start-ver=1.4 cd-journal=joma no-vol=4 cd-vols= no-issue= article-no= start-page=27 end-page=29 dt-received= dt-revised= dt-accepted= dt-pub-year=2024 dt-pub=20240331 dt-online= en-article= kn-article= en-subject= kn-subject= en-title=Report: International Symposium organized by Graduate school of interdisciplinary science and engineering in health systems kn-title=ヘルスシステム統合科学研究科国際シンポジウム報告 en-subtitle= kn-subtitle= en-abstract= kn-abstract= en-copyright= kn-copyright= en-aut-name=KIWAToshihiko en-aut-sei=KIWA en-aut-mei=Toshihiko kn-aut-name=紀和利彦 kn-aut-sei=紀和 kn-aut-mei=利彦 aut-affil-num=1 ORCID= en-aut-name=KANAYAMANaoki en-aut-sei=KANAYAMA en-aut-mei=Naoki kn-aut-name=金山直樹 kn-aut-sei=金山 kn-aut-mei=直樹 aut-affil-num=2 ORCID= 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=3 ORCID= en-aut-name=HAKAMADARei en-aut-sei=HAKAMADA en-aut-mei=Rei kn-aut-name=袴田玲 kn-aut-sei=袴田 kn-aut-mei=玲 aut-affil-num=4 ORCID= affil-num=1 en-affil=Faculty of Interdisciplinary Science and Engineering in Health Systems, 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=Faculty of Interdisciplinary Science and Engineering in Health Systems, 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=Faculty of Interdisciplinary Science and Engineering in Health Systems, 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=Faculty of Interdisciplinary Science and Engineering in Health Systems, 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=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=525 cd-vols= no-issue=1 article-no= start-page=251 end-page=257 dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200423 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells en-subtitle= kn-subtitle= en-abstract= kn-abstract=Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5′AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property. en-copyright= kn-copyright= en-aut-name=TakabatakeShota en-aut-sei=Takabatake en-aut-mei=Shota kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=FukumotoYusei en-aut-sei=Fukumoto en-aut-mei=Yusei 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=SakagamiHiroyuki en-aut-sei=Sakagami en-aut-mei=Hiroyuki 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=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=Department of Anatomy, Kitasato University School of Medicine 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=pplied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=CaMKKβ kn-keyword=CaMKKβ en-keyword=Phosphorylation kn-keyword=Phosphorylation en-keyword=Dephosphorylation kn-keyword=Dephosphorylation en-keyword=PP1 kn-keyword=PP1 en-keyword=PP2A kn-keyword=PP2A en-keyword=Okadaic acid kn-keyword=Okadaic acid END start-ver=1.4 cd-journal=joma no-vol=1863 cd-vols= no-issue=4 article-no= start-page=672 end-page=680 dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20190117 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Regulation of Ca2+/calmodulin-dependent protein kinase kinase beta by cAMP signaling en-subtitle= kn-subtitle= en-abstract= kn-abstract=BACKGROUND:
Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear.
METHODS:
Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ.
RESULTS:
Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme.
CONCLUSION:
cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells.
GENERAL SIGNIFICANCE:
Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation. en-copyright= kn-copyright= en-aut-name=TakabatakeShota en-aut-sei=Takabatake en-aut-mei=Shota 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=SugawaraTakeyuki en-aut-sei=Sugawara en-aut-mei=Takeyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 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=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=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=Department of Anatomy, Kitasato University School of Medicine 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=Calmodulin kn-keyword=Calmodulin en-keyword=Intracellular Ca(2+) kn-keyword=Intracellular Ca(2+) en-keyword=PKA kn-keyword=PKA en-keyword=Phosphorylation kn-keyword=Phosphorylation en-keyword=Signal transduction kn-keyword=Signal transduction END start-ver=1.4 cd-journal=joma no-vol=485 cd-vols= no-issue=2 article-no= start-page=261 end-page=266 dt-received= dt-revised= dt-accepted= dt-pub-year=2017 dt-pub=201704 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus en-subtitle= kn-subtitle= en-abstract= kn-abstract=　Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSF1-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSF1-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, overexpression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSF1-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C-terminus-dependent manner. en-copyright= kn-copyright= en-aut-name=KawaguchiYuka en-aut-sei=Kawaguchi en-aut-mei=Yuka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NarikiHiroaki en-aut-sei=Nariki en-aut-mei=Hiroaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KawamotoNaoko en-aut-sei=Kawamoto en-aut-mei=Naoko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KanehiroYuichi en-aut-sei=Kanehiro en-aut-mei=Yuichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MiyazakiSatoshi en-aut-sei=Miyazaki en-aut-mei=Satoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=SuzukiMari en-aut-sei=Suzuki en-aut-mei=Mari kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 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=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= en-aut-name=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=2 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=3 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=4 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=5 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=6 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=7 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=8 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=9 en-affil=Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University kn-affil= en-keyword=AID kn-keyword=AID en-keyword=DT40 kn-keyword=DT40 en-keyword=Gene conversion kn-keyword=Gene conversion en-keyword=Ig kn-keyword=Ig en-keyword=SRSF1 kn-keyword=SRSF1 en-keyword=Somatic hypermutation kn-keyword=Somatic hypermutation 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