phosphorylated c-Src was enhanced 6 hour after pcDNA3.1- Piwil2 transfection, while the level of pY416 c-Src decreased after Piwil2-siRNA transfection (Fig. 4B). A triple protein-protein complex is formed when Piwil2 recruits STAT3 to c-Src The effect of Piwil2 on STAT3 phosphorylation was investi- gated by co-immunoprecipitation (coIP) assay, revealing that the ,110 kD Piwil2 protein was physically associated with STAT3 and c-Src, while STAT3 was also observed associating with c-Src (Fig. 5A). Meanwhile, the absence of STAT3 blocked the binding between Piwil2 and c-Src (Fig. 5B), whereas Piwil2 knockdown led to decreased c-Src signal detected in STAT3 immunoprecipitates (Fig. 5C). The results suggested that Piwil2 played an essential role in the assocaiation between STAT3 and non-receptor tyrosine kinase c-Src. Thus they formed a triple protein-protein complex and STAT3 was phosphorylated and activated. To further investigate the interaction between Piwil2, STAT3 and c-Src, we constructed a series of Piwil2 deletion mutants to identify the functional domains required for phosphorylating STAT3. Two Piwil2 mutants, one harbored a deletion of PAZ conserved domain while the other lacks both PAZ and PIWI domains, failed to bind with STAT3, while other mutants retained the ability (Fig. 6A and B). Notably, the PAZ-deleted mutants failed to induce STAT3 phosphorylation (Fig. 6C) and inhibit cell apoptosis (Fig. 6D), confirming the potential role of PAZ domain of Piwil2. Figure 5. Interaction between Piwil2, c-Src and STAT3. A, cell lysates were prepared and subjected to coIP assays. TP, total protein lysates as positive control; NC, protein A+G agarose incubated with total protein lysate to detect nonspecific interaction (negative control, detected with corresponding antibodies). B, In STAT3-silenced cells (STAT32, transfected with STAT3-specific siRNA), the binding between Piwil2 and c-Src was repressed in contrast to lysates of control cells (STAT3+, transfected with nonspecific siRNA). C, the binding between c- Src and STAT3 was repressed in Piwil2-silenced cells (Piwil2-, transfected with Piwil2-specific siRNA). doi:10.1371/journal.pone.0030999.g005 Piwil2 Activates STAT3 and Inhibits P53 Discussion It has been reported that Piwil2 transcripts were widely detected in various tumors or cancer cell lines, and played important roles in tumorigenesis and apoptosis inhibition [13,24–26]. However, the mechanism underlying piwil2-mediated tumor development re- mained unclear yet. To investigate the signaling pathway through which Piwil2 suppresses the apoptosis of cancer cells, we studied the relationship between Piwil2 and its potential target genes. Our research demonstrated that Piwil2 can efficiently activate STAT3 by phosphorylating STAT3 at residue Tyr705 (Fig. 2), which leads to dimerization of STAT3 and downstream targets control [18,20]. We next examined whether Piwil2 also enhanced STAT3 DNA binding activity. Our results indicated that induced by Piwil2 overexpression, STAT3 DNA binding activity to P53 promoter was significantly increased, leading to epigenetic modification and silencing of P53 gene (Fig. 3B and C). This founding was supported by previous study on roles of STAT3 in regulating P53 by Niu et al [19]. Activation of STAT3 required tyrosine kinases activity, which Piwi family proteins have not yet been reported to possess. JAK/ STAT3 pathway is well-known to respond extracellular signal and regulate expression of various genes [27–29]. However, AG490, a specific JAK/STAT3 pathway inhibitor, showed little effort in blocking Piwil2-induced STAT3 phosphorylation (Fig. 4A), sug- gesting that JAK family kinases may not be involved in Piwil2- STAT3-P53 pathway. So we next analyzed another candidate Src, a family of non-receptor tyrosine kinases that has been reported to constitutively activate STAT3 during oncogenic transformation [30–33]. Western blot experiments revealed that the overexpres- sion of Piwil2 in PP2-treated HeLa cells failed to increase pYSTAT3 level, while the knockdown of Piwil2 made no significant decrease of pYSTAT3 compared to the control. Further experiments revealed that overexpressed Piwil2 can increase the phosphorylation level on Tyr416 of c-Src, which indicates tyrosine kinase activity of the protein (Fig. 4B). Immunoprecipitation assay demonstrated that Piwil2, STAT3 and c-Src can physically associated with each other. However, silencing of Piwil2 or STAT3 may lead to significantly repressed binding level between c-Src and the other protein(Fig. 5), suggesting that Piwil2 played an essential role in the association between STAT3 and non-receptor tyrosine kinase c-Src. Thus they formed a triple protein-protein complex and STAT3 was phosphorylated and activated. Based on our results and previous finding about STAT and P53, we demonstrated that Piwil2 inhibits P53 involved apoptosis through Src-STAT3 pathway in HeLa cells (Fig. 7). Our immunoprecipitation analysis shows that Piwil2 can directly associate with STAT3 via its PAZ domain and form a Piwil2/ STAT3/c-Src triple protein-protein complex. As shown in Figure 1 and Figure 2, our present study shows that the activation of STAT3 phosphorylated by c-Src at residue Tyr705 is significantly increased in Piwil2 overexpressed HeLa cells. The phosphorylated STAT3 proteins dimerize and translocate to nucleus as reviewed by Burdon et al [22]. Furthermore, dimerized STAT3 proteins bind to the promoter region of P53 gene and repress P53 expression. To study the function of human Piwil2 gene, alias HILI in Hela cells, we analyzed its relationship with P53 and apoptosis. As variant deletions or mutations of P53 have been observed in a great number of tumors, the underlying mechanism of anti- apoptosis may differ among variant cancer cells. However, our results demonstrated that Piwil2 functioned to induce STAT3 phosphorylation and prevent expression of P53 at least in two cell lines (HeLa and HepG2). These investigations will provide a novel insight into roles of Piwil2 in tumor cells. PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30999