Abstract
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.
Highlights
Type p53 response element in the 5Ј-flanking region (Ϫ74 to Ϫ122) [6], confirming the observation that BTG2 expression is dependent on wild type p53 function [5]
It has been known that tis21/BTG2/pc3 interacts with transcription factor caf1 [10], protein arginine methyltransferase PRMT1 [11], and homeogene Hoxb9 [12], which can regulate cell cycle and differentiation
We demonstrate in the present study that tis21 protein interacts with Pin-1 after phosphorylation of its Ser147 residue by p-Erk1/2, which was induced by epidermal growth factor (EGF) and inhibition of cyclin B1-associated Cdc2 activity
Summary
S147A mutant (a439gc 3 g439cc) P148A mutant (c442cc 3 g442cc) S149A mutant (t445cg 3 g445cg). 5Ј-gg agc agc ccc gcg aag aac tat g-3Ј response gene induced by TPA or various growth factors [1,2,3], neither TPA nor high cell densities are able to increase BTG2 expression in the tumor cells to the level observed in normal renal tissues [32]. It should be noted that all of these counterpart normal tissues constitutively express tis mRNAs. The above described findings strongly support tumor suppressive function of tis in carcinogenic process and indicate that down-regulation of BTG2 is a pivotal event involved in cancer development. Mitochondrial depolarization and increased cell death occurred. We have found a mechanism of how EGF can be anti-proliferative through Pin-1 binding to tis21/BTG2/pc, resulting in increased mitochondrial depolarization
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