Thomas Elaine C, Zhe Yang, Molero Juan Carlos, Schmitz-Peiffer Carsten, Ramm Georg, James David E, Whitehead Jonathan P
Centre for Diabetes and Endocrine Research, Princess Alexandra Hospital, University of Queensland, Brisbane, Qld 4102, Australia.
Int J Biochem Cell Biol. 2006;38(10):1686-99. doi: 10.1016/j.biocel.2006.03.009. Epub 2006 Apr 3.
Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim(-/-) cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton.
有效的胰岛素作用需要信号级联反应在空间和时间上的协调。典型的胰岛素受体底物IRS-1在胰岛素信号传导中起核心作用。通过亚细胞分级分离，IRS-1在一种颗粒级分中富集，称为高速沉淀（HSP），其从该级分中的重新分布与信号衰减和胰岛素抵抗相关。有证据表明细胞骨架可能是IRS-1定位于HSP的基础。在本研究中，我们采用了系统的方法来研究细胞骨架是否有助于IRS-1的亚细胞分级分离特性和功能。通过标准显微镜或免疫沉淀，我们无法检测到支持IRS-1与3T3-L1脂肪细胞或CHO.IR.IRS-1细胞中的主要细胞骨架成分肌动蛋白（微丝）、波形蛋白（中间丝）和微管蛋白（微管）之间存在特异性相互作用的证据。单独或联合使用药物破坏微丝和微管，对两种细胞类型中IRS-1的亚细胞分布或胰岛素刺激的酪氨酸磷酸化均无影响。在3T3-L1脂肪细胞中，Akt的磷酸化适度降低（20-35%），但在CHO.IR.IRS-1细胞中没有降低。在缺乏中间丝（Vim(-/-)）的细胞中，IRS-1的表达、分布和胰岛素刺激的磷酸化看起来正常。即使在微丝和微管解聚后，Vim(-/-)细胞中胰岛素刺激的IRS-1和Akt磷酸化仍得以维持。综上所述，这些数据表明，IRS-1的特征性亚细胞分级分离特性和功能不太可能由细胞骨架网络介导，并且近端胰岛素信号传导不需要完整的细胞骨架。
