CAMP-DEPENDENT PROTEIN KINASES IN NEURONAL MODULATION

cAMP依赖性蛋白激酶（PKA）是细胞内cAMP依赖性蛋白激酶的组分。 在许多神经系统疾病的例子中， 可塑性，包括感觉运动突触的突触前易化 在失智症中，一种简单形式学习和记忆的细胞相关物。 这种酶能传递神经调节递质所携带的信号， 因为动物的经历而被释放到 在目标神经元内改变它们的性质和相互作用 与其他神经元。 PKA参与了促进的几个方面 在空间上和时间上是分开的。 例如，在短- 术语易化，PKA介导钾通道的关闭， 延长动作电位，导致神经递质增加 release. 在长时程易化中，突触生长被激活， 需要通过PKA磷酸化转录因子。 怎么能 一种单一的酶控制着如此多样的事件，而不会引起混乱。 通过不加选择的磷酸化答案很多在于 Aphasia PKA的复杂性，它由至少五个调节蛋白组成， (R)和四个催化（C）亚基，产生多种全酶 （R2C2）。 该实验室最近的研究表明，这些形式的PKA 不同的底物特异性，调节和亚细胞位置。 我们的假设是，这解释了生理上的多功能性， PKA，通过提供具有多种生理作用的酶， 重叠和不同。 为了建立这一思想， 将检测失智症神经元PKA的各种R和C亚基， 在体外和完整的感觉神经元和细胞提取物中，强调 不同形式的亚单位之间的行为差异。 在细胞研究中，诱导短- 将确定长期便利化。 以下问题将 解决：1，R和C的多样性的本质是什么？ PKA亚单位在感觉神经元中的作用？目前编码 未鉴定的亚基将被克隆和测序。 2. 有哪些 C亚基的底物？底物特异性将在 体外，在感觉神经元和匀浆，并通过电生理 录制. 3. PKA在失智症神经元中是如何调节的？特别 将注意通过R亚基和通过 “自磷酸化”以及通过其他激酶的磷酸化。 4. PKA亚单位在失智症神经元中的位置以及它们是否改变了位置？ R和C亚基的单个形式的亚细胞位置将 在诱导易化的治疗之前和之后， 使用亚细胞分级分离，免疫荧光显微镜， 荧光成像 很可能， 这些研究将适用于荧光成像。 很可能 这些研究揭示的一般原则将适用于 脊椎动物 因此，这项工作导致更好地了解 正常和疾病大脑的可塑性。

cAMP-dependent protein kinase (PKA) is a component of an intracellular signalling pathway that is implicated in many examples of neural plasticity including presynaptic facilitation of the sensorimotor synapse in Aplysia, a cellular correlate of a simple form of learning and memory. The enzyme transduces signals carried by neuromodulatory transmitters, which are released as the result of an animal's experience, to sites within target neurons altering their properties and their interactions with other neurons. PKA is involved in several aspects of facilitation that are spatially and temporally separated. For example, during short- term facilitation, PKA mediates the closure of potassium channels, prolonging the action potential, which results in increased transmitter release. In long-term facilitation, synaptic growth is activated and requires the phosphorylation of transcription factors by PKA. How can a single enzyme control such diverse events, without causing chaos through indiscriminate phosphorylation? The answer many lie in the complexity of Aplysia PKA, which is composed of at least five regulatory (R) and four catalytic (C) subunits that generate multiple holoenzymes (R2C2). Recent work from this laboratory shows that these forms of PKA differ in substrate specificity, regulation, and subcellular location. Our hypothesis is that this accounts for the physiological versatility of PKA, by providing enzymes with multiple physiological roles, both overlapping and distinct. To establish this idea, the properties of the various R and C subunits of Aplysia neuronal PKA will be examined both in vitro and in intact sensory neurons and cell extracts, with emphasis on differences in behavior between the various forms of the subunits. In the cellular studies, the effects of treatments that induce short- and long-term facilitation will be determined. The following questions will be addressed: 1, What is the nature of the diversity of the R and C subunits of PKA in Aplysia sensory neurons? cDNAs encoding presently uncharacterized subunits will be cloned and sequenced. 2. What are the substrates of the C subunits? Substrate specificity will be examined in vitro, in sensory neurons and homogenates, and by electrophysiological recording. 3. How is PKA regulated in Aplysia neurons? Particular attention will be given to regulation by R subunits and by "autophosphorylation" as well as phosphorylation by other kinases. 4. Where are PKA subunits in Aplysia neurons and do they change location? The subcellular locations of individual forms of R and C subunits will be determined, before and after treatments that induce facilitation, by using subcellular fractionation, immunofluorescence microscopy and fluorescence imaging. It is likely that general principles revealed in these studies will be applicable and fluorescence imaging. It is likely that general principles revealed in these studies will be applicable to vertebrates. Thus, this work lead to a better understanding of plasticity in normal and disease brain.