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

通过磷酸化修饰细胞蛋白质被认为是外部刺激控制细胞内事件的主要调控机制。我们证明了细菌脂多糖(LPS)诱导小鼠腹腔巨噬细胞中一组独特的磷酸化蛋白(PP)，并且这些蛋白特异性地定位于胞浆和/或膜组分。通过Sephacryl S-200层析、阴离子交换层析和羟基磷灰石高效液相色谱分离纯化得到一种磷酸化程度最高的底物蛋白，其相对分子质量为65 kDa(Pp65)，经SDSPAGE分析和放射自显影分析，均一。我们的pp65显然是第一个纯化的内毒素诱导的PP。对pp65蛋白氨基酸序列的分析表明，pp65蛋白是一个与人纤溶酶原蛋白同源的新蛋白。Pp65上的丝氨酸残基被发现完全被磷酸化，这表明内毒素诱导的丝氨酸激酶(S)起到了作用。有趣的是，在脂多糖无反应的C3H/HeJ品系小鼠的巨噬细胞中没有观察到内毒素诱导的pp65的磷酸化，尽管他们的巨噬细胞含有与正常巨噬细胞相同数量的非磷酸化的p65，用多克隆抗pp65抗体进行Western印迹分析。这表明在pp65磷酸化之前，C3H/HeJ巨噬细胞的功能缺陷存在于某个过程中。综上所述，pp65似乎在巨噬细胞激活中起着至关重要的作用，pp65的结构和功能应该有助于我们理解内毒素激活巨噬细胞的机制。

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.