Egawa K, Sharma P M, Nakashima N, Huang Y, Huver E, Boss G R, Olefsky J M
Department of Medicine, Division of Endocrinology and Metabolism, and the Whittier Diabetes Institute, University of California, San Diego, La Jolla, California 92093, USA.
J Biol Chem. 1999 May 14;274(20):14306-14. doi: 10.1074/jbc.274.20.14306.
Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. We have investigated whether targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes is sufficient and necessary to induce PI 3-kinase dependent signaling responses, characteristic of insulin action. We overexpressed Myc-tagged, membrane-targeted p110 (p110(CAAX)), and wild-type p110 (p110(WT)) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibited approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates, that further increased to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulated p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) had little or no effect on these downstream effects. Interestingly p110(CAAX) did not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) did not increase basal glycogen synthase activity, and inhibited insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibited insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulated IRS-1 Ser/Thr phosphorylation, and inhibited IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, our studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact.
磷脂酰肌醇（PI）3激酶在多种胰岛素刺激的生物学反应中发挥重要作用，包括葡萄糖转运、糖原合成和蛋白质合成。然而，PI 3激酶与这些生物学反应之间的分子联系仍不清楚。我们研究了将PI 3激酶的催化性p110亚基靶向细胞膜对于诱导PI 3激酶依赖性信号反应（胰岛素作用的特征性反应）是否足够且必要。我们通过腺病毒介导的基因转移在3T3-L1脂肪细胞中过表达了Myc标签的、靶向膜的p110（p110(CAAX)）和野生型p110（p110(WT)）。过表达的p110(CAAX)在p110免疫沉淀物中的基础激酶活性增加了约2倍，胰岛素刺激后进一步增加至约4倍。即使在这种亚最大PI 3激酶活性水平下，p110(CAAX)也能充分刺激p70 S6激酶、Akt、2-脱氧葡萄糖摄取和Ras，而p110(WT)对这些下游效应几乎没有影响。有趣的是，尽管p110(CAAX)刺激了p21(ras)，但它并未激活丝裂原活化蛋白激酶。令人惊讶的是，p110(CAAX)并未增加基础糖原合酶活性，反而抑制了胰岛素刺激的活性，这表明细胞对胰岛素的这种作用产生了抗性。p110(CAAX)还抑制了胰岛素刺激的丝裂原活化蛋白激酶磷酸化，但不抑制血小板衍生生长因子刺激的丝裂原活化蛋白激酶磷酸化，这表明p110(CAAX)诱导的丝裂原活化蛋白激酶和胰岛素信号抑制是特异性的，并非对细胞产生了某种毒性或非特异性影响。此外，p110(CAAX)刺激了胰岛素受体底物-1（IRS-1）的丝氨酸/苏氨酸磷酸化，并抑制了与IRS-1相关的PI 3激酶活性，而不影响胰岛素受体酪氨酸磷酸化，这表明它可能作为胰岛素信号的负调节因子发挥重要作用。总之，我们的研究表明靶向膜的PI 3激酶可以模拟胰岛素通常诱导的许多生物学效应。此外，p110(CAAX)表达诱导的PI 3激酶持续激活导致特定信号通路脱敏。有趣的是，细胞胰岛素抵抗状态并非全局性的，因为胰岛素的一些作用受到抑制，而其他作用则保持完整。
