Nahas N, Molski T F, Fernandez G A, Sha'afi R I
Department of Physiology, University of Connecticut Health Center, Farmington 06030, USA.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):247-53. doi: 10.1042/bj3180247.
The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine-coupled receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha). The effect of low concentrations of PAF is greatly increased in cells pretreated with LPS. The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the protein translation blocker anisomycin. While anisomycin is known to increase the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated protein kinase), this is the first report that shows that anisomycin also tyrosine phosphorylates the p38 MAP kinase. Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those that do not affect the erk1 and erk2 MAP kinases. The possible role of the p38 MAP kinase in the phosphorylation of cytosolic phospholipase A2 is discussed.
已证实在人中性粒细胞（一种终末分化细胞）中，当用低浓度脂多糖（LPS）与血清联合刺激这些细胞时，存在一种新的38 kDa蛋白，该蛋白发生酪氨酸磷酸化。这种38 kDa蛋白被鉴定为酵母中HOG1的哺乳动物同源物，即p38丝裂原活化蛋白（MAP）激酶。这一结论基于以下实验结果：抗磷酸酪氨酸（抗PY）抗体免疫沉淀出一种能被抗p38 MAP激酶抗体识别的38 kDa蛋白，反之，抗p38 MAP激酶抗体免疫沉淀出一种能被抗PY抗体识别的38 kDa蛋白。此外，发现这种酪氨酸磷酸化蛋白完全与胞质溶胶相关。还发现，在用低浓度LPS与血清联合刺激这些细胞后，这种p38 MAP激酶被激活。这一结论基于三个实验结果。第一，从LPS刺激的细胞中分离出的可溶性组分在体外试验中使热休克蛋白27（hsp27）磷酸化，并且这种效应不受蛋白激酶C和蛋白激酶A抑制剂肽的抑制。这种效应类似于市售的磷酸化和活化的MAPKAP激酶-2（丝裂原活化蛋白激酶激活的蛋白激酶-2）产生的效应。第二，在用放射性磷酸盐预标记的完整人中性粒细胞经LPS刺激后，一种与抗hsp27抗体识别的蛋白相对应的27 kDa蛋白发生磷酸化。最后，使用[γ-32P]ATP和激活转录因子2（ATF2）作为底物的免疫复合物蛋白激酶试验显示，LPS刺激的人中性粒细胞中p38 MAP激酶活性增加。这种p38 MAP激酶的磷酸化和激活可受到诸如血小板活化因子（PAF）等G蛋白偶联受体以及诸如粒细胞-巨噬细胞集落刺激因子（GM-CSF）和肿瘤坏死因子α（TNF-α）的细胞因子偶联受体等非G蛋白偶联受体的影响。低浓度PAF的效应在用LPS预处理的细胞中大大增强。p38 MAP激酶的酪氨酸磷酸化不限于通过膜相关受体介导其作用的刺激，而是可受到诸如蛋白翻译阻断剂茴香霉素之类绕过膜相关受体的试剂的影响。虽然已知茴香霉素会增加54 kDa应激激活蛋白激酶（SAPK）的酪氨酸磷酸化，但这是首次表明茴香霉素也使p38 MAP激酶发生酪氨酸磷酸化的报告。增加erk1和erk2 MAP激酶酪氨酸磷酸化和激活的细胞因子受体对这种p38 MAP激酶的影响小于那些不影响erk1和erk2 MAP激酶的细胞因子受体。文中讨论了p38 MAP激酶在胞质磷脂酶A₂磷酸化中的可能作用。
