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Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
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Introduction |
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, and c-Cbl (Okutani et al. 1994; Sakaguchi et al. 1998; Chattopadhyay et al. 1999; Rodrigues et al. 2000). For example, phosphorylated tyrosine residue 1068 of EGFR recruits Grb2 and Gab1, and triggers the activation of the Ras-mitogen activated protein kinase (MAPK) pathway and phosphatidylinositol-3-kinase (PI3K)-Akt pathway, respectively, resulting in cell proliferation or cell survival (Lowenstein et al. 1992; Li et al. 1993; Rodrigues et al. 2000; Mattoon et al. 2004). Phosphorylated and activated EGFR is then down-regulated by negative regulatory mechanisms. These include the rapid internalization of phosphorylated EGFR from the cell surface through clathrin-mediated endocytosis and the degradation of EGFR in lysosomes. The phosphorylated EGFR recruits c-Cbl via direct interaction with or by Grb2-mediated interaction with EGFR (Waterman et al. 2002). The recruited c-Cbl is phosphorylated and activated by EGFR. c-Cbl, in turn, monoubiquitinates EGFR. Although the function of this monoubiquitination is controversial, in some cases at least it allows EGFR to associate with Eps15, which contains UIM (ubiquitin interacting motifs). This would guide the activated EGFRs to clathrin-coated pits and induce endocytosis (de Melker et al. 2004a,b; Le Roy & Wrana 2005). After its internalization into the early endosomes, EGFR can form a complex with hepatocyte-growth-factor-regulated tyrosine-kinase substrate (HRS) or signal transduction adaptor molecule (STAM), resulting in its delivery to the multivesicular body and degradation (Le Roy & Wrana 2005). Alternatively, some populations of internalized EGFR are recycled to the plasma membrane. In addition, c-Cbl has also been shown to promote the internalization of activated EGFR via a different mechanism from the ubiquitination of EGFR. It associates with the adaptor molecule CIN85, which then rapidly recruits endophilin to the activated EGFR (Soubeyran et al. 2002). These complexes further associate with other proteins including dynamin and synaptojanin2, resulting in the formation of clathrin-coated vesicles and induction of endocytosis (Ringstad et al. 1997; Rusk et al. 2003; Schmidt & Dikic 2005).
The activation status of EGFR can also be regulated by protein tyrosine phosphatases (PTPs), including PTP-1B TC-PTP, SHP2 and RPTP
(Tiganis et al. 1998, 1999; Klingler-Hoffmann et al. 2001; Haj et al. 2002; 2003; Agazie & Hayman 2003; Vijayvargia et al. 2004; Xu et al. 2005). The mechanism of dephosphorylation of EGFR by PTP-1B is relatively well characterized (Flint et al. 1997; Liu & Chernoff 1997). PTP-1B localizes at the endoplasmic reticulum and dephosphorylates endocytosed EGFR (Burke et al. 2001; Haj et al. 2002). Fibroblasts derived from PTP-1B knockout mice showed an increased and sustained phosphorylation of EGFR (Haj et al. 2003). In addition, phosphatase activity is also believed to be involved in controlling EGFR phosphorylation at the cell membrane. Phosphatases located on the plasma membrane continuously dephosphorylate EGFR in the absence of EGF stimulation to prevent the spontaneous autophosphorylation and activation (Reynolds et al. 2003; Offterdinger et al. 2004).
Vinexin is a focal adhesion protein involved in the regulation of the actin cytoskeleton and cell spreading (Kioka et al. 1999). Vinexin belongs to an adaptor protein family, which consists of vinexin, CAP/ponsin, and ArgBP2 (Wang et al. 1997; Ribon et al. 1998; Kioka et al. 1999, 2002; Mandai et al. 1999). Vinexin is expressed in several isoforms, including vinexin 
and vinexin ß (Kioka et al. 2002; Matsuyama et al. 2005). Vinexin contains a sorbin homology (SoHo) domain at the N-terminus and three SH (src homology) 3 domains at the C-terminus and shows tissue-specific expression, whereas vinexin ß contains only three SH3 domains and shows an ubiquitous expression (Kioka et al. 1999; Kawauchi et al. 2001). Both vinexins bind to vinculin, a major focal adhesion protein, and localize at focal adhesions. Furthermore, vinexin induces the accumulation of F-actin at focal adhesions (Kioka et al. 1999; Takahashi et al. 2005). We have also reported that vinexin ß promotes EGF-induced activation of c-Jun N-teminal kinase (JNK) as well as anchorage-independent activation of extracellular signal-regulated kinase (ERK) 2 in NIH3T3 cells (Akamatsu et al. 1999; Suwa et al. 2002). In addition, ERK2 activated by EGF stimulation or cell adhesion interacts with and phosphorylates vinexin ß (Mitsushima et al. 2004). These observations suggest that vinexin controls growth factor-mediated signaling as well as the cytoskeletal organization.
In this study, we show that exogenous expression of vinexin ß promoted the phosphorylation of EGFR in Cos7 cells. Treatment with AG1478, an EGFR-specific inhibitor, before or after stimulation of the cells with EGF completely inhibited the effect, suggesting that the kinase activity of EGFR is required for the sustained phosphorylation induced by vinexin ß. The first and third SH3 domains of vinexin ß were both required for this effect. Interestingly, expression of vinexin ß induced a sustained phosphorylation of EGFR on the plasma membrane. Moreover, the knockdown of endogenous vinexin ß using siRNA led to a decrease in the phosphorylation of EGFR on the plasma membrane. Interestingly, vinexin ß associated with and modulated the subcellular distribution of c-Cbl. Furthermore, over-expression of c-Cbl suppressed the sustained phosphorylation of EGFR induced by vinexin ß. These results suggest that vinexin ß plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl.
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Results |
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We previously reported that vinexin ß regulates the EGF-induced activation of JNK and anchorage-dependent activation of ERK2 in NIH3T3 cells. In this study, we examined whether the expression of vinexin ß affects other molecules activated by EGF stimulation. Control vector or FLAG-tagged vinexin ß was transfected into NIH3T3 cells and Cos-7 cells. After incubation with or without EGF for 30 min, tyrosine-phosphorylated proteins were detected by Western blotting using the anti-phosphotyrosine antibody PY20. The expression of vinexin ß promoted the tyrosine phosphorylation of 180-kDa protein(s) significantly in Cos-7 cells (Fig. 1A). Because EGFR is a major tyrosine-phosphorylated protein of around 180 kDa after EGF stimulation, we next determined the tyrosine phosphorylation of EGFR. Tyrosine 1068 (Y1068) of EGFR is a well known autophosphorylation site, which leads to activation of the Ras-MAPK pathway. Thus, we first determined the phosphorylation of tyrosine 1068 using anti-phospho-EGFR (Y1068) antibody. As expected, expression of vinexin ß promoted the phosphorylation of tyrosine residues of EGFR, including tyrosines 1068, 992, 1045 and 845, without altering the amount of EGFR in cell lysates (Fig. 1B). Immunodepletion of EGFR in vinexin ß-transfected cells resulted in a loss of tyrosine-phosphorylated 180-kDa protein(s) (Fig. 1C), suggesting that EGFR is the major tyrosine-phosphorylated protein of 180 kDa. Interestingly, the expression of vinexin ß did not affect the phosphorylation of EGFR in NIH3T3 cells (data not shown). This difference between Cos-7 and NIH3T3 cells may be derived from cell type-specific effect, such as expression level of EGFR. We therefore focused on Cos-7 cells in the following experiments
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We next examined which region of vinexin ß was responsible for inducing the sustained phosphorylation of EGFR using deletion and point mutants described in Fig. 3A. Full-length vinexin ß efficiently induced the sustained phosphorylation of EGFR, but neither the N-terminal nor C-terminal of vinexin ß did (Fig. 3B). This result indicated that both the N-terminal and C-terminal of vinexin ß were required for inducing the sustained phosphorylation of EGFR. Vinexin ß contains three SH3 domains. Thus, we next examined whether these SH3 domains are required for inducing a sustained phosphorylation of EGFR. Figure 3C shows that the wild-type vinexin ß and 2ndWF mutant, which contains a point mutation in the second SH3 domain, were able to sustain the phosphorylation, whereas the 1stWF and 3rdWF mutants, which contain a point mutation in the first and third SH3 domains, respectively, were not. Furthermore, the 3rdYV mutant, in which a tyrosine residue within the third SH3 domain of vinexin ß was substituted with valine, did not induce the sustained phosphorylation of EGFR, either (Fig. 3D). These results indicated that both the first and third SH3 domains of vinexin ß were required for inducing the sustained phosphorylation of EGFR. Interestingly, EGFR contains potential SH3 recognition sequences (PXXP) in its cytoplasmic region. However, no co-immunoprecipitation of vinexin ß with EGFR was detected either in EGF-stimulated or unstimulated cells (data not shown), suggesting that the binding partner(s) for the first and third SH3 domains may not be EGFR per se.
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We next determined the subcellular distribution of phosphorylated EGFR. The control vector or FLAG-tagged vinexin ß was transfected into Cos-7 cells and the cells were stimulated with EGF for given periods. Phosphorylated EGFR was detected both at the plasma membrane and in the cytosol 5 min after the stimulation with EGF in FLAG-vinexin ß-expressing and control cells (Fig. 4A). Phosphorylated EGFR was still detected at the plasma membrane in vinexin ß-expressing cells 60 min after the stimulation with EGF, whereas it was completely disappeared in control cells (Fig. 4A). To confirm that phosphorylated EGFR is located at the cell surface, we determined the EGFR distribution using a biochemical method. Cell surface proteins of FLAG-vinexin ß-expressing and control Cos-7 cells were biotinylated before or after stimulation with EGF. Biotinylated proteins were then precipitated with avidin-agarose beads as a cell surface fraction, and the supernatants were immunoprecipitated with anti-EGFR antibody as a cytosolic fraction. The amount of EGFR 60 min after the stimulation with EGF was greatly reduced because of its rapid endocytosis (Fig. 4C) and degradation. Same reduction was confirmed by different two antibodies against EGFR (data not shown). Phosphorylated EGFR was only slightly detected in both the cell surface and cytosolic fractions of control cells (Fig. 4B). Interestingly, expression of vinexin ß increased the phosphorylated EGFR on the cell surface without altering the phosphorylation of EGFR in the cytosol after the stimulation with EGF (Fig. 4B). These results are consistent with the result of the immunofluorescence analysis (Fig. 4A). Expression of vinexin ß also slightly increased the phosphorylation of EGFR on the cell surface even before the stimulation with EGF. This may be a result of the promoted phosphorylation of basally activated EGFR under the low serum condition. In addition to the sustained phosphorylation of EGFR, the amount of EGFR on the plasma membrane was also increased by the expression of vinexin ß slightly but significantly (Fig. 4B). To confirm the sustained phosphorylation of EGFR on the plasma membrane and the delayed endocytosis of EGFR, we investigated the time course of the endocytosis and dephosphorylation of EGFR on the plasma membrane (Fig. 4C). As expected, the phosphorylation of EGFR on the plasma membrane in vinexin ß-expressing cells was sustained until at least 6 h after the stimulation with EGF, whereas the phosphorylation in control cells ceased 4 h after the stimulation. Furthermore, the expression of vinexin ß delayed the endocytosis of EGFR significantly. To examine whether recycling of EGFR is involved in this sustained phosphorylation and delayed endocytosis, we utilized monensin, an inhibitor for recycling endosomes. We found that monensin did not affect the phosphorylation or the amount of EGFR on the plasma membrane (data not shown), suggesting that recycling of EGFR makes little contribution in Cos-7 cells. Altogether, these results suggested that the expression of vinexin ß affects not only the phosphorylation, but also the endocytosis of EGFR.
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To confirm the importance of endogenous vinexin to the regulation of EGFR, we examined the effect of siRNA-mediated down-regulation of endogenous vinexin on the phosphorylation of EGFR. HeLa cells were transfected with vinexin-specific siRNA or control siRNA. The vinexin-specific siRNA reduced the amount of vinexin ß by 95%, whereas the control siRNA had no effect (Fig. 5, bottom). The knockdown of endogenous vinexin did not affect the phosphorylation or amount of EGFR on the plasma membrane before the stimulation with EGF. Treatment with EGF increased the phosphorylation of EGFR transiently, then this phosphorylation decreased to or rather below the basal level on the plasma membrane 30 min after the stimulation in control HeLa cells (Fig. 5). Time course of the down-regulation of the phosphorylation of EGFR in HeLa cells may be different from those in Cos-7 cells (see Fig. 4C). As expected, the phosphorylation of EGFR on the plasma membrane was further reduced slightly but significantly in vinexin-knockdown cells 30 min after the stimulation (Fig. 5), suggesting that dephosphorylation of EGFR was promoted in vinexin-knockdown cells. This relatively weak effect of loss of vinexin expression might be a result of redundant function of vinexin/CAP/ArgBP2 family proteins in the regulation of dephosphorylation of EGFR, because other members, including ArgBP2, is expressed in HeLa cells (Yuan et al. 2005). Together, these results support the hypothesis that vinexin ß plays a role in the regulation of the phosphorylation of EGFR on the plasma membrane.
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It is reported that activated EGFR is ubiquitinated by c-Cbl, which acts as an E3 ubiquitin ligase, upon EGF stimulation and internalized by clathrin-mediated endocytosis (Waterman et al. 2002; de Melker et al. 2004a,b). Most endocytosed EGFRs are thought to be degraded in lysosomes. Interestingly, vinexin was reported to interact with CBLC/CBL3, a c-Cbl related protein, in a yeast two-hybrid system (Courbard et al. 2002). Therefore, one can assume that the expression of vinexin ß affects the phosphorylation and endocytosis of EGFR through the control of c-Cbl. Thus, we first examined whether vinexin ß interacts with c-Cbl in Cos-7 cells. The control vector or FLAG-vinexin ß was co-transfected with HA-c-Cbl into Cos-7 cells. FLAG-vinexin ß was precipitated using anti-FLAG antibody, and co-precipitated c-Cbl was detected with anti-HA antibody. As expected, HA-c-Cbl was co-precipitated with FLAG-vinexin ß (Fig. 6). To determine which region of vinexin ß is required for the interaction with c-Cbl, a co-immunoprecipitation assay using a series of point mutants of vinexin ß was performed. We found that the mutation of two or three SH3 domains decreased the interaction of vinexin ß with c-Cbl and that a mutation in the third SH3 domain had the greatest effect (Fig. 6), suggesting that the third SH3 domain, which was required for the induction of the sustained phosphorylation of EGFR, mainly interacts with c-Cbl, although all SH3 domains contribute to the interaction.
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We next examined the subcellular distribution of c-Cbl using biochemical fractionation. The control vector or the wild-type or the 3rdWF mutant of vinexin ß was transfected into Cos-7 cells. After the cells were stimulated with or without EGF for 5 min, the cytosolic fraction and the membrane fraction were obtained. N-cadherin was detected only in membrane fraction and p130cas as well as vinculin mainly in cytosolic fraction (Fig. 7A), showing that membrane and cytosolic proteins were separated by this procedure. In this condition, the wild-type as well as 3rdWF mutant of vinexin ß was detected in both the cytosolic and the membrane fractions (Fig. 7A). The expression of the wild-type vinexin ß decreased the cytosolic pool of c-Cbl and increased the amount of the membrane-associated c-Cbl. We further noticed that the expression of vinexin ß caused a retardation of the mobility of c-Cbl on SDS-PAGE (Fig. 7A). This retardation is attributed to the increased phosphorylation of c-Cbl because it disappeared after CIAP treatment (data not shown). On the other hand, expression of the 3rdWF mutant of vinexin ß had less of an effect on the distribution and phosphorylation of c-Cbl. These observations suggest that vinexin ß regulates the subcellular distribution and the phosphorylation of c-Cbl in an interaction-dependent manner.
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Discussion |
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The phosphorylation of EGFR resulted in its degradation in lysosomes in a Cbl-ubiquitin-dependent manner. c-Cbl binds to phosphorylated tyrosine residues of EGFR through its PTB domain and mediates the ubiquitination of EGFR (Thien & Langdon 2001). We showed here that expression of vinexin ß decreased the amount of c-Cbl in the cytosolic pool and increased the amount in the membrane fraction. The membrane fraction includes the plasma membrane as well as the intracellular membranes such as cytosolic vesicles. Although further analysis is necessary to determine which fraction is important, the decrease of c-Cbl in the cytosolic fraction may play a role in sustaining the phosphorylation of EGFR induced by vinexin ß, since over-expression of c-Cbl suppressed the sustained phosphorylation and the delayed endocytosis of EGFR induced by vinexin ß (Fig. 7C,D). Furthermore, we demonstrated that vinexin ß interacted with c-Cbl mainly through the third SH3 domain (Fig. 6). A point mutation in the third SH3 domain of vinexin ß dramatically reduced the ability to alter the subcellular distribution of c-Cbl and to induce the sustained phosphorylation of EGFR as well as to associate with c-Cbl. Thus, these observations suggest that the expression of vinexin ß induces the sustained phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl, possibly by altering the subcellular distribution of c-Cbl. In addition to the third SH3 domain, we demonstrated that the first SH3 domain of vinexin ß also contributes to the sustained phosphorylation of EGFR induced by vinexin ß (Fig. 3C). Although the first SH3 domain binds to c-Cbl slightly, the second SH3 domain, which is dispensable for the sustained phosphorylation, also interacts with c-Cbl with comparable affinity (Fig. 6). Therefore, other proteins that associate with the first SH3 domain of vinexin ß may contribute to the sustained phosphorylation of EGFR.
How did vinexin ß sustain the phosphorylation of EGFR on the plasma membrane? It is reported that the phosphorylation of EGFR is amplified on the plasma membrane, a process termed "lateral propagation" (Verveer et al. 2000; Ichinose et al. 2004). One possibility is that expression of vinexin ß promotes this lateral propagation. Sawano et al. (2002) reported that over-expression and a blockade of the endocytosis of EGFR triggered the lateral propagation of phosphorylated EGFR on the plasma membrane. We showed here that the expression of vinexin ß delayed the endocytosis of EGFR and increased the amount of phosphorylated EGFR on the plasma membrane (Fig. 4C). Furthermore, inhibiting the kinase activity of EGFR by adding AG1478 after the stimulation with EGF prevented the sustained phosphorylation of EGFR induced by vinexin ß (Fig. 2D), indicating that kinase activity is required for the sustained phosphorylation of EGFR induced by vinexin ß even after the stimulation with EGF. These results support the idea that expression of vinexin ß delays the endocytosis of activated (phosphorylated) EGFR and this delay increases the activated EGFR on the plasma membrane and enables it to phosphorylate adjacent unphosphorylated EGFRs, resulting in lateral propagation of phosphorylated EGFR on the plasma membrane. This mechanism could explain why expression of vinexin ß did not induce the sustained phosphorylation of EGFR in NIH3T3 cells, in which expression of EGFR is much lower than Cos-7 cells. Lateral propagation is also known to be induced by inhibiting PTP activities. The inhibition of PTP by reactive oxygen species (ROS) or the addition of orthovanadate, a major inhibitor of PTP, has been reported to stimulate the phosphorylation of EGFR and enable EGFR to phosphorylate neighboring unphosphorylated EGFRs, resulting in lateral propagation (Gamou & Shimizu 1995; Bae et al. 1997; De Wit et al. 2001; Reynolds et al. 2003). Thus, we can not exclude the possibility that the expression of vinexin ß attenuates certain PTP activity, leading to the lateral propagation of EGFR phosphorylation. These possibilities should be examined.
We have previously reported that the expression of vinexin ß stimulates EGF-induced activation of JNK and anchorage-independent activation of ERK2 in NIH3T3 cells. However, these events seem to be independent of the regulation of EGFR by vinexin ß, which we reported in this study. First, the expression of vinexin ß did not induce the sustained phosphorylation of EGFR in NIH3T3 cells (data not shown) as discussed above. Second, we have recently found that the binding of vinexin to ERK2 directly induces the anchorage-independent activation of ERK2 in NIH3T3 cells (manuscript in preparation). Finally, the sustained phosphorylation of EGFR induced by vinexin ß did not affect the downstream signals for the activation of ERK2 in Cos-7 cells. Thus, these observations indicate that vinexin ß can regulate EGF-induced signals via at least two mechanisms, by regulating the down-regulation of phosphorylation of EGFR and by controlling the downstream signals directly.
Various adaptor proteins have been reported to regulate EGFR function. Alix/AIP is reported to be involved in the regulation of the endocytosis of EGFR (Schmidt & Hoeller 2004). ASAP1 is reported to promote the recycling of EGFR through interaction with CIN85 another adaptor protein (Kowanetz et al. 2004). NHERF, which was initially identified as a cofactor for cAMP-dependent inhibition of NHE3, has been reported to stabilize EGFR on the plasma membrane (Lazar et al. 2004). In the present study, we showed that vinexin ß sustained the phosphorylation of EGFR on the plasma membrane. Thus, various adaptor proteins are involved in the regulation of EGFR. Future studies will reveal the physiological significances of these adaptor proteins in the regulation of EGFR.
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Experimental procedures |
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Monoclonal antibodies against EGFR, phosphotyrosine (pY20), p130cas, and N-cadherin were purchased from BD Transduction Laboratories. Polyclonal antibodies against EGFR, phospho-EGFR (pY845, pY992, pY1045, and pY1068) were obtained from Cell Signaling Technologies. Polyclonal antibodies against EGFR, c-Cbl, and HA epitope were purchased from Santa Cruz Biotechnologies. Both monoclonal (M2) and polyclonal antibodies against FLAG epitope were purchased from Sigma. Polyclonal anti-vinexin antibody was previously described (Mitsushima et al. 2006). EGF was purchased from Sigma. EZ-LinkTM Sulfo-NHS-Biotin and monomeric-avidin conjugated agarose beads were purchased from Pierce. AG1478 were purchased from Calbiochem.
Expression vectors
FLAG-tagged vinexin ß and deletion or point mutants thereof were previously described (Akamatsu et al. 1999; Kioka et al. 1999; Suwa et al. 2002). pAlterMAX-c-Cbl was a gift from Dr A. Y. Tsygankov.
Immunostaining
Transfected Cos-7 cells were serum-starved for 16 h, EGF was added to the medium, and cells were further incubated for the periods indicated. Cells were fixed with 3.7% formaldehyde, and then permeabilized with 0.4% Triton-X100, and immunostained as previously described (Mitsushima et al. 2004). The fluorescence images were taken with BX51 (OLYMPUS).
Biotinylation
Cos-7 cells were transfected with the control vector or FLAG-tagged vinexin ß and then serum-starved for 16 h. Cells were stimulated with EGF and washed with ice-cold PBS. Cells were incubated with 1 mg/mL of Sulfo-NHS-Biotin/PBS at 4 °C. Cells were then washed with ice-cold PBS three times and lyzed with RIPA buffer containing protease inhibitors and phosphatase inhibitors as previously described (Suwa et al. 2002; Mitsushima et al. 2004). Equal amounts of protein were incubated with monomeric-avidin-conjugated agarose beads for 1 h at 4 °C. Beads were washed with 1% NP-40 three times and subjected to SDS-PAGE.
Small interfering RNA (siRNA)
A pair of single strand RNA was synthesized by Japan Bio Services Co.,LTD. (Saitama, JAPAN). The target sequence was GACCCAGAAAUUCGGAACGUUTT for vinexin, and UGUCGCUAUA-AGACAAGCGCATT for the control. These oligonucleotides were annealed for 60 min at 37 °C. Annealed siRNA (25 nM) was transfected into HeLa cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Subcellular fractionation
Cytosolic and membrane fractions were separately obtained with a ProteoExtractTM Subcellular Proteome Extraction kit (Calbiochem) according to the manufacturer's instructions.
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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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References |
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Received: 20 April 2006
Accepted: 18 May 2006
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