Roles of Tyrosine Phosphorylation in The Development and Plasticity of The Central Nervous System

我们进行了三种类型的研究，即(1)寻找在中枢神经系统中表达的新的酪氨酸激酶，(2)鉴定分别参与Lyn和ALK酪氨酸激酶介导的中枢神经系统信号通路的蛋白质，(3)鉴定与离子型谷氨酸受体相关的蛋白-酪氨酸磷酸酶PTPMEG。本研究的主要内容如下：(1)通过对RT-PCR产物进行差异性筛选和电子筛选，我们发现了一种新的蛋白激酶，其中包括一种名为Brek(脑浓缩蛋白)的蛋白。我们还鉴定了BREK的一个同源物，称为BREK2。BREK和BREK2类似于AATYK，AATYK是一种酪氨酸激酶，参与调节神经元的凋亡性死亡。与AATYK不同的是，Brek和BREK2都是显示出较强的丝氨酸/苏氨酸和较弱的酪氨酸酶活性的双特异性激酶。Brek在中枢神经系统中高表达，如大脑皮层和嗅球。在NGF刺激培养的神经细胞后，Brek被磷酸化，提示Brek参与了轴突的延伸。(2)通过过滤酶试验和酵母双杂交筛选，我们分别确定了BANK和SNT2为Lyn和ALK的底物。BANK是一种连接Lyn和IP_3受体(IP_3R)的接头蛋白。我们发现，LYN/BANK/IP_3R的形成导致IP_3R的酪氨酸磷酸化，从而上调IP_3R的通道活性。关于SNT2，我们发现SNT2在ALK介导的信号转导中起着至关重要的作用。(3)我们还鉴定了与离子型谷氨酸受体相关的各种信号分子。其中一种是与GluRd2和NMDA受体亚基NR2a相关的PTPMEG。PTPMEG在丘脑中高表达，并影响NR2A的酪氨酸磷酸化水平，提示PTPMEG参与控制突触的可塑性。

We performed three types of studies that are (1) the search of the novel tyrosine kinases expressed in the central nervous system, (2) identification of proteins that are respectively involved in Lyn and ALK tyrosine kinases-mediated signaling pathways in the central nervous system, (3) characterization of a protein-tyrosine phosphatase PTPMEG that associates with ionotropic glutamate receptors. The followings are the summary of the study.(1) By performing differential screening of RT-PCR products and in silico screening, we identified novel kinases that included the one termed BREK (brain enriched kinase). We also identified a homologue of BREK termed BREK2. BREK and BREK2 are similar to AATYK that has been reported to be a tyrosine kinase involved in regulation of apoptotic death of neurons. Unlike AATYK, both BREK and BREK2 are dual specificity kinases showing strong serine/threonine and weak tyrosine kinase activities. Expression of BREK is high in the central nervous system, such as in cerebral cortex and olfactory bulb. BREK becomes phosphorylated upon NGF stimulation of cultured neuronal cells, suggesting that BREK is involved in neurite extension.(2) By employing filter kinase assay and yeast two-hybrid screening, we identified BANK and SNT2 as substrates of Lyn and ALK, respectively. BANK is an adaptor protein that links Lyn to IP_3 receptor (IP_3R). We showed that formation of Lyn/BANK/IP_3R resulted in tyrosine phosphorylation of IP_3R, which is known to upregulate the channel activity of IP_3R. Regarding SNT2, we showed that SNT2 was critically important for ALK-mediated signaling.(3) We also identified various signaling molecules that were associated with ionotropic glutamate receptors. One of them is PTPMEG that associates with GluRd2 and NMDA receptor subunits NR2A. PTPMEG is expressed high in the thalamus and affects the level of tyrosine phosphorylation of NR2A, suggesting that PTPMEG is involved in controlling synaptic plasticity.