PIAS1 Negatively Regulates EMT (48 h), alone or together were subjected to PIAS1 immunoprecipitation followed by sequential ubiquitin and PIAS1 immunoblotting. Scans in C to F are representative of experiments that were carried out at least 3 independent times. doi:10.1371/journal.pone.0013971.g001 suppression of E-cadherin reporter gene promoter activity (Figure 2C). Collectively, these data suggest that the SUMO E3 PIAS1 inhibits TGFb-repression of E-cadherin expression. We next used biochemical and cell-based approaches to test the possibility that PIAS1 negatively regulates TGFb-induced EMT. We first generated NMuMG cells that stably expressed the wild type or SUMO ligase mutant PIAS1, or that were stably transfected with the control vector. Immunoblotting analyses confirmed that the wild type and SUMO E3 ligase mutant PIAS1 were expressed at equivalent level in these cells (Figure 3A). Comparison of the level of exogenous PIAS1 in these cells relative to control transfectants showed that expression of the wild type or SUMO E3 ligase mutant PIAS1 was only modestly increased relative to endogenous levels of PIAS1 (Figure 3A). Immunoblot- ting analyses using an antibody to E-cadherin revealed that expression of PIAS1 decreased the ability of TGFb to suppress the levels of E-cadherin protein in NMuMG cells (Figure 3B). Quantitative analysis confirmed that the ability of PIAS1 to reverse TGFb downregulation of E-cadherin levels was statistically significant (Figure 3C). In contrast, we found that mutation of the SUMO ligase activity significantly mitigated the ability of PIAS1 to inhibit TGFb-repression of E-cadherin protein level (Figures 3B and 3C). Together, these data show that PIAS1 protects E- cadherin protein from TGFb-dependent downregulation. We also examined the effect of PIAS1 expression on different EMT parameters using cell-based assays. At the basal epithelial state, expression of PIAS1 did not appreciably affect the localization or expression of E-cadherin (Figure 3D). Similarly, we found that wild type or SUMO ligase mutant PIAS1 did not alter significantly the cortical actin organization or cuboidal cell shape in untreated cells (Figures S3A and S3B). In contrast, expression of wild type but not the SUMO ligase mutant PIAS1 drastically inhibited the ability of two different concentrations of TGFb to induce a loss in E-cadherin levels (Figures 3D and 3E). Expression of PIAS1 suppressed the ability of TGFb to induce the formation of actin stress fibers and fibroblastic cell phenotype (Figures S3A and S3B). In contrast, the SUMO ligase mutant PIAS1 did not reduce the ability of TGFb to change actin reorganization and cell shape (Figures S3A and S3B). Collectively, these findings suggest that the SUMO E3 ligase activity of PIAS1 plays a critical role in suppressing the ability of TGFb to induce EMT. The PIAS1 substrate SnoN contributes to PIAS1 inhibition of EMT The finding that the SUMO E3 ligase activity is required for the ability of PIAS1 to antagonize EMT suggested that specific SUMO substrates might mediate the effect of PIAS1 on this fundamental cellular process. The transcriptional regulator SnoN plays important roles in TGFb signaling and responses, including antagonizing TGFb-induced EMT in cancer cells [39,49]. Recently, we and others showed that SnoN is sumoylated and that Lysines 50 and 383 on SnoN are major sites for SUMO conjugation [38,50]. We also identified SnoN as a PIAS1 SUMO substrate [38,50]. We therefore tested the hypothesis that sumoylation contributes to the ability of SnoN to inhibit TGFb- induced EMT and that TGFb triggers a reduction in the levels of PIAS1 and hence sumoylated SnoN to mediate EMT. SUMO- conjugated proteins can be difficult to detect due to their desumoylation by SUMO-proteases that might be activated during lysis [37,51]. The general SUMO-protease inhibitor N- ethylmaleimide (NEM) is typically included in the lysis buffer to preserve the SUMO-conjugated species in the cellular extracts [52,53]. Using SDS-PAGE analysis, the apparent molecular mass of these NEM-sensitive SUMO-conjugated species is 20 kDa or more as compared to the unmodified protein [37,54]. We prepared NMuMG cellular extracts in the absence or presence of NEM and subjected these lysates to SnoN immunoprecipitation followed by SnoN immunoblotting. We first confirmed that endogenous SnoN was sumoylated in NMuMG cells as indicated by the appearance of several specific slow migrating NEM- sensitive SnoN immunoreactive species as compared to unmod- ified SnoN in SnoN immunoprecipitates (Figure 4A). Next, we examined the status of SnoN sumoylation in cells undergoing EMT. Interestingly, we found that TGFb stimulation led to a reduction in the proportion of NEM-sensitive sumoylated-SnoN species in cells undergoing EMT (Figure 4B). In other experi- ments, inhibition of TbRI blocked the ability of TGFb to reduce sumoylated-SnoN in these cells (Figure 4C). These data suggest that TGFb signaling reduces the level of SnoN sumoylation in cells undergoing EMT. The finding that TGFb decreases SnoN sumoylation suggested that this modification may contribute to SnoN’s ability to inhibit EMT. To test this possibility, we compared the effect of stable expression of wild type SnoN or a SUMO loss of function SnoN mutant, in which Lysines 50 and 383 were converted to arginine residues (SnoN (KdR)), on EMT [38]. Wild type and mutant SnoN were expressed at equivalent levels (Figure 5A). Upon immunoblotting, we found that wild type SnoN inhibited the ability of TGFb to suppress E-cadherin expression (Figures 5B and 5C). In contrast, expression of the SUMO loss of function SnoN mutant (KdR) had little or no effect on the ability of TGFb to reduce E-cadherin expression (Figures 5B and 5C). Likewise, expression of wild type SnoN but not SnoN (KdR) antagonized TGFb-inhibition of E-cadherin in the NMuMG cells as deter- mined by immunocytochemistry (Figures 5D and 5E). These data suggest that sumoylation may be important for SnoN to inhibit TGFb-induced EMT. In addition to sumoylation, lysine residues can undergo other modifications including acetylation and methylation [37,55]. To exclude the possibility that the lack of effect of the SnoN (KdR) on E-cadherin suppression by TGFb is due to impairment of potential modifications other than sumoylation, we generated a distinct SUMO loss of function SnoN mutant (SnoN (EdA)) in which Glutamates 52 and 385 within SUMO consensus motifs (yKXE) in SnoN were converted to alanine residues (Figure 6A). The acidic amino acid in position +3 of the SUMO consensus motif is critical for covalent linkage of the lysine residue with SUMO [37]. Accordingly, we found that replacement of Glutamates 52 and 383 with alanines abrogated the ability of SnoN to undergo sumoylation at Lysines 50 and 383 (Figure 6B). We next compared the effect of stable expression of wild type and the SUMO loss of function SnoN (EdA) on EMT. Wild type SnoN and the SnoN (EdA) mutant were expressed at comparable levels in these cells (Figure 6C). Immunoblotting analyses showed that while wild type SnoN blocked the ability of TGFb to reduce E-cadherin expression, the SnoN (EdA) mutant was ineffective in counteracting the TGFb response (Figures 6D and 6E). Likewise, immunofluorescence studies showed that although wild type SnoN blocked downregulation of E-cadherin in NMuMG cells under- PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e13971