Elsevier BV Acta Medica Okayama 0143-4160 135 2026 Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca2+/calmodulin 103134 EN Naoyuki Washida Moe Kataoka Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Anna R. Brun Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Uryu Takezaki Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ko Hijikawa Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Haruki Yamauchi Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ryo Morishita CellFree Sciences Co., Ltd. Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University We conducted a genome-wide calmodulin (CaM) interaction screening of 462 GST-fused human protein kinases to identify novel CaM-dependent protein kinases (CaMKs). In addition to known CaMKs, including myosin light chain kinases, CaMK2γ, and death-associated kinase 2, we identified the brain-specific protein kinase 2 (BRSK2, also known as SAD-A) as a novel CaM interactant. Proximity biotinylation and CaM–sepharose chromatography assays revealed that rat BRSK isoforms (BRSK1/2) interact with CaM in a Ca2+-dependent manner in vitro. We found that CaM suppresses the activation-loop phosphorylation of BRSK1 (at Thr189) and BRSK2 (at Thr175) by liver kinase B1 (LKB1), an activating kinase, in a Ca2+-dependent manner (IC50 of ∼7 µM), thereby inhibiting BRSK activation. LKB1-catalyzed phosphorylation of the catalytic domain mutant of BRSK1 (residues 1–294) at Thr189 was suppressed by the addition of Ca2+/CaM, consistent with direct CaM binding of the kinase domain, as well as wild-type BRSK1. We confirmed that the LKB1 activity was not directly suppressed by Ca2+/CaM, supporting the hypothesis that the direct interaction of Ca2+/CaM with the kinase domain blocks the phosphorylation/activation of BRSK1/2 by LKB1. The kinase activity and PP2Cα-catalyzed dephosphorylation of LKB1-phosphorylated BRSK1 were not altered by Ca2+/CaM, although it was demonstrated to bind to Ca2+/CaM like that of unphosphorylated BRSK1. This unrecognized mechanism of BRSK1/2 regulation, involving the direct role of Ca2+/CaM binding, which inhibits phosphorylation/activation by LKB1, may open a new Ca2+ signal transduction pathway in neurons. No potential conflict of interest relevant to this article was reported.

岡山大学大学院ヘルスシステム統合科学研究科 Acta Medica Okayama 2436-3227 6 2026 2025 年度における「先進病院実習」の取り組み 49 57 EN Mizuki MORITA Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki MAGARI Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Jin Wang Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toyohiko WATANABE Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masataka OITA Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Nahoko HARADA Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Keisuke SHISHIDO Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University 10.18926/interdisciplinary/70330 No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 0006-2960 64 20 2025 Characterization of Autonomous and Ca2+/Calmodulin-Dependent Activities of CaMKK Isoforms In Vitro and in Mouse Tissues 4309 4317 EN Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yerun Chen Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Teruhiko Ishikawa Department of Science Education, Graduate School of Education, Okayama University Hiroyuki Sakagami Department of Anatomy, Kitasato University School of Medicine Futoshi Suizu Clinical Examination Department, Kagawa Prefectural University of Health Sciences Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ca2+/CaM-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream kinases, including CaMKI, CaMKIV, PKB, and AMPK, regulating various cellular functions such as neuronal morphogenesis, metabolic control, and pathophysiological pathways, such as cancer progression. CaMKKα/1 is tightly regulated by an autoinhibitory mechanism. CaMKKβ/2 activity is highly Ca2+/CaM-independent (autonomous activity) in vitro and Ca2+/CaM-dependent in cultured cells. Whether these two activity states of CaMKKβ/2 exist in vivo and the detailed regulatory mechanisms for the transition of both activity states remain unclear due to the difficulty in distinguishing the two activity states. In this study, we detected Ca2+-dependent and autonomous CaMKK activity in HeLa cells and successfully separated both activity states of CaMKKβ/2 in mouse brain and testis extracts using a recently developed CaMKK inhibitor (TIM-063)-coupled sepharose, which binds to the catalytic domain in the active state but not in the autoinhibited state. Furthermore, lambda protein phosphatase treatment converted the Ca2+/CaM-dependent form to the autonomous form of CaMKKβ/2, which was not affected by Ala mutation of Ser128, Ser132, and Ser136. The two activity forms of CaMKKβ/2 had equivalent Ca2+/CaM-binding ability. The findings demonstrate the presence of autonomous and Ca2+/CaM-dependent forms of CaMKKβ/2 independently in mouse tissues and cultured cells. The transition of these states of CaMKKβ/2 may be dynamically regulated by the phosphorylation/dephosphorylation of serine residues in the N-terminal regulatory domain. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0014-5793 599 13 2025 Characterization of molecular mechanisms of CaMKKα/1 oligomerization 1914 1924 EN Shun Uenoyama Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hayato Nitta Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Futoshi Suizu Department of Medical Technology, Kagawa Prefectural University of Health Sciences Hiroshi Tokumitsu Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is an activating kinase for calcium/calmodulin-dependent protein kinase type 1 (CaMKI), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), RAC-alpha serine/threonine-protein kinase (PKB), and AMP-activated protein kinase (AMPK) that has been reported to form an active oligomer in cells. Glutathione S-transferase (GST) pulldown assay from the extracts of COS-7 cells expressing GST- and His6-CaMKKα/1 mutants showed that the C-terminal region containing the autoinhibitory and calmodulin (CaM)-binding sequence (residues 438–463) is required for CaMKKα/1 homo-oligomerization. This was confirmed by the fact that the GST-CaMKKα/1 C-terminal domain (residues 435–505) directly interacted with EGFP-CaMKKα/1 residues 435–505 as well as with wild-type CaMKKα/1. Notably, once oligomerized in cells, CaMKKα/1 is neither exchangeable between the oligomeric complexes nor dissociated by Ca2+/CaM binding. These results support stable oligomerization of CaMKK in the cells by intermolecular self-association of its C-terminal region containing a regulatory domain. No potential conflict of interest relevant to this article was reported.

岡山大学大学院ヘルスシステム統合科学研究科 Acta Medica Okayama 2436-3227 5 2025 イノベーションループの実践：2024年度先進病院実習報告と未来課題 39 45 EN Nahoko HARADA Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Satoshi TAKAHASHI Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Tomoaki MORI Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Haruka HIKASA Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Keisuke SHISHIDO Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Masaki MAGARI Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Toyohiko WATANABE Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Jin Wang Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university Mizuki MORITA Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university 10.18926/interdisciplinary/68625 No potential conflict of interest relevant to this article was reported.

American Association for Cancer Research (AACR) Acta Medica Okayama 0008-5472 85 6 2025 Myeloid Cells Induce Infiltration and Activation of B Cells and CD4+ T Follicular Helper Cells to Sensitize Brain Metastases to Combination Immunotherapy 1082 1096 EN Toshifumi Ninomiya Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naoya Kemmotsu Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Fumiaki Mukohara Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ai Miyamoto Medical Protein Engineering, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Youki Ueda Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takamasa Ishino Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Joji Nagasaki Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomohiro Fujiwara Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hidetaka Yamamoto Department of Pathology and Oncology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hidetoshi Hayashi Department of Medical Oncology, Kindai University Faculty of Medicine Kota Tachibana Department of Dermatology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Joji Ishida Department of Neurological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoshihiro Otani Department of Neurological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shota Tanaka Department of Neurological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shinichi Toyooka Department of General Thoracic Surgery, Breast and Endocrinological Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Science, Okayama University Isamu Okamoto Department of Respiratory Medicine, Graduate School of Medical Sciences, Kyushu University Yosuke Togashi Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Brain metastasis is a poor prognostic factor in patients with cancer. Despite showing efficacy in many extracranial tumors, immunotherapy with anti–PD-1 mAb or anti–CTLA4 mAb seems to be less effective against intracranial tumors. Promisingly, recent clinical studies have reported that combination therapy with anti–PD-1 and anti–CTLA4 mAbs has a potent antitumor effect on brain metastasis, highlighting the need to elucidate the detailed mechanisms controlling the intracranial tumor microenvironment (TME) to develop effective immunotherapeutic strategies. In this study, we analyzed the tumor-infiltrating lymphocytes in murine models of brain metastasis that responded to anti–CTLA4 and anti–PD-1 mAbs. Activated CD4+ T follicular helper (TFH) cells with high CTLA4 expression characteristically infiltrated the intracranial TME, which were activated by combination anti–CTLA4 and anti–PD-1 treatment. The loss of TFH cells suppressed the additive effect of CTLA4 blockade on anti–PD-1 mAb. B-cell–activating factor belonging to the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) produced by abundant myeloid cells, particularly CD80hiCD206lo proinflammatory M1-like macrophages, in the intracranial TME induced B-cell and TFH-cell infiltration and activation. Furthermore, the intracranial TME of patients with non–small cell lung cancer featured TFH- and B-cell infiltration as tertiary lymphoid structures. Together, these findings provide insights into the immune cell cross-talk in the intracranial TME that facilitates an additive antitumor effect of CTLA4 blockade with anti–PD-1 treatment, supporting the potential of a combination immunotherapeutic strategy for brain metastases.<br> Significance: B-cell and CD4+ T follicular helper cell activation via BAFF/APRIL from abundant myeloid cells in the intracranial tumor microenvironment enables a combinatorial effect of CTLA4 and PD-1 blockade in brain metastases. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 14 1 2024 Development of a novel AAK1 inhibitor via Kinobeads-based screening 6723 EN Akari Yoshida Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Fumiya Matsumoto Department of Science Education, Graduate School of Education, Okayama University Tomoyuki Miyagawa Department of Science Education, Graduate School of Education, Okayama University Rei Okino Department of Science Education, Graduate School of Education, Okayama University Yumeya Ikeda Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Natsume Tada Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Gotoh Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoya Hatano Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ryo Morishita CellFree Sciences Co. Ltd Ayano Satoh Organelle Systems Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yukinari Sunatsuki Graduate School of Natural Science and Technology, Okayama University Ulf J. Nilsson Department of Chemistry, Lund University Teruhiko Ishikawa Department of Science Education, Graduate School of Education, Okayama University Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKK alpha/1 and beta/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 mu M), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 mu M) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 mu M), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 mu M TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1742-464X 289 19 2022 Substrate recognition by Arg/Pro‐rich insert domain in calcium/calmodulin‐dependent protein kinase kinase for target protein kinases 5971 5984 EN Riku Kaneshige Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yuhei Harada Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Issei Kawamata Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoki Kanayama Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoya Hatano Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hiroyuki Sakagami Department of Anatomy, Kitasato University School of Medicine Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+-signaling pathways. Mammalian cells expressing CaMKK alpha and CaMKK beta lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPK alpha, CaMKI alpha, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKI alpha and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKK alpha and CaMKK beta inserted between kinase subdomains II and III acquired CaMKI alpha and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKI alpha at Thr177, HA-CaMKIV at Thr196, and HA-AMPK alpha at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKK alpha and CaMKK beta interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 0006-2960 61 7 2022 Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063 545 553 EN Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Taisei Okumura Department of Science Education, Graduate School of Education, Okayama University Yuna Τabuchi Department of Science Education, Graduate School of Education, Okayama University Tomoyuki Miyagawa Department of Science Education, Graduate School of Education, Okayama University Naoki Kanayama Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Naoya Hatano Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Hiroyuki Sakagami Department of Anatomy, Kitasato University School of Medicine Futoshi Suizu Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University Teruhiko Ishikawa Department of Science Education, Graduate School of Education, Okayama University Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), a Ca2+/CaM-dependent enzyme that phosphorylates and activates multifunctional kinases, including CaMKI, CaMKIV, protein kinase B/Akt, and 5'AMP-activated protein kinase, is involved in various Ca2+-signaling pathways in cells. Recently, we developed an ATP competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo-[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, Ohtsuka et al. Biochemistry 2020, 59, 1701-1710). To gain mechanistic insights into the interaction of CaMKK with TIM-063, we prepared TIM-063-coupled sepharose (TIM-127-sepharose) for association/dissociation analysis of the enzyme/inhibitor complex. CaMKK alpha/beta in transfected COS-7 cells and in mouse brain extracts specifically bound to TIM-127-sepharose and dissociated following the addition of TIM-063 in a manner similar to that of recombinant GST-CaMKK alpha/beta, which could bind to TIM-127sepharose in a Ca2+/CaM-dependent fashion and dissociate from the sepharose following the addition of TIM-063 in a dose dependent manner. In contrast to GST-CaMKK alpha, GST-CaMKK beta was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST-CaMKK beta without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKK alpha prevented the inhibitor from interacting with the catalytic domain as the GST-CaMKK alpha mutant (residues 126-434) lacking the regulatory domain (residues 438-463) interacted with TIM-127-sepharose regardless of the presence or absence of Ca2+/CaM. Furthermore, CaMKK alpha bound to TIM-127-sepharose in the presence of Ca2+/ CaM completely dissociated from TIM-127-sepharose following the addition of excess EGTA. These results indicated that TIM-063 interacted with and inhibited CaMKK in its active state but not in its autoinhibited state and that this interaction is likely reversible, depending on the concentration of intracellular Ca2+. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0006-291X 587 2022 Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase 160 165 EN Yusei Fukumoto Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yuhei Harada Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Satomi Ohtsuka Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoki Kanayama Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoya Hatano Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hiroyuki Sakagami Department of Anatomy, Kitasato University School of Medicine Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ca2+/calmodulin-dependent protein kinase kinases (CaMKKα and β) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His–CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG–CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST–CaMKKα/His–CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Nisepharose and was observed in a Ca2+/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG–CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2218-273X 11 4 2021 Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target 510 EN Maho Yamamoto Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Rina Kondo Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Haruka Hozumi Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Seita Doi Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Miwako Denda Cell Free Sciences Co., Ltd. Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoki Kanayama Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoya Hatano Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ryo Morishita Cell Free Sciences Co., Ltd. Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-Delta C) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation. No potential conflict of interest relevant to this article was reported.

Faculty of Engineering, Okayama University Acta Medica Okayama 0475-0071 36 2 2002 Regulatory Role for Complement Receptors (CD21/CD35) in the Recombination Activating Gene Expression in Mouse Peripheral B Cells 51 60 EN Masaki Hikida Masaki Magari Yasunori Nakayama Naoki Kanayama Hitoshi Ohmori 10.18926/47025 A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect in immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. Consistent with these observations, RAG-2 expression was lower in the draining lymph nodes of the quasi-monoclonal mice when they were immunized with a high-affinity antigen than with a low-affinity one. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells. No potential conflict of interest relevant to this article was reported.

Faculty of Engineering, Okayama University Acta Medica Okayama 0475-0071 38 1-2 2004 Generation of IgM and IgG1 monoclonal antibodies with identical variable regions: comparison of avidity 91 96 EN Naoki Kanayama Kimi Yamakoshi Masaaki Kiyomi Masaki Magari Hitoshi Ohmori 10.18926/46955 Generally, IgM antibodies (Abs) produced in a primary immune response show lower affinity for an inducing antigen (Ag) compared with the corresponding IgG Abs that are major switched isotypes formed in the secondary response. An IgM molecule is a pentamer with 10 Ag-binding sites that will contribute to an increase of avidity for an Ag. To estimate the contribution of the pentameric structure to the avidity of an IgM Ab, we generated IgM and IgG1 monoclonal Abs (mAbs) with identical V regions that are specific for 4-hydroxy-3-nitrophenylacetyl (NP) by in vitro class switching of B cells followed by the cell fusion with a mouse myeloma cell line. Compared with an anti-NP IgG1 mAb, the corresponding IgM mAb showed much higher avidity for NP-conjugated bovine serum albumin, which was drastically reduced after being dissociated into monomers. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2002 Studies on the biological role of recombination activating gene products in the generation of new B cell repertoire in the periphery EN No potential conflict of interest relevant to this article was reported.