title: Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET(2).
abstract: Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET(2) pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP(2). This BRET(2) sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET(2) sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate β-arrestin2, decreased the BRET(2) signal by up to 50%. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50%. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET(2) sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.
authors.forename: Gerda M
authors.initials: GM
authors.lastname: Hübner
authors.forename: Jane Nøhr
authors.initials: JN
authors.lastname: Larsen
authors.forename: Barbara
authors.initials: B
authors.lastname: Guerra
authors.forename: Karsten
authors.initials: K
authors.lastname: Niefind
authors.forename: Milka
authors.initials: M
authors.lastname: Vrecl
authors.forename: Olaf-Georg
authors.initials: OG
authors.lastname: Issinger
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D047390:Casein Kinase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066329:Protein Aggregates; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D020035:Holoenzymes; D066329:Protein Aggregates; D000071557:beta-Arrestins; D047390:Casein Kinase II