Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis.

Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.


Proteomic identification of Bcl2-associated athanogene 2 as a novel MAPK-activated protein kinase 2 substrate.

The p38 MAPK cascade is activated by various stresses or cytokines. Downstream of p38 MAPKs, there are diversification and extensive branching of signaling pathways. Fluorescent two-dimensional difference gel electrophoresis of phosphoprotein-enriched samples from HeLa cells in which p38 MAPK activity was either suppressed or activated enabled us to detect approximately 90 candidate spots for factors involved in p38-dependent pathways. Among these candidates, here we identified four proteins including Bcl-2-associated athanogene 2 (BAG2) by peptide mass fingerprintings. BAG family proteins are highly conserved throughout eukaryotes and regulate Hsc/Hsp70-mediated molecular chaperone activities and apoptosis. The results of two-dimensional immunoblots suggested that the phosphorylation of BAG2 was specifically controlled in a p38 MAPK-dependent manner. Furthermore, BAG2 was directly phosphorylated at serine 20 in vitro by MAPK-activated protein kinase 2 (MAPKAP kinase 2), which is known as a primary substrate of p38 MAPK and mediates several p38 MAPK-dependent processes. We confirmed that MAPKAP kinase 2 is also required for phosphorylation of BAG2 in vivo. Thus, p38 MAPK-MAPKAP kinase 2-BAG2 phosphorylation cascade may be a novel signaling pathway for response to extracellular stresses.


Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord.

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.


Combinatorial roles of Olig2 and Neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons.

Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. Our results further revealed crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. We suggest that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube.


Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow.

The small GTPase Rho and one of its targets, Rho-kinase (also termed ROK or ROCK), are implicated in various cellular functions including stress fiber formation, smooth muscle contraction, tumor cell invasion and cell motility. We have previously reported that Rho-kinase accumulates at the cleavage furrow during cytokinesis in several cultured cells. Here, using Rho-kinase inhibitors, Y-27632 and HA1077, we found that Rho-kinase is responsible for the phosphorylation of myosin regulatory light chain at Ser19 in the cleavage furrow during cytokinesis. On the other hand, phosphorylation of ezrin/radixin/moesin (ERM) proteins at the cleavage furrow was enhanced by the addition of the above Rho-kinase inhibitors. Treatment with Y-27632 strongly enhanced the accumulation of Rho-kinase but not RhoA and citron kinase at the cleavage furrow. Furthermore, the furrow ingression in cytokinesis was significantly prolonged in the presence of Y-27632. These results suggest that Rho-kinase is involved in the progression of cytokinesis through the phosphorylation of several proteins including myosin light chain at the cleavage furrow.


Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.


Regulation of intermediate filament organization during cytokinesis: possible roles of Rho-associated kinase.

Intermediate filaments (IFs), which form the structural framework of cytoskeleton, have been found to be dramatically reorganized during mitosis. Some protein kinases activated in mitosis are thought to control spatial and temporal IF reorganization through phosphorylation of IF proteins. Rho-associated kinase (Rho-kinase), one of the putative targets of the small GTPase Rho, does phosphorylate IF proteins, specifically at the cleavage furrow during cytokinesis. This cleavage furrow-specific phosphorylation plays an important role in the local IF breakdown and efficient separation of IF networks. Recent studies on Rho signaling pathways have introduced new models about the molecular mechanism of rearrangements of cytoskeletons including IFs during cytokinesis.


Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.


Distribution of Rho-kinase in the bovine brain.

Biochemical and Biophysical Research Communications


Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments.


Phosphorylation of vimentin by Rho-associated kinase at a unique amino-terminal site that is specifically phosphorylated during cytokinesis.


Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase.


Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN.


Multiple roles of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase cascade in Xenopus laevis.


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


Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes.


Mitogen-activated protein kinase kinase is required for the Mos-induced metaphase arrest.


Characterization of recombinant Xenopus MAP kinase kinases mutated at potential phosphorylation sites.


Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.


Regulation and function of the MAP kinase cascade in Xenopus oocytes.


cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates.


Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection.


Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation.


Xenopus MAP kinase activator: identification and function as a key intermediate in the phosphorylation cascade.


In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase.

The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.


Sulfoxide-mediated Cys-Trp-selective bioconjugation that enables protein labeling and peptide heterodimerization


ボルバキアがアワノメイガで引き起こすオス殺しの原因因子Oscarに関する研究

ボルバキアがアワノメイガで引き起こすオス殺しの原因因子Oscarに関する研究

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集


アワノメイガオス化因子OfMascの分解に関与するユビキチン化の解析

アワノメイガオス化因子OfMascの分解に関与するユビキチン化の解析

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集


オス殺しWolbachiaによる「性決定ハイジャック」の分子的証拠の提出

オス殺しWolbachiaによる「性決定ハイジャック」の分子的証拠の提出

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集


カイコのオス化カスケードにおいてMascと相互作用するタンパク質の探索

カイコのオス化カスケードにおいてMascと相互作用するタンパク質の探索

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集

蚕糸・昆虫機能利用学術講演会・日本蚕糸学会大会講演要旨集


新規LUBAC結合E3による複合型ユビキチン鎖形成を介したネクロプトーシスの制御

日本Cell Death学会学術集会プログラム抄録集

日本Cell Death学会学術集会プログラム抄録集


Identification and functional analysis of host proteins that regulate the entry process of SARS-CoV-2

SARS-CoV-2の侵入過程を制御する宿主タンパク質の同定および機能解析

日本ウイルス学会学術集会プログラム・予稿集(Web)

日本ウイルス学会学術集会プログラム・予稿集(Web)


Protein-Protein interactome analysis of Plasmodium parasites using AirID, the novel proximity biotinylation enzyme

新規ビオチン化酵素AirIDを用いたマラリア原虫のタンパク質インタラクトーム解析

日本寄生虫学会大会プログラム・抄録集

日本寄生虫学会大会プログラム・抄録集


液胞アミノ酸トランスポーターAvt4N末端領域の機能とその分子機構解明

液胞アミノ酸トランスポーターAvt4N末端領域の機能とその分子機構解明

日本農芸化学会中四国支部講演会講演要旨集(Web)

日本農芸化学会中四国支部講演会講演要旨集(Web)


Development of technology for identification of proteins interacting with a target protein in plants using a proximity biotinylation enzyme, AirID

新規近位依存性ビオチン化酵素AirIDを利用した植物体内での相互作用タンパク質解析技術の開発

日本植物生理学会年会(Web)

日本植物生理学会年会(Web)