Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing

Abstract

Objective To analyze the biological processes regulated by protein kinase WNK2 in ovarian cancer cells based on protein phosphorylation modified mass spectrometry. Methods Ovarian cancer cell line SKOV3 was transfected with lentiviral vector to knockdown WNK2 and the obtained cells were further screened by drug screening. Then the total protein was extracted for protein phosphorylation modification mass spectrometry analysis. Bioinformatics analysis was adopted to detect the phosphorylation changes before and after WNK2 knockdown, and differential proteins were screened out. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were employed to explore the functions of differentially phosphorylated proteins, and experiments were performed to verify the biological processes regulated by WNK2. Results Phosphorylation modified mass spectrometry indicated that WNK2 knockdown significantly decreased the phosphorylation of 745 proteins with 1 078 altered protein sites, and increased the phosphorylation of 328 proteins with 415 altered protein sites. KEGG and GO enrichment analyses showed WNK2 influences biological processes such as cytoskeleton organization, signal transduction, cell composition, and regulation of kinase activity. Phalloidin staining indicated after WNK2 knockdown resulted in broken and disordered cytoskeleton, while its overexpression promoted cytoskeleton remodeling. Conclusion WNK2 is involved in the cytoskeleton formation in ovarian cancer cells by regulating the phosphorylation of vimentin and other proteins.

Keywords

• ovarian cancer
• wnk2
• phosphorylation modification
• vimentin
• cytoskeleton