Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase
Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase
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DOI：
10.1074/jbc.271.14.8488
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发表时间：
1996-04-05
影响因子：
4.8
通讯作者：
Young, PR
中科院分区：
文献类型：
--
作者：
McLaughlin, MM;Kumar, S;Young, PR
CSBP p38是一种丝裂原活化蛋白激酶，其响应于应激、内毒素、白细胞介素1和肿瘤坏死因子而活化。利用人CSBP 2的无催化活性突变体（D168 A）作为诱饵在酵母双杂交筛选中，我们鉴定并克隆了一种新的激酶，其与促分裂原活化蛋白激酶活化蛋白激酶（MAPKAP激酶）-2共享相似的70%氨基酸同一性，因此被命名为MAPKAP激酶-3。CSBP与MAPKAP激酶-3的结合在体外通过细菌表达的GST-MAPKAP激酶-3融合蛋白沉淀表位标记的CSBP 1、CSBP 2和CSBP 2（D168 A）和来自哺乳动物细胞的内源性CSBP来证实，并且在体内通过共沉淀HeLa细胞中共表达的表位标记的蛋白来证实。MAPKAP激酶-3被CSBP 1和CSBP 2磷酸化，然后能够在体外磷酸化HSP 27。用山梨醇或TNF处理HeLa细胞导致CSBP和MAPKAP激酶3的激活，并且MAPKAP激酶3的激活可以通过用CSBP激酶活性的特异性抑制剂SB 203580预孵育细胞来阻断。这些数据表明，MAPKAP激酶-3被激活的压力和细胞因子，是一种新的底物CSBP在体外和体内。
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 similar to 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.