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=23 cd-vols= no-issue=19 article-no= start-page=11025 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20220920 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction en-subtitle= kn-subtitle= en-abstract= kn-abstract=Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is the activating kinase for multiple downstream kinases, including CaM-kinase I (CaMKI), CaM-kinase IV (CaMKIV), protein kinase B (PKB/Akt), and 5'AMP-kinase (AMPK), through the phosphorylation of their activation-loop Thr residues in response to increasing the intracellular Ca2+ concentration, as CaMKK itself is a Ca2+/CaM-dependent enzyme. The CaMKK-mediated kinase cascade plays important roles in a number of Ca2+-dependent pathways, such as neuronal morphogenesis and plasticity, transcriptional activation, autophagy, and metabolic regulation, as well as in pathophysiological pathways, including cancer progression, metabolic syndrome, and mental disorders. This review focuses on the molecular mechanism underlying CaMKK-mediated signal transduction in normal and pathophysiological conditions. We summarize the current knowledge of the structural, functional, and physiological properties of the regulatory kinase, CaMKK, and the development and application of its pharmacological inhibitors. en-copyright= kn-copyright= en-aut-name=TokumitsuHiroshi en-aut-sei=Tokumitsu en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 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=2 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 Anatomy, Kitasato University School of Medicine kn-affil= en-keyword=CaMKK kn-keyword=CaMKK en-keyword=CaM-kinase cascade kn-keyword=CaM-kinase cascade en-keyword=Ca2+ signaling kn-keyword=Ca2+ signaling en-keyword=phosphorylation kn-keyword=phosphorylation 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=96 cd-vols= no-issue= article-no= start-page=102404 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20216 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins en-subtitle= kn-subtitle= en-abstract= kn-abstract=To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dosedependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization. en-copyright= kn-copyright= en-aut-name=DoiSeita en-aut-sei=Doi en-aut-mei=Seita kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=FujiokaNaoki en-aut-sei=Fujioka en-aut-mei=Naoki 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=KondoRina en-aut-sei=Kondo en-aut-mei=Rina kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YamamotoMaho en-aut-sei=Yamamoto en-aut-mei=Maho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 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=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=KanayamaNaoki en-aut-sei=Kanayama en-aut-mei=Naoki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 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=9 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=10 ORCID= en-aut-name=HasegawaTakafumi en-aut-sei=Hasegawa en-aut-mei=Takafumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 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=12 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=Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=6 en-affil=CellFree Sciences Co., Ltd 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=CellFree Sciences Co., Ltd kn-affil= affil-num=11 en-affil=Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine kn-affil= affil-num=12 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=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