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Originally published In Press as doi:10.1074/jbc.M402304200 on June 7, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34570-34577, August 13, 2004
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Extracellular Signal-regulated Kinase Activated by Epidermal Growth Factor and Cell Adhesion Interacts with and Phosphorylates Vinexin*

Masaru Mitsushima, Akira Suwa, Teruo Amachi, Kazumitsu Ueda, and Noriyuki Kioka{ddagger}

From the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received for publication, March 1, 2004 , and in revised form, May 10, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular signal-regulated kinase 1/2 (ERK1/2) is activated by various extracellular stimuli including growth factors and cytokines and plays a pivotal role in regulating cell proliferation and differentiation by phosphorylating nuclear transcription factors. Recently, it was reported that activated ERK1/2 also concentrates at adhesion sites and regulates cell spreading and migration. Vinexin is a focal adhesion protein regulating both cell spreading and growth factor signaling. We show here that vinexin was directly phosphorylated by ERK1/2 upon stimulation with growth factors. ERK1/2 phosphorylated the linker region of vinexin between the second and third SH3 domains. Site-directed mutagenesis revealed that ERK2 mainly phosphorylated the serine 189 residue of vinexin {beta}. Furthermore, vinexin {beta} interacted with ERK1/2 both in vitro and in vivo. Vinexin interacted with the active but not inactive form of ERK1/2. A putative DEF (docking for ERK FXFP) domain located in the linker region of vinexin was required for the interaction with ERK1/2 and efficient phosphorylation of vinexin {beta} by ERK2. Finally, we showed that cell adhesion to fibronectin also induced the association of vinexin {beta} with ERK2 and the phosphorylation of vinexin {beta}. Furthermore, vinexin and ERK were co-localized to the periphery of cells during cell spreading on fibronectin. Together, these results suggest that vinexin is a novel substrate of ERK2 and may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitogen-activated protein kinase (MAPK)1 consists of four subfamilies, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase/stress-activated kinase, p38MAPK, and ERK5. ERK1/2 is mainly activated by various growth factors and regulates a diverse array of cellular events including cell proliferation, survival, and differentiation (1, 2). Many extracellular signals activate membrane receptors, including receptor tyrosine kinases, G protein-coupled receptors, or integrins, leading to the activation of Raf. Activated Raf, in turn, activates MEK (MAPK/ERK kinase) by the direct phosphorylation of dual serine residues. Activated MEK also directly phosphorylates and activates ERK1/2. Once activated, ERK1/2 enters the nucleus and phosphorylates nuclear transcription factors, including Elk-1, Sap1, c-Myc, and c-Fos (3).

Recently, ERK has been shown to play crucial roles in regulating cell adhesion and cell migration independent of its nuclear functions (4–7). ERK is involved in the migration of breast cancer cells stimulated by urokinase-type tissue plasminogen activator. Neither de novo gene transcription nor protein synthesis is required for this process (5). Activation of ERK is also suggested to be involved in the chemotaxis or the extension of pseudopodia, probably through the phosphorylation of cytoplasmic proteins (8, 9). Furthermore, ERK is well known to be activated by integrin engagement and to be localized to adhesion sites (7, 10–14). Although some cytoplasmic proteins, including myosin light chain kinase (MLCK), integrin {beta} subunits, and paxillin, have been reported to mediate the ERK-dependent stimulation of cell motility or cell adhesion (5, 6, 15–17), details of the function of ERK or its substrates at adhesion sites remain to be determined.

Vinexin was identified as a binding partner of vinculin, one of the major focal adhesion proteins (18, 19). Vinexin is localized at focal adhesion sites as well as cell-cell contact sites. Vinexin is expressed as two alternative forms, {alpha} and {beta}. Both vinexin {alpha} and {beta} contain three SH3 domains, but vinexin {alpha} has an extended N-terminal region. We previously showed that expression of vinexin {alpha} affected the actin cytoskeletal organization in NIH3T3 cells, and that expression of vinexin {beta} promoted the spreading of C2C12 cells (18). Moreover, vinexin {beta} regulates the EGF-induced activation of JNK and the anchorage-dependent activation of ERK2 induced by EGF (20, 21). Furthermore, it was reported that vinexin interacted with scaffold attachment factor B2 or estrogen receptor, both of which are implicated in the response to steroid hormone (22, 23). These observations suggested that vinexin is involved not only in the regulation of cell adhesion/cytoskeletal organization, but also in the regulation of signal transduction.

In the present study, we show that vinexin was a downstream target for ERK2 upon stimulation with EGF as well as cell adhesion. Vinexin was phosphorylated by ERK2 both in vitro and in vivo. Moreover, we show that vinexin interacted with the active but not inactive form of ERK2. This interaction required FPFP sequence, a putative docking sequence for ERK2, of vinexin. Finally, we show that vinexin was co-localized with activated ERK to the periphery of NIH3T3 cells during cell spreading on fibronectin. These findings suggest that vinexin is a novel substrate of ERK and that vinexin may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—NIH3T3 cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum and COS7 cells, A431 cells, HT1080 cells, and HeLa cells were cultured with DMEM supplemented with 10% fetal bovine serum. NIH3T3 cells were transfected with each plasmids using LipofectAMINE Plus (Invitrogen) according to the manufacturer's instructions.

Materials—Two MEK inhibitors, U0126 and PD98059, and active ERK2 were purchased from Cell Signaling Technology. Inactive ERK2 was purchased from New England Biolabs Inc. Fibronectin purified from bovine serum, anisomycin, EGF, PDGF, LPA, TPA, and insulin were all purchased from Sigma.

Antibodies—Anti-FLAG epitope monoclonal antibody M2 was purchased from Sigma. Anti-phospho-ERK1/2, anti-phospho-JNK1/2, antiphospho-p38MAPK antibodies were all purchased from Cell Signaling Technology. Anti-ERK2 polyclonal (C-14), anti-ERK2 monoclonal (D-2), anti-JNK1 polyclonal (C-17), and anti-GFP polyclonal (FL) antibodies were purchased from Santa Cruz Biotechnology. Anti-pan ERK antibody was purchased from BD Transduction Laboratories.

Plasmids—FLAG-tagged vinexin {beta} and mutants were all introduced into the mammalian expression vector p401F (18). GST-tagged vinexin {beta} and mutants were all introduced into pGEX-4T1 (Amersham Biosciences). GFP-ERK2 was described previously (21). GFP-vinexin {beta} was constructed by introducing vinexin {beta} cDNA into pEGFPC2 (Clontech). Constitutive active and dominant negative MEK plasmids were generous gifts from Dr. N. Ahn.

Site-directed Mutagenesis—Mutants of vinexin {beta} were constructed using a QuikChange Site-directed Mutagenesis Kit (Stratagene), according to the manufacturer's instructions. Mutagenesis was confirmed by DNA sequencing.

Phosphatase Treatment—FLAG-LINKER was transfected into NIH3T3 cells, and cells were serum-starved for 16 h. Cells were then stimulated with or without EGF (100 ng/ml) for 5 min and lysed. Cell lysates were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were resuspended with reaction buffer containing 50 units of calf intestine alkaline phosphatase (CIAP) (TaKaRa, Japan) and incubated at 37 °C for 90 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.

Immunoprecipitation—Immunoprecipitation was performed as described previously (20). Briefly, cells were serum-starved for 16 h. They were then stimulated with serum (10%), EGF (100 ng/ml), PDGF (50 ng/ml), TPA (50 µg/ml), LPA (0.5 mg/ml), or insulin (100 µM) for 5 min and lysed with lysis buffer (1% Triton X-100, 0.02 mg/ml aprotinin, 0.1 mg/ml p-amidinophenyl methanesulfonyl fluoride hydrochloride, 5 µg/ml leupeptin, 5 mM benzamidine, 1 µg/ml pepstatin A, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 40 mM {beta}-glycerophosphate, and 20 nM calyculin A in PBS). Equal amounts of cell lysate were subjected to immunoprecipitation with anti-FLAG antibody at 4 °C. The immunoprecipitates were washed four times using ice-cold 1% Triton X-100/PBS. Co-precipitated proteins were separated by SDS-PAGE and subjected to immunoblotting with specific antibodies.

In Vitro Binding Assay—GST or GST-fused proteins (5 µg) were incubated with active or inactive ERK2 (25 pmol) in 1% Triton X-100/PBS at 4 °C for 60 min. Glutathione-Sepharose 4B beads (Amersham Biosciences) were added, and the mixture was incubated for 45 min. The glutathione beads were then precipitated by centrifugation at 500 rpm and washed with ice-cold 1% Triton X-100/PBS four times. For the pull-down assay, cell lysates stimulated with or without EGF (100 ng/ml) for 5 min were incubated with GST-fused proteins and glutathione-Sepharose 4B beads at 4 °C for 60 min. The glutathione beads were precipitated by centrifugation at 500 rpm and washed with ice-cold 1% Triton X-100/PBS four times. Co-precipitated proteins were separated by SDS-PAGE and subjected to immunoblotting with specific antibodies.

In Vitro Kinase Assay—NIH3T3 cells were transfected with GFP-ERK2 or GFP alone and then serum-starved for 16 h. After treatment with or without EGF (100 ng/ml) for 5 min, the cells were lysed with modified radioimmune precipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 0.02 mg/ml aprotinin, 0.1 mg/ml p-amidinophenyl methanesulfonyl fluoride hydrochloride, 5 µg/ml leupeptin, 5 mM benzamidine, 1 µg/ml pepstatin A, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 40 mM {beta}-glycerophosphate, and 20 nM calyculin A, 50 mM Tris, pH 7.5). Cell lysates were immunoprecipitated with anti-GFP antibody. Immunocomplexes were then resuspended in 100 µl of kinase reaction buffer with GST-fused proteins (5 µg) and 1 µCi of [{gamma}-32P]ATP (6000 Ci/mmol; Amersham Biosciences). After incubation at 30 °C for 20 min, the reaction was stopped by addition of Laemmli sample buffer. Phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography. In some experiments, active ERK2 (New England BioLabs) was used instead of immunoprecipitated GFP-ERK2 as a kinase.

In Vivo Cell Labeling—NIH3T3 cells were serum-starved by incubation with DMEM supplemented with 0.5% calf serum for 16 h. These cells were further incubated with phosphate-free DMEM (Invitrogen) containing 0.5% dialyzed calf serum for 3 h. [32P]orthophosphate (200 µCi/ml; Amersham Biosciences) was added to the medium, and the cells were incubated for an hour. The cells were stimulated with or without EGF (100 ng/ml) for 5 min and lysed with modified radioimmune precipitation assay buffer as described above. Cell lysates were immunoprecipitated with anti-vinexin antibody or preimmune serum. Phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography.

Immunostaining—NIH3T3 cells were serum-starved for 16 h and trypsinized. Cells were then maintained in suspension for 30 min, replated on fibronectin-coated coverslips (BD Bioscience), and incubated for the indicated times. Cells were fixed in 10% formalin for 15 min at room temperature and then permeabilized by 0.4% Triton X-100. The cells were blocked with 10% goat serum/TBS-T (TBS with 0.1% Triton X-100) for 1 h, then incubated with anti-vinexin antibody and anti-panERK antibody, or anti-phospho-ERK1/2 antibody at 4 °C overnight. They were then stained with Alexa 568-labeled goat anti-rabbit IgG (Molecular Probes) and Alexa 488-labeled goat anti-mouse IgG (Molecular Probes), for 1 h. Fluorescence images were taken with LSM 5 PASCAL confocal microscopy system (Carl Zeiss Co., Ltd).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Vinexin Is Phosphorylated Downstream of the MEK-ERK Cascade upon EGF Stimulation—We previously showed that vinexin {beta} regulates the anchorage-dependence of ERK2 activation induced by EGF (21). It is reported that activated ERK phosphorylates some upstream components (24–26). Therefore, we examined whether vinexin is a substrate localized at adhesion site for ERK. First we determined mobility shifts of vinexin on SDS-PAGE upon stimulation with EGF and found that this treatment retarded the mobility of vinexin {beta} in various cell lines, including NIH3T3, A431, HT1080, and HeLa cells (Fig. 1A). Interestingly, these mobility shifts were prevented by pretreatment of U0126, a MEK1/2-specific inhibitor, suggesting that vinexin was modified downstream of the MEK-ERK cascade. To confirm that vinexin was phosphorylated downstream of the MEK-ERK cascade, NIH3T3 cells were labeled with [32P]orthophosphate and stimulated with EGF for 5 min. Vinexin was immunoprecipitated with anti-vinexin antibody then subjected to SDS-PAGE. Both vinexin {alpha} and {beta} were weakly phosphorylated under quiescent conditions (Fig. 1B). Stimulation with EGF increased the phosphorylation of both vinexin {alpha} and {beta}. This increase was also prevented by pretreatment with U0126 similar to the mobility shifts, suggesting that both vinexin {alpha} and {beta} were phosphorylated downstream of the MEK-ERK cascade.



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  		FIG. 1.
Vinexin is phosphorylated by ERK2. A, NIH3T3, A431, HT1080, and HeLa cells were serum-starved for 16 h. Cells were treated with or without U0126 for 3 h and left unstimulated or stimulated with EGF (100 ng/ml) for 5 min and then lysed. Cell lysates were immunoblotted with anti-vinexin (top panel) and with anti-phospho-ERK1/2 (bottom panel) antibodies. B, NIH3T3 cells were serum-starved for 16 h, then incubated in phosphate-free Dulbecco's modified Eagle's medium for 1 h. Cells were next incubated in medium containing [32P]orthophosphate for another 3 h with or without U0126 (10 µM). After treatment with EGF (100 ng/ml) for 5 min, the cells were lysed and immunoprecipitated with anti-vinexin antibody or preimmune serum, and subjected to SDS-PAGE. Phosphorylated proteins were visualized by autoradiography. C, GFP-ERK2 or GFP alone was transfected into NIH3T3 cells. The cells were then serum-starved for 16 h. After the treatment with EGF for 5 min, cell lysates were immunoprecipitated with anti-GFP antibody. Precipitated immunocomplexes were then subjected to an ERK2 kinase assay using GST or GST-vinexin {beta} as a substrate. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography. D, in vitro kinase assay of purified active ERK2 using GST or GST-vinexin {beta} as a substrate. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography.

 
To determine which kinase phosphorylated vinexin, we performed an immunocomplex kinase assay using GST-vinexin {beta} as a substrate. Immunoprecipitated ERK2 from EGF-stimulated NIH3T3 cells efficiently phosphorylated GST-vinexin {beta} (Fig. 1C), whereas immunoprecipitated MEK1 did not (data not shown). GST alone was not phosphorylated by either MEK1 or ERK2. ERK2 was reported to make a complex with other kinases under certain conditions. To exclude the possibility that other kinases, which might be co-precipitated with ERK2, phosphorylated vinexin {beta}, we examined whether the active ERK2 purified from Escherichia coli could phosphorylate vinexin {beta} in vitro. Similar to the result of the immunocomplex kinase assay, vinexin was directly phosphorylated by the purified ERK2 (Fig. 1D). These findings suggested that vinexin was directly phosphorylated by ERK2.

Linker Region of Vinexin Is Phosphorylated by ERK2—To identify the region of vinexin {beta} phosphorylated by ERK2, we carried out in vitro kinase assays using various deletion mutants of vinexin {beta} (Fig. 2A). Wild-type vinexin {beta} (amino acids 1–328) and C-terminal half (amino acids 173–328) and LINKER (amino acids 173–271) mutants were efficiently phosphorylated by purified ERK2 (Fig. 2B). In contrast, the N-terminal half (amino acids 1–172) and {Delta}-linker (amino acids 1–172, 252–328) mutants, both of which lack the linker region between the second and third SH3 domains of vinexin {beta}, were scarcely phosphorylated. These results suggested that the linker region of vinexin {beta} contained the major phosphorylation sites for ERK2.



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  		FIG. 2.
The linker region of vinexin is phosphorylated by ERK2. A, a schematic diagram of deletion mutants of vinexin {beta}. B, deletion mutants, as described in A, purified from E. coli were utilized as substrates for the in vitro ERK2 kinase assay. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography (top panel). The purified deletion mutants were stained with Coomassie Brilliant Blue (CBB) to verify that equal amounts of protein were present (bottom panel). C, putative residues phosphorylated by ERK2 in the linker region of vinexin {beta}. D, in vitro ERK2 kinase assay using mutants of vinexin {beta} purified from E. coli as substrates. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography (top panel). The purified proteins were stained with CBB to verify that equal amounts of protein were present (bottom panel). E, FLAG-tagged wild type or S189A mutant of vinexin {beta} were transfected into NIH3T3 cells with constitutively active or dominant negative MEK1. Cells were then labeled by [32P] orthophosphate, and cell lysates were immunoprecipitated with anti-FLAG antibody. Phosphorylated proteins were visualized by autoradiography.

 
ERK2 phosphorylates serine/threonine residue adjacent to a proline residue, and hence is called a "proline-directed kinase." There are three serines and one threonine residue adjacent to prolines in the linker region (Fig. 2C). To examine whether these residues were phosphorylated by ERK2, we substituted each serine/threonine residue with alanine (S189A, S219A, T243A, or S264A). In vitro kinase assays revealed that S219A, T243A, and S264A mutants were phosphorylated as strongly as the wild type of vinexin {beta} by purified ERK2 (Fig. 2D). In contrast, the S189A mutant was markedly less phosphorylated by ERK2. These results indicated that ERK2 mainly phosphorylates serine 189 of vinexin {beta} in vitro.

To confirm that the serine 189 residue of vinexin {beta} is phosphorylated by ERK2 in vivo, we co-transfected FLAG-tagged wild-type vinexin {beta} or S189A mutant with constitutively active MEK1 into NIH3T3 cells to activate ERK specifically, and examined the phosphorylation of these proteins. As expected, the S189A mutant was significantly less phosphorylated than the wild-type vinexin {beta} (Fig. 2E). The remaining phosphorylation of the S189A mutant might be the results of phosphorylation of other residues by ERK or by other kinases downstream of MEK-ERK cascade, such as mitogen-activated protein kinase-activated protein kinase. Both wild-type and S189A mutant of vinexin {beta} were barely phosphorylated in cells transfected with dominant negative MEK1. These results suggested that the serine 189 residue in the linker region of vinexin {beta} was at least one of major phosphorylation sites for MEK-ERK cascade in vivo.

Phosphorylation of Serine 189 Residue of Vinexin Causes a Mobility Shift on SDS-PAGE—We found that deletion mutants of vinexin containing the linker region, including C-half, {Delta}3SH3, and LINKER, showed mobility shifts on SDS-PAGE upon stimulation with EGF (Fig. 3A, and data not shown). Interestingly, this shift was prevented by pretreatment with U0126 similar to endogenous vinexin {beta}. To confirm that phosphorylation by ERK induced the mobility shifts, we first examined whether phosphorylation of vinexin leads to a retardation of mobility on SDS-PAGE. FLAG-tagged LINKER was immunoprecipitated from NIH3T3 cells stimulated with or without EGF, and then immunoprecipitates were subjected to a phosphatase treatment. FLAG-LINKER showed a shift in mobility upon stimulation with EGF and this shift disappeared on treatment with CIAP but not buffer (Fig. 3B). We next examined whether phosphorylation of serine 189 was involved in the mobility shift on SDS-PAGE. The wild type, S219A, T247A, and S268A mutants of LINKER showed a change in mobility upon EGF stimulation, but the S189A mutant did not (Fig. 3C). Furthermore, in vitro kinase assay revealed that the wild type as well as the S219A, T247A, and S268A mutants of GST-LINKER were well phosphorylated by ERK2 and showed a mobility shift, but the S189A mutant was less phosphorylated and did not show a shift (Fig. 3D). Together, these findings suggested that phosphorylation of the serine 189 residue of vinexin {beta} by ERK results in a retardation of mobility on SDS-PAGE probably because of the conformational change of vinexin.



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  		FIG. 3.
Phosphorylated vinexin shows a mobility shift on SDS-PAGE. A, FLAG-LINKER was transfected into NIH3T3 cells. The cells were serum-starved for 16 h, then treated with U0126 (10 µM) or Me2SO (DMSO) for 3 h. After the treatment with EGF (100 ng/ml) for 5 min, cells were lysed. Cell lysates were immunoblotted with anti-FLAG (top panel) and with anti-phospho-ERK1/2 (bottom panel) antibodies. B, lysates obtained in A were immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were subjected to a phosphatase treatment. C, FLAG-LINKER mutants were transfected into NIH3T3 cells. Cells were serum-starved for 16 h, then left untreated or treated with EGF (100 ng/ml) for 5 min. Cell lysates were immunoblotted with anti-FLAG (top panel) and with anti-phospho-ERK1/2 (bottom panel) antibodies. D, GST-LINKER mutants were purified from E. coli. In vitro kinase assay of purified active ERK2 using these mutants as substrates was performed. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography.

 
Vinexin Interacts with ERK2—ERK2 forms a stable interaction, a "docking interaction," with its substrates to phosphorylate them efficiently (27–34). We, therefore, examined whether vinexin interacted stably with ERK2. NIH3T3 cells were transfected with FLAG-tagged vinexin {beta} and then stimulated with EGF for 5 min. Cell lysates were immunoprecipitated using anti-FLAG antibody, and co-immunoprecipitated ERK2 was detected by Western blotting using anti-ERK2 antibody. Fig. 4A shows that endogenous ERK2 was co-precipitated with FLAG-tagged wild type or S189A mutant of vinexin {beta} when cells were stimulated with EGF. Interestingly, these interactions were not observed in serum-starved NIH3T3 cells. Furthermore, the interactions were completely inhibited by pretreatment with U0126 (data not shown), suggesting that the interaction was dependent on EGF-induced activation of MEKERK cascade. To confirm the interaction of endogenous vinexin with ERK2, lysates from COS7 cells stimulated with or without EGF were immunoprecipitated using anti-vinexin antibody or preimmune serum. Endogenous ERK2 was also co-immunoprecipitated with endogenous vinexin upon EGF stimulation (Fig. 4B). Together, these observations suggested that vinexin {beta} interacted with ERK2 in an EGF-dependent manner.



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  		FIG. 4.
Vinexin interacts specifically with ERK2. A, FLAG-tagged wild type or S189A vinexin {beta} or empty vector was transfected into NIH3T3 cells, and cells were serum-starved for 16 h. After treatment with EGF (100 ng/ml) for 5 min, cells were lysed, and the lysates were immunoprecipitated with anti-FLAG antibody. Immunocomplexes were subjected to immunoblotting with anti-ERK2 antibody (top panel). Cell lysates were immunoblotted with anti-phospho-ERK1/2 or anti-FLAG antibody to verify the activation of ERK1/2 (middle panel) or equal expression of FLAG-vinexin (bottom panel), respectively. B, COS7 cells were serum-starved, left untreated, or stimulated with EGF (100 ng/ml) for 5 min, and then lysed. Cell lysates were immunoprecipitated with anti-vinexin antibody or preimmune serum. Immunocomplexes were subjected to immunoblotting with anti-ERK2 monoclonal antibody. C, FLAG-vinexin {beta} or empty vector was transfected into NIH3T3 cells, and cells were serum-starved for 16 h. Cells were then left untreated or treated with EGF (100 ng/ml) or anisomycin (aniso) (500 ng/ml) for 5 min or 30 min, respectively. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-ERK2 antibody (c-1, top panel), anti-JNK1 antibody (c-2, top panel), or anti-p38MAPK antibody (c-3, top panel). Lysates were also immunoblotted with anti-phospho-ERK1/2 antibody (c-1, bottom panel), anti-phospho-JNK1/2 antibody (c-2, bottom panel), and anti-phospho-p38MAPK antibody (c-3, bottom panel) to confirm the equal loading and activation of each kinase.

 
We, next, examined whether other MAP kinases (JNK and p38MAPK) interact with vinexin {beta}. NIH3T3 cells were transfected with FLAG-tagged vinexin {beta} and then stimulated with anisomycin, which stimulates both JNK and p38MAPK, for 30 min. Although the stimulation with anisomycin induced the activation of both JNK and p38MAPK, no interaction of vinexin {beta} with either JNK or p38MAPK was observed (Fig. 4C). These results indicated that vinexin {beta} specifically interacted with ERK2 among MAPK family members.

Vinexin {beta} Interacts Specifically with Phosphorylated ERK2— The finding that vinexin {beta} bound to ERK2 only in cells stimulated with EGF raised the possibility that vinexin {beta} interacts only with the active (phosphorylated) form of ERK2. To examine this possibility, we determined the time course of vinexin-ERK2 interaction after the stimulation with EGF. Endogenous ERK2 was phosphorylated within 1 min of the stimulation (Fig. 5A, bottom panel). The phosphorylation of ERK2 was maintained until 5 min, and then the gradually decreased and disappeared within 30 min after EGF stimulation. Interestingly, consistent with the time dependence of ERK2 activation and inactivation, interaction of vinexin {beta} with ERK2 was observed within 1 min of the EGF stimulation and diminished gradually to the basal level within 30 min (Fig. 5A, top panel). This result suggested that vinexin {beta} bound specifically to the active form of ERK2.



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  		FIG. 5.
Vinexin interacts with the active but not inactive form of ERK2. A, FLAG-vinexin {beta} was transfected into NIH3T3 cells, and the cells were serum-starved for 16 h. Cells were then stimulated with EGF (100 ng/ml) for the period indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunocomplexes were subjected to immunoblotting with anti-ERK2 antibody (top panel). To observe the time dependence of ERK activation, cell lysates were immunoblotted with anti-phospho-ERK1/2 antibody (bottom panel). B, NIH3T3 cells transfected with FLAG-vinexin {beta} were serum-starved for 16 h. Cells were left untreated or stimulated with 10% calf serum, EGF (100 ng/ml), PDGF (50 ng/ml), TPA (50 µg/ml), LPA (0.5 mg/ml), or insulin (100 µM) for 5 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunocomplexes were subjected to immunoblotting with anti-ERK2 antibody (top panel). To confirm the activation of ERK1/2, cell lysates were immunoblotted with anti-phospho-ERK1/2 antibody (bottom panel). C, purified active or inactive ERK2 was incubated with GST or GST-vinexin {beta} at 4 °C. GST or GST-vinexin {beta} was precipitated using glutathione-Sepharose 4B beads, and co-precipitated ERK2 was detected with anti-ERK2 antibody.

 
We next examined whether the vinexin {beta}-ERK2 interaction was stimulated by other factors. NIH3T3 cells transfected with FLAG-tagged vinexin {beta} were treated with serum, EGF, PDGF, TPA, LPA, or insulin. Stimulations with serum, EGF, PDGF, and TPA activated ERK2 strongly, while the treatment with LPA and insulin activated ERK2 only moderately (Fig. 5B, bottom panel). Interestingly, vinexin {beta} interacted strongly with ERK2 after the stimulations with serum, EGF, PDGF, or TPA, whereas vinexin {beta} interacted only weakly with ERK2 after the treatment with LPA or insulin (Fig. 5B, upper panel). Furthermore, Western blotting using the phosphospecific ERK1/2 antibody revealed that phosphorylated ERK2 was co-precipitated with vinexin {beta} in the immunoprecipitation assay, and that phosphorylated ERK1 was also co-precipitated with vinexin {beta} (data not shown). These results supported the hypothesis that the active form of ERK2 interacted with vinexin {beta}.

To confirm this finding, an in vitro binding assay using purified GST-vinexin {beta} and recombinant phosphorylated or non-phosphorylated ERK2 was performed. Phosphorylated ERK2 was co-precipitated with vinexin {beta}, whereas non-phosphorylated ERK2 was not (Fig. 5C). Together, these results strongly suggested that vinexin {beta} interacted directly with the active form of ERK2.

ERK2 Interacts with the Linker Region of Vinexin {beta}—Vinexin {beta} contains three SH3 domains, of which the first and second bind to vinculin and the third binds to Sos and lp-dlg (20, 35). No other functional domains have been identified. Therefore, we first examined whether these SH3 domains were involved in the interaction with ERK2. Immunoprecipitation assays using mutants of vinexin {beta}, which have one amino acid substitution (tryptophan to phenylalanine) in each SH3 domain (21), showed that all mutants interacted with ERK2 as the wild-type vinexin {beta} did (data not shown), suggesting that none of the SH3 domains were required for the interaction with ERK2. We thus performed pull-down assays using various deletion mutants of vinexin {beta} to determine the region required for the interaction with ERK2 (Fig. 2A). ERK2 was co-precipitated with full-length vinexin {beta} efficiently and with the C-half and the LINKER slightly but significantly, whereas ERK2 was not co-precipitated with the N-half or the {Delta}linker (Fig. 6A). These results indicated that the linker region between the second and third SH3 domains of vinexin {beta} was indispensable for the interaction with ERK2. To confirm the interaction of the linker region with the active form of ERK2 in vivo, co-immunoprecipitation experiments were performed. ERK2 was efficiently coimmunoprecipitated with the LINKER but not the {Delta}linker mutant (Fig. 6B). These results indicated that the linker region of vinexin {beta} was required for the interaction with ERK2.



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  		FIG. 6.
The linker region of vinexin interacts with ERK2. A, COS7 cells were serum-starved for 16 h, and left untreated or stimulated with EGF (100 ng/ml) for 5 min. Cell lysates were incubated with GST or GST fusion proteins of vinexin {beta}. Co-precipitated ERK2 was detected with anti-ERK2 antibody. B, FLAG-tagged deletion mutants of vinexin {beta} were transfected into NIH3T3 cells, and the cells were serum-starved for 16 h. Cells were then stimulated with EGF (100 ng/ml) for 5 min and the lysates were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were subjected to immunoblotting with anti-ERK2 antibody (top panel) or with anti-FLAG antibody (bottom panel).

 
The FPFP Sequence Is Required for the Interaction with ERK2—ERK2 is known to interact with substrates, activators, and some scaffold proteins through at least two consensus sequences, the D domain and the DEF domain (31, 33, 34). The former consists of a cluster of basic amino acid residues followed by a cluster of hydrophobic residues, whereas the latter consists of the amino acid sequence FXFP. The linker region of vinexin contains these two consensus sequences: RRXXXXXX-ITL for the D domain and FPFP for the DEF domain (Fig. 7A). To examine whether these sequences were required for the vinexin-ERK2 interaction, we generated a RRAA mutant in which the arginine residues at positions 221 and 222 were substituted with alanines, and a FFAA mutant in which the phenylalanine residues at positions 225 and 227 were substituted with alanines (Fig. 7A). These mutants were assayed for the interaction with ERK2 in co-immunoprecipitation experiments. The RRAA mutant interacted with ERK2 as strongly as the wild-type vinexin {beta} (Fig. 7B). However, the FFAA mutant failed to interact with ERK2 (Fig. 7B), suggesting that the FPFP sequence of vinexin, but not the D domain-like sequence, was required for interacting with ERK2. To confirm the importance of this sequence, we examined if the FFAA mutant was phosphorylated by ERK2 in vitro (Fig. 7C). The FFAA mutant that failed to interact with ERK2 was significantly less phosphorylated than the wild-type vinexin {beta}, indicating that the FPFP sequence is important not only for the interaction with ERK2 but also for efficient phosphorylation by ERK2.



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  		FIG. 7.
FPFP sequence in the linker region of vinexin is required for the interaction with ERK2. A, sequences of wild-type vinexin {beta} (top), FFAA mutant (middle), and RRAA mutant (bottom), between serine 219 and arginine 235 of the linker region. B, FLAG-tagged wild-type vinexin {beta}, FFAA mutant, RRAA mutant, or empty vector was transfected into NIH3T3 cells and the cells were serum-starved for 16 h. Cells were then stimulated with EGF (100 ng/ml) for 5 min, and the lysates were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were subjected to immunoblotting with anti-ERK2 antibody (top panel), anti-phospho-ERK1/2 antibody (middle panel), and anti-FLAG antibody (bottom panel). C, in vitro ERK2 kinase assay using GST-vinexin {beta} or FFAA mutant as a substrate. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography (left panel). Both proteins were stained with CBB to verify that equal amounts of protein were present (right panel).

 
Vinexin Is Co-localized with ERK at the Periphery of NIH3T3 Cells Adhering to Fibronectin—It is reported that ERK is activated by cell adhesion on the extracellular matrix and localized at the periphery of the cells at an early phase of cell spreading (7, 36). We first examined whether the vinexin-ERK2 complex is formed at an early phase of cell spreading. NIH3T3 cells were transfected with FLAG-tagged vinexin {beta}, and cell lysate was prepared from suspended cells or cells adhered to fibronectin after 10–30 min. Immunoprecipitation using anti-FLAG antibody revealed that vinexin interacted with ERK2 in the cells adherent to fibronectin, when ERK2 was activated, but not in suspension, when ERK2 was inactivated (Fig. 8A). Because vinexin interacted with activated ERK upon cell adhesion, we next examined where vinexin and activated ERK localizes during cell spreading on fibronectin. Serum-starved NIH3T3 cells transfected with GFP-vinexin {beta} were maintained in suspension and seeded on fibronectin-coated coverslips. Activated ERK was localized at the periphery of the cells 15–45 min after the adhesion to fibronectin in addition to central region of the cells (Fig. 8B, middle). Interestingly, vinexin was co-localized at the periphery of the cells with ERK during this period (Fig. 8B, top). Furthermore, endogenous vinexin was also localized at the periphery of the cells as well as at central region of the cells (Fig. 8C). Together, these observations suggested that vinexin was co-localized with ERK at the periphery of cells during cell spreading.



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  		FIG. 8.
Vinexin is co-localized with ERK at periphery of NIH3T3 cells at an early phase of cell spreading. A, NIH3T3 cells transfected with FLAG-vinexin {beta} were serum-starved for 16 h. Cells were incubated in suspension for 15 min and re-plated on fibronectin-coated dishes for the periods indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody and the immunocomplexes were subjected to immunoblotting with anti-ERK2 antibody (top panel). Cell lysates were also immunoblotted with anti-phospho-ERK1/2 antibody to confirm ERK activation (bottom panel). B, NIH3T3 cells transfected with GFP-vinexin {beta} were serum-starved for 16 h and incubated in suspension for 30 min. Cells were re-plated on fibronectin-coated coverslip and incubated for the periods indicated. Cells were then fixed and immunostained using anti-phospho-ERK1/2 antibody as described under "Experimental Procedures." Individual images of GFP-vinexin {beta} or phosphorylated ERK1/2 and merged images (GFP-vinexin, green; phosphorylated ERK1/2, red) are shown. The arrowheads indicate the co-localization of vinexin and phosphorylated ERK1/2 at the periphery of NIH3T3 cells. Bar, 10 µm. Higher magnification views are shown in the inset. Bar, 5 µm. C, NIH3T3 cells were plated on fibronectin-coated coverslip as described in B. Cells were then immunostained using anti-panERK antibody and anti-vinexin antibody. The arrowheads indicate the coimmunostaining of ERK and vinexin at the periphery of NIH3T3 cells. D, A431 cells were serum-starved for 16 h. Cells were incubated in suspension for 15 min and re-plated on fibronectin-coated dishes for the periods indicated and then lysed. Cell lysates were immunoblotted with anti-vinexin (top panel) and with anti-phospho-ERK1/2 (bottom panel) antibodies.

 
Finally, we examined whether vinexin was phosphorylated by ERK2 during cell spreading on fibronectin. Because phosphorylated vinexin showed a clear mobility shift in A431 cells (Fig. 1A), we determined the mobility shift of vinexin {beta} in A431 cells during spreading on fibronectin, instead of examining the phosphorylation levels of vinexin directly. Serum-starved A431 cells were maintained in suspension and then seeded on fibronectin-coated dishes. ERK1/2 were transiently activated in A431 cells 15–30 min after the adhesion to fibronectin (Fig. 8D). As expected, vinexin {beta} showed a clear mobility shift in A431 (Fig. 8D) with a similar time course to the activation of ERK1/2. These results further supported the idea that ERK activated by cell adhesion to fibronectin interacts with and phosphorylates vinexin.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we showed that vinexin was phosphorylated downstream of the MEK-ERK cascade upon stimulation with EGF, and that vinexin {beta} was phosphorylated directly by ERK2 in vitro. Site-directed mutagenesis revealed that the serine 189 residue of vinexin {beta} was the phosphorylation site for ERK2. We also demonstrated that the mobility of LINKER as well as endogenous vinexin on SDS-PAGE was decreased by the ERK-dependent phosphorylation. Furthermore, cell adhesion to fibronectin caused this mobility shift of vinexin {beta} and induced co-localization of vinexin and ERK at periphery. Together, these findings suggest that vinexin is a novel ERK2 substrate localized at adhesion sites and that the serine 189 residue of vinexin {beta} is the major phosphorylation site for ERK2.

We presented evidence that vinexin interacted with the active form of ERK both in vitro and in vivo. ERK2 is known to interact with substrates, activators, and scaffold proteins through the D domain and the DEF domain (28, 29, 32, 37, 38). Vinexin contains both a putative D domain and DEF domain (FPFP sequence), like other physiological substrates, including Elk-1 and Tob (28, 39). We demonstrated here that the RRAA mutant, which has mutations in the putative D domain, still interacted with ERK2 (Fig. 6B), whereas the FFAA mutant, which has mutations in the FPFP sequence, did not. Furthermore, vinexin interacted with the ERK2 D321,324 N mutant (kindly provided by Dr. E. Nishida), which cannot interact with D domain (40), as strongly as with wild-type ERK2.2 These results suggested that the FPFP sequence of vinexin was indispensable for the interaction with ERK2.

The D domain has been reported to interact with the CD (common docking) domain of ERK2 (37, 40). In contrast, the binding site for the DEF domain in ERK2 is still unclear. In this study, we showed that the FPFP sequence of vinexin mediated the interaction with the active form of ERK2, raising two possibilities. One possibility is that the FPFP sequence of vinexin recognizes a dual-phosphorylated TEY sequence in the activation loop of ERK2. The unpublished observation that phenylphosphate, which mimics phosphorylated tyrosine residues (41), prevented the vinexin {beta}-ERK2 interaction in a dose-dependent manner may support this possibility. The other possibility is that the FPFP sequence of vinexin interacts with the surfaces that newly emerge with the conformational change induced by the phosphorylation of the TEY sequence. Future studies will resolve this issue.

Vinexin belongs to a novel protein family, which consists of vinexin, c-Cbl-associated protein (CAP)/ponsin, and Arg-binding protein 2 (ArgBP2) (19, 42–44). These proteins share one sorbin homology domain in the N-terminal region and three SH3 domains in the C-terminal region. Interestingly, the DEF domain is conserved among orthologs of human, mouse, and rat vinexin. In contrast, neither CAP/ponsin nor ArgBP2 contain this domain in the linker region between the second and third SH3 domains. Furthermore, the Pro-Ser-Ser189-Pro sequence, whose serine residue is the phosphorylation site for ERK2, of vinexin {beta} was also conserved among orthologs, whereas there is no PX(S/T)P sequence, a consensus sequence phosphorylated by ERK2, in the linker region of CAP/ponsin or ArgBP2. These observations suggest that ERK may exclusively interact with vinexin and phosphorylate the linker region of vinexin. Thus, vinexin might have unique functions or unique regulatory mechanisms among family members.

We demonstrated that vinexin interacted with ERK2 activated by cell adhesion and that vinexin was co-localized with ERK at the periphery of NIH3T3 cells during cell spreading on fibronectin. Recently, paxillin was also reported to be co-localized with ERK and regulate the spreading of mIMCD-3 epithelial cells induced by hepatocyte growth factor (16, 45). Furthermore, both vinexin and paxillin interact with ERK through the DEF domain (FPFP) or the DEF domain-like sequence (YSFP) (16), respectively. However, the interaction of paxillin with ERK is a quite different from that of vinexin {beta} with ERK. Paxillin preferentially interacts with the inactive form of ERK in vitro (16). Paxillin, furthermore, interacts with both Raf and MEK. Because of these observations, paxillin is proposed to serve as a scaffold protein to recruit the inactive form of ERK to the adhesion sites and mediate activation of ERK at these sites (16). In contrast, vinexin interacts with the active but not inactive form of ERK both in vitro and in vivo. Furthermore, we could not detect any interactions of vinexin {beta} with either Raf or MEK.2 Therefore, although both vinexin and paxillin bind to ERK at the periphery of cells during cell spreading, they might play different roles in cell spreading.

What is the function of the vinexin-ERK interactions? Vinexin has been suggested to function as a scaffold or anchoring protein because of its structure. Thus, one possibility is that vinexin links the active form of ERK to its substrates as a scaffold protein. We and others reported that vinexin bound to Sos (20) and estrogen receptor (23), both of which are phosphorylated and regulated by ERK (24, 46, 47), through the third SH3 domain and the N-terminal half of vinexin {alpha}, respectively. Vinexin bound to the active form of ERK through the linker region between the second and third SH3 domains. Thus, vinexin might form a ternary complex with these proteins and the active form of ERK. Another possibility is that vinexin anchors ERK activated by growth factors or cell adhesion in the cytosol, especially at adhesion sites. In support of this, the exogenous expression of vinexin {beta} reduced the phosphorylation of Elk-1, a nuclear substrate of ERK, stimulated with EGF.2 These possibilities remain to be examined.

In conclusion, we showed here that vinexin specifically interacted with ERK1/2 activated by various extracellular stimuli, including growth factors and cell adhesion. The FPFP sequence of vinexin was required for the interaction. Vinexin was directly phosphorylated by ERK2 upon stimulation with EGF at the serine 189 of vinexin {beta}. We provided evidences that vinexin was co-localized with ERK at the periphery of NIH3T3 cells and that vinexin was phosphorylated during cell spreading. These observations suggests that vinexin is a novel ERK2 substrate localized at adhesion sites and may play roles in ERK-dependent regulation during cell spreading as well as in growth factor-induced responses.


   FOOTNOTES
 
Note Added in Proof—The binding surface of ERK for the DEF domain was determined by Lee et al. (48). The surface was reported to be exposed after ERK is activated. This may explain why vinexin binds only to activated ERK.

* This work was supported in part by The Asahi Glass Foundation and a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Fax: 81-75-753-6104; E-mail: nkioka{at}kais.kyoto-u.ac.jp.

1 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; EGF, epidermal growth factor; JNK, c-Jun N-terminal kinase; GFP, green fluorescence protein; GST, glutathione S-transferase; SH3, Src homology 3; PDGF, platelet-derived growth factor; LPA, lysophosphatidic acid; TPA, phorbol-12-myristate-13 acetate; PBS, phosphate-buffered saline. Back

2 M. Mitsushima and N. Kioka, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. N. G. Ahn and E. Nishida for the valuable materials.



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 DISCUSSION
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