Protein Interaction Network of Human Protein Kinase D2 Revealed by Chemical Cross-Linking/Mass Spectrometry.
Protein Interaction Network of Human Protein Kinase D2 Revealed by Chemical Cross-Linking/Mass Spectrometry.
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DOI：
10.1021/acs.jproteome.6b00513
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发表时间：
2016-09
影响因子：
4.4
通讯作者：
Björn Häupl;C. Ihling;A. Sinz
中科院分区：
文献类型：
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作者：
Björn Häupl;C. Ihling;A. Sinz
我们研究了人PKD 2在细胞质和高尔基体富集的亚细胞蛋白组分的相互作用网络，结合化学交联/质谱（MS）的亲和富集策略。亚蛋白质组的分析揭示了不同的蛋白质在胞质和高尔基体组分的存在。通过化学交联的瞬时或弱相互作用物的共价固定允许捕获在常规下拉实验期间否则可能消失的相互作用伴侣。总共鉴定了PKD 2的31种相互作用伴侣，包括糖原合成酶激酶-3 β（GSK 3 B）、14-3-3蛋白γ（YWHAG）和蛋白磷酸酶2A的55 kDa调节亚基B的α同种型（PPP 2 R2 A）。值得注意的是，整个七亚基Arp 2/3复合物（ARPC 1B，ARPC 2，ARPC 3，ARPC 4，ARPC 5，ACTR 3，ACTR 2）以及ARPC 1A和ARPC 5L，它们是ARPC 1B和ARPC 5的假定替代品，被鉴定出来。我们提供了PKD 2和Arp 2/3之间直接蛋白质-蛋白质相互作用的证据。我们的研究结果将为进一步研究PKD 2复合物的结构和功能，特别是PKD 2/Arp 2/3相互作用，阐明PKD 2在trans-Golgi网络转运过程中的作用铺平道路。数据可通过ProteomeXchange获得，标识符为PXD 003909（从胞质组分富集）、PXD 003913（从高尔基体组分富集）和PXD 003917（亚细胞分级分离）。
We investigated the interaction network of human PKD2 in the cytosol and in Golgi-enriched subcellular protein fractions by an affinity enrichment strategy combined with chemical cross-linking/mass spectrometry (MS). Analysis of the subproteomes revealed the presence of distinct proteins in the cytosolic and Golgi fractions. The covalent fixation of transient or weak interactors by chemical cross-linking allowed capturing interaction partners that might otherwise disappear during conventional pull-down experiments. In total, 31 interaction partners were identified for PKD2, including glycogen synthase kinase-3 beta (GSK3B), 14-3-3 protein gamma (YWHAG), and the alpha isoform of 55 kDa regulatory subunit B of protein phosphatase 2A (PPP2R2A). Remarkably, the entire seven-subunit Arp2/3 complex (ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, ACTR3, ACTR2) as well as ARPC1A and ARPC5L, which are putative substitutes of ARPC1B and ARPC5, were identified. We provide evidence of a direct protein-protein interaction between PKD2 and Arp2/3. Our findings will pave the way for further structural and functional studies of PKD2 complexes, especially the PKD2/Arp2/3 interaction, to elucidate the role of PKD2 for transport processes at the trans-Golgi network. Data are available via ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic fractions), PXD003913 (enrichment from Golgi fractions), and PXD003917 (subcellular fractionation).