Assaying Protein Kinase A Activity Using a FRET-Based Sensor Purified from Mammalian Cells

Abstract

Protein Kinase A (PKA) is the major intracellular receptor for cAMP. Research into this prototype kinase is supported by kinase assays that are typically performed in vitro using radio-labelled ATP. For in vivo studies, genetically-encoded FRET-based sensors have become popular for monitoring PKA activity. Here, we show that it is also possible to apply such reporters in vitro. We describe how to express and purify milligram quantities of a FRET-based PKA activity reporter using cultured human embryonic kidney cells. We demonstrate how to utilize the purified reporter in a plate reader to determine the IC50 for the widely-utilized PKA inhibitor H89 in the presence of a physiologicallyrelevant concentration of ATP. The protocol takes advantage of the economical transfection reagent polyethylenimine, and can be performed in a standard cell culture facility. Whereas assays based on radiolabelling are more sensitive, the approach presented here has several advantages: it enables continuous measurement of changes in substrate phosphorylation; a single preparation produces enough reporter for thousands of recordings; the reporter has a long shelf life; and it avoids the safety considerations that arise when working with radioactive material.

Regulator of G-Protein Signaling-4 Attenuates Cardiac Adverse Remodeling and Neuronal Norepinephrine Release-Promoting Free Fatty Acid Receptor FFAR3 Signaling

“…Indeed, pretreatment of H9c2 cardiomyocytes with either isoproterenol, which stimulates both β 1 - and β 2 ARs, or salbutamol (albuterol), which is a β 2 AR-selective agonist [ 27 , 28 , 29 ], markedly reduced propionic acid-elicited Giα activity in control and wild-type H9c2 cardiomyocytes ( Figure 3 A), but both catecholamines failed to do so in RGS4-depleted cells ( Figure 3 B). Furthermore, in the presence of H89, a well-characterized PKA inhibitor [ 30 ], neither isoproterenol nor salbutamol, had any detectable effect on propionic acid-dependent Giα activation in the native, wild-type H9c2 cardiomyocytes ( Figure 3 B). Taken together, these results strongly suggest that catecholamines via both β 1 - and β 2 ARs induce PKA-dependent activation of RGS4, which subsequently impedes FFAR3 signaling in cardiac myocytes.…”

Molecular basis of interactions between CaMKII and α-actinin-2 that underlie dendritic spine enlargement

“…N-terminally 6His-tagged mouse CaMKIIα was cloned into pcDNA3.1 using primers EcoRI_6HisCaMKIIa and CaMKIIa_Term_XhoI for expression in adherent HEK293T cells. Cells were transfected at 60–70% confluency with 10 μg DNA and 60 μg polyethlyenimine (MW 25000) applied to each 10 cm dish ( Curtis et al, 2022 ). The media was replaced with DMEM supplemented with 10% FBS the morning after transfection, and cells were harvested 3 days after transfection.…”

Molecular basis of interactions between CaMKII and α-actinin-2 that underlie dendritic spine enlargement

“…N-terminally 6His-tagged mouse CaMKIIα was cloned into pcDNA3.1 using primers EcoRI_6HisCaMKIIα & CaMKIIα_Term_XhoI for expression in adherent HEK293T cells. Cells were transfected at 60-70% confluency with 10 μg DNA and 60 μg polyethlyenimine (MW 25000) applied to each 10-cm dish (Curtis et al, 2022). The media was replaced with DMEM supplemented with 10% FBS the morning after transfection, and cells were harvested 3 days after transfection.…”