Purification and Characterization of Phosphoproteins which are Induced in Murine Macrophages Stimulated with 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）诱导一组不同的磷酸化蛋白（PP）在小鼠腹腔巨噬细胞，和LPS诱导的PP具体位于胞质溶胶和/或膜组分。通过SDS-PAGE分析和Sephacryl S-200上的连续层析、HPLC阴离子交换和羟基磷灰石HPLC的放射自显影将分子量为65 kDa（pp 65）的最严重磷酸化的底物蛋白之一纯化至均一。我们的pp 65显然是第一个纯化的LPS诱导的pp。pp 65的蛋白质氨基酸序列的分析表明，pp 65是一种新的蛋白质，具有同源性与人类的plastin。发现pp 65上的丝氨酸残基仅被磷酸化，表明LPS诱导的丝氨酸激酶的贡献。有趣的是，LPS诱导的磷酸化的pp 65没有观察到在巨噬细胞从一个LPS无反应的C3 H/HeJ小鼠株，虽然他们的巨噬细胞有大约相同数量的非磷酸化的p65作为正常的巨噬细胞whnn下检测到的蛋白质印迹分析使用多克隆抗pp 65抗体。这表明C3 H/HeJ巨噬细胞在pp 65磷酸化之前的过程中存在功能缺陷。考虑到这些观察结果，pp 65似乎在巨噬细胞活化中起着至关重要的作用，pp 65的结构和功能应该导致我们对LPS激活巨噬细胞机制的理解取得进展。

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.