Identification of pHis Modified Substrates Regulated by NME1 and its Function in Hepatocellular Carcinoma Cells
Objective:Study the function of Nucleoside diphosphate kinase A(NME1)in hepatocellular carcinoma(HCC)cell line HCCLM3 and the regulated pHis substrates of NME1.Methods:The wound healing assay,Transwell chamber assay,and proteomics and phosphoproteomics analysis methods were used to analyze the phenotype and molecular changes of cells after NME1 knockout.Re-sults:Herein,our results showed that histidine kinase NME1 depletion in HCC cell lines inhibited cell motility.Moreover,the proteome analysis found that the differentially expressed proteins after NME1 knockout were primarily involved in the regulation of cell adhesion,cell cycle,and regulation of protein kinase activity.Western blot analysis shows that knockout of NME1 decreases the overall level of pHis modification.Subsequently,using the strategy for unbiased histidine phosphoproteome profiling of dimethyl labeling,Strong cation exchange(SCX)separation of modified and non-modified naked peptides,Cu-iminodiacetic acid(Cu-IDA)enrichment,and mass spec-trometry detection,we identified a total of 242 pHis-peptides,corresponding to 206 proteins in the HCC cell line.About 25％of the pHis-modified proteins were reported for the first time,among which about 30 proteins with significantly down-regulated pHis modifica-tion levels in response to NME1 knockout include Cell adhesion molecule 3(CADM3),Cytochrome C(CYCS),Glyceraldehyde-3-phos-phate dehydrogenase(GAPDH),Phosphofructokinase(PFKM),etc.Conclusions:These findings suggest that NME1 can influence the phenotype changes of tumor cells,such as cell motility,and the dysregulation of the intracellular pHis modification may be widely related to disease procession.
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