Alessi D R, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings B A
MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, UK.
EMBO J. 1996 Dec 2;15(23):6541-51.
Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).
胰岛素可使L6肌管中的内源性蛋白激酶Bα（也称为RAC/Akt激酶）活性提高12倍，而转染至293细胞后，蛋白激酶Bα分别对胰岛素和胰岛素样生长因子-1（IGF-1）的反应活性提高了20倍和50倍。在这两种细胞中，蛋白激酶Bα的激活均伴随着其苏氨酸308（Thr308）和丝氨酸473（Ser473）位点的磷酸化，并且与激活一样，磷脂酰肌醇3激酶抑制剂渥曼青霉素可阻止这两个位点的磷酸化。将苏氨酸308和/或丝氨酸473突变为丙氨酸（Ala）或天冬氨酸（Asp），并在转染至293细胞后分析突变型蛋白激酶Bα分子的活性。在丝裂原活化蛋白激酶相关激酶-2（MAPKAP激酶-2）对丝氨酸473进行化学计量磷酸化后，还在体外测定了野生型和突变型蛋白激酶Bα的活性。这些实验表明，胰岛素或胰岛素样生长因子-1（IGF-1）对蛋白激酶Bα的激活源于苏氨酸308和丝氨酸473的磷酸化，这两个位点的磷酸化对于产生高水平的蛋白激酶Bα活性至关重要，并且体内苏氨酸308的磷酸化不依赖于丝氨酸473的磷酸化，反之亦然。我们提出了一个模型，即蛋白激酶Bα在胰岛素/IGF-1刺激的细胞中被上游激酶磷酸化并激活。
