REGULATION OF PROTEIN DEPHOSPHORYLATION

蛋白磷酸酶2A(PP2A)是一种普遍存在的丝氨酸/苏氨酸 磷酸酶是信号通路的关键组成部分 调节细胞功能。PP2A分子的异二聚体核心 具有多种调节亚基，这些亚单位有助于特异性 以及酶的亚细胞定位。PP2A还与一种 不被认为是常规调控蛋白的数量 酶的亚基。调查人员期待着这一身份证明 PP2A的其他互动合作伙伴将提供新的见解 进入酶的细胞组织，这种组织 决定了酶作用的特异性。 PP2A的一个亚群与神经元中的微管有关。 Tau和map2是神经元微管相关蛋白(MAP)， 在体内高度磷酸化，是许多蛋白质的靶标 体外试验中的激活酶。Tau和map2的磷酸化状态决定了 它们与微管结合从而稳定微管的能力。结果 来自研究人员实验室的研究表明，PP2A能使tau脱磷 和MAP2在体内，表明该酶调节神经元 细胞骨架。这项工作的一个重要方面是， 阿尔茨海默病的病理特征包括 Tau蛋白过度磷酸化和轴突微管破坏。 该提案有三个具体目标。1)第一个目标将测试 假设促进PP2A靶向的锚定蛋白 微管是tau和tau有效去磷酸化所必需的。 Map2.PP2A与微管相互作用的分子基础 我们将探索细胞骨架。推测的锚定蛋白(S)将是 纯化、鉴定和克隆cDNA。锚定在中的作用 对PP2A的监管将在假设直接 蛋白质-蛋白质相互作用定义了PP2A对 神经元图。2)目标2的基本假设是 抑制完整动物神经元中的PP2A将导致增加 Tau蛋白的磷酸化与神经元变性。SV40小T抗原， 一种PP2A的特异性抑制剂将在转基因小鼠中表达 神经元特异性启动子。PP2A活性降低的影响将 通过测量tau磷酸化和神经元形态来评估。 3)在这个目标中要检验的假设是两个新的PP2A- 相互作用的蛋白质影响PP2A的活性和功能。 PP2A相互作用蛋白p31和p17的特性研究 在双混合屏幕上确定，将以高度优先的方式追求。 这些蛋白质对活性、亚细胞定位和 PP2A的体内功能将被确定。其他候选人 在双杂交筛选中确定的相互作用蛋白也将是 特色化的。

Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that is a critical component of signaling pathways regulating cell function. The heterodimeric core of PP2A associates with a variety of regulatory subunits that contribute to the specificity and subcellular localization of the enzyme. PP2A also interacts with a number of proteins that are not considered to be conventional regulatory subunits of the enzyme. The investigators anticipate that identification of additional interacting partner for PP2A will provide new insights into the cellular organization of the enzyme and that such organization dictates specificity of enzyme action. One sub-population of PP2A is associated with microtubules in neurons. Tau and MAP2 are neuronal microtubule-associated proteins (MAPs) that are highly phosphorylated in vivo and are targets for numerous protein kinases in vitro. The phosphorylation state of tau and MAP2 determines their abilities to bind to and thus stabilize microtubules. Results from investigators' laboratory indicate that PP2A dephosphorylates tau and MAP2 in vivo, suggesting the enzyme regulates the neuronal cytoskeleton. An important aspect of this work is that one of the pathological hallmarks of Alzheimer's Disease includes hyperphosphorylation of tau and disruption of axonal microtubules. The proposal has three specific aims. 1) The first aim will test the hypothesis that an anchoring protein that promotes targeting of PP2A to microtubules is required for efficient dephosphorylation of tau and MAP2. The molecular basis of PP2A interaction with the microtubule cytoskeleton will be explored. Putative anchoring protein(s) will be purified, characterized, and cDNAs cloned. The role of anchoring in regulating PP2A will be examined with the assumption that direct protein-protein interactions define the specificity of PP2A toward neuronal MAPs. 2) The underlying hypothesis of aim 2 is that suppression of PP2A in neurons of intact animals will lead to increased phosphorylation of tau and neuronal degeneration. SV40 small-t antigen, a specific inhibitor of PP2A, will be expressed in transgenic mice using neuron-specific promoters. The effects of reduced PP2A activity will be assessed by measuring tau phosphorylation and neuronal morphology. 3) The hypothesis to be tested in this aim is that two novel PP2A- interacting protein influence the activity and function of PP2A. Characterization of p31 and p17, two PP2A-interacting proteins identified in a two-hybrid screen, will be pursued with high priority. The effects of these proteins on activity, subcellular localization, and in vivo function of PP2A will be determined. Additional candidate interacting proteins, identified in the two-hybrid screen will also be characterized.