Dissecting protein kinase A regulation of neurons using synthetic approaches

Cells throughout the body present surface receptors that enable them to respond to external stimuli such as hormones and neurotransmitters. A common signal transduction mechanism is for these external primary messengers to trigger accumulation of the 'second messenger' cyclic AMP (cAMP) within cells. The major receptor for cAMP - protein kinase A (PKA) - responds to cAMP elevations to bring about physiological changes by phosphorylating proteins. The myriad processes controlled by PKA phosphorylation include sympathetic stimulation of heart rate, control of water reuptake in the kidney, and control of the excitability and shape of brain cells called neurons. Research in recent years has revealed that cAMP signalling in cells is organised in 'nanodomains' sometimes with a diameter of less than 100 nanometres. The precise location of a copy of PKA in a cell therefore dictates whether it will be activated by a given stimulus. Anchoring proteins position PKA at different sub-cellular locations, and these anchoring proteins are thought to direct the kinase to phosphorylate different sub-sets of substrates linked to different functions. Furthermore, targeting of individual PKA anchoring sites is considered a promising strategy for selective disruption of pathological processes supported by PKA phosphorylation such as neuronal excitability underlying epilepsy. However, fundamental aspects of our current understanding of PKA anchoring have not been resolved, and the precise role that different PKA anchoring proteins play in neuronal excitability is yet to be disentangled. These areas would benefit from new technologies for manipulating PKA activity in time and space.

In this study, we will develop two innovative technologies inspired by the field of synthetic biology that may be applied to direct PKA to specific anchoring proteins, and to dictate when the kinase is activated under the control of blue light. We will then utilise these technologies in combination with existing methods to investigate fundamentals of PKA signalling in nanodomains, and the specific roles of different PKA anchoring proteins in controlling the shape and excitability of neurons. To enable specific anchoring of PKA to individual anchoring proteins, we will take advantage of protein domains that enable molecular 'gluing' of proteins in living cells. This work will involve the development of two cell lines using gene editing technologies. To develop a photo-activatable form of PKA, we will perform high-throughput screening with a library of PKA regulatory and catalytic subunits in which the elements that normally respond to cAMP are replaced with ones that respond to blue light. The most promising combinations will be optimised and validated using protein binding and activity assays. Our investigations of cAMP nanodomain fundamentals will include determining how individual PKA-anchoring protein complexes respond to different primary stimuli using targeted fluorescent reporters of PKA activity and quantitative proteomics. The final component of our study will focus on clarifying how changes in cAMP and PKA are linked to epilepsy using a slice model preparation. We will also measure changes in excitability and morphology in cultured neurons to determine how different PKA anchoring sites control these aspects of neuronal function.

We have assembled a team of investigators with complementary expertise in techniques ranging from protein engineering to electrophysiology, and in fields including cAMP signalling and epilepsy. The proposed research will benefit from collaboration with experts in photoactivation and quantitative proteomics. In addition to advancing fundamental knowledge of nanodomain cAMP signalling in neurons, the new technologies developed during this research will benefit researchers focusing on the many other roles played by PKA throughout the body.

Elevations in the second messenger cAMP may occur in sub-cellular nanodomains of 100 nm radius and smaller. Anchoring proteins for the major cAMP receptor, protein kinase A (PKA), are thought to play key roles in cAMP signalling processes by dictating when the kinase is activated and what it phosphorylates. Individual anchoring sites are promising targets for selectively intervening in pathological cAMP signalling processes including heart disease and epilepsy. The aim of this proposal is to develop and apply novel technologies derived from synthetic biology that will enable us to advance basic understanding of cAMP signalling mechanics, and to specifically explore the role of PKA targeting underlying neuronal excitability and epilepsy. We will develop two new cell lines using CRISPR knockdown and lentiviral replacement methods that can be applied to direct PKA to individual anchoring proteins in live cells through isopeptide linking. In a second branch of technology development, we will engineer a photo-activatable PKA (paPKA) using high-throughput screening in live cells with PKA subunits in which the cAMP-responsive elements are replaced by blue light-responsive domains. Ideal paPKA variants will be identified and optimised, using radioactive kinase activity assays and isothermal titration calorimetry with purified subunits. Our fundamental investigation of cAMP nanodomains will take advantage of recordings using ratiometric FRET sensors and quantitative phospho-proteomics following tandem mass tagging. We will explore the role of cAMP signalling in epilepsy using viral delivery approaches in a slice model system, and through imaging and patch clamp recordings with neuronal cultures. In this way, we aim to contribute fundamental breakthroughs to the understanding of PKA signalling in neuronal cAMP nanodomains, while developing tools that will assist researchers in the wider cAMP research community.