title: Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2.
abstract: Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli.
authors.affiliation: Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Forskningsparken, University of Oslo, Oslo University Hospitals, 0349 Oslo, Norway.
authors.forename: Lisa
authors.initials: L
authors.lastname: Gerner
authors.affiliation: Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Forskningsparken, University of Oslo, Oslo University Hospitals, 0349 Oslo, Norway.
authors.forename: Steffi
authors.initials: S
authors.lastname: Munack
authors.affiliation: European Molecular Biology Laboratory Hamburg, Notkestrasse 85, 22603 Hamburg, Germany; European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Koen
authors.initials: K
authors.lastname: Temmerman
authors.affiliation: European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Ann-Marie
authors.initials: AM
authors.lastname: Lawrence-Dörner
authors.affiliation: European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Hüseyin
authors.initials: H
authors.lastname: Besir
authors.affiliation: European Molecular Biology Laboratory Hamburg, Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilmanns
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Jan Kristian
authors.initials: JK
authors.lastname: Jensen
authors.affiliation: Department of Biosciences, University of Oslo, Norway.
authors.forename: Bernd
authors.initials: B
authors.lastname: Thiede
authors.affiliation: Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Forskningsparken, University of Oslo, Oslo University Hospitals, 0349 Oslo, Norway. Electronic address: ian.mills@ncmm.uio.no.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Mills
authors.affiliation: Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Forskningsparken, University of Oslo, Oslo University Hospitals, 0349 Oslo, Norway. Electronic address: j.p.morth@ncmm.uio.no.
authors.forename: Jens Preben
authors.initials: JP
authors.lastname: Morth
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001562:Benzimidazoles / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D053644:Naphthalimides / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D001562:Benzimidazoles; D002147:Calmodulin; D005456:Fluorescent Dyes; D053644:Naphthalimides; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C458525:STO 609; D000255:Adenosine Triphosphate; C517698:CAMKK2 protein, human; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase
keywords: CaMKK2; Drug screening; Fluorescent probe; Kinome; Phosphorylation; STO-609