start-ver=1.4 cd-journal=joma no-vol=599 cd-vols= no-issue=13 article-no= start-page=1914 end-page=1924 dt-received= dt-revised= dt-accepted= dt-pub-year=2025 dt-pub=20250525 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Characterization of molecular mechanisms of CaMKKα/1 oligomerization en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=UenoyamaShun en-aut-sei=Uenoyama en-aut-mei=Shun kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NittaHayato en-aut-sei=Nitta en-aut-mei=Hayato kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OhtsukaSatomi en-aut-sei=Ohtsuka en-aut-mei=Satomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=SuizuFutoshi en-aut-sei=Suizu en-aut-mei=Futoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=TokumitsuHiroshi en-aut-sei=Tokumitsu en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University kn-affil= affil-num=3 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Department of Medical Technology, Kagawa Prefectural University of Health Sciences kn-affil= affil-num=6 en-affil= kn-affil= en-keyword=calmodulin kn-keyword=calmodulin en-keyword=calmodulin-kinase cascade kn-keyword=calmodulin-kinase cascade en-keyword=CaMKKa/ kn-keyword=CaMKKa/ en-keyword=oligomerization kn-keyword=oligomerization en-keyword=protein–protein interaction kn-keyword=protein–protein interaction en-keyword=regulatory domain kn-keyword=regulatory domain END start-ver=1.4 cd-journal=joma no-vol=5 cd-vols= no-issue= article-no= start-page=39 end-page=45 dt-received= dt-revised= dt-accepted= dt-pub-year=2025 dt-pub=20250331 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Practicing the Innovation Loop: 2024 Report on Advanced Hospital Practicums and Future Challenges kn-title=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 en-subtitle= kn-subtitle= en-abstract= kn-abstract= en-copyright= kn-copyright= en-aut-name=HARADANahoko en-aut-sei=HARADA en-aut-mei=Nahoko kn-aut-name=原田奈穂子 kn-aut-sei=原田 kn-aut-mei=奈穂子 aut-affil-num=1 ORCID= en-aut-name=TAKAHASHISatoshi en-aut-sei=TAKAHASHI en-aut-mei=Satoshi kn-aut-name=髙橋智 kn-aut-sei=髙橋 kn-aut-mei=智 aut-affil-num=2 ORCID= en-aut-name=MORITomoaki en-aut-sei=MORI en-aut-mei=Tomoaki kn-aut-name=森友明 kn-aut-sei=森 kn-aut-mei=友明 aut-affil-num=3 ORCID= en-aut-name=HIKASAHaruka en-aut-sei=HIKASA en-aut-mei=Haruka kn-aut-name=日笠晴香 kn-aut-sei=日笠 kn-aut-mei=晴香 aut-affil-num=4 ORCID= en-aut-name=SHISHIDOKeisuke en-aut-sei=SHISHIDO en-aut-mei=Keisuke kn-aut-name=宍戸圭介 kn-aut-sei=宍戸 kn-aut-mei=圭介 aut-affil-num=5 ORCID= en-aut-name=MAGARIMasaki en-aut-sei=MAGARI en-aut-mei=Masaki kn-aut-name=曲正樹 kn-aut-sei=曲 kn-aut-mei=正樹 aut-affil-num=6 ORCID= en-aut-name=WATANABEToyohiko en-aut-sei=WATANABE en-aut-mei=Toyohiko kn-aut-name=渡邉豊彦 kn-aut-sei=渡邉 kn-aut-mei=豊彦 aut-affil-num=7 ORCID= en-aut-name=WangJin en-aut-sei=Wang en-aut-mei=Jin kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=MORITAMizuki en-aut-sei=MORITA en-aut-mei=Mizuki kn-aut-name=森田瑞樹 kn-aut-sei=森田 kn-aut-mei=瑞樹 aut-affil-num=9 ORCID= affil-num=1 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=2 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=3 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=4 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=5 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=6 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=7 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=8 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 affil-num=9 en-affil=Academic Field of Interdisciplinary Science and Engineering in Health Systems Health Sciences, Okayama university kn-affil=岡山大学学術研究院ヘルスシステム統合科学学域 END start-ver=1.4 cd-journal=joma no-vol=85 cd-vols= no-issue=6 article-no= start-page=1082 end-page=1096 dt-received= dt-revised= dt-accepted= dt-pub-year=2025 dt-pub=20250314 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Myeloid Cells Induce Infiltration and Activation of B Cells and CD4+ T Follicular Helper Cells to Sensitize Brain Metastases to Combination Immunotherapy en-subtitle= kn-subtitle= en-abstract= kn-abstract=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.
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. en-copyright= kn-copyright= en-aut-name=NinomiyaToshifumi en-aut-sei=Ninomiya en-aut-mei=Toshifumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KemmotsuNaoya en-aut-sei=Kemmotsu en-aut-mei=Naoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MukoharaFumiaki en-aut-sei=Mukohara en-aut-mei=Fumiaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MiyamotoAi en-aut-sei=Miyamoto en-aut-mei=Ai kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=UedaYouki en-aut-sei=Ueda en-aut-mei=Youki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=IshinoTakamasa en-aut-sei=Ishino en-aut-mei=Takamasa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=NagasakiJoji en-aut-sei=Nagasaki en-aut-mei=Joji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=FujiwaraTomohiro en-aut-sei=Fujiwara en-aut-mei=Tomohiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=YamamotoHidetaka en-aut-sei=Yamamoto en-aut-mei=Hidetaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=HayashiHidetoshi en-aut-sei=Hayashi en-aut-mei=Hidetoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=TachibanaKota en-aut-sei=Tachibana en-aut-mei=Kota kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=IshidaJoji en-aut-sei=Ishida en-aut-mei=Joji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=OtaniYoshihiro en-aut-sei=Otani en-aut-mei=Yoshihiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= en-aut-name=TanakaShota en-aut-sei=Tanaka en-aut-mei=Shota kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=15 ORCID= en-aut-name=ToyookaShinichi en-aut-sei=Toyooka en-aut-mei=Shinichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=16 ORCID= en-aut-name=OkamotoIsamu en-aut-sei=Okamoto en-aut-mei=Isamu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=17 ORCID= en-aut-name=TogashiYosuke en-aut-sei=Togashi en-aut-mei=Yosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=18 ORCID= affil-num=1 en-affil=Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Medical Protein Engineering, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=6 en-affil=Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=7 en-affil=Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=8 en-affil=Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=9 en-affil=Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=10 en-affil=Department of Pathology and Oncology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=11 en-affil=Department of Medical Oncology, Kindai University Faculty of Medicine kn-affil= affil-num=12 en-affil=Department of Dermatology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=13 en-affil=Department of Neurological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=14 en-affil=Department of Neurological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=15 en-affil=Department of Neurological Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=16 en-affil=Department of General Thoracic Surgery, Breast and Endocrinological Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Science, Okayama University kn-affil= affil-num=17 en-affil=Department of Respiratory Medicine, Graduate School of Medical Sciences, Kyushu University kn-affil= affil-num=18 en-affil=Department of Tumor Microenvironment, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=14 cd-vols= no-issue=1 article-no= start-page=6723 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2024 dt-pub=20240320 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Development of a novel AAK1 inhibitor via Kinobeads-based screening en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=YoshidaAkari en-aut-sei=Yoshida en-aut-mei=Akari kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=OhtsukaSatomi en-aut-sei=Ohtsuka en-aut-mei=Satomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=MatsumotoFumiya en-aut-sei=Matsumoto en-aut-mei=Fumiya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MiyagawaTomoyuki en-aut-sei=Miyagawa en-aut-mei=Tomoyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=OkinoRei en-aut-sei=Okino en-aut-mei=Rei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=IkedaYumeya en-aut-sei=Ikeda en-aut-mei=Yumeya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=TadaNatsume en-aut-sei=Tada en-aut-mei=Natsume kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=GotohAkira en-aut-sei=Gotoh en-aut-mei=Akira kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=HatanoNaoya en-aut-sei=Hatano en-aut-mei=Naoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=MorishitaRyo en-aut-sei=Morishita en-aut-mei=Ryo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=SatohAyano en-aut-sei=Satoh en-aut-mei=Ayano kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=SunatsukiYukinari en-aut-sei=Sunatsuki en-aut-mei=Yukinari kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=NilssonUlf J. en-aut-sei=Nilsson en-aut-mei=Ulf J. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= en-aut-name=IshikawaTeruhiko en-aut-sei=Ishikawa en-aut-mei=Teruhiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=15 ORCID= en-aut-name=TokumitsuHiroshi en-aut-sei=Tokumitsu en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=16 ORCID= affil-num=1 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=4 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=5 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=6 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=7 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=8 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=9 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=10 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=11 en-affil=CellFree Sciences Co. Ltd kn-affil= affil-num=12 en-affil=Organelle Systems Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=13 en-affil=Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=14 en-affil=Department of Chemistry, Lund University kn-affil= affil-num=15 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=16 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=289 cd-vols= no-issue=19 article-no= start-page=5971 end-page=5984 dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20220517 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Substrate recognition by Arg/Pro‐rich insert domain in calcium/calmodulin‐dependent protein kinase kinase for target protein kinases en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KaneshigeRiku en-aut-sei=Kaneshige en-aut-mei=Riku kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=OhtsukaSatomi en-aut-sei=Ohtsuka en-aut-mei=Satomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HaradaYuhei en-aut-sei=Harada en-aut-mei=Yuhei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KawamataIssei en-aut-sei=Kawamata en-aut-mei=Issei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=HatanoNaoya en-aut-sei=Hatano en-aut-mei=Naoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=SakagamiHiroyuki en-aut-sei=Sakagami en-aut-mei=Hiroyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=TokumitsuHiroshi en-aut-sei=Tokumitsu en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University kn-affil= affil-num=5 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=6 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=7 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=8 en-affil=Department of Anatomy, Kitasato University School of Medicine kn-affil= affil-num=9 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=AMP-activated protein kinase kn-keyword=AMP-activated protein kinase en-keyword=Arg/Pro-rich insert domain (RP-domain) kn-keyword=Arg/Pro-rich insert domain (RP-domain) en-keyword=calcium/calmodulin-dependent protein kinase kn-keyword=calcium/calmodulin-dependent protein kinase en-keyword=calcium/calmodulin-dependent protein kinase kinase kn-keyword=calcium/calmodulin-dependent protein kinase kinase en-keyword=substrate recognition kn-keyword=substrate recognition END start-ver=1.4 cd-journal=joma no-vol=61 cd-vols= no-issue=7 article-no= start-page=545 end-page=553 dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20220311 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063 en-subtitle= kn-subtitle= en-abstract= kn-abstract=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+. en-copyright= kn-copyright= en-aut-name=OhtsukaSatomi en-aut-sei=Ohtsuka en-aut-mei=Satomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=OkumuraTaisei en-aut-sei=Okumura en-aut-mei=Taisei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=ΤabuchiYuna en-aut-sei=Τabuchi en-aut-mei=Yuna kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MiyagawaTomoyuki en-aut-sei=Miyagawa en-aut-mei=Tomoyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=HatanoNaoya en-aut-sei=Hatano en-aut-mei=Naoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=SakagamiHiroyuki en-aut-sei=Sakagami en-aut-mei=Hiroyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=SuizuFutoshi en-aut-sei=Suizu en-aut-mei=Futoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=IshikawaTeruhiko en-aut-sei=Ishikawa en-aut-mei=Teruhiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=TokumitsuHiroshi en-aut-sei=Tokumitsu en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= affil-num=1 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=3 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=4 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=5 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University kn-affil= affil-num=6 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University kn-affil= affil-num=7 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University kn-affil= affil-num=8 en-affil=Department of Anatomy, Kitasato University School of Medicine kn-affil= affil-num=9 en-affil=Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University kn-affil= affil-num=10 en-affil=Department of Science Education, Graduate School of Education, Okayama University kn-affil= affil-num=11 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=587 cd-vols= no-issue= article-no= start-page=160 end-page=165 dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20221 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=FukumotoYusei en-aut-sei=Fukumoto en-aut-mei=Yusei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=HaradaYuhei en-aut-sei=Harada en-aut-mei=Yuhei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OhtsukaSatomi en-aut-sei=Ohtsuka en-aut-mei=Satomi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=HatanoNaoya en-aut-sei=Hatano en-aut-mei=Naoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SakagamiHiroyuki en-aut-sei=Sakagami en-aut-mei=Hiroyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=TokumitsuHiroshi en-aut-sei=Tokumitsu en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=6 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=7 en-affil=Department of Anatomy, Kitasato University School of Medicine kn-affil= affil-num=8 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=CaMKK kn-keyword=CaMKK en-keyword=oligomerization kn-keyword=oligomerization en-keyword=Ca2+-signaling kn-keyword=Ca2+-signaling en-keyword=phosphorylation kn-keyword=phosphorylation en-keyword=CaM kinase cascade kn-keyword=CaM kinase cascade END start-ver=1.4 cd-journal=joma no-vol=11 cd-vols= no-issue=4 article-no= start-page=510 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210330 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=YamamotoMaho en-aut-sei=Yamamoto en-aut-mei=Maho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KondoRina en-aut-sei=Kondo en-aut-mei=Rina kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HozumiHaruka en-aut-sei=Hozumi en-aut-mei=Haruka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=DoiSeita en-aut-sei=Doi en-aut-mei=Seita kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=DendaMiwako en-aut-sei=Denda en-aut-mei=Miwako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=HatanoNaoya en-aut-sei=Hatano en-aut-mei=Naoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=MorishitaRyo en-aut-sei=Morishita en-aut-mei=Ryo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=TokumitsuHiroshi en-aut-sei=Tokumitsu en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= affil-num=1 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University kn-affil= affil-num=4 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Cell Free Sciences Co., Ltd. kn-affil= affil-num=6 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=7 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=8 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=9 en-affil=Cell Free Sciences Co., Ltd. kn-affil= affil-num=10 en-affil=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=S100 protein kn-keyword=S100 protein en-keyword=HMG20A kn-keyword=HMG20A en-keyword=protein-protein interaction kn-keyword=protein-protein interaction en-keyword=Ca2+-signal transduction kn-keyword=Ca2+-signal transduction en-keyword=genome-wide screening kn-keyword=genome-wide screening END start-ver=1.4 cd-journal=joma no-vol=36 cd-vols= no-issue=2 article-no= start-page=51 end-page=60 dt-received= dt-revised= dt-accepted= dt-pub-year=2002 dt-pub=200203 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Regulatory Role for Complement Receptors (CD21/CD35) in the Recombination Activating Gene Expression in Mouse Peripheral B Cells en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=HikidaMasaki en-aut-sei=Hikida en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NakayamaYasunori en-aut-sei=Nakayama en-aut-mei=Yasunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=OhmoriHitoshi en-aut-sei=Ohmori en-aut-mei=Hitoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=2 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=3 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=4 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=5 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University END start-ver=1.4 cd-journal=joma no-vol=38 cd-vols= no-issue=1-2 article-no= start-page=91 end-page=96 dt-received= dt-revised= dt-accepted= dt-pub-year=2004 dt-pub=200403 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Generation of IgM and IgG1 monoclonal antibodies with identical variable regions: comparison of avidity en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YamakoshiKimi en-aut-sei=Yamakoshi en-aut-mei=Kimi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KiyomiMasaaki en-aut-sei=Kiyomi en-aut-mei=Masaaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MagariMasaki en-aut-sei=Magari en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=OhmoriHitoshi en-aut-sei=Ohmori en-aut-mei=Hitoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=2 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=3 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=4 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University affil-num=5 en-affil= kn-affil=Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University END start-ver=1.4 cd-journal=joma no-vol= cd-vols= no-issue= article-no= start-page= end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2002 dt-pub=20020930 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=末梢リンパ組織でのrecombination activating gene産物による新規B細胞レパトワ形成の生理的役割に関する研究 kn-title=Studies on the biological role of recombination activating gene products in the generation of new B cell repertoire in the periphery en-subtitle= kn-subtitle= en-abstract= kn-abstract= en-copyright= kn-copyright= en-aut-name= en-aut-sei= en-aut-mei= kn-aut-name=曲正樹 kn-aut-sei=曲 kn-aut-mei=正樹 aut-affil-num=1 ORCID= affil-num=1 en-affil= kn-affil=岡山大学 END