Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils
Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils
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DOI：
10.4049/jimmunol.174.11.7257
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发表时间：
2005-06-01
影响因子：
4.4
通讯作者：
McLeish, KR
中科院分区：
文献类型：
--
作者：
Lominadze, G;Rane, MJ;McLeish, KR
介导中性粒细胞功能反应的p38MAPK通路的靶点在很大程度上是未知的。为了确定p38MAPK靶点，我们应用了一种蛋白质组学方法，将重组的活性p38MAPK和[P-32]ATP加入到未受刺激的人中性粒细胞的裂解物中。用双向凝胶电泳法分离蛋白质，用放射自显影和MALDI-TOF鉴定磷蛋白。髓系相关蛋白-14(MRP-14)是p38MAPK的候选底物。纯化的MRP-14/MRP-8复合体的体外激酶反应证实了p38MAPK对MRP-14的磷酸化作用。经串联质谱仪和定点突变鉴定，MRP-14被p38MAPK磷酸化的位点为Thr(113)。在p38 MAPK抑制剂SB203580存在和不存在的情况下，通过[P-32]正磷酸负载证实完整中性粒细胞中的MRP-14被p38 MAPK磷酸化，随后fMLP刺激。Triton X-100渗透性中性粒细胞共聚焦显微镜显示，未刺激的细胞中有少量MRP-14与皮质F-肌动蛋白相关。FMLP刺激导致片状脂基底部p38MAPK依赖的MRP-14染色增加。免疫印迹分析显示，MRP-14存在于细胞质膜/分泌囊泡、明胶酶和特定颗粒中，而不存在于嗜天青颗粒中。经fMLP刺激后，质膜/分泌囊泡和明胶酶颗粒组分的MRP-14含量增加，且依赖于p38MAPK。P38MAPK直接磷酸化MRP-14/MRP-8复合体，使体外肌动蛋白结合量增加2倍。这些结果表明，MRP-14是人中性粒细胞p38MAPK依赖的功能反应的潜在介体。
The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [P-32]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [P-32]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.