Regulation of β-Catenin Nuclear Dynamics by GSK-3β Involves a LEF-1 Positive Feedback Loop
Regulation of β-Catenin Nuclear Dynamics by GSK-3β Involves a LEF-1 Positive Feedback Loop
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DOI：
10.1111/j.1600-0854.2011.01207.x
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发表时间：
2011-08-01
期刊：
影响因子：
4.5
通讯作者：
Henderson, Beric R.
中科院分区：
文献类型：
--
作者：
Jamieson, Cara;Sharma, Manisha;Henderson, Beric R.
β -连环蛋白的核定位是其在Wnt信号传导和癌症中的作用不可或缺的一部分。Wnt或氯化锂（LiCl）刺激细胞使糖原合成酶激酶-3 β (GSK-3 β)失活，引起β -连环蛋白的核积累和转化细胞的基因的反激活。-连环蛋白是一种穿梭蛋白；然而，GSK-3 β调节β -连环蛋白核动力学的机制尚不清楚。本文采用光漂白后荧光恢复法测定GSK-3 β抑制前后NIH 3T3细胞β -catenin-绿色荧光蛋白动态。我们首次表明，LiCl和Wnt3a在活细胞和固定细胞中洗涤剂提取后引起β -连环蛋白核保留的特异性增加。此外，LiCl降低了核输出速率，但不影响核输入，从而使β -连环蛋白的转运偏向细胞核。有趣的是，S45A突变阻断了GSK-3 β对β -连环蛋白的磷酸化，但没有改变核保留或转运，这意味着GSK-3 β通过一个独立的调节因子起作用。我们比较了五种核结合伙伴，确定了LEF-1是Wnt3a和licl诱导的β -连环蛋白核保留的关键介质。因此，Wnt刺激触发了一个LEF-1正反馈回路，使核染色质保留的β -连环蛋白池增加了100-300%。这些发现为β -连环蛋白核动力学的调控提供了新的思路。
Nuclear localization of beta-catenin is integral to its role in Wnt signaling and cancer. Cellular stimulation by Wnt or lithium chloride (LiCl) inactivates glycogen synthase kinase-3 beta (GSK-3 beta), causing nuclear accumulation of beta-catenin and transactivation of genes that transform cells. beta-catenin is a shuttling protein; however, the mechanism by which GSK-3 beta regulates beta-catenin nuclear dynamics is poorly understood. Here, fluorescence recovery after photobleaching assays were used to measure the beta-catenin-green fluorescent protein dynamics in NIH 3T3 cells before and after GSK-3 beta inhibition. We show for the first time that LiCl and Wnt3a cause a specific increase in beta-catenin nuclear retention in live cells and in fixed cells after detergent extraction. Moreover, LiCl reduced the rate of nuclear export but did not affect import, hence biasing beta-catenin transport toward the nucleus. Interestingly, the S45A mutation, which blocks beta-catenin phosphorylation by GSK-3 beta, did not alter nuclear retention or transport, implying that GSK-3 beta acts through an independent regulator. We compared five nuclear binding partners and identified LEF-1 as the key mediator of Wnt3a and LiCl-induced nuclear retention of beta-catenin. Thus, Wnt stimulation triggered a LEF-1 positive feedback loop to enhance the nuclear chromatin-retained pool of beta-catenin by 100-300%. These findings shed new light on regulation of beta-catenin nuclear dynamics.