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1.
Biochem Biophys Res Commun ; 476(2): 102-7, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27178209

RESUMO

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.


Assuntos
Benzimidazóis/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Corantes Fluorescentes/farmacologia , Naftalimidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Benzimidazóis/metabolismo , Sítios de Ligação , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Calmodulina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/metabolismo , Humanos , Naftalimidas/metabolismo , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência/métodos
2.
Exp Dermatol ; 22(11): 754-6, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24433183

RESUMO

Epidermal barrier acquisition during late mammalian development is a prerequisite for terrestrial existence. Over a 24-h period, the epidermis goes from being a barrier-deficient, dye permeable epithelium to a barrier-competent epithelium. We have previously shown that Akt signalling is necessary for barrier acquisition in the mouse and that the protein phosphatase 2A regulatory subunit Ppp2r2a causes barrier acquisition by dephosphorylation of cJun. Here, we demonstrate that there is transient interaction between the gap junction protein Connexin 43 (Cx43) and Zonula occludins-1 (Zo-1) during epidermal barrier acquisition. Ppp2r2a knockdown prevented plasma membrane co-localisation and interaction between the two proteins. Ppp2r2a knockdown also increased phosphorylation at Serine 368 of Connexin 43. Cx43 phosphorlyation at Serine368 occurred just prior to the interaction between Connexin 43 and Zo-1. We therefore propose a model in which Ppp2r2a is required both for the initial interaction between Zo-1 and Cx43 and the consequent dephosphorylation of Connexin 43, preventing interaction of Zo-1 and allowing Zo-1 to initiate tight junction formation and barrier acquisition.


Assuntos
Conexina 43/química , Epiderme/patologia , Regulação da Expressão Gênica , Proteína Fosfatase 2/fisiologia , Animais , Membrana Celular/metabolismo , Conexinas/metabolismo , Epiderme/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/citologia , Proteínas de Membrana/metabolismo , Camundongos , Fosforilação , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais , Proteína da Zônula de Oclusão-1/metabolismo
3.
Cell Death Dis ; 12(11): 1040, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34725334

RESUMO

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.


Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Complexo de Golgi/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Vesículas Transportadoras/metabolismo , Ácidos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Autofagia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Linhagem Celular Tumoral , Proliferação de Células , Complexo I de Proteína do Envoltório/metabolismo , Sequência Conservada , Complexo de Golgi/ultraestrutura , Homeostase , Humanos , Lisossomos/metabolismo , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Interferente Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Resposta a Proteínas não Dobradas
4.
Data Brief ; 8: 733-40, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27508226

RESUMO

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2" [1].

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