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 CaMKKƒÀ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKƒÀ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKƒÀ at Thr144 in intact cells and in vivo remains unclear.
METHODS:
Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKƒÀ 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 CaMKKƒÀ.
RESULTS:
Our data suggest that the phosphorylation of Thr144 in CaMKKƒÀ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKƒÀ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKƒÀ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKƒÀ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKƒÀ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKƒÀ into Ca2+/CaM-dependent enzyme.
CONCLUSION:
cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKƒÀ-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/CaMKKƒÀ 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