Filippa N, Sable C L, Filloux C, Hemmings B, Van Obberghen E
Institut National de la Santé et de la Recherche Médicale Faculté de Médecine, 06107 Nice Cedex 2, France.
Mol Cell Biol. 1999 Jul;19(7):4989-5000. doi: 10.1128/MCB.19.7.4989.
Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.
生长因子和激素对蛋白激酶B（PKB）的激活已被证明是通过磷脂酰肌醇3激酶（PI3激酶）进行的。在本报告中，我们表明PKB也可以被蛋白激酶A（环磷酸腺苷[cAMP]依赖性蛋白激酶）通过一条不依赖PI3激酶的途径激活。虽然这种激活需要PKB的磷酸化，但PKB不太可能是PKA的生理底物，因为PKB唯一的PKA共有磷酸化位点发生突变并没有消除PKA诱导的PKB激活。此外，从机制上讲，这种激活与生长因子的激活不同，因为它不需要S473残基的磷酸化，而S473残基的磷酸化对于胰岛素诱导的PKB完全激活至关重要。PKB的S473残基突变为丙氨酸并不妨碍它被福斯可林激活，这一事实支持了这些数据。此外，来自COS细胞的过表达PKB的磷酸肽图谱显示，胰岛素刺激的细胞和福斯可林刺激的细胞之间存在差异，这表明根据使用的是胰岛素还是cAMP，PKB的激活机制不同。我们研究了PKB下游的事件，发现PKA对PKB的激活导致糖原合酶激酶-3（GSK-3）活性的磷酸化和抑制，GSK-3是PKB已知的体内底物。在胰岛素处理和福斯可林处理的细胞中，显性负性PKB的过表达导致GSK-3抑制作用的丧失，这表明在这两种情况下PKB都负责这种抑制作用。最后，我们通过共聚焦显微镜显示，福斯可林与胰岛素相似，能够诱导PKB向质膜转位。这一过程受到高浓度渥曼青霉素（300 nM）的抑制，表明福斯可林诱导的PKB移动可能需要磷脂，而这些磷脂可能不是由I类或III类PI3激酶产生的。然而，高浓度的渥曼青霉素并没有消除PKB的激活，这表明转位本身对于PKA诱导的PKB激活并不重要。
