Abstract
GTSE-1 (G2 and S phase-expressed-1) protein is specifically expressed during S and G2 phases of the cell cycle. It is mainly localized to the microtubules and when overexpressed delays the G2 to M transition. Here we report that human GTSE-1 (hGTSE-1) protein can negatively regulate p53 transactivation function, protein levels, and p53-dependent apoptosis. We identified a physical interaction between the C-terminal regulatory domain of p53 and the C-terminal region of hGTSE-1 that is necessary and sufficient to down-regulate p53 activity. Furthermore, we provide evidence that hGTSE-1 is able to control p53 function in a cell cycle-dependent fashion. hGTSE-1 knock-down by small interfering RNA resulted in a S/G2-specific increase of p53 levels as well as cell sensitization to DNA damage-induced apoptosis during these phases of the cell cycle. Altogether, this work suggests a physiological role of hGTSE-1 in apoptosis control after DNA damage during S and G2 phases through regulation of p53 function.
Highlights
The murine GTSE-11 (G2 and S phase-expressed) gene, previously named B99, was cloned in our laboratory during a screening of p53-inducible genes from a murine cell line that stably expresses a temperature-sensitive p53 allele [6]
When p53 transactivation function was assessed using the pG13-Luc reporter, we observed that human GTSE-1 (hGTSE-1) silencing could significantly enhance p53 activity even in cells treated with ET (Fig. 3F), concluding that increased p53 levels due to hGTSE-1 silencing should synergize with DNA damage-dependent p53 stabilization and activation, providing a more robust p53 response when compared with cells expressing normal levels of hGTSE-1 protein
In this work we have presented evidence that hGTSE-1 protein participates in the cellular response to DNA-damaging agents by regulating p53 function and stability during the S and G2 phases of the cell cycle
Summary
U2OS (wt-p53) and MG-63 (p53 null) human osteosarcoma cell lines were cultured at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 g/ml). Cells treated with methyl methanesulfonate (MMS) (Fluka) were incubated for 4 h with the drug, followed by extensive washing and medium replacement until the end of the treatment. The N-terminal hGTSE-1 construct (NT-hGTSE-1) encodes for a deletion mutant of hGTSE-1 from amino acids 1– 476 inserted into pcDNA3 (Invitrogen). The Cterminal hGTSE-1 construct (CT-hGTSE-1) encodes for a deletion mutant of hGTSE-1 from amino acids 476 –720 inserted into pcDNA3.1 with a His N-terminal tag (Invitrogen). GFP1⁄7hGTSE-1 contains the full-length hGTSE-1 fused to GFP (pEGFP vector, Clontech). PcDNA3-p53wt contains the full-length human wt-p53 cDNA. GFP1⁄7p53 contains human wild-type p53 fused to the GFP protein
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