NEURONAL MAP INTERACTIONS WITH MICROTUBULES

MAP-2a和MAP-2b是高分子量神经元微管， 与树突状微管（MT）相互作用的相关蛋白，以及 MAP-2c是一种相应的低分子量MAP， 发育中的轴突 这两种蛋白质都具有高度保守的N-末端， C-末端区域分别为约150和310个氨基酸， 后者含有三个18个氨基酸的重复序列， 以构成MT结合结构域。 我们建议进一步定义 MT结合片段的微管结合相互作用，依赖于 新的程序，准备片段和建设我们的研究， 三个不同的重复氨基酸区域。 我们还计划 表征完整MAP-2的m2-肽置换， 微管 为了确定负责 肽-M2与微管的结合并评估最小序列 需要促进小管组装和/或MAP-2置换。 我们计划 微管长度再分布动力学研究 在不存在或存在肽-M2的情况下由纯微管蛋白组装而成。 我们建议完成的氨基酸序列的测定 使用PCR衍生的牛脑cDNA的牛MT结合片段 克隆 在一个平行的努力，我们将研究磷酸化的 牛MAP-2的微管结合片段，包括：（a）评价 磷酸化微管结合片段对微管的亲和力 肽置换测定;（B）m2-肽的表征 使用直接合成的M2类似物的磷酸化作用 （c）磷酸化位点的定位 在使用酶和化学切割的MT结合片段中， 然后进行碱性梯度电泳， 磷酸酶处理 最后，我们将进行显微注射 使用MT结合片段和合成肽的实验， 研究它们在置换MAP-2和tau蛋白中的功效，以及 它们在改变MAP-2和tau的细胞分布中的作用，以及 细胞形态的任何变化。

MAP-2a and MAP-2b are high-molecular-weight neuronal microtubule- associated proteins that interact with dendritic microtubules (MTs), and MAP-2c is a corresponding lower-molecular-weight MAP abundant in developing axons. Both proteins share highly conserved N-terminal and C-terminal regions of about 150 and 310 amino acids, respectively, with the latter containing the three 18-amino-acid repeated sequences thought to constitute the MT-binding domain. We propose to further define microtubule binding interactions of the MT-binding fragment, relying on new procedures for preparing the fragment and building on our studies of the three non-identical repeated amino-acid regions. We also plan to characterize m2-peptide displacement of intact MAP-2 from assembled microtubules. To define the amino acid side-chains responsible for binding of peptide-m2 to microtubules and to assess the minimal sequence needed to promote tubule assembly and/or MAP-2 displacement. We plan to study microtubule length redistribution kinetics of microtubules assembled from pure tubulin in the absence or presence of the peptide-m2. We propose to complete the determination of the amino acid sequence of the bovine MT-binding fragment using PCR-derived bovine brain cDNA clones. In a parallel effort, we will investigate phosphorylation of tubule-binding fragment of bovine MAP-2, including: (a) evaluation of the affinity of phosphorylated-tubule-binding fragment for microtubules with the peptide displacement assay; (b) characterization of m2-peptide phosphorylation effects using direct synthesis of m2 analogues containing p-Ser and p-Thr residues; and (c) localization of phosphorylated sites in the MT-binding fragment using enzymatic and chemical cleavage, followed by the diagonal electrophoresis with intervening alkaline phosphatase treatment. Finally, we will carry out microinjection experiments using the MT-binding fragment and synthetic peptides to investigate their efficacy in displacing MAP-2 and tau proteins, and their action in altering cellular distribution of MAP-2 and tau, as well as any changes in cell morphology.