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).