Proteomic and Phosphoproteomic Profiling Reveals the Oncogenic Role of Protein Kinase D Family Kinases in Cholangiocarcinoma
Abstract
:1. Introduction
2. Materials and Methods
2.1. Collection of Clinical Samples and Information
2.2. Extraction and Digestion of Protein
2.3. TMT Labelling
2.4. HPLC Fractionation
2.5. Enrichment of Phosphopeptides
2.6. LC-MS/MS Analysis
2.7. Standard Database Search
2.8. Proteomic Analysis
2.9. Phosphoproteomic Analysis
2.10. Public P-Site Resources
2.11. GO Functional Enrichment Analysis
2.12. KEGG Functional Enrichment Analysis
2.13. The CifPK Pipeline
2.14. Cell Culture and Transfection
2.15. Western Blotting
2.16. Cell Proliferation and Drug Cytotoxic Assay In Vitro
2.17. Wound Healing
2.18. Transwell Assay
3. Results
3.1. The Procedure for Analysis of CCA-Associated PKs
3.2. The Proteomic and Phosphoproteomic Profiling of CCAs
3.3. The Landscapes of Protein Expression and Phosphorylation for CCAs
3.4. The Integrated Pipeline for the Prediction of CCA-Associated PKs
3.5. Screening of PK Candidates
3.6. The Inhibition of PKDs Suppressed the Migration and Invasion of CCA
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BRAF | Serine/threonine-protein kinase B-raf |
CCA | Cholangiocarcinoma |
CifPK | Comprehensive inference of functional protein kinases |
CCK-8 | Cell Counting Kit-8 |
DEPs | Differentially expressed proteins |
DRPs | Differentially regulated p-sites |
DPPs | Differentially phosphorylated proteins |
EMT | Epithelial mesenchymal transition |
eCCA | Extrahepatic cholangiocarcinoma |
FGFR2 | Fibroblast growth factor receptor 2 |
FDA | Food and Drug Administration |
HCC | Hepatocellular carcinoma |
IC50 | Half-maximal inhibitory concentration |
iCCA | Intrahepatic cholangiocarcinoma |
LC-MS/MS | Liquid chromatography with tandem mass spectrometry |
mTOR | Mammalian target of rapamycin |
NATs | Normal adjacent tissues |
PKs | Protein kinases |
PKD/PRKD | Protein kinase D |
PI3K | Phosphoinositide 3-kinase |
PRKCI | Atypical protein kinase C iota |
RTK | Receptor tyrosine kinase |
Sp1 | Specificity protein 1 |
siRNAs | Small interfering RNAs |
1-NA-PP1 | 1-naphthyl PP1 |
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Lu, Y.; Li, X.; Zhao, K.; Shi, Y.; Deng, Z.; Yao, W.; Wang, J. Proteomic and Phosphoproteomic Profiling Reveals the Oncogenic Role of Protein Kinase D Family Kinases in Cholangiocarcinoma. Cells 2022, 11, 3088. https://doi.org/10.3390/cells11193088
Lu Y, Li X, Zhao K, Shi Y, Deng Z, Yao W, Wang J. Proteomic and Phosphoproteomic Profiling Reveals the Oncogenic Role of Protein Kinase D Family Kinases in Cholangiocarcinoma. Cells. 2022; 11(19):3088. https://doi.org/10.3390/cells11193088
Chicago/Turabian StyleLu, Yun, Xiangyu Li, Kai Zhao, Yuanxin Shi, Zhengdong Deng, Wei Yao, and Jianming Wang. 2022. "Proteomic and Phosphoproteomic Profiling Reveals the Oncogenic Role of Protein Kinase D Family Kinases in Cholangiocarcinoma" Cells 11, no. 19: 3088. https://doi.org/10.3390/cells11193088