Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Central South University, 87 Xiangya Road, Changsha 410008, China.
Medical Science and Technology Innovation Center, Shandong First Medical University, 6699 Qingdao Road, Jinan 250117, China.
Cells. 2021 Aug 27;10(9):2225. doi: 10.3390/cells10092225.
To investigate the biological role of protein phosphorylation in human nonfunctional pituitary neuroendocrine tumors (NF-PitNETs), proteins extracted from NF-PitNET and control tissues were analyzed with tandem mass tag (TMT)-based quantitative proteomics coupled with TiO enrichment of phosphopeptides. A total of 595 differentially phosphorylated proteins (DPPs) with 1412 phosphosites were identified in NF-PitNETs compared to controls ( < 0.05). KEGG pathway network analysis of 595 DPPs identified nine statistically significant signaling pathways, including the spliceosome pathway, the RNA transport pathway, proteoglycans in cancer, SNARE interactions in vesicular transport, platelet activation, bacterial invasion of epithelial cells, tight junctions, vascular smooth muscle contraction, and protein processing in the endoplasmic reticulum. GO analysis revealed that these DPPs were involved in multiple cellular components (CCs), biological processes (BPs), and molecule functions (MFs). The kinase analysis of 595 DPPs identified seven kinases, including GRP78, WSTF, PKN2, PRP4, LOK, NEK1, and AMPKA1, and the substrate of these kinases could provide new ideas for seeking drug targets for NF-PitNETs. The randomly selected DPP calnexin was further confirmed with immunoprecipitation (IP) and Western blot (WB). These findings provide the first DPP profiling, phosphorylation-mediated molecular network alterations, and the key kinase profiling in NF-PitNET pathogenesis, which are a precious resource for understanding the biological roles of protein phosphorylation in NF-PitNET pathogenesis and discovering effective phosphoprotein biomarkers and therapeutic targets and drugs for the management of NF-PitNETs.
为了研究蛋白质磷酸化在人类无功能垂体神经内分泌肿瘤（NF-PitNET）中的生物学作用，我们采用基于串联质量标签（TMT）的定量蛋白质组学结合 TiO2 富集磷酸肽的方法，对 NF-PitNET 和对照组织中提取的蛋白质进行了分析。与对照组相比，NF-PitNET 中鉴定出 595 种差异磷酸化蛋白（DPP）和 1412 个磷酸化位点（<0.05）。对 595 个 DPP 的KEGG 通路网络分析确定了 9 个具有统计学意义的信号通路，包括剪接体通路、RNA 转运通路、癌症中的蛋白聚糖、囊泡运输中的 SNARE 相互作用、血小板激活、细菌入侵上皮细胞、紧密连接、血管平滑肌收缩和内质网中的蛋白质加工。GO 分析表明，这些 DPP 参与了多种细胞成分（CC）、生物过程（BP）和分子功能（MF）。对 595 个 DPP 的激酶分析确定了 7 种激酶，包括 GRP78、WSTF、PKN2、PRP4、LOK、NEK1 和 AMPKA1，这些激酶的底物为 NF-PitNET 的药物靶点提供了新的思路。随机选择的 DPP 钙网蛋白（calnexin）通过免疫沉淀（IP）和 Western blot（WB）进一步得到了验证。这些发现提供了 NF-PitNET 发病机制中 DPP 谱分析、磷酸化介导的分子网络改变和关键激酶谱分析的首例报道，这是理解蛋白质磷酸化在 NF-PitNET 发病机制中的生物学作用和发现有效的磷酸蛋白生物标志物以及用于 NF-PitNET 管理的治疗靶点和药物的宝贵资源。
