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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
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Abstract |
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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
) can inhibit Wnt5a-induced phosphorylation of Ror2. Immunoprecipitated Ror2 can also be phosphorylated by purified GSK-3 or GSK-3ß
in vitro, and ectopic co-expression of Ror2 and GSK-3 (mainly GSK-3) 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 (and GSK-3ß) expression, further emphasizing the role(s) of GSK-3 in Wnt5a-induced signaling. |
Introduction |
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It has been shown that Ror2 associates with and is phosphorylated on serine/threonine residues by CKI, 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 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 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 (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, is involved in Wnt5a-induced phosphorylation of Ror2. Treatment of cultured cells with small interfering RNA (siRNA) for GSK-3 (or GSK-3ß) also results in inhibition of Ror2 phosphorylation induced by Wnt5a. We further show that Ror2 can be phosphorylated by GSK-3 (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 and GSK-3ß expressions. Collectively, these results indicate that GSK-3 is involved in Wnt5a-induced phosphorylation of Ror2 and cell migration.
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Results |
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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.
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Wnt5a-induced phosphorylation of Ror2 is inhibited by GSK-3 inhibitors or siRNA for GSK-3 (or GSK-3ß)
It has been shown that Ror2 is phosphorylated on serine/threonine residues by CKI when both Ror2 and CKI 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 and/or GSK-3ß on Wnt5a-induced phosphorylation of Ror2 in HeLa-S3 cells. As shown in Fig. 2C, suppression of GSK-3 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 siRNA than GSK-3ß siRNA, it is likely that GSK-3 rather than GSK-3ß is more responsible for Wnt5a-induced Ror2 phosphorylation. These results indicate that GSK-3 (GSK-3 and possibly GSK-3ß) is required for phosphorylation of Ror2 induced by Wnt5a.
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We then tested whether or not GSK-3 and/or GSK-3ß can phosphorylate Ror2 in vitro. Ror2-HA and HA-GSK-3 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 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 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 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 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 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 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 and possibly GSK-3ß) is involved in Wnt5a-induced phosphorylation of Ror2.
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It has been shown that GSK-3 (GSK-3 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 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 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.
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Discussion |
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, 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 when both molecules are over-expressed in cultured cells. However, unexpectedly, it was found that GSK-3 (GSK-3 and/or GSK-3ß) rather than CKI, 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 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 and GSK-3ß) are usually constitutively active by phosphorylation of GSK-3 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 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 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.
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Experimental procedures |
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pcDNA-Ror2-HA and pcDNA-Ror2-FLAG were constructed as described (Oishi et al. 2003). pCGN-HA-GSK-3 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 (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 (5'-GAAGGUUCUCCAGGACAAGTT-3'; Kobayashi et al. 2006), human GSK-3ß (5'-GUAAUCCACCUCUGGCUACTT-3'; Liao et al. 2003), mouse GSK-3
(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 
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.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: minami{at}kobe-u.ac.jp
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References |
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Received: 26 April 2007
Accepted: 23 July 2007
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