Purification and Characterization of Phosphoproteins which are Induced in Murine Macrophages Stimulated with LPS.

LPS 刺激的鼠巨噬细胞诱导的磷蛋白的纯化和表征。

基本信息

批准号：
01570243
负责人：
SHINOMIYA Hiroto
金额：
$ 1.15万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial lipopolysaccharide (LPS) induced a distinct set of phosphorylated prtein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp. Analysis of the protein amino acid sequence of the pp65 revealed that pp65 is a novel protein having homology with human plastin. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase(s). Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages whnn detected under Western blot analysis using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Considering these observation, the pp65 seems to play a crucial role in macrophage activation, and the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.

已知通过磷酸化修饰细胞蛋白质是外部刺激控制细胞内事件的主要调节机制。我们证明细菌脂多糖（LPS）在小鼠腹腔巨噬细胞中诱导一组独特的磷酸化蛋白质（pp），并且LPS诱导的pp特异性地位于细胞质和/或膜部分中。分子量为 65kDa (pp65) 的最严重磷酸化的底物蛋白之一通过 SDS-PAGE 分析和放射自显影法通过 Sephacryl S-200 上的顺序色谱、HPLC 阴离子交换和羟基磷灰石 HPLC 纯化至同质。我们的pp65显然是第一个纯化的LPS诱导的pp。对pp65的蛋白质氨基酸序列的分析表明，pp65是一种与人塑性蛋白具有同源性的新型蛋白质。发现 pp65 上的丝氨酸残基完全被磷酸化，表明 LPS 诱导型丝氨酸激酶的贡献。有趣的是，在来自 LPS 无反应的 C3H/HeJ 品系小鼠的巨噬细胞中没有观察到 LPS 诱导的 pp65 磷酸化，尽管使用多克隆抗 pp65 进行蛋白质印迹分析时，其巨噬细胞具有与正常巨噬细胞大致相同数量的未磷酸化 p65抗体。这表明C3H/HeJ巨噬细胞的功能缺陷存在于pp65磷酸化之前的过程中。考虑到这些观察结果，pp65 似乎在巨噬细胞激活中发挥着至关重要的作用，pp65 的结构和功能应该有助于我们对 LPS 激活巨噬细胞机制的理解取得进展。