Phosphorylation of Serine 147 of tis21/BTG2/pc3 by p-Erk1/2 Induces Pin-1 Binding in Cytoplasm and Cell Death

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.

TIS21 /BTG2/PC3 as a link between ageing and cancer: cell cycle regulator and endogenous cell death molecule

“…Interestingly, the constitutive expression of TIS21 mRNA in renal proximal tubule and prostate acini was lost in the renal cell carcinoma (Struckmann et al 2004) and the early stage of carcinogenesis in prostate (Ficazzola et al 2001), respectively. It is of an interest to note that Pin1 is a potential target for recurrent prostate cancer treatment (Ayala et al 2003;Ryo et al 2005) and one of the binding proteins of TIS21, when U937 tumor cells were stimulated by EGF (Hong et al 2005). These findings strongly support our hypothesis that cell death by TIS21 is induced through its binding to Pin1, resulting in the loss of mitochondrial membrane potential difference (Hong et al 2005).…”

“…Indeed, employing the wt and mt TIS21 proteins prepared, it was confirmed that S 147 in TIS21 was phosphorylated by p-ERK 1/2 , whereas S 149 residue was phosphorylated by p38 MAPK . Furthermore, pull-down Pin-1 and pre-phosphorylation of S 147 residue in TIS21 molecule by the treatment of U937 cells with EGF confirmed the interaction of TIS21 with Pin-1, and the P 148 residue was found to be essential for TIS21-Pin-1 binding (Hong et al 2005). Normally, Pin-1 is in nuclei, however, the treatment of U937 cells with EGF clearly induced translocation of Pin-1 to cytoplasm in 40 min.…”

“…We recently reported that TIS21 binds to Cdc2 kinase (Ryu et al 2004), and p-ERK 1/2 proteins can phosphorylate TIS21 serine 147 residue, resulting in the interaction with Pin-1 (Lu et al 1996), proline directed cis/trans isomerase. Therefore, p-ERK 1/2 can be regarded as a kinase for serine 147 of TIS21 protein (Hong et al 2005). In addition to p-ERK 1/2 , active p38…”

“…It is of an interest to note that Pin1 is a potential target for recurrent prostate cancer treatment (Ayala et al 2003;Ryo et al 2005) and one of the binding proteins of TIS21, when U937 tumor cells were stimulated by EGF (Hong et al 2005). These findings strongly support our hypothesis that cell death by TIS21 is induced through its binding to Pin1, resulting in the loss of mitochondrial membrane potential difference (Hong et al 2005). Treatment of prostate cancer cells with flutamide (androgen receptor antagonist) significantly up-regulates cell cycle regulators such as BTG1 and BTG2 /TIS21 (Wang et al 2005).…”

Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer

“…Although the most striking finding was the loss of expression in CRPC, we also found reduced expression of BTG2 in a subset of PC specimens. BTG2 seems to be involved in several cellular processes, including cell cycle control and apoptosis (Hong et al, 2005;Winkler, 2010). BTG2 belongs to a BTG/Tob family of six proteins and has been demonstrated to have antiproliferative activities (Winkler, 2010).…”