PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

神经丝（nf）是神经元特异性的中间丝，是大髓鞘轴突的主要细胞骨架成分。在体内，高分子量NF蛋白（NF- m和NF- h）尾部区域的赖氨酸-丝氨酸-脯氨酸（KSP）重复序列在神经元轴突室中被选择性地广泛磷酸化。这种尾部区域的磷酸化被认为在介导神经元特异性特性（包括轴突直径和传导速度）中起重要作用。我们实验室最近的研究表明，有丝分裂原活化蛋白激酶（MAP激酶，或细胞外信号调节激酶，Erk1和Erk2）在体外磷酸化NF-M和NF-H尾结构域衍生的肽底物中的KSP基序。然而，目前尚不清楚MAP激酶途径的激活是否能在体内磷酸化这些结构域。为了回答这个问题，我们将有丝分裂原激活的Erk激活激酶（MEK1）的组成活性形式与NF-M表达构建体共转染到NIH 3T3细胞中。激活突变体，而不是显性阴性突变体，诱导NF-M磷酸化。此外，研究表明，在NIH 3T3细胞中诱导MAP激酶级联的表皮生长因子（EGF）也激活了转染细胞内源性ERk1、Erk2和NF-M尾结构域磷酸化。这些结果提供了直接证据，证明Erk1和Erk2的体内激活足以在转染细胞中实现NF-M尾部结构域磷酸化。我们还证明了内源性Erk1/2通过膜去极化和钙通过l型钙通道内流激活，导致PC12细胞nf -尾结构域磷酸化。这种磷酸化在硝苯地平（一种l型钙通道阻滞剂）和PD98059（一种特异性MEK1抑制剂）的存在下被抑制。这些研究提示了MAP激酶信号转导与神经丝磷酸化的联系机制。这些发现为神经丝磷酸化的机制提供了重要的新见解。-神经丝，磷酸化，MAP激酶，地形学，调控，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