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Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β
Diabetic nephropathy is the most common chronic kidney disease in the world and the main cause of end-stage renal disease (ESRD). The structural integrity of podocytes is fundamental to the normal function of the glomerulus, and the role of glycogen synthase kinase 3β (GSK-3β) in podocytes is compli...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059835/ https://www.ncbi.nlm.nih.gov/pubmed/33897769 http://dx.doi.org/10.3389/fgene.2021.657140 |
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author | Hu, Mingyang Fang, Jiuyuan Wang, Huijuan Zhou, Sijie |
author_facet | Hu, Mingyang Fang, Jiuyuan Wang, Huijuan Zhou, Sijie |
author_sort | Hu, Mingyang |
collection | PubMed |
description | Diabetic nephropathy is the most common chronic kidney disease in the world and the main cause of end-stage renal disease (ESRD). The structural integrity of podocytes is fundamental to the normal function of the glomerulus, and the role of glycogen synthase kinase 3β (GSK-3β) in podocytes is complicated. A thorough understanding of GSK-3β is crucial to understand the mechanism of diabetic nephropathy. To analyze the roles of GSK-3β in podocytes, GSK-3β knockdown lentivirus by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas)9 was applied to establish stable cell lines. Mass spectrometry was utilized to search for differentially expressed proteins. Consequently, we found 34 proteins with higher levels and 115 proteins with lower levels in GSk-3β knockdown cells than in control cells and identified 581 phosphosites with higher phosphorylation levels and 288 phosphosites with lower phosphorylation levels. We performed functional enrichment analysis of these proteins and phosphorylated proteins based on public databases. Enrichment analysis revealed that GSK-3β participates in the spliceosome, Hippo signaling pathway, actin binding, structural molecule activity, and other pathways. Then, we used motif analysis of phosphate sites to determine 89 conserved motifs based on 1,068 phosphoserine (pS) sites and 15 conserved motifs in view of 104 phosphothreonine (pT) sites. Additionally, protein–protein interaction network analysis was carried out using the STRING database. Cytoscape’s add-on Molecular Complex Detection (MCODE) was used to analyze key and core protein groups. In quantitative differential protein analysis, four MCODEs were obtained, and 22 MCODEs were obtained in the analysis of the phosphoproteome of differentially expressed proteins. Finally, we analyzed the kinase regulatory network in podocytes after GSK-3β knockdown and identified 299 protein kinases and 3,460 significantly changed phosphorylation modification sites on 1,574 proteins. These results will be valuable for further research on GSK-3β. |
format | Online Article Text |
id | pubmed-8059835 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-80598352021-04-22 Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β Hu, Mingyang Fang, Jiuyuan Wang, Huijuan Zhou, Sijie Front Genet Genetics Diabetic nephropathy is the most common chronic kidney disease in the world and the main cause of end-stage renal disease (ESRD). The structural integrity of podocytes is fundamental to the normal function of the glomerulus, and the role of glycogen synthase kinase 3β (GSK-3β) in podocytes is complicated. A thorough understanding of GSK-3β is crucial to understand the mechanism of diabetic nephropathy. To analyze the roles of GSK-3β in podocytes, GSK-3β knockdown lentivirus by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas)9 was applied to establish stable cell lines. Mass spectrometry was utilized to search for differentially expressed proteins. Consequently, we found 34 proteins with higher levels and 115 proteins with lower levels in GSk-3β knockdown cells than in control cells and identified 581 phosphosites with higher phosphorylation levels and 288 phosphosites with lower phosphorylation levels. We performed functional enrichment analysis of these proteins and phosphorylated proteins based on public databases. Enrichment analysis revealed that GSK-3β participates in the spliceosome, Hippo signaling pathway, actin binding, structural molecule activity, and other pathways. Then, we used motif analysis of phosphate sites to determine 89 conserved motifs based on 1,068 phosphoserine (pS) sites and 15 conserved motifs in view of 104 phosphothreonine (pT) sites. Additionally, protein–protein interaction network analysis was carried out using the STRING database. Cytoscape’s add-on Molecular Complex Detection (MCODE) was used to analyze key and core protein groups. In quantitative differential protein analysis, four MCODEs were obtained, and 22 MCODEs were obtained in the analysis of the phosphoproteome of differentially expressed proteins. Finally, we analyzed the kinase regulatory network in podocytes after GSK-3β knockdown and identified 299 protein kinases and 3,460 significantly changed phosphorylation modification sites on 1,574 proteins. These results will be valuable for further research on GSK-3β. Frontiers Media S.A. 2021-04-07 /pmc/articles/PMC8059835/ /pubmed/33897769 http://dx.doi.org/10.3389/fgene.2021.657140 Text en Copyright © 2021 Hu, Fang, Wang and Zhou. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Genetics Hu, Mingyang Fang, Jiuyuan Wang, Huijuan Zhou, Sijie Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β |
title | Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β |
title_full | Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β |
title_fullStr | Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β |
title_full_unstemmed | Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β |
title_short | Proteome and Phosphoproteome Analyses Reveal the Kinase Regulatory Network Involved in Glycogen Synthesis Kinase 3β |
title_sort | proteome and phosphoproteome analyses reveal the kinase regulatory network involved in glycogen synthesis kinase 3β |
topic | Genetics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059835/ https://www.ncbi.nlm.nih.gov/pubmed/33897769 http://dx.doi.org/10.3389/fgene.2021.657140 |
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