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1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
2 Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Introduction |
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contains an additional N-terminal region including a sorbin homology (SoHo) domain (Kioka et al. 1999, 2002; Matsuyama et al. 2005). Vinexin belongs to an adaptor protein family that includes ArgBP2/nArgBP2 and CAP/ponsin (Kioka et al. 2002). Members of this family share the same domain structure and are likely to function as scaffolding or adaptor proteins to put binding partners close to each other (Wang et al. 1997; Kawabe et al. 1999; Kioka et al. 1999, 2002; Mandai et al. 1999; Baumann et al. 2000). Indeed, vinexin binds to signaling molecules including ERK and Sos and modulates EGF-induced JNK and ERK signaling (Akamatsu et al. 1999; Suwa et al. 2002; Mitsushima et al. 2004). Furthermore, vinexin enhances cell spreading in myoblasts and induces the accumulation of F-actin at focal adhesions in fibroblasts (Kioka et al. 1999), although the mechanism regulating cell adhesion and the cytoskeleton is not clear. The reorganization of the actin cytoskeleton plays an important role in cell morphogenesis, cell spreading and cell motility. The Wiskott-Aldrich syndrome protein (WASP) family including WASP, neuronal-WASP (N-WASP) and WAVE1, 2 and 3 play crucial roles in actin polymerization through activation of the actin-related protein (Arp) 2/3 complex (Miki & Takenawa 2003; Bompard & Caron 2004; Stradal et al. 2004). WASP family proteins contain the verprolin (V) homology, cofilin (C) homology and acidic (A) regions at the C-terminus. This VCA region associates with and stimulates the activity of the Arp2/3 complex. Regulatory mechanisms of N-WASP and WASP have been elucidated. They are known to exist as inactive conformations through intramolecular interaction between the GTPase-binding domain or basic region and VCA region (Rohatgi et al. 2000; Miki & Takenawa 2003; Bompard & Caron 2004). The binding of CDC42, PIP2, or SH3-containing proteins such as WISH and NCK with N-WASP/WASP disrupts this intramolecular interaction and activates N-WASP/WASP (Miki et al. 1998a; Rohatgi et al. 1999, 2001; Fukuoka et al. 2001). Furthermore, tyrosine phosphorylation has been shown to be involved in the activation and degradation of N-WASP/WASP proteins (Cory et al. 2002; Suetsugu et al. 2002; Torres & Rosen 2003; Wu et al. 2004; Park et al. 2005). In contrast to N-WASP/WASP, WAVE proteins appear to be constitutively active in vitro and different mechanisms seem to regulate their functions. Recent studies have shown that WAVE proteins are included in the protein complex PIR121/NAP1/Abi/HSPC300 (Eden et al. 2002; Kunda et al. 2003; Echarri et al. 2004; Innocenti et al. 2004; Steffen et al. 2004). Association with this complex increases the stability of WAVEs (Kunda et al. 2003; Echarri et al. 2004; Innocenti et al. 2004; Steffen et al. 2004). One report showed that the formation of this complex inhibits a WAVE activity and this inhibition was rescued by GTP-Rac (Eden et al. 2002). Other reports showed that WAVEs in the complex are still active and GTP-Rac induces the translocation of the complex to the cell cortex (Innocenti et al. 2004; Steffen et al. 2004). Furthermore, WAVE protein is phosphorylated and its mobility in SDS-PAGE is delayed after growth factor stimulation inducing activation of ERK (Miki et al. 1999).
In this study, we show that vinexin ß increases the amount of WAVE2 and induces the phosphorylation of WAVE2. Vinexin ß associates with WAVE2 through the first and second SH3 domains. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin ß on WAVE2. Inhibition of PKA activity suppressed the vinexin-induced increase in expression of WAVE2 and the activation of PKA increased WAVE2 expression without vinexin ß. These results suggest that vinexin ß regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.
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Results |
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We have reported that the focal adhesion protein vinexin promotes the reorganization of actin cytoskeleton in NIH3T3 cells and cell spreading in myoblasts (Kioka et al. 1999); however, just how it affects the actin cytoskeleton is not clear. Thus, we first determined whether expression of vinexin ß affects the quantity of several actin regulatory molecules. After FLAG- or GFP-tagged vinexin ß was co-transfected with several actin regulatory proteins, including vinculin, FAK, and WAVE2, into NIH3T3 cells, cell lysates were isolated and subjected to immunoblotting. Expression of vinexin ß did not affect the amount of vinculin or FAK (data not shown). In contrast, FLAG- or GFP-tagged vinexin ß increased the amount of MycHis-tagged WAVE2 in serum-starved NIH3T3 cells (Fig. 1). Furthermore, co-expression of vinexin ß retarded the mobility of MycHis-WAVE2 on SDS-PAGE (Fig. 1, and see also Fig. 2A and Fig. 3). These observations suggest that vinexin ß is involved in the regulation of WAVE2.
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Vinexin ß contains three SH3 domains, which are known to associate with proline-rich sequences, and functions as an adaptor molecule. On the other hand, WAVE2 contains proline-rich sequences in addition to the VCA region and WHD domains, and associates with several SH3-containing proteins through the proline-rich sequences. Thus, we first examined whether vinexin ß interacts with WAVE2 through its SH3 domain(s). Cos7 cells were used for co-precipitation experiments, because the effect of vinexin ß on the amount of WAVE2 was less in Cos7 cells than in NIH3T3 cells, possibly due to higher levels of WAVE2, and it is easier to have negative controls in Cos7 cells. After FLAG-tagged vinexin ß or SH3 mutants were co-transfected with MycHis-WAVE2 into COS7 cells, cell lysates were immunoprecipitated using anti-FLAG antibody. As shown in Fig. 2A, MycHis-tagged WAVE2 was co-precipitated with wild-type vinexin ß. Interestingly, only slower migrating WAVE2 was co-precipitated with vinexin ß but faster migrating WAVE2 was not. Mutation in the first SH3 domain reduced the amount of WAVE2 co-precipitated with vinexin ß. Mutation in the second SH3 domain reduced the co-precipitated WAVE2 moderately. Expression of these mutants induced only the partial retardation of WAVE2. In contrast, mutation in the third SH3 domain did not affect the association or mobility of WAVE2. Mutations in both the first and second SH3 domains completely inhibited the co-precipitation and suppressed the mobility retardation of WAVE2. We next determined whether the proline-rich region of WAVE2 is involved in the interaction with vinexin ß. Wild-type WAVE2 or a deletion mutant (P-rich), which contains only the proline-rich region (273-406), were co-transfected with vinexin ß into Cos7 cells and a co-immunoprecipitation assay was performed. As shown in Fig 2B, a P-rich mutant was co-precipitated with vinexin ß at levels comparable with wild-type WAVE2, suggesting that the proline-rich region of WAVE2 is sufficient for the interaction with vinexin ß. Furthermore, endogenous WAVE2 was also co-immunoprecipitated with endogenous vinexin in NIH3T3 cells (Fig. 2C). Under these conditions, control immunoprecipitation with rabbit IgG did not precipitate WAVE2. Reciprocal immunoprecipitation experiments were also carried out with anti-WAVE2 antibody, followed by immunoblotting with anti-vinexin antibodies. As expected, vinexin ß as well as vinexin were detected in WAVE2 immunoprecipitates (data not shown). Interestingly, vinexin was more co-precipitated with WAVE2 than vinexin ß. However we could detect only the marginal association of exogenously expressed vinexin with WAVE2, possibly by the much lower solubility of exogenously expressed vinexin . Thus, we focused on the vinexin ß in this study. Together, these results suggest that vinexin ß as well as vinexin interact with WAVE2 and both the first and second SH3 domains of vinexin contribute to this interaction.
Other WASP family members, including WAVE1 and N-WASP, also contain a proline-rich region and interact with several SH3 domains (Rivero-Lezcano et al. 1995; Miki et al. 1996, 2000; Anton et al. 1998; Westphal et al. 2000). Thus, we next determined whether vinexin ß interacts with WAVE1 and N-WASP through its SH3 domain(s). As shown in Fig. 2D, WAVE1 was also co-immunoprecipitated with wild-type vinexin ß and the 3rdWF mutant. Interestingly, the introduction of a mutation into either the first or second SH3 domain reduced the co-precipitation of WAVE1, suggesting that vinexin ß associates with WAVE1 and 2 in a similar fashion. On the other hand, N-WASP was co-immunoprecipitated with wild-type vinexin ß, the 1stWF mutant, and the 2ndWF mutant at comparable levels (Fig. 2E). A point mutation in the third SH3 domain of vinexin ß disrupted the co-precipitation completely, suggesting that vinexin ß can interact with N-WASP and WAVE1/2 using different SH3 domains. No increase in the amount of WAVE1 or N-WASP was detected after the expression of vinexin ß in this condition.
Phosphorylation is involved in the effect of vinexin ß on the mobility of WAVE2
To determine the mechanism by which vinexin ß retards the mobility of WAVE2, the wild-type or 1st2ndWF mutant of vinexin ß was co-expressed with MycHis-tagged WAVE2 in NIH3T3 cells. Expression of the wild-type reduced the mobility of WAVE2, as well as increased the amount of WAVE2. In contrast, 1st2ndWF vinexin ß did not affect the mobility or the amount of WAVE2 (Figs 3 and 4A, and data not shown). To determine whether this retardation is a result of the phosphorylation of WAVE2, an equal amount of WAVE2 protein, estimated by immunoblotting using anti-c-Myc antibody in advance, was precipitated using Ni-NTA agarose and these beads were subjected to phosphatase treatment. Treatment with CIAP for 60 min rescued the retardation of mobility on SDS-PAGE induced by co-expression of vinexin ß. Treatment with the buffer alone did not affect the mobility of WAVE2 (Fig. 3). These findings suggest that expression of vinexin ß alters the phosphorylation status of WAVE2, possibly through interaction with WAVE2.
Loss of interaction of vinexin ß with WAVE2 impaired the ability of vinexin ß to increase the amount of WAVE2
To examine whether the interaction of vinexin ß with WAVE2 is involved in the stabilization of WAVE2, wild-type or mutants that contain mutations in SH3 domains were co-transfected with MycHis-WAVE2 into NIH3T3 cells. Expression of wild-type vinexin ß or the 3rdWF mutant efficiently increased the amount of WAVE2, whereas the 1st2ndWF mutant, which failed to interact with WAVE2, did not (Fig. 4A). Interestingly, the 1stWF or 2ndWF mutant, which showed decreased but significant affinity for WAVE2, still had the ability to increase the amount of WAVE2 (Fig. 4B). These observations suggest that a weak association of vinexin ß with WAVE2 is enough for the increase in the amount of WAVE2 and a tight association of vinexin ß with WAVE2 is not necessary.
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Since exogenously expressed WAVE2 mRNA is transcribed from a constitutively active CMV promoter, vinexin-induced increase in WAVE2 seems to be mediated post-transcriptionally. To confirm this, we determined the effect of vinexin ß on the stability of MycHis-WAVE2 (Fig. 4C). After co-transfection of MycHis-WAVE2 with FLAG-tagged vinexin ß or 1st2ndWF mutant, cellular protein synthesis was inhibited by treatment with cycloheximide to block the supply of newly synthesized WAVE2. After the inhibition of cellular protein synthesis, more than 50% of WAVE2 protein was degraded in 2 h in cells expressing 1st2ndWF, which did not increase the amount of WAVE2. In contrast, when wild-type vinexin ß was co-expressed, most WAVE2 proteins still remained even after treatment with cycloheximide for 5 h. These results suggest that expression of vinexin ß decreases the degradation of WAVE2, resulting in the increase in WAVE2 protein.
N-WASP and WAVE1/2 proteins have been reported to be regulated by ubiquitin-proteasome-dependent and calpain-dependent degradation (Kunda et al. 2003; Echarri et al. 2004; Innocenti et al. 2004; Steffen et al. 2004; Oda et al. 2005; Yamazaki et al. 2005). We therefore next determined the effect of proteasome inhibitors as well as a calpain inhibitor on WAVE2 expression under serum-starved conditions to elucidate the mechanism by which vinexin stabilizes the WAVE2 protein. Treatment with proteasome inhibitors, MG132 and lactacystin, increased the amount of MycHis-WAVE2 protein under serum-starved conditions (Fig. 4D). In contrast, the calpain inhibitor calpeptin did not increase the WAVE2 expression. We next examined the effect of proteasome inhibitors on vinexin ß-transfected cells, in which WAVE2 expression was already increased. Interestingly, the proteasome inhibitors did not have additive effects, i.e. they did not increase the amount of WAVE2 protein further in vinexin ß-transfected cells (Fig. 4D). Addition of 10% serum also increased the expression of WAVE2 protein. These observations suggest that MycHis-WAVE2 is degraded at least partially by a proteasome-dependent pathway and that expression of vinexin ß allows WAVE2 to escape this degradation process.
c-Cbl has been reported to function as an E3 ubiquitin ligase and associate with other members of the vinexin adaptor family, CAP and ArgBP2 (Ribon et al. 1998; Joazeiro et al. 1999; Levkowitz et al. 1999; Waterman et al. 1999; Soubeyran et al. 2003). Thus, we determined the effects of co-expression of c-Cbl on WAVE2 and found that co-expression of c-Cbl significantly reduced the vinexin ß-induced increase in WAVE2 (data not shown). This result may support the hypothesis that degradation of WAVE2 is mediated by a proteasome-dependent pathway and that expression of vinexin ß allows WAVE2 to escape this degradation.
PKA is involved in the regulation of WAVE2 degradation
Phosphorylation has been reported to be involved in the regulation of proteasome-dependent degradation in various cases (Busino et al. 2004; Ang & Wade Harper 2005). Thus, we examined the effects of kinase inhibitors on the vinexin ß-induced increase in WAVE2. As shown in Fig. 5A, treatment with inhibitors for Src (PP2), phosphatidylinositol-3 kinase (wortmannin, LY294002), ERK1/2 (U0126), JNK (SP600125), or PKC (Go6976) did not affect WAVE2 expression (Fig. 5A and data not shown), suggesting that c-Src, phosphatidylinositol-3 kinase, ERK, JNK, and PKC are not involved in the vinexin ß-induced increase in WAVE2. However, treatment with a PKA specific inhibitor, H89, dramatically suppressed the vinexin ß-induced increase in a dose-dependent manner (Fig. 5B). Expression of WAVE2 completely disappeared after the treatment with 50 µM of H89. In contrast, the activation of PKA by adding 8-bromo-cyclic AMP (Fig. 5C) or forskolin with 3-isobutyl-1-methylxanthine (data not shown) increased WAVE2 expression in serum-containing and starved medium. PKA has been reported to bind to WAVE1 and co-localized with WAVE1/2 proteins at lamellipodia in PDGF-stimulated Swiss 3T3 cells (Westphal et al. 2000; Wong & Scott 2004). Furthermore, CAP/ponsin, another member of the vinexin adaptor family, was also recently reported to associate with PKA (Matson et al. 2005). Thus, we examined whether PKA can associate with vinexin ß and WAVE2. After transfection of FLAG-tagged vinexin ß or WAVE2 into Cos7 cells, cell lysates were subjected to immunoprecipitation using anti-FLAG antibody. Interestingly, PKA was co-precipitated with both FLAG-vinexin ß and FLAG-WAVE2 (Fig. 5D). Thus, these results suggest that PKA can associate with vinexin ß and WAVE2 and is involved in the vinexin ß-induced increase in the expression of WAVE2. In addition, we also noted that none of these kinase inhibitors, including H89, affected the vinexin ß-induced mobility retardation of WAVE2. Thus, the phosphorylation responsible for the retardation of mobility may be mediated by other kinase(s).
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To confirm the association of vinexin with WAVE2 in vivo, we examined the immunolocalization of endogenously expressed vinexin and WAVE2 (Fig. 6). MDA-MB-231 cells were plated on fibronectin-coated coverslips and stained with anti-vinexin or anti-WAVE2 antibody. As previously shown, vinexin was localized at focal adhesions, where another vinexin binding protein, vinculin, was accumulated (arrow heads). In addition, both vinexin and vinculin were also localized at the peripheral membrane of the leading edge (arrows). As expected, WAVE2 was also localized at the peripheral membrane of the leading edge and co-localized with vinexin. Interestingly, WAVE2 was not detected at the focal adhesions. Similar co-localization of vinexin and WAVE2 at the peripheral membrane of the leading edge was also observed in NIH3T3 cells (data not shown), although cells having protruding membranes were much less common than in MDA-MB-231 cells. Together, these observations support the hypothesis that the interaction of vinexin with WAVE2 plays a role in regulating the membrane protrusion at the leading edge.
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Discussion |
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Vinexin retarded the mobility of WAVE2. This effect was reversed by the phosphatase treatment, suggesting that vinexin induced the phosphorylation of WAVE2, resulting in the retardation of the mobility. Both the phosphorylation and stabilization of WAVE2 require either the first or second SH3 domain of vinexin ß and seem to be dependent on the interaction with vinexin ß. However, the PKA inhibitor suppressed the increase in WAVE2 protein but not the mobility retardation. Thus, phosphorylation induced by vinexin itself may be necessary but was not sufficient for the stabilization of WAVE2. Another issue of interest is which kinase is involved in the vinexin-induced phosphorylation of WAVE2. Activation of ERK2 by growth factors or constitutively active MAPK/ERK kinase is known to induce the phosphorylation and retard the mobility of WAVE2 (Miki et al. 1999). Nonreceptor-type tyrosine kinase Abl was recently reported to phosphorylate tyrosine residue Y150 of WAVE2 and stimulate actin polymerization (Leng et al. 2005). However, neither of these kinases seem to be involved in the mobility retardation of WAVE2 induced by vinexin ß. Inhibition of ERK by U0126 or introduction of point mutation in Y150 did not suppress the effect on the mobility of WAVE2 or increase the amount of protein (Fig. 5A and data not shown). Furthermore, inhibition of src, JNK, PKC or PI3K did not affect the change in mobility (Fig. 5A and data not shown). Future study will define the kinase responsible for the retardation of mobility induced by vinexin ß.
We could not detect the increase in the amount of WAVE1 after expression of vinexin ß in the cells we used. However, we observed the vinexin ß-induced mobility retardation of WAVE1 in NIH3T3 cells (data not shown). Thus, WAVE1 and WAVE2 seem to be regulated at least partially by the same mechanism, but regulation of stability may be different. It is reported that regulatory subunit of PKA or profilin shows higher affinity to WAVE1 than to WAVE2 but IRSp53 prefers WAVE2 (Miki et al. 2000; Oda et al. 2005). One possible explanation for the different regulation of the stability is that the difference of accessibility or association of other regulatory molecules with WAVEs may affect the stability. Future study will show whether these associated molecules contribute to the different regulation of the stability.
Vinexin associated with WAVE2 through the first and second SH3 domains. We previously reported that vinexin interacts with vinculin, another focal adhesion protein, through the same first and second SH3 domains (Kioka et al. 1999). It is plausible that vinculin competes with WAVE2 to bind with vinexin ß, although we did not detect competition in a co-immunoprecipitation assay in preliminary experiments. Vinexin may associate with WAVE2 and vinculin at different compartments within cells, such as at the leading edge and at focal adhesions, respectively.
ArgBP2/nArgBP2, another member of the vinexin adaptor family, was recently reported to associate with WAVE1/2 as well as vinculin and c-Cbl by Cestra et al. (2005). Interestingly, they also showed that a C-terminal-truncated form of nArgBP2, which contains a SoHo domain but not any SH3 domains, inhibited the ubiquitination of WAVE2, although it did not affect the amount of WAVE2 in HEK293T cells. Thus, both vinexin and nArgBP2 may form a similar protein complex and have related regulatory effects on WAVE2.
Recently WAVE2 was reported to be involved in HGF-induced membrane ruffling in C2C12 cells. Exogenous expression of vinexin ß stimulates the spreading of C2C12 cells. Thus, it is interesting to examine the coordination of vinexin and WAVE in C2C12 cells. In conclusion, we have shown that vinexin ß associated with and increased the amounts of WAVE2. We also demonstrated that treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin ß on WAVE2. Furthermore, inhibition of PKA activity suppressed the vinexin-induced increase in expression of WAVE2. These results suggest that vinexin ß regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.
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Anti-FLAG (M2) mouse monoclonal antibody was purchased from Sigma. Anti-c-Myc (A-14), anti-PKA catalytic subunit (C-20) and anti-WAVE2 (D-16) polyclonal antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against WAVE2 and vinexin were previously described (Miki et al. 1998b; Kioka et al. 1999). Proteasome inhibitors, MG132, and lactacystin, were purchased from Calbiochem.
Expression vectors
WAVE2 cDNA from pEGFF-WAVE2 (Oikawa et al. 2004) was subcloned into pcDNA3.1(-)/myc-His (Invitrogen) to generate Myc-His-tagged WAVE2. pEFBOS-myc-N-WASP was previously described. Myc-tagged P-rich mutant of WAVE2 (amino acids 273–406) were generated by PCR using human WAVE2 cDNA as a template and subcloned into pcDNA3.1(-)/myc-His. A mutation of the first SH3 domain (substitution of the tryptophan residue at 76 to phenylalanine; 1stWF), the second SH3 domain (substitution of the tryptophan residue at 150 to phenylalanine; 2ndWF), or the third SH3 domain (substitution of the tryptophan residue at 306 to phenylalanine; 3rdWF) of vinexin ß was previously described (Suwa et al. 2002). The dual first and second SH3 domain mutant (1st2ndWF) was constructed using restriction enzymes. pAlter-max-HA-c-Cbl (Feshchenko et al. 1998) was kindly provided by Dr A. Tsygankov (Temple University School of Medicine).
Immunoblotting and immunoprecipitation
Immunoprecipitation assays were performed as previously described (Wakabayashi et al. 2003; Mitsushima et al. 2004). Briefly, cells were lyzed with cell lysis buffer (1% NP-40, 0.02 mg/mL aprotinin, 0.1 mg/mL p-amidinophenyl methanesulfonyl fluoride hydrochloride, 5 µg/mL leupeptin, 5 mM benzamidine, and 1 µg/mL pepstatin A in PBS). Equal amounts of cell lysates were subjected to immunoprecipitation with each antibody at 4 °C. Immunoprecipitates were washed four times using ice-cold 1% NP-40/PBS. In some cases, precipitated proteins were eluted by 50 mM Tris-HCl, pH 7.5, containing 500 µg/mL FLAG-peptide (Sigma). Co-precipitated proteins were separated by SDS-PAGE and subjected to immunoblotting with specific antibodies.
Immunofluorescence
Cells were cultured on fibronectin-coated coverslips. Cells were fixed with 10% formalin for 15 min at room temperature and permeabilized in 0.2% TritonX-100/PBS for 5 min. They were blocked with 10% goat or donkey serum/TBS-T (TBS containing 0.1% TritonX-100) for 1 h, then incubated with anti-vinexin antibody and anti-WAVE2 antibody at 4 °C overnight. The cells were stained with Alexa 568-labeled donkey anti-rabbit IgG and Alexa 488-labeled donkey anti-goat IgG (Molecular Probes), for 1 h. Fluorescence images were taken with a PASCAL confocal microscopy system (Carl Zeiss Co., Ltd).
Phosphatase treatment
Phosphatase treatment was performed as previously described (Mitsushima et al. 2004). In brief, MycHis-tagged WAVE2 was co-transfected with FLAG-tagged wild-type or 1st2ndWF mutant of vinexin ß into NIH3T3 cells. Cell lysates were precipitated with Ni-NTA agarose beads (Qiagen). The amounts of protein used for the phosphatase assay were normalized by immunoblotting. Precipitated beads were resuspended with reaction buffer containing 50 units of calf intestine alkaline phosphatase (CIAP) (TaKaRa, Japan) and incubated at 37 °C for 60 min. The reaction was stopped by adding Laemmli sample buffer and boiling at 97.5 °C for 5 min. Proteins were separated by SDS-PAGE and subjected to immunoblotting.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: nkioka{at}kais.kyoto-u.ac.jp
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Received: 2 September 2005
Accepted: 20 November 2005
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