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Development of a medium throughput whole-cell microtiter plate Thr286 autophosphorylation assay for CaMKIIα using ELISA.

التفاصيل البيبلوغرافية
العنوان: Development of a medium throughput whole-cell microtiter plate Thr286 autophosphorylation assay for CaMKIIα using ELISA.
المؤلفون: Palmelund LB; University of Copenhagen, Department of Drug Design and Pharmacology, Universitetsparken 2, 2100 Copenhagen, Denmark., van Woerden GM; Erasmus University Medical Center, Department of Neuroscience and Department of Clinical Genetics, 3015, CN, Rotterdam, the Netherlands., Bräuner-Osborne H; University of Copenhagen, Department of Drug Design and Pharmacology, Universitetsparken 2, 2100 Copenhagen, Denmark., Wellendorph P; University of Copenhagen, Department of Drug Design and Pharmacology, Universitetsparken 2, 2100 Copenhagen, Denmark. Electronic address: pw@sund.ku.dk.
المصدر: Journal of pharmacological and toxicological methods [J Pharmacol Toxicol Methods] 2022 Nov-Dec; Vol. 118, pp. 107226. Date of Electronic Publication: 2022 Sep 27.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: Elsevier Country of Publication: United States NLM ID: 9206091 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-488X (Electronic) Linking ISSN: 10568719 NLM ISO Abbreviation: J Pharmacol Toxicol Methods Subsets: MEDLINE
أسماء مطبوعة: Original Publication: New York, NY : Elsevier, c1992-
مواضيع طبية MeSH: Calcium-Calmodulin-Dependent Protein Kinase Type 2*/metabolism , Signal Transduction*, Humans ; Phosphorylation ; HEK293 Cells ; Enzyme-Linked Immunosorbent Assay
مستخلص: Ca 2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα) is a multifunctional Ser/Thr kinase involved in several neuronal signaling pathways including synaptic plasticity. CaMKIIα autonomous activity is highly dependent on Thr286 autophosphorylation (pThr286), which is widely used as a readout for its enzymatic activity. To readily characterise compounds and potential drug candidates targeting CaMKIIα, a simple, generic cell-based assay for quantification of pThr286 levels is needed. In this study, we present a cell-based assay using an adapted ELISA as a suitable and higher throughput alternative to Western blotting. In this 96-well plate-based assay, we use whole HEK293T cells recombinantly expressing CaMKIIα and apply a phospho-specific antibody to detect pThr286 levels by chemiluminescence. In parallel, total CaMKIIα expression levels are detected by fluorescence using an Alexa488-conjugated anti-myc antibody targeting a C-terminal myc-tag. By multiplexing chemiluminescence and fluorescence, phosphorylation levels are normalised to CaMKIIα total expression within each well. The specificity of the assay was confirmed using a phosphodead mutant (T286A) of CaMKIIα. By applying Ca 2+ or known CaMKIIα inhibitors (KN93, tatCN21 and AS100105) and obtaining concentration-response curves, we demonstrate high sensitivity and validity of the assay. Lastly, we demonstrate the versatility of the assay by determining autophosphorylation levels in CaMKIIα patient-related mutations, known to possess altered pThr286 responses (E109D, E183V and H282R). The established assay for CaMKIIα is a reproducible, easily implemented, and facile ELISA-based assay that allows for reliable quantification of pThr286 levels.
Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest.
(Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
فهرسة مساهمة: Keywords: CaMKIIα patient-related mutations; Calcium/calmodulin-dependent protein kinase II; Fluorescence; Luminescence; Methods; Multiplex detection; Pharmacological inhibitors; Stimulation
المشرفين على المادة: EC 2.7.11.17 (Calcium-Calmodulin-Dependent Protein Kinase Type 2)
تواريخ الأحداث: Date Created: 20220929 Date Completed: 20221129 Latest Revision: 20221129
رمز التحديث: 20240628
DOI: 10.1016/j.vascn.2022.107226
PMID: 36174932
قاعدة البيانات: MEDLINE
الوصف
تدمد:1873-488X
DOI:10.1016/j.vascn.2022.107226