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.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses that is linked to learning and memory. In this study, we focused on understanding how interactions between CaMKIIα and the actin crosslinking protein α-actinin-2 underlie long-lasting changes in dendritic spine architecture. We found that association of the two proteins was unexpectedly elevated following stimulation of NMDA receptors to trigger structural LTP in primary hippocampal neurons. Furthermore, disruption of interactions between the two proteins prevented the accumulation of enlarged mushroom-type dendritic spines following NMDA receptor activation. α-actinin-2 binds to the regulatory segment of CaMKII. Calorimetry experiments, and a crystal structure of α-actinin-2 EF hands 3 and 4 in complex with the CaMKII regulatory segment, indicate that the regulatory segment of autoinhibited CaMKII is not fully accessible to α-actinin-2. Pull-down experiments show that occupation of the CaMKII substrate binding groove by GluN2B markedly increases α-actinin-2 access to the CaMKII regulatory segment. Overall, our study provides new mechanistic insight into the molecular basis of structural LTP and reveals an added layer of sophistication to the function of CaMKII.