PROLINE DIRECTED KINASES IN ALZHEIMERS DISEASE

这项建议的长期目标是了解病原学和 阿尔茨海默病(AD)的发病机制。越来越多的证据表明 在阿尔茨海默病中，几种神经元蛋白的磷酸化状态发生改变。 这种异常的磷酸化显然会导致不同的 细胞功能，从而最终导致神经元死亡。尤其是 微管相关蛋白tau已被证明存在于 AD患者成对螺旋丝(PHF)中的过度磷酸化状态 大脑。最近的证据表明，PHF tau的过度磷酸化 可能在很大程度上被催化。但并不是唯一的，是由脯氨酸引导的蛋白质 蛋白激酶(PDPKs)。目前已知的PDPKs有三种：MAP激酶、CDC2激酶、 和糖原合成酶激酶3。PDPKs和非PDPKs的身份 负责体内PHF tau过度磷酸化的是目前 不清楚。这项建议的具体目的是：1)比较 三种PDPKs对tau的磷酸化作用。磷酸化动力学 而tau上的磷酸化位点将通过肽图谱和 通过不同的磷酸化位点特异性的抗tau抗体；2) 研究非PDPKs在tau过度磷酸化中的作用。六个非- PDPKs将被用来制备六种磷酸化tau。这个 将比较PDPKs对这些tau物种的进一步磷酸化作用； 3)使用不同的含有已知磷酸化的合成肽 Tau的位点来确定负责的蛋白激酶的身份 用于它们的磷酸化；以及4)测量PDPKs的水平。 对照组和阿尔茨海默病患者大脑的胞浆和颗粒部分。两者都有 将测量激活度和激活度蛋白水平。这些研究 应该有助于澄清负责这一事件的激酶(S)的身份 PHF tau的过度磷酸化。

The long term objective of this proposal is to understand the etiology and pathogenesis of Alzheimer disease (AD). Increasing evidence suggests that in AD the phosphorylation state of several neuronal proteins are altered. Such abnormal phosphorylation apparently lead to a disruption of different cellular functions and hence eventual death of neurons. In particular the microtubule-associated protein tau has been shown to be present in a hyperphosphorylated state in the paired helical filaments (PHF) of AD brain. Recent evidence suggests that the hyperphosphorylation of PHF tau may be catalyzed largely. but not exclusively, by proline-directed protein kinases (PDPKs). There are three well known PDPKs: MAP kinase, cdc2 kinase, and glycogen synthase kinase 3. The identities of the PDPKs and non-PDPKs responsible for the in vivo hyperphosphorylation of PHF tau are presently unclear. The specific aims of this proposal are: 1) To compare the phosphorylation of tau by the three PDPKs. The kinetics of phosphorylation and the sites phosphorylated on tau will be compared by peptide mapping and by different phosphorylation site-specific anti-tau antibodies; 2) To investigate the role of non-PDPKs in tau hyperphosphorylation. Six non- PDPKs will be used to prepare six species of phosphorylated tau. The further phosphorylation of these tau species by the PDPKs will be compared; 3) To use different synthetic peptides containing known phosphorylation sites of tau to establish the identities of the protein kinases responsible for their phosphorylation; and 4) To measure the levels of the PDPKs in both the cytosol and particulate fractions of control and AD brains. Both kinase activity and kinase protein levels will be measured. These studies should help clarify the identities of the kinase(s) responsible for the hyperphosphorylation of PHF tau.