Identification And Function Study Of UHRF2 Differ Phosphorylation Level Sites In L02 And HepG2 Cells

ObjectiveThis article aims to identify differ phosphorylation level sites of UHRF2 and study its biological function in Human liver carcinoma cells(HepG2)and Human normal liver cells(L02).MethodsIn this paper,these different phosphorylation level sites of UHRF2 protein were identified by LC-MS-MS in HepG2 and L02 cells;The expression of UHRF2 phospho-mutants and TIP60-related proteins were detected by Western blotting(WB);The effects of UHRF2 mutants on cell cycle were detected by Flow cytometry(FCM);The cellular localization of UHRF2 mutant proteins were verified by Immunofluorescence(IF);The ability of ubiquitination of UHRF2 mutants were detected by in vivo Ubiquitination assay.ResultsLC-MS-MS results showed that the serine 643(S643)and the serine 683(S683)of UHRF2 protein have different phosphorylation levels in HepG2 and L02 cells;After constructed the UHRF2 mutant plasmids,their sequences were detected by DNA sequencing;The UHRF2 mutants were able to be expressed normally in HepG2 and L02 cells detected by WB;The effects of UHRF2 mutants on cell cycle were detected by FCM.In L02 cells,the cells in S phase were decreased significantly in the mutation groups compared with the WT UHRF2 group,while cells in G1 and G2 phase were increased significantly.In HepG2 cells,the cells in the S phase were decreased significantly in the UHRF2 S643 A and UHRF2 S643A/S683 A groups compared with the WT UHRF2 group,while the cells in G1 and G2 phase were increased significantly in the UHRF2 S643A/S683 A groups;The expression of UHRF2 mutants and TIP60-associated proteins were detected by WB.The ubiquitination ability of UHRF2 mutants were higher than UHRF2 detected by in vivo Ubiquitination assay.ConclusionUHRF2 have different phosphorylation level sites in Human normal liver cells(L02)and Human liver carcinoma cells(HepG2).The UHRF2 phosphorylation sites do not affect its own sublocalization,but have significance influence on its ubiquitination ability,cell cycle progression and the expression of TIP60-related proteins.