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Originally published In Press as doi:10.1074/jbc.M310974200 on October 14, 2003

J. Biol. Chem., Vol. 278, Issue 52, 52116-52123, December 26, 2003
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Regulation of SPIN90 Phosphorylation and Interaction with Nck by ERK and Cell Adhesion*

Chol Seung Lim{ddagger}§, Sung Hyun Kim{ddagger}§, Jin Gyoung Jung{ddagger}§, Jin-Kyu Kim¶, and Woo Keun Song{ddagger}||

From the {ddagger}Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712 and the Department of Microbiology, Changwon National University, Changwon 641-773, Korea

Received for publication, October 6, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation. We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, {beta}PIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins. It appears that {beta}PIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix. The {beta}PIX·WASP·SPIN90·Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes. The SPIN90·{beta}PIX·WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90·Nck and SPIN90·{beta}PIX interactions, resulting in detachment of cells from extracellular matrix. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90·{beta}PIX·WASP complex and Nck. It thus appears that the interaction of the {beta}PIX·WASP·SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adhesion to ECM1 plays a critical role in many important biological processes, including embryonic development, cell adhesion and migration, and cell growth and differentiation (1, 2). Interactions between cells and components of the ECM are primarily mediated by integrins, which in turn leads to recruitment of a variety of other cellular proteins to focal adhesions, thereby providing a structural link between the ECM and actin cytoskeleton and serving as a signal transducer for temporal and spatial regulation of cellular events (3-5).

Among the proteins recruited to focal adhesions, many possess SH3 domains and are therefore able to bind to Pro-rich motifs (minimally containing a consensus PXXP motif), which are found mainly in kinase-regulated signal transduction molecules and cytoskeletal proteins (6-10). Proteins containing SH3 domains are essential in several well characterized signaling pathways, in particular the Ras/MAPK pathway, which is involved in cell division and differentiation and cytoskeletal reorganization in response to growth factor receptor activation (11). Among these, Nck, which contains three SH3 domains and one SH2 domain, is ubiquitously expressed in a variety of cell types, mediating receptor tyrosine signaling affecting growth factor signaling cascades, integrin-mediated cell adhesion signaling, and the organization of actin cytoskeleton (12-14). Effector molecules known to interact with the SH3 domains of Nck include Abl protein-tyrosine kinase, son of sevenless, Nck-associated kinase, PAK, Rho effector protein kinase N-related kinase 2, Nck-interacting kinase, and WASP (15-21).

SPIN90 is another Nck-binding protein that contains an SH3 domain, three Pro-rich motifs, and a serine/threonine-rich region. SPIN90 is known to participate in sarcomere assembly during cardiac myocyte differentiation, to function as an important regulator of actin dynamics (22), and, in concert with Nck, to participate in downstream signaling triggered by integrin {beta}1A (23). Moreover, the SH3 domain of SPIN90 is completely conserved among Fyn, Yes, and c-Src, suggesting SPIN90 may interact with a variety of proteins containing Pro-rich motifs, including signaling molecules, enzymes, and structural proteins. The SH3 domain of SPIN90 is also very similar to that of WISH, which has been identified as a WASP-binding protein (24) that is able to strongly enhance N-WASP-induced Arp2/3 complex activation, resulting in rapid actin polymerization. Thus, SPIN90 may play a key role not only in signal pathway from cell adhesion but also in the reorganization of the actin cytoskeleton. But to date few in vivo ligands for the SH3 domain or Pro-rich region of SPIN90 have been identified.

With the aim of understanding better the function of SPIN90, we set out to identify proteins that interact with SPIN90 using in vitro binding assays and yeast two-hybrid screening. We found that SPIN90 has the capacity to mediate formation of protein complexes containing {beta}PIX, WASP, and Nck and that this effect is highly dependent on cell adhesion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotic/antimycotics, complete Freund's adjuvant, incomplete Freund's adjuvant, and trypsin-EDTA were all obtained from Invitrogen. Anti-ERK1 monoclonal antibody was from Zymed Laboratories Inc. and BD Transduction Laboratories; anti-phospho-ERK1 monoclonal antibody was from Cell Signaling Technology; and anti-phospho-Ser antibody was from Upstate Biotechnology. Horseradish peroxidase-labeled anti-mouse immunoglobulin G (IgG) and fluorescein isothiocyanate- or tetramethyl rhodamine isothiocyanate-conjugated goat anti-mouse IgG were from Jackson ImmunoResearch Laboratory. Protein A-Sepharose and glutathione-agarose 4B fast flow were from Amersham Biosciences. Spectra/Por (molecularporous membrane tubing) was from Spectrum Medical Industries, Inc. Primers for PCR and sequencing were synthesized at Genotech. TNT T7-coupled reticulocyte lysate system was from Promega. [{gamma}-32P]ATP, [35S]methionine ([35S]Met) and H3[32P]O4 were from PerkinElmer Life Sciences. Rat brain MATCHMAKER cDNA library was from Clontech. PDGF-BB was from Sigma.

HeLa Cell Culture—HeLa cells were grown in DMEM supplemented with 10% FBS and 100 units/ml antibiotic/antimycotic and maintained at 37 °C in 5% CO2 incubator. For suspension cultures, cells were detached from plates using trypsin-EDTA and collected in DMEM containing 1% bovine serum albumin (BSA) and 2 mg/ml soybean trypsin inhibitor. The collected cells were resuspended in DMEM-BSA, and incubated at 37 °C in suspension with gentle rotation for the indicated times. For replating after 2 h in suspension, cells were placed onto dishes coated with 20 µg/ml FN for the indicated times. In some cases, cells were serum-starved for 18 h in serum-free DMEM and then exposed to 20 ng/ml PDGF for indicated times.

Yeast Two-hybrid Screening—Yeast two-hybrid screening assays were performed according to the manufacturer's instructions with some previously described modification (25). The full-length SPIN90 cDNA (amino acids 1-722) was amplified using PCR and subcloned into pGBKT7 (a bait vector containing a GAL4 DNA-binding domain; GAL4 BD), after which a rat brain cDNA library in pACT2 (GAL4 activating domain-containing vector; GAL4 AD) was screened. Briefly, cells cotransfected with pGBKT7/SPIN90 bait vector and pACT2/GAL4 AD library were plated on Trp-, Leu- plates and Trp-, Leu-, Ade-, His--plates, and colonies were allowed to grow at 30 °C for 3-5 day. Positive colonies were selected based on their large size on Trp-, Leu-, Ade-, His- plates. To confirm the real interaction between SPIN90 and ERK1, pGBKT7/SPIN90 bait vector was co-transfected into yeast cells with pACT2/ERK1 vector. The transfectants were then plated as described above and analyzed.

GST Pull-down Assays—To assess in vitro binding, full-length inserts of SPIN90, {beta}PIX, or WASP were subcloned into pRSET, a bacterial expression vector, and translated in vitro using the TNT T7-coupled reticulocyte lysate system. The radiolabeled products were incubated with purified glutathione S-transferase (GST) or GST fusion proteins (GST-Nck, {beta}1 integrin, or SPIN90) bound to glutathione beads. All incubations were carried out in binding buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.2% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 µg/ml aprotinin, 50 µg/ml leupeptin, 50 µg/ml pepstatin) for 6 h at 4 °C. The glutathione beads were then washed four times in PBS containing 1% Triton X-100, and the radiolabeled proteins bound to the beads were solubilized by addition of an SDS sample buffer in the presence of reducing agent and subjected to 8% SDS-PAGE.

Purification GST Fusion Proteins and Generation of Anti-Nck and Anti-{beta}PIX Antibodies—To generate antibodies, the cDNA corresponding to the three SH3 domains of Nck (amino acids 1-251) or the SH3 domain of {beta}PIX (amino acids 1-63) were amplified by PCR and subcloned, in-frame, into pGEX4T-1 vector for GST fusion protein expression. GST-Nck three SH3 or GST-{beta}PIX SH3 fusion proteins were then overexpressed in bacteria and purified using glutathione-agarose 4B beads, after which the purified proteins were used as an immunogen for antibody production. After the fourth injection into rabbits, the serum specificity was tested by immunoblot analysis, after which it was further purified by affinity chromatography.

Immunoblot Analysis—Cells were lysed by boiling in a lysis buffer (1% SDS, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM Tris-HCl (pH 7.4), 1 mM PMSF, 10 mM leupeptin, 1.5 mM pepstatin, and 1 mM aprotinin), after which detergent-insoluble materials were removed by centrifugation at 12,000 x g for 10 min. Protein concentrations in the soluble fraction were measured using a bicinchoninic acid (BCA) Protein Assay Reagent kit (Pierce). Constant amounts of protein were then separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were then blocked for 1 h with 5% nonfat dry milk or 3% BSA (for phospho-Ser or phospho-ERK1 antibodies) in 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20, after which they were incubated first with the respective primary antibodies and then with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG. The antigen·antibody complexes were detected with the Enhanced Chemiluminescence (ECL) reagents (Amersham Biosciences). In some cases, blots were stripped by heating to 60 °C for 30 min in stripping buffer (100 mM {beta}-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) and reprobed.

Co-immunoprecipitation—Cells were washed three times with cold PBS and extracted for 1 h at 4 °C in extraction buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM CaCl2, 1 mM MgCl2) supplemented with protease inhibitors. The extracts were then clarified by centrifugation for 10 min at 12,000 x g, and the protein concentrations in the supernatants were determined using BCA. Samples containing 1 mg of total protein were then taken for subsequent immunoprecipitation overnight, followed by an additional 4-h incubation at 4 °C with protein A-Sepharose beads. The immunoprecipitates were extensively washed with the same extraction buffer and then subjected to SDS-PAGE and immunoblot analysis.

Immunokinase Assay for Active ERK1—To assay ERK1 kinase activity (26), HeLa cells were washed with cold PBS and extracted by sonication in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerolphosphate, 1 mM vanadate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin, 1 mM PMSF), after which the cell lysates were collected by centrifugation for 10 min at 4 °C, and the protein content was determined with BCA. Phospho-ERK1 was then immunoprecipitated from samples containing 400 µg of protein by incubation with 2 µg of monoclonal anti-phospho-ERK1 antibody overnight with gentle rocking at 4 °C, after which 30 µl of 50% protein A beads were added to the immunocomplexes and incubated for 2 h at 4 °C. After centrifuging the mixture for 1 min at 1000 x g, the beads were washed twice with 500 µl of lysis buffer, and then twice with kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM {beta}-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM vanadate, and 10 mM MgCl2, 1 mM PMSF). After centrifuging again for 1 min at 1000 x g, immunocomplexes were incubated in 30 µl of kinase buffer contained 5 µCi/µl [{gamma}-32P]ATP and 2 µg of substrate proteins (GST, myelin basic protein (MBP), or GST-SPIN90) for 30 min at 30 °C. The samples were then boiled and centrifuged, and the supernatants were mixed with 4x SDS sample buffer, boiled for 3 min, and subjected to SDS-PAGE (8-12%). The gels were then dried and exposed in a MAC BAS (Fuji) as indirect autoradiography to check band intensity for 6 h or on x-ray film (Kodak) overnight.

In Vivo Labeling—HeLa cells were metabolically labeled under exponential growth conditions. After washing the cells in PBS, they were incubated for 6 h at 37 °C in 6 ml of phosphate-free DMEM containing 5% FBS. Thereafter, 1 mCi of 32Pi (10 mCi/ml) was added to the medium (200 µCi/ml), and the cells were incubated for 4 h at 37 °C to metabolically label the proteins. In some experiments, labeled cells were washed in PBS, harvested, placed in suspension for 2 h, and then replated onto FN-coated dishes in the same 32P-labeling medium and allowed to attach for 2 h. Cells were extracted in extraction buffer and further analyzed as described above.

Transfection and Cell Adhesion Assays—cDNAs encoding the SPIN90 SH3 domain (HA-SPIN90 SH3; amino acids 1-61), Pro-rich motifs containing the Ser/Thr-rich region (HA-SPIN90 PR; amino acids 146-279), or C-terminal region (HA-SPIN90 C-term; amino acids 280-722) were amplified by PCR and subcloned into pcDNA3-HA vector for HA-tagged mammalian expression. Each construct was transfected into HeLa cells using LipofectAMINE according to the manufacturer's protocol (Invitrogen). For cell adhesion assays, transfected cells were detached, incubated in suspension for 2 h, and replated onto FN-coated dishes and allowed to adhere for 2 h at 37 °C. Non-adherent cells were removed by washing twice with PBS, after which the adherent cells were fixed for 5 min in methanol and counted under a microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Proteins Associating with SPIN90 in Vitro and in Vivo—Using in vitro and in vivo binding assays, we have identified several proteins that interact with SPIN90. GST pull-down analysis carried out with in vitro-translated proteins showed that SPIN90 co-precipitated with GST-Nck,-{beta}PIX, and -WASP, but not GST-{beta}1 integrin (Fig. 1, A-D). Subsequent co-immunoprecipitation analysis confirmed that SPIN90 does indeed associate with Nck, {beta}1 integrin, {beta}PIX, and WASP (Fig. 1, E-H), suggesting that SPIN90 participates in {beta}1 integrin-mediated signaling via protein-protein interactions. SPIN90 is unlikely to bind to {beta}1 integrin directly, however, because amino acid sequence analysis revealed these proteins each to contain multiple Pro-rich motifs and SH3 domains (Fig. 1I).



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  		FIG. 1.
Association of SPIN90 with Nck, {beta}PIX and WASP in vitro and in vivo. Recombinant GST-Nck (A), {beta}1 integrin (B), or SPIN90 (C and D) fusion proteins were immobilized on glutathione-agarose beads, after which GST pull-down assays were carried out using [35S]Met-labeled, in vitro-translated SPIN90 (A and B), {beta}PIX (C), and WASP (D). Bound proteins were visualized by autoradiography. SPIN90 was bound directly to Nck (A), {beta}PIX (C), and WASP (D) but not {beta}1 integrin (B). To confirm the interactions in vivo, HeLa cell lysates were immunoprecipitated with anti-Nck (E), anti-SPIN90 (F and G), or anti-WASP (H) antibody, and the precipitates were immunoblotted with anti-SPIN90 (E and H), anti-{beta}1 integrin (F), or anti-{beta}PIX (G) antibody. I, schematic structures of SPIN90, Nck, {beta}PIX, and WASP. SH3, Src homology 3; SH2, Src homology 2; PR, proline-rich motif; STR, Ser/Thr-rich region; DH, Dbl homology; PH, Pleckstrin homology; EVH, Ena/VASP homology; and GBD, GTPase-binding domain.

 
Interaction between SPIN90 and Nck Is Adhesion-dependent—To test the dependence of the SPIN90·Nck interaction on cell adhesion, lysates from adherent or detached cells were subjected to co-immunoprecipitation assays. SPIN90 immunoprecipitates from stably adherent cells contained readily detectable levels of Nck, but no Nck associated with SPIN90 was detected in suspended or cytochalasin D-treated cells (Fig. 2A). Moreover, when lysates from cells placed in suspension for various times were subjected to co-immunoprecipitation, dissociation of SPIN90 from Nck was found to be more than 90% complete within 30 min after cell detachment, and their association was undetectable within 60 min. Notably, this effect was rapidly reversed by replating the cells on FN-coated dishes (Fig. 2B).



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  		FIG. 2.
Cell adhesion-dependent interaction of SPIN90 with Nck. A, SPIN90 immunoprecipitates from adherent, suspended, and adherent cells pretreated for 30 min with 5 µM cytochalasin D were immunoblotted with anti-SPIN90 or anti-Nck antibody. B, SPIN90 immunoprecipitates from adherent and suspended cells and cells replated on FN-coated dishes for the indicated times were immunoblotted anti-SPIN90, anti-Nck, anti-{beta}PIX, or anti-WASP antibody. C and D, WASP (C) or Nck (D) immunoprecipitates from suspended or replated cells were immunoblotted with anti-{beta}PIX or anti-Nck antibody.

 
By contrast, SPIN90, {beta}PIX, and WASP remained associated in suspended cells. Likewise, the interaction between {beta}PIX and WASP was apparent in both suspended and replated cells. The interaction between Nck and WASP was not detected in suspended cells and only weakly detected in replated cells (Fig. 2C); the interaction between {beta}PIX and Nck was also absent in suspended cells, but was strongly detected in replated cells (Fig. 2D). Taken together, these results suggest that SPIN90, {beta}PIX, and WASP make up a molecular complex that interacts with Nck in a manner dependent on cell adhesion.

SPIN90·Nck Interaction Is Dynamically Regulated by ERK1 Activation—Among the proteins isolated by yeast two-hybrid screening using a rat brain cDNA library and full-length SPIN90 as bait was ERK1, which was identified as binding to SPIN90 (Fig. 3A). GST pull-down assays and co-immunoprecipitation analysis confirmed that ERK1 does indeed bind to SPIN90 both in vitro and in vivo (Fig. 3, B and C). Furthermore, consistent with earlier reports that ERK activity is regulated by cell adhesion (27), we found that phospho-ERK1/2 was undetectable in suspended cells but that levels of phospho-ERK1/2 increased for up to 120 min after replating the cells on FN (Fig. 4A). Pre-treating the cells with PD98059, an ERK inhibitor, before replating blocked the increases in ERK1/2 activation (Fig. 4B).



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  		FIG. 3.
Interaction of SPIN90 with ERK1. A, ERK1 was isolated by yeast two-hybrid screening using a rat brain cDNA library with SPIN90 as bait. B, [35S]Met-labeled,invitro-translatedERK1 was incubated with purified GST-SPIN90 or GST protein immobilized on glutathione-agarose beads, after which GST pull-down assays were carried out. Bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. C, to confirm the association of SPIN90 and ERK1, in vivo, HeLa cell lysates were immunoprecipitated with anti-SPIN90 antibody, and immunoblotted with anti-SPIN90 or anti-ERK antibody.

 



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  		FIG. 4.
Interaction of SPIN90 and Nck is dependent on SPIN90 phosphorylation by ERK. A, adherent HeLa cells were detached, incubated in suspension for 2 h, and replated on FN-coated dishes for the indicated times, after which the lysates were immunoblotted with anti-ERK1/2 or anti-phospho-ERK1/2 antibody. B, treatment with PD98059 (10 or 30 µM), a specific MEK inhibitor, suppressed ERK activation by cell adhesion. C, the lysates from adherent, suspended, and replated cells, with or without 30 µM PD98059 pretreatment, were immunoprecipitated with anti-SPIN90 antibody and then immunoblotted with anti-phospho-Ser antibody. For in vivo labeling, HeLa cells were metabolically labeled with [32P]orthophosphate and treated as described above. 32P-Labeled SPIN90 was immunoprecipitated with anti-SPIN90 antibody, separated by 8% SDS-PAGE, and detected by autoradiography. D, cells treated as described above were lysed and immunoprecipitated with anti-Nck antibody, and the precipitants were immunoblotted with anti-Nck or SPIN90 antibody.

 
We next examined SPIN90 phosphorylation in adherent, detached, and replated cells in the absence and presence of 30 µM PD98059. Using in vivo labeling, we detected SPIN90 phosphorylation in both adherent and replated cells, but weak phosphorylation of SPIN90 was detected in detached or PD98059-treated cells. In addition, Ser-phosphorylation of SPIN90 was routinely detected in SPIN90 precipitants from adherent cells, but was abolished by cell detachment or PD98059 treatment (Fig. 4C). We were not able to test for the presence of Thrphosphorylation because of the absence of available antibodies. In parallel experiments, a strong interaction between SPIN90 and Nck was detected in adherent and replated cells, whereas their interaction almost disappeared in detached and PD98059-treated cells (Fig. 4D).

That ERK1 phosphorylated SPIN90 was confirmed by in vitro kinase assays showing that immunoprecipitates obtained using anti-phospho-ERK1 antibody in adherent or replated cells were able to phosphorylate purified GST-SPIN90 protein and MBP, and this effect was blocked by detaching the cells or pretreating them with PD98059 (Fig. 5, A and B). Thus, the interaction of SPIN90 and Nck apparently coincides with an ERK1-catalyzed change in the phosphorylation state of SPIN90 during cell adhesion.



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  		FIG. 5.
SPIN90 is phosphorylated by ERK1-immuncomplex in vitro. A, adherent, suspended, and replated cells, with or without PD98059 (10 or 30 µM) pretreatment, were lysed and immunoprecipitated with anti-phospho-ERK1 antibody. Immunoprecipitants were then incubated in kinase buffer containing 5 µCi/µl [{gamma}-32P]ATP and 2 µg of substrate protein (GST-SPIN90, GST, or MBP). Samples were then separated by SDS-PAGE and visualized by autoradiography. B, relative band intensities (GST-SPIN90 and MBP) were determined by densitometry.

 
Relationship between SPIN90·Nck Interaction and ERK1 Activation by PDGF—It is known that ERK1 can be activated by a variety of growth factors, including PDGF (28). We found that activation of ERK1 by 20 ng/ml PDGF reached to the maximum within 10 min and decreased thereafter (Fig. 6A) and that the interaction of SPIN90 and Nck followed the same time course (Fig. 6B). AG1295 is a specific PDGF receptor antagonist that reversibly inhibits PDGF by competitively binding it its receptor. We found that AG1295 inhibited ERK1 activation by PDGF, as well as the interaction of SPIN90 and Nck (Fig. 6C). Likewise, 30 µM PD98059 specifically inhibited both PDGF-induced ERK1 activation and SPIN90·Nck interaction (Fig. 6D). By contrast, AG1478, an epidermal growth factor (EGF) receptor antagonist, was less able to inhibit the interaction of SPIN90 and Nck (Fig. 6C). These findings clearly indicate that SPIN90 can be a substrate for ERK1 and that its phosphorylation by ERK1 likely modulates SPIN90·Nck interaction.



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  		FIG. 6.
Modulation of SPIN90·Nck interaction by ERK1 activated by PDGF. A, serum-starved HeLa cells were incubated with 20 ng/ml PDGF for the indicated times, after which the lysates were immunoblotted with anti-ERK1, anti-phospho-ERK1/2, or anti-SPIN90 antibody. B, lysates from cells treated with PDGF for the indicated times were immunoprecipitated with anti-Nck antibody and immunoblotted with anti-Nck or SPIN90 antibody. C, PDGF-treated cells were incubated with AG1478, an EGF receptor inhibitor, or AG1295, a PDGF receptor inhibitor, after which the lysates were immunoblotted with anti-ERK1 or phospho-ERK1 antibody. The same cell lysates were immunoprecipitated with anti-Nck antibody and immunoblotted with anti-Nck or SPIN90 antibody. D, cells were treated with 10 or 30 µM PD98059 in the presence or absence of PDGF, after which the lysates were immunoblotted with anti-ERK1 or phospho-ERK1 antibody. To test the effect of PD98059 on SPIN90·Nck interaction, the same cell lysates cells immunoprecipitated with anti-Nck antibody and immunoblotted with anti-Nck or SPIN90 antibody. E, to identify ERK1 activation by PDGF, adherent and suspended cells, pretreated with or without 20 ng/ml PDGF, were lysed, immunoprecipitated with anti-PDGF receptor antibody, and immunoblotted with anti-p-Tyr, anti-PDGF receptor, anti-Nck, or anti-SPIN90 antibody. Then the same cell lysates were immunoprecipitated with anti-Nck antibody and immunoblotted with anti-Nck or SPIN90 antibody.

 
Interestingly, phosphorylation/activation of PDGF receptors in suspended cells led to recruitment of Nck to the receptor, but not SPIN90. And in the presence of PDGF, non-adherent cells exhibited the induced interaction of SPIN90 and Nck (Fig. 6E), suggesting PDGF can induce SPIN90·Nck interaction and likely potentiates their interaction during cell adhesion.

Modulation of Cell Adhesion by Specific Domains of SPIN90 —SPIN90 contains three functional domains or motifs known to mediate protein-protein interaction. To further analyze these interacting motifs, its SH3 domain (HA-SPIN90 SH3), Pro-rich motifs (HA-SPIN90 PR), and C-terminal region (HA-SPIN90 C-term) were, respectively, cloned into an HA-tagged mammalian expression vector (Fig. 7A) and expressed in HeLa cells (Fig. 7B). Fig. 7C shows that overexpression of SPIN90 Pro-rich motifs dramatically suppressed the interaction between SPIN90 and Nck, suggesting that Nck binds to Pro-rich motifs of SPIN90 protein via SH3 domains. The interaction of SPIN90 and {beta}PIX was suppressed by overexpression of either the SPIN90 SH3 domain or Pro-rich motifs, although the inhibitory effect of the former was much stronger, suggesting {beta}PIX binds to the SH3 domain of SPIN90 via Pro-rich motifs.



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  		FIG. 7.
Inhibition of cell adhesion by SPIN90 SH3 and Pro-rich domains. A and B, SPIN90 deletion constructs (HA-SPIN90 SH3 (SH3 domain; amino acids 1-61), HA-SPIN90 PR (Pro-rich motifs with Ser/Thr-rich region; amino acids 146-279), and HA-SPIN90 C-term (C terminus region; amino acids 280-722)) were amplified by PCR, subcloned into pcDNA3-HA vector, and transiently overexpressed in HeLa cells as described under "Experimental Procedures." C, SPIN90 deletion mutants were transfected into HeLa cells, after which the lysates were immunoprecipitated with anti-Nck or anti-{beta}PIX antibody and, respectively, immunoblotted with anti-SPIN90 and anti-Nck or anti-SPIN90 and anti-{beta}PIX antibodies. D, cells overexpressing SPIN90 mutants were detached, replated on FN-coated dishes, and washed with PBS to remove unbound cells. The remaining adherent cells were then counted under a microscope.

 
The fact that the loss of SPIN90·Nck interaction after detachment was rapidly reversed by replating the cells on FN-coated dishes suggests their interaction plays a key role in the process of cell adhesion. To test this idea, cell adhesion assays were carried out using cells overexpressing the HA-SPIN90 SH3 domain, Pro-rich motifs, or C-terminal region. Overexpression of the HA-SPIN90 SH3 domain or Pro-rich motifs diminished cell adhesion by up to 60-70%, whereas cell adhesion of cells transfected with HA-SPIN90 C-term was indistinguishable from that in HA vector-transfected cells (Fig. 7D). Apparently, the interactions between SPIN90 and other cellular proteins are essential for stable cell adhesion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We previously showed SPIN90 to be a Nck-binding protein widely expressed in a variety of tissues and cell types, and to participate in sarcomere assembly during cardiac myocyte differentiation (22). This protein has a well conserved SH3 domain and three separated Pro-rich motifs capable of mediating protein-protein interactions. Moreover, between two of the Prorich motifs, there is a serine/threonine-rich region, indicating the potential for phosphorylation by serine/threonine kinases. Still, the function of SPIN90 remains largely unknown, and few in vivo ligands have been identified.

Particular interactions of a protein with other protein or specific phosphorylation are crucial in a variety of biological processes, including signal transduction. In the present study, therefore, we carried out a series of in vitro binding assays and co-immunoprecipitation analyses with the aim of identifying proteins that bind to SPIN90 and used a yeast two-hybrid screening system to identify kinases capable of phosphorylating it. We found that SPIN90 associates with Nck, {beta}PIX, and WASP. Each of these proteins contains more than one Pro-rich motif or SH3 domain through which they are able to participate in the formation of a protein complex. Indeed, the Nck·SPIN90 interaction is likely mediated through the first and third SH3 domains of Nck (22), which bind to a Pro-rich region of SPIN90. On the other hand, the SH3 domain of SPIN90 likely mediates its interaction with {beta}PIX.

Nck contains three SH3 domains and one SH2 domain that mediate protein-protein interactions. The former are able to bind to a variety of downstream proteins, including SOS, WASP, dynamin, and Cbl, which are all involved in reorganization of actin cytoskeleton (16, 21, 29, 30). {beta}PIX is a Rho family guanine nucleotide exchange factor that interacts with PAK through its SH3 domain and has a role in nuclear signaling by MAPK activation as well as in actin cytoskeleton regulation (31, 32). Members of the PAK family of serine/threonine kinases serve as targets for the small GTP-binding proteins Cdc42 and Rac and have been implicated in a wide range of biological activities related to the modulation of actin cytoskeletal organization (33, 34), including neurite formation, axonal guidance, development of cell polarity, and motile responses (35, 36). The interaction of PAK and Nck is reported to be dynamically regulated by cell adhesion (37). WASP has been shown to regulate the cortical actin cytoskeleton as a downstream effector of Rac and Cdc42 and to integrate signaling cascades leading to actin polymerization (38). In addition, SPIN90 has a SH3 domain very similar to that of WISH, a WASP-binding protein that can enhance N-WASP-induced Arp2/3 complex activation, resulting in rapid actin polymerization (24). Based on these observations and the results of preliminary experiments, we suggest that SPIN90 also plays a role in the actin cytoskeleton rearrangement, including formation of filopodia or lamellipodia, through its interactions with Nck, {beta}PIX, and WASP. Indeed, overexpression of SPIN90 in HeLa cells readily promoted the filopodia formation, and this effect was potentiated by PDGF.2

Ectopic expression of SPIN90 SH3 domain or Pro-rich motifs abolished the interaction of SPIN90 with Nck and {beta}PIX, thereby promoting cell detachment from the ECM. The interaction of SPIN90 and Nck appears to be cell adhesion-dependent and to be reversibly diminished by placing cells in suspension or cytochalasin D treatment, in which cytochalasin D induced actin cytoskeleton disruption and promoted cell detachment and rounding. By contrast, the association between SPIN90 and {beta}PIX or WASP remains intact in suspended cells. These findings suggest that SPIN90 first mediates the formation of the SPIN90·{beta}PIX·WASP complex; the interaction between the complex and Nck is subsequently mediated by SPIN90 in a manner dynamically regulated by cell adhesion.

In the correlation with the regulation of SPIN90·Nck interaction, the reverse mechanism of SPIN90·Nck interaction and dissociation reflects that the interaction may be regulated by a post-translational modification, such as phosphorylation. Herein, we also found that ERK1 is able to bind to and efficiently phosphorylate SPIN90. Activation of ERK1 is regulated by cell adhesion as well as a variety of growth factors, including PDGF, insulin, EGF, and fibroblast growth factor (27, 28, 39-41); once activated, ERK is able to phosphorylate numerous proteins, including RSK, c-Fos, c-Jun, c-Myc, c-raf, MAP2, and Mek (42-44). SPIN90 also appears to be phosphorylated by ERK1 in adherent cells, and its phosphorylation may, in turn, facilitate assembly of signaling complexes containing integrin {beta}1, SPIN90, and ERK1, which likely transmit a cell adhesion signal to the cytoskeletal network. It is noteworthy that ERK1/MAPK is targeted to newly forming cell-matrix adhesion by integrin engagement (45), which suggests that the interaction of ERK1 and SPIN90 is closely related in the spatial formation of focal adhesions and that phosphorylation of SPIN90 by ERK1 is a crucial step via which the SPIN90 complex is recruited to Nck for stable cell adhesion.

The function of adhesion receptors is regulated by growth factors and other agonists (4). Because the signaling pathways activated by integrins and growth factors share many common elements (46), there are many opportunities for integrin signals to modulate growth factor signals and vice versa. For example, adhesion to ECM proteins induces transient tyrosine phosphorylation of EGF and PDGF receptors, which in turn leads to ERK1/MAPK activation. In addition to cell adhesion, ERK1 activity can be induced by EGF, nerve growth factor, and PDGF. Our data indicate that cell adhesion-mediated ERK1 activation is potentiated by PDGF, which in turn enhances SPIN90·Nck interaction. Indeed, PDGF treatment was sufficient to induce tyrosine phosphorylation of PDGF receptors and its binding to Nck, and the interaction of SPIN90 with Nck, even in non-adherent cells.

It thus appears that integrin- and growth factor receptor-mediated signaling can act in concert to directly to trigger specific downstream signaling cascades, which has broad implications for the regulation of protein-protein interactions and anchorage-dependent protein localization.


   FOOTNOTES
 
* This study was supported in part by grants from the National Research Laboratory (The Ministry of Science & Technology). 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

§ Supported by the Brain Korea 21 Program. Back

|| To whom correspondence should be addressed. Tel.: 82-62-970-2487; Fax: 82-62-970-2484; E-mail: wksong{at}kjist.ac.kr.

1 The abbreviations used are: ECM, extracellular matrix; SPIN90, SH3 protein interacting with Nck, 90 kDa; WASP, Wiscott-Aldrich syndrome protein; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; ERK1, extracellular signal-regulated kinase 1; FBS, fetal bovine serum; FN, fibronectin; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; PAK, p21-activated kinase; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PIX, PAK-interacting exchange factor; PMSF, phenylmethylsulfonyl fluoride; SH3/SH2 domain, Src homology 3/2 domain; MBP, myelin basic protein; HA, hemagglutinin. Back

2 C. S. Lim, S. H. Kim, J. G. Jung, J.-K. Kim, and W. K. Song, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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