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是一种主要的调节机制，外部刺激控制细胞内事件。我们证明了细菌脂多糖（LPS）在鼠腹膜巨噬细胞中诱导了一组独特的磷酸化prtein（PP），并且LPS诱导的PP专门位于细胞质和/或膜级分中。通过SDS-PAGE分析和SEPHACRYL S-200，HPLC Anion Exchange和Hydroxyapatite HPLC，通过SDS-PAGE分析和放射自显影将最高磷酸化的底物蛋白之一纯化为65KDA（PP65）（PP65）（PP65）。我们的PP65显然是第一个纯化的LPS诱导的PP。对PP65的蛋白氨基酸序列的分析表明，PP65是一种与人类塑料同源的新型蛋白质。发现PP65上的丝氨酸残基被完全磷酸化，表明LPS诱导丝氨酸激酶（S）的贡献。有趣的是，尽管LPS-非反应性C3H/HEJ菌株的巨噬细胞中未观察到LPS诱导的PP65的磷酸化，尽管它们的巨噬细胞具有正常的巨噬细胞，其巨噬细胞的巨噬细胞具有正常的巨噬细胞，该巨噬细胞是正常的巨噬细胞，在使用Polyclonal Anti-Ppp65 antibibys anti-Ppp65 antibibsies中，在正常的巨噬细胞中被检测到WHNN。这表明C3H/HEJ巨噬细胞的功能缺陷存在于PP65磷酸化之前的某个地方。考虑到这些观察，PP65似乎在巨噬细胞激活中起着至关重要的作用，PP65的结构和功能应导致我们对LPS巨噬细胞激活机制的理解。