Molecular and Cellular Analysis of Brain Enriched PTPs

描述（由申请人提供）：拟议调查的中心目标是研究称为STEP的蛋白酪氨酸磷酸酶家族的结构和功能。STEP在中枢神经系统中特异性表达，并在纹状体、杏仁核、伏隔核、海马和大脑皮层的神经元中发现。蛋白质由细胞质和膜相关的同工异构体组成。它在神经元突触后密度中的存在表明STEP参与了信号转导通路，这是我们在上一个资助周期中建立的。在过去的四年中，我们发现STEP调节关键信号蛋白的功能，包括谷氨酸受体的NMDA亚型和丝裂原活化蛋白激酶和酪氨酸激酶家族成员。这些发现开辟了一个新的研究方向。我们计划继续通过生化、分子、免疫组织化学和电生理实验来表征STEP的调控和功能。我们将在STEP中识别新的磷酸化位点，识别调节这些位点的激酶和磷酸酶，并确定这些位点磷酸化的功能意义。初步数据表明，除了我们迄今发现的三种蛋白质外，还有其他蛋白质与STEP相关。我们将鉴定这些与step相互作用的蛋白质，并确定这些相互作用的功能意义。然而，STEP调节NMDA电流和MAPK家族的事实表明，它参与了LTP以及学习和记忆的各个方面。我们将通过STEP的功能破坏来检验新的假设。这将在原代细胞培养和体内通过使用无活性的TAT-STEP蛋白以及STEP敲除小鼠来完成。这些研究将阐明大脑中step调控信号的生理和功能意义，并可能提供与学习和记忆功能改变相关的神经精神疾病的信息。

DESCRIPTION (provided by applicant): The central goals of the proposed investigation are to study the structure and function of a family of protein tyrosine phosphatases, termed STEP. STEP is specifically expressed within the CNS, and is found within neurons of the striatum, amygdala, nucleus accumbens, hippocampus, and cerebral cortex. The proteins consist of both cytosolic and membrane-associated isoforms. Its presence in the postsynaptic densities of neurons suggested that STEP was involved in signal transduction pathways, which we established over the last cycle of the grant. Over the past four years, we have discovered that STEP regulates the function of key signaling proteins, including the NMDA subtype of glutamate receptor and members of the mitogen-activated protein kinase and tyrosine kinase families. These findings have opened a new direction of research. We plan to continue characterizing the regulation and function of STEP through biochemical, molecular, immunohistochemical, and electrophysiological experiments. We will identify novel phosphorylation sites in STEP, identify the kinases and phosphatases that regulate these sites, and determine the functional significance of phosphorylation at these sites. Preliminary data suggests that other proteins associate with STEP, in addition to the three we have discovered to date. We will identify these STEP-interacting proteins and determine the functional significance of these interactions. However, the fact that STEP regulates both NMDA currents and the MAPK family suggests it is involved in LTP as well as aspects of learning and memory. We will test the new hypothesis through functional disruption of STEP. This will be accomplished in primary cell cultures and in vivo by using inactive TAT-STEP proteins as well as the STEP knockout mouse. These studies will clarify the physiological and functional significance for STEPregulated signaling in the brain and may provide information about neuropsychiatric disorders associated with alterations in learning and memory function.