Huang C K, Zhan L, Ai Y, Jongstra J
Department of Pathology, University of Connecticut Health Center, Farmington 06030-3105, USA.
J Biol Chem. 1997 Jan 3;272(1):17-9. doi: 10.1074/jbc.272.1.17.
In intact cells, mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 is rapidly activated by various cytokines, stresses, and chemotactic factors. The small heat shock protein p27 has been shown to be a substrate for MAPKAP kinase 2. Recently, we identified a novel substrate, designated p60, for MAPKAP kinase 2 in human neutrophils (Zu, Y.-L., Ai, Y., Gilchrist, A., Labadia, M. E., Sha'afi, R. I., and Huang, C.-K. (1996) Blood 87, 5287-5296). To further understand the signaling pathway of MAPKAP kinase 2, we have purified p60 from a heat-treated neutrophil lysate by DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis. Microsequencing of five peptides derived from purified p60 indicates that p60 is lymphocyte-specific protein 1 (LSP1). Furthermore antibodies specific for human and mouse LSP1 react with human and mouse p60. The sequence of human LSP1 indicates two serine residues at positions 204 and 252 as potential phosphorylation sites. The amino acid sequences surrounding these two sites are in agreement with the consensus sequence (Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa) for phosphorylation by MAPKAP kinase 2. Both serine residues in human LSP1 and the corresponding conserved serine residues in mouse LSP1 are in the basic C-terminal F-actin binding domain. Various fusion proteins of wild type and truncated mouse LSP1 with glutathione S-transferase were tested for their capacity to be phosphorylated by MAPKAP kinase 2. The results indicate that LSP1 is a substrate for MAPKAP kinase 2 in vitro and that the phosphorylation sites are located in the basic C-terminal domain of LSP1. Because both the small heat shock proteins and LSP1 are F-actin binding proteins, these results suggest a role for MAPKAP kinase 2 in the regulation of cytoskeletal structure or function.
在完整细胞中，丝裂原活化蛋白激酶激活的蛋白（MAPKAP）激酶2可被多种细胞因子、应激和趋化因子迅速激活。小热休克蛋白p27已被证明是MAPKAP激酶2的底物。最近，我们在人中性粒细胞中鉴定出一种名为p60的MAPKAP激酶2的新底物（Zu，Y.-L.，Ai，Y.，Gilchrist，A.，Labadia，M.E.，Sha'afi，R.I.和Huang，C.-K.（1996年）《血液》87，5287 - 5296）。为了进一步了解MAPKAP激酶2的信号通路，我们通过DEAE - 纤维素色谱和SDS - 聚丙烯酰胺凝胶电泳从热处理的中性粒细胞裂解物中纯化了p60。对从纯化的p60衍生的五个肽段进行微测序表明，p60是淋巴细胞特异性蛋白1（LSP1）。此外，针对人和小鼠LSP1的特异性抗体与人及小鼠的p60发生反应。人LSP1的序列表明在第204和252位有两个丝氨酸残基作为潜在的磷酸化位点。这两个位点周围的氨基酸序列与MAPKAP激酶2磷酸化的共有序列（Xaa - Xaa - Hyd - Xaa - Arg - Xaa - Xaa - Ser - Xaa - Xaa）一致。人LSP1中的两个丝氨酸残基以及小鼠LSP1中相应的保守丝氨酸残基都位于碱性C末端F - 肌动蛋白结合结构域。测试了野生型和截短型小鼠LSP1与谷胱甘肽S - 转移酶的各种融合蛋白被MAPKAP激酶2磷酸化的能力。结果表明，LSP1在体外是MAPKAP激酶2的底物，且磷酸化位点位于LSP1的碱性C末端结构域。由于小热休克蛋白和LSP1都是F - 肌动蛋白结合蛋白，这些结果表明MAPKAP激酶2在细胞骨架结构或功能的调节中起作用。
