Macfarlane W M, Smith S B, James R F, Clifton A D, Doza Y N, Cohen P, Docherty K
Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.
J Biol Chem. 1997 Aug 15;272(33):20936-44. doi: 10.1074/jbc.272.33.20936.
Insulin upstream factor 1 (IUF1), a transcription factor present in pancreatic beta-cells, binds to the sequence C(C/T)TAATG present at several sites within the human insulin promoter. Here we isolated and sequenced cDNA encoding human IUF1 and exploited it to identify the signal transduction pathway by which glucose triggers its activation. In human islets, or in the mouse beta-cell line MIN6, high glucose induced the binding of IUF1 to DNA, an effect mimicked by serine/threonine phosphatase inhibitors, indicating that DNA binding was induced by a phosphorylation mechanism. The glucose-stimulated binding of IUF1 to DNA and IUF1-dependent gene transcription were both prevented by SB 203580, a specific inhibitor of stress-activated protein kinase 2 (SAPK2, also termed p38 mitogen-activated protein kinase, reactivating kinase, CSBP, and Mxi2) but not by several other protein kinase inhibitors. Consistent with this finding, high glucose activated mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase-2) (a downstream target of SAPK2) in MIN6 cells, an effect that was also blocked by SB 203580. Cellular stresses that trigger the activation of SAPK2 and MAPKAP kinase-2 (arsenite, heat shock) also stimulated IUF1 binding to DNA and IUF1-dependent gene transcription, and these effects were also prevented by SB 203580. IUF1 expressed in Escherichia coli was unable to bind to DNA, but binding was induced by incubation with MgATP, SAPK2, and a MIN6 cell extract, which resulted in the conversion of IUF1 to a slower migrating form. SAPK2 could not be replaced by p42 MAP kinase, MAPKAP kinase-2, or MAPKAP kinase-3. The glucose-stimulated activation of IUF1 DNA binding and MAPKAP kinase-2 (but not the arsenite-induced activation of these proteins) was prevented by wortmannin and LY 294002 at concentrations similar to those that inhibit phosphatidylinositide 3-kinase. Our results indicate that high glucose (a cellular stress) activates SAPK2 by a novel mechanism in which a wortmannin/LY 294002-sensitive component plays an essential role. SAPK2 then activates IUF1 indirectly by activating a novel IUF1-activating enzyme.
胰岛素上游因子1（IUF1）是一种存在于胰腺β细胞中的转录因子，它与人类胰岛素启动子内多个位点上的C（C/T）TAATG序列结合。在此，我们分离并测序了编码人类IUF1的cDNA，并利用它来确定葡萄糖触发其激活的信号转导途径。在人类胰岛或小鼠β细胞系MIN6中，高糖诱导IUF1与DNA结合，丝氨酸/苏氨酸磷酸酶抑制剂可模拟这一效应，表明DNA结合是由磷酸化机制诱导的。IUF1与DNA的葡萄糖刺激结合以及IUF1依赖性基因转录均被SB 203580（一种应激激活蛋白激酶2的特异性抑制剂，应激激活蛋白激酶2也称为p38丝裂原活化蛋白激酶、再激活激酶、CSBP和Mxi2）所抑制，但未被其他几种蛋白激酶抑制剂所抑制。与这一发现一致，高糖激活了MIN6细胞中的丝裂原活化蛋白激酶激活的蛋白激酶2（MAPKAP激酶-2，是SAPK2的下游靶点），这一效应也被SB 203580所阻断。触发SAPK2和MAPKAP激酶-2激活的细胞应激（亚砷酸盐、热休克）也刺激了IUF1与DNA的结合以及IUF1依赖性基因转录，这些效应同样被SB 203580所抑制。在大肠杆菌中表达的IUF1无法与DNA结合，但与MgATP、SAPK2和MIN6细胞提取物一起孵育可诱导其结合，这导致IUF1转变为迁移较慢的形式。p42丝裂原活化蛋白激酶、MAPKAP激酶-2或MAPKAP激酶-3无法替代SAPK2。wortmannin和LY 294002在与抑制磷脂酰肌醇3-激酶相似的浓度下可阻止葡萄糖刺激的IUF1 DNA结合激活和MAPKAP激酶-2激活（但不能阻止亚砷酸盐诱导的这些蛋白的激活）。我们的结果表明，高糖（一种细胞应激）通过一种新机制激活SAPK2，其中wortmannin/LY 294002敏感成分起关键作用。然后，SAPK2通过激活一种新的IUF1激活酶间接激活IUF1。
