研究者詳細 - 柴田　秀樹

論文 - 柴田　秀樹

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Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding Open Access

Amemiya, Y; Ioi, Y; Araki, M; Kontani, K; Maki, M; Shibata, H; Takahara, T

JOURNAL OF BIOLOGICAL CHEMISTRY 301 巻 ( 2 ) 頁： 108122 - 108122 2025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：Journal of Biological Chemistry

The mechanistic target of rapamycin complex 1 (mTORC1) functions as a master regulator of cell growth and proliferation. We previously demonstrated that intracellular calcium ion (Ca2+) concentration modulates the mTORC1 pathway via binding of the Ca2+ sensor protein calmodulin (CaM) to tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTORC1. However, the precise molecular mechanism by which Ca2+/CaM modulates mTORC1 activity remains unclear. Here, we performed a binding assay based on nano-luciferase reconstitution, a method for detecting weak interactions between TSC2 and its target, Ras homolog enriched in the brain (Rheb), an activator of mTORC1. CaM inhibited the binding of TSC2 to Rheb in a Ca2+-dependent manner. Live-cell imaging analysis indicated increased interaction between the CaM-binding region of TSC2 and CaM in response to elevated intracellular Ca2+ levels. Furthermore, treatment with carbachol, an acetylcholine analog, elevated intracellular Ca2+ levels and activated mTORC1. Notably, carbachol-induced activation of mTORC1 was inhibited by CaM inhibitors, corroborating the role of Ca2+/CaM in promoting the mTORC1 pathway. Consistent with the effect of Ca2+/CaM on the TSC2-Rheb interaction, increased intracellular Ca2+ concentration promoted the dissociation of TSC2 from lysosomes without affecting Akt-dependent phosphorylation of TSC2, suggesting that the regulatory mechanism of TSC2 by Ca2+/CaM is distinct from the previously established action mechanism of TSC2. Collectively, our findings offer mechanistic insights into TSC2-Rheb regulation mediated by Ca2+/CaM, which links Ca2+ signaling to mTORC1 activation.

DOI： 10.1016/j.jbc.2024.108122

Cytoprotective Role of Autophagy in CDIP1 Expression-Induced Apoptosis in MCF-7 Breast Cancer Cells 査読有り国際誌 Open Access

Inukai, R; Mori, K; Maki, M; Takahara, T; Shibata, H

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 25 巻 ( 12 ) 2024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：International Journal of Molecular Sciences

Cell death-inducing p53-target protein 1 (CDIP1) is a proapoptotic protein that is normally expressed at low levels and is upregulated by genotoxic and endoplasmic reticulum stresses. CDIP1 has been reported to be localized to endosomes and to interact with several proteins, including B-cell receptor-associated protein 31 (BAP31) and apoptosis-linked gene 2 (ALG-2). However, the cellular and molecular mechanisms underlying CDIP1 expression-induced apoptosis remain unclear. In this study, we first demonstrated that CDIP1 was upregulated after treatment with the anticancer drug adriamycin in human breast cancer MCF-7 cells but was degraded rapidly in the lysosomal pathway. We also demonstrated that treatment with the cyclin-dependent kinase 5 (CDK5) inhibitor roscovitine led to an increase in the electrophoretic mobility of CDIP1. In addition, a phosphomimetic mutation at Ser-32 in CDIP1 resulted in an increase in CDIP1 expression-induced apoptosis. We also found that CDIP1 expression led to the induction of autophagy prior to apoptosis. Treatment of cells expressing CDIP1 with SAR405, an inhibitor of the class III phosphatidylinositol 3-kinase VPS34, caused a reduction in autophagy and promoted apoptosis. Therefore, autophagy is thought to be a defense mechanism against CDIP1 expression-induced apoptosis.

DOI： 10.3390/ijms25126520

New Insights into the Regulation of mTOR Signaling via Ca<SUP>2+</SUP>-Binding Proteins 査読有り国際誌 Open Access

Amemiya, Y; Maki, M; Shibata, H; Takahara, T

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 24 巻 ( 4 ) 2023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：International Journal of Molecular Sciences

Environmental factors are important regulators of cell growth and proliferation. Mechanistic target of rapamycin (mTOR) is a central kinase that maintains cellular homeostasis in response to a variety of extracellular and intracellular inputs. Dysregulation of mTOR signaling is associated with many diseases, including diabetes and cancer. Calcium ion (Ca2+) is important as a second messenger in various biological processes, and its intracellular concentration is tightly regulated. Although the involvement of Ca2+ mobilization in mTOR signaling has been reported, the detailed molecular mechanisms by which mTOR signaling is regulated are not fully understood. The link between Ca2+ homeostasis and mTOR activation in pathological hypertrophy has heightened the importance in understanding Ca2+-regulated mTOR signaling as a key mechanism of mTOR regulation. In this review, we introduce recent findings on the molecular mechanisms of regulation of mTOR signaling by Ca2+-binding proteins, particularly calmodulin (CaM).

DOI： 10.3390/ijms24043923

Hepatitis C Virus-Induced ROS/JNK Signaling Pathway Activates the E3 Ubiquitin Ligase Itch to Promote the Release of HCV Particles via Polyubiquitylation of VPS4A 査読有り国際誌 Open Access

Deng, L; Liang, YJ; Ariffianto, A; Matsui, C; Abe, T; Muramatsu, M; Wakita, T; Maki, M; Shibata, H; Shoji, I

JOURNAL OF VIROLOGY 96 巻 ( 6 ) 頁： e0181121 2022年3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：Journal of Virology

We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. However, the roles of ROS/JNK activation in the HCV life cycle remain unclear. We sought to identify a novel role of the ROS/JNK signaling pathway in the HCV life cycle. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of Itch, a HECT-type E3 ubiquitin ligase, leading to activation of Itch. The small interfering RNA (siRNA) knockdown of Itch significantly reduced the extracellular HCV infectivity titers, HCV RNA, and HCV core protein without affecting intracellular HCV infectivity titers, HCV RNA, and HCV proteins, suggesting that Itch is involved in the release of HCV particles. HCV-mediated JNK/Itch activation specifically promoted polyubiquitylation of an AAA-type ATPase, VPS4A, but not VPS4B, required to form multivesicular bodies. Site-directed mutagenesis revealed that two lysine residues (K23 and K121) on VPS4A were important for VPS4A polyubiquitylation. The siRNA knockdown of VPS4A, but not VPS4B, significantly reduced extracellular HCV infectivity titers. Coimmunoprecipitation analysis revealed that HCV infection specifically enhanced the interaction between CHMP1B, a subunit of endosomal sorting complexes required for transport (ESCRT)-III complex, and VPS4A, but not VPS4B, whereas VPS4A K23R/K121R greatly reduced the interaction with CHMP1B. HCV infection significantly increased ATPase activity of VPS4A, but not VPS4A K23R/K121R or VPS4B, suggesting that HCV-mediated polyubiquitylation of VPS4A contributes to activation of VPS4A. Taken together, we propose that the HCV-induced ROS/JNK/Itch signaling pathway promotes VPS4A polyubiquitylation, leading to enhanced VPS4ACHMP1B interaction and promotion of VPS4A ATPase activity, thereby promoting the release of HCV particles.

DOI： 10.1128/jvi.01811-21

Amino Acid-Mediated Intracellular Ca<SUP>2+</SUP> Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca<SUP>2+</SUP>/Calmodulin 査読有り国際誌 Open Access

Amemiya, Y; Nakamura, N; Ikeda, N; Sugiyama, R; Ishii, C; Maki, M; Shibata, H; Takahara, T

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 22 巻 ( 13 ) 2021年7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：International Journal of Molecular Sciences

Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+ /calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+ /CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+ /CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.

DOI： 10.3390/ijms22136897

The Novel ALG-2 Target Protein CDIP1 Promotes Cell Death by Interacting with ESCRT-I and VAPA/B 査読有り Open Access

Inukai, R; Mori, K; Kuwata, K; Suzuki, C; Maki, M; Takahara, T; Shibata, H

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 22 巻 ( 3 ) 頁： 1 - 25 2021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：International Journal of Molecular Sciences

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EFhand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

DOI： 10.3390/ijms22031175

The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination 査読有り国際誌 Open Access

Zhang, W; Muramatsu, A; Matsuo, R; Teranishi, N; Kahara, Y; Takahara, T; Shibata, H; Maki, M

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 21 巻 ( 17 ) 頁： 1 - 21 2020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：International Journal of Molecular Sciences

ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca2+ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca2+-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.

DOI： 10.3390/ijms21176315

Amino acid-dependent control of mTORC1 signaling: a variety of regulatory modes 招待有り査読有り国際誌 Open Access

Takahara, T; Amemiya, Y; Sugiyama, R; Maki, M; Shibata, H

JOURNAL OF BIOMEDICAL SCIENCE 27 巻 ( 1 ) 頁： 87 - 87 2020年8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：Journal of Biomedical Science

The mechanistic target of rapamycin complex 1 (mTORC1) is an essential regulator of cell growth and metabolism through the modulation of protein and lipid synthesis, lysosome biogenesis, and autophagy. The activity of mTORC1 is dynamically regulated by several environmental cues, including amino acid availability, growth factors, energy levels, and stresses, to coordinate cellular status with environmental conditions. Dysregulation of mTORC1 activity is closely associated with various diseases, including diabetes, cancer, and neurodegenerative disorders. The discovery of Rag GTPases has greatly expanded our understanding of the regulation of mTORC1 activity by amino acids, especially leucine and arginine. In addition to Rag GTPases, other factors that also contribute to the modulation of mTORC1 activity have been identified. In this review, we discuss the mechanisms of regulation of mTORC1 activity by particular amino acids.

DOI： 10.1186/s12929-020-00679-2

ATeam technology for detecting early signs of viral cytopathic effect Open Access

Doysabas, KCC; Oba, M; Ishibashi, T; Shibata, H; Takemae, H; Shimoda, H; Tarigan, R; Mizutani, T; Iida, A; Hondo, E

JOURNAL OF VETERINARY MEDICAL SCIENCE 82 巻 ( 3 ) 頁： 387 - 393 2020年3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：公益社団法人日本獣医学会

Adenosine 5’-triphosphate (ATP), the major energy currency of the cell, is involved in many cellular processes, including the viral life cycle, and can be used as an indicator of early signs of cytopathic effect (CPE). In this study, we demonstrated that CPE can be analyzed using an FRET-based ATP probe named ATP indicator based on Epsilon subunit for Analytical Measurements (ATeam). The results revealed that as early as 3 hr, the virus infected cells showed a significantly different Venus/cyan fluorescent protein (CFP) ratio compared to the mock-infected cells. The ATeam technology is therefore useful to determine the early signs of ATP-based CPE as early as 3 hr without morphology-based CPE by light microscopy, and enables high throughput determination of the presence of microorganisms in neglected samples stored in laboratories.

DOI： 10.1292/jvms.20-0021

SH3YL1 cooperates with ESCRT-I in the sorting and degradation of the EGF receptor 査読有り国際誌 Open Access

Hasegawa, J; Jebri, I; Yamamoto, H; Tsujita, K; Tokuda, E; Shibata, H; Maki, M; Itoh, T

JOURNAL OF CELL SCIENCE 132 巻 ( 19 ) 2019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：Journal of Cell Science

Ubiquitinated membrane proteins such as epidermal growth factor receptor (EGFR) are delivered to early endosomes and then sorted to lysosomes via multivesicular bodies (MVBs) for degradation. The regulatory mechanismunderlying formation of intralumenal vesicles en route to generation of MVBs is not fully understood. In this study, we found that SH3YL1, a phosphoinositide-binding protein, had a vesicular localization pattern overlapping with internalized EGF in endosomes in the degradative pathway. Deficiency of SH3YL1 prevents EGF trafficking from early to late endosomes and inhibits degradation of EGFR. Moreover, we show that SH3YL1 mediates EGFR sorting intoMVBs in a manner dependent on its C-terminal SH3 domain, which is necessary for the interaction with an ESCRT-I component, Vps37B. Taken together, our observations reveal an indispensable role of SH3YL1 in MVB sorting and EGFR degradation mediated by ESCRT complexes.

DOI： 10.1242/jcs.229179

カルシウム依存的相互作用因子から探るpenta-EF-handファミリーの機能査読有り

牧正敏, 高原照直, 柴田秀樹

生化学 91 巻 ( 2 ) 頁： 191 - 209 2019年4月

記述言語：日本語出版者・発行元：公益社団法人日本生化学会

典型的カルパイン分子中に見いだされたpenta-EF-hand（PEF）ドメインは，EF-handを連続して五つ持ち，C末端のEF5どうしが一対になって二量体を形成する．PEFファミリーには，sorcinやALG-2のように触媒ドメインを持たないメンバーが存在し，さまざまなタンパク質と相互作用してその機能を発揮する．動物細胞においてALG-2二量体はカルシウム依存性分子アダプターとして働き，異なる分子どうしの連結や標的複合体を安定化をする．ESCRTシステムにおける初期補助因子としての役割や，小胞体–ゴルジ体間における小胞輸送調節が注目されている．一方，PEFドメインを持たない非典型的カルパインであるcalpain-7は，N末端にMITドメインを持ち，ESCRT因子と相互作用して活性化され，エンドソーム・リソソーム経路で働いている．

DOI： 10.14952/seikagaku.2019.910191

カルシウム依存的相互作用因子から探るpenta-EF-handファミリーの機能

牧正敏, 高原照直, 柴田秀樹

生化学 91 巻 ( 2 ) 頁： 191 - 209 2019年4月

Adaptor functions of the Ca<SUP>2+</SUP>-binding protein ALG-2 in protein transport from the endoplasmic reticulum 招待有り査読有り国際誌 Open Access

Shibata, H

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 83 巻 ( 1 ) 頁： 20 - 32 2019年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioscience, Biotechnology and Biochemistry

Apoptosis-linked gene 2 (ALG-2) is a Ca2+-binding protein with five repetitive EF-hand motifs, named penta-EF-hand (PEF) domain. It interacts with various target proteins and functions as a Ca2+-dependent adaptor in diverse cellular activities. In the cytoplasm, ALG-2 is predominantly localized to a specialized region of the endoplasmic reticulum (ER), called the ER exit site (ERES), through its interaction with Sec31A. Sec31A is an outer coat protein of coat protein complex II (COPII) and is recruited from the cytosol to the ERES to form COPII-coated transport vesicles. I will overview current knowledge of the physiological significance of ALG-2 in regulating ERES localization of Sec31A and the following adaptor functions of ALG-2, including bridging Sec31A and annexin A11 to stabilize Sec31A at the ERES, polymerizing the Trk-fused gene (TFG) product, and linking MAPK1-interacting and spindle stabilizing (MISS)like (MISSL) and microtubule-associated protein 1B (MAP1B) to promote anterograde transport from the ER.

DOI： 10.1080/09168451.2018.1525274

High Sensitive Quantitative Binding Assays Using a Nanoluciferase-Fused Probe for Analysis of ALG-2-Interacting Proteins 査読有り国際誌

Zhang, W; Matsuo, R; Takahara, T; Shibata, H; Maki, M

CALCIUM-BINDING PROTEINS OF THE EF-HAND SUPERFAMILY 1929 巻頁： 501 - 516 2019年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Methods in Molecular Biology

Many non-catalytic cellular proteins exert biological functions by formation of stable or transient complexes with other proteins. Analysis of the signal-induced physical interactions is important to understand their physiological roles in cells. Here we describe a biochemical method for assessing the binding of ALG-2 (gene name, PDCD6) to its target proteins that are immunoprecipitated from cell lysates. Application of nanoluciferase (Nluc)-fused ALG-2 enables a rapid quantitative evaluation of Ca 2+ -dependent interactions of target proteins with ALG-2 in vitro binding assays.

DOI： 10.1007/978-1-4939-9030-6_31

Cellular Ca<SUP>2+</SUP>-Responding Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-palindromic NFAT-Response Elements 査読有り国際誌

Zhang, W; Takahara, T; Achiha, T; Shibata, H; Maki, M

CALCIUM-BINDING PROTEINS OF THE EF-HAND SUPERFAMILY 1929 巻頁： 95 - 109 2019年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Methods in Molecular Biology

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca 2+ elevation. However, Ca 2+ mobilization agent (e.g., ionomycin) alone is not adequate to activate the currently often employed reporter gene that contains the NFAT-RE found in the IL2 promoter. In addition to activation of NFAT through the Ca 2+ -calmodulin/calcineurin pathway, activation of AP-1 as a partner transcription factor is essential for the IL2-based NFAT-RE system. Here, we describe a detailed method for the recently developed new reporter gene system containing the NFAT-RE from the IL8 promoter. This system enables us to monitor endpoint effects of Ca 2+ -mobilizing agonists independent of AP-1 activation.

DOI： 10.1007/978-1-4939-9030-6_7

Topics from special edition 小胞体からの分子輸送におけるALG-2の役割

柴田秀樹, 高原照直, 京卓志, 牧正敏

細胞 50 巻 ( 8 ) 頁： 441 - 445 2018年7月

A microtubule-associated protein MAP1B binds to and regulates localization of a calcium-binding protein ALG-2 査読有り国際誌

Takahara, T; Arai, Y; Kono, Y; Shibata, H; Maki, M

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 497 巻 ( 2 ) 頁： 492 - 498 2018年3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：Biochemical and Biophysical Research Communications

MAP1B (microtubule-associated protein 1B) binds to microtubules and regulates microtubule dynamics. Previously, we showed calcium-dependent interaction between MAP1B and a calcium-binding protein ALG-2 (apoptosis-linked gene 2), which is involved in regulation of the protein secretion pathway. Although ALG-2 generally binds to proteins through two consensus binding motifs such as ABM-1 and ABM-2, the absence of these motifs in MAP1B suggests a unique binding mode between MAP1B and ALG-2. Here, we identified the region of mouse MAP1B responsible for binding to ALG-2, and found point mutations that abrogated binding of MAP1B to ALG-2. Furthermore, interaction between MAP1B and ALG-2 selectively prevented ALG-2 from binding to proteins with ABM-2 such as Sec31A, suggesting competition between MAP1B and ABM-2-containing proteins for binding to ALG-2. Consistently, in MAP1B knockout cells, co-localization of ALG-2 with Sec31A was increased. Moreover, overexpression of wild-type MAP1B, but not the MAP1B mutant defective in ALG-2 binding, altered localizations of ALG-2 and Sec31A into dispersed distributions, suggesting that MAP1B regulates localizations of ALG-2 and Sec31A in the cells. Finally, we found two cancer-associated mutations of human MAP1B located near ALG-2 binding sites. The introduction of the corresponding mutations in mouse MAP1B dramatically reduced the binding ability to ALG-2. Thus, these results suggest that MAP1B plays a role in regulation of ALG-2 and Sec31A localizations, and that dysregulation of calcium-dependent binding of ALG-2 to MAP1B might influence pathological conditions such as cancers.

DOI： 10.1016/j.bbrc.2018.02.048

Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca<SUP>2+</SUP> Mobilization 査読有り国際誌 Open Access

Zhang, W; Takahara, T; Achiha, T; Shibata, H; Maki, M

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 19 巻 ( 2 ) 2018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：International Journal of Molecular Sciences

NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+-response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca2+-induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization. Ionomycin treatment enhanced the relative luciferase activity (RLA), which was suppressed by calcineurin inhibitors. HEK293 cells that stably express human STIM1 and Orai1, components of the store-operated calcium entry (SOCE) machinery, gave a much higher RLA by stimulation with thapsigargin, an inhibitor of sarcoplasmic/endoplamic reticulum Ca2+-ATPase (SERCA). HEK293 cells deficient in a penta-EF-hand Ca2+-binding protein ALG-2 showed a higher RLA value than the parental cells by stimulation with an acetylcholine receptor agonist carbachol. The novel reporter gene system is found to be useful for applications to cell signaling research to monitor biological endpoint effects of cellular Ca2+ mobilization.

DOI： 10.3390/ijms19020605

小胞体からの分子輸送におけるALG-2の役割

柴田秀樹

月刊「細胞」 50 巻頁： 441 - 445 2018年

The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins. 査読有り

Terunao Takahara, Kuniko Inoue, Yumika Arai, Keiko Kuwata, Hideki Shibata, Masatoshi Maki

Journal of Biological Chemistry 292 巻 ( 41 ) 頁： 17057-17072 2017年10月

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