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首页> 外文期刊>Molecular and Cellular Biology >Adenovirus-mediated overexpression of IRS-1 interacting domains abolishes insulin-stimulated mitogenesis without affecting glucose transport in 3T3-L1 adipocytes.
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Adenovirus-mediated overexpression of IRS-1 interacting domains abolishes insulin-stimulated mitogenesis without affecting glucose transport in 3T3-L1 adipocytes.

机译：腺病毒介导的IRS-1相互作用域的过表达消除了胰岛素刺激的有丝分裂，而不影响3T3-L1脂肪细胞中的葡萄糖转运。

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Activated insulin receptor (IR) interacts with its substrates, IRS-1, IRS-2, and Shc via the NPXY motif centered at Y960. This interaction is important for IRS-1 phosphorylation. Studies using the yeast two-hybrid system and sequence identity analysis between IRS-1 and IRS-2 have identified two putative elements, the PTB and SAIN domains, between amino acids 108 and 516 of IRS-1 that are sufficient for receptor interaction. However, their precise function in mediating insulin's bioeffects is not understood. We expressed the PTB and SAIN domains of IRS-1 in HIRcB fibroblasts and 3T3-L1 adipocytes utilizing replication-defective adenoviral infection to investigate their role in insulin signalling. In both cell types, overexpression of either the PTB or the SAIN protein caused a significant decrease in insulin-induced tyrosine phosphorylation of IRS-1 and Shc proteins, IRS-1-associated phosphatidylinositol 3-kinase (PI 3-K) enzymatic activity, p70s6k activation, and p44 and p42 mitogen-activated protein kinase (MAPK) phosphorylation. However, epidermal growth factor-induced Shc and MAPK phosphorylation was unaffected by the overexpressed proteins. These findings were associated with a complete inhibition of insulin-stimulated cell cycle progression. In 3T3-L1 adipocytes, PTB or SAIN expression extinguished IRS-1 phosphorylation with a corresponding 90% decrease in IRS-1-associated PI 3-K activity. p70s6k is a downstream target of PI 3-K, and insulin-stimulated p70s6k was inhibited by PTB or SAIN expression. Interestingly, overexpression of either PTB or SAIN protein did not affect insulin-induced AKT activation or insulin-stimulated 2-deoxyglucose transport, even though both of these bioeffects are inhibited by wortmannin. Thus, interference with the IRS-1-IR interaction inhibits insulin-stimulated IRS-1 and Shc phosphorylation, PI 3-K enzymatic activity, p70s6k activation, MAPK phosphorylation and cell cycle progression. In 3T3-L1 adipocytes, interference with the IR-IRS-1 interaction did not cause inhibition of insulin-stimulated AKT activation or glucose transport. These results indicate a bifurcation or subcompartmentalization of the insulin signalling pathway whereby some targets of PI 3-K (i.e., p70s6k) are dependent on IRS-1-associated PI 3-K and other targets (i.e., AKT and glucose transport) are not. IR-IRS-1 interaction is not essential for insulin's effect on glucose transport, and alternate, or redundant, pathways exist in these cells.
机译：活化的胰岛素受体（IR）通过以Y960为中心的NPXY基序与其底物IRS-1，IRS-2和Shc相互作用。这种相互作用对于IRS-1磷酸化很重要。使用酵母双杂交系统进行的研究以及IRS-1和IRS-2之间的序列同一性分析，已经确定了IRS-1氨基酸108和516之间两个足以与受体相互作用的推定元素，即PTB和SAIN结构域。但是，它们在介导胰岛素的生物效应中的确切功能尚不清楚。我们利用复制缺陷型腺病毒感染，在IRRC-1成纤维细胞和3T3-L1脂肪细胞中表达了IRS-1的PTB和SAIN结构域，以研究其在胰岛素信号传导中的作用。在这两种细胞类型中，PTB或SAIN蛋白的过度表达都会导致胰岛素诱导的IRS-1和Shc蛋白的酪氨酸磷酸化，IRS-1相关的磷脂酰肌醇3-激酶（PI 3-K）的酶活性显着降低， p70s6k激活，以及p44和p42丝裂原激活的蛋白激酶（MAPK）磷酸化。但是，表皮生长因子诱导的Shc和MAPK磷酸化不受过表达的蛋白质的影响。这些发现与胰岛素刺激的细胞周期进程的完全抑制有关。在3T3-L1脂肪细胞中，PTB或SAIN表达消除了IRS-1磷酸化，IRS-1相关的PI 3-K活性相应降低了90％。 p70s6k是PI 3-K的下游靶标，胰岛素刺激的p70s6k被PTB或SAIN表达抑制。有趣的是，即使这两种生物效应都被渥曼青霉素抑制，PTB或SAIN蛋白的过表达也不影响胰岛素诱导的AKT激活或胰岛素刺激的2-脱氧葡萄糖转运。因此，对IRS-1-IR相互作用的干扰会抑制胰岛素刺激的IRS-1和Shc磷酸化，PI 3-K酶活性，p70s6k活化，MAPK磷酸化和细胞周期进程。在3T3-L1脂肪细胞中，对IR-IRS-1相互作用的干扰并未引起胰岛素刺激的AKT激活或葡萄糖转运的抑制。这些结果表明胰岛素信号通路的分支或亚室化，由此PI 3-K的某些靶标（即p70s6k）依赖于IRS-1相关的PI 3-K，而其他靶标（即AKT和葡萄糖转运）则不。 IR-IRS-1相互作用对于胰岛素对葡萄糖转运的作用不是必需的，并且这些细胞中存在替代或冗余的途径。

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