The Epithelial Cell Transforming Sequence 2, a Guanine Nucleotide Exchange Factor for Rho GTPases, Is Repressed by p53 via Protein Methyltransferases and Is Required for G1-S Transition

Abstract

The epithelial cell transforming sequence 2 (ECT2), a member of the Dbl family of guanine nucleotide exchange factor for Rho GTPases, is required for cytokinesis. The tumor suppressor p53 plays a crucial role in coordinating cellular processes, such as cell cycle arrest and apoptosis, in response to stress signals. Here, we showed that ECT2 is negatively regulated by wild-type p53 but not tumor-derived mutant p53 or other p53 family members. In addition, ECT2 is down-regulated in multiple cell lines by DNA damage agents and Nutlin-3, an MDM2 antagonist, in a p53-dependent manner. We also showed that the activity of the ECT2 promoter is repressed by wild-type p53, and to a lesser extent, by p21. In addition, the second activation domain in p53 is necessary for the efficient repression of ECT2. Importantly, we found that the ECT2 gene is bound by p53 in vivo in response to DNA damage and Nutlin-3 treatment. Furthermore, we provided evidence that inhibition of protein methyltransferases, especially arginine methyltransferases, relieve the repression of ECT2 induced by DNA damage or Nutlin-3 in a p53-dependent manner. Finally, we generated multiple cell lines in which ECT2 is inducibly knocked down and found that ECT2 knockdown triggers cell cycle arrest in G 1 . Taken together, we uncovered a novel function for ECT2 and provided a novel mechanism by which p53 represses gene expression via protein methyltransferases. (Cancer Res 2006; 66(12): 6271-9)

The Lysine-specific Demethylase 1 Is Required for Cell Proliferation in Both p53-dependent and -independent Manners

“…MCF7-LSD1-KD-11 cells were uninduced or induced to knockdown LSD1 for 4 days. The percentage of cells in each phase of the cell cycle was quantified by fluorescence-activated cell sorter analysis as described previously (12).…”

“…Section 1734 solely to indicate this fact. 1 Cell Culture-The MCF7-pTR-7 cell line was generated previously by transfection of MCF7 cells with pcDNA6 vector that expresses a tetracycline repressor (12). To generate MCF7 cell lines inducibly expressing LSD1, MCF7-pTR-7 cells were transfected with pcDNA4/HA-LSD1 as well as a pBabe vector for puromycin selection.…”

“…Cells were collected and stained with propidium iodide as previously described and examined by fluorescence-activated cell sorter (FACS calibur) (12).…”

“…Interestingly, lysine methylation has varied effect on p53 function: methylation at lysine 372 by Set9 activates p53 transcriptional activity and methylation at lysine 370 by Smyd2 represses it (9,11). Furthermore, our earlier study indicates that p53 represses specific target genes via protein methyltransferases (12). The effect of LSD1 on non-histone protein function is yet unknown.…”

Wild-type p53 and p73 negatively regulate expression of proliferation related genes

“…Although most of the work has been focused on WT p53-mediated transactivation, p53 has also been shown to repress transcription of cellular and viral genes el-Deiry, 1998;Kubicka et al, 1999;Lee et al, 1999;Murphy et al, 1999;Krause et al, 2000;Maxwell and Davis, 2000;Sun et al, 2000;Zhang et al, 2000;Kannan et al, 2001;Koumenis et al, 2001;Aldaz et al, 2002;Hoffman et al, 2002;Maeda et al, 2006;Moskovits et al, 2006). Additionally, WT p53 inhibits transcription of genes promoting cell survival and genes involved in G 2 /M transition (Murphy et al, 1996(Murphy et al, , 1999Lee et al, 1999;Johnsen et al, 2000;Krause et al, 2000;Manni et al, 2001;Hoffman et al, 2002;Chae et al, 2005;Ho et al, 2005;Imbriano et al, 2005;Sengupta et al, 2005;Scoumanne and Chen, 2006;Spurgers et al, 2006;Van Bodegom et al, 2006).…”