long-lived daf-2 and age-1 mutants and is undetectable in a short-lived daf-16 mutant. SCL-1 is a putative secretory protein with an SCP domain and is homologous to the mammalian cysteine-rich secretory protein (CRISP) family. scl-1 is required for the extension of the life span of daf-2 and age-1 mutants, and downregulation of scl-1 reduces both life span and stress resistance of this animal. SCL-1, whose expression is dependent on DAF-16, is the first example of a putative secretory protein that positively regulates longevity and stress resistance.

Aging↗
PubMed2003

Molecular recognitions in the MAP kinase cascades.

The mitogen-activated protein kinase (MAPK) cascades play a pivotal role in many aspects of cellular functions, and are evolutionarily conserved from yeast to mammals. In mammals, there are four subfamily members in the MAPKs. Each MAPK has its own activators, substrates and inactivators. In order to achieve normal cellular functions, the MAPK cascades should transduce signals with high efficiency and fidelity. However, the molecular basis for the mechanism underlying the specific reactions in the MAPK cascades has not been fully understood. The MAPKs form a globular structure without a distinct domain specific for protein-protein interactions. Recent studies revealed two mechanisms regulating the signalling, the docking interaction and the scaffolding. The docking interaction is achieved through the common docking domain (the CD domain) on MAPKs, and is different from a transient enzyme-substrate interaction through the active centre of the enzymes. Almost all the MAPK-interacting molecules have a conserved motif interacting with the CD domain. The scaffolding usually utilizes a third molecule to tether several components of the MAPK cascades. Both of them are thought to regulate the enzymatic specificity and efficiency.

Amino Acid Motifs↗
PubMed2003

Regulation of c-Fos and Fra-1 by the MEK5-ERK5 pathway.

BACKGROUND: ERK5 is the newest subfamily member of the mitogen-activated protein kinase (MAPK) family, and is activated by various extracellular signals including growth factors. MEK5 is a specific activator of ERK5. c-Fos and Fra-1, well-known immediate early gene products, are members of the AP-1 family. We previously reported that activation of the MEK5-ERK5 pathway is able to induce expression of c-Fos. RESULTS: We have found that activation of the MEK5-ERK5 pathway causes the phosphorylation and stabilization of c-Fos and Fra-1. Phosphorylation of c-Fos appears to be mediated by ERK5 and a kinase(s) lying downstream of ERK5, and the MEK5-ERK5 pathway-dependent phosphorylation sites on c-Fos are different from the ERK1/2 pathway-dependent ones. Interestingly, activation of the MEK5-ERK5 pathway, but not that of the ERK1/2 pathway, is found to markedly increase the transactivation activity of c-Fos. Furthermore, our results show that the C-terminal half of ERK5 is necessary for the maximal activation of the transactivation activity of c-Fos and Fra-1. CONCLUSION: These results reveal a role of the MEK5-ERK5 pathway in modulating the function of the Fos family proteins which is different from the role of the ERK1/2 pathway.

3T3 Cells↗
PubMed2003

AKRL1 and AKRL2 activate the JNK pathway.

BACKGROUND: c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, is activated by specific cytokines and various environmental stresses. MKK4 and MKK7 are shown to be direct activators of JNK. Although several upstream components of the JNK pathway, including members of the MAPKKK family have been described, the components lying between the receptors or sensors and JNK have not been fully characterized. RESULTS: We have identified AKRL1 and AKRL2 (Akr1p-like 1 and 2) as novel activators of the JNK pathway. AKRL1 and AKRL2 proteins have a considerable sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae. Expression of AKRL1 or AKRL2 activates JNK and its activators MKK4 and MKK7. This AKRL1/2-induced JNK activation is significantly suppressed by the expression of a kinase-negative mutant of TAK1, a member of the MAPKKK family. AKRL1 and AKRL2 localize to the Golgi. Both the N-terminal half and the C-terminal transmembrane domain of AKRL1/2 are required for the JNK activation. The C-terminal transmembrane domain of AKRL1/2 is required for localization to the Golgi. CONCLUSION: AKRL1 and AKRL2 are localized to Golgi and the novel activators of the JNK pathway.

Acyltransferases↗
PubMed2003

Temperature compensation and temperature resetting of circadian rhythms in mammalian cultured fibroblasts.

BACKGROUND: Circadian rhythms control many physiological processes. One of characteristic properties of circadian rhythms is insensitivity to temperature, called temperature compensation. Although this temperature-insensitive property has repeatedly been observed mainly in circadian output rhythms, temperature effect on autoregulatory feedback loops of clock gene expression, the rhythm-generating mechanisms, has not been fully investigated. RESULTS: We show first that the circadian oscillation of clock gene expression in NIH3T3 fibroblasts, which is induced by TPA (12-O-tetradecanoylphorbol-13-acetate) treatment, is strongly temperature-compensated over the temperature range of 33-42 degrees C. We then show that heat treatment at 42 degrees C is able to trigger circadian oscillation of clock gene expression in NIH3T3 cells. This 42 degrees C heat treatment, unlike serum shock or TPA treatment, did not induce immediate expression of mPer1 mRNA, suggesting the existence of several different resetting mechanisms. CONCLUSIONS: This is the first demonstration of temperature compensation of the rhythm-generating core feedback loops of clock gene expression in mammalian cultured cells. It is possible that cells in the periphery could sense the change of ambient temperature as a resetting cue and that the whole organism thus could be entrained rapidly at dawn, in cooperation with the resetting mechanism by light.

Animals↗