Dissecting protein kinase A regulation of neurons using synthetic approaches

全身的细胞都有表面受体，使它们能够对激素和神经递质等外部刺激做出反应。一种常见的信号转导机制是这些外部初级信使触发细胞内“第二信使”环AMP（cAMP）的积累。cAMP的主要受体-蛋白激酶A（PKA）-响应cAMP升高，通过磷酸化蛋白质引起生理变化。PKA磷酸化控制的无数过程包括心率的交感神经刺激，肾脏中水再摄取的控制，以及称为神经元的脑细胞的兴奋性和形状的控制。近年来的研究表明，细胞中的cAMP信号传导是在“纳米结构域”中组织的，有时直径小于100纳米。因此，PKA拷贝在细胞中的精确位置决定了它是否会被给定的刺激激活。锚定蛋白将PKA定位在不同的亚细胞位置，这些锚定蛋白被认为指导激酶磷酸化与不同功能相关的底物的不同子集。此外，针对单个PKA锚定位点被认为是选择性破坏PKA磷酸化支持的病理过程（如癫痫相关的神经元兴奋性）的有前途的策略。然而，我们目前对PKA锚定的理解的基本方面还没有得到解决，不同的PKA锚定蛋白在神经元兴奋性中所起的确切作用还有待解开。这些领域将受益于新的技术来操纵PKA的活动在时间和空间。在这项研究中，我们将开发两个创新的技术，灵感来自合成生物学领域，可能会被应用到直接PKA特定的锚定蛋白，并指示当激酶激活的蓝光控制下。然后，我们将利用这些技术与现有方法相结合，研究纳米结构域中PKA信号传导的基本原理，以及不同PKA锚定蛋白在控制神经元形状和兴奋性方面的具体作用。为了使PKA能够特异性锚定到单个锚定蛋白，我们将利用蛋白质结构域，使活细胞中的蛋白质分子“粘合”。这项工作将涉及使用基因编辑技术开发两种细胞系。为了开发PKA的光活化形式，我们将使用PKA调节和催化亚基库进行高通量筛选，其中通常对cAMP响应的元件被对蓝光响应的元件取代。最有前途的组合将使用蛋白质结合和活性测定进行优化和验证。我们对cAMP纳米结构域基本原理的研究将包括确定单个PKA锚定蛋白复合物如何使用PKA活性的靶向荧光报告分子和定量蛋白质组学对不同的初级刺激做出反应。我们研究的最后一部分将集中在阐明cAMP和PKA的变化如何与癫痫使用切片模型制备。我们还将测量培养的神经元的兴奋性和形态学的变化，以确定不同的PKA锚定位点如何控制神经元功能的这些方面。我们已经组建了一个研究团队，他们在蛋白质工程到电生理学等技术领域以及cAMP信号传导和癫痫等领域具有互补的专业知识。拟议的研究将受益于与光活化和定量蛋白质组学专家的合作。除了推进神经元中nanodomain cAMP信号传导的基础知识外，本研究期间开发的新技术将使研究人员受益，重点关注PKA在整个身体中发挥的许多其他作用。

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.