Fig. 4

FYN directly binds TOPK and phosphorylates TOPK at Y272. A Ni–NTA-His-TOPK bound with endogenous FYN of MGC803 cells. B COIP experiments confirm that FYN can bind to TOPK endogenously. C Colocalization of FYN and TOPK was visualized by confocal microscope in MGC803 cells. Cytoplasmic and nuclear staining of FYN and TOPK was mostly merged together. D Active FYN phosphorylated inactive TOPK in vitro in the presence of [γ-32P] ATP as visualized by autoradiograph. E Potential phosphorylated tyrosine sites of TOPK were predicted by NetPhos 3.1 software program. F FYN phosphorylated TOPK at Y272 in peptide mapping. Five synthesized peptides containing potential tyrosine sites were used as substrates in an in vitro kinase assay with active FYN in the presence of [γ-32P] ATP and the results were visualized by autoradiography. G Wild type His-TOPK (WT), single mutant His-TOPK (74F), single mutant His-TOPK (272F) or double mutant His-TOPK (FF) were used as substrates in an in vitro kinase assay with active FYN in the presence of [γ-32P] ATP and the results were visualized by autoradiography. H Validation of anti phospho-TOPK (Y272) (p-TOPK (Y272)) in an in vitro kinase assay. Wild type His-TOPK (WT), single mutant His-TOPK (74F), single mutant His-TOPK (272F) or double mutant His-TOPK (FF) as shown was used as substrate for active FYN. Reactive products were resolved by SDS-PAGE and visualized by Western blot with p-TOPK (Y272). I Co-transfection of Flag-FYN and HA-TOPK-WT,HA-TOPK-Y272F in 293 T cells. J-K Detection of changes in TOPK, p-TOPK(Y272) levels after knockdown of FYN expression in SGC7901 and MGC803 cells