PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

神经丝蛋白虽然是在神经元胞体中合成的， 通常在翻译后的轴突中被磷酸化。在一些 然而，神经退行性疾病(例如，肌萎缩侧索硬化症)是不正常的 在细胞体中过度磷酸化。我们一直在研究这些因素 调节神经丝(NF)的这些拓扑型 磷酸化。我们实验室发现了一种名为CDK5的激酶， 特异性地磷酸化KSPXK基序，它占到 高分子量核因子亚基的总重复数，核因子-H。剩下的 80%的重复序列是KSPXXXK基序激酶(S)使这些基序磷酸化 都是未知的。一种人工合成的KSPXXXK肽，KSPAEAKSPAAAKS，它 使用在非KSP残基上有微小变化的重复41次作为 一种鉴定大鼠脑中磷酸化核因子-2的激酶的底物 H.在生化、免疫化学、药理学和 氨基酸序列分析表明，纯化后的蛋白为MEK- 激活的MAP激酶。为了验证这一说法，我们演示了 细菌表达的MAP激酶磷酸化表达的大鼠核因子-H 添加KSPXXXK、KSPXXK和KSPXK多肽。这项研究表明 神经元型MAPK能磷酸化大鼠神经营养因子-H和 可能是体内使丝氨酸残基磷酸化的主要激酶 在KSP中，在神经丝尾部区域重复。由于CDK5特别是 磷酸化KSPXK多肽而不是KSPXXXK，我们研究了 通过分析构象，这些基元的结构差异 四个具有KSPXK或KSPXXXK基序的多肽。KSPXXXK 多肽的CD光谱表明为螺旋构象； 在相同条件下，KSPXK多肽主要表现为 扩展的贝塔转角构象。这些差异揭示了一个 该酶底物的结构特异性。使用两个 三维核磁共振方法和分子模拟。 我们还在研究CDK5的激活机制。不过，CDK5是 与其他cdc2激酶类似，它的调节完全不同。 来自有丝分裂的cdc2激酶；例如，cdk5只在有丝分裂后才活跃。 它的活性不依赖于它的磷酸化，它的活性也不是 受细胞周期蛋白结合调节的活性。相反，神经元的联想 特定的蛋白质P35和P67负责其激活。 这些调节蛋白与细胞周期蛋白没有同源性。我们已经开始了 使用各种突变定位P35中的激活域 P35基因在体外实验中的截断。这些研究表明， 激酶激活的潜在残基位于氨基酸残基中 N-末端和C-末端的尾区分别为137-167和291-263 35页。进一步的突变和截断研究正在进行中。

Neurofilament (NF) proteins, though synthesized in neuronal cell bodies, are normally posttranslationally phosphorylated in axons. In some neuro-degenerative disorders (e.g., ALS), however, they are abnormally hyperphosphorylated in cell bodies. We have been studying the factors regulating these topographic pattern of neurofilament (NF) phosphorylation. One kinase, cdk5, identified in our laboratory, specifically phosphorylates KSPXK motifs which constitutes 20% of the total repeats in high molecular weight NF-subunit, NF-H. The remaining 80% repeats are KSPXXXK motifs kinase(s) phosphorylating these motifs are not known. A synthetic KSPXXXK peptide, KSPAEAKSPAEAKS, which repeats 41 times with minor variations at non-KSP residues was used as a substrate to identify the kinase in rat brain that phospshorylates NF- H. On the basis of biochemical, immunochemical, pharmacological and amino acid sequence analysis, the purified kinase appeared to be MEK- activated MAP kinase. To verify this statement, we demonstrated that bacterially expressed MAP kinase phosphorylated expressed rat NF-H in addition to KSPXXXK, KSPXXK and KSPXK peptides. This study suggests that neuronal MAPK can phosphsorylate all KSP repeats in rat NF-H and may be the principal kinase in vivo that phosphorylates serine residues in KSP repeats in neurofilament tail domains. Since cdk5 specifically phosphorylates KSPXK peptides and not KSPXXXK, we investigated the structural differences of these motifs, by analyzing the conformation of four peptides with either KSPXK or KSPXXXK motifs. The KSPXXXK peptide exhibited a CD spectrum indicative of a helical conformation; under the same conditions, however, the KSPXK peptide showed a mainly extended conformation with beta-turns. These differences revealed a structural specificity for the substrate of this kinase. using two dimensional NMR methods and molecular modeling. We are also studying the mechanism of cdk5 activation. Though, cdk5 is similar to other cdc2 kinases, its regulation is completely different from mitotic cdc2 kinases; e.g., cdk5 is active only in postmitotic neurons, its activity is independent of its phosphorylation, nor is its activity regulated by cyclin binding. Instead association of neuron specific proteins, P35 and P67 are responsible for its activation. These regulator proteins share no homology with cyclins. We have begun to locate the activation domains in P35 using various mutations and truncations of P35 in in vitro assays. These studies suggest the potential residues for kinase activation reside in amino acid residues 137-167 and 291-263 of N- and C-terminal tail domains respectively of P35. Further mutational and truncational studies are in progress.