Protein kinase PKN1 represses Wnt/β-catenin signaling in human melanoma cells.
Protein kinase PKN1 represses Wnt/β-catenin signaling in human melanoma cells.
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DOI：
10.1074/jbc.m113.500314
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发表时间：
2013-11-29
期刊：
影响因子：
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通讯作者：
Moon RT
中科院分区：
文献类型：
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作者：
James RG;Bosch KA;Kulikauskas RM;Yang PT;Robin NC;Toroni RA;Biechele TL;Berndt JD;von Haller PD;Eng JK;Wolf-Yadlin A;Chien AJ;Moon RT
背景：Wnt/β-catenin信号通路抑制黑色素瘤细胞活力。结果如下：磷酸蛋白质组学和RNA干扰筛选的整合查询Wnt/β-catenin途径揭示了蛋白激酶N1作为信号传导的抑制剂。结论：蛋白激酶N1抑制Wnt/β-catenin信号通路和黑色素瘤细胞凋亡。意义：本研究鉴定了一种抑制Wnt/β-catenin信号传导的激酶，这是一种对黑色素瘤细胞活力至关重要的途径。磷酸蛋白质组学的进展使监测信号传导不同步骤发生的蛋白质磷酸化变化成为可能，并有助于识别新的途径成分。在本研究中，我们应用该技术来推进我们对黑色素瘤细胞对分泌配体WNT 3A启动的信号传导的反应的理解。我们首先比较用WNT 3A处理不同时间段的细胞的磷酸肽模式。接下来，我们将这些数据集与靶向蛋白激酶的siRNA筛选结果进行了整合。这种siRNA筛选和蛋白质组学的整合使我们能够鉴定出四种激酶，它们响应于WNT 3A而表现出改变的磷酸化，并调节β-连环蛋白响应性转录的荧光素酶报告基因（β-连环蛋白激活的报告基因）。我们专注于这些激酶之一，一个非典型的PKC激酶，蛋白激酶N1（PKN 1）。用siRNA降低PKN 1水平显著增强β-连环蛋白激活的报告基因的激活，并增加黑素瘤细胞系的凋亡。然后使用亲和纯化和质谱分析，我们发现PKN 1存在于与WNT 3A受体Frizzled 7以及与Frizzled 7共纯化的蛋白质的蛋白质复合物中。这些数据证实蛋白激酶PKN 1抑制Wnt/β-连环蛋白信号传导并使黑素瘤细胞对WNT 3A刺激的细胞死亡敏感。
Background: Wnt/β-catenin signaling inhibits melanoma cell viability. Results: Integration of phosphoproteomics and RNA interference screens querying the Wnt/β-catenin pathway reveals protein kinase N1 as an inhibitor of signaling. Conclusion: Protein kinase N1 inhibits Wnt/β-catenin signaling and apoptosis in melanoma cells. Significance: This study identifies a kinase that inhibits Wnt/β-catenin signaling, a pathway critical to melanoma cell viability. Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.