PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

神经元系统中蛋白质磷酸化和细胞骨架的调节

• 批准号: 3760272
• 负责人: H C PANT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3760272
• 关键词: animal tissue; cell cycle proteins; cerebellar Purkinje cell; cytoskeleton; developmental neurobiology; gene expression; genetic transcription; immunocytochemistry; in situ hybridization; molecular weight; neurofilament; neurofilament proteins; phosphoprotein phosphatase; phosphorylation; protein kinase; protein sequence; protein structure function

We are studying nervous system specific kinases and phosphatases to understand the role of neurofilament phosphorylation. Previously, we have shown that second messenger dependent and independent kinases can phosphorylate the neurofilament proteins (NFPs) in vitro; however, the kinases involved in phosphorylation in vivo are unknown. In order to determine the kinases involved in this phosphorylation, we have analyzed the endogenous phosphorylation sites in carboxy-terminal tail domain of high molecular weight neurofilament protein (NF-H), where most of the in vivo phosphorylation takes place. These studies showed that serines (s) in all three KSP motifs: KSPXK, KSPXXK and KSPXXXK are phosphorylated. Cyclin-dependent kinase, Cdk-5, from rat spinal cord or bovine brain, specifically phosphorylated KSPXK motifs. Purification of this kinase from rat spinal cord showed that it is associated with a protein of 67KDa (p67). Removal of this protein from the complex resulted in a considerable loss of the kinase activity which could be restored by adding back the purified or bacterially expressed p67. The complete amino acid sequence of p67 deduced from a number of cDNA clones from rat brain cDNA libraries appeared to be identical to Munc-18, a synaptic vesicle complex protein. The p67 transcript expression begins early in development and is restricted to the nervous tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody indicated that p67 is expressed exclusively in neurons. Localization in brain tissue and in cultured rat hippocampal neurons demonstrated that p67 is highly enriched in axons. We also investigated the expression of cdk-5, p67 and phosphorylated NF-H during development of the rat cerebellum from postnatal day 2 to adult using specific antibodies to these molecules. We could demonstrate that at all stages the epitopes for Cdk-5 and phosphorylated NF-H were colocalized in developing afferent fibers (mossy and climbing fibers) as well as basket cell fibers surrounding the Purkinje cells. Purkinje cell bodies, on the other hand, contained high levels of dephosphorylated NF-H and NF-M. These results are consistent with the hypothesis that cdk5 regulates the phosphorylation of some of the KSP motifs in neurofilament proteins.

我们正在研究神经系统特异性激酶和磷酸酶， 了解神经丝磷酸化的作用。此前，我们有 显示第二信使依赖性和非依赖性激酶可以 在体外磷酸化神经丝蛋白（NFP）;然而， 参与体内磷酸化的激酶是未知的。为了 确定参与这种磷酸化的激酶，我们分析了 内源性磷酸化位点的羧基末端尾部结构域 高分子量神经丝蛋白（NF-H），其中大部分的神经丝蛋白（NF-H）， 发生体内磷酸化。 这些研究表明，丝氨酸（S） 在所有三个KSP基序中： KSPXK、KSPXXK和KSPXXXK被磷酸化。 细胞周期蛋白依赖性激酶，Cdk-5，来自大鼠脊髓或牛脑， 特别是磷酸化的KSPXK基序。 该激酶的纯化 从大鼠脊髓中分离到一个67 KDa的蛋白质 （第67页）。 从复合物中去除这种蛋白质导致 激酶活性的相当大的损失，可以通过 再加入纯化的或细菌表达的p67。 完整的 大鼠p67基因cDNA克隆的氨基酸序列分析 脑cDNA文库似乎与Munc-18相同， 囊泡复合蛋白p67转录本的表达开始于 发育，仅限于神经组织。免疫组 用氨基末端肽特异性抗体染色表明， p67仅在神经元中表达。 脑组织定位 在体外培养的大鼠海马神经元中，p67在海马神经元中的表达水平明显高于对照组。 富含轴突 我们还研究了cdk-5，p67和 磷酸化NF-H在大鼠小脑发育过程中的作用 从出生后第2天到成年，使用针对这些分子的特异性抗体。 我们可以证明，在所有阶段，Cdk-5和 磷酸化NF-H共定位于发育中的传入纤维（苔藓纤维）中， 和攀缘纤维）以及周围的篮状细胞纤维。 浦肯野细胞。另一方面，浦肯野细胞体含有高浓度的 去磷酸化NF-H和NF-M的水平。这些结果是一致 假设CDK 5调节一些 神经丝蛋白中的KSP基序。