Phosphorylation of serine 147 of tis21/BTG2/pc3 by p-Erk1/2 induces Pin-1 binding in cytoplasm and cell death

Treatment of U937 cells with epidermal growth factor (EGF) induces phosphorylation of tis21 and subsequent interaction of tis21 with Pin-1, resulting in the increased cell death with mitochondrial depolarization. Ser147 and Ser149 residues of tis21 were strongly phosphorylated by p-Erk1/2 and p-p38(MAPK), respectively, but not by JNK. To investigate the significance of phosphorylation of the Ser147 residue, Pin-1, one of the mitotic regulators that binds to the Ser(P)/Thr(P)-Pro region, was employed. Wild type tis21 phosphorylated by p-Erk1/2 clearly increased its binding to Pin-1, but not the P148A mutant, indicating that Pin-1 was bound to the Ser(P)147-Pro148 region of tis21. Transfection of tis21 significantly enhanced EGF-induced Pin-1 diffusion to cytoplasm, compared with that in the vector-transfected cells. Knockdown of tis21 expression by using shRNAi significantly inhibited EGF-induced Pin-1 diffusion, and analysis by flow cytometry after JC-1 stain and confocal microscope revealed that EGF aggravated tis21-induced mitochondrial depolarization and cell death. Furthermore, tis21 was bound to cyclin B1 and Cdc2 and inhibited its activity in vivo and in vitro. In summary, treatment of U937 cells with EGF activates ERK1/2, which in turn phosphorylates Ser147 of tis21 and induces tis21 and Pin-1 binding and mitochondrial depolarization. These data suggest, for the first time, a mechanism of how EGF can be antiproliferative in human tumor cells: binding of tis21/BTG2/pc3 to Pin-1 or cyclin B1-Cdc2 complex and induction of mitochondrial depolarization.