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© The Rockefeller University Press,
0021-9525/2001//585 $5.00
The Journal of Cell Biology, Volume 153, Number 3,
, 2001 585-598
Original Article |
A New Focal Adhesion Protein That Interacts with Integrin-Linked Kinase and Regulates Cell Adhesion and Spreading
carywu{at}imap.pitt.edu
Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti–CH-ILKBP antibodies, and green fluorescent protein–CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP–ILK–PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.
Key Words: focal adhesion • integrin-linked kinase • calponin homology • integrins • cell adhesion
© 2001 The Rockefeller University Press
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Introduction |
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-actinin, talin, and paxillin are recruited to FAs in response to cell adhesion (Parsons et al. 1994; Ilic et al. 1997; Turner 1998). Consistent with the fundamental roles of cell matrix adhesions in control of cell behavior, many components of the FAs are well conserved in different organisms ranging from nematodes and insects to vertebrates (Hynes and Zhao 2000). Integrin-linked kinase (ILK) is an evolutionally conserved FA protein that is involved in the integrin-mediated processes (Hannigan et al. 1996; Dedhar et al. 1999; Li et al. 1999; Wu 1999). ILK participates in the regulation of cell adhesion, growth, gene expression, differentiation, cell shape change, and ECM assembly. Furthermore, recent genetic studies in Caenorhabditis elegans [Mackinnon, A.C., and B.D. Williams. 2000. 40th American Society for Cell Biology Annual Meeting. 2664. (Abstr.)] and Drosophila (Zervas et al. 2001) have demonstrated that lack of ILK expression results in phenotypes resembling that of integrin-null mutants, suggesting that ILK is indispensable for integrin function during embryonic development.
Although it is clear that ILK is an important component of FAs, how ILK functions in FA is not completely understood. At the molecular level, ILK consists of three structurally distinct motifs. At the NH2 terminus of ILK lie four ankyrin (ANK) repeats, which are responsible for binding to PINCH (Tu et al. 1999). The PINCH binding is required for FA localization of ILK (Li et al. 1999). In addition, it connects ILK to other proteins including Nck-2, a SH2/3-containing adaptor protein involved in the growth factor and small GTPase signaling (Tu et al. 1998). The importance of the ILK–PINCH interaction is underscored by recent genetic studies in C. elegans (Hobert et al. 1999), showing that lack of PINCH causes a phenotype that is identical to that of null mutation of β-integrin/pat-3 or ILK/pat-4. COOH-terminal to the ANK repeat domain is a pleckstrin homology-like motif that likely binds PtdIns(3,4,5)P3 and participates in the regulation of the kinase activity (Delcommenne et al. 1998). ILK COOH-terminal domain exhibits significant homology to other kinase catalytic domains (Dedhar et al. 1999). ILK phosphorylates several key proteins including PKB/Akt and glycogen synthase kinase 3 and regulates their activation (Delcommenne et al. 1998). In addition to catalyzing serine/threonine phosphorylation, the multidomain structure and FA localization of ILK suggest that it could potentially function as a scaffolding protein mediating multiple protein–protein interactions. To test this hypothesis, we have carried out studies aimed at identifying additional ILK-binding proteins. We report here the identification of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP) that is widely expressed and highly conserved among different organisms. Furthermore, we have characterized the interaction between ILK and CH-ILKBP and show that CH-ILKBP, ILK, and PINCH form a complex in cells. Additionally, we demonstrate that CH-ILKBP localizes to FAs and the ILK-binding activity is required for the FA localization. Finally, we provide functional evidence showing that CH-ILKBP is critically involved in the regulation of cell adhesion and spreading.
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Northern Blot
A 32P-labeled CH-ILKBP cDNA probe was prepared by labeling a human CH-ILKBP cDNA fragment (encoding residues 1–229) using a random-primed DNA labeling kit (Boehringer). A blot containing equal amount (2 µg /lane) of polyA+ RNA from different human tissues (CLONTECH Laboratories, Inc.) was hybridized with the 32P-labeled CH-ILKBP probe following the manufacturer's protocol.
Site-directed Mutagenesis
A QuickChangeTM site-directed mutagenesis system (Stratagene) was used to change F at position 271 to D. The point mutation was confirmed by DNA sequencing.
Generation of Glutathione-S-Transferase– and Maltose-binding Protein–CH-ILKBP Fusion Proteins
DNA fragments encoding CH-ILKBP sequences were prepared by PCR and inserted into the EcoRI/XhoI sites of a pGEX-5x-1 vector (Amersham Pharmacia Biotech) or the EcoRI/SalI sites of a pMAL-C2 vector (New England BioLabs, Inc.). The recombinant vectors were used to transform Escherichia coli cells. The expression of the glutathione S-transferase (GST) and maltose-binding protein (MBP) fusion proteins was induced with IPTG, and they were purified by affinity chromatography using glutathione-Sepharose 4B and amylose-agarose (Tu et al. 1999), respectively.
GST Fusion Protein Pull-Down Assays
Mouse C2C12 cells were lysed with 1% Triton X-100 in 20 mM Tris HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 2 mM AEBSF, 5 µg/ml pepstatin A, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. The lysates were precleared and then incubated with equal amounts of GST–CH-ILKBP fusion proteins or GST for 2 h at 4°C. GST and GST fusion proteins were precipitated with glutathione-Sepharose beads and ILK was detected by Western blotting with anti-ILK antibody 65.1 (5 µg/ml).
Generation of Monoclonal Anti–CH-ILKBP Antibodies
Mouse monoclonal anti–CH-ILKBP antibodies were prepared using GST fusion protein containing CH-ILKBP residues 29–372 as an antigen based on a previously described method (Tu et al. 1998). Hybridoma supernatants were initially screened for anti–CH-ILKBP activities by ELISA and Western blotting using MBP–CH-ILKBP29–372. Antibodies that recognize MBP–CH-ILKBP29–372 were selected and further tested by Western blotting using GST–CH-ILKBP fusion proteins and mammalian cell lysates.
Expression of FLAG– and Green Fluorescent Protein–CH-ILKBP Fusion Proteins in Mammalian Cells
DNA fragments encoding CH-ILKBP sequences were cloned into the EcoRI/KpnI sites of the p3XFLAG–CMV-14 vector (Sigma-Aldrich) or those of the pEGFP-N1 vector (CLONTECH Laboratories, Inc.). C2C12 cells, rat mesangial cells, and CHO K1 cells were transfected with the vectors using LipofectAmine PLUS (Life Technologies) (Huang et al. 2000).
Coimmunoprecipitation Assays
To immunoprecipitate endogenous CH-ILKBP, C2C12 cells were lysed with 1% Triton X-100 in the Hepes buffer (50 mM Hepes, pH 7.1, 150 mM NaCl, 10 mM Na4P2O7, 2 mM Na3VO4, 100 mM NaF, 10 mM EDTA) containing protease inhibitors. The cell lysates (750 µg) were incubated with 750 µl of hybridoma culture supernatant containing anti–CH-ILKBP antibody 1D4 or 750 µl of unconditioned medium as a control for 2 h. The samples were then mixed with 50 µl of UltraLink immobilized protein G (Pierce Chemical Co.). After incubation for 2 h, the beads were washed four times, and the proteins bound were released from the beads by boiling in 75 µl of SDS-PAGE sample buffer for 5 min. The samples (10 µl/lane) were analyzed by Western blotting with anti–CH-ILKBP antibody 3B5, anti-ILK antibody 65.1, antipaxillin antibody (clone 349; Transduction Laboratories), and rabbit polyclonal anti-PINCH antibodies, respectively.
To immunoprecipitate FLAG-tagged, wild-type, or mutant forms of CH-ILKBP, lysates (prepared as described above) of the C2C12 transfectants (500 µg lysates) or the CHO transfectants (750 µg lysates) were mixed with 70 µl agarose beads conjugated with anti-FLAG antibody M2 (Sigma-Aldrich). The precipitated proteins were released from the beads by boiling in 50 µl of SDS-PAGE sample buffer for 5 min and analyzed by Western blotting (10 µl/lane).
To immunoprecipitate FLAG-PINCH, C2C12 cells were transfected with pFLAG–CMV-2/PINCH, which was generated by inserting the full-length PINCH cDNA into the BglII/SalI sites of the pFLAG–CMV-2 vector (Sigma-Aldrich) using LipofectAmine2000 (Life Technologies). 24 h after transfection, the cells were lysed with 1% Triton X-100 in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM Na3VO4, and 100 mM NaF containing protease inhibitors. The lysates (1 mg) were mixed with 30 µl agarose beads conjugated with anti-FLAG antibody M2. The precipitated proteins were released from the beads by boiling in 60 µl of SDS-PAGE sample buffer for 5 min and analyzed by Western blotting (10 µl/lane).
To immunoprecipitate paxillin, C2C12 lysates (500 µg) were mixed with 2 µg of antipaxillin antibody or 2 µg of irrelevant mouse IgG. The immune complexes were precipitated with 50 µl of UltraLink immobilized protein G, and the proteins bound were released from the beads by boiling in 50 µl of SDS-PAGE sample buffer for 5 min. The samples (10 µl/lane) were analyzed by Western blotting.
Immunofluorescence Staining of Cells
Immunofluorescence staining was performed as described (Li et al. 1999). In brief, cells were plated on fibronectin-coated coverslips, fixed, and stained with mouse anti–CH-ILKBP antibody 1D4 (hybridoma supernatant), mouse anti-FLAG antibody M2 (4 µg/ml), rabbit anti-FAK antibody (4 µg/ml) (Santa Cruz Biotechnology, Inc.), and/or rabbit anti–β-catenin antiserum (1:2,000 dilution) (Sigma-Aldrich). The mouse and rabbit antibodies were detected with a fluorescein-conjugated anti–mouse IgG antibody (28 µg/ml) and a Rhodamine redTX-conjugated anti–rabbit IgG antibody (10 µg/ml), respectively. In control experiment, no specific staining was observed with unconditioned medium or with ID4 supernatants that were preincubated with 1 µM GST–CH-ILKBP for 1 h. For staining of cells expressing green fluorescent protein (GFP)–CH-ILKBP fusion proteins, anti-ILK and FAK antibodies were detected with Rhodamine redTX-conjugated anti–mouse and anti–rabbit IgG antibodies, respectively. Actin stress fibers were visualized by staining the cells with rhodamine-labeled phalloidin.
Isolation of Cytoskeleton Fractions
Cytoskeleton fractions were isolated from C2C12 cells as described (Yang et al. 1994). In brief, C2C12 cell monalayers were extracted with 0.7 ml of cold PBS containing 1% Trion X-100 and protease inhibitors. The Triton X-100–insoluble material (cytoskeletal fractions) remaining on the dishes was extracted with equal volume (0.7 ml) of PBS containing 1% SDS and protease inhibitors. The distributions of CH-ILKBP and other FA proteins were analyzed by Western blotting (30 µl/lane).
Cell Adhesion and Spreading Assays
For assays using CHO cells, the cells were transfected with FLAG vectors encoding CH-ILKBP, CH2 (residues 222–372), CH-ILKBP F271
D point mutant, CH2 deletion mutant (residues 1–229), or a FLAG vector lacking CH-ILKBP sequence using LipofectAmine PLUS. Cell adhesion assays were performed as described (Wu et al. 1995). In brief, the transfectants were harvested with trypsin 1 d after transfection and washed twice with -MEM containing 10% FBS, twice with serum-free Opti-MEM (Life Technologies), and kept in suspension for 30 min. The cells (3 x 104/well) were seeded in collage IV–coated 96-well plates (Becton Dickinson). After incubation at 37°C under a 5% CO2–95% air atmosphere for 90 min, the wells were washed three times with PBS. The numbers of adhered cells were quantified by measuring N-acetyl-β-D-hexosaminidase activity (Landegren 1984).
For assays using C2C12 cells, the cells were transfected with FLAG vectors encoding CH-ILKBP (FLAG–CH-ILKBP), CH2 (residues 222–372) (FLAG-CH2), CH2 deletion mutant (residues 1–229) (FLAG-
CH2), or a FLAG control vector using LipofectAmine PLUS. C2C12 cells stably expressing the FLAG-tagged proteins were selected with 1 mg/ml of G418 (Life Technologies). Two independent clones expressing each form of CH-ILKBP were isolated. C2C12 cells expressing FLAG-CHILKBP (clones A28 and A31), FLAG-CH2 (clones B20 and B45), FLAG-CH2 (clones C31 and C46), and the vector control cells were harvested with trypsin. The cells were washed twice with DME containing 10% FBS and twice with DME containing 1% BSA. The cells were kept in suspension for 30 min and then seeded in 12-well plates coated with collagen I (Becton Dickinson). The cells were allowed to adhere at 37°C for 30 min, and four randomly selected fields were photographed. The plates were then washed twice with PBS, and four randomly selected fields were photographed after the wash. The numbers of the total cells (before wash) and the adhered cells (after wash) from the four randomly selected fields were counted manually. The percentage of cell adhesion is presented as the number of adhered cells divided by the number of total cells. In addition to manual counting, we also measured cell adhesion using the hexosaminidase method and obtained similar results.
For cell spreading, C2C12 cells expressing different forms of CH-ILKBP were prepared as described above and seeded in 12-well plates coated with collagen I. The plates were incubated at 37°C under a 5% CO2-95% air atmosphere for different periods of time. The cell morphology (phase–contrast image) was recorded with a DVC-1310C MagnafireTM digital camera (Optronics). Unspread cells were defined as round phase–bright cells, whereas spread cells were defined as cells with extended processes, lacking a rounded morphology and not phase–bright (Komoriya et al. 1991; Richardson et al. 1997). The percentage of cells adopting spread morphology was quantified by analyzing 
300 cells from three randomly selected fields (>100 cells/field).
Online Supplemental Material
Figure S1: Association of FLAG–CH-ILKBP, FLAG-F271D, and FLAG-CH2 with Triton X-100–insoluble cytoskeleton fractions. Figure S2: CH-ILKBP binds to ILK but not paxillin in rat embryo fibroblasts (REF-52). Figure S3: Immunofluorescence staining of GFP–CH-ILKBP–expressing cells with monoclonal antipaxillin antibody. Supplemental figures available at http://www.jcb.org/cgi/content/full/153/3/585/DC1.
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-actinin, filamin, and other actin-binding proteins. Proteins that are structurally closely related are present in other organisms including C. elegans (Fig. 1 A), suggesting that CH-ILKBP, like ILK and PINCH, represents an ancient protein.
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The CH2 Domain Mediates the Interaction with ILK
To confirm the interaction between CH-ILKBP and ILK, we generated GST fusion proteins containing the full-length (Fig. 2 A, lane 1) and the COOH-terminal region (N-ter, residues 29–372) (Fig. 2 A, lane 7) of CH-ILKBP and tested their ability to bind ILK. Both GST–CH-ILKBP (Fig. 2 B, lane 1) and GST–N-ter (29–372) (Fig. 2 B, lane 7), but not GST alone (Fig. 2 B, lane 8), interacted with ILK. These results are consistent with those obtained in the yeast two-hybrid binding assays and confirm that CH-ILKBP forms a complex with ILK in vitro and in yeast cells.
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CH-ILKBP Associates with ILK in Mammalian Cells
To facilitate studies on CH-ILKBP in mammalian cells, we generated mouse monoclonal anti–CH-ILKBP antibodies. Two antibodies (1D4 and 3B5), which recognize GST–CH-ILKBP (Fig. 3A and Fig. B, lane 2) and MBP–CH-ILKBP29–372 (data not shown) but not GST (Fig. 3A and Fig. B, lane 1), were further characterized. 1D4 recognizes an epitope located within the link region between the two CH domains of CH-ILKBP (Fig. 3 A), whereas 3B5 recognizes an epitope located within the NH2-terminal region (residues 1–229) (Fig. 3 B). To test whether ILK interacts with CH-ILKBP in mammalian cells, we immunoprecipitated CH-ILKBP from C2C12 cell lysates with anti–CH-ILKBP antibody 1D4. Western blotting analyses showed that ILK (Fig. 3 C, lane 2) was coprecipitated with CH-ILKBP (Fig. 3 D, lane 2). Probing the same samples with an antipaxillin antibody failed to detect paxillin in the anti–CH-ILKBP immunoprecipitates (Fig. 3 E, lane 2), despite the presence of abundant paxillin in the cell lysate (Fig. 3 E, lane 1). In additional control experiments, neither CH-ILKBP nor ILK was precipitated in the absence of the anti–CH-ILKBP antibody (Fig. 3C and Fig. D, lane 4). Thus, consistent with the ILK–CH-ILKBP interaction detected in yeast cells and in vitro, ILK and CH-ILKBP form a complex in mammalian cells.
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To analyze the subcellular localization of endogenous CH-ILKBP, we stained mammalian cells with monoclonal anti–CH-ILKBP antibodies. The results showed that endogenous CH-ILKBP (Fig. 6 A) like FLAG–CH-ILKBP (Fig. 5E and Fig. G) was clustered in FAs where FAKs were also detected (Fig. 6 B). Clusters of CH-ILKBP were concentrated at the ends of actin stress fibers (Fig. 6C and Fig. D). In epithelial cells that had formed cell–cell contacts, CH-ILKBP localized to cell matrix FAs but not to cell–cell adhesions where abundant β-catenin was detected (Fig. 6E and Fig. F). In control experiments, incubation of the anti–CH-ILKBP antibody with GST–CH-ILKBP eliminated the staining (data not shown), confirming the specificity of the antibody staining. The observation that CH-ILKBP is concentrated at the ends of actin stress fibers suggested a possibility that CH-ILKBP is physically associated with the cytoskeleton fractions. To test this, we isolated cytoskeleton fractions from C2C12 cells. Under the conditions used, 
5–10% of CH-ILKBP was found in the Triton X-100–insoluble cytoskeleton fractions (Fig. 6 G). As a comparison, we found that a similar percentage of FAK (Fig. 6 H), an abundant component of FAs, and no detectable amount of extracellular signal–regulated kinase (ERK) (Fig. 6 I), a more dynamic component of FAs (Fincham et al. 2000), was present in the cytoskeleton fractions.
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CH2), and the CH2 fragment (GFP-CH2) in mammalian cells. Consistent with the results obtained with anti–CH-ILKBP antibody (Fig. 6) and FLAG–CH-ILKBP (Fig. 5), GFP–CH-ILKBP localized to FAs where ILK (Fig. 7A and Fig. B) and FAK (data not shown) were clustered. Deletion of the ILK-binding CH2 domain eliminated its ability to localize to FAs (Fig. 7C and Fig. D), indicating that the ILK-binding CH2 domain is required for the FA localization. Furthermore, GFP fusion protein containing the CH2 fragment was able to cocluster with ILK in FAs (Fig. 7E and Fig. F), albeit the level of fluorescence was lower than that of GFP–CH-ILKBP (Fig. 7, compare A and E). Taken together, these results suggest that the ILK-binding CH2 domain mediates the FA localization of CH-ILKBP.
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CH2 (lane 5), formed a complex with ILK (Fig. 9 B) and PINCH (Fig. 9 D) in CHO cells. Consistent with the results obtained with C2C12 cells (Fig. 3 E and Fig. 5 C), paxillin was not associated with the FLAG-tagged wild-type or mutant forms of CH-ILKBP in CHO cells (Fig. 9 C, lanes 2–5), despite the presence of abundant paxillin in these cells (Fig. 9 C, lanes 6–9). To analyze the effect of overexpression of the different forms of CH-ILKBP on cell adhesion, we plated the CHO cells on collagen IV–coated 96-well plates and quantified the number of adhered cells. The results showed that overexpression of FLAG–CH-ILKBP did not significantly alter cell adhesion (Fig. 9 E). However, overexpression of the ILK-binding defective CH-ILKBP point mutant F271D or the ILK-binding CH2 fragment, but not that of the NH2-terminal fragment lacking CH2, significantly reduced cell adhesion (Fig. 9 E), suggesting that CH-ILKBP, through interactions with ILK and other proteins, plays an important role in the regulation of cell adhesion.
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CH2 in these cells was confirmed by Western blotting (Fig. 10 A). Analyses of the cell adhesion showed that although C2C12 cells overexpressing CH-ILKBP adhered to collagen I as efficiently as the vector control cells, C2C12 cells overexpressing the ILK-binding CH2 fragment adhered much less efficiently (Fig. 10 B). By contrast, overexpression of the CH2 deletion NH2-terminal fragment did not significantly alter the efficiency of cell adhesion (Fig. 10 B). Noticeably, the spreading of the C2C12 cells overexpressing the ILK-binding CH2 fragment was much slower than that of the vector control cells or those overexpressing FLAG–CH-ILKBP or FLAG-CH2 (Fig. 10C and Fig. d). Staining of the CH2-overexpressing cells with phalloidin reveled that the actin stress fiber formation was retarded (Fig. 10 E). Thus, consistent with the ILK-binding activity and FA localization of CH-ILKBP, CH-ILKBP is critically involved in the regulation of cell adhesion, actin cytoskeleton organization, and cell shape change.
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CH2 or F271D) with the Triton X-100–insoluble cytoskeleton was significantly reduced (our unpublished preliminary data). Using cells that were either transiently or stably overexpressing mutant forms of CH-ILKBP, we have obtained important functional evidence supporting this model. Cells overexpressing the ILK-binding CH2 fragment exhibited significant reductions in cell matrix adhesion, actin stress fiber formation, and spreading. However, it is interesting to note that the adhesion and spreading of the cells overexpressing the ILK-binding CH2 fragment were only partially impaired. Besides the technical considerations (for example, the transfection efficiency or the expression level of the CH2 fragment), this likely reflects the fact that there are multiple protein complexes, including those containing talin, filamin, or -actinin, that can physically link the ECM and transmembrane receptors to the intracellular actin cytoskeleton (Jockusch et al. 1995; Burridge and Chrzanowska-Wodnicka 1996; Calderwood et al. 2000).
Cell adhesion is an essential cellular function of all multicellular organisms. Recent studies suggest that cells in different organisms ranging from nematodes to human use strikingly similar strategies to mediate this fundamental process (Hynes and Zhao 2000). In genetic model system C. elegans, mutations in genes encoding integrin (pat-2) or β (pat-3) cause a PAT phenotype characterized by defects in muscle dense body and M-lines, which resemble FAs in mammalian cells (Williams and Waterston 1994; Gettner et al. 1995). Recent genetic studies have shown that lack of PAT-4–ILK [Mackinnon, A.C., and B.D. Williams. 2000. 40th American Society for Cell Biology Annual Meeting. 2664. (Abstr.)]or UNC-97–PINCH (Hobert et al. 1999) in C. elegans results in a phenotype that is identical to that of the integrins. These studies provide strong genetic evidence for a crucial role of the ILK and its binding partner PINCH in the integrin-mediated cell adhesion. Like ILK and PINCH, CH-ILKBP is an ancient protein with homologues found in different organisms including C. elegans. Human CH-ILKBP shares significant sequence similarity with a predicted C. elegans protein (T21D12.4 protein) (Fig. 1 A). Recently, Lin and Williams [Lin, X., and B.D. Williams. 2000. 40th American Society for Cell Biology Annual Meeting. 2666. (Abstr.)] presented genetic evidence showing that C. elegans gene T21D12.4 corresponds to another PAT gene, pat-6. Thus, the CH-ILKBP–ILK–PINCH complex described in this paper likely represents an evolutionally well-conserved link between cell adhesion receptors and the actin cytoskeleton. Zervas et al. 2001 reported recently that mutations in Drosophila ILK cause defects similar to loss of integrin adhesion, and the actin filaments were detached from the membrane at the muscle attachment sites in these mutants. Given the fact that homologues of ILK, CH-ILKBP, and PINCH are present in Drosophila, a CH-ILKBP–ILK–PINCH complex that is analogous to the complex in mammalian cells likely exists in Drosophila and provides an essential connection linking the actin cytoskeleton to the adhesion sites in Drosophila and in other organisms.
The data obtained in this and other studies suggest that ILK is a protein with dual functions. The first function is to provide a molecular scaffold for the assembly of the PINCH–ILK–CH-ILKBP complex as shown in this study, which together with other interactive proteins at FAs links ECM to the actin cytoskeleton. The second function is to serve as a protein kinase in the regulation of gene expression, survival, differentiation, and proliferation (Hannigan et al. 1996; Radeva et al. 1997; Delcommenne et al. 1998; Novak et al. 1998; Wu et al. 1998; Huang et al. 2000; Persad et al. 2000). Given the importance of compartmentalization in kinase action during signal transduction, it is likely that the protein–protein interactions mediated by ILK, including that between ILK and CH-ILKBP, are involved not only in cell adhesion, actin cytoskeleton organization, and spreading as shown in this study but also in other ILK-regulated processes.
Northern blotting analyses indicate that CH-ILKBP is a member of a family of structurally related proteins (Fig. 1 B). When this manuscript was in preparation, Nikolopoulos and Turner 2000 described a new paxillin- and actin-binding protein termed actopaxin. Actopaxin is structurally related to CH-ILKBP (human actopaxin is 98% identical to human CH-ILKBP at the amino acid level and 90% identical at the cDNA level). Because the difference between actopaxin and CH-ILKBP cDNA sequences is significant and occurs throughout the sequences, they are likely encoded by two different genes. However, the striking similarity in protein sequences suggests that they may share certain common functions. Actopaxin contains a potential ILK-binding sequence that is identical to that in CH-ILKBP, suggesting that actopaxin may also function as an ILK-binding protein. However, it is important to note that although CH-ILKBP contains a potential paxillin-binding sequence as defined by Nikolopoulos and Turner 2000, we could not detect an association between paxillin and CH-ILKBP in vivo, despite using several different cell types, including mouse C2C12 cells (Fig. 3 and Fig. 5), CHO cells (Fig. 9), human kidney 293 cells, and rat embryo fibroblasts (REF-52) (our unpublished results), and multiple experimental approaches (immunoprecipitation of endogenous or epitope-tagged CH-ILKBP, PINCH, or paxillin). On the other hand, in all of the cell types analyzed, we have readily detected a stable CH-ILKBP–ILK–PINCH complex. Thus, although our results do not rule out the possibility that paxillin could bind to CH-ILKBP in vitro or under certain other conditions, they do suggest that CH-ILKBP functions primarily as an ILK-binding protein in cells.
In summary, we have identified a new ILK-binding protein and have shown that it plays an important role in cell adhesion and spreading. The data presented here provide new insights into the molecular mechanism by which ILK functions at adhesion sites. The CH-ILKBP–ILK–PINCH complex described in this report likely functions as an important scaffold in the assembly and signaling through cell matrix adhesion sites.
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Submitted: 30 January 2001
Revised: 6 March 2001
Accepted: 21 March 2001
The online version of this article contains supplemental material.
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