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A Critical Role of the PINCH-Integrin-linked Kinase Interaction in the Regulation of Cell Shape Change and Migration

2002; Elsevier BV; Volume: 277; Issue: 1 Linguagem: Inglês

10.1074/jbc.m108257200

ISSN

1083-351X

Autores

Yongjun Zhang, Lida Guo, Ka Chen, Chuanyue Wu,

Tópico(s)

Hippo pathway signaling and YAP/TAZ

Resumo

The interaction of cells with extracellular matrix recruits multiple proteins to cell-matrix contact sites (e.g. focal and fibrillar adhesions), which connect the extracellular matrix to the actin cytoskeleton and regulate cell shape change, migration, and other cellular processes. We previously identified PINCH, an adaptor protein comprising primarily five LIM domains, as a binding protein for integrin-linked kinase (ILK). In this study, we show that PINCH co-localizes with ILK in both focal adhesions and fibrillar adhesions. Furthermore, we have investigated the molecular basis underlying the targeting of PINCH to the cell-matrix contact sites and the functional significance of the PINCH-ILK interaction. We have found that the N-terminal LIM1 domain, which mediates the ILK binding, is required for the targeting of PINCH to the cell-matrix contact sites. The C-terminal LIM domains, although not absolutely required, play an important regulatory role in the localization of PINCH to cell-matrix contact sites. Inhibition of the PINCH-ILK interaction, either by overexpression of a PINCH N-terminal fragment containing the ILK-binding LIM1 domain or by overexpression of an ILK N-terminal fragment containing the PINCH-binding ankyrin domain, retarded cell spreading, and reduced cell motility. These results suggest that PINCH, through its interaction with ILK, is crucially involved in the regulation of cell shape change and motility. The interaction of cells with extracellular matrix recruits multiple proteins to cell-matrix contact sites (e.g. focal and fibrillar adhesions), which connect the extracellular matrix to the actin cytoskeleton and regulate cell shape change, migration, and other cellular processes. We previously identified PINCH, an adaptor protein comprising primarily five LIM domains, as a binding protein for integrin-linked kinase (ILK). In this study, we show that PINCH co-localizes with ILK in both focal adhesions and fibrillar adhesions. Furthermore, we have investigated the molecular basis underlying the targeting of PINCH to the cell-matrix contact sites and the functional significance of the PINCH-ILK interaction. We have found that the N-terminal LIM1 domain, which mediates the ILK binding, is required for the targeting of PINCH to the cell-matrix contact sites. The C-terminal LIM domains, although not absolutely required, play an important regulatory role in the localization of PINCH to cell-matrix contact sites. Inhibition of the PINCH-ILK interaction, either by overexpression of a PINCH N-terminal fragment containing the ILK-binding LIM1 domain or by overexpression of an ILK N-terminal fragment containing the PINCH-binding ankyrin domain, retarded cell spreading, and reduced cell motility. These results suggest that PINCH, through its interaction with ILK, is crucially involved in the regulation of cell shape change and motility. integrin-linked kinase ankyrin calponin homology domain-containing ILK-binding protein Chinese hamster ovary green fluorescent protein Cell-extracellular matrix interactions are critically involved in the embryonic development and many physiological and pathological processes including injury repair, inflammation and metastasis. Upon adhesion to extracellular matrix, cells recruit a highly selective group of membrane and cytoplasmic proteins to the cell-extracellular matrix contact sites, where they connect the extracellular matrix to the actin cytoskeleton and regulate cell shape change, migration, and signal transduction (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar, 2Jockusch B.M. Bubeck P. Giehl K. Kroemker M. Moschner J. Rothkegel M. Rudiger M. Schluter K. Stanke G. Winkler J. Annu. Rev. Cell Dev. Biol. 1995; 11: 379-416Crossref PubMed Scopus (430) Google Scholar, 3Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1660) Google Scholar, 4Yamada K.M. Miyamoto S. Curr. Opin. Cell Biol. 1995; 7: 681-689Crossref PubMed Scopus (588) Google Scholar, 5Zamir E. Katz B.Z. Aota S. Yamada K.M. Geiger B. Kam Z. J. Cell Sci. 1999; 112: 1655-1669Crossref PubMed Google Scholar, 6Calderwood D.A. Shattil S.J. Ginsberg M.H. 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Fibrillar adhesions are more elongated (typical axial ratio, >7) and are rich in integrins, fibronectin, and tensin, but they are deficient in paxillin and several other components of focal adhesions (5Zamir E. Katz B.Z. Aota S. Yamada K.M. Geiger B. Kam Z. J. Cell Sci. 1999; 112: 1655-1669Crossref PubMed Google Scholar, 10Zamir E. Katz M. Posen Y. Erez N. Yamada K.M. Katz B.Z. Lin S. Lin D.C. Bershadsky A. Kam Z. Geiger B. Nat Cell Biol. 2000; 2: 191-196Crossref PubMed Scopus (462) Google Scholar, 11Pankov R. Cukierman E. Katz B.Z. Matsumoto K. Lin D.C. Lin S. Hahn C. Yamada K.M. J. Cell Biol. 2000; 148: 1075-1090Crossref PubMed Scopus (384) Google Scholar). ILK1 is a common component of both focal adhesions (12Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (967) Google Scholar, 13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar, 14Mulrooney J. 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In previous studies, we have shown that PINCH binds to ILK (18Wu C. J. Cell Sci. 1999; 112: 4485-4489Crossref PubMed Google Scholar, 20Tu Y. Li F. Goicoechea S. Wu C. Mol. Cell. Biol. 1999; 19: 2425-2434Crossref PubMed Scopus (246) Google Scholar) and forms a ternary complex with ILK and CH-ILKBP (21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar), an ILK C-terminal domain-binding protein, in cells. Mutational studies have shown that the formation of the PINCH-ILK complex is mediated by a direct interaction between the second zinc finger located within the PINCH LIM1 domain and the N-terminal ANK domain of ILK (13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar, 20Tu Y. Li F. Goicoechea S. Wu C. Mol. Cell. Biol. 1999; 19: 2425-2434Crossref PubMed Scopus (246) Google Scholar). The three-dimensional structure of the PINCH LIM1 domain has recently been solved. It folds into a globular structure consisting of two zinc fingers, each of which comprises two antiparallel β-sheets (22Velyvis A. Yang Y. Wu C. Qin J. J. Biol. Chem. 2001; 276: 4932-4939Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Consistent with the mutational studies, chemical mapping studies have revealed that many residues in the second zinc finger of the PINCH LIM1 domain undergo large chemical shift changes upon ILK binding (22Velyvis A. Yang Y. Wu C. Qin J. J. Biol. Chem. 2001; 276: 4932-4939Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). PINCH has been detected in integrin-rich cell-matrix adhesion sites in cells that are spreading on fibronectin (13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar). The current study is aimed at determining (i) whether PINCH co-localizes with ILK in both focal adhesions and fibrillar adhesions, (ii) the molecular basis underlying the targeting of PINCH to the cell-matrix contact sites, and (iii) the function of the PINCH-ILK interaction in cell spreading and migration. Mouse C2C12 cells (from American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. Rat embryo fibroblasts (REF-52, kindly provided by Dr. James Pipas, the University of Pittsburgh) were cultured in minimal essential medium supplemented with 10% fetal bovine serum. CHO K1 cells were cultured in α-minimal essential medium supplemented with 10% fetal bovine serum. DNA fragments encoding the FLAG-tagged full-length or mutant forms (as specified in each experiment) of human PINCH were cloned into the pEGFP-C2 expression vector (CLONTECH). C2C12 cells and CHO K1 cells were transfected with the GFP expression vectors using LipofectAMINE PLUS (Invitrogen) as described (21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar, 23Huang Y. Li J. Zhang Z. Wu C. J. Cell Biol. 2000; 150: 861-871Crossref PubMed Scopus (68) Google Scholar). CHO K1 cells stably expressing GFP and GFP fusion proteins were selected with 1 mg/ml of G418 (Invitrogen) and cloned as described previously (23Huang Y. Li J. Zhang Z. Wu C. J. Cell Biol. 2000; 150: 861-871Crossref PubMed Scopus (68) Google Scholar, 24Li J. Mayne R. Wu C. J. Cell Biol. 1999; 147: 1391-1397Crossref PubMed Scopus (52) Google Scholar). The adenoviral expressing vector encoding a FLAG-tagged N-terminal fragment of ILK (residues 1–230) was generated based on a previously described protocol (25He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3253) Google Scholar). Briefly, the cDNA fragment encoding the FLAG-tagged N-terminal fragment of ILK was cloned into theSalI/XbaI sites of the pAdTrack-CMV shuttle vector. The shuttle vector plasmid was linearized with PmeI, purified by phenol/chloroform extraction and ethanol precipitation, and mixed with supercoiled pADEsay-1. The vectors were transferred intoEscherichia coli BJ5183 by electroporation using a Bio-Rad Gene Pulser electroporator. The bacteria were immediately placed in 1 ml of LB Broth (10 g/l tryptone, 5 g/l yeast extract, and 5 g/l NaCl) (Fisher) and grown at 37 °C for 1 h. The bacteria were then inoculated onto agar containing LB Broth supplemented with 50 μg/ml of kanamycin. After 16–20 h of growth, colonies were picked and grown in 2 ml of LB Broth containing 50 μg/ml of kanamycin. The clones were screened by digestions with restriction endonucleasesPacI and BamHI. The positive supercoiled plasmids were transformed into DH10B cells by electroporation for large scale amplification. The plasmid DNA was digested with PacI and ethanol-precipitated and was used to transfect 293 cells using LipofectAMINE PLUS. The transfected cells were harvested 10 days after transfection. The cells were lysed by three cycles of freezing in a methanol/dry ice bath and rapid thawing at 37 °C, and the lysates containing the recombinant adenovirus were collected. The control adenoviral expression vector encoding β-galactosidase was kindly provided by Drs. Tong-Chuan He and Bert Vogelstein (Howard Hughes Medical Institute, the Johns Hopkins Oncology Center, Baltimore, MD). The recombinant adenoviral expressing vector encoding the FLAG-tagged N-terminal ILK fragment and the control vector were used to infect REF-52 cells. The infection efficiency was monitored by the expression of GFP encoded by the viral vectors and typically reached 80–90% within 48 h. The expression of the FLAG-tagged N-terminal ILK fragment in the infected cells was further confirmed by Western blotting with monoclonal anti-FLAG antibody M5. Immunofluorescence staining was performed as described (13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar, 21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar, 26Wu C. Keivens V.M. TE O.T. McDonald J.A. Ginsberg M.H. Cell. 1995; 83: 715-724Abstract Full Text PDF PubMed Scopus (300) Google Scholar). Briefly, cells (as specified in each experiment) were plated in complete medium on fibronectin- or vitronectin-coated coverslips or Lab-Tek eight-chamber culture slides and cultured for 24 h. The cells were then fixed with 3.7% paraformaldehyde in phosphate-buffered saline and stained with mouse monoclonal anti-ILK antibody 65.1 (13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar) or monoclonal anti-paxillin antibody (clone 349; Transduction Laboratories). The mouse antibodies were detected with a Rhodamine RedTM-conjugated anti-mouse antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA). Similar results were obtained with cells plated on fibronectin- or vitronectin-coated surfaces. The cells were cultured in complete medium in 60- or 100-mm culture plates. Cell monolayers were rinsed twice with phosphate-buffered saline and directly lysed on the plates with 1% Triton X-100 in 50 mm Tris-HCl (pH 7.4) containing 150 mm NaCl, 10 mmNa4P2O7, 2 mmNa3VO4, 100 mm NaF, and protease inhibitors. The protocol for immunoprecipitation with monoclonal anti-CH-ILKBP antibody was previously described (21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar). Briefly, cell lysates (500 μg) were mixed with 500 μl of hybridoma culture supernatant containing monoclonal anti-CH-ILKBP antibody 1D4. The samples were incubated for 3 h, mixed with 40 μl of UltraLink immobilized protein G (Pierce), and then incubated for an additional 1.5 h. The beads were washed four times, and the proteins bound were released from the beads by boiling in SDS-PAGE sample buffer for 5 min. The samples were analyzed by Western blotting with anti-CH-ILKBP antibody 3B5, anti-ILK antibody 65.1, or rabbit polyclonal anti-PINCH antibodies as specified in each experiment. For immunoprecipitation of GFP-FLAG-ΔLIM5 (PINCH residues 1–249), GFP-FLAG-ΔLIM1 (PINCH residues 63–325), and GFP-FLAG-LIM1–2 (PINCH residues 1–130), the cells expressing the corresponding GFP- and FLAG-tagged PINCH mutants or those expressing GFP as a control were lysed as described above. The lysates (300 μg) were mixed with 30 μl of agarose beads conjugated with anti-FLAG antibody M2 (Sigma). 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 (8 μl/lane) with antibodies as specified in each experiment. The cells (as specified in each experiment) were plated on 96-well plates coated with vitronectin (BD Biosciences, Bedford, MS) in Opti-MEM I serum-free medium (Invitrogen). The plates were incubated at 37 °C under a 5% CO2, 95% air atmosphere for different periods of time (as specified in each experiment). The cell morphology were observed under an Olympus IX70 fluorescence microscope equipped with an Hoffman Modulation Contrast system and recorded with a DVC-1310C MagnafireTM digital camera (Optronics). Unspread cells were defined as round cells, whereas spread cells were defined as cells with extended processes as described (21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar, 27Komoriya A. Green L.J. Mervic M. Yamada S.S. Yamada K.M. Humphries M.J. J. Biol. Chem. 1991; 266: 15075-15079Abstract Full Text PDF PubMed Google Scholar, 28Richardson A. Malik R.K. Hildebrand J.D. Parsons J.T. Mol. Cell. Biol. 1997; 17: 6906-6914Crossref PubMed Scopus (289) Google Scholar). The percentage of cells adopting spread morphology was quantified by analyzing at least 300 cells from three randomly selected fields (>100 cells/field) (21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar, 27Komoriya A. Green L.J. Mervic M. Yamada S.S. Yamada K.M. Humphries M.J. J. Biol. Chem. 1991; 266: 15075-15079Abstract Full Text PDF PubMed Google Scholar, 28Richardson A. Malik R.K. Hildebrand J.D. Parsons J.T. Mol. Cell. Biol. 1997; 17: 6906-6914Crossref PubMed Scopus (289) Google Scholar). Cell migration was assessed by the ability of the cells to migrate into a cell-free area as previously described (29Giancotti F.G. Ruoslahti E. Cell. 1990; 60: 849-859Abstract Full Text PDF PubMed Scopus (695) Google Scholar, 30Chen P. Xie H. Sekar M.C. Gupta K. Wells A. J. Cell Biol. 1994; 127: 847-857Crossref PubMed Scopus (285) Google Scholar). Briefly, the cells were plated in complete medium on 24-well plates and grown for 24 h to reach confluence. The monolayers were then wounded by scratching with a plastic pipette tip. After washing, the cells were incubated in complete medium for the indicated times and observed under an Olympus IX70 microscope equipped with an Hoffman Modulation Contrast system. Images of three different segments of the cell-free area were recorded with a DVC-1310C MagnafireTMdigital camera (Optronics), and the distances traveled by the cells at the front in three different segments of the wound were measured. To test whether PINCH co-localizes with ILK in both focal adhesions and fibrillar adhesions, we expressed a GFP-tagged PINCH in mouse C2C12 cells. Cells expressing GFP-PINCH were stained with antibodies that recognize ILK (Fig.1, A and B), fibronectin (as a marker for fibrillar adhesions) (Fig. 1, Cand D), and paxillin (as a marker for focal adhesions) (Fig.1, E and F), respectively. GFP-PINCH was clustered in fibrillar adhesions that were rich in fibronectin (Fig. 1,C and D, arrowheads) but deficient in paxillin (Fig. 1, E and F,arrowheads). In addition, it localized to focal adhesions where abundant paxillin (Fig. 1, E and F,arrows) but little or no fibronectin clusters (Fig. 1,C and D, arrows) were detected. Analyses of cells stained with the monoclonal anti-ILK antibody showed that GFP-PINCH co-localized with ILK in both types of cell-matrix contact sites (Fig. 1, A and B, arrowsand arrowheads). We next analyzed the PINCH domains that are involved in the localization of PINCH to the cell-matrix contact sites. Because PINCH binds to ILK (13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar, 20Tu Y. Li F. Goicoechea S. Wu C. Mol. Cell. Biol. 1999; 19: 2425-2434Crossref PubMed Scopus (246) Google Scholar), and PINCH and ILK co-localize in cell-matrix adhesion sites (Fig. 1), we hypothesized that the ILK-binding is required for the localization of PINCH to the cell-matrix contact sites. To test this, we expressed PINCH mutants in which either the N-terminal-most ILK-binding LIM1 domain or the C-terminal-most LIM5 domain is deleted in C2C12 cells. The PINCH mutants were tagged with GFP and a FLAG epitope to facilitate cellular and biochemical analyses. The expression of the GFP- and FLAG-tagged ΔLIM1 (Fig. 2A,lane 2) and ΔLIM5 (Fig. 2A, lane 1) PINCH mutants was confirmed by Western blotting with an anti-GFP antibody. To test whether the PINCH mutants bind to ILK, we immunoprecipitated the GFP- and FLAG-tagged PINCH mutants from the cell lysates with a monoclonal anti-FLAG antibody (Fig. 2A,lanes 4 and 5). Western blotting analyses of the ΔLIM1 and ΔLIM5 immunoprecipitates showed that ILK was co-precipitated with ΔLIM5 (Fig. 2B, lane 4) but not with ΔLIM1 (Fig. 2B, lane 5). In control experiments, neither GFP (Fig. 2A, lane 6) nor ILK (Fig. 2B, lane 6) was precipitated with the anti-FLAG antibody from lysates of control cells that express GFP, confirming the specificity of the immunoprecipitation assay. Taken together, these results show that the GFP-FLAG-ΔLIM5, but not GFP-FLAG-ΔLIM1, binds to ILK in cells. To determine the ability of the PINCH mutants to localize to cell-matrix contact sites, we plated the C2C12 cells expressing GFP-FLAG-ΔLIM1 or GFP-FLAG-ΔLIM5 on fibronectin-coated coverslips and stained them with monoclonal anti-ILK antibody 65.1. The ILK-binding defective LIM1 deletion mutant (Fig. 2C), unlike the full-length PINCH (Fig. 1), was unable to localize to cell-matrix contact sites where abundant ILK was detected (Fig. 2D), suggesting that the ILK binding is essential for the localization of PINCH to the cell-matrix contact sites. By contrast, the LIM5 deletion mutant was able to co-localize with ILK in cell-matrix contact sites (Fig. 2, E and F), indicating that the LIM5 domain, unlike the LIM1 domain, is not required for the localization of PINCH. The level of the LIM5 deletion mutant (Fig. 2E) that was detected in the cell-matrix contact sites, however, appeared lower than that of the full-length PINCH (Fig. 1), suggesting that deletion of LIM5 decreases the efficiency or the stability of the PINCH localization to the cell-matrix contact sites. To further analyze this, we expressed a PINCH mutant in which LIM3–5 were deleted in C2C12 cells. The expression of the PINCH mutant (LIM1–2) was confirmed by Western blotting with a monoclonal anti-FLAG antibody (Fig. 3A, lane 4). As expected, ILK (Fig. 3B, lane 2) was readily co-immunoprecipitated with GFP-FLAG-LIM1–2 (Fig.3A, lane 2). In control experiment, no ILK (Fig.3B, lane 1) was precipitated by the anti-FLAG antibody in the absence of GFP-FLAG-LIM1–2 (Fig. 3A,lane 1), confirming the specificity of the co-immunoprecipitation experiment. Fluorescence microscopic analyses showed that GFP-FLAG-LIM1–2 distributed rather diffusely in cells (Fig. 3, C, E, and G). A closer examination of the cells revealed that in some but not all cells, a very low level of GFP-FLAG-LIM1–2 clusters was present in cell adhesion sites where ILK was clustered (Fig. 3, E andF). Taken together, these results suggest that deletion of the LIM3–5 domains greatly impairs the ability of PINCH to localize to cell-matrix contact sites. Thus, although the C-terminal LIM3–5 domains were not absolutely required for the targeting of PINCH to cell-matrix contact sites, they do play important roles in the regulation of PINCH localization. Intriguingly, although we have detected ILK clusters in cells overexpressing GFP-FLAG-LIM1–2 (Fig. 3, D andF), they often appeared smaller and less well organized than those in cells that overexpress GFP-FLAG-PINCH (Fig. 1B), GFP-FLAG-ΔLIM1 (Fig. 2D) or GFP alone (data not shown). Abundant paxillin clusters were detected in the GFP-FLAG-LIM1–2 overexpressing cells under the same condition (Fig. 3, G andH). These results suggest that overexpression of LIM1–2, which binds to ILK but fails to localize to the cell-matrix adhesion sites efficiently, negatively influences the localization of ILK to the adhesion sites. We next sought to assess the functional significance of the PINCH-ILK interaction. To do this, we generated reagents that allow us to modulate the PINCH-ILK interaction in cells. Because the LIM1–2 fragment binds efficiently to ILK in cells (Fig. 3), we postulated that it could potentially function as a dominant negative inhibitor of the PINCH-ILK interaction. To test this, we transfected CHO cells, which have been widely used in gene transfer experiments because of their high transfection efficiency, with expression vectors encoding either GFP-FLAG-LIM1–2 or GFP alone (as a control). Under the experimental conditions used, the transfection efficiency was ∼80% based on the percentages of CHO cells that expressed GFP or GFP-FLAG-LIM1–2. The expression of GFP (Fig.4A, lane 2) and GFP-FLAG-LIM1–2 (Fig. 4A, lane 3) in the corresponding transfectants was confirmed by Western blotting. To test the effect of LIM1–2 overexpression on the complex formation, we immunoprecipitated the PINCH-ILK-CH-ILKBP complex from lysates of CHO cells overexpressing GFP-FLAG-LIM1–2 and those of the GFP control cells, respectively, with monoclonal anti-CH-ILKBP antibody 1D4. Analyses of the immunoprecipitates showed that similar amounts of CH-ILKBP (Fig. 4B, lanes 3 and 4) and ILK (Fig. 4C, lanes 3 and 4) were precipitated from the GFP-FLAG-LIM1–2 overexpressing cells and the control cells. The amount of PINCH associated with ILK and CH-ILKBP was noticeably reduced in cells overexpressing LIM1–2 (Fig. 4D, compare lanes 3 and 4), suggesting that overexpression of LIM1–2 significantly inhibits the PINCH-ILK interaction. To facilitate studies on the functions of the PINCH-ILK interaction, we selected cells that stably express GFP-FLAG-LIM1–2 by culturing the transfectants in the presence of G418. Three independent clones (7.2, 8.1, and 9.1) were obtained. To investigate whether the PINCH-ILK interaction is involved in the cellular control of cell shape change, we plated the CHO cells stably expressing GFP-FLAG-LIM1–2, CHO cells stably expressing GFP-FLAG-PINCH, the control GFP-expressing cells, and the parental CHO cells on vitronectin-coated surfaces. Most of the parental CHO cells and GFP control cells spread within 30 min of plating (Fig. 5). Overexpression of GFP-FLAG-PINCH did not alter cell spreading (Fig. 5). By contrast, most of the GFP-FLAG-LIM1–2-expressing cells exhibited a round cell shape during the same period of time (Fig. 5), although they were able to spread after longer incubation time (Fig. 5A). Taken together, these results suggest that inhibition of the PINCH-ILK interaction significantly delays the change of cell shape. Cell migration is a pathologically and physiologically important process that involves dynamic changes of cell shape. The finding that the PINCH-ILK interaction is involved in the cellular control of cell shape change prompted us to test whether it plays a role in cell migration. To do this, we analyzed the migration of cells overexpressing GFP-FLAG-LIM1–2 using a "wound" assay, a well established in vitro system for measuring cell motility (29Giancotti F.G. Ruoslahti E. Cell. 1990; 60: 849-859Abstract Full Text PDF PubMed Scopus (695) Google Scholar,30Chen P. Xie H. Sekar M.C. Gupta K. Wells A. J. Cell Biol. 1994; 127: 847-857Crossref PubMed Scopus (285) Google Scholar). The results showed that the cells overexpressing GFP-FLAG-LIM1–2 migrated much more slowly than the cells overexpressing GFP-FLAG-PINCH, the parental cells, and the GFP control cells (Fig.6). Thus, consistent with the retardation in cell spreading, overexpression of the LIM1–2 fragment significantly impairs the cell motility. Because the N-terminal ANK domain of ILK mediates the binding to PINCH, we hypothesized that overexpressing the ILK N-terminal ANK fragment might also inhibit the PINCH-ILK interaction. To test this, we generated an adenoviral vector encoding a FLAG-tagged ILK N-terminal ANK fragment, which allowed us to express the ILK fragment with high efficiency (typically 80–90%). We infected rat embryo fibroblasts (REF-52) with the recombinant adenovirus encoding the ANK fragment or that encoding an irrelevant protein (β-galactosidase) as a control. The expression of the FLAG-tagged ILK N-terminal ANK fragment in the REF-52 cells that were infected with the adenovirus encoding the ANK fragment (Fig.7A, lane 3) but not in the uninfected REF-52 (Fig. 7A, lane 1) nor in the control cells that were infected with the β-galactosidase adenovirus (Fig. 7A, lane 2) was confirmed by Western blotting. To determine the effect of overexpression of the ANK fragment on the complex formation between PINCH, ILK, and CH-ILKBP, we immunoprecipitated the PINCH-ILK-CH-ILKBP complex from lysates of the ANK-expressing cells as well as those of the control cells with monoclonal anti-CH-ILKBP antibody 1D4. Similar amount of ILK (Fig.7C, lanes 3 and 4) was co-precipitated with CH-ILKBP (Fig. 7B, lanes 3 and 4) either in the presence or absence of the ANK fragment, indicating that the interaction between ILK and CH-ILKBP was not altered by the overexpression of the ANK fragment. By contrast, the amount of PINCH associated with ILK was markedly reduced in cells overexpressing the ANK fragment (Fig. 7D, lanes 3 and 4), confirming that overexpression of the ILK N-terminal ANK fragment inhibits the complex formation between PINCH and ILK. To test whether overexpression of the ILK ANK fragment, like that of the PINCH LIM1–2 fragment, also inhibits cell spreading, we plated cells infected with the ANK adenovirus, cells infected with the control β-galactosidase adenovirus, or the uninfected REF-52 cells on vitronectin-coated surfaces. The viral infection efficiency was ∼80–90% under the condition used. Analyses of the cells at different time points after the plating revealed that the spreading of the cells overexpressing the ILK ANK fragment was significantly slower than that of the control cells (Fig. 8,A and B). We next analyzed the migration of cells overexpressing the ILK ANK fragment using the wound assay. The results showed that cells overexpressing the ILK ANK fragment migrated much more slowly than the parental REF-52 and the β-galactosidase vector control cells (Fig. 8,C and D). Taken together, these results indicate that inhibition of the PINCH-ILK interaction, either by overexpression of the PINCH LIM1–2 fragment or by overexpression of the ILK ANK fragment, significantly retards cell spreading and reduces cell motility. In this study, we have shown that PINCH co-localizes with ILK in both focal adhesions and fibrillar adhesions. Furthermore, we have mapped the domains of PINCH that are involved in the localization of PINCH to the cell-matrix contact sites. Our results indicate that the ILK-binding LIM1 domain is required for the localization of PINCH to the cell-matrix contact sites, whereas the C-terminal LIM domains are involved in the modulation of this process. Finally, we have investigated the function of the PINCH-ILK interaction in cell spreading and migration. We have developed two different dominant negative inhibitors of the PINCH-ILK interaction and demonstrated that down-regulation of the PINCH-ILK interaction by overexpression of either inhibitor results in a significant reduction of cell spreading and migration. The results reported in this paper, together with those of previous studies, shed light on the mechanism by which PINCH and ILK localize to cell-matrix contact sites. The finding that the ILK-binding LIM1 domain is indispensable for the localization of PINCH to cell-matrix adhesion sites (Fig. 2) suggests an essential role of ILK in this process. One explanation for this observation is that ILK simply provides a PINCH-docking site at the cell-matrix adhesion sites. However, two pieces of evidence suggest that the PINCH-ILK interaction is not only crucial for the localization of PINCH but also important for the localization of ILK. First, we have found in previous studies that the PINCH-binding ANK repeat is required for the localization of ILK to cell-matrix adhesion sites (13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar). Second, we have observed in this study that overexpression of the PINCH LIM1–2 fragment, which reduced but did not completely eliminate the PINCH-ILK interaction, partially inhibited the localization of ILK to cell-matrix contact sites (Fig. 3). Thus, a more likely possibility is that ILK and PINCH form a complex before reaching the cell-matrix adhesion sites and that the formation of such a complex is a prerequisite for the efficient localization of both proteins. The formation of the PINCH-ILK complex could allow multiple, simultaneous interactions mediated by other domains of PINCH and ILK and therefore promote efficient localization of both proteins to the cell-matrix adhesion sites. This model explains why the PINCH-ILK interaction is crucial for the localization of both PINCH and ILK to the cell-matrix adhesion sites. Furthermore, it implies that there exist other PINCH- and/or ILK-mediated interactions that are important for the localization of both proteins. The C-terminal domain of ILK is capable of interacting with several additional components of the cell-matrix adhesion structures including β1 integrins (12Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (967) Google Scholar), CH-ILKBP (21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar), paxillin (15Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), and affixin (31Yamaji S. Suzuki A. Sugiyama Y. Koide Y. Yoshida M. Kanamori H. Mohri H. Ohno S. Ishigatsubo Y. J. Cell Biol. 2001; 153: 1251-1264Crossref PubMed Scopus (170) Google Scholar). Deletion of the C-terminal domain abolished the ability of ILK to localize to cell-matrix adhesion sites (13Li F. Zhang Y. Wu C. J. Cell Sci. 1999; 112: 4589-4599Crossref PubMed Google Scholar), suggesting that at least one and, more likely, multiple interactions mediated by this domain are required in this process. Mutations in an ILK C-terminal sequence that is 15–33% identical to the paxillin binding sequences in vinculin, focal adhesion kinase, and actopaxin disrupt the localization of ILK to cell-matrix contact sites, suggesting that the interaction with paxillin is involved in this process (15Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). However, because paxillin is deficient in fibrillar adhesions (5Zamir E. Katz B.Z. Aota S. Yamada K.M. Geiger B. Kam Z. J. Cell Sci. 1999; 112: 1655-1669Crossref PubMed Google Scholar, 10Zamir E. Katz M. Posen Y. Erez N. Yamada K.M. Katz B.Z. Lin S. Lin D.C. Bershadsky A. Kam Z. Geiger B. Nat Cell Biol. 2000; 2: 191-196Crossref PubMed Scopus (462) Google Scholar, 11Pankov R. Cukierman E. Katz B.Z. Matsumoto K. Lin D.C. Lin S. Hahn C. Yamada K.M. J. Cell Biol. 2000; 148: 1075-1090Crossref PubMed Scopus (384) Google Scholar), to which both ILK (16Guo L. Sanders P.W. Woods A. Wu C. Am. J. Pathol. 2001; 159: 1735-1742Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and PINCH (this report) localize, other ILK C-terminal binding proteins such as β1 integrins, CH-ILKBP/actopaxin/α-parvin, and affixin/β-parvin are likely also involved in this process (12Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (967) Google Scholar, 21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar,32Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (170) Google Scholar, 33Olski T.M. Noegel A.A. Korenbaum E. J. Cell Sci. 2001; 114: 525-538Crossref PubMed Google Scholar, 34Wu C. J. Cell Sci. 2001; 114: 2549-2550PubMed Google Scholar, 35Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (350) Google Scholar). It is worth noting in this regard that CH-ILKBP forms a ternary complex with ILK and PINCH in cells (21Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (192) Google Scholar), and therefore it is attractive to propose that the formation of the PINCH-ILK-CH-ILKBP complex is a crucial step in the localization of these three proteins to cell-matrix contact sites. The notion that PINCH, ILK, and CH-ILKBP assemble into a complex and thereby localize and function interdependently at the cell-matrix adhesion sites is consistent with recent genetic studies in model organisms such as Caenorhabditis elegans showing that lack of PINCH/UNC-97 (36Hobert O. Moerman D.G. Clark K.A. Beckerle M.C. Ruvkun G. J. Cell Biol. 1999; 144: 45-57Crossref PubMed Scopus (178) Google Scholar), ILK/PAT-4 (44Mackinnon A.C. Williams B.D. Mol. Biol. Cell. 2000; 11: 515aGoogle Scholar), or CH-ILKBP/PAT-6 (45Lin X. Williams B.S. Mol. Biol. Cell. 2000; 11: 515aGoogle Scholar) resulted in an identical defect in the assembly of muscle attachment sites. In addition to the interactions involving ILK, interactions mediated by the PINCH C-terminal LIM domains likely also contribute to the localization of PINCH, and consequently ILK and CH-ILKBP, to cell-matrix contact sites. Deletion of the LIM3–5 domains greatly impairs the ability of PINCH to localize to cell-matrix contact sites. Deletion of the LIM5 domain also decreased, although to a lesser extent, the efficiency of the localization of PINCH to cell-matrix contact sites. Because the LIM1-containing PINCH N-terminal fragments bind efficiently to ILK (Figs. 2 and 3), the impairment in the localization of the PINCH C-terminal deletion mutants is likely caused by the loss of interactions mediated by the C-terminal LIM domains. It is interesting to note that one of the C-terminal domains, LIM4, interacts with Nck-2 (also known as Nckβ (37Chen M. She H. Davis E.M. Spicer C.M. Kim L. Ren R. Le Beau M.M. Li W. J. Biol. Chem. 1998; 273: 25171-25178Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) or Grb4 (38Braverman L.E. Quilliam L.A. J. Biol. Chem. 1999; 274: 5542-5549Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar)), a Src homology 2 domain- and Src homology 3 domain-containing adaptor protein (39Tu Y. Li F. Wu C. Mol. Biol. Cell. 1998; 9: 3367-3382Crossref PubMed Scopus (161) Google Scholar). Because Nck-2 is present in the cell-matrix contact sites in spreading cells, 2S. M. Goicoechea and C. Wu, unpublished observations. it could represent one of the interactions mediated by the PINCH C-terminal domains that are involved in the localization of PINCH to these sites. The observation that deletion of LIM3–4 further reduces the efficiency of the localization to cell-matrix contact sites (compare Fig.2E with Figs. 3, C–G) provides evidence supporting, albeit not proving, this possibility. Clearly, future studies are required to test this possibility and to identify other interactions that are involved in this process. Cell migration is a complex process that is critically involved in embryonic development and many physiological and pathological processes including injury repair, inflammation, and metastasis. Using two different dominant negative inhibitors of the PINCH-ILK interaction, we have demonstrated in this study that PINCH, through its interaction with ILK, plays an important role in the regulation of cell migration. Because cell migration requires coordinated changes of cell shape and inhibition of the PINCH-ILK interaction impairs this process, the defect in cell migration is most likely caused by, at least in part, the disregulation of cell shape change. At the molecular level, this could reflect a need for the PINCH-ILK interaction in physically connecting the transmembrane receptors such as integrins to the actin cytoskeleton. Additionally, PINCH could participate in other events that are critical for cell migration. For example, PINCH, through interactions mediated by Nck-2, could potentially bring proteins that are involved in the actin polymerization into proximity of the cell-matrix adhesion sites and therefore promote cell migration. Nck-2 is known to interact with p21-activated kinase and Wiskott-Aldrich syndrome protein (37Chen M. She H. Davis E.M. Spicer C.M. Kim L. Ren R. Le Beau M.M. Li W. J. Biol. Chem. 1998; 273: 25171-25178Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 38Braverman L.E. Quilliam L.A. J. Biol. Chem. 1999; 274: 5542-5549Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), proteins that are directly involved in the regulation of actin polymerization. Although a direct role for Nck-2 in the regulation of actin polymerization remains to be tested, it has recently been shown that Nck-1/Nckα, which is structurally closely related to Nck-2, cooperates with phosphatidylinositol 4,5-bishposphate to dramatically activate neural-Wiskott-Aldrich syndrome protein/Arp2/3-mediated actin nucleation (40Rohatgi R. Nollau P. Ho H.Y. Kirschner M.W. Mayer B.J. J. Biol. Chem. 2001; 276: 26448-26452Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Additional Nck-2-binding proteins include DOCK180 (41Tu Y. Kucik D.F. Wu C. FEBS Lett. 2001; 491: 193-199Crossref PubMed Scopus (39) Google Scholar), a Rac-activating protein that is involved in the regulation of membrane ruffling and cell migration (42Kiyokawa E. Hashimoto Y. Kobayashi S. Sugimura H. Kurata T. Matsuda M. Genes Dev. 1998; 12: 3331-3336Crossref PubMed Scopus (384) Google Scholar, 43Cheresh D.A. Leng J. Klemke R.L. J. Cell Biol. 1999; 146: 1107-1116Crossref PubMed Scopus (234) Google Scholar). Delineation of the functions of PINCH and its associated proteins will be of considerable value to the understanding of the molecular mechanism by which cells regulate shape change, movement, and other processes involving cell-matrix interactions. We thank Drs. Tong-Chuan He and Bert Vogelstein for the pAdTrack-CMV and pADEsay-1 vectors and Dr. James Pipas for the REF-52 cells.

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