Croucher David R, Iconomou Mary, Hastings Jordan F, Kennedy Sean P, Han Jeremy Z R, Shearer Robert F, McKenna Jessie, Wan Adrian, Lau Joseph, Aparicio Samuel, Saunders Darren N
The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. St. Vincent's Hospital Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia. School of Medicine, University College Dublin, Belfield, Dublin D4, Ireland.
The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
Sci Signal. 2016 Jul 12;9(436):ra69. doi: 10.1126/scisignal.aaf0793.
The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells.
多蛋白复合物的动态组装是许多细胞信号通路的核心机制。这一过程对于维持准确且适应性强的细胞反应所需的时空特异性至关重要，这种反应是针对快速变化的细胞条件的。我们描述了一种技术，用于特异性分离任意两种相互作用的蛋白质并对其进行下游蛋白质组学表征，同时排除它们各自的部分和竞争性结合伴侣。我们将这种方法称为双分子互补亲和纯化（BiCAP），因为它将构象特异性纳米抗体的使用与蛋白质片段互补分析和亲和纯化相结合。使用BiCAP，我们表征了表皮生长因子受体（EGFR）家族成员ERBB2以同二聚体形式或与EGFR或ERBB3形成异二聚体形式时的特异性相互作用组。我们确定了关键衔接蛋白的二聚体特异性相互作用模式，并鉴定了许多先前未知 的相互作用伴侣。对这些新鉴定的伴侣之一进行功能分析，揭示了一种非经典的细胞外信号调节激酶（ERK）激活机制，该机制特定于ERBB2：ERBB3异二聚体，并通过乳腺癌细胞中的衔接蛋白FAM59A起作用。
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