研究者詳細 - 曲　正樹

論文 - 曲　正樹

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Ca2+/Calmodulin結合型転写因子の網羅的同定

太尾田泰成, 大西和貴, 古谷雄穂, 傳田美和子, 金山直樹, 曲正樹, 森下了, 徳光浩

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [3P0185] - [3P0185] 2015年12月

インターラクトーム解析法を用いたヒトS100A6標的分子の探索

坂根恭平, 西口みゆ, 古谷雄穂, 傳田美和子, 山口文徳, 曲正樹, 金山直樹, 森下了, 徳光浩

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1P0204] - [1P0204] 2015年12月

Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells 査読

Yuya Fujiwara, Yuri Hiraoka, Tomohito Fujimoto, Naoki Kanayama, Masaki Magari, Hiroshi Tokumitsu

BIOCHEMISTRY 54 ( 25 ) 3969 - 3977 2015年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

To assess the isoform specificity of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKK alpha mutant (Ala292Thr/Leu233Phe) and a CaMKK beta mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wildtype or the STO-609-resistant mutant of CaMKK alpha was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKK beta mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKK beta is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK. isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

DOI： 10.1021/acs.biochem.5b00149

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Compartmentalized AMPK Signaling Illuminated by Genetically Encoded Molecular Sensors and Actuators 査読

Takafumi Miyamoto, Elmer Rho, Vedangi Sample, Hiroki Akano, Masaki Magari, Tasuku Ueno, Kirill Gorshkov, Melinda Chen, Hiroshi Tokumitsu, Jin Zhang, Takanari Inoue

CELL REPORTS 11 ( 4 ) 657 - 670 2015年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CELL PRESS

AMP-activated protein kinase (AMPK), whose activity is a critical determinant of cell health, serves a fundamental role in integrating extracellular and intracellular nutrient information into signals that regulate various metabolic processes. Despite the importance of AMPK, its specific roles within the different intracellular spaces remain unresolved, largely due to the lack of real-time, organelle-specific AMPK activity probes. Here, we present a series of molecular tools that allows for the measurement of AMPK activity at the different subcellular localizations and that allows for the rapid induction of AMPK inhibition. We discovered that AMPK alpha 1, not AMPK alpha 2, was the subunit that preferentially conferred spatial specificity to AMPK, and that inhibition of AMPK activity at the mitochondria was sufficient for triggering cytosolic ATP increase. These findings suggest that genetically encoded molecular probes represent a powerful approach for revealing the basic principles of the spatiotemporal nature of AMPK regulation.

DOI： 10.1016/j.celrep.2015.03.057

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ヒトCalmodulin標的分子の網羅的同定

古谷雄穂, 西口みゆ, 傳田美和子, 曲正樹, 金山直樹, 森下了, 徳光浩

日本生化学会大会プログラム・講演要旨集 87回 [4T14a - 01] 2014年10月

A Missense Mutation in Rev7 Disrupts Formation of Polζ, Impairing Mouse Development and Repair of Genotoxic Agent-induced DNA Lesions 査読

Maryam Khalaj, Abdolrahim Abbasi, Hiroshi Yamanishi, Kouyou Akiyama, Shuso Wakitani, Sotaro Kikuchi, Michiko Hirose, Misako Yuzuriha, Masaki Magari, Heba A. Degheidy, Kuniya Abe, Atsuo Ogura, Hiroshi Hashimoto, Tetsuo Kunieda

The Journal of Biological Chemistry 289 ( 6 ) 3811 - 3824 2014年2月

CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34 査読

Fumihiro Yamane, Yumiko Nishikawa, Kazue Matsui, Miki Asakura, Eriko Iwasaki, Koji Watanabe, Hikaru Tanimoto, Hiroki Sano, Yuki Fujiwara, E. Richard Stanley, Naoki Kanayama, Neil A. Mabbott, Masaki Magari, Hitoshi Ohmori

JOURNAL OF LEUKOCYTE BIOLOGY 95 ( 1 ) 19 - 31 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FEDERATION AMER SOC EXP BIOL

Occurrence of a CSF-1 receptor-dependent monocyte differentiation process driven by IL-34, but not CSF-1. With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b(+) monocytic cells (F4/80(+), Gr-1(-), Ly6C(-), I-A/E-/lo, CD11c(-), CD115(+), CXCR4(+), CCR2(+), CX(3)CR1(-)) when cultured with a Lin(-)c-kit(+) population from mouse spleen cells. The developed CD11b(+) cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

DOI： 10.1189/jlb.0613311

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In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5 '-triphosphate as a phosphate donor 査読

Saki Yurimoto, Tomohito Fujimoto, Masaki Magari, Naoki Kanayama, Ryoji Kobayashi, Hiroshi Tokumitsu

BMC BIOCHEMISTRY 13 ( 27 ) 2012年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOMED CENTRAL LTD

Background: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases -including CaMKI, CaMKIV, and AMPK- to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
Results: Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKI alpha (Thr(177)) and of AMPK (Thr(172)) in vitro. Kinetic analysis indicated that the K-m values of CaMKK isoforms for GTP (400-500 mu M) were significantly higher than those for ATP (similar to 15 mu M), and a 2- to 4-fold decrease in V-max was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of similar to 45 kDa and similar to 35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
Conclusions: These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.

DOI： 10.1186/1471-2091-13-27

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Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1 査読

Yuichi Kanehiro, Kagefumi Todo, Misaki Negishi, Junji Fukuoka, Wenjian Gan, Takuya Hikasa, Yoshiaki Kaga, Masayuki Takemoto, Masaki Magari, Xialu Li, James L. Manley, Hitoshi Ohmori, Naoki Kanayama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 ( 4 ) 1216 - 1221 2012年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

DOI： 10.1073/pnas.1120368109

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B細胞免疫記憶 AIDに依存したIgV変異には、SRタンパク質SRSF1のスプライスアイソフォームが必要である(AID-dependent IgV hypermutation requires a splice isoform of the SR protein SRSF1)

金山直樹, 金広優一, 藤堂景史, 根岸美咲, 福岡純司, 曲正樹, Li Xialu, Manley James L., 大森斉

日本免疫学会総会・学術集会記録 40 172 - 172 2011年11月

IL-21-Dependent B Cell Death Driven by Prostaglandin E-2, a Product Secreted from Follicular Dendritic Cells 査読

Masaki Magari, Yumiko Nishikawa, Yasumasa Fujii, Yumi Nishio, Koji Watanabe, Michiya Fujiwara, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF IMMUNOLOGY 187 ( 8 ) 4210 - 4218 2011年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC IMMUNOLOGISTS

In germinal centers (GCs), B cells are selected through interaction with follicular dendritic cells bearing immune complexes and follicular helper T (Tfh) cells secreting Tfh cytokines, including IL-21. To analyze these cellular interactions, we have explored culture conditions that can simulate GC B cell selection in vitro using a mouse follicular dendritic cell line, FL-YB. FL-YB cells efficiently enhanced viability of cocultured mouse B cells in a BAFF-dependent fashion. Interestingly, we found that addition of IL-21, a major Tfh cytokine, readily induced death of B cells that were cocultured with FL-YB cells, whereas IL-21 alone sustained viability of B cells in the absence of FL-YB cells. The IL-21-induced death was dependent on a low m.w. soluble factor that was released from FL-YB cells, which was finally identified as PGE(2). Treatment of B cells with IL-21 plus PGE(2), but not either alone, resulted in enhanced expression of a proapoptotic protein Bim and the upstream transcription factor Foxo1. A PGE(2) receptor isoform, EP4, was responsible for IL-21/PGE(2)-induced B cell death. Thus, PGE(2) is an endogenous chemical mediator that can switch pleiotropic actions of IL-21 on B cells. IL-21/PGE(2)-induced B cell death was rescued if B cells were costimulated via CD40. In immunized mice, deficiency of IL-21R in B cells led to a significant decrease in the frequency of activated caspase-3-positive GC B cells concomitant with impaired affinity maturation of Abs. Taken together, results implicate a physiological role of IL-21/PGE(2)-induced B cell death in GC B cell selection. The Journal of Immunology, 2011, 187: 4210-4218.

DOI： 10.4049/jimmunol.1100934

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AIDに依存した体細胞突然変異には、SRタンパク質SRSF1のスプライスアイソフォームが必要である

金山直樹, 金広優一, 藤堂景史, 根岸美咲, 福岡純司, 曲正樹, Li Xialu, Manley James L., 大森斉

日本生化学会大会プログラム・講演要旨集 84回 3T15a - 15 2011年9月

変異能力を有するニワトリB細胞株DT40-SWを用いたマウスモノクローナル抗体の親和性成熟

鴨下佳代子, 小島聡史, 藤井忍, 北村幸一, 井上和恵, 岡山展久, 松田修一, 小嶋宏侑, 池田美香, 金広優一, 藤堂景史, 曲正樹, 大森齊, 金山直樹

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 2T6 - 10 2010年12月

抗体遺伝子への体細胞突然変異におけるsplicing factor ASF/SF2の寄与

金広優一, 藤堂景史, 根岸美咲, 福岡純司, 曲正樹, 大森齊, 金山直樹

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 2P - 0478 2010年12月

Efficient affinity maturation of antibodies in an engineered chicken B cell line DT40-SW by increasing point mutation 査読

Masamichi Kajita, Takahiro Okazawa, Mika Ikeda, Kagefumi Todo, Masaki Magari, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 110 ( 3 ) 351 - 358 2010年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

The chicken B cell line DT40 undergoes hypermutation of immunoglobulin variable region (IgV) genes during culture, thereby constituting an antibody (Ab) library. We previously established an in vitro Ab generation system using an engineered line DT40-SW whose hypermutation machinery can be switched on and off. Abs for various antigens (Ags) can be obtained from the DT40-SW library and the specificity of the Ag-specific clones can be stabilized by stopping hypermutation. Furthermore, the affinity of obtained monoclonal Abs (mAbs) can be improved through further mutation followed by selection, a process analogous to "affinity maturation" that occurs in vivo. Although gene conversion dominantly diversifies the IgV genes in DT40 cells, point mutation is considered to be more favorable for fine-tuning Ab properties during affinity maturation. Here, we examined whether affinity maturation occurs more efficiently when the hypermutation pattern was transformed from gene conversion into point mutation in DT40-SW cells. To this end, we disrupted the XRCC3 gene that is essential for gene conversion. It was found that hemizygous disruption of the XRCC3 gene was sufficient to increase the point mutation frequency. Since hemizygous disruption is conducted more easily, we tested whether the XRCC3 (+/-) mutant generates high-affinity Abs through affinity maturation more efficiently than the wild type. Using this affinity maturation technique, we generated an improved 4-hydroxy-3-nitrophenylacetyl-specific mAb with similar to 600-fold lower K(D)) than that of the original mAb. Taken together, hemizygous disruption of the XRCC3 gene is considered to be useful for obtaining high-affinity mAbs from DT40-SW cells though affinity maturation. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2010.03.006

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変異能力を有するニワトリB細胞株DT40-SWを用いたマウスモノクローナル抗体の親和性成熟

金山直樹, 小島聡史, 藤井忍, 北村幸一, 井上知恵, 岡山展久, 松田修一, 鴨下佳代子, 藤堂景史, 池田美香, 曲正樹, 大森齊

日本生物工学会大会講演要旨集平成22年度 81 - 81 2010年9月

Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire 査読

Masaki Magari, Yuichi Kanehiro, Kagefumi Todo, Mika Ikeda, Naoki Kanayama, Hitoshi Ohmori

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 396 ( 2 ) 353 - 358 2010年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SW Delta C, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SW Delta C cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SW Delta C cells, although the protein might be highly susceptible to degradation. In DT40-SW Delta C cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SW Delta C cells may be useful for constructing AID libraries for efficient screening of mAbs in vitro. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.04.096

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Conditional transformation of immunoglobulin mutation pattern from gene conversion into point mutation by controlling XRCC3 expression in the DT40 B cell line 査読

Masamichi Kajita, Masaki Magari, Kagefumi Todo, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 109 ( 4 ) 407 - 410 2010年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

A hypermutating B cell line DT40 is useful for screening antibodies and improving affinity of the selected antibodies in vitro. To perform affinity maturation efficiently, we generated an engineered DT40 line whose immunoglobulin mutation pattern can be transformed from gene conversion into point mutation by conditional suppression of XRCC3 expression. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.09.050

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Enhancement of antibody production from a chicken B cell line DT40 by reducing Pax5 expression 査読

Masaki Magari, Takahiro Aya, Mika Ikeda, Kagefumi Todo, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 107 ( 2 ) 206 - 209 2009年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

We developed a novel in vitro antibody (Ab) generation system using a hypermutating chicken B cell line (DT40-SW). We suppressed the expression of the Pax5 transcription factor by targeted disruption of the gene to increase Ab production in isolated clones and produce the desired Abs. This single genetic manipulation resulted in a significant enhancement of Ab production without significantly affecting maximum cell density. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2008.09.014

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変異導入能力を有する培養B細胞株を用いたin vitro抗体作製システムの高機能化

金山直樹, 曲正樹, 梶田真道, 金広優一, 藤堂景史, 大森斉

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 81回・31回 1T9 - 7 2008年11月

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