McLaughlin M M, Kumar S, McDonnell P C, Van Horn S, Lee J C, Livi G P, Young P R
Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
J Biol Chem. 1996 Apr 5;271(14):8488-92. doi: 10.1074/jbc.271.14.8488.
CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.
CSBP p38是一种丝裂原活化蛋白激酶，可在应激、内毒素、白细胞介素1和肿瘤坏死因子的作用下被激活。在酵母双杂交筛选中，我们使用人CSBP2的催化失活突变体（D168A）作为诱饵，鉴定并克隆了一种新型激酶，它与丝裂原活化蛋白激酶激活的蛋白激酶（MAPKAP激酶）-2具有约70%的氨基酸同一性，因此被命名为MAPKAP激酶-3。通过细菌表达的GST-MAPKAP激酶-3融合蛋白从哺乳动物细胞中沉淀表位标记的CSBP1、CSBP2和CSBP2（D168A）以及内源性CSBP，在体外证实了CSBP与MAPKAP激酶-3的结合；在体内，通过共沉淀在HeLa细胞中共表达的表位标记蛋白也证实了这种结合。MAPKAP激酶-3可被CSBP1和CSBP2磷酸化，然后能够在体外磷酸化HSP27。用山梨醇或TNF处理HeLa细胞会导致CSBP和MAPKAP激酶-3的激活，并且用CSBP激酶活性的特异性抑制剂SB203580对细胞进行预孵育可阻断MAPKAP激酶-3的激活。这些数据表明，MAPKAP激酶-3在应激和细胞因子作用下被激活，并且在体外和体内都是CSBP的新型底物。
