A biosensor for MAPK-dependent Lin28 signaling.
A biosensor for MAPK-dependent Lin28 signaling.
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DOI：
10.1091/mbc.e17-08-0500
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发表时间：
2018-05-15
影响因子：
3.3
通讯作者：
Meffert MK
中科院分区：
文献类型：
--
作者：
Oldach LM;Gorshkov K;Mills WT 4th;Zhang J;Meffert MK
RNA结合蛋白和多能性因子Lin28a的细胞内水平受到严格控制，以控制细胞和生物体的生长。Lin28a在转录后水平被广泛调控，并且可以通过与RNA沉默因子HIV TAR RNA结合蛋白（TRBP）的磷酸化依赖性稳定相互作用，在分化细胞中经历促分裂原活化蛋白激酶（MAPK）介导的从低基础水平的升高。然而，这种关键控制机制的分子和时空细节仍然未知。在这项工作中，我们解剖了Lin28a和TRBP蛋白的相互作用区域，并开发了生物传感器来可视化这种相互作用。我们确定了截短的结构域的Lin28a和TRBP，足以支持共缔合和相互升高的蛋白质水平，并要求MAPK依赖的磷酸化TRBP在假定的ERK靶丝氨酸152，以及Lin28a丝氨酸200磷酸化，介导增加Lin28a蛋白的TRBP。利用Lin28a和TRBP截短构建体的磷酸化依赖性缔合来开发基于荧光共振能量转移（FRET）的传感器，用于动态监测Lin28a和TRBP相互作用。我们展示了双分子和单分子FRET传感器对活细胞中生长因子刺激的响应，并对Erk激活进行了共成像，以进一步了解MAPK信号在Lin28a调控中的作用。
Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)–mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.