Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets
Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets
复制标题
研究雷帕霉素激酶抑制靶点对莱茵衣藻磷酸蛋白质组的影响：从已知同源物到新靶点
DOI：
10.1111/nph.15339
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发表时间：
2018
期刊：
影响因子：
9.4
通讯作者：
Hicks, Leslie M.
中科院分区：
文献类型：
--
作者：
Werth, Emily G.;McConnell, Evan W.;Couso Lianez, Inmaculada;Perrine, Zoee;Crespo, Jose L.;Umen, James G.;Hicks, Leslie M.
雷帕霉素靶蛋白激酶（TOR）是一种保守的细胞生长调节因子，其活性受营养、能量和应激的调节。模式光合微藻莱茵衣藻（Chlamyeliumreinhardtii）的TOR激酶信号通路中的关键蛋白是保守的，但TOR激酶的底物和下游信号网络尚不清楚。我们的研究为研究衣原体TOR激酶通路调控的磷酸化网络提供了新的资源。我们使用定量磷酸化蛋白质组学研究抑制衣原体TOR激酶对蛋白质磷酸化动态的影响。我们用TOR特异性化学抑制剂（雷帕霉素、AZD 8055和Torin 1）处理野生型和AZD不敏感的衣原体菌株，然后鉴定出受差异影响的磷酸化位点。我们的定量磷酸化蛋白质组学数据集包含来自1432种不同蛋白质的2547个独特磷酸化位点。抑制TOR激酶导致258个磷酸化位点的显著定量变化，来自219个独特的磷酸肽。我们的结果包括TOR信号相关蛋白的衣原体同源物，包括RPS 6上磷酸化减少的位点。此外，还鉴定了参与翻译和类胡萝卜素生物合成的蛋白质上的磷酸化位点。由番茄红素β/β环化酶中的这些磷酸蛋白质组学发现指导的后续实验表明，类胡萝卜素水平受到TORC 1抑制的影响，并且类胡萝卜素的产生在藻类中受到TOR控制。
Target of rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is modulated in response to nutrients, energy and stress. Key proteins involved in the pathway are conserved in the model photosynthetic microalgaChlamydomonas reinhardtii, but the substrates of TOR kinase and downstream signaling network have not been elucidated. Our study provides a new resource for investigating the phosphorylation networks governed by the TOR kinase pathway in Chlamydomonas.We used quantitative phosphoproteomics to investigate the effects of inhibiting Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild‐type and AZD‐insensitive Chlamydomonas strains were treated with TOR‐specific chemical inhibitors (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were identified.Our quantitative phosphoproteomic dataset comprised 2547 unique phosphosites from 1432 different proteins. Inhibition of TOR kinase caused significant quantitative changes in phosphorylation at 258 phosphosites, from 219 unique phosphopeptides.Our results include Chlamydomonas homologs of TOR signaling‐related proteins, including a site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins involved in translation and carotenoid biosynthesis were identified. Follow‐up experiments guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR control in algae.