Weinstein S L, Gold M R, DeFranco A L
Department of Microbiology and Immunology, George Williams Hooper Foundation, San Francisco, CA.
Proc Natl Acad Sci U S A. 1991 May 15;88(10):4148-52. doi: 10.1073/pnas.88.10.4148.
Lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, stimulates immune responses by activating macrophages, B lymphocytes, and other cells of the immune system. The mechanisms by which LPS activates these cells are poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signaling event that mediates cellular responses, we examined whether LPS alters tyrosine phosphorylation in macrophages. We found that Escherichia coli K235 LPS increased tyrosine phosphorylation of several proteins in the RAW 264.7 murine macrophage cell line and in resident peritoneal macrophages from C3H/HeSNJ mice. Changes in tyrosine phosphorylation were detectable by 4-5 min, reached a maximum by 15 min, and declined after 30-60 min. Protein tyrosine phosphorylation increased following stimulation with LPS at 100 pg/ml and was maximal with 10 ng/ml. Similar changes in tyrosine phosphorylation were induced by Salmonella minnesota R595 LPS and by the biologically active domain of LPS, lipid A, but not by the inactive lipid A derivative N2-monoacylglucosamine 1-phosphate. Phorbol 12-myristate 13-acetate also stimulated protein tyrosine phosphorylation, but some of the modulated proteins were different than those phosphorylated by LPS. Treatment of RAW 264.7 cells with a tyrosine kinase inhibitor, herbimycin A, inhibited both LPS-stimulated tyrosine phosphorylation and LPS-stimulated release of arachidonic acid metabolites. Thus, increased protein tyrosine phosphorylation is a rapid LPS-activated signaling event that may mediate release of arachidonic acid metabolites in RAW 264.7 cells.
脂多糖（LPS）是革兰氏阴性菌的一种膜成分，通过激活巨噬细胞、B淋巴细胞和免疫系统的其他细胞来刺激免疫反应。LPS激活这些细胞的机制目前还不清楚。由于蛋白酪氨酸磷酸化似乎是介导细胞反应的主要细胞内信号事件，我们研究了LPS是否会改变巨噬细胞中的酪氨酸磷酸化。我们发现，大肠杆菌K235 LPS增加了RAW 264.7小鼠巨噬细胞系和C3H/HeSNJ小鼠腹腔巨噬细胞中几种蛋白质的酪氨酸磷酸化。酪氨酸磷酸化的变化在4-5分钟时可检测到，15分钟时达到最大值，30-60分钟后下降。用100 pg/ml的LPS刺激后，蛋白酪氨酸磷酸化增加，10 ng/ml时达到最大值。明尼苏达沙门氏菌R595 LPS和LPS的生物活性结构域脂多糖A也诱导了类似的酪氨酸磷酸化变化，但无活性的脂多糖A衍生物N2-单酰氨基葡萄糖1-磷酸没有这种作用。佛波醇12-肉豆蔻酸酯13-乙酸盐也刺激蛋白酪氨酸磷酸化，但一些被调节的蛋白质与LPS磷酸化的蛋白质不同。用酪氨酸激酶抑制剂赫比霉素A处理RAW 264.7细胞，可抑制LPS刺激的酪氨酸磷酸化和LPS刺激的花生四烯酸代谢产物的释放。因此，蛋白酪氨酸磷酸化增加是LPS快速激活的信号事件，可能介导RAW 264.7细胞中花生四烯酸代谢产物的释放。
