Gnudi L, Frevert E U, Houseknecht K L, Erhardt P, Kahn B B
Division of Endocrinology, Dana Farber Cancer Institute, Boston, Massachusetts 02215, USA.
Mol Endocrinol. 1997 Jan;11(1):67-76. doi: 10.1210/mend.11.1.9866.
Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in mediating the actions of insulin.
最近的研究表明，ras-丝裂原活化蛋白激酶（MAP激酶）和磷脂酰肌醇-3激酶（PI3激酶）级联反应相互汇聚。我们试图确定在经典胰岛素靶细胞的胰岛素信号传导中，PI3激酶是否位于ras的下游。我们通过将人H-ras cDNA（在氨基酸17处有丝氨酸到天冬酰胺的替换，即ras（asn17））克隆到pACCMVpLpA载体中，并与含有腺病毒基因组的pJM17质粒共转染293细胞，构建了编码显性负性ras的重组腺病毒。通过用表达β-半乳糖苷酶（β-gal）的重组腺病毒感染完全分化的3T3L1脂肪细胞来评估基因转移效率；超过70%的细胞被感染。用ras（asn17）感染脂肪细胞导致其表达量比内源性ras高10倍。这种高效的基因转移使得能够进行生化分析。在表达ras（asn17）的细胞中，胰岛素刺激ras-GTP形成受到抑制。MAP激酶凝胶迁移率变动分析显示，胰岛素（1微摩尔）或表皮生长因子（100纳克/毫升）可使未感染细胞和表达β-gal的细胞中出现p42 MAP激酶的超磷酸化形式，但在表达ras（asn17）的细胞中则未出现。相反，胰岛素使对照细胞中与胰岛素受体底物-1（IRS-1）相关的PI3激酶活性增加约10倍，而ras（asn17）的高水平过表达并未削弱这种作用。同样，胰岛素和表皮生长因子对胞质溶胶和细胞膜总PI3激酶活性（无免疫沉淀）的激活以及胰岛素对葡萄糖转运的刺激不受显性负性ras表达的影响。因此，腺病毒介导的基因转移对于研究完全分化的胰岛素靶细胞中的胰岛素信号传导是有效的。ras激活的抑制消除了胰岛素刺激的MAP激酶磷酸化，但不影响胰岛素对PI3激酶活性的刺激。在正常细胞生理学中，在介导胰岛素作用方面，PI3激酶似乎并不位于ras的下游。
