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Eisuke Nishida

Publications and source records attributed to Eisuke Nishida.

50 records · Page 3Linked to original sources
PubMed2003

Mammalian cultured cells as a model system of peripheral circadian clocks.

The mammalian circadian system consists of multiple oscillators with basically hierarchical relationship, in which the hypothalamic suprachiasmatic nucleus (SCN) is the master pacemaker and the other oscillators in the periphery are subordinate. Although peripheral oscillators have been preceded by the SCN in circadian studies, accumulating data have revealed the importance and characteristics of peripheral oscillators. Cultured cell lines have also provided valuable information about intracellular mechanisms of circadian rhythms. This review outlines the properties of peripheral clocks in several perspectives such as the mechanisms of autonomous oscillations, the clock resetting, and the clock outputs, and describes the usefulness of immortalized cultured cells as a model system of mammalian circadian clocks by introducing some fruits of related works.

Animals↗
PubMed2003

Activation of p38 kinase in the gerbil hippocampus showing ischemic tolerance.

Ischemic tolerance is a phenomenon in which brief episodes of ischemia protect against the lethal effects of subsequent periods of prolonged ischemia. The authors investigated the activation of p38 mitogen-activated protein kinase (p38) in the gerbil hippocampus by Western blotting and immunohistochemistry to clarify the role of p38 kinase in ischemic tolerance. After the 2-minute global ischemia, immunoreactivity indicating active p38 was enhanced at 6 hours of reperfusion and continuously demonstrated 72 hours after ischemia in CA1 and CA3 neurons. Pretreatment with SB203580, an inhibitor of active p38 (0-30 micromol/l), 30 minutes before the 2-minute ischemia reduced the ischemic tolerance effect in a dose-dependent manner. Immunoblot analysis indicated that alteration of the phosphorylation pattern of p38 kinase in the hippocampus after subsequent lethal ischemia was induced by the preconditioning. These findings suggest that lasting activation of p38 may contribute to ischemic tolerance in CA1 neurons of the hippocampus and that components of the p38 cascade can be target molecules to modify neuronal survival after ischemia.

Animals↗
PubMed2002

Essential role of the transcription factor Ets-2 in Xenopus early development.

The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate that Xenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription.

Animals↗
PubMed2002

Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal.

Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila Polo, is a serine-threonine protein kinase implicated in the regulation of multiple aspects of mitosis. The protein level, activity, and localization of Plk1 change during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Although localization of Plk1 to the centrosome has been established, nuclear localization or nucleocytoplasmic translocation of Plk1 has not been fully addressed. Here we show that Plk1 accumulates in both the nucleus and the cytoplasm in addition to its localization to the centrosome during S and G(2) phases. Our results identify a conserved region in the kinase domain of Plk1 (residues 134-146) as a functional bipartite nuclear localization signal (NLS) sequence that regulates nuclear translocation of Plk1. The identified NLS is necessary and sufficient for directing nuclear localization of Plk1. This bipartite NLS has an unusually short spacer sequence between two clusters of basic amino acids but is sensitive to RanQ69L, a dominant negative form of Ran, similar to ordinary bipartite NLS. Remarkably, the expression of an NLS-disrupted mutant of Plk1 during S phase was found to arrest the cells in G(2) phase. These results suggest that the bipartite NLS-dependent nuclear localization of Plk1 before mitosis is important for ensuring normal cell cycle progression.

Amino Acid Sequence↗
PubMed2002

Identification of the Anti-proliferative protein Tob as a MAPK substrate.

Mitogen-activated protein kinases (MAPKs) regulate a wide variety of cellular functions by phosphorylating their specific substrates. Here we have identified Tob as a novel substrate of MAPK. Tob, a member of the Tob and B-cell translocation gene anti-proliferative protein family, is shown to negatively regulate the proliferation of osteoblasts and T cells. In this study, our two-hybrid screening has identified Tob as an ERK2-interacting protein. Biochemical analyses have then shown that ERK MAPK (ERK2) and JNK/SAPK (JNK2) bind to and phosphorylate Tob in vitro. ERK catalyzes the phosphorylation more efficiently than JNK. When the ERK pathway is activated in cells, phosphorylation of Tob is induced. An ERK-binding or -docking site locates in the N-terminal portion of Tob, and phosphorylation sites reside in the C-terminal stretch region. The docking is crucial for efficient phosphorylation. Mutant forms of Tob, in which serines are replaced by glutamic acids to mimic phosphorylation, show a much reduced ability to inhibit the cell cycle progression to S phase from G(0)/G(1) phase, as compared with wild-type Tob, indicating that ERK phosphorylation negatively regulates the anti-proliferative function of Tob.

Amino Acid Sequence↗
PubMed2002

Modular structure of a docking surface on MAPK phosphatases.

Mitogen-activated protein kinases (MAPKs) must be precisely inactivated to achieve proper functions in the cells. Ten members of dual specificity phosphatases specifically acting on MAPKs, termed MAPK phosphatases (MKPs), have been reported. Each member has its own substrate specificity that should be tightly regulated. However, the molecular mechanism underlying the regulation of the specificity is largely unknown. In the MAPK signaling pathways, docking interactions, which are different from transient enzyme-substrate interaction, are known to regulate the enzymatic specificity. Here we have identified and characterized a docking surface of MKPs. Our results show that a docking surface is composed of a tandem alignment of three subregions (modules): a cluster of positively charged amino acids, a cluster of hydrophobic amino acids, and a cluster of positively charged amino acids (positive-hydrophobic-positive). This modular structure well fits the docking groove on MAPKs that we have previously identified and may contribute to regulating the docking specificity of the MKP family. The position, number, and species of charged amino acids in each module including the central hydrophobic subregion are important factors in regulation of docking to specific MAPKs. This modular structure in the docking interaction may define a novel model of protein-protein interaction that would also regulate other systems.

Amino Acid Sequence↗
PubMed2002

The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo.

The GTPase Ran is known to regulate transport of proteins across the nuclear envelope. Recently, Ran has been shown to promote microtubule polymerization and spindle assembly around chromatin in Xenopus mitotic extracts and to stimulate nuclear envelope assembly in Xenopus or HeLa cell extracts. However, these in vitro findings have not been tested in living cells and do not necessarily describe the generalized model of Ran functions. Here we present several lines of evidence that Ran is indispensable for correct chromosome positioning and nuclear envelope assembly in C. elegans. Embryos deprived of Ran by RNAi showed metaphase chromosome misalignment and aberrant chromosome segregation, while astral microtubules seemed unaffected. Depletion of RCC1 or RanGAP by RNAi resulted in essentially the same defects. The immunofluorescent staining showed that Ran localizes to kinetochore regions of metaphase and anaphase chromosomes, suggesting the role of Ran in linking chromosomes to kinetochore microtubules. Ran was shown to localize to the nuclear envelope at telophase and during interphase in early embryos, and the depletion of Ran resulted in failure of nuclear envelope assembly. Thus, Ran is crucially involved in chromosome positioning and nuclear envelope assembly in C. elegans.

Animals↗
PubMed2002

Plk1 promotes nuclear translocation of human Cdc25C during prophase.

The nuclear accumulation of active M-phase promoting factor (MPF) during prophase is thought to be essential for coordinating M-phase events in vertebrate cells. The protein phosphatase Cdc25C, an activator of MPF, enters the nucleus to keep MPF active in the nucleus during prophase. However, the molecular mechanisms that control nuclear translocation of Cdc25C during prophase are unknown. We show that phosphorylation of a serine residue (Ser198) in a nuclear export signal sequence of human Cdc25C occurs during prophase and promotes nuclear localization of Cdc25C. We also show that Polo-like kinase 1 (Plk1) is responsible for this phosphorylation and that constitutively active Plk1 promotes nuclear localization of Cdc25C. Remarkably, a mutant Cdc25C in which Ser198 is replaced by alanine remains in the cytoplasm when wild-type Cdc25C accumulates in the nucleus during prophase. These results suggest that Plk1 phosphorylates Cdc25C on Ser198 and regulates nuclear translocation of Cdc25C during prophase.

Cell Cycle Proteins↗
PubMed2002

Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway.

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Control of intracellular dynamics of mammalian period proteins by casein kinase I epsilon (CKIepsilon) and CKIdelta in cultured cells.

Recent studies have shown that casein kinase I epsilon (CKIepsilon) is an essential regulator of the mammalian circadian clock. However, the detailed mechanisms by which CKIepsilon regulates each component of the circadian negative-feedback loop have not been fully defined. We show here that mPer proteins, negative limbs of the autoregulatory loop, are specific substrates for CKIepsilon and CKIdelta. The CKI phosphorylation of mPer1 and mPer3 proteins results in their rapid degradation, which is dependent on the ubiquitin-proteasome pathway. Moreover, CKIepsilon and CKIdelta are able to induce nuclear translocation of mPer3, which requires its nuclear localization signal. The mutation in potential phosphorylation sites on mPer3 decreased the extent of both nuclear translocation and degradation of mPer3 that are stimulated by CKIepsilon. CKIepsilon and CKIdelta affected the inhibitory effect of mPer proteins on the transcriptional activity of BMAL1-CLOCK, but the inhibitory effect of mCry proteins on the activity of BMAL1-CLOCK was unaffected. These results suggest that CKIepsilon and CKIdelta regulate the mammalian circadian autoregulatory loop by controlling both protein turnover and subcellular localization of mPer proteins.

ARNTL Transcription Factors↗
PubMed2001

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates.

Recent genetic studies in Drosophila identified a novel non-canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.

Amino Acid Sequence↗
PubMedDate not supplied

Docking interactions in the mitogen-activated protein kinase cascades.

Regulation of cellular functions and responses utilizes a number of the signal transduction pathways. Each pathway should transduce signals with high efficiency and fidelity to avoid unnecessary crosstalks. The mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular functions, including cell proliferation, differentiation, and stress responses. MAPK is activated by MAPK kinase; phosphorylates various targets, including transcription factors and MAPK-activated protein kinases; and is inactivated by several phosphatases. Recent studies have provided a cue to understand the molecular mechanism underlying the signal transduction through the MAPK cascades. In the MAPK cascades, docking interactions, which are achieved through a site outside the catalytic domain of MAPKs, regulate the efficiency and specificity of the enzymatic reactions. The docking interaction is different from a transient enzyme-substrate interaction through the active center. It has been shown that activators, substrates, and inactivators of MAPKs utilize a common site on MAPKs in the docking interaction. Then, the docking interaction may regulate not only the efficiency and specificity of the cascades, but also the ordered and integrated signaling.

Animals↗
PubMedDate not supplied

Regulatory mechanisms and functions of MAP kinase signaling pathways.

Mitogen-activated protein kinase (MAPK) pathways play central roles in controlling diverse cellular functions. They are finely regulated by several mechanisms, including scaffolding of their components, and phosphorylation/dephosphorylation and compartmentalization of MAPKs. A number of molecules have been identified as regulators involved in these mechanisms. They modulate the magnitude and the specificity of MAPK signaling, and thereby regulate the wide variety of signaling outputs. Recent studies have identified novel functions of the MAPK signaling pathways. It is becoming clear that strict regulation of the MAPK pathways underlies their manifold functions in numerous biological processes.

Animals↗