Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 β controls effector activity, stability and processing. However, a deeper and more comprehensive understanding of phosphorylation in the signal transduction remains unclear, particularly during early response processes involved in SMO activation and preceding GLI target gene regulation. We applied temporal quantitative phosphoproteomics to reveal phosphorylation dynamics underlying the short-term chemical activation and inhibition of early hedgehog signaling in HH responsive human medulloblastoma cells. Medulloblastoma cells were treated for 5.0 and 15 min with Smoothened Agonist (SAG) to induce and with vismodegib to inhibit the HH pathway. Our phosphoproteomic profiling resulted in the quantification of 7700 and 10,000 phosphosites after 5.0 and 15 min treatment, respectively. The data suggest a central role of phosphorylation in the regulation of ciliary assembly, trafficking, and signal transduction already after 5.0 min treatment. ERK/MAPK signaling, besides Protein Kinase A signaling and mTOR signaling, were differentially regulated after short-term treatment. Activation of Polo-like Kinase 1 and inhibition of Casein Kinase 2A1 were characteristic for vismodegib treatment, while SAG treatment induced Aurora Kinase A activity. Distinctive phosphorylation of central players of HH signaling such as SMO, SUFU, GLI2 and GLI3 was observed only after 15 min treatment. This study provides evidence that phosphorylation triggered in response to SMO modulation dictates the localization of hedgehog pathway components within the primary cilium and affects the regulation of the SMO-SUFU-GLI axis. The data are relevant for the development of targeted therapies of HH-associated cancers including sonic HH-type medulloblastoma. A deeper understanding of the mechanisms of action of SMO inhibitors such as vismodegib may lead to the development of compounds causing fewer adverse effects and lower frequencies of drug resistance.

中文翻译：

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人髓母细胞瘤细胞中短期刺猬信号的磷酸化蛋白质组学。

异常刺猬 (HH) 信号与各种癌症实体（如髓母细胞瘤）的发展有关。GLI 转录因子的激活被揭示为通路激活的驱动力。包括蛋白激酶 A、酪蛋白激酶 1 和糖原合酶激酶 3 β 在内的激酶对重要效应子（例如平滑 (SMO) 和 GLI 蛋白）的磷酸化增加控制效应子活性、稳定性和加工。然而，对信号转导中磷酸化的更深入和更全面的理解仍不清楚，特别是在 SMO 激活和之前的 GLI 靶基因调控的早期反应过程中。我们应用时间定量磷酸化蛋白质组学来揭示 HH 反应性人髓母细胞瘤细胞中短期化学激活和早期刺猬信号抑制的磷酸化动力学。髓母细胞瘤细胞用平滑激动剂 (SAG) 处理 5.0 和 15 分钟以诱导并用 vismodegib 抑制 HH 通路。我们的磷酸化蛋白质组学分析分别在 5.0 和 15 分钟处理后量化了 7700 和 10,000 个磷酸位点。数据表明磷酸化在 5.0 分钟处理后已经在纤毛组装、运输和信号转导的调节中发挥核心作用。除了蛋白激酶 A 信号和 mTOR 信号之外，ERK/MAPK 信号在短期治疗后受到差异调节。Polo 样激酶 1 的激活和酪蛋白激酶 2A1 的抑制是 vismodegib 治疗的特征，而 SAG 治疗诱导极光激酶 A 活性。仅在处理 15 分钟后才观察到 HH 信号传导的核心参与者（如 SMO、SUFU、GLI2 和 GLI3）的独特磷酸化。这项研究提供的证据表明，响应 SMO 调制而触发的磷酸化决定了刺猬通路成分在初级纤毛内的定位，并影响了 SMO-SUFU-GLI 轴的调节。这些数据与 HH 相关癌症（包括声波 HH 型髓母细胞瘤）的靶向治疗的开发相关。