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Originally published In Press as doi:10.1074/jbc.M708300200 on February 5, 2008

J. Biol. Chem., Vol. 283, Issue 15, 10089-10096, April 11, 2008
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Cell Adhesion-dependent Cofilin Serine 3 Phosphorylation by the Integrin-linked Kinase·c-Src Complex*

Yong-Bae Kim{ddagger}, Suyong Choi{ddagger}, Moon-Chang Choi{ddagger}, Min-A Oh{ddagger}, Sin-Ae Lee§, Moonjae Cho, Kensaku Mizuno||, Sung-Hoon Kim**1, and Jung Weon Lee{ddagger}§2

From the Departments of {ddagger}Tumor Biology and §Molecular & Clinical Oncology, Cancer Research Institute, Cell Dynamics Research Center, College of Medicine, Seoul National University, Seoul 110-799, Korea, the Department of Medicine, Cheju National University, Jeju 690-756, Korea, the ||Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan, and the **College of Oriental Medicine, Kyunghee University, Seoul 131-701, Korea

Received for publication, October 5, 2007 , and in revised form, February 1, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser3 phosphorylation (pS3cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS3cofilin, we examined the effects of ILK on pS3cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS3cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS3cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin-mediated interaction with the extracellular matrix triggers intracellular signal cascades that regulate the activity and localization of numerous signaling molecules that consequently modulate diverse cellular functions including actin reorganization (1, 2). The recruitment of adaptor and signaling molecules to the cytoplasmic tails of integrin subunits occurs at focal adhesions and allows the activation of diverse signaling pathways during cell adhesion (3). Integrin-mediated cell adhesion activates FAK,3 c-Src family kinase, Erk, Akt, and the Rho GTPase family (4). The Rho GTPase family mediates actin reorganization via downstream effectors, including ROCKs, myosin light-chain kinase, LIMKs, and cofilin (5). The actin polymerization/depolymerization status depends on the Ser3 phosphorylation level of cofilin, an actin-severing protein (6). Cofilin severs actin filaments when it is dephosphorylated by Slingshot phosphatase or chronophin, whereas its severing activity can be inhibited when phosphorylated by LIMKs or testicular protein kinases (TESKs) (7).

ILK is a Ser/Thr kinase located at focal adhesions by binding to the β1 integrin cytoplasmic tail, the overexpression of which inhibits intestinal epithelial cell adhesion to integrin substrates (8). ILK expression was suppressed epigenetically upon adhesion of gastric carcinoma cells, and ILK overexpression reduced adhesion abilities and enhanced anchorage-independent growth (9). In contrast, ILK expression also correlated with an increased adhesion of prostate cancer cells (10), and ILK was transiently activated upon adhesion of intestinal epithelial cells to fibronectin (Fn) (11). Although the roles of ILK in cell adhesion-mediated actin reorganization have thus been elucidated, depending on the cell type or signaling contexts, the mechanistic roles of ILK in the regulation of cofilin phosphorylation remain largely unknown. In this study, we revealed a new functional linkage of ILK to cofilin by showing that ILK-associated c-Src affected ILK activity to phosphorylate cofilin during normal RIE1 cell adhesion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—The integrin {alpha}5-expressing normal rat intestinal epithelial cell line (RIE1-{alpha}5) was described previously (12).

Cell Conditions, Treatments, and Immunoblots—Cells were transfected with the indicated plasmid (see below) or with siRNAs against ILK (Dharmacon) or LIMK1 (Bioneer Corp., Daejeon, Korea) for 24 h using the MagnetofectionTM system (polyMag, OZ Biosciences) following the manufacturer's protocols, and/or cells were infected with control or ILK-encoding adenovirus (kind gifts from Dr. H.-S. Kim, Seoul National University Hospital, Seoul, Korea) for 24 h. The plasmid used for transfection was pcDNA3 with ILK(WT), ILK(E359K), ILK(S343A), kinase-dead ROCK1 (DA-1A, a kind gift from Dr. S. Narumiya, Kyoto University, Japan), kinase-dead TESK1(D170A) or TESK2(D176A) (13), or kinase-inactive c-Src(Y416F) or c-Src(Y527F) (14). ILK constructs were prepared from WT ILK (a kind gift from Dr. R. Juliano, University of North Carolina, Chapel Hill, NC) via polymerase chain reactions (GeneAll, Seoul), and their sequences were confirmed by direct sequence analysis. The cells were then harvested or treated with Me2SO or pharmacological inhibitors for 30 min before being kept in suspension or replated on Fn (10 µg/ml; Chemicon)-precoated or polylysine (10 µg/ml; Sigma)-precoated dishes for 1 h. The inhibitors include LY294002 (20 µM; LC Laboratories), PP2 (10 µM; A.G. Scientific, Inc.), PP3 (10 µM; A.G. Scientific, Inc.), U0126 (40 µM; LC Laboratories), p18 peptide (5 µM; Calbiochem), and Y27632 (15 µM; Calbiochem). In some cases, EGF (50 ng/ml) was treated for the last 5 min of the replating time. Whole cell lysates were prepared using a modified radioimmune precipitation assay buffer (50 mM HEPES, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mM Na2VO4, 1 mM nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µM pepstatin A, and 10 µM leupeptin) and used in immunoblotting with pY397FAK, pY416c-Src, ILK, and c-Src (Santa Cruz Biotechnology); Akt, pS473Akt, Erk1/2, phospho-Erk1/2, pT508LIMK1, LIMK1, cofilin, and pS3cofilin (Cell Signaling Technology); FAK, GST, ROCK1, and {alpha}-tubulin (BD Transduction Laboratories); and phospho-Ser/Thr (Abcam, Cambridge, UK) antibodies.

Immunofluorescence Microscopy—Cells on Fn-precoated coverglasses were manipulated with or without pretreatment with 10 µM PP2 (a c-Src family kinase inhibitor) or treatment with EGF (50 ng/ml) as above or alternatively transfected with pEGFP-ILK. Immunofluorescence for ILK or pY416c-Src or staining of actin using rhodamine-conjugated phalloidin was performed using confocal (MRC-500, Bio-Rad) or fluorescent (BX51, Olympus) microscopy as described previously (15).

Coimmunoprecipitation—Cells were transfected with inactive c-Src(Y416F) or kinase-dead ROCK1 (KD-1A) for 48 h. Cell lysates were prepared and immunoprecipitated with anti-ILK antibody as described previously (15) and probed for c-Src or ILK by immunoblotting.

Expression of Fusion Proteins and in Vitro Pulldown Assay—Diverse human ILK (NP_004508 [GenBank] ) gene constructs were prepared and cloned into the pGEX4T-3 vector (GE Healthcare) at EcoRI and XhoI cloning sites. Their sequences were directly confirmed. Each construct, GST-cofilin (a kind gift from Dr. T. Nakamura, Osaka University) or GST-c-Src in Escherichia coli BL21, was induced by 1 mM isopropyl 1-thio-β-D-galactopyranoside for 4 h. The E. coli extracts (with 50 mM Tris-HCl, pH 8.2, 2 mM MgCl2, 0.2 mM Na2S2O5, 10% glycerol, 20% sucrose, 2 mM dithiothreitol, 1 mM Na2VO4, and protease inhibitors) were then incubated with glutathione-Sepharose beads (Amersham Biosciences) for 6 h at 4 °C. GST fusion proteins bound to beads were washed once with the extracting buffer and three times with ice-cold phosphate-buffered saline and mixed with RIE1-{alpha}5 cell extracts (1 mg of protein/condition) for 4 h at 4 °C with or without 10 µM PP2 treatment. After washings as above, proteins were eluted via the addition of SDS-PAGE sample buffer and boiling. Eluted proteins were resolved, transferred, and immunoblotted for anti-ILK, anti-GST, or anti-c-Src antibody.

In Vitro Kinase Assay—In vitro ILK kinase assay was performed as described previously (16). ILK immunoprecipitates were prepared overnight using whole cell extracts, mixed with protein A/G beads (Upstate Biotechnology) for 2 h at 4 °C, and washed three times with ice-cold phosphate-buffered saline. An equal amount of ILK immunoprecipitates was then incubated with myelin basic protein (MBP) (3 µg/condition; Sigma) or recombinant GST-cofilin (3 µg/condition) for 30 min at 25 °C in the reaction buffer including 2 µM dithiothreitol, 100 µM ATP, 50 mM HEPES, pH 7.0, 10 mM MnCl2, 10 mM MgCl2, and 2 mM NaF. The reactions were stopped with the addition of SDS-PAGE sample buffer, and they were then used in immunoblotting for the indicated molecules or Ponceau S staining for MBP.

Statistical Analysis—The relative pS3cofilin under diverse experimental conditions was calculated for graphic presentation (mean ± S.D.) or for -fold difference presentation (with raising fractions not lower than 0.05-0.1 for a concise representation) after the normalization of pS3cofilin over cofilin band intensities as measured by densitometry. Student's t test was performed for comparison of mean values to see if the difference was significant. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ILK-mediated Cofilin Phosphorylation upon Cell Adhesion—Although ILK and cofilin are both known to be involved in actin organization, their functional linkage remains largely unknown. While investigating integrin-mediated actin organization, we found that RIE1-{alpha}5 cells replated on Fn showed a higher pS3cofilin than suspended cells did, and exogenous overexpression of ILK further enhanced the adhesion-dependent pS3cofilin (Fig. 1A). In contrast, cells held in suspension, adherent on polylysine, or in a normal serum-containing condition showed no significant pS3cofilin even upon ILK overexpression (Fig. 1A). Over time, after being suspended or replated on polylysine, pS3colofin was not observed or was minor, respectively, whereas cells adherent on Fn showed significant pS3cofilin levels when ILK was overexpressed. The ILK-enhanced pS3cofilin was not correlated with the activities of FAK, Akt, or GSK3β (Fig. 1A and data not shown), which are downstream of integrins or of ILK (17); they did not show adhesion dependence and/or ILK overexpression dependence, unlike pS3cofilin levels in Fn-adherent cells overexpressing ILK. However, Tyr118-phosphorylated paxillin was enhanced by cell adhesion, although exogenous ILK did not additionally enhance it (Fig. 1A). The ILK(E359K) mutant, which is paxillin and parvin binding-deficient (18) and putatively kinase-dead with residual kinase activity (19), did not alter the ILK-enhanced pS3cofilin, whereas the ILK(S343A) mutant with abolished kinase activity (20) reduced the ILK effects (Fig. 1C). However, cell adhesion-dependent pY397FAK was not altered by the expression of different ILK constructs, indicating again that the active ILK could enhance pS3cofilin in a FAK-independent manner (Fig. 1C). Consistent with actin polymerization supported by Ser3-phosphorylated cofilin (7), the green fluorescent protein-ILK transfected cells formed more stress fibers compared with neighboring untransfected cells (Fig. 1D).


Figure 1
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  		FIGURE 1.
ILK-enhanced cofilin Ser3 phosphorylation upon integrin-mediated cell adhesion. A, RIE1-{alpha}5 cells were infected with control (Ad-Cont) or ILK-encoding (Ad-ILK) adenovirus for 24 h, trypsinized, washed twice with serum-free medium containing 1% bovine serum albumin, rolled over (60 rpm) for 1 h at 37 °C to null-out the basal signaling activity (N), and then held in suspension (S) or replated on Fn- or polylysine (PL)-precoated dishes for 1 h. *, p < 0.05. B, cells were infected with ILK-encoding adenovirus for 24 h (0) and manipulated to be suspended (Sus) or Fn-adherent as in A for the indicated periods. C and D, cells were transfected with WT ILK, ILK(E359K), or ILK(S343A) (C) or with green fluorescent protein (GFP)-ILK (D) for 48 h and manipulated as above. Whole cell lysates were prepared and used for immunoblotting for the indicated molecules (A-C), or cells on Fn-precoated coverglasses were stained for actin (D). The relative pS3cofilin was calculated for graphic presentation (mean ± S.D.) after normalization of pS3cofilin over cofilin band intensities measured by densitometry. Data shown represent three different experiments.

 
ILK-mediated pS3cofilin Depends on c-Src Activity—Next we examined the molecular basis of the ILK effects on cell adhesion. To address this, ILK-infected cells were suspended or replated on Fn for 1 h with or without pretreatment with diverse pharmacological inhibitors. Pretreatment with inhibitors including LY294002 (a specific inhibitor against phosphatidylinositol 3-kinase and thus its downstream Akt), PP2 (a selective c-Src family kinase inhibitor), U0126 (a specific inhibitor against mitogen-activated protein kinase/extracellular signal-regulated kinases (MEKs) and thus downstream Erk1/2), p18 peptide (a selective myosin light-chain kinase inhibitor), or Y27632 (a selective ROCK inhibitor) had been done 30 min before cells were held in suspension or replated on Fn. Immunoblots of whole cell lysates from these treatments showed that inhibition of c-Src or ROCK (Fig. 2A) abolished the ILK-enhanced pS3cofilin. Furthermore, pS3cofilin levels in Fn-adherent cells with diverse treatments were correlated with pY416c-Src levels, but not with pY397FAK or pS473Akt levels (Fig. 2A). Therefore, these data indicate that c-Src activity might be important for pS3cofilin in the RIE1-{alpha}5 cells. Although it is known that ROCK1 phosphorylates LIMKs, which in turn phosphorylate cofilin (21, 22), pT508LIMK1 levels under these treatments were not correlated with the pS3cofilin levels (Fig. 2A). Suppression of LIMK1 by its siRNA transfection nearly abolished the adhesion-mediated pS3cofilin in control virus-infected cells, but not in ILK overexpression-enhanced pS3cofilin of Fn-adherent cells (Fig. 2B), indicating that LIMK1 does not play a significant role in the ILK overexpression-enhanced pS3cofilin. When kinase-dead ROCK1 (KD-1A) was expressed, the basal cell adhesion-dependent pS3cofilin was completely abolished, but ILK overexpression-mediated pS3cofilin was still significantly maintained (Fig. 2C), indicating that ILK overexpression-mediated pS3cofilin might be independent in part of ROCK1. However, the siRNA against ILK completely abolished the ILK effects (Fig. 2C). Cofilin is also phosphorylated by the Ser/Thr kinase TESKs (13). Therefore, we evaluated whether kinase-inactive TESK1-(D170A) and TESK2(D176A) could block the ILK effects. Transfection of the TESK mutants abolished the basal, but not ILK overexpression-enhanced, pS3cofilin of Fn-adherent cells (Fig. 2D). These observations indicate that ILK enhanced pS3cofilin in a TESK-independent manner. Pharmacological inhibition of c-Src by PP2 inhibited the ILK effect on pS3cofilin (Fig. 2A). Inhibition of ROCK by Y27632 decreased pY416c-Src, as did the c-Src inhibitor PP2 (Fig. 2A). Therefore, it is likely that the ROCK inhibition-mediated effect might be a result of nonspecific c-Src inhibition. To confirm that c-Src was required for the ILK effect, we examined pS3cofilin in suspended or Fn-adherent SYF-/- (c-Src/Yes/Fyn-deficient) fibroblasts after ILK-encoding adenovirus infection or Y27632 pretreatment. These conditions did not significantly alter pS3cofilin, supporting that c-Src was required for the ILK effects (Fig. 2E). Meanwhile, the SYF-/- cells appeared to regulate pS3cofilin via LIMK1 because LIMK1 suppression reduced pS3cofilin (Fig. 2F).

ILK Binds c-Src—Because ILK increased pS3cofilin in a c-Src activity-dependent manner, we examined whether ILK might bind active c-Src (i.e. pY416c-Src). We did this by microscopic visualization of their colocalization in Fn-adherent cells using a specific c-Src inhibitor, PP2. ILK was colocalized with pY416c-Src on the periphery of Fn-adherent cells, and PP2 pretreatment abolished their colocalization (Fig. 3A). We next performed an in vitro pulldown assay using recombinant GST-c-Src proteins and RIE1-{alpha}5 extracts to determine whether ILK directly interacts with c-Src. ILK from only Fn-adherent (but not suspended) cells bound to the WT GST-c-Src, which was abolished by PP2 treatment (Fig. 3B, left panel). This observation indicates that the binding depended on c-Src activity. c-Src has an SH2 and SH3 domain at its N-terminal end (23). Recombinant GST-SH3 (of c-Src), but not GST alone, showed significant binding to ILK from Fn-adherent (but not suspended) cells, although GST-SH2 (of c-Src) showed just a detectable binding (Fig. 3B, right panel). Next we determined which part of ILK binds to c-Src. Because ILK bound to GST-SH3 (of c-Src), the proline-rich regions of ILK could interface with the c-Src SH3 domain. Human ILK (NP_004508 [GenBank] ) has three proline-rich regions in its kinase domain. The in vitro pulldown assay of the recombinant GST-ILK proteins (Fig. 3C) clearly showed a direct interaction between the intact kinase domain of ILK and c-Src; GST-ILK proteins without the whole or any part of the kinase domain of ILK failed to bind c-Src, whereas GST-ILK proteins with the WT or intact kinase domain itself of ILK bound to c-Src (Fig. 3D). In addition, coimmunoprecipitation of c-Src was next examined from ILK immunoprecipitates of RIE1-{alpha}5 extracts. ILK clearly coimmunoprecipitated c-Src in cells adherent on Fn, but not suspended, and the association was abolished by inactive c-Src(Y416F) expression (Fig. 3E). These observations indicate that ILK can associate with active c-Src. However, kinase-dead ROCK1 expression did not completely abolish the cell adhesion-dependent ILK association with c-Src, indicating again that the ILK·c-Src complex formation is at least in part irrelevant to the ROCK1 activity (Fig. 3E).


Figure 2
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  		FIGURE 2.
The ILK effect depends on c-Src activity. A, ILK-encoding adenovirus (Ad-ILK)-infected RIE1-{alpha}5 cells were pretreated with Me2SO (DMSO) or pharmacological inhibitors 30 min before the cell manipulation to be held in suspension (Sus) or replated on Fn as in Fig. 1A. In the middle of the rolling-over process prior to the cell manipulation, the Me2SO or inhibitor was added directly into the replating medium (serum-free medium with 1% bovine serum albumin) at 20 µM for LY294002, 10 µM for PP2, 40 µM for U0126, 5 µM for p18 peptide, and 15 µM for Y27632. B-D, 1 day after RIE1-{alpha}5 cells were transfected with siRNA against LIMK1 (siLIMK1; B), mock or kinase-dead ROCK1 plasmid (KD-1A; C), siRNA against ILK (siILK; C), or kinase-dead TESK1(D170A) or TESK2(D176A) plasmid (D) for 24 h, the cells were infected with control (Ad-Cont) or ILK-encoding adenovirus for 24 h before the manipulation of cells as in Fig. 1A. A reduced extraction of LIMK1 was observed in Fn-adherent cells, presumably because of less solubilization of LIMK1 upon cell adhesion. E, SYF-/- fibroblasts infected with control (Cont; Y27632) or ILK-encoding adenovirus for 48 h were manipulated without (control) or with pretreatment with the specific ROCK inhibitor Y27632 (15 µM) 30 min before the cell manipulation as in Fig. 1A. F, 1 day after SYF-/- cells were transfected with siRNA against LIMK1, the cells were infected with control or ILK-encoding adenovirus for an additional 24 h before manipulation of the cells as above. Whole cell lysates were prepared and immunoblotted for the indicated molecules. The relative pS3cofilin for diverse experimental conditions was calculated for graphic presentation (mean ± S.D.) after normalization of pS3cofilin over cofilin band intensities measured by densitometry. *, p < 0.05; **, p ≥ 0.05. Data shown represent three isolated experiments.

 
Active c-Src-associated ILK Phosphorylates Cofilin—ILK-enhanced pS3cofilin depended on c-Src activity because pretreatment with PP2 to inhibit c-Src family kinase abolished ILK-enhanced pS3cofilin, whereas treatment with PP3 (a negative control compound of PP2) did not (Figs. 2A and 4A). In addition, ILK bound directly to c-Src (Fig. 3). We thus examined if ILK kinase activity might be modulated via c-Src association by performing an in vitro kinase assay of ILK immunoprecipitates using MBP or GST-cofilin as substrates. ILK immunoprecipitates from adherent, but not suspended, cells efficiently phosphorylated Ser/Thr residues of MBP (Fig. 4B) and Ser3 of recombinant GST-cofilin (Fig. 4C), which was abolished by PP2 treatment of the reaction mixture (Fig. 4, B and C). In addition, ILK immunoprecipitates from adherent cells had a higher pY416c-Src compared with those from suspended cells, which was inhibited by PP2 treatment (Fig. 4C). These observations suggest that active c-Src-associated-ILK caused pS3cofilin. Because interaction of ILK with GST-SH2 (of c-Src) was just detectable (Fig. 3B, right panel), it is likely that the ILK-associated c-Src might phosphorylate and affect the ILK activity. However, we could not observe Tyr phosphorylation in ILK (data not shown), indicating that active c-Src might activate ILK indirectly through an unidentified molecule(s).


Figure 3
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  		FIGURE 3.
The kinase domain of ILK directly binds to the SH3 domain of c-Src. A, RIE1-{alpha}5 cells replated on Fn-precoated coverglasses for 1 h with or without pretreatment with PP2 (10 µM), as explained in the legend to Fig. 2A, were immunostained for ILK and pY416c-Src prior to confocal analysis. Scale bar = 10 µm. B, RIE1-{alpha}5 cell lysates or extracts were prepared from cells manipulated to be suspended (Sus) or replated onto Fn as in Fig. 1A. EGF (50 ng/ml) was treated for the last 5 min of the replating 1-h incubation. An equal amount of protein was incubated with recombinant GST or GST-c-Src proteins bound to glutathione-Sepharose beads at 4 °C with rolling. After three washings with ice-cold phosphate-buffered saline, proteins were eluted from the beads before immunoblotting for the indicated molecules and Ponceau S staining for MBP. GST-SH2 and GST-SH3 are the recombinant GST-fused SH2 and SH3 domains of c-Src, respectively. C, recombinant GST-ILK proteins are shown. AR, ankyrin repeats; PH, pleckstrin homology. Kin or ase depicts the N- or C-terminal part of the ILK kinase domain, which includes two (PR12) or one (PR3) proline-rich region, respectively. D, recombinant GST-ILK proteins were incubated with whole cell lysates (WCL) from Fn-adherent RIE1-{alpha}5 cells. The pulled-down proteins and whole cell lysates were immunoblotted against GST or c-Src as explained under "Experimental Procedures." * indicates proteolytic products. AP, K, or APPR12 depicts the ankyrin repeats plus the pleckstrin homology, the kinase domain, or the ankyrin repeats plus pleckstrin homology and the PR12 of ILK, respectively. E, 1 day after RIE1-{alpha}5 cells were transfected with control (Mock) or inactive c-Src(Y416F) or kinase-dead ROCK1 (KD-1A) plasmid for 24 h, the cells were infected with ILK-encoding adenovirus for an additional 24 h. The cells were then manipulated to be suspended or replated onto Fn as above. An equal amount of whole cell lysates was immunoprecipitated with anti-ILK antibody before immunoblotting of the ILK immunoprecipitates (IP) and whole cell lysates for c-Src and ILK. Data shown represent three independent experiments. WB, Western blot.

 
EGF Treatment Blocks ILK-enhanced pS3cofilin—We next evaluated if extracellular stimulation by EGF affected the adhesion-dependent ILK effects. EGF treatment of cells replated on Fn was performed for the last 5 min of the replating period before cell harvesting and immunoblotting. EGF treatment abolished the basal and ILK overexpression-enhanced pS3cofilin in Fn-adherent cells (Fig. 5A). EGF treatment and/or inactive c-Src(Y416F) expression abolished the basal and ILK-enhanced pS3cofilin in Fn-adherent cells, although EGF-mediated Erk activation in the adherent cells was irrelevant to ILK infection (Fig. 5, A and B). Expression of c-Src(Y527F) (in which the inhibitory phosphorylation site was mutated) did not alter pS3cofilin levels compared with the control vector-transfected cells (Fig. 5C), indicating that cell adhesion-mediated activation of endogenous c-Src could afford ILK/cofilin in the absence of EGF treatment and the EGFR/Erk pathway in the presence of EGF treatment (see "Discussion"). EGF treatment of adherent cells abolished the colocalization between ILK and pY416c-Src (Fig. 5D), ILK binding to WT GST-c-Src or GST-SH3 (of c-Src) (Fig. 3B), and ILK activity for MBP substrate (Fig. 4B). Therefore, the EGF treatment data indicate that the linkage from ILK to cofilin is biologically functional.


Figure 4
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  		FIGURE 4.
Active c-Src-associated ILK phosphorylates cofilin. A, cells infected with either control (Ad-Cont) or ILK-encoding (Ad-ILK) adenovirus for 48 h were pretreated with or without 10 µM PP2 or PP3 (a negative control of PP2) 30 min before the cell manipulation to be suspended (Sus) or replated on Fn as above. Whole cell lysates from RIE1-{alpha}5 cells under diverse conditions were immunoblotted for the indicated molecules. B, RIE1-{alpha}5 cells were infected with control (C) or ILK-encoding (+) adenovirus for 48 h before the cell manipulation for suspension or replating on Fn with or without PP2 pretreatment or EGF treatment as above. C, alternatively, RIE1-{alpha}5 cells were manipulated to be suspended or replated on Fn with Me2SO (DMSO) or PP2 pretreatment as above. The whole cell lysates from the cells were prepared and used in immunoprecipitation with anti-ILK antibody. An equal amount of the ILK immunoprecipitates was mixed with MBP (B) or recombinant GST-cofilin (C) as substrate for in vitro kinase assay. The resultant reaction mixtures and lysates were resolved and immunoblotted for the indicated molecules. MBP was stained with Ponceau S, and GST-cofilin was immunoblotted with anti-GST antibody for equal amounts of substrates. The relative pS3cofilin for diverse experimental conditions was calculated for graphic presentation (mean ± S.D.) after normalization of pS3cofilin over cofilin band intensities measured by densitometry. *, p < 0.05; **, p ≥ 0.05. Data shown represent three isolated experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Integrin-mediated cell adhesion triggers diverse intracellular signals leading to actin polymerization. ILK binds to integrin β subunits by forming a modular platform for protein complexes to rearrange actin filaments (24). Meanwhile, cofilin severs actin filaments, depending on its Ser3 phosphorylation status (7, 25). Although ILK and cofilin are both involved in the actin organization, their functional linkage is largely unknown. Our observations in this study provide evidence for a new functional linkage of ILK to cofilin for actin polymerization, showing that association between ILK and c-Src affected ILK activity to phosphorylate cofilin during cell adhesion.

Although integrin-mediated cell adhesion is suggested to involve ILK·paxillin·FAK complexes at the focal adhesions (26), ILK-enhanced pS3cofilin was irrelevant to FAK, paxillin, and Akt phosphorylation. The ILK(E359K) mutant deficient for paxillin and parvin binding (18) and putatively kinase-dead with residual kinase activity (19) still supported the ILK-enhanced pS3cofilin, but the ILK(S343A) mutant with abolished kinase activity (20) blocked the ILK-mediated effects. Therefore, the ILK effect on cofilin phosphorylation appeared to require its activity.

Cofilin is known to be phosphorylated by LIMKs or TESKs (13, 22). Furthermore, ROCK1 phosphorylates LIMKs (21). Interestingly, the adhesion-dependent pS3cofilin in RIE1-{alpha}5 cells without exogenous ILK expression in this study could be regulated by ROCK1, LIMK1, TESKs, and ILK as well; transient transfection of kinase-dead ROCK1 or TESKs or of siRNAs against LIMK1 or ILK abolished cell adhesion-dependent pS3cofilin. However, our results indicate that ILK overexpression-mediated pS3cofilin did not significantly involve ROCK1, LIMK1, and TESKs. The basal cell adhesion-dependent pS3cofilin was abolished by expression of kinase-dead ROCK1, but ILK overexpression-mediated pS3cofilin was still significantly maintained. Kinase-dead ROCK1 expression did not disrupt the association between ILK and c-Src. It is thus likely that ILK affects pS3cofilin, at least partially, in a ROCK1-independent manner. In addition, pT508LIMK1 was not correlated with the ILK effects, and furthermore, LIMK1 suppression did not significantly inhibit the ILK overexpression-mediated pS3cofilin. These observations indicate that ILK overexpression-mediated pS3cofilin in RIE1-{alpha}5 cells might be irrelevant to LIMK1. Meanwhile, {alpha}-parvin (actopaxin) binds ILK, paxillin (27), and TESK1 to cause cellular spreading on Fn (28), indicating that ILK can be relevant to TESKs. However, transfection of the kinase-inactive TESK1 and TESK2 mutants abolished the basal, but not ILK overexpression-enhanced, pS3cofilin of Fn-adherent cells, indicating that ILK enhanced pS3cofilin in a TESK-independent manner. However, the ILK overexpression-mediated pS3cofilin appeared to be dependent on c-Src activity in adherent cells; pharmacological inhibitor treatment or kinase-inactive c-Src transfection abolished ILK-mediated pS3cofilin in Fn-adherent cells. On the other hand, pS3cofilin in c-Src-deficient SYF-/- cells was not dependent on ILK (overexpression) but required LIMK1. Therefore, these observations suggest that active c-Src may be involved in the ILK-mediated pS3cofilin.

ILK binds diverse molecules to rearrange actin filaments during integrin-mediated cell adhesion (26), although the interaction of ILK with c-Src or cofilin remains relatively unknown. Consistent with the observations that ILK enhanced pS3cofilin in a c-Src activity-dependent manner, in vitro pulldown analysis revealed that the kinase domain of ILK interacted directly with the SH3 domain of c-Src. The pY416c-Src of ILK immuno-precipitates from adherent cells was observed to be higher than that from suspended cells. Furthermore, in vitro kinase assays also supported that ILK could interact with and phosphorylate GST-cofilin, which was abolished by the inhibition of c-Src that was presumably associated with ILK. Although the kinase domain of ILK is involved in many protein interactions for actin reorganization (19), the interaction of ILK with c-Src is for the first time revealed and indicates a new signaling linkage to regulate cofilin phosphorylation and actin polymerization during adhesion of RIE1-{alpha}5 cells. The ILK-enhanced pS3cofilin seems not to be biologically functional or significant in the signal contexts that c-Src is not significantly activated or regulated, however, because we observed that pS3cofilin in c-Src/Yes/Fyn-deficient SYF-/- cells was regulated by LIMK1, but not by ILK·c-Src linkage.


Figure 5
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  		FIGURE 5.
EGF treatment abolishes ILK-enhanced pS3cofilin upon cell adhesion. A, RIE1-{alpha}5 cells were infected with either control (Ad-Cont) or ILK-encoding (Ad-ILK) adenovirus for 48 h and then manipulated to be suspended (Sus) or replated on Fn with or without EGF treatment as above. B and C, 1 day after cells were transfected with control (Mock), inactive c-Src(Y416F) (B and C), or active c-Src(Y527F) (C) plasmid, the cells were infected with either control or ILK-encoding adenovirus for an additional 24 h before the cell manipulation to be suspended and replated on Fn with or without EGF treatment as above. Whole cell lysates were prepared and used in immunoblotting for the indicated molecules (A-C). The -fold differences in pS3cofilin for diverse experimental conditions were calculated after normalization of pS3cofilin over cofilin band intensities measured by densitometry, with raising fractions not lower than 0.05-0.1 for a concise representation. D, RIE1-{alpha}5 cells replated on Fn-precoated coverglasses with or without EGF treatment as above were doubly immunostained for ILK and pY416c-Src prior to confocal microscopy analysis. Scale bar = 10 µm. Data shown represent three different experiments.

 
Interestingly, EGF treatment abolished the basal and ILK overexpression-enhanced pS3cofilin in Fn-adherent RIE1-{alpha}5 cells. Signaling cross-talks between the integrin and the growth factor receptor (29) can be regulated by ILK (30). This finding was thus unexpected because EGF treatment is mitogenic and positive for Akt and GSK3β activation (31) and because EGF signaling requires c-Src (32). Previous reports showed that EGF treatment or EGFR activity was negatively or positively correlated with pS3cofilin, indicating contradictory roles of EGFR signaling in pS3cofilin (33, 34). The observation of no significant change in Erks even with c-Src(Y416F) expression, which led to the blockade of ILK-mediated pS3cofilin, supports the idea that there can be two distinct pools of active c-Src involved in either ILK-mediated pS3cofilin or the EGFR/Erk pathway; cell adhesion-mediated activation of endogenous c-Src could support ILK/cofilin in the absence of EGF treatment and the EGFR/Erk pathway in the presence of EGF treatment (Fig. 5C). We may thus speculate that EGF treatment-dependent intracellular signal pathways might extract active c-Src from the ILK·c-Src·cofilin complex or linkage, leading to down-regulation of ILK-mediated Ser3 phosphorylation of cofilin.

In neutrophils, cofilin was dephosphorylated by treatment with diverse chemoattractants via phosphoinositide-specific phospholipase C-mediated protein kinase C and Ca2+ pathways (35). EGF can act as a chemoattractant for spatial and temporal regulation of the actin cytoskeleton via modulation of actin binding proteins, such as cofilin. ILK is a Ser/Thr kinase binding to the β1 integrin cytoplasmic tail at focal adhesions (8). With dynamic focal adhesion turnover and membrane ruffling around the leading edge of a migrating cell, integrin-mediated adhesion may lead to activation of ILK to regulate cofilin phosphorylation and activity. EGF treatment caused the activation of cofilin to sever actin filaments to result in early generation of free barbed ends that participate in the nucleation of actin polymerization at the leading edges of migratory MTLn3 cells (36). Therefore, it may not be ruled out that ILK·c-Src complex-mediated regulation of cofilin phosphorylation and activity participates in the severing of actin filaments to generate their free barbed ends at the leading edges of RIE1-{alpha}5 cells, although regulation of cofilin activity by phospholipid was recently shown to occur at the leading edge of carcinoma MTLn3 cells (37). More detailed understanding of the significance of the ILK·c-Src complex-mediated cofilin phosphorylation in dynamic actin remodeling can have important implications for our comprehension of physiological and pathological processes, such as cell adhesion/migration and cancer metastasis.


   FOOTNOTES
 
* This work was supported by the Korea Science and Engineering Foundation Grant from the Korean Government (Ministry of Science and Technology) (Grant R13-2007-019-00000-0 to S.-H. K. and Cell Dynamics Research Center Grant R11-2007-007-01004-0 to J. W. L.). 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

1 To whom correspondence may be addressed. Tel.: 82-31-201-2179; Fax: 82-31-205-1074; E-mail: sungkim7{at}khu.ac.kr.

2 To whom correspondence may be addressed: Cancer Research Inst., College of Medicine, Seoul National University, Seoul 110-799, Korea. Tel.: 82-2-3668-7030; Fax: 82-2-766-4487; E-mail: jwl{at}snu.ac.kr.

3 The abbreviations used are: FAK, focal adhesion kinase; WT, wild-type; ILK, integrin-linked kinase; Fn, fibronectin; TESK, testicular protein kinase; GST, glutathione S-transferase; EGF, epidermal growth factor; EGFR, EGF receptor; siRNA, small interfering RNA; MBP, myelin basic protein; SH, Src homology; pS3cofilin, Ser3-phosphorylated cofilin; pY397FAK, Tyr397-phosphorylated FAK; pY416c-Src, Tyr416-phosphorylated Src; pS473Akt, Ser473-phosphorylated Akt; pT508LIMK1, Thr508-phosphorylated LIMK1; ROCK, Rho-associated kinase; LIMK, LIM kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Sarah Short (Children's Hospital Boston and Harvard Medical School) for editorial help and discussion.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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