Correspondence to: Chuanyue Wu, 707B Scaife Hall, Dept. of Pathology, Univ. of Pittsburgh, 3550 Terrace St., Pittsburgh, PA 15261. Tel:(412) 648-2350 Fax:(509) 561-4062 E-mail:carywu{at}imap.pitt.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 antiCH-ILKBP antibodies, and green fluorescent proteinCH-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-ILKBPILKPINCH 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
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell adhesion to the extracellular matrix (ECM)1 is mediated primarily by integrins (-actinin, talin, and paxillin are recruited to FAs in response to cell adhesion (
Integrin-linked kinase (ILK) is an evolutionally conserved FA protein that is involved in the integrin-mediated processes (
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 (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast Two-Hybrid Assays
A cDNA fragment encoding human ILK residues 136452 was inserted into the EcoRI/XhoI sites of a pLexA vector (CLONTECH Laboratories, Inc.). The resulting construct (pLexA/ILK136452) was used as a bait to screen a human heart MATCHMAKER LexA cDNA library (>3 x 106independent clones) (CLONTECH Laboratories, Inc.) as described (
Northern Blot
A 32P-labeled CH-ILKBP cDNA probe was prepared by labeling a human CH-ILKBP cDNA fragment (encoding residues 1229) 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 ProteinCH-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 (
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 GSTCH-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 AntiCH-ILKBP Antibodies
Mouse monoclonal antiCH-ILKBP antibodies were prepared using GST fusion protein containing CH-ILKBP residues 29372 as an antigen based on a previously described method (
Expression of FLAG and Green Fluorescent ProteinCH-ILKBP Fusion Proteins in Mammalian Cells
DNA fragments encoding CH-ILKBP sequences were cloned into the EcoRI/KpnI sites of the p3XFLAGCMV-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) (
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 antiCH-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 antiCH-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 pFLAGCMV-2/PINCH, which was generated by inserting the full-length PINCH cDNA into the BglII/SalI sites of the pFLAGCMV-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 (
Isolation of Cytoskeleton Fractions
Cytoskeleton fractions were isolated from C2C12 cells as described (
Cell Adhesion and Spreading Assays
For assays using CHO cells, the cells were transfected with FLAG vectors encoding CH-ILKBP, CH2 (residues 222372), CH-ILKBP F271D point mutant, CH2 deletion mutant (residues 1229), or a FLAG vector lacking CH-ILKBP sequence using LipofectAmine PLUS. Cell adhesion assays were performed as described (
-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 IVcoated 96-well plates (Becton Dickinson). After incubation at 37°C under a 5% CO295% air atmosphere for 90 min, the wells were washed three times with PBS. The numbers of adhered cells were quantified by measuring N-acetyl-ß-D-hexosaminidase activity (
For assays using C2C12 cells, the cells were transfected with FLAG vectors encoding CH-ILKBP (FLAGCH-ILKBP), CH2 (residues 222372) (FLAG-CH2), CH2 deletion mutant (residues 1229) (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 (phasecontrast image) was recorded with a DVC-1310C MagnafireTM digital camera (Optronics). Unspread cells were defined as round phasebright cells, whereas spread cells were defined as cells with extended processes, lacking a rounded morphology and not phasebright (300 cells from three randomly selected fields (>100 cells/field).
Online Supplemental Material
Figure S1: Association of FLAGCH-ILKBP, FLAG-F271D, and FLAG-CH2 with Triton X-100insoluble cytoskeleton fractions. Figure S2: CH-ILKBP binds to ILK but not paxillin in rat embryo fibroblasts (REF-52). Figure S3: Immunofluorescence staining of GFPCH-ILKBPexpressing cells with monoclonal antipaxillin antibody. Supplemental figures available at http://www.jcb.org/cgi/content/full/153/3/585/DC1.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of a Novel ILK-binding Protein by Yeast Two-Hybrid Screens
To identify proteins that interact with the COOH-terminal region of ILK, we screened a human heart LexA cDNA library with a bait construct encoding ILK residues 136452. 48 positive clones were obtained. PCR analyses showed that 39 out of the 48 positive clones contained cDNA inserts with an identical size (1.5 kb). Four clones (clones 10, 12, 35, and 36) from this major positive group were selected and sequenced. The results showed that they contained an identical cDNA insert. BLAST searches of cDNA database revealed that this insert overlaps with several EST clones and a cDNA encoding an "Unnamed Protein Product" (sequence data available from Genbank/EMBL/DDBJ under accession numbers
AA057458,
AA316121, and
AK001655). A cDNA encoding the full-length ILKBP was isolated from the heart cDNA library by PCR. It encodes a protein of 372 residues comprising two CH domains at the COOH-terminal region (Fig 1 A) and was therefore designated as CH domaincontaining ILK-binding protein or CH-ILKBP. The CH domains of CH-ILKBP share significant homology with those of -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.
|
Northern blot analyses with a 32P-labeled CH-ILKBP cDNA probe showed three positive bands (4.4, 3.5, and 1.7 kb) in almost all human tissues tested, with strong signals detected in the heart, skeletal muscle, and kidney (Fig 1 B). Additional bands, which likely represent tissue-specific CH-ILKBP isoforms or other related transcripts, were also detected in the heart and the skeletal muscle. The presence of multiple bands suggests that there likely exists a family of structurally related proteins. To confirm the interaction between ILK and CH-ILKBP, we cotransformed yeast cells with purified ILK and CH-ILKBP expression vectors and found that they readily bind to each other (Table 1). In control experiments, replacement of either binding partner sequence with those of other proteins (for example, PINCH and lamin C) abolished the binding (Table 1), confirming the specificity of the assay.
|
The ILK COOH-terminal Domain but not the NH2-terminal Domain Mediates the Interaction with CH-ILKBP
We next sought to identify regions of ILK involved in the CH-ILKBP binding. To do this, we expressed the full-length and various ILK mutants in yeast cells and analyzed the CH-ILKBPbinding activities (Fig 1 C). The full-length ILK, like the ILK COOH-terminal fragment (136452), readily binds to CH-ILKBP (Fig 1 C). By contrast, the NH2-terminal ANK domain (1163), which binds PINCH (Fig 1 C) (
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 29372) (Fig 2 A, lane 7) of CH-ILKBP and tested their ability to bind ILK. Both GSTCH-ILKBP (Fig 2 B, lane 1) and GST
N-ter (29372) (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.
|
CH-ILKBP contains two tandem CH domains (Fig 1 A). To define the ILK-binding site on CH-ILKBP, we generated a series of GST fusion proteins containing different regions of CH-ILKBP (Fig 2 A) and analyzed their ILK-binding activities. Deletion of 221 residues from the NH2 terminus did not reduce the ILK-binding activity (Fig 2 B, lane 2), indicating that the NH2-terminal region including CH1 is not required for the ILK binding. Furthermore, a GST fusion protein containing only the CH2 domain bound to ILK (Fig 2 B, lane 4). By contrast, deletion of either the entire or part of CH2 abolished the ILK binding (Fig 2 B, lanes 3, 5, and 6). We conclude from these results that the CH2 domain mediates the interaction with ILK.
CH-ILKBP Associates with ILK in Mammalian Cells
To facilitate studies on CH-ILKBP in mammalian cells, we generated mouse monoclonal antiCH-ILKBP antibodies. Two antibodies (1D4 and 3B5), which recognize GSTCH-ILKBP (Fig 3A and Fig B, lane 2) and MBPCH-ILKBP29372 (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 1229) (Fig 3 B). To test whether ILK interacts with CH-ILKBP in mammalian cells, we immunoprecipitated CH-ILKBP from C2C12 cell lysates with antiCH-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 antiCH-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 antiCH-ILKBP antibody (Fig 3C and Fig D, lane 4). Thus, consistent with the ILKCH-ILKBP interaction detected in yeast cells and in vitro, ILK and CH-ILKBP form a complex in mammalian cells.
|
CH-ILKBP, ILK, and PINCH Form a Ternary Complex in Mammalian Cells
We next tested whether the CH-ILKBPILK complex associates with PINCH, an FA protein that interacts with the NH2-terminal ANK domain of ILK (
|
CH-ILKBP Localizes to FAs and Associates with the Cytoskeleton
Previous studies have shown that ILK is a component of FAs (
|
To test whether FLAGCH-ILKBP localizes to FAs, we stained C2C12 cells and rat mesangial cells that express FLAGCH-ILKBP with anti-FLAG antibodies. The results showed that FLAGCH-ILKBP is targeted to FAs (Fig 5 E), where clusters of FAK (a marker of FAs) were also detected (Fig 5 F). Clusters of FLAGCH-ILKBP were concentrated at the ends of actin stress fibers (Fig 5G and Fig H). In control experiments, no specific staining was observed in cells transfected with a control FLAG vector (Fig 5I and Fig J), confirming the specificity of the staining. In additional experiments, we expressed GFPCH-ILKBP in mammalian cells and found that it also localized to FAs (see below).
To analyze the subcellular localization of endogenous CH-ILKBP, we stained mammalian cells with monoclonal antiCH-ILKBP antibodies. The results showed that endogenous CH-ILKBP (Fig 6 A) like FLAGCH-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 cellcell contacts, CH-ILKBP localized to cell matrix FAs but not to cellcell adhesions where abundant ß-catenin was detected (Fig 6E and Fig F). In control experiments, incubation of the antiCH-ILKBP antibody with GSTCH-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, 510% of CH-ILKBP was found in the Triton X-100insoluble 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 signalregulated kinase (ERK) (Fig 6 I), a more dynamic component of FAs (
|
The FA Localization of CH-ILKBP Is Mediated by the ILK-binding CH2 Domain
We next sought to determine the domain of CH-ILKBP that mediates the FA localization of CH-ILKBP. To do this, we expressed GFP fusion proteins containing the full-length CH-ILKBP (GFPCH-ILKBP), the CH2 deletion mutant (GFP-CH2), and the CH2 fragment (GFP-CH2) in mammalian cells. Consistent with the results obtained with antiCH-ILKBP antibody (Fig 6) and FLAGCH-ILKBP (Fig 5), GFPCH-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 GFPCH-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.
|
A Point Mutation That Disrupts the ILK-binding Impairs the FA Localization of CH-ILKBP
To further analyze the ILK-binding activity and FA localization of CH-ILKBP, we introduced a point mutation (F271D) into the ILK-binding CH2 domain of CH-ILKBP. GSTCH-ILKBP fusion proteins bearing the F271D point mutation were generated (Fig 8 A), and their ILK-binding activities were determined (Fig 8 B). Neither the full-length CH-ILKBP containing the F271D point mutation (Fig 8 B, lane 6) nor the CH2 domain containing the F271D point mutation (Fig 8 B, lane 2) bound to ILK. In control experiments, both GSTCH-ILKBP (Fig 8 B, lane 5) and the GST fusion protein containing the CH2 fragment (Fig 8 B, lane 3), but not GST alone (Fig 8 B, lane 4), bound to ILK. These results indicate that the F271D point mutation ablated the ILK-binding activity. We next determined the effect of disrupting the ILK-binding on FA localization of CH-ILKBP. To do this, we expressed a GFPCH-ILKBP protein bearing the F271D point mutation (GFP-F271D) in mammalian cells. In contrast to GFPCH-ILKBP, which was coclustered with ILK in FAs (Fig 7A and Fig B), GFP-F271D was unable to localize to FAs where abundant ILK was detected (Fig 8C and Fig D). Thus, ablation of the ILK-binding activity by a single amino acid substitution impairs the FA localization of CH-ILKBP.
|
CH-ILKBP Is Involved in the Regulation of Cell Adhesion and Spreading
The findings that CH-ILKBP interacts with ILK and localizes to FAs prompted us to investigate whether CH-ILKBP plays a role in cell adhesion. To do this, we transfected CHO cells with FLAG vectors encoding the full-length CH-ILKBP, the ILK-binding defective F271D point mutant, the ILK-binding CH2 fragment, the CH2 deletion mutant, or a FLAG vector lacking CH-ILKBP sequence as a control (Fig 9 A). As expected, the FLAG-tagged CH-ILKBP (Fig 9 A, lane 2) and CH2 domain (lane 4), but neither FLAG-F271D (lane 3) nor FLAG-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 25), despite the presence of abundant paxillin in these cells (Fig 9 C, lanes 69). To analyze the effect of overexpression of the different forms of CH-ILKBP on cell adhesion, we plated the CHO cells on collagen IVcoated 96-well plates and quantified the number of adhered cells. The results showed that overexpression of FLAGCH-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.
|
To confirm that CH-ILKBP is involved in cell adhesion, we expressed FLAG-tagged CH-ILKBP, the ILK-binding CH2 fragment, and the CH2 deletion mutant, respectively, in a different cell type (C2C12 cells). Because of the relative low efficiency of transient transfection in C2C12 cells, we isolated C2C12 clones stably expressing different forms of CH-ILKBP. Two independent clones were isolated from each transfection, and the expression of FLAGCH-ILKBP, FLAG-CH2, or FLAG-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 FLAGCH-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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell matrix adhesion is a fundamental process that regulates gene expression, differentiation, proliferation, and morphological changes. FAs are important cell adhesion sites through which the ECM is physically linked to the actin cytoskeleton (for reviews see CH2 or F271D) with the Triton X-100insoluble 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 (
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 ( (pat-2) or ß (pat-3) cause a PAT phenotype characterized by defects in muscle dense body and M-lines, which resemble FAs in mammalian cells (
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 PINCHILKCH-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 (
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,
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-ILKBPILKPINCH complex described in this report likely functions as an important scaffold in the assembly and signaling through cell matrix adhesion sites.
![]() |
Footnotes |
---|
The online version of this article contains supplemental material.
Y. Tu and Y. Huang contributed equally to this work.
1 Abbreviations used in this paper: ANK, ankyrin; CH, calponin homology; CH-ILKBP, CH domain-containing ILK-binding protein; ECM, extracellular matrix; ERK, extracellular signalregulated kinase; FA, focal adhesion; FAK, FA kinase; GFP, green fluorescent protein; GST, glutathione S-transferase; ILK, integrin-linked kinase; MBP, maltose-binding protein.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work was supported by National Institutes of Health grant DK54639 and American Cancer Society research project grant 98-220-01-CSM (to C. Wu).
Submitted: 30 January 2001
Revised: 6 March 2001
Accepted: 21 March 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|