Oncogene (2010) 29, 3124–3133 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Role of CK1 in GSK3b-mediated phosphorylation and degradation of Snail

YXu1,4, S-H Lee2,4, HS Kim3, NH Kim3, S Piao1, S-H Park1, YS Jung2, JI Yook3, B-J Park2 and N-C Ha1

1Department of Manufacturing Pharmacy, College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan, Republic of Korea; 2Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan, Republic of Korea and 3Department of Oral Pathology, Oral Cancer Research Institute, College of Dentistry, Yonsei University, Seoul, Republic of Korea

The epithelial to mesenchymal transition (EMT) that occurs Snail binds to the E-box motifs of its target gene during embryonic development has begun to attract attention promoters through a C-terminal Zn-finger domain and as a potential mechanism for tumor cell metastasis. Snail represses their transcription by associating with histone is a well-known Zn-finger transcription factor that promotes deacetylase activity through its N-terminal region EMT by repressing E-cadherin expression. It is known (Batlle et al., 2000; Peinado et al., 2004). that Snail is phosphorylated by GSK3b and degraded The transforming growth factor-b signaling has been by b-TrCP-mediated ubiquitination. Here we described shown to increase Snail transcription during the wound- another protein kinase, CK1, whose phosphorylation of healing process (Peinado et al., 2003). However, Snail Snail is required for the subsequent GSK3b phosphorylation. mRNA is constitutively present in many cells, even Specific inhibition or depletion of CK1e inhibits the in the absence of the activation of this signaling path- phosphorylation and degradation of Snail and promotes cell way (Zhou et al., 2004; Lee et al., 2009). Snail is also migration, suggesting a central role of CK1e in the EMT regulated at the protein level in many cells and is process. Furthermore, our study uncovered distinct roles and constantly degraded by proteasome, depending on the steps of Snail phosphorylation by CK1e and GSK3b.Taken phosphorylation status (Zhou et al., 2004). As the half- together, we identified CK1e as a new component of the life of Snail is very short in cells, the detection of Snail Snail-mediated EMT process, providing insight into the protein in cells is difficult (Zhou et al., 2004). mechanism of human cancer metastasis. Snail has many parallels with a transcriptional co- Oncogene (2010) 29, 3124–3133; doi:10.1038/onc.2010.77; activator, b-catenin. Cytosolic b-catenin is phospho- published online 22 March 2010 rylated by GSK3b at serine or threonine residues in its N-terminal domain (Liu et al., 2002). The phospho- Keywords: snail; casein kinase 1; EMT; GSK3b;metastasis rylated b-catenin is recognized by the b-TrCP-a F-box protein of the Skp1–Cul1–F-box-protein E3 ubiquitin ligase complex (Winston et al., 1999), then polyubiqui- tinated and thus targeted for proteasomal degradation Introduction (Aberle et al., 1997; Orford et al., 1997). Likewise, Snail is phosphorylated by GSK3b at Ser/Thr residues The epithelial to mesenchymal transition (EMT) is a key (Figure 1a), which is then recognized by b-TrCP event in cancer cell invasion, which accounts for about for degradation (Zhou et al., 2004). Wnt stimulation 90% of cancer deaths (Pantel and Brakenhoff, 2004; inhibits the GSK3b activity towards b-catenin (Liu Zhou and Hung, 2005). The EMT process is associated et al., 2002; Piao et al., 2008) and Snail (Yook et al., with the functional loss of E-cadherin, an essential 2005, 2006) through distinct mechanisms, thereby step towards the invasive phase of carcinoma (Nieto, inducing the accumulation of both proteins in cells. 2002; Thiery, 2002). The loss of E-cadherin is due to the It is well established that b-catenin requires CK1 as a transcriptional down-regulation rather than gene loss or priming kinase for GSK3b phosphorylation, which mutation in many metastatic cancer cells (Birchmeier requires a phosphorylated Ser/Thr residue at the p þ 4 et al., 1991; Cavallaro and Christofori, 2004), and position of the substrate (Liu et al., 2002). CK1 E-cadherin expression has been associated with Snail, phosphorylates b-catenin at Ser45, providing a priming a Zn-finger transcription factor (Batlle et al., 2000). site for the subsequent GSK3b phosphorylation of b-catenin (Thr41, Ser37 and Ser33) (Liu et al., 2002). Correspondence: N-C Ha, Department of Manufacturing Pharmacy, However, it remains to be elucidated whether GSK3b College of Pharmacy and Research Institute for Drug Development, requires a priming kinase for phosphorylating Snail. The Pusan National University, 30 Jangjeon-dong, Geumjeong-gu, Busan similarities between the two proteins prompted us to 609-735, Republic of Korea. examine the involvement of CK1 in the phosphorylation E-mail: [email protected] of Snail. In this study, we provided evidence that the 4These authors contributed equally to this work. Received 16 September 2009; revised 20 January 2010; accepted 9 phosphorylation of Snail is primed by CK1, which is February 2010; published online 22 March 2010 analogous to the phosphorylation of b-catenin. Phosphorylation of Snail by CK1 YXuet al 3125 Results this antibody was used as the primary antibody in western blot. The bands corresponding to the Snail Sequence analysis predicted the presence of two GSK3b proteins disappeared, whereas the GSK3b band that target regions in Snail: site I (residues 96–104) and site II was not recognized by the antibody was not affected (residues 107–119) (Zhou et al., 2004). Site I comprises (pS96 þ ; Figure 1b right). Moreover, the band intensity the destruction box that is recognized by b-TrCP, of the fully phosphorylated b-catenin fragment (1–133) containing two serine residues (Ser96 and Ser100). Site that contains the natural target sites of the antibody was II covers the Ser/Thr-rich cluster ranging from Ser107 to only partially reduced by the treatment of the synthetic Ser119 and is involved in the nuclear export of Snail, phospho-peptide (Figure 1b lane 9). These results when phosphorylated by GSK3b (Dominguez et al., exclude the possibility that the synthetic phospho- 2003; Zhou et al., 2004) (Figure 1a). As Snail is peptide pS96 non-specifically block phospho-specific degraded via b-TrCP when Ser96 and Ser100 residues antibodies in the experiment using the Snail fragments. are phosphorylated, the detection of the phosphoryla- This result shows that the polyclonal anti-phospho-b- tion at Ser96 and Ser100 is important for monitoring the catenin (Ser33/37/Thr41) antibody contains antibodies stability of Snail in cells. In particular, the phosphoryla- that specifically recognize the phosphorylated Ser96 tion at Ser96 has an important role in regulating the residue in Snail. The specificity of the antibody was stability of Snail, because Ser96 is the final phosphor- verified using the Snail S96A mutant protein. This ylation site that is required for the b-TrCP binding. mutant was not recognized by the antibody due to the lack of the phosphorylation recognition site (Figure 1c). Given the specificity of the anti-phospho-b-catenin Anti-phospho-b-catenin (S33/37/T41) antibody (Ser33/37/Thr41) antibody, we used it to investigate can recognize phosphorylated Ser96 in Snail the phosphorylation of Snail at Ser96 in this study. To investigate how Snail is phosphorylated by GSK3b, we developed an in vitro kinase assay for the The Snail phosphorylated by CK1 and GSK3b can bind phosphorylation of Snail at Ser96, using purified to b-TrCP proteins and a phospho-specific antibody. As no To assess the Snail fragment phosphorylated by the co- phospho-specific antibody for this site is available, we treatment of CK1 and GSK3b using a cellular attempted to find an alternative antibody that recog- component, the phosphorylated protein was incubated nizes this phosphorylation site. Given the similarity with the cell lysate containing b-TrCP that recognizes in sequence identity between the signature sequence the phosphorylated Snail. Only the phosphorylated (Asp–Ser–Gly) of the b-TrCP target site in Snail and form of Snail specifically interacted with the b-TrCP that of b-catenin (Figure 1a), a polyclonal anti-phospho- protein in the cell lysate (Figure 1d). This finding b-catenin (Ser33/37/Thr41) antibody was tested for this indicates that the kinases phosphorylate Snail at the purpose in western blot analysis. This antibody was Ser96 residue, presumably also at the Ser100 residue, generated from a synthetic phospho-peptide corre- because b-TrCP efficiently binds to the DSGxxS motif sponding to the residues surrounding the Ser33, Ser37 when both Ser residues are phosphorylated in this motif and Thr41 of human b-catenin (SYLDSGIHSGAT (Winston et al., 1999; Frescas and Pagano, 2008). In this TAPC, where S or T represents phosphorylated S or T), experiment, the binding of p53 to the Snail fragment and it recognizes the phosphorylation of b-catenin at (Lee et al., 2009) was examined as a positive control, Ser33, Ser37 or Thr41 (Liu et al., 2002). because the Snail fragment contains a p53-binding Two glutathione-S-transferase (GST)-fused Snail region. The phosphorylation of Snail by the co- fragments (residues 91–112 and residues 91–128) were treatment of GSK3b and CK1 did not affect its binding used as substrates (Figure 1a). The smaller fragment to p53 in the cell lysate (Figure 1d). (Snail-S) is composed of the conserved serine residues that are present in Snail from Drosophila to human and Both CK1 and GSK3b activities are required includes the b-TrCP recognition site; the longer one to phosphorylate Snail at Ser96 (Snail-L) has the additional Ser/Thr-rich cluster at- To analyze the role of CK1, we treated the Snail-MC tached to the Snail-S fragment (Figure 1a). Western blot fragment (residues 91–264, containing the Zn-finger analysis showed strong bands when the recombinant domain) that is more physiologically relevant than kinase domains of CK1e and GSK3b were incubated the other shorter fragments with CK1e and GSK3b, with the Snail fragments, indicating that Snail is respectively, or in combination. As shown in Figure 2a, phosphorylated by the combination of CK1e and GSK3b could not phosphorylate Ser96 in any tested GSK3b. In addition, the results suggest that the anti- Snail fragments in the absence of CK1. However, the phospho-b-catenin (Ser33/37/Thr41) antibody is a can- Snail fragments were strongly phosphorylated at Ser96 didate to detect the phosphorylation of Snail at Ser96 when both GSK3b and CK1 were present (Figure 2a). (Figure 1b). The Snail-MC fragment was phosphorylated more To examine whether this anti-phospho-b-catenin efficiently by the co-treatment of GSK3b and CK1 (Ser33/37/Thr41) antibody specifically recognized the (Figure 2a) than the other shorter fragments (Figure 1), phosphorylation of Snail at Ser96, we added a synthetic even though a much smaller amount was used, indicat- phospho-peptide (pS96; Figure 1) containing the phos- ing that the Snail-MC fragment is a better substrate than phorylated Ser96 residue and its adjacent residues, when the other shorter Snail fragments. These results suggest

Oncogene Phosphorylation of Snail by CK1 YXuet al 3126 that Snail requires CK1, as well as GSK3b, in the responsible for the phosphorylation of Snail in coopera- phosphorylation of Snail, as in the case of b-catenin (Liu tion with GSK3b. Likewise, the phosphorylation rate et al., 2002), and that the Zn-finger domain of Snail may was significantly inhibited by a potent GSK3b inhibitor, have a role in the phosphorylation by CK1. lithium chloride, demonstrating that GSK3b catalytic To rule out the possibility that CK1 may function as activity is also important, which has been previously an allosteric activator of GSK3b or Snail, we generated reported (Zhou et al., 2004; Figure 2c). a non-catalytic CK1e mutant protein and measured Ser96 phosphorylation in the presence of both GSK3b CK1 is essential for the phosphorylation of Snail by and the mutant CK1e protein. As shown in Figure 2b, GSK3b and interacts with the Zn-finger domain of Snail the non-catalytic CK1 protein was not involved in the Given the importance of the Zn-finger domain of the phosphorylation at Ser96, even in the presence of active Snail-MC fragment in phosphorylation, we tested the GSK3b, demonstrating that the CK1 kinase activity is involvement of Zn2 þ in the phosphorylation of Snail at

Oncogene Phosphorylation of Snail by CK1 YXuet al 3127 Ser96. As shown in Figure 3a, the phosphorylation rate substrate Snail-MC fragment by EDTA before the of the Snail-MC fragment by the kinases was signifi- enzymatic reaction. Consistent with this observation, cantly decreased when Zn2 þ was removed from the the GST-fused Snail-MC fragment specifically bound to CK1e in cells, while the GST-fused Snail fragments (residues 1–90 or 91–128) lacking the Zn-finger domain failed to interact with CK1e (Figure 3b). These results suggest that the Zn-finger domain of Snail provides a docking site for CK1e, and that CK1e phosphorylates a residue that may not be Ser96 but is crucial for the phosphorylation of Snail at Ser96. We next examined which kinase activity determines the overall rate of phosphorylation of Snail. We observed the increased phosphorylation of Snail at Ser96 with increasing doses of CK1 in the presence of GSK3b, suggesting that the CK1 activity regulates the phosphorylation rate of Snail (Figure 3c). In contrast, we did not see the dose-dependent effect of GSK3b in the presence of CK1, even though GSK3b activity is indispensable for the phosphorylation of Snail (Figure 3d and Supplementary Figure S3). This result suggests that the kinase activity of GSK3b toward Snail Figure 2 Cooperative activity of CK1 with GSK3b and the is saturated even at a low concentration, and that CK1 interaction of CK1 with Snail. (a) Snail Ser96 is strongly activity may be a limiting factor for the phosphorylation phosphorylated by the co-treatment with CK1 and GSK3b in vitro. The Snail-MC fragment (40 mg/ml) was incubated with of Snail. the kinase domain of CK1e and/or GSK3b in kinase reaction buffer, and subsequently immunoblotted using the anti-phospho-b- catenin (S33/37/T41) antibody to detect the phosphorylation of CK1 regulates the stability of Snail protein in cells Snail at Ser96. The Snail fragment is strongly phosphorylated only To examine the role of CK1 in the phosphorylation of in the presence of both kinases. Recombinant kinase domains of Snail in cells, we treated a prostate cancer cell line, PC3, GSK3b (4 mg/ml) and CK1e (4 mg/ml) were used. (b) A CK1 kinase and a lung cancer cell line, A549, with a CK1 d/e- inactive mutant, CK1e (D128N), has no effect on the phospho- rylation of Snail at Ser96. The catalytically inactive mutant of CK1e selective inhibitor, IC261 (Mashhoon et al., 2000), or a (D128N) was used at concentrations from 8 to 40 mg/ml. CK1e and GSK3b inhibitor, lithium chloride. We found that both GSK3b were used at 8 mg/ml in this experiment. (c) A GSK3b IC261 and lithium chloride elevated the endogenous inhibitor, lithium chloride, inhibits the phosphorylation of Snail at Snail protein level (Figure 4a). These observations Ser96 by the two kinases. The phosphorylation at Ser96 of Snail was performed and detected as shown in Figure 3a. The result is indicate that CK1, in cooperation with GSK3b, can consistent with the previous observations that lithium chloride regulate the stability of Snail by regulating the increased the cellular Snail level (Zhou et al., 2004). phosphorylation level of Snail. However, the increase

Figure 1 Snail phosphorylation at Ser96 by GSK3b and CK1 and its detection using anti-phospho-b-catenin (S33/37/T41). (a) Comparison of Snail with b-catenin and Snail fragments. Schematic representations of b-catenin and Snail are shown at the top and bottom. b-Catenin comprises an N-terminal (N), armadillo (ARM) and C-terminal (c) domain, in which Ser45, Thr41, Ser37 and Ser33 are phosphorylated by CK1 and GSK3b as indicated. Snail consists of an N-terminal (N), middle (M) and Zn-finger domain. The amino-acid sequences for the Snail fragments (Snail-S, Snail-L, Snail-N and Snail-MC) used in this study are shown between the schematic representations. The destruction boxes that are recognized by b-TrCP are shown in violet. The sequence in the green box represents the Ser/Thr-rich cluster. The bold and underlined S in the pS96 peptide represents the phosphorylated serine residue. All serine or threonine residues are underlined. The Snail Ser96 and Ser100 residues are considered to be phosphorylated by GSK3b (Zhou et al., 2004). (b) Left (pS96À): the GST-fused Snail-S fragment (GST-Snail-S) and the GST-fused Snail-L fragment (GST-Snail-L; 1 mg/ml) were phosphorylated by co-treatment with GSK3b and CK1e and were detected by the anti-phospho-b-catenin (S33/37/T41) in western blotting analysis (indicated by *; lanes 1–6). GSK3b was non-specifically recognized by the antibody, indicated by the blot of GSK3b. In a control, the b-catenin fragment (1–133) was phosphorylated by CK1e or/and GSK3b, and then was immunoblotted with the antibody (lanes 7–8). The phosphorylated b-catenin fragment by the co-treatment of CK1e and GSK3b was strongly recognized by the antibody (indicated by # lanes 7–9). Interestingly, CK1e alone was able to phosphorylate the b-catenin fragment at Ser33, Ser37 or Ser41, indicated by the phospho-specific antibody (lane 8). Coomassie blue-stained PVDF membranes used for this experiment are shown at the bottom. Right (pS96 þ ): the same procedure was performed, except that 1 mg/ml of the pS96 peptide was co-incubated with the anti-phospho-b-catenin (S33/37/T41) antibody in western blotting. Anti-pSnail (S96) represents the antibody that was used in the western blot analysis as a primary antibody. (c) Confirmation of the specificity of the anti-phospho-b-catenin (S33/37/T41) antibody toward the phosphorylated Ser96 of Snail. Only the wild-type (WT) GST-fused Snail-L fragment (1 mg/ml) was recognized by the anti-phospho-b-catenin (S33/37/T41) antibody in western blot, when CK1e and GSK3b were co-incubated. The GSK3b band, non-specifically recognized by the antibody, was weakly shown over the Snail phosphorylated Ser96 band, indicated by *. (d) Phosphorylation-dependent binding of Snail to b-TrCP in MDA-MB 468 cell lysates. To detect the interaction between Snail and b-TrCP, GST pull-down assays were performed using whole-cell lysates (WCL) from MDA-MB 468 cells. Only the phosphorylated GST-fused Snail-L fragment (p-GST-Snail-L) interacted with b-TrCP, while the unphosphorylated fragment (GST-Snail-L) did not. p53 protein also bound to the GST-fused Snail fragments regardless of their phosphorylation status, consistent with our previous report (Lee et al., 2009). b-TrCP, p53 and GST were visualized using the corresponding antibodies in western blot.

Oncogene Phosphorylation of Snail by CK1 YXuet al 3128

Figure 3 The importance of the Zn-finger domain of Snail in phosphorylation by CK1 and a more determinant role of CK1 activity than GSK3b activity in vitro.(a) Importance of the Zn2 þ of Snail in the phosphorylation. The Snail-MC fragment was pre-treated ( þ ) or untreated (À) with 2 mM EDTA, and then the EDTA was removed by dialysis. (b) GST pull-down shows that the Snail Zn-finger domain specifically interacts with CK1e in cells. Glutathione-coupled resin bound to the individual GST-fused Snail fragment (Snail- N, Snail-L or Snail-MC) was incubated with the whole-cell lysate (WCL) from 293 cells, analyzed by SDS-PAGE, and immunoblotted with the indicated antibodies. (c) CK1 enhances the phosphorylation of Snail at Ser96 in the presence of GSK3b in a dose-dependent manner. Snail-MC fragment (40 mg/ml) was used as a substrate. GSK3b was used at 8 mg/ml and CK1e was used as indicated (4–24 mg/ml). (d) GSK3b alone cannot phosphorylate Snail at Ser96, and the phosphorylation by the two kinases is not sensitive to the amount of GSK3b. Snail-MC fragment (40 mg/ml) was used as a substrate. CK1e was used at 8 mg/ml, and GSK3b was used as indicated (0.8–16 mg/ml). A similar result was obtained when the GST-fused Snail-L fragment was used (Supplementary Figure S3).

Figure 4 CK1-dependent Snail degradation, mediated by ubiquitination in cells. (a) The PC3 cell line (left) or A549 cell line (right) was treated with 50 mM lithium chloride, 20 mM IC261, or 5 mM ALLN (a proteasome inhibitor), and then the cell lysate was analyzed using each indicated antibody. Actin or tubulin is shown as a loading control. (b) HA-tagged ubiquitin expressing vector, FLAG- tagged Snail (Snail) expressing vector or the control empty vector (EV) was co-transfected in 293 cells with the indicated siRNAs (Ctl for control). After the Snail proteins were immunoprecipitated with an anti-Snail antibody, the ubiquitination of Snail was examined by western blot. (c) Pulse-chase analysis shows that the silencing of CK1e can extend the half-life of Snail regardless of the pre- treatment of a proteasome inhibitor, ALLN. The 293 cells were transfected with indicated siRNAs for 24 h and incubated with or without ALLN for 2 h. After washing the cells with serum-free medium, we blocked the de novo protein synthesis using cycloheximide (CHX; 5 mg/ml) for the indicated time and measured the expression of Snail and CK1e.

Oncogene Phosphorylation of Snail by CK1 YXuet al 3129 in the Snail protein level by the treatment of lithium chloride or IC261 was barely detectable in a pancreatic cancer cell line, Capan-1 cells (Supplementary Figure S1), suggesting that other mechanisms may exist in the regulation of the Snail protein level in this cell line. To confirm whether the phosphorylation by CK1 is required for the ubiquitin-mediated Snail degradation, Snail-overexpressing cells were treated with CK1e small interfering RNA (siRNA) and the ubiquitinated Snail was measured by western blot analysis. As shown in Figure 4b, the ubiquitination of Snail was apparently reduced by silencing CK1e. Indeed, pulse-chase analysis using a protein biosynthesis inhibitor, cycloheximide, further demonstrated that inhibiting CK1e by siRNA extended the half-life of Snail in cells (Figure 4c). Taken together, these observations demonstrate that CK1 is required for the Snail degradation mediated by phos- phorylation of Snail in cells. Our findings are in agreement with the previous observation of GSK3b phosphorylation of the destruc- tion box of Snail in cells or cell extracts (Zhou et al., 2004), because the CK1 activity is constitutively present in cells. Our observations showed a critical role of CK1 in GSK3b-mediated phosphorylation of the b-TrCP recognition site and thus highlight its important role in vivo.

CK1e is responsible for the phosphorylation and degradation of Snail in cells CK1 has a, g, d and e isoforms that are biochemically related but have different physiological roles in cells Figure 5 The CK1e isoform is involved in the Snail phosphoryla- (Gross and Anderson, 1998; Knippschild et al., 2005). tion. (a) To examine the effect of the CK1 isoform-specific siRNAs, Snail was transfected into the PC3 cell line. Transfection of CK1e As CK1g is localized on the cytoplasmic membrane and siRNA dramatically elevated the protein level of Snail in the cells, CK1d is structurally and functionally similar to CK1e, whereas CK1a siRNA resulted in a slight increase in Snail protein only CK1a and e isoforms were examined in this study. level in the cells. IGF-1 treatment resulted in an increase in cellular To examine which CK1 isoform has a dominant role Snail level. The specificities of these siRNAs are shown in Supplementary Figure S2. (b) Only CK1e, but not CK1a, can in the phosphorylation of Snail in cells, we depleted the phosphorylate Snail in vitro. The recombinant CK1a concentration endogenous CK1a or CK1e in PC3 cells using isoform- was determined by comparison with the activity of CK1e on b- specific siRNAs. CK1e siRNA dramatically increased catenin at Ser45. GSK3b and CK1e were used at 4 mg/ml, and the protein level of Snail in cells, whereas CK1a siRNA CK1a was used at 4 or 20 mg/ml in this experiment. The Snail-MC showed only a marginal effect (Figure 5a). To examine fragment substrate was used at 20 mg/ml. (c) Inability of CK1a to interact with Snail. Glutathione-associated GST-fused Snail-MC their relationship with GSK3b activity, we reduced the (GST-Snail-MC) proteins (wild type, S104A or S107A mutant) cellular GSK3b activity by co-treatment of siRNA and were incubated with 293 cell lysate, and the bound proteins were insulin-like growth factor-1 in cells (Figure 5a). Co- analyzed by western blot after washing. Only CK1e was able to treatment with insulin-like growth factor-1 resulted in a bind to Snail. CK1e showed reduced binding to the S104A and moderate increase in Snail level in the cells transfected S107A Snail mutants. with control siRNA or CK1a siRNA. However, because CK1e siRNA may have raised the cellular Snail level to its maximum, co-treatment with insulin-like growth the CK1e isoform is a primary kinase for the phospho- factor-1 did not induce a further increase in Snail level. rylation and degradation of Snail protein in cells. Consistent with our observations in cells, we observed that CK1a failed to phosphorylate Snail at Ser96 when cells were co-treated with GSK3b in our purified system Inhibition of CK1e down-regulates E-cadherin and (Figure 5b). We found that the CK1a siRNA used in the promotes cell migration previous publications also reduced the level of CK1e We next assessed the ability of Snail regulated by CK1e transcript (Supplementary Figure S2), which could to induce the EMT process in cells. The treatment of a explain the moderate increase in the cellular Snail level CK1e inhibitor or the transfection of CK1e siRNA caused by this CK1a siRNA. Furthermore, unlike significantly reduced the level of E-cadherin protein, an CK1e, CK1a failed to bind to Snail in vitro, suggesting established marker for EMT, which is repressed by Snail a limited role of CK1a in the phosphorylation of Snail (Batlle et al., 2000; Peinado et al., 2004; Figure 6a). (Figure 5c). Taken together, our findings indicate that Moreover, the silencing of CK1e by siRNA repressed

Oncogene Phosphorylation of Snail by CK1 YXuet al 3130

Figure 6 Inhibition of CK1e correlates with the promotion of EMT in cells. (a) A549 cells were treated with 20 mM IC261 (left) or transfected with siRNA for CK1e (right). The whole-cell levels of endogenous Snail and E-cadherin were assessed by immuno- blotting. Tublin was used for a loading control. mRNA expressions of Claudin and Fibronectin were determined by RT-PCR (right). (b) Promotion of motility by silencing CK1e in PC3 cells. Cells grown to confluence on cell culture dishes were treated with control siRNA, CK1e siRNA and/or Snail siRNA. A scratch was made in the cell layer, and photographs of the wound area were taken 48 h later.

Claudin transcription, which is also repressed by Snail (Ikenouchi et al., 2003), and up-regulated Fibronectin (Lee et al., 2006); both phenomena are EMT characteri- Figure 7 Sequential phosphorylation of Snail by CK1e and stics (Figure 6a). Consistent with these observations, cell GSK3b (a) The phosphorylation of Snail at Ser96 depends on migration was dramatically increased by silencing Ser100, Ser104 and Ser107. The Snail-MC fragment and its CK1e, whereas the co-silencing of CK1e and Snail by variants were phosphorylated by the kinase domains of CK1e siRNAs inhibited cell migration (Figure 6b), indicating (4 mg/ml) and GSK3b (4 mg/ml). A mutation at Ser100, Ser104 or Ser107 abolished the phosphorylation at Ser96 by the co-treatment that cell migration is promoted by Snail, in response to of GSK3b and CK1e. The Snail-MC fragment substrate was used the suppression of CK1e in cells. Our findings collec- at 40 mg/ml. (b) Snail Ser104 and Ser107 residues are important in tively suggest that CK1e depletion may promote the the ubiquitin-dependent degradation of Snail. Recombinant EMT process in cells by stabilizing Snail. proteins of the wild-type or mutant GST-fused Snail-MC fragments, bound to glutathione agarose, were incubated with 293 cell lysate for the indicated time. The bound proteins were analyzed by immunoblotting. (c) A model of the phosphorylation Phosphorylation of Ser104 by CK1e may serve as and recognition of Snail by b-TrCP. CK1e phosphorylates Snail at a priming site for the subsequent phosphorylation Ser104 and Ser107, and the phosphorylation of Ser104 allows of Snail by GSK3b GSK3b to phosphorylate Ser100, then Ser96 and Ser92. The phosphorylation at Ser96 and Ser100 creates the recognition site To examine how CK1e facilitates the GSK3b phosphor- for b-TrCP. It has been proposed that the canonical Wnt signaling ylation of Snail, mass spectrometric analysis was inhibits the phosphorylation and degradation of Snail by seques- performed to detect phosphorylation using the diverse tering GSK3b in the nucleus in breast cancer cells (Yook et al., Snail fragments in combination with either one or both 2006). of the two kinases. Results of this experiment were inconclusive, perhaps due to the multiple phosphoryla- tion sites and the inappropriate length of the tryptic proteins in a purified system suggests a prolonged fragments of interest. Instead of the direct detection of half-life of S104A and S107A mutants. To evaluate the phosphorylation, we applied an indirect approach using stability of these mutants, the GST-fused Snail-MC the Snail-MC fragment variants in which Ser100, Ser104 fragments containing the mutations were incubated with or Ser107 were replaced with alanine. Using the mutant cell lysate. As expected, the ubiquitination of S104A Snail proteins, we found that all the tested serine and S107A Snail mutants was reduced and, accordingly, residues were critical for the phosphorylation at Ser96, the stability of these mutants was increased, whereas the when both GSK3b and CK1e were present (Figure 7a). wild-type Snail fragment was rapidly degraded by the None of the mutated Snail proteins were phosphory- components of the cell lysate (Figure 7b). This result lated at Ser96 by the co-incubation of the two kinases. confirmed the key role of the Ser104 and Ser107 residues In agreement with our results, Snail Ser100 and Ser96 in the phosphorylation of Snail by CK1e and GSK3b have been considered to be GSK3b target sites (Zhou and the cellular stability of Snail. et al., 2004; Yook et al., 2005). Although the Ser104 and Ser107 residues have been The inability of the co-incubated GSK3b and CK1 to known to have a critical role in the GSK3b-mediated phosphorylate the S104A and S107A mutant Snail phosphorylation of Snail in cells (Yook et al., 2005),

Oncogene Phosphorylation of Snail by CK1 YXuet al 3131 it remains elusive which kinase directly phosphorylates stimulus to induce the translocation of CK1e to the cell Ser104 and Ser107 in cells. The phosphorylation at membrane with an enhanced activity (Swiatek et al., Ser104 is a pre-requisite for the phosphorylation at 2004; Knippschild et al., 2005). Ser100 by GSK3b, because GSK3v prefers substrates The Wnt/b-catenin signaling pathway has been with a phosphorylated Ser/Thr residue as the fourth implicated as a dominant regulator in the EMT process residue after the C-terminal of the target site (Frame of the mammary gland, which is critical for morpho- et al., 2001). Based on our analysis of the mutants, we genesis (Chu et al., 2004; Cowin et al., 2005; Reya suggest that Ser104 is phosphorylated by CK1e, which and Clevers, 2005). Axin2 is strongly induced by Wnt provides the priming site for the subsequent phosphor- stimulation to decrease the duration of the Wnt/ ylation at Ser100 and Ser96 by GSK3b (Figure 7c). The b-catenin signaling pathway (Jho et al., 2002) and, requirement of an intact Ser107 may have a role in the similar to Axin, it mediates the phosphorylation and phosphorylation at Ser104 by CK1. We speculate that degradation of b-catenin by binding to GSK3b,CK1 the Ser104 and Ser107 residues located in the proline- and b-catenin (Behrens et al., 1998). Yook et al. (2006) rich sequence,102PPSPPSP, which is strictly evolutiona- reported that nuclear GSK3b is depleted via the rily conserved, may be a novel class of the sub-optimal nucleocytoplasmic shuttling of GSK3b by Axin2, target sequence of CK1. Although the PPSPPSP seq- resulting from the nuclear export signal of Axin2 and uence is not a canonical CK1 target site, it may be effici- leading to the nuclear retention and stabilization of ently phosphorylated by CK1e, owing to the induced Snail. CK1e is a key positive regulator of Wnt/b-catenin proximity effect mediated by the Snail Zn-finger domain signaling (Peters et al., 1999) and CK1e is diffusely and CK1e. distributed in the cytoplasm and nuclear region in the Moreover, we observed an affinity between the absence of Wnt stimulation (Kishida et al., 2001). As substrate sequence, PPSPPSP, and the enzyme, CK1e CK1e can associate with Axin2, similar to GSK3b (Figure 5c). When Ser104 or Ser107 in the sequence was (Behrens et al., 1998; Sakanaka et al., 1999; Jho et al., mutated, the Snail-MC fragment showed a reduced 2002), it is expected that nuclear CK1e is also exported binding to CK1e. As Ser104 and Ser107 are located in to the cytoplasm through the Axin2 binding induced by the putative substrate site, this interaction may reflect Wnt stimulation. This nuclear depletion of CK1e via the interaction between the substrate and the enzyme Axin2 may have a cumulative effect on the nuclear active site. Taken together, our observations suggest depletion of GSK3b and thus have an important role in that the Ser104 and Ser107 residues of Snail are the stabilization and/or nuclear retention of Snail. As an phosphorylated by cellular CK1, but not by GSK3b, alternative mechanism to the Axin2-mediated export, which serve as the priming sites for the subsequent nuclear CK1e could be depleted as an initial event phosphorylation of Snail by GSK3b. In addition, the during the activation of the Wnt signaling pathway. characteristic kinase domain and/or C-terminal tail of Nuclear CK1e would be exported to the cytoplasmic CK1e (Dahlberg et al., 2009) might contribute to the membrane, because Wnt stimulation immediately in- CK1e-specific phosphorylation of Snail. duces the re-localization of CK1e to the Dvl puncta on the cytoplasmic membrane (Kishida et al., 2001; Bilic et al., 2007; Schwarz-Romond et al., 2007). Further Discussion investigation is warranted to gain insight into which mechanism is more important in the stabilization of To date, only GSK3b has been characterized as a kinase Snail, in response to Wnt stimulation. responsible for the phosphorylation of Snail. Here we In conclusion, our study uncovered distinct roles and revealed CK1e as a new player in the phosphorylation of steps of Snail phosphorylation and identified CK1e as a the b-TrCP recognition region of Snail, in collaboration new component of the Snail-mediated EMT process. It with GSK3b. Given the interaction between CK1e and also shed light on the elucidation of the molecular the Snail Zn-finger domain, CK1e is thought to more mechanism of Snail regulation, which is an important efficiently phosphorylate the non-consensus sequence axis for the regulation of EMT process in normal and around the b-TrCP recognition region, providing a cancerous status. priming site for GSK3b. Consistently, we further observed that cellular Snail protein was markedly stabilized and that the EMT process was promoted Materials and methods when the transcription of CK1e was suppressed in cells. Although CK1 family members are constitutively Experimental procedures for transfection and protein analysis, active in many different tissues and cell lines (Tuazon phosphorylation of Snail fragments, western blotting, immuno- and Traugh, 1991), a number of effectors are able precipitation and RT-PCR are described in Supplementary to modulate CK1 expression and activity through Information. different mechanisms (Knippschild et al., 2005). Treat- ing cells with insulin (Cobb and Rosen, 1983), Wnt Plasmids and siRNA stimuli (Swiatek et al., 2004), topoisomerase inhibitors DNA fragments encoding human Snail fragments were (Knippschild et al., 1997) or g-radiation (Santos et al., obtained from pCR3.1-Snail-Flag (Yook et al., 2006) by 1996) results in elevated levels of CK1 activity and/or PCR and inserted into the pGEX-TEV vector for expression as protein expression. Of these, Wnt is a well-characterized GST-fusion proteins in Escherichia coli. DNA fragments

Oncogene Phosphorylation of Snail by CK1 YXuet al 3132 encoding human CK1a were obtained from a human cDNA b-catenin was expressed in E. coli and purified as described library by PCR and inserted into the pET21d vector previously (Piao et al., 2008). (Invitrogen, Carlsbad, CA, USA) using NcoI and XhoI sites, yielding pET21d-hCK1a. For the expression in E. coli, site- Peptide synthesis directed mutagenesis was performed using the QuikChange Peptide pS96 was prepared by solid-phase synthesis. The method. The mammalian expression vector for Snail has been amino-acid sequence of pS96 peptide is shown in Figure 1a. described previously (Yook et al., 2006; Lee et al., 2009). The The N- and C-termini of all peptides were modified by siRNAs for CK1a and CK1e were prepared as previously formylation and amidation, respectively. The identity and described (Liu et al., 2002). The HA-Ubiquitin expression purity of the synthesized peptide were confirmed by mass vector was provided by S Kim (Seoul National University, spectrometry. Republic of Korea; Kim et al., 2003). GST pull-down assay Antibodies Glutathione agarose beads bound to each GST-fused protein The anti-phospho-b-catenin (Ser33/37/Thr41) antibody was were incubated with whole-cell lysates for 2 h at 4 1C. After purchased from Cell Signaling (Beverly, MA, USA). The anti- washing with phosphate-buffered saline and RIPA, the bound Snail, anti-b-TrCP, anti-p53, anti-actin, anti-b-catenin, anti- proteins were subjected to SDS–PAGE and western blotting. GST, anti-tubulin, anti-HA, anti-ubiquitin and anti-CK1 antibodies were purchased from Santa Cruz (Santa Cruz, Cell migration assay CA, USA). The antibody against FLAG peptide (M2) was To assess the effect of CK1e siRNA on cell motility, wound- obtained from Sigma (St Louis, MO, USA) and the antibody healing experiments were conducted. In brief, cells were grown against E-cadherin was purchased from Zymed Laboratories to confluence on 30-mm cell culture dishes coated with rat inc. (South San Francisco, CA, USA) tail collagen (20 mg/ml, BD Biosciences, Bedford, MA, USA), and were treated with control siRNA, CK1e siRNA, or a Expression and purification of recombinant proteins combination of CK1e siRNA and Snail. Then, a scratch was The kinase domains of mouse GSK3b (residues 27–393) and made in the cell layer using a razor blade. At 24 h after washing CK1e (residues 1–319) were expressed and purified as with phosphate-buffered saline, photographs of the scratched previously described (Ha et al., 2004). The b-catenin (1–133) area were taken. fragment was obtained as described previously (Piao et al., 2008). To obtain the recombinant human CK1a protein, pET21d-hCK1a was expressed in E. coli and purified using the Conflict of interest same procedure as for the purification of the kinase domain of CK1e and its variant. The Snail fragments and its variants The authors declare no conflict of interest. were expressed in E. coli as GST-fusion proteins and purified by glutathione affinity. The final buffer for the GST-fusion proteins was changed to 20 mM Tris buffer (pH 8.0), contain- ing 150 mM NaCl and 2 mM 2-mercaptoethanol, using Cen- Acknowledgements triprep (10 kDa cutoff; Millipore, Billerica, MA, USA). For the Snail-MC fragment (91–264) and its variants, the GST tag We thank Xiao Ling Jin for assisting in DNA construction and was cleaved by treatment of the tobacco etch virus protease protein purification. This study was supported by a grant from and the protein mixture was dialyzed against 20 mM Tris buffer the National R&D Program for Cancer Control, Ministry for (pH 8.0) containing 150 mM NaCl, 0.2 mM ZnCl2 and 2 mM Health, Welfare and Family affairs, Republic of Korea 2-mercaptoethanol. The N-terminal region (residues 1–133) of (0920140).

References

Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. (1997). Beta- Chu EY, Hens J, Andl T, Kairo A, Yamaguchi TP, Brisken C et al. catenin is a target for the ubiquitin-proteasome pathway. EMBO J (2004). Canonical WNT signaling promotes mammary placode 16: 3797–3804. development and is essential for initiation of mammary gland Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J morphogenesis. Development 131: 4819–4829. et al. (2000). The transcription factor snail is a repressor of E- Cobb MH, Rosen OM. (1983). Description of a protein kinase derived cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2: from insulin-treated 3T3-L1 cells that catalyzes the phosphorylation 84–89. of ribosomal protein S6 and casein. J Biol Chem 258: 12472–12481. Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Cowin P, Rowlands TM, Hatsell SJ. (2005). Cadherins and catenins in Wirtz R et al. (1998). Functional interaction of an axin homolog, breast cancer. Curr Opin Cell Biol 17: 499–508. conductin, with beta-catenin, APC, and GSK3beta. Science 280: Dahlberg CL, Nguyen EZ, Goodlett D, Kimelman D. (2009). 596–599. Interactions between Casein kinase Iepsilon (CKIepsilon) and two Bilic J, Huang YL, Davidson G, Zimmermann T, Cruciat CM, substrates from disparate signaling pathways reveal mechanisms for Bienz M et al. (2007). Wnt induces LRP6 signalosomes and substrate-kinase specificity. PLoS ONE 4: e4766. promotes dishevelled-dependent LRP6 phosphorylation. Science Dominguez D, Montserrat-Sentis B, Virgos-Soler A, Guaita S, Grueso 316: 1619–1622. J, Porta M et al. (2003). Phosphorylation regulates the subcellular Birchmeier W, Behrens J, Weidner KM, Frixen UH, Schipper J. location and activity of the snail transcriptional repressor. Mol Cell (1991). Dominant and recessive genes involved in tumor cell Biol 23: 5078–5089. invasion. Curr Opin Cell Biol 3: 832–840. Frame S, Cohen P, Biondi RM. (2001). A common phosphate binding Cavallaro U, Christofori G. (2004). Cell adhesion and signalling by site explains the unique substrate specificity of GSK3 and its cadherins and Ig-CAMs in cancer. Nat Rev Cancer 4: 118–132. inactivation by phosphorylation. Mol Cell 7: 1321–1327.

Oncogene Phosphorylation of Snail by CK1 YXuet al 3133 Frescas D, Pagano M. (2008). Deregulated proteolysis by the F-box Peinado H, Ballestar E, Esteller M, Cano A. (2004). Snail mediates proteins SKP2 and beta-TrCP: tipping the scales of cancer. Nat Rev E-cadherin repression by the recruitment of the Sin3A/histone Cancer 8: 438–449. deacetylase 1 (HDAC1)/HDAC2 complex. Mol Cell Biol 24: Gross SD, Anderson RA. (1998). Casein kinase I: spatial organization 306–319. and positioning of a multifunctional protein kinase family. Cell Peinado H, Quintanilla M, Cano A. (2003). Transforming growth Signal 10: 699–711. factor beta-1 induces snail transcription factor in epithelial cell lines: Ha NC, Tonozuka T, Stamos JL, Choi HJ, Weis WI. (2004). mechanisms for epithelial mesenchymal transitions. J Biol Chem Mechanism of phosphorylation-dependent binding of APC to 278: 21113–21123. beta-catenin and its role in beta-catenin degradation. Mol Cell 15: Peters JM, McKay RM, McKay JP, Graff JM. (1999). Casein kinase I 511–521. transduces Wnt signals. Nature 401: 345–350. Ikenouchi J, Matsuda M, Furuse M, Tsukita S. (2003). Regulation of Piao S, Lee SH, Kim H, Yum S, Stamos JL, Xu Y et al. (2008). tight junctions during the epithelium-mesenchyme transition: direct Direct inhibition of GSK3beta by the phosphorylated cytoplasmic repression of the gene expression of claudins/occludin by Snail. domain of LRP6 in Wnt/beta-catenin signaling. PLoS ONE 3: J Cell Sci 116: 1959–1967. e4046. Jho EH, Zhang T, Domon C, Joo CK, Freund JN, Costantini F. Reya T, Clevers H. (2005). Wnt signalling in stem cells and cancer. (2002). Wnt/beta-catenin/Tcf signaling induces the transcription of Nature 434: 843–850. Axin2, a negative regulator of the signaling pathway. Mol Cell Biol Sakanaka C, Leong P, Xu L, Harrison SD, Williams LT. (1999). 22: 1172–1183. Casein kinase iepsilon in the wnt pathway: regulation of beta- Kim MJ, Park BJ, Kang YS, Kim HJ, Park JH, Kang JW et al. (2003). catenin function. Proc Natl Acad Sci USA 96: 12548–12552. Downregulation of FUSE-binding protein and c-myc by tRNA Santos JA, Logarinho E, Tapia C, Allende CC, Allende JE, Sunkel synthetase cofactor p38 is required for lung cell differentiation. Nat CE. (1996). The casein kinase 1 alpha gene of Drosophila Genet 34: 330–336. melanogaster is developmentally regulated and the kinase Kishida M, Hino S, Michiue T, Yamamoto H, Kishida S, Fukui A activity of the protein induced by DNA damage. J Cell Sci 109 et al. (2001). Synergistic activation of the Wnt signaling pathway by (Pt 7): 1847–1856. Dvl and casein kinase Iepsilon. J Biol Chem 276: 33147–33155. Schwarz-Romond T, Metcalfe C, Bienz M. (2007). Dynamic recruit- Knippschild U, Gocht A, Wolff S, Huber N, Lohler J, Stoter M. ment of axin by Dishevelled protein assemblies. J Cell Sci 120: (2005). The casein kinase 1 family: participation in multiple cellular 2402–2412. processes in eukaryotes. Cell Signal 17: 675–689. Swiatek W, Tsai IC, Klimowski L, Pepler A, Barnette J, Yost HJ et al. Knippschild U, Milne DM, Campbell LE, DeMaggio AJ, Christenson (2004). Regulation of casein kinase I epsilon activity by Wnt E, Hoekstra MF et al. (1997). p53 is phosphorylated in vitro and signaling. J Biol Chem 279: 13011–13017. in vivo by the delta and epsilon isoforms of casein kinase 1 and Thiery JP. (2002). Epithelial-mesenchymal transitions in tumour enhances the level of casein kinase 1 delta in response to progression. Nat Rev Cancer 2: 442–454. topoisomerase-directed drugs. Oncogene 15: 1727–1736. Tuazon PT, Traugh JA. (1991). Casein kinase I and II–multipotential Lee JM, Dedhar S, Kalluri R, Thompson EW. (2006). The epithelial- serine protein kinases: structure, function, and regulation. Adv mesenchymal transition: new insights in signaling, development, and Second Messenger Phosphoprotein Res 23: 123–164. disease. J Cell Biol 172: 973–981. Winston JT, Strack P, Beer-Romero P, Chu CY, Elledge SJ, Harper Lee SH, Lee SJ, Jung YS, Xu Y, Kang HS, Ha NC et al. (2009). JW. (1999). The SCFbeta-TRCP-ubiquitin ligase complex associates Blocking of p53-Snail binding, promoted by oncogenic K-Ras, specifically with phosphorylated destruction motifs in IkappaBal- recovers p53 expression and function. Neoplasia 11: 22–31 and 6 pha and beta-catenin and stimulates IkappaBalpha ubiquitination pages following 31. in vitro. Genes Dev 13: 270–283. Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y et al. (2002). Yook JI, Li XY, Ota I, Fearon ER, Weiss SJ. (2005). Wnt-dependent Control of beta-catenin phosphorylation/degradation by a dual- regulation of the E-cadherin repressor snail. J Biol Chem 280: kinase mechanism. Cell 108: 837–847. 11740–11748. Mashhoon N, DeMaggio AJ, Tereshko V, Bergmeier SC, Egli M, Yook JI, Li XY, Ota I, Hu C, Kim HS, Kim NH et al. (2006). A Wnt- Hoekstra MF et al. (2000). Crystal structure of a conformation- Axin2-GSK3beta cascade regulates Snail1 activity in breast cancer selective casein kinase-1 inhibitor. J Biol Chem 275: 20052–20060. cells. Nat Cell Biol 8: 1398–1406. Nieto MA. (2002). The snail superfamily of zinc-finger transcription Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M et al. (2004). Dual factors. Nat Rev Mol Cell Biol 3: 155–166. regulation of Snail by GSK-3beta-mediated phosphorylation Orford K, Crockett C, Jensen JP, Weissman AM, Byers SW. (1997). in control of epithelial-mesenchymal transition. Nat Cell Biol 6: Serine phosphorylation-regulated ubiquitination and degradation of 931–940. beta-catenin. J Biol Chem 272: 24735–24738. Zhou BP, Hung MC. (2005). Wnt, hedgehog and snail: sister pathways Pantel K, Brakenhoff RH. (2004). Dissecting the metastatic cascade. that control by GSK-3beta and beta-Trcp in the regulation of Nat Rev Cancer 4: 448–456. metastasis. Cell Cycle 4: 772–776.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene