Park K, Kim K H
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
J Biol Chem. 1993 Aug 25;268(24):17811-9.
Insulin induction of acetyl-CoA carboxylase (ACC) and differentiation of 30A5 preadipocytes into adipocytes requires a brief exposure of the cells to cAMP. Using the techniques of DNase I footprinting, DNA band shift, and analysis of the point and deletion mutations, a region from -113 to -95 has been identified as the site through which cAMP sensitizes the cell for the response to insulin. One sequence-specific DNA-protein complex, b3, is formed in the DNA-mobility shift assay when nuclear extract from 30A5 cells is mixed with the oligonucleotide representing this region. Purified human AP-2 also generates the complex corresponding to b3 with the same ACC PII probe or with the AP-2 consensus sequence probe from SV40 promoter. Substitution of A for G in the sequence GGGGCTGGG abolishes the formation of b3 sequence-specific complex. Stably transfected 30A5 cells with the same mutations in the plasmid no longer respond to insulin in spite of their exposure to cAMP. These results establish that the 21 base pair region in ACC promoter II and the binding of AP-2 protein to this sequence are required for cAMP action. cAMP-dependent protein kinase phosphorylates AP-2 both in vitro and in vivo and the phosphorylation of AP-2 does not affect its binding activity.
胰岛素诱导乙酰辅酶A羧化酶（ACC）以及30A5前脂肪细胞分化为脂肪细胞需要细胞短暂暴露于环磷酸腺苷（cAMP）。运用脱氧核糖核酸酶I足迹法、DNA条带迁移以及点突变和缺失突变分析技术，已确定从-113至-95的区域是cAMP使细胞对胰岛素反应敏感的位点。当将30A5细胞的核提取物与代表该区域的寡核苷酸混合时，在DNA迁移率变动分析中会形成一种序列特异性DNA-蛋白质复合物b3。纯化的人AP-2与相同的ACC PII探针或来自SV40启动子的AP-2共有序列探针也会产生与b3对应的复合物。在序列GGGGCTGGG中将G替换为A会消除b3序列特异性复合物的形成。尽管稳定转染了具有相同突变质粒的30A5细胞暴露于cAMP，但它们不再对胰岛素产生反应。这些结果表明，ACC启动子II中的21个碱基对区域以及AP-2蛋白与该序列的结合是cAMP发挥作用所必需的。环磷酸腺苷依赖性蛋白激酶在体外和体内均使AP-2磷酸化，且AP-2的磷酸化不影响其结合活性。
