Faculty Profiles - SHIBATA, Hideki

Amino Acid-Mediated Intracellular Ca2+ Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca2+/Calmodulin. Reviewed International journal Open Access

Yuna Amemiya, Nao Nakamura, Nao Ikeda, Risa Sugiyama, Chiaki Ishii, Masatoshi Maki, Hideki Shibata, Terunao Takahara

International journal of molecular sciences Vol. 22 ( 13 ) 2021.7

Language：English Publishing type：Research paper (scientific journal)

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 Reviewed Open Access

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol. 22 ( 3 ) page： 1 - 25 2021.2

The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination. Reviewed International journal Open Access

Wei Zhang, Ayaka Muramatsu, Rina Matsuo, Naoki Teranishi, Yui Kahara, Terunao Takahara, Hideki Shibata, Masatoshi Maki

International journal of molecular sciences Vol. 21 ( 17 ) page： 1 - 21 2020.9

Language：English Publishing type：Research paper (scientific journal)

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. Invited Reviewed International journal Open Access

Terunao Takahara, Yuna Amemiya, Risa Sugiyama, Masatoshi Maki, Hideki Shibata

Journal of biomedical science Vol. 27 ( 1 ) page： 87 - 87 2020.8

Language：English Publishing type：Research paper (scientific journal)

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 Karla Cristine C., OBA Mami, ISHIBASHI Tomoki, SHIBATA Hideki, TAKEMAE Hitoshi, SHIMODA Hiroshi, TARIGAN Ronald, MIZUTANI Tetsuya, IIDA Atsuo, HONDO Eiichi

Journal of Veterinary Medical Science Vol. 82 ( 3 ) page： 387 - 393 2020.3

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE SOCIETY OF VETERINARY SCIENCE

<p>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.</p>

DOI： 10.1292/jvms.20-0021

SH3YL1 cooperates with ESCRT-I in the sorting and degradation of the EGF receptor. Reviewed International journal Open Access

Junya Hasegawa, Imen Jebri, Hikaru Yamamoto, Kazuya Tsujita, Emi Tokuda, Hideki Shibata, Masatoshi Maki, Toshiki Itoh

Journal of cell science Vol. 132 ( 19 ) 2019.10

Language：English Publishing type：Research paper (scientific journal)

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 mechanism underlying 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 into MVBs 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

Exploring functions of the penta-EF-hand family by searching for calcium-dependent interacting factors Reviewed

Maki Masatoshi, Takahara Terunao, Shibata Hideki

Vol. 91 ( 2 ) page： 191 - 209 2019.4

Exploring functions of the penta-EF-hand family by searching for calcium-dependent interacting factors

Vol. 91 ( 2 ) page： 191 - 209 2019.4

Adaptor functions of the Ca2+-binding protein ALG-2 in protein transport from the endoplasmic reticulum. Invited Reviewed International journal Open Access

Hideki Shibata

Bioscience, biotechnology, and biochemistry Vol. 83 ( 1 ) page： 20 - 32 2019

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal

Wei Zhang, Rina Matsuo, Terunao Takahara, Hideki Shibata, Masatoshi Maki

Methods in molecular biology (Clifton, N.J.) Vol. 1929 page： 501 - 516 2019

Language：English Publishing type：Research paper (scientific journal)

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 Ca2+-dependent interactions of target proteins with ALG-2 in vitro binding assays.

DOI： 10.1007/978-1-4939-9030-6_31

Cellular Ca2+-Responding Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-palindromic NFAT-Response Elements. Reviewed International journal

Wei Zhang, Terunao Takahara, Takuya Achiha, Hideki Shibata, Masatoshi Maki

Methods in molecular biology (Clifton, N.J.) Vol. 1929 page： 95 - 109 2019

Language：English Publishing type：Research paper (scientific journal)

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca2+ elevation. However, Ca2+ 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 Ca2+-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 Ca2+-mobilizing agonists independent of AP-1 activation.

DOI： 10.1007/978-1-4939-9030-6_7

Roles of ALG-2 in molecular transport from the endoplasmic reticulum

Vol. 50 ( 8 ) page： 441 - 445 2018.7

A microtubule-associated protein MAP1B binds to and regulates localization of a calcium-binding protein ALG-2. Reviewed International journal

Terunao Takahara, Yumika Arai, Yuta Kono, Hideki Shibata, Masatoshi Maki

Biochemical and biophysical research communications Vol. 497 ( 2 ) page： 492 - 498 2018.3

Language：English Publishing type：Research paper (scientific journal)

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 Ca2+ Mobilization. Reviewed International journal Open Access

Wei Zhang, Terunao Takahara, Takuya Achiha, Hideki Shibata, Masatoshi Maki

International journal of molecular sciences Vol. 19 ( 2 ) 2018.2

Language：English Publishing type：Research paper (scientific journal)

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」 Vol. 50 page： 441 - 445 2018

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

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

Journal of Biological Chemistry Vol. 292 ( 41 ) page： 17057-17072 2017.10

The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins. Reviewed International journal Open Access

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

The Journal of biological chemistry Vol. 292 ( 41 ) page： 17057 - 17072 2017.10

Language：English Publishing type：Research paper (scientific journal)

Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that MAPK1-interacting and spindle-stabilizing (MISS)-like (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of secreted alkaline phosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B.

DOI： 10.1074/jbc.M117.800201

Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis Reviewed Open Access

Bradley N. Smith, Simon D. Topp, Claudia Fallini, Hideki Shibata, Han-Jou Chen, Claire Troakes, Andrew King, Nicola Ticozzi, Kevin P. Kenna, Athina Soragia-Gkazi, Jack W. Miller, Akane Sato, Diana Marques Dias, Maryangel Jeon, Caroline Vance, Chun Hao Wong, Martina de Majo, Wejdan Kattuah, Jacqueline C. Mitchell, Emma L. Scotter, Nicholas W. Parkin, Peter C. Sapp, Matthew Nolan, Peter J. Nestor, Michael Simpson, Michael Weale, Monkel Lek, Frank Baas, J. M. Vianney de Jong, Anneloor L. M. A. ten Asbroek, Alberto Garcia Redondo, Jesus Esteban-Perez, Cinzia Tiloca, Federico Verde, Stefano Duga, Nigel Leigh, Hardev Pall, Karen E. Morrison, Ammar Al-Chalabi, Pamela J. Shaw, Janine Kirby, Martin R. Turner, Kevin Talbot, Orla Hardiman, Jonathan D. Glass, Jacqueline de Belleroche, Masatoshi Maki, Stephen E. Moss, Christopher Miller, Cinzia Gellera, Antonia Ratti, Safa Al-Sarraj, Robert H. Brown, Vincenzo Silani, John E. Landers, Christopher E. Shaw

SCIENCE TRANSLATIONAL MEDICINE Vol. 9 ( 388 ) page： eaad9157 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC ADVANCEMENT SCIENCE

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

DOI： 10.1126/scitranslmed.aad9157

The calcium-binding protein ALG-2 promotes endoplasmic reticulum exit site localization and polymerization of Trk-fused gene (TFG) protein. Reviewed International journal Open Access

Takashi Kanadome, Hideki Shibata, Keiko Kuwata, Terunao Takahara, Masatoshi Maki

The FEBS journal Vol. 284 ( 1 ) page： 56 - 76 2017.1

Language：English Publishing type：Research paper (scientific journal)

Apoptosis-linked gene 2 (ALG-2), which is a gene product of PDCD6, is a 22-kDa Ca2+ -binding protein. Accumulating evidence points to a role for ALG-2 as a Ca2+ -responsive adaptor protein. On binding to Ca2+ , ALG-2 undergoes a conformational change that facilitates its interaction with various proteins. It also forms a homodimer and heterodimer with peflin, a paralog of ALG-2. However, the differences in cellular roles for the ALG-2 homodimer and ALG-2/peflin heterodimer are unclear. In the present study, we found that Trk-fused gene (TFG) protein interacted with the ALG-2 homodimer. Immunostaining analysis revealed that TFG and ALG-2 partially overlapped at endoplasmic reticulum exit sites (ERES), a platform for COPII-mediated protein transport from the endoplasmic reticulum. Time-lapse live-cell imaging demonstrated that both green fluorescent protein-fused TFG and mCherry-fused ALG-2 are recruited to ERES after thapsigargin treatment, which raises intracellular Ca2+ levels. Furthermore, overexpression of ALG-2 induced the accumulation of TFG at ERES. TFG has an ALG-2-binding motif and deletion of the motif decreased TFG binding to ALG-2 and shortened its half-life at ERES, suggesting a critical role for ALG-2 in retaining TFG at ERES. We also demonstrated, by in vitro cross-linking assays, that ALG-2 promoted the polymerization of TFG in a Ca2+ -dependent manner. Collectively, the results suggest that ALG-2 acts as a Ca2+ -sensitive adaptor to concentrate and polymerize TFG at ERES, supporting a potential role for ALG-2 in COPII-dependent trafficking from the endoplasmic reticulum.

DOI： 10.1111/febs.13949