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

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII alpha) is a multifunctional Ser/Thr kinase involved in several neuronal signaling pathways including synaptic plasticity. CaMKII alpha 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 CaMKII alpha, 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 CaMKII alpha and apply a phospho-specific antibody to detect pThr286 levels by chemiluminescence. In parallel, total CaMKII alpha 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 CaMKII alpha total expression within each well. The specificity of the assay was confirmed using a phosphodead mutant (T286A) of CaMKII alpha. By applying Ca2+ or known CaMKII alpha inhibitors (KN93, tatCN21 and AS100105) and obtaining concentrationresponse curves, we demonstrate high sensitivity and validity of the assay. Lastly, we demonstrate the versatility of the assay by determining autophosphorylation levels in CaMKII alpha patient-related mutations, known to possess altered pThr286 responses (E109D, E183V and H282R). The established assay for CaMKII alpha is a reproducible, easily implemented, and facile ELISA-based assay that allows for reliable quantification of pThr286 levels.

Automated summary

In this study, a cell-based ELISA assay was developed to quantitate pThr286 levels of CaMKII alpha. The assay showed high sensitivity and specificity, and was validated using known inhibitors and patient-related mutations. This assay provides a reproducible and easily implemented method for studying CaMKII alpha and evaluating potential drug candidates.