Eisuke Nishida

Activation of the Estrogen Receptor Through Phosphorylation by Mitogen-Activated Protein Kinase

The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

Identification of a Member of the MAPKKK Family as a Potential Mediator of TGF-β Signal Transduction

The mitogen-activated protein kinase (MAPK) pathway is a conserved eukaryotic signaling module that converts receptor signals into various outputs. MAPK is activated through phosphorylation by MAPK kinase (MAPKK), which is first activated by MAPKK kinase (MAPKKK). A genetic selection based on a MAPK pathway in yeast was used to identify a mouse protein kinase (TAK1) distinct from other members of the MAPKKK family. TAK1 was shown to participate in regulation of transcription by transforming growth factor-β (TGF-β). Furthermore, kinase activity of TAK1 was stimulated in response to TGF-β and bone morphogenetic protein. These results suggest that TAK1 functions as a mediator in the signaling pathway of TGF-β superfamily members.

CRM1 is responsible for intracellular transport mediated by the nuclear export signal

The discovery of nuclear export signals (NESs) in a number of proteins revealed the occurrence of signal-dependent transport of proteins from the nucleus to the cytoplasm. Although the consensus motif of the NESs has been shown to be a leucine-rich, short amino-acid sequence,,, its receptor has not been identified. A cytotoxin leptomycin B (LMB) has recently been suggested to inhibit the NES-mediated transport of Rev protein. Here we show that LMB is a potent and specific inhibitor of the NES-dependent nuclear export of proteins. Moreover, we have found a protein of relative molecular mass 110K (p110) in Xenopus oocyte extracts that binds to the intact NES but not to the mutated, non-functional NES. The binding of p110 to NES is inhibited by LMB. We show that p110 is CRM1, which is an evolutionarily conserved protein originally found as an essential nuclear protein in fission yeast and known as a likely target of LMB. We also show that nuclear export of a fission yeast protein, Dsk1, which has a leucine-rich NES, is disrupted in wild-type yeast treated with LMB or in the crm1 mutant. These results indicate that CRM1 is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells.

Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization

Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at 3 (refs 11, 12); however, the kinases responsible for this phosphorylation have not been identified. Here we show that LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, our results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin.

THE MAP KINASE CASCADE IS ESSENTIAL FOR DIVERSE SIGNAL TRANSDUCTION PATHWAYS

Mitogen-activated protein (MAP) kinases are activated by combined tyrosine and threonine phosphorylation catalysed by MAP kinase kinase, a novel class of protein kinases with dual specificity for both tyrosine and serine/threonine. MAP kinase kinase is turned on by serine/threonine phosphorylation catalysed by an immediate upstream kinase. The MAP kinase cascade appears to be conserved during evolution and thus might play an essential role in diverse intracellular signaling processes from yeasts to vertebrates.

Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF‐3 and CBP/p300

It has been hypothesized that certain viral infections directly activate a transcription factor(s) which is responsible for the activation of genes encoding type I interferons (IFNs) and interferon‐stimulated genes (ISGs) via interferon regulatory factor (IRF) motifs present in their respective promoters. These events trigger the activation of defense machinery against viruses. Here we demonstrate that IRF‐3 transmits a virus‐induced signal from the cytoplasm to the nucleus. In unstimulated cells, IRF‐3 is present in its inactive form, restricted to the cytoplasm due to a continuous nuclear export mediated by nuclear export signal, and it exhibits few DNA‐binding properties. Virus infection but not IFN treatment induces phosphorylation of IRF‐3 on specific serine residues, thereby allowing it to complex with the co‐activator CBP/p300 with simultaneous nuclear translocation and its specific DNA binding. We also show that a dominant‐negative mutant of IRF‐3 could inhibit virus‐induced activation of chromosomal type I IFN genes and ISGs. These findings suggest that IRF‐3 plays an important role in the virus‐inducible primary activation of type I IFN and IFN‐responsive genes.

A conserved docking motif in MAP kinases common to substrates, activators and regulators.

Mitogen-activated protein kinases (MAPKs) are specifically phosphorylated and activated by the MAPK kinases, phosphorylate various targets such as MAPK-activated protein kinases and transcription factors, and are inactivated by specific phosphatases. Recently, docking interactions via the non-catalytic regions of MAPKs have been suggested to be important in regulating these reactions. Here we identify docking sites in MAPKs and in MAPK-interacting enzymes. A docking domain in extracellular-signal-regulated kinase (ERK), a MAPK, serves as a common site for binding to the MAPK kinase MEK1, the MAPK-activated protein kinase MNK1 and the MAPK phosphatase MKP3. Two aspartic acids in this domain are essential for docking, one of which is mutated in the sevenmaker mutant of Drosophila ERK/Rolled. A corresponding domain in the MAPKs p38 and JNK/SAPK also serves as a common docking site for their MEKs, MAPK-activated protein kinases and MKPs. These docking interactions increase the efficiency of the enzymatic reactions. These findings reveal a hitherto unidentified docking motif in MAPKs that is used in common for recognition of their activators, substrates and regulators.

Activation of the Protein Kinase ERK5/BMK1 by Receptor Tyrosine Kinases IDENTIFICATION AND CHARACTERIZATION OF A SIGNALING PATHWAY TO THE NUCLEUS

ERK5 (also known as BMK1), a member of the mitogen-activated protein kinase (MAPK) superfamily, was known to be activated strongly by oxidant and osmotic stresses. Here we have found that ERK5 is strongly activated by epidermal growth factor and nerve growth factor, whose receptors are tyrosine kinases. The activation of ERK5 was inhibited by expression of dominant-negative Ras and induced by expression of active Ras in PC12 cells, indicating a requirement for Ras in ERK5 activation. The epidermal growth factor-induced activation of ERK5 was found to be inhibited by PD98059 and U0126 inhibitors, which were previously thought to act specifically on classical MAPK kinase (also known as MEK1) and readily reversed by CL100 and MKP-3 dual-specificity phosphatases for which classical MAPKs were previously shown to serve as preferred substrates. The reporter assays demonstrated that the serum-induced enhancement of transcription from serum response element was significantly inhibited by expression of a dominant-negative form of MEK5, which was a direct and specific activator for ERK5 and that transcription from serum response element mediated by the Ets-domain transcription factor Sap1a, but not by Elk1, was stimulated by coexpression of ERK5 and active MEK5. In addition, Sap1a was shown to be phosphorylated by ERK5 in vitro and by the activation of the ERK5 pathway in cells. Moreover, the serum-induced c-Fos expression was markedly inhibited by expression of dominant-negative MEK5. These results reveal a novel signaling pathway to the nucleus mediated by ERK5 that functions downstream of receptor tyrosine kinases to induce immediate early genes, in parallel with the classical MAPK cascade.

TAB1: An Activator of the TAK1 MAPKKK in TGF-β Signal Transduction

Transforming growth factor-β (TGF-β) 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-β 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-β, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-β signal transduction.

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.