Phosphorylation-Mediated Molecular Pathway Changes in Human Pituitary Neuroendocrine Tumors Identified by Quantitative Phosphoproteomics.

Siqi Wen,Na Li,Jiajia Li,Biao Li,Xianquan Zhan

doi:10.3390/cells10092225

Siqi Wen, Na Li + Show 3 more

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https://doi.org/10.3390/cells10092225

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Abstract

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 TiO2 enrichment of phosphopeptides. A total of 595 differentially phosphorylated proteins (DPPs) with 1412 phosphosites were identified in NF-PitNETs compared to controls (p < 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.

Highlights

Human genomics has made great advancements in recent years, and ca. 20,300 genes have been deciphered by means of sequencing techniques [1]
595 differentially phosphorylated proteins (DPPs) with 1412 phosphosites were identified in NF-pituitary neuroendocrine tumor (PitNET) compared to controls (p < 0.05) (Supplementary Table S2)
The phosphorylation site was localized to the amino acid residue Ser641, and the phosphorylation level was significantly increased in NF-PitNETs compared to controls (Supplementary Table S2)

Summary

Introduction

Human genomics has made great advancements in recent years, and ca. 20,300 genes have been deciphered by means of sequencing techniques [1]. When certain determinable factors of a disease such as cancer are obtained from only the gene level for the prediction, prevention, diagnosis, therapy, and prognostic assessment of cancer, the effort often fails. This is because many diseases, including cancers, are very complex, involving a series of molecule alterations at the genome, transcriptome, proteome, and metabolome levels, and these molecules interact mutually and function in a molecular network system [2]. PTMs, including phosphorylation, acetylation, ubiquitylation, nitration, and glycosylation, result in proteomic variations and the diversity of protein function to a great extent. The phosphorylation proportion of residues of S, T, and Y in humans was 86.4%, 11.8%, and 1.8%, respectively [5,6,7]

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