Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate:: Role in dimer formation and ligand binding
Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate:: Role in dimer formation and ligand binding
复制标题
DOI：
10.1128/mcb.23.15.5376-5387.2003
复制
发表时间：
2003-08-01
影响因子：
5.3
通讯作者：
McLeish, KR
中科院分区：
文献类型：
--
作者：
Powell, DW;Rane, MJ;McLeish, KR
丝裂原活化蛋白激酶（MAPK）活化蛋白激酶2（MAPKAPK 2）介导多种p38 MAPK依赖性炎症反应。为了确定MAPKAPK 2激活的信号转导途径，我们通过使用功能蛋白质组学方法鉴定了潜在的MAPKAPK 2底物，该方法包括：通过活性重组MAPKAPK 2体外磷酸化中性粒细胞裂解物，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离蛋白质（SDS-PAGE），基质辅助激光解吸电离质谱肽质量指纹鉴定磷蛋白（MALDI-MS）和蛋白质数据库分析。八种候选MAPKAPK 2底物之一是衔接蛋白14-3- 3 zeta。我们证实MAPKAPK 2在体外和HEK 293细胞中与14-3- 3 zeta相互作用并磷酸化。化学引诱物甲酰-甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）刺激人中性粒细胞中14-3- 3 zeta蛋白的p38-MAPK依赖性磷酸化。突变分析显示MAPKAPK 2在Ser-58处磷酸化14-3- 3 zeta。计算模型和理论结合能的计算预测，在Ser-58的磷酸化和Ser-58突变为Asp（S58 D）都损害了14-3- 3 zeta二聚化的能力。实验上，S58 D突变显著损害14-3- 3 zeta二聚化和与Raf-1的结合。这些数据表明，MAPKAPK 2介导的磷酸化调节14-3- 3 zeta功能，这种MAPKAPK 2活性可能代表一种新的途径介导p38 MAPK依赖性炎症。
Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3zeta. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3zeta in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylaianine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3zeta proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3zeta at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3zeta to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3zeta dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3zeta functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.