Kim S J, Kahn C R
Joslin Diabetes Center, Research Division, Room 620, One Joslin Place, Boston, MA 02215, USA.
Biochem J. 1997 May 1;323 ( Pt 3)(Pt 3):621-7. doi: 10.1042/bj3230621.
After insulin receptor activation, many cytoplasmic enzymes, including mitogen-activated protein (MAP) kinase, MAP kinase kinase (MEK) and casein kinase II (CKII) are activated, but exactly how insulin signalling progresses to the nucleus remains poorly understood. In Chinese hamster ovary cells overexpressing human insulin receptors [CHO(Hirc)], MEK, CKII and the MAP kinases ERK I and ERK II can be detected by immunoblotting in the nucleus, as well as in the cytoplasm, in the unstimulated state. Nuclear localization of MAP kinase is also observed in 3T3-F442A adipocytes, NIH-3T3 cells and Fao hepatoma cells, whereas MEK is found in the nucleus only in Fao and CHO cells. Insulin treatment for 5-30 min induces a translocation of MEK from the cytoplasm to the nucleus, whereas the MAP kinases and CKII are not translocated into the nucleus in response to insulin during this period. However, nuclear MAP kinase and CKII activities increase by 2-3-fold within 1-10 min after stimulation with insulin. By using gel-shift assays, it has been shown that insulin also stimulates nuclear protein binding to an AP-1 site with kinetics similar to MEK translocation and MAP kinase and CKII activation. Treatment of the extracts in vitro with protein phosphatase 2A or treatment of the intact cells with 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole, a cell-permeable inhibitor of CKII, almost completely blocks the insulin-induced DNA-binding activity, whereas incubation of cells with a MEK inhibitor produces only a slight decrease. These results suggest that insulin signalling results in the activation of serine kinases in the nucleus via two pathways: (1) insulin stimulates the nuclear translocation of some kinases, such as MEK, which might directly phosphorylate nuclear protein substrates or activate other nuclear kinases, and (2) insulin activates nuclear kinases without translocation. The latter is true of CKII, which seems to regulate the binding of nuclear proteins to the AP-1 site, possibly by phosphorylation of AP-1 transcription factors.
胰岛素受体激活后，包括丝裂原活化蛋白（MAP）激酶、MAP激酶激酶（MEK）和酪蛋白激酶II（CKII）在内的许多细胞质酶被激活，但胰岛素信号如何传导至细胞核仍知之甚少。在过表达人胰岛素受体的中国仓鼠卵巢细胞[CHO（Hirc）]中，在未受刺激状态下，通过免疫印迹法可在细胞核以及细胞质中检测到MEK、CKII以及MAP激酶ERK I和ERK II。在3T3 - F442A脂肪细胞、NIH - 3T3细胞和Fao肝癌细胞中也观察到MAP激酶的核定位，而MEK仅在Fao细胞和CHO细胞的细胞核中被发现。胰岛素处理5 - 30分钟会诱导MEK从细胞质转位至细胞核，而在此期间，MAP激酶和CKII不会因胰岛素而转位至细胞核。然而，胰岛素刺激后1 - 10分钟内，细胞核内的MAP激酶和CKII活性增加2 - 3倍。通过凝胶迁移分析表明，胰岛素还能刺激核蛋白与AP - 1位点结合，其动力学与MEK转位以及MAP激酶和CKII激活相似。用蛋白磷酸酶2A体外处理提取物或用CKII的细胞可渗透抑制剂5,6 - 二氯 - 1 - β - D - 呋喃核糖基苯并咪唑处理完整细胞，几乎完全阻断胰岛素诱导的DNA结合活性，而用MEK抑制剂孵育细胞只会导致轻微下降。这些结果表明，胰岛素信号通过两条途径导致细胞核内丝氨酸激酶的激活：（1）胰岛素刺激某些激酶的核转位，如MEK，其可能直接磷酸化核蛋白底物或激活其他核激酶；（2）胰岛素在不发生转位的情况下激活核激酶。CKII就是后者这种情况，它似乎通过磷酸化AP - 1转录因子来调节核蛋白与AP - 1位点的结合。
