Differential 14-3-3 affinity capture reveals new downstream targets of phosphatidylinositol 3-kinase signaling.
Differential 14-3-3 affinity capture reveals new downstream targets of phosphatidylinositol 3-kinase signaling.
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DOI：
10.1074/mcp.m800544-mcp200
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发表时间：
2009-11
期刊：
影响因子：
--
通讯作者：
MacKintosh C
中科院分区：
文献类型：
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作者：
Dubois F;Vandermoere F;Gernez A;Murphy J;Toth R;Chen S;Geraghty KM;Morrice NA;MacKintosh C
我们设计了一种14 - 3 - 3亲和捕获与释放、胰蛋白酶消化产物的同位素差异（d0/d4）二甲基标记以及磷酸肽表征的策略，以鉴定胰岛素/胰岛素样生长因子1/磷脂酰肌醇3 -激酶信号传导的新靶点。值得注意的是，四种已知的受胰岛素调节的蛋白质（磷酸果糖激酶 - 2、PRAS40、AS160和MYO1C）具有较高的d0/d4值，这意味着在胰岛素刺激的细胞中，它们在14 - 3 - 3结合蛋白中的含量比未受刺激的细胞中更高。在新的候选蛋白中，胰岛素受体底物2、促凋亡的CCDC6、E3泛素连接酶ZNRF2和信号转导衔接蛋白SASH1被证实可在胰岛素样生长因子1/磷脂酰肌醇3 -激酶信号传导的作用下与14 - 3 - 3结合。胰岛素受体底物2、ZNRF2和SASH1也可通过p90RSK受佛波酯调节，而CCDC6和PRAS40则不受其调节。相比之下，肌动蛋白相关蛋白血管舒张剂刺激磷蛋白和脂解刺激脂蛋白受体的d0/d4分数较低，它们无论是否有胰岛素样生长因子1和佛波酯刺激都会与14 - 3 - 3结合。ZNRF2的磷酸化丝氨酸19（RTRAYpS19GS）、SASH1的磷酸化丝氨酸90（RKRRVpS90QD）以及脂解刺激脂蛋白受体的磷酸化丝氨酸493（RPRARpS493LD）为这些蛋白质各自提供了一个14 - 3 - 3结合位点。差异14 - 3 - 3捕获为确定特定信号通路的下游调节机制提供了一种强有力的方法。
We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d0/d4) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d0/d4 values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d0/d4 scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser19 of ZNRF2 (RTRAYpS19GS), phospho-Ser90 of SASH1 (RKRRVpS90QD), and phospho- Ser493 of lipolysis-stimulated lipoprotein receptor (RPRARpS493LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.