Overexpressed Melk Promotes the Stability of EZH2 through Phosphorylation in Natural Killer/T Cell Lymphoma (NKTL)

In this study, we examined EZH2 protein turnover mechanisms in the NKTL context.The serine/threonine kinase Melk is one of the overexpressed genes in NKTL patient samples and cell lines, and the interaction between Melk and EZH2 was established by co-immunoprecipitation. Inhibition of Melk using inhibitor or siRNA both resulted in a decrease of EZH2 protein levels in NKTL cells, whereas there was no change in the mRNA level of EZH2, suggesting that Melk regulated EZH2 at the protein level. Next, we observed a change of EZH2 ubiquitination upon manipulation of Melk expression.Next, in order to confirm that Melk truly affect EZH2 ubiquitination and to identify its (de)ubiquitination site, we used a SILAC-based mass spectrometry (MS) approach. We overexpressed all-lysine-mutated ubiquitin plus EZH2 with or without Melk overexpression in 293T cells, and pulled down EZH2 for MS analysis. The MS data found a decrease of K48-linked ubiquitin peptide upon Melk overexpression, which corresponded to the impact of Melk on EZH2 protein stability, as well as two possible sites of EZH2 (de)ubiquitination. One of these two sites were confirmed in later experiments as a critical site (K222).As Melk is a kinase, it is possible that the regulation of EZH2 ubiquitination is phosphorylation-based. A re-analysis of the EZH2 post-translational modification from the MS data identified S220-phosphorylated EZH2 upon Melk overexpression. And by comparing the forward and reverse H/L ratio of S220-phosp...
Source: Blood - Category: Hematology Authors: Tags: 622. Lymphoma Biology-Non-Genetic Studies: Poster II Source Type: research