REGULATION OF PROTEIN DEPHOSPHORYLATION

蛋白质去磷酸化的调控

基本信息

批准号：
6180478
负责人：
Marc C. MUMBY
金额：
$ 25.61万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2002-07-31
项目状态：
已结题

项目摘要

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.

蛋白质磷酸酶2a（PP2A）是无处不在的丝氨酸/苏氨酸磷酸酶是信号通路的关键成分调节细胞功能。 PP2A同事的异二聚体核心具有多种有助于特异性的监管亚基和酶的亚细胞定位。 pp2a也与不被认为是常规调节的蛋白质数量酶的亚基。调查人员预计该身份证明 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和此类组织决定酶作用的特异性。 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（地图）在体内高度磷酸化，是众多蛋白质的靶标体外激酶。 Tau和Map2的磷酸化状态确定它们与微管结合并因此稳定的能力。结果从研究人员的实验室表明pp2a脱磷酸化tau 和MAP2在体内，表明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氏病的病理标志包括 tau的高磷酸化和轴突微管的破坏。该提案具有三个具体目标。 1）第一个目标将测试假设促进PP2A靶向靶向的锚定蛋白微管是有效的tau和 MAP2。 PP2A与微管相互作用的分子基础将探索细胞骨架。假定的锚定蛋白将是纯化，特征和cDNA克隆。锚定在调节PP2A将通过直接的假设进行检查蛋白质 - 蛋白质相互作用定义了PP2A的特异性神经元图。 2）目标的基本假设2是完整动物神经元中PP2A的抑制将导致增加 tau和神经元变性的磷酸化。 SV40小型抗原， PP2A的特定抑制剂将在转基因小鼠中表达神经元特异性启动子。降低PP2A活性的影响将通过测量tau磷酸化和神经元形态来评估。 3）在此目的中要检验的假设是两个新型的PP2A- 相互作用蛋白会影响PP2A的活性和功能。 p31和p17的表征，两个pp2a相互作用蛋白在两个杂交屏幕上确定，将以高优先级进行。这些蛋白质对活性，亚细胞定位和将确定PP2A的体内功能。其他候选人在两个杂交屏幕上鉴定的相互作用蛋白也将是特征。