PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

我们在了解神经丝的结构和功能方面的进展， (NFs)，它们的磷酸化，并确定特定的激酶， 所涉及的磷酸酶如下：（1）我们已经表明，第二， 信使依赖性蛋白激酶（PK）磷酸化头部结构域 和第二信使非依赖性PKs，酪蛋白激酶I和II， 与神经丝相关的微管相关PK样活性 制备磷酸化C-末端尾部结构域中的丝氨酸残基 神经丝蛋白（NFP）在体外，但不是多个重复 在中（NF~M）和高（NF~H）NFP中存在lys~ser~pro（KSP）基序。(2)的 KSP重复序列的磷酸化状态的分析， 化学和酶消化的组合，反相 高压色谱法、Edman微量测序和电喷雾 质谱分析表明，NF~H中的大部分KSP基序是 在体内磷酸化;含有不间断KSP的结构域 重复序列对蛋白质水解具有高度抗性，并且可以在 去磷酸化(3)我们已经从大鼠脊髓中鉴定并分离出 编码磷酸化特定KSP序列的蛋白激酶（KSPXK） 在NF~M和NF~H中。 这种酶的特性揭示了一个密切的 与细胞周期依赖性激酶（CDK）的关系最密切， CDK5。从大鼠脊髓中分离纯化CDK 5样激酶， 显示它与62 kDa的蛋白质（p62）强烈相关。 从激酶中分离出这种蛋白质， 这种激酶活性的降低可以通过增加 纯化的p62。 推导了p62的氨基酸序列 从大鼠脑文库中的大量cDNA克隆中。没有相似性 与当前蛋白质序列数据库中的任何已知蛋白质一起存在。 小鼠胚胎和成体的Ribo~探针原位杂交实验 已经证明p62转录本的表达在早期就开始了， 发育，仅限于神经系统， 在神经元中表达，在周围胶质细胞中不表达。 这些研究 提示CDK样激酶及相关调节因子（p62）参与了 在NF~M和NF~H的KSP位点的磷酸化中， 神经元的生长和分化以及轴突的稳定性 结构. (4)我们还证明， CDK 5样激酶介导的NF~H被蛋白磷酸酶2A去磷酸化; 并在大鼠脊髓中发现了这种磷酸酶。

Our progress to understand the structure and function of neurofilaments, (NFs), their phosphorylation and to identify the specific kinases and phosphatases involved is as follows: (1) We have shown that second messenger~dependent protein kinases (PK) phosphorylate the head domains and second messenger~independent PKs, casein kinase I and II and microtubule~associated PK~like activities associated with neurofilament preparation phosphorylate serine residues in the C~terminal tail domain of neurofilament proteins (NFPs) in vitro but not the multiple repeat lys~ser~pro (KSP) motifs in middle (NF~M) and high (NF~H) NFPs. (2) The analysis of the phosphorylation state of KSP repeats by means of a combination of chemical and enzymatic digestion, reverse phase high~pressure chromatography, Edman microse~quencing and electrospray mass spectrometry showed that most of the KSP motifs in NF~H are phos~phorylated in vivo; and that the domain containing uninterrupted KSP repeats is highly resistant to proteolysis and can be proteolysed after dephosphorylation. (3) We have identified and isolated from rat spinal cord a protein kinase that phosphorylates a specific KSP sequence (KSPXK) in NF~M and NF~H. Characterization of this enzyme revealed a close relationship to the cell~cycle dependent kinases (CDK), most closely to CDK5. Purification of this CDK5~like kinase from rat spinal cord has shown that it is strongly associated with a protein of 62 kDa (p62). Separation of this protein from the kinase resulted in a considerable decrease in this kinase activity which could be restored by adding back the purified p62. The complete amino acid sequence of p62 was deduced from a number of cDNA clones from rat brain libraries. No similarity exists with any known protein in the current protein sequence data banks. Ribo~probe in situ hybridization experiments of mouse embryos and adults have demonstrated that p62 transcript expression begins early in development and is restricted to the nervous system, exclusively expressed in neurons and absent from surrounding glia. These studies suggest that CDK~like kinases and related regulators (p62) are involved in phosphorylation of KSP sites in NF~M and NF~H, and may be involved in neuronal growth and differentiation as well as in the stability of axonal structures. (4) We have also demonstrated that the phosphorylation of NF~H by CDK5~like kinase is dephosphorylated by protein phosphatase 2A; and identified such a phosphatase in the rat spinal cord.