Fig. 5: MST4 interacts with NPM1 and phosphorylates NPM1 at Thr95.

A Venn diagram showing the overlapping of mass spectrometry results. e.g., yellow region represents proteins specifically pulled down by the MST4 antibody in KYSE150 cells, blue region represents proteins specifically pulled down by the IgG antibody in KYSE150 cells, while the mixed-color region indicates common proteins identified across these two groups. Mass spectrometry analysis revealed that MST4 and NPM1 were specifically detected only in MST4 antibody-treated KYSE150 and KYSE450 cell lines, as indicated by the arrows in the figure. B, C Direct interaction of MST4 and NPM1 was checked by western blot. D Co-IP of Flag-MST4 WT (wild type) or Flag-MST4 mutant T178A with HA-NPM1 in HEK293T cells. E In vitro kinase assay was performed in the presence of [γ-³²P] ATP and the phosphorylation signal was visualized by autoradiography. F In vitro kinase assay of the NPM1 by MST4. The phosphorylated protein was detected using p-NPM1 (T95) and p-NPM1 (T199) antibody. G Active MST4 was incubated with the indicated NPM1 mutate protein in kinase reaction buffer. Phosphorylation signals were detected by western blot. The gray value was calculated using Image J software. H MST4 knock down decreased p-NPM1 (T95) expression in indicated cells, but did not affect total NPM1 expression. I In vitro kinase assay of the NPM1 by LIMK2. The phosphorylated protein was detected using p-NPM1 (T95) antibody. J In vitro kinase assay was performed in the presence of [γ-³²P] ATP, phosphorylation signal was visualized by autoradiography.