Kotani K, Ogawa W, Matsumoto M, Kitamura T, Sakaue H, Hino Y, Miyake K, Sano W, Akimoto K, Ohno S, Kasuga M
Second Department of Internal Medicine, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan.
Mol Cell Biol. 1998 Dec;18(12):6971-82. doi: 10.1128/MCB.18.12.6971.
Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
磷酸肌醇（PI）3激酶参与多种生物学作用，包括胰岛素刺激脂肪细胞中的葡萄糖转运。Akt（蛋白激酶B）是一种具有普列克底物蛋白同源结构域的丝氨酸 - 苏氨酸激酶，蛋白激酶C的非典型亚型（PKCζ和PKCλ）都被认为是PI 3激酶的下游效应器。现已表明，3T3 - L1脂肪细胞或CHO细胞中的内源性或转染的PKCλ可被胰岛素以对PI 3激酶抑制剂（渥曼青霉素和PI 3激酶的显性负性突变体）敏感的方式激活。通过腺病毒介导的基因转移实现PKCλ激酶缺陷突变体（λKD或λΔNKD）的过表达，导致PKCλ的胰岛素激活受到抑制，表明这些突变体发挥显性负性作用。λKD或λΔNKD以剂量依赖性方式抑制胰岛素刺激的葡萄糖摄取以及葡萄糖转运蛋白GLUT4向质膜的转位，但不抑制生长激素或高渗诱导的葡萄糖摄取。PKCλ显性负性突变体对胰岛素诱导的葡萄糖摄取的最大抑制约为50%至60%。这些突变体不抑制胰岛素诱导的Akt激活。缺乏假底物结构域的PKCλ突变体（λΔPD）相对于野生型酶表现出明显增加的激酶活性，并且在静止的3T3 - L1脂肪细胞中表达λΔPD导致葡萄糖摄取的刺激和GLUT4的转位，但不导致Akt的激活。此外，生长因子靶向的磷酸化位点被丙氨酸取代的Akt突变体的过表达导致胰岛素诱导的Akt激活受到抑制，但不抑制PKCλ。这些结果表明，通过PI 3激酶传递的胰岛素引发的信号随后至少分为两条独立的途径，一条Akt途径和一条PKCλ途径，并且后一条途径至少部分地有助于胰岛素刺激3T3 - L1脂肪细胞中的葡萄糖摄取。
