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Genes to Cells (2007) 12, 1215-1223. doi:10.1111/j.1365-2443.2007.01128.x
© 2007 Blackwell Publishing or its licensors
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Wnt5a modulates glycogen synthase kinase 3 to induce phosphorylation of receptor tyrosine kinase Ror2

 Hiroyuki Yamamoto1, Sa Kan Yoo1, Michiru Nishita1, Akira Kikuchi2 and Yasuhiro Minami1,*

1 Department of Physiology and Cell Biology, Faculty of Medical Sciences, Graduate School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
2 Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The receptor tyrosine kinase Ror2 plays important roles in mediating non-canonical Wnt5a signaling by activating the Wnt–JNK pathway and inhibiting the ß-catenin–TCF pathway. It has been shown that Ror2 is phosphorylated and activated by casein kinase I{varepsilon} when both molecules are over-expressed in cultured cells. However, it remains unknown whether or not Ror2 is phosphorylated upon Wnt5a stimulation. Here we show that Ror2 is phosphorylated on serine/threonine residues upon stimulation of cultured cells, expressing Ror2 endogenously, with Wnt5a, but not Wnt3a. It was found that treatment of cells with glycogen synthase kinase-3 (GSK-3) inhibitors (LiCl and SB216763) or small interfering RNAs (siRNAs) for GSK-3 (mainly GSK-3{alpha}) can inhibit Wnt5a-induced phosphorylation of Ror2. Immunoprecipitated Ror2 can also be phosphorylated by purified GSK-3{alpha} or GSK-3ß in vitro, and ectopic co-expression of Ror2 and GSK-3 (mainly GSK-3{alpha}) in cultured cells results in Ror2 phosphorylation, irrespective of Wnt5a, that is sensitive to SB216763. These results indicate that GSK-3 is involved in Wnt5a-induced phosphorylation of Ror2. Moreover, it was found that Wnt5a-induced cell migration can be inhibited by SB216763 or by siRNA-mediated suppression of GSK-3{alpha} (and GSK-3ß) expression, further emphasizing the role(s) of GSK-3 in Wnt5a-induced signaling.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Ror2 belongs to the Ror-family of receptor tyrosine kinases, characterized by extracellular Frizzled-like cysteine-rich domains (CRDs) and intracellular tyrosine kinase domains, resembling those of the Trk-family RTKs, and proline-rich domains (PRDs) (Masiakowski & Carroll 1992; Oishi et al. 1999; Forrester 2002; Yoda et al. 2003). Ror2 is expressed mainly in neural crest-derived and mesenchymal cells during mouse embryogenesis (Matsuda et al. 2001), and has been shown to play crucial roles in developmental morphogenesis (DeChiara et al. 2000; Takeuchi et al. 2000). Mice lacking Ror2 expression exhibit skeletal, cardiovascular, respiratory and genital abnormalities (DeChiara et al. 2000; Takeuchi et al. 2000; Nomi et al. 2001; Oishi et al. 2003), reflecting partially disrupted convergent extension movements (CE) during mouse development. Interestingly, it has been shown that Ror2 acts as an alternative receptor or co-receptor for Wnt5a, a representative non-canonical Wnt protein (Oishi et al. 2003; Mikels & Nusse 2006; Nishita et al. 2006). Ror2 mediates Wnt5a signaling by activating the Wnt–JNK pathway and/or inhibiting the ß-catenin–TCF pathway (Oishi et al. 2003; Mikels & Nusse 2006). Furthermore, it has recently been reported that Ror2 mediates Wnt5a-induced migration of cultured cells by associating with the actin-binding protein filamin A (FLNa) (Nishita et al. 2006).

It has been shown that Ror2 associates with and is phosphorylated on serine/threonine residues by CKI{varepsilon}, a critical regulator of the canonical Wnt signaling, when both molecules are over-expressed in cultured cells, and that this serine/threonine phosphorylation of Ror2 by CKI{varepsilon} results in autophosphorylation of tyrosine residues in Ror2, leading to activation of Ror2 tyrosine kinase (Kani et al. 2004). Database analysis has revealed that the intracellular region of Ror2 also possesses multiple Ser/Thr-X-X-X-Ser/Thr (X is any amino acid, but often Pro), the consensus sequence for glycogen synthase kinase-3 (GSK-3), suggesting that GSK-3 is another candidate protein serine/threonine kinase which phosphorylates Ror2. GSK-3 was named for its role in regulating glycogen synthase, and has subsequently been shown to regulate many cellular responses (Cohen & Frame 2001; Harwood 2001; Doble & Woodgett 2003; Jope & Johnson 2004). There are two members of GSK-3{alpha} and GSK-3ß in mammals (Woodgett 1990), yet little is known about isoform-specific functions. GSK-3 is usually constitutively active and various biological stimuli lead to inactivation of GSK-3 via phosphorylation of Ser9 in GSK-3ß and Ser21 in GSK-3{alpha} (Doble & Woodgett 2003; Jope & Johnson 2004). GSK-3 has also been shown to play a crucial inhibitory role in canonical Wnt signaling. In the destruction complex, GSK-3 phosphorylates ß-catenin, adenomatous polyposis coli (APC) and Axin efficiently, and thereby induces ubiquitination of ß-catenin, resulting in its degradation (Ikeda et al. 1998; Hinoi et al. 2000). However, it remains largely unknown whether or not Ror2 is phosphorylated on serine/threonine and/or tyrosine residues upon Wnt5a stimulation, and how Ror2 transmits Wnt5a signals to cell interior.

In this study, we first examined whether or not Wnt5a stimulation can induce phosphorylation of Ror2, and found that Ror2 is phosphorylated on serine/threonine residues upon stimulation with Wnt5a, but not Wnt3a. This Wnt5a-induced phosphorylation of Ror2 is inhibited by GSK-3 inhibitors, LiCl and SB216763 (Klein & Melton 1996; Stambolic et al. 1996; Etienne-Manneville & Hall 2003; Liao et al. 2003), but not by a CKI inhibitor CKI-7 (Lee et al. 2001; Gao et al. 2002), suggesting that GSK-3, but not CKI{varepsilon}, is involved in Wnt5a-induced phosphorylation of Ror2. Treatment of cultured cells with small interfering RNA (siRNA) for GSK-3{alpha} (or GSK-3ß) also results in inhibition of Ror2 phosphorylation induced by Wnt5a. We further show that Ror2 can be phosphorylated by GSK-3{alpha} (and GSK-3ß) both in vitro and in vivo. Interestingly, it was also found that Wnt5a-induced migration of cultured cells can be inhibited by treatment of cell with the GSK-3 inhibitor SB216763 or by siRNA-mediated suppression of GSK-3{alpha} and GSK-3ß expressions. Collectively, these results indicate that GSK-3 is involved in Wnt5a-induced phosphorylation of Ror2 and cell migration.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Ror2 is phosphorylated on serine/threonine residues upon Wnt5a stimulation

We first examined whether or not Ror2 can be phosphorylated upon Wnt5a stimulation. It has been appreciated that phosphorylation of dishevelleds (Dvls), as revealed by their electrophoretic mobility shifts, is a reliable surrogate marker for Wnt5a signaling in mammals (Gonzalez-Sancho et al. 2004; Schulte et al. 2005; Takada et al. 2005; Masckauchán et al. 2006). Thus, NIH3T3 and HeLa-S3 cells, both expressing Ror2 and Dvl3 endogenously, were stimulated with either control conditioned medium (neo CM), CM containing Wnt3a (Wnt3a CM) or CM containing Wnt5a (Wnt5a CM), and electrophoretic mobilities of Ror2 or Dvl3, respectively, were monitored by anti-Ror2 or anti-Dvl3 immunoblotting of whole cell lysates (WCLs) as described in Experimental procedures. As expected, treatment of NIH3T3 and HeLa-S3 cells with Wnt5a CM or Wnt3a CM, but not neo CM, resulted in phosphorylation of Dvl3 as assessed by its electrophoretic mobility shift (see Supplementary Fig. S1). This Wnt5a-induced phosphorylation of Dvl3 occurred in a time-dependent manner (Fig. 1A). Interestingly, electrophoretic mobility shift of Ror2 upon stimulation with Wnt5a CM, but not neo CM or Wnt3a CM, was observed (see Supplementary Fig. S1), and this Wnt5a-induced phosphorylation of Ror2 also occurred in a time-dependent manner with a somewhat slower kinetics compared to that of Dvl3 (Fig. 1A, data not shown), suggesting the Wnt5a-induced modification of Ror2.


Figure 1
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  		Figure 1  Wnt5a-stimulation induces phosphorylation of Ror2. (A) NIH3T3 or HeLa-S3 cells were stimulated with Wnt5a CM for indicated periods. Expression levels of endogenous Ror2 and Dvl3 proteins, respectively, were determined by anti-Ror2 (upper panel) and anti-Dvl3 (lower panel) immunoblotting of the respective WCLs. (B) NIH3T3 cells were stimulated with neo CM or Wnt5a CM for 2 h. The respective WCLs were incubated with or without alkaline phosphatase (0.2 U/µL) for 30 min at 37 °C, and analyzed by immunoblotting as in (A). (C) NIH3T3 cells were stimulated with neo CM or Wnt5a CM for 2 h. Anti-Ror2 immunoprecipitates from the WCLs were separated by SDS-PAGE (7.5% PAG), followed by immunoblotting with anti-phosphoserine/threonine (top panel), anti-phosphotyrosine (middle panel) or anti-Ror2 antibodies (bottom panel), respectively.

 
We then tested whether or not Wnt5a-induced modification of Ror2 reflects its phosphorylation by treatment with calf intestinal alkaline phosphatase (CIP). Electrophoretic mobility shift of Ror2 induced by stimulation with Wnt5a CM, but not neo CM or Wnt3a CM, was inhibited by CIP treatment (Fig. 1B, see Supplementary Fig. S1), indicating that Ror2 is indeed phosphorylated upon Wnt5a stimulation. To determine whether serine/threonine or tyrosine residues in Ror2 are phosphorylated, anti-Ror2 immunoprecipitates of the WCLs from NIH3T3 cells, treated with either neo CM or Wnt5a CM, were immunoblotted with anti-phosphoserine/threonine or anti-phosphotyrosine antibodies as described in Experimental procedures. As shown in Fig. 1C, phosphorylation of serine/threonine residues, but not tyrosine residues in Ror2 was detected in response to Wnt5a stimulation.

Wnt5a-induced phosphorylation of Ror2 is inhibited by GSK-3 inhibitors or siRNA for GSK-3{alpha} (or GSK-3ß)

It has been shown that Ror2 is phosphorylated on serine/threonine residues by CKI{varepsilon} when both Ror2 and CKI{varepsilon} are over-expressed in cultured cells (Kani et al. 2004). Thus, we examined the effect of a CKI inhibitor CKI-7 on the Wnt5a-induced phosphorylation of Ror2, but failed to detect any inhibition of Ror2 phosphorylation by CKI-7 (200 µM) (see Supplementary Fig. S2). To identify a protein serine/threonine kinase(s) responsible for Wnt5a-induced phosphorylation of Ror2, we monitored the effects of a series of protein serine/threonine kinase inhibitors on Wnt5a-induced phosphorylation of Ror2. It was found that serine/threonine phosphorylation of Ror2 induced by Wnt5a CM, but not neo CM or Wnt3a CM, were inhibited by the presence of GSK-3 inhibitors, LiCl (50 mM) or SB216763 (20 µM), as assessed by anti-phosphoserine/threonine (top panels) of anti-Ror2 immunoprecipitates, respectively (Fig. 2A,B), suggesting that GSK-3 is involved in Wnt5a-induced phosphorylation of Ror2. To further confirm the role of GSK-3 in regulating phosphorylation of Ror2 induced by Wnt5a, we examined the effect of siRNA-mediated suppression of GSK-3{alpha} and/or GSK-3ß on Wnt5a-induced phosphorylation of Ror2 in HeLa-S3 cells. As shown in Fig. 2C, suppression of GSK-3{alpha} and/or GSK-3ß expression resulted in the significant inhibition of Wnt5a-induced Ror2 phosphorylation as assessed by anti-phosphoserine/threonine immunoblotting. Since Wnt5a-induced Ror2 phosphorylation was more strongly inhibited by GSK-3{alpha} siRNA than GSK-3ß siRNA, it is likely that GSK-3{alpha} rather than GSK-3ß is more responsible for Wnt5a-induced Ror2 phosphorylation. These results indicate that GSK-3 (GSK-3{alpha} and possibly GSK-3ß) is required for phosphorylation of Ror2 induced by Wnt5a.


Figure 2
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  		Figure 2  GSK-3 is involved in Wnt5a-induced phosphorylation of Ror2. (A) HeLa-S3 cells were stimulated with neo CM, Wnt3a CM or Wnt5a CM in the absence or presence of NaCl (50 mM) or LiCl (50 mM) for 2 h. WCLs or anti-Ror2 immunoprecipitates from the WCLs were separated by SDS-PAGE (7.5% PAG), followed by immunoblotting with anti-phosphoserine/threonine (top panel), anti-Ror2 (middle panel) or anti-ß-catenin antibodies (bottom panel), respectively. (B) HeLa-S3 cells were pre-treated with DMSO (–) or SB216763 (20 µM; +) for 12 h and then stimulated with neo CM, Wnt3a CM or Wnt5a CM, containing DMSO (–) or SB216763 (20 µM; +) for 2 h. Cells were lysed and analyzed as in (A). (C) HeLa-S3 cells were transfected with the indicated siRNAs, and stimulated with neo CM or Wnt5a CM for 2 h. Anti-Ror2 immunoprecipitates from the WCLs were separated by SDS-PAGE (7.5% PAG), followed by immunoblotting with anti-phosphoserine/threonine or anti-Ror2 antibodies (top and second panels), respectively. The WCLs were also immunoblotted with anti-GSK-3{alpha} (third panel), anti-GSK-3ß (fourth panel) or anti-ß-actin antibodies (bottom panel), respectively. Inhibition of electrophoretic mobility shift of Ror2 by GSK-3 inhibitors or GSK-3 siRNAs was rather modest or unclear due to an unknown reason(s).

 
Ror2 is phosphorylated by GSK-3 both in vitro and in vivo

We then tested whether or not GSK-3{alpha} and/or GSK-3ß can phosphorylate Ror2 in vitro. Ror2-HA and HA-GSK-3{alpha} or HA-GSK-3ß proteins, purified from HEK293T cells expressing the respective proteins with anti-HA-conjugated Sepharose, were subjected to in vitro kinase assay in the absence (DMSO alone) or presence of the GSK-3 inhibitor SB216763 (10 µM) as described in Experimental procedures. As shown in Fig. 3A, both HA-GSK-3{alpha} and HA-GSK-3ß could phosphorylate Ror2-HA in vitro, and this GSK-3-mediated phosphorylation of Ror2 was inhibited by the presence of SB216763. We also examined whether GSK-3 (GSK-3{alpha} or GSK-3ß) can phosphorylate serine/threonine residues in Ror2 when these molecules are co-expressed in cultured cells. To this end, HEK293T cells were transfected transiently with either pCGN-HA-GSK-3{alpha} or pCGN-HA-GSK-3ß along with pcDNA-Ror2-FLAG as described in Experimental procedures. Anti-FLAG immunoprecipitates of WCLs from HEK293T cells, expressing Ror2-FLAG along with HA-tagged GSK-3{alpha} or GSK-3ß were subjected to anti-phosphoserine/threonine immunoblotting. As shown in Fig. 3B, Ror2-FLAG could be phosphorylated on serine/threonine residues by either HA-GSK-3{alpha} or HA-GSK-3ß in HEK293T cells when these molecules were over-expressed, and this GSK-3-mediated phosphorylation of Ror2 in vivo was inhibited by the presence of SB216763. On the other hand, GSK-3-mediated tyrosine auto-phosphorylation of Ror2 was undetected under this experimental condition (data not shown). Furthermore, it was found that anti-FLAG immunoprecipitates contained GSK-3 (GSK-3{alpha} or GSK-3ß) as revealed by anti-HA immunoblotting (Fig. 3C), suggesting that Ror2 associates with GSK-3. These results further support the idea that GSK-3 (GSK-3{alpha} and possibly GSK-3ß) is involved in Wnt5a-induced phosphorylation of Ror2.


Figure 3
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  		Figure 3  Ror2 is phosphorylated by GSK-3 (GSK-3{alpha} and/or GSK-3ß) (A) HA-tagged Ror2 and GSK-3 (GSK-3{alpha} or GSK-3ß) proteins were prepared by anti-HA immunoprecipitation of WCLs from HEK293T cells transfected with pCGN-HA-GSK-3{alpha} or pCGN-HA-GSK-3ß along with pcDNA-Ror2-HA, respectively. Subsequently, in vitro kinase assay was performed in the absence (DMSO alone; –) or presence of SB216763 (10 µM; +) as described in Experimental procedures. (B) HEK293T cells were transfected with pCGN-HA-GSK-3{alpha} or pCGN-GSK-3ß along with pcDNA-Ror2-FLAG as described in Experimental procedures. Transfected cells were cultured in the absence (DMSO alone; –) or presence of SB216763 (20 µM; +) for 12 h prior to harvest them, and then solubilized with lysis buffer. WCLs or anti-FLAG immunoprecipitates from the respective WCLs were separated by SDS-PAGE (7.5% PAG), followed by immunoblotting with anti-phosphoserine/threonine (top panel), anti-FLAG (middle panel) or anti-HA antibodies (bottom panel), respectively. The electrophoretic mobility shift of Ror2 seen in the middle panel was found to be not significant from our repeated experiments. (C) FLAG-tagged Ror2 protein was expressed transiently in HEK293T cells with or without HA-tagged GSK-3 (GSK-3{alpha} or GSK-3ß) proteins, as shown in the panel. WCLs or anti-FLAG immunoprecipitates from the respective WCLs were separated by SDS-PAGE (7.5% PAG), followed by immunoblotting with anti-HA (top and bottom panels) or anti-FLAG antibodies (middle panel), respectively.

 
GSK-3 is involved in Wnt5a-induced cell migration

It has been shown that GSK-3 (GSK-3{alpha} and/or GSK-3ß) regulates cell migration either positively or negatively (Kim et al. 2001; Koivisto et al. 2003, 2006; Yoon et al. 2005; Kobayashi et al. 2006), presumably depending on receptor and/or cellular contexts (see Discussion). Since it has recently been reported that Wnt5a induces migration of several cultured cells (Weeraratna et al. 2002; Nishita et al. 2006), and that Ror2 is required for this Wnt5a-induced cell migration (Nishita et al. 2006), we examined whether or not GSK-3 (GSK-3{alpha} and/or GSK-3ß) is involved in Wnt5a-induced cell migration by wound-healing assay (see Experimental procedures). Wounded monolayers of NIH3T3 cells were treated with either neo CM or Wnt5a CM in the absence (DMSO alone) or presence of the GSK-3 inhibitor SB216763 (0.3 µM) for 8–12 h. As shown in Fig. 4A, enhanced wound closure by Wnt5a stimulation was inhibited drastically by the presence of SB216763. Furthermore, siRNA-mediated suppression of GSK-3{alpha} and/or GSK-3ß expression results in the drastic inhibition of Wnt5a-induced wound closure (Fig. 4B). Taken together, these results indicate that GSK-3 is also involved in Wnt5a-induced cell migration.


Figure 4
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  		Figure 4  GSK-3 is involved in Wnt5a-induced migration of NIH3T3 cells. (A) Confluent monolayers of NIH3T3 cells on fibronectin-coated coverslips were scratched, and wounded monolayers were treated with either neo CM (left panels) or Wnt5a CM (right panels) in the absence (DMSO alone, top panels) or presence of SB216763 (0.3 µM) (bottom panels) for 8–12 h. The length of the wounds was measured and the data are expressed as the means ± SD. Statistical analyses were carried out using a Student's t-test (n = 3). (B) NIH3T3 cells were transfected with the indicated siRNAs, and plated onto fibronectin-coated coverslips. Confluent monolayers of transfected NIH3T3 cells [control siRNA (upper panels) or GSK-3{alpha} and GSK-3ß siRNAs (lower panels)] on coverslips were scratched, and wounded monolayers were treated with either neo CM (left panels) or Wnt5a CM (right panels) for 8–12 h. The length of the wounds was measured and the data are expressed as the means ± SD. Statistical analyses were carried out using a Student's t-test (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
It has been appreciated that Wnt5a activates non-canonical Wnt pathway by regulating planar cell polarity (PCP) and convergent extension (CE) movements during developmental morphogenesis (Moon et al. 1993; Kilian et al. 2003; Qian et al. 2007). Analyses using cultured cells have revealed that Wnt5a stimulation results in the activation of JNK (Yamanaka et al. 2002; Oishi et al. 2003) and phosphorylation of Dvls (Gonzalez-Sancho et al. 2004; Schulte et al. 2005; Takada et al. 2005; Masckauchán et al. 2006), and that Wnt5a induces migration, differentiation and survival of cultured cells, depending on cell-types (Weeraratna et al. 2002; Schulte et al. 2005; Kurayoshi et al. 2006; Masckauchán et al. 2006; Nishita et al. 2006). Interestingly, it has recently been shown that Ror2 acts as an alternative receptor or co-receptor for Wnt5a, and that Ror2 mediates cell migration, activation of the Wnt–JNK pathway and inhibition of the ß-catenin–TCF pathway, induced by Wnt5a (Oishi et al. 2003; Mikels & Nusse 2006; Nishita et al. 2006). Although it has been reported that Ror2 associates with CKI{varepsilon}, a critical regulator of the canonical Wnt signaling, and Dlxin-1, the melanoma-associated antigen family protein (Matsuda et al. 2003; Kani et al. 2004), it is still unknown about the signaling mechanisms mediated by Ror2 upon Wnt5a stimulation.

In the present study, we show that Ror2 is phosphorylated on serine/threonine residues upon stimulation of cultured cells with Wnt5a, but not Wnt3a (Fig. 1, see Supplementary Fig. S1). The biological significance of Ror2 phosphorylation in Wnt5a-induced signaling mediated by Ror2 is currently unclear, yet our observations suggest that Ror2 phosphorylation can be a suitable biochemical marker for Wnt5a-induced signaling. It has previously been shown that Ror2 is phosphorylated and activated by CKI{varepsilon} when both molecules are over-expressed in cultured cells. However, unexpectedly, it was found that GSK-3 (GSK-3{alpha} and/or GSK-3ß) rather than CKI{varepsilon}, is involved in Wnt5a-induced phosphorylation of Ror2 (Figs 2 and 3, see Supplementary Fig. S2). Furthermore, we show that GSK-3 is involved in Wnt5a-induced cell migration (Fig. 4). It has been well established that GSK-3ß is a critical regulator involved in the canonical Wnt-ß-catenin–TCF pathway. However, our present data indicate that GSK-3{alpha} as well as GSK-3ß are also involved in non-canonical Wnt5a-induced signaling.

It has generally been appreciated that functions of GSK-3 can be regulated by phosphorylation, association with its binding partners, and subcellular distribution (Cohen & Frame 2001; Harwood 2001; Doble & Woodgett 2003; Jope & Johnson 2004). GSK-3 (GSK-3{alpha} and GSK-3ß) are usually constitutively active by phosphorylation of GSK-3{alpha} at Tyr279 and GSK-3ß at Tyr216 via their auto-tyrosine phosphorylation activities, respectively (Cole et al. 2004), and their functions have been assumed to be regulated primarily by inhibition of their respective activities via phosphorylation of GSK-3{alpha} at Ser21 and GSK-3ß at Ser9 by other protein kinases in response to various biological stimuli (Doble & Woodgett 2003; Jope & Johnson 2004). For example, insulin stimulation induces the activation of protein kinase B (PKB) to phosphorylate Ser9 of GSK-3ß, resulting in the inhibition of GSK-3ß (Cross et al. 1995). Nevertheless, in this study we show that Wnt5a stimulation induces phosphorylation of Ror2 via GSK-3. With this respect, it should be noted that decrease in phosphorylation of GSK-3ß at Ser9 was detected at 30–60 min after stimulation with Wnt5a CM, but not neo CM (data not shown), prior to the detection of Wnt5a-induced phosphorylation of Ror2 (see Fig. 1). Further study will be required to elucidate the molecular mechanism underlying Wnt5a-induced phosphorylation of Ror2 by GSK-3.

Previous studies demonstrate that GSK-3 (GSK-3{alpha} and/or GSK-3ß) is involved in the regulation of cell migration, yet the exact roles of GSK-3 in cell migration are controversial. Under several circumstances, GSK-3 has been shown to regulate cell migration negatively. For example, it has been reported that integrin mediates the inhibition of GSK-3 by activating integrin-linked kinase and PKB, thereby promoting cell migration (Kim et al. 2001), and that hypoxia stimulates tumor cell invasion by inhibiting GSK-3 (Yoon et al. 2005). On the other hand, it has been shown that cell migration mediated by epidermal growth factor receptor or induced by serum can be inhibited by suppressing the activity or expression of GSK-3, indicating that GSK-3 regulates cell migration positively (Koivisto et al. 2003, 2006; Kobayashi et al. 2006). Here we show by wound-healing assay that suppressed activity or expression of GSK-3 results in the inhibition of Wnt5a-induced cell migration (Fig. 4), indicating that GSK-3 regulates Wnt5a-induced cell migration positively. It will be of interest to clarify the functions of GSK-3 in regulating cell migration positively upon Wnt5a stimulation. It will also be of importance to address an important question of whether or not Ror2 phosphorylation by GSK-3 is involved in Wnt5a-induced cell migration. Future study aiming to identify GSK-3-mediated phosphorylation sites in Ror2 will provide a clue to clarify this issue.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmids, antibodies and chemical reagents

pcDNA-Ror2-HA and pcDNA-Ror2-FLAG were constructed as described (Oishi et al. 2003). pCGN-HA-GSK-3{alpha} and pCGN-HA-GSK-3ß were constructed as described (Ikeda et al. 1998; Tanji et al. 2002). An anti-mouse Ror2 antibody was prepared as described (Kani et al. 2004). Antibodies against GSK-3{alpha} (Cell Signaling Technology, Danvers, MA), GSK-3ß (BD Transduction Laboratories, San Jose, CA), phospho-GSK-3ß (Ser9) (Cell Signaling Technology), Dvl3 (Santa Cruz Biotechnology, Santa Cruz, CA), ß-catenin (Sigma, St. Louis, MO), ß-actin (Sigma), HA (BABCO, Cumberland, VA), FLAG (Sigma), phosphoserine (Zymed Laboratories, San Francisco, CA), phosphothreonine (Cell Signaling Technology) and phosphotyrosine (Cell Signaling Technology or Upstate Biotechnology, Lake Placid, CA) were purchased commercially. An alkaline phosphatase CAP-101 was purchased from TOYOBO (Osaka, Japan). GSK-3 inhibitors, LiCl and SB216763, were purchased from Nacalai tesque (Kyoto, Japan) and Tocris (Ellisville, CA), respectively. Casein kinase I inhibitor CKI-7 was purchased from D. Western Therapeutics Institute (Nagoya, Japan). The siRNAs targeting the human GSK-3{alpha} (5'-GAAGGUUCUCCAGGACAAGTT-3'; Kobayashi et al. 2006), human GSK-3ß (5'-GUAAUCCACCUCUGGCUACTT-3'; Liao et al. 2003), mouse GSK-3{alpha} (5'-GAAGGUUCUUCAGGACAAATT-3'), mouse GSK-3ß (5'-GAAGUCUAGCCUAUAUCCATT-3'; Kobayashi et al. 2006) and the control siRNA (5'-GTACCGCACGTCATTCGTA-3') were used.

Cells, transfection and conditioned media

NIH3T3, HeLa-S3 and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Nissui, Tokyo, Japan) supplemented with 10% (v/v) fetal calf serum (FCS). Cells were transfected using Lipofectamine 2000 according to the manufacturer's instruction. Wnt3a, Wnt5a and control (neo) conditioned media (CM) were harvested from confluent monolayers of Wnt3a/L, Wnt5a/L and neo/L cells (Takada et al. 2005) that had been cultured for 72 h in DMEM supplemented with 5% (v/v) FCS, respectively.

Immunoprecipitation and immunoblotting analyses

Cells were solubilized with lysis buffer [50 mM Tris–HCl (pH 7.4), 0.5% (v/v) Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethyl sulfonyl fluoride, 10 µg/mL leupeptin and 10 µg/mL aprotinin], and whole cell lysates (WCLs) were prepared by centrifugation at 12 000 g for 15 min. The WCLs were pre-cleared for 1 h at 4 °C with protein A-Sepharose (Amersham Biosciences, Little Chalfont, England). The pre-cleared supernatants were then immunoprecipitated with anti-Ror2, anti-HA or anti-FLAG antibody conjugated to protein A-Sepharose beads for 2 h at 4 °C. The immunoprecipitates were washed 5 times with 1 mL of the above lysis buffer and eluted with Laemmli sample buffer. Immunoprecipitates or WCLs were separated by SDS-PAGE (7.5% PAG), and transferred to polyvinylidene difluoride membrane filters (Immobilon, Millipore, Bedford, MA). The membranes were immunoblotted with the respective antibodies, and the bound antibodies were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies using chemiluminescence reagents (Western Lightning, PerkinElmer Life Sciences, Boston, MA) as described (Matsuda et al. 2003).

In vitro kinase assay

Thirty six hours after transfection, HEK293T cells were solubilized with lysis buffer. WCLs were immunoprecipitated with anti-HA antibody. The immunoprecipitates were washed 3 times with lysis buffer, then once with kinase reaction buffer [20 mM HEPES (pH 7.4), 2 mM EGTA, 15 mM MgCl2]. The immunoprecipitates were resuspended in 30 µL of kinase reaction buffer containing 10 µCi {gamma}32P-ATP (3000 Ci/mmol, Amersham Biosciences) in the absence (DMSO alone) or presence of SB216763 (10 µM), and incubated for 20 min at 37 °C. The reaction was terminated by the addition of Laemmli sample buffer and samples were separated by SDS-PAGE (7.5% PAG). Subsequently, the gels were subjected to autoradiography.

Wound-healing assay

NIH3T3 cells were plated onto fibronectin-coated coverslips. The confluent monolayer cells were then scratched manually with a plastic pipette tip, and after being washed once with phosphate-buffered saline, wounded monolayers of the cells were allowed to heal for 8–12 h. Cells cultured on coverslips were then fixed in 4% paraformaldehyde, and stained with rhodamine-phalloidin (Invitrogen, Carlsbad, CA) to visualize F-actin. The relative migrating distance of the wound edge was shown as the mean ± SD of three independent experiments, with the migrating distance of neo CM (DMSO or control siRNA)-treated cells set as 1.0.


   Acknowledgements
 
We thank A. Yoda for critical reading of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research in Priority Areas and a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Uehara Memorial Foundation, and the Naito Foundation. HY is a research fellow of the Japan Society for the Promotion of Sciences.


   Footnotes
 
Communicated by: Tadashi Yamamoto

* Correspondence: E-mail: minami{at}kobe-u.ac.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 26 April 2007
Accepted: 23 July 2007




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