Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors.
Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors.
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DOI：
10.1371/journal.pone.0044471
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Jones RB
中科院分区：
文献类型：
--
作者：
Hause RJ Jr;Leung KK;Barkinge JL;Ciaccio MF;Chuu CP;Jones RB
Src同源性2 （SH2）结构域与受体酪氨酸激酶（RTK）磷酸位点的第一代相互作用图谱先前已使用蛋白质微阵列（PM）技术生成。在这里，我们开发了一种大规模的荧光偏振（FP）方法，能够以更高的保真度和灵敏度表征SH2结构域和ErbB受体磷酸化位点之间的相互作用，而不是以前用pm实现的。我们使用FP分析查询了89个ErbB受体胞内酪氨酸位点对应的合成磷酸肽与93个人类SH2结构域和2个磷酸酪氨酸结合（PTB）结构域的相互作用。从358,944个偏振测量中，确定了1,405个独特的生物相互作用的亲和力，其中83%是新的。与先前报告的数据相反，我们的分析表明，ErbB2并不比其他ErbB受体更具混杂性。我们的研究结果表明，每种受体在募集的SH2结构域的亲和力和位置上表现出独特的偏好，这可能导致下游信号传导潜力的差异。ErbB1相对于其他受体富集，用于从RAS gef中募集结构域，而ErbB2富集，用于从酪氨酸和磷脂酰肌醇磷酸酶中募集结构域。ErbB3是激酶失活的ErbB受体家族成员，可预见地富集于从磷脂酰肌醇激酶募集结构域，令人惊讶的是，富集于从酪氨酸激酶、细胞骨架调节蛋白和RHO gef募集结构域，但缺乏从磷脂酰肌醇磷酸酶募集结构域。许多新的相互作用也被观察到与ErbB受体酪氨酸对应的磷酸化肽之间的相互作用，这些磷酸化肽之前没有被质谱法磷酸化过，这表明存在许多与RTK相关的生物位点，这些位点可能被磷酸化，但低于标准质谱法的检测阈值。该数据集代表了关于ErbB受体生物学机制的可测试假设的丰富来源。
First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.
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影响因子：
--
作者：
Hsieh, Ming-yu;Yang, Shujie;Wilson, Bridget S.
通讯作者：
Wilson, Bridget S.
影响因子：
--
作者：
Kaushansky, Alexis;Gordus, Andrew;MacBeath, Gavin
通讯作者：
MacBeath, Gavin
影响因子：
5.3
作者：
FELDER, S;ZHOU, M;SCHLESSINGER, J
通讯作者：
SCHLESSINGER, J
影响因子：
5.4
作者：
Kaimachnikov, Nikolai P.;Kholodenko, Boris N.
通讯作者：
Kholodenko, Boris N.
影响因子：
11.4
作者：
GrausPorta, D;Beerli, RR;Hynes, NE
通讯作者：
Hynes, NE