Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains
Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains
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DOI：
10.1091/mbc.e08-05-0449
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发表时间：
2008-10-01
影响因子：
3.3
通讯作者：
Zhang, Jin
中科院分区：
文献类型：
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作者：
Gao, Xinxin;Zhang, Jin
Akt作为磷脂酰肌醇3-激酶途径中的一种中心蛋白，已被广泛研究，但Akt在不同膜微域中的时空调控仍不清楚。为了检测活细胞膜微区中Akt的动态活性，我们通过对不同底物和荧光蛋白的系统检测，开发了一种特异和灵敏的基于荧光共振能量转移的Akt活性报告器Aktar。靶向Aktar报告了更高的Akt活性，与质膜的非RAFT区域相比，脂筏内的激活动力学更快。阻断RAFT可减弱血小板衍生生长因子(PDGF)刺激的RAFT中Akt活性，而不影响非RAFT区域的活性。然而，在胰岛素样生长因子-1(IGF-1)刺激下，非RAFT区的Akt信号依赖于RAFT区的Akt信号。因此，胆固醇消耗会降低这两个区域的Akt活性。因此，Akt的活性在不同的膜微区中受到不同的调节，并且这一致癌途径的整体活性依赖于RAFT功能。鉴于某些癌细胞中脂筏的数量增加，PDGF和IGF-1在有效性和RAFT依赖性方面的独特Akt激活特征表明，不同的生长因子信号通路具有跨质膜转导差异致癌信号的能力。
As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor ( PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.