Function of the neuronal tyrosine kinase N2-Src in synaptic vesicle exocytosis

蛋白质的酪氨酸磷酸化(将磷酸盐加到氨基酸酪氨酸上)是由称为酪氨酸激酶的酶执行的，在人体所有细胞中用作信号机制。蛋白质的磷酸化导致蛋白质的形状和电荷的局部变化，并根据发生磷酸化的位置，通过改变蛋白质的功能来传递信号，例如。通过改变其与其他蛋白质结合的亲和力。酪氨酸激酶C-Src在脑细胞(神经元)中高度丰富，被认为调节大脑功能的许多方面，如发育、学习和记忆。在神经元中，C-Src集中在突触小泡上，突触小泡是储存神经元相互交流释放的化学神经递质的隔室。在大脑中还发现了另一种类型的C-Src，称为N2-Src，它在蛋白质中调节其相互作用的部分有所不同，并可以确定它磷酸化了哪些蛋白质。因此，N2-Src不与通常由C-Src结合的蛋白质结合，这表明它具有替代功能。为了支持这一点，我们发现N2-Src(而不是C-或N1-Src)与两种蛋白质Munc18和NSF结合，这两种蛋白质是神经元释放神经递质所必需的。此外，Munc18和NSF的酪氨酸磷酸化对身体其他部位的激素释放也很重要。我们假设，N2-Src通过与Munc18和NSF结合并使其酪氨酸磷酸化来控制神经元神经递质的释放。为了解决这一假设，我们将首先研究N2-Src与Munc18或NSF在试管中的相互作用，以找到结合所需的蛋白质的特定区域。我们还将发现Munc18和NSF上的哪些酪氨酸被N2-Src磷酸化，以及磷酸化如何影响它们的功能。从这些实验中获得的信息将使我们能够设计出Munc18和NSF的形式，这些形式已经失去了与N2-Src结合或被酪氨酸磷酸化的能力。通过测试这些工程蛋白对活神经元神经递质释放的影响，我们将发现N2-Src是否通过改变Munc18或NSF的功能来调节这一过程。由于大多数激酶可以磷酸化数十种蛋白质，因此Munc18和NSF不太可能是其唯一的靶标。我们将寻找在神经元中被N2-Src磷酸化的其他蛋白质，以获得其在大脑中功能的更完整的图景。神经递质的释放是正常大脑功能所必需的基本过程，但也会受到神经系统疾病和药物滥用的影响。因此，对神经递质释放是如何由N2-Src酪氨酸激酶调节的特征将有助于对健康和患病大脑功能的研究。更具体地说，有证据表明，在小鼠癫痫样发作后，神经元Src激酶的水平会发生变化，因此来自该项目的数据将有助于更好地了解癫痫导致的神经传递的变化。

Tyrosine phosphorylation (the addition of phosphate to the amino acid tyrosine) of proteins is performed by enzymes called tyrosine kinases and is used as a signalling mechanism in all cells of the body. Phosphorylation of proteins causes a localised change in the shape and charge of the protein and, depending upon where phosphorylation occurs, can transmit a signal by changing the function of a protein, eg. by altering its affinity for binding to other proteins. The tyrosine kinase C-Src is highly enriched in brain cells (neurons) and is thought to regulate many aspects of brain function, such as development and learning and memory. In neurons, C-Src is concentrated on synaptic vesicles, compartments which store the chemical neurotransmitters that are released by neurons to communicate with each other. An additional type of C-Src is found in the brain, called N2-Src, that differs in a part of the protein which regulates its interactions and can determine which proteins it phosphorylates. Consequently, N2-Src does not bind to the proteins usually bound by C-Src suggesting it has an alternative function. In support of this, we found that N2-Src (but not C- or N1-Src) binds to two proteins, Munc18 and NSF, that are necessary for the release of neurotransmitter by neurons. Furthermore, the tyrosine phosphorylation of Munc18 and NSF has been shown to be important for the release of hormones in other parts of the body. We hypothesise that N2-Src controls the release of neurotransmitter from neurons by binding to and tyrosine phosphorylating Munc18 and NSF. To address the hypothesis, we will first study the interactions between N2-Src and Munc18 or NSF in the test tube to find the specific regions of the proteins which are necessary for binding. We will also discover which tyrosines on Munc18 and NSF are phosphorylated by N2-Src and how phosphorylation affects their function. The information gained from these experiments will allow us to engineer forms of Munc18 and NSF that have lost the ability to bind N2-Src or to be tyrosine phosphorylated. By testing the effects of these engineered proteins upon neurotransmitter release from living neurons we will find out whether N2-Src regulates this process by altering the function of Munc18 or NSF. Since most kinases can phosphorylate tens of proteins, it is unlikely that Munc18 and NSF are its only targets. We will search for other proteins that are phosphorylated in neurons by N2-Src to gain a more complete picture of its function in the brain. Neurotransmitter release is a fundamental process required for normal brain function but is also affected by neurological disease and drugs of abuse. Thus the characterisation of how neurotransmitter release is regulated by the N2-Src tyrosine kinase will aid research into the function of the healthy and diseased brain. More specifically, there is evidence that the levels of neuronal Src kinases are altered following epileptic-like seizures in mice and hence the data from this project will help to better understand the changes in neurotransmission resulting from epilepsy.