PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

神经丝是神经元特有的中间丝，是大型有髓轴突的主要细胞骨架成分。赖氨酸-丝氨酸-脯氨酸(KSP)重复序列位于高相对分子质量的核因子蛋白(NF-M和NF-H)的尾部，在体内被广泛的磷酸化，选择性地存在于神经元的轴突间。这种尾部区域的磷酸化被认为在调节神经元的特异性特性方面发挥了重要作用，包括轴突口径和传导速度。本实验室最近的研究表明，在体外，丝裂原激活的蛋白激酶(MAP激酶，或细胞外信号调节激酶，ERK1和ERK2)磷酸化来自于NF-M和NF-H尾部结构域的多肽底物上的KSP基序。然而，目前尚不清楚MAP激酶通路的激活是否能在体内将这些结构域磷酸化。为了回答这个问题，我们将活性形式的丝裂原激活的ERK激活蛋白(MEK1)与核因子-M表达载体共转染入NIH3T3细胞。激活的突变体诱导核因子-M的磷酸化，而不是显性的负性突变体。此外，在NIH3T3细胞中诱导MAP激酶级联的表皮生长因子(EGF)也激活了内源性ERK1和ERK2，并激活了NIH3T3细胞中的核因子-M尾部结构域磷酸化。这些结果直接证明，在体内激活ERK1和ERK2足以使转基因细胞中的核因子-M尾部结构域磷酸化。我们还证实，通过膜去极化激活内源性ERK1/2和通过L型钙通道的钙内流导致了PC12细胞中核因子-尾部结构域的磷酸化。这种磷酸化在L类钙通道阻滞剂硝苯地平和特异性MEK1抑制剂PD98059的存在下被抑制。这些研究表明，MAP激酶信号转导与神经细丝的磷酸化有关。这些发现为神经丝磷酸化的机制提供了重要的新见解。-神经细丝，磷酸化，MAP激酶，局部，调节，磷酸酶

Neurofilaments (NFs) are neuron-specific intermediate filaments, and are the major cytoskeletal component in large myelinated axons. Lysine- serine-proline (KSP) repeats in the tail domains of high molecular weight NF proteins (NF-M and NF-H) are extensively phosphorylated in vivo, selectively in the axonal compartment of neurons. This phosphorylation in the tail domain has been postulated to play an important role in mediating neuron-specific properties, including axon caliber and conduction velocity. Recent studies from our laboratory have shown that the mitogen-activated protein kinases (MAP kinases, or extracellular signal regulated kinases, Erk1 and Erk2) phosphorylate KSP motifs in peptide substrates derived from the NF-M and NF-H tail domains in vitro. However, it is not clear whether activation of the MAP kinase pathway can phosphorylate these domains in vivo. To answer this question, a constitutively active form of mitogen-activated Erk activating kinase (MEK1) was cotransfected with an NF-M expression construct into NIH 3T3 cells. The activated mutant, but not the dominant negative mutant, induced phosphorylation of NF-M. In addition, it was shown that epidermal growth factor (EGF), which induced the MAP kinase cascade in NIH 3T3 cells, also activated endogenous ERk1 and Erk2 and NF-M tail domain phosphorylation in the transfected cells. These results present direct evidence that in vivo activation of Erk1 and Erk2 is sufficient for NF-M tail domain phosphorylation in transfected cells. We have also demonstrated that activation of endogenous Erk1/2 by membrane depolarization and calcium influx through L-type calcium channels resulted in phosphorylation of the NF-tail domain in PC12 cells. This phosphorylation was inhibited in the presence of nifedipine, an L-type calcium channel blocker, and PD98059, a specific MEK1 inhibitor. These studies suggest a mechanism linking MAP kinase signal transduction to phosphorylation of neurofilaments. These findings provide significant new insights into mechanisms involved in neurofilament phosphorylation. - neurofilaments, phosphorylation, MAP kinases, topographic,regulation,phosphatases