Solution structure of the focal adhesion adaptor PINCH LIM1 domain and characterization of its interaction with the integrin-linked kinase ankyrin repeat domain

PINCH is a recently identified adaptor protein that comprises an array of five LIM domains. PINCH functions through LIM-mediated protein-protein interactions that are involved in cell adhesion, growth, and differentiation. The LIM1 domain of PINCH interacts with integrin-linked kinase (ILK), thereby mediating focal adhesions via a specific integrin/ILK signaling pathway. We have solved the NMR structure of the PINCH LIM1 domain and characterized its binding to ILK. LIM1 contains two contiguous zinc fingers of the CCHC and CCCH types and adopts a global fold similar to that of functionally distinct LIM domains from cysteine-rich protein and cysteine-rich intestinal protein families with CCHC and CCCC zinc finger types. Gel-filtration and NMR experiments demonstrated a 1:1 complex between PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift mapping experiment identified regions in PINCH LIM1 that are important for interaction with ILK. Comparison of surface features between PINCH LIM1 and other functionally different LIM domains indicated that the LIM motif might have a highly variable mode in recognizing various target proteins.     

Integrin-linked kinase regulates N-WASp-mediated actin polymerization and tension development in tracheal smooth muscle

The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a beta-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and alpha-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and alpha-parvin form a stable cytosolic complex and that the ILK.PINCH.alpha-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with beta-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK.PINCH.alpha-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle.     

The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.     

Structural and functional insights into PINCH LIM4 domain-mediated integrin signaling

PINCH is an adaptor protein found in focal adhesions, large cellular complexes that link extracellular matrix to the actin cytoskeleton. PINCH, which contains an array of five LIM domains, has been implicated as a platform for multiple protein-protein interactions that mediate integrin signaling within focal adhesions. We had previously characterized the LIM1 domain of PINCH, which functions in focal adhesions by binding specifically to integrin-linked kinase. Using NMR spectroscopy, we show here that the PINCH LIM4 domain, while maintaining the conserved LIM scaffold, recognizes the third SH3 domain of another adaptor protein, Nck2 (also called Nckbeta or Grb4), in a manner distinct from that of the LIM1 domain. Point mutation of LIM residues in the SH3-binding interface disrupted LIM-SH3 interaction and substantially impaired localization of PINCH to focal adhesions. These data provide novel structural insight into LIM domain-mediated protein-protein recognition and demonstrate that the PINCH-Nck2 interaction is an important component of the focal adhesion assembly during integrin signaling.     

Characterization of PINCH-2, a new focal adhesion protein that regulates the PINCH-1-ILK interaction,cell spreading,and migration

Integrin-linked kinase (ILK) is a multidomain protein that plays important roles at cell-extracellular matrix (ECM) adhesion sites. We describe here a new LIM-domain containing protein (termed as PINCH-2) that forms a complex with ILK. PINCH-2 is co-expressed with PINCH-1 (previously known as PINCH), another member of the PINCH protein family, in a variety of human cells. Immunofluorescent staining of cells with PINCH-2-specific antibodies show that PINCH-2 localizes to both cell-ECM contact sites and the nucleus. Deletion of the first LIM (LIM1) domain of PINCH-2 abolished the ability of PINCH-2 to form a complex with ILK. The ILK-binding defective LIM1-deletion mutant, unlike the wild type PINCH-2 or the ILK-binding competent LIM5-deletion mutant, was incapable of localizing to cell-ECM contact sites, suggesting that ILK binding is required for this process. Importantly, the PINCH-2-ILK and PINCH-1-ILK interactions are mutually exclusive. Overexpression of PINCH-2 significantly inhibited the PINCH-1-ILK interaction and reduced cell spreading and migration. These results identify a novel nuclear and focal adhesion protein that associates with ILK and reveals an important role of PINCH-2 in the regulation of the PINCH-1-ILK interaction, cell shape change, and migration.     

Structural Basis of Focal Adhesion Localization of LIM-only Adaptor PINCH by Integrin-linked Kinase

The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK·PINCH complex (28 kDa, K_D ∼ 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.Cell-extracellular matrix (ECM)³ adhesion, migration, and survival are essential for the development and maintenance of tissues and organs in living organisms. They are mediated by integrin transmembrane receptors, which function by adhering to ECM proteins via their large extracellular domains while connecting to the actin cytoskeleton via their small cytoplasmic tails (20-70 residues) (1). The integrin-actin connection supports strong cell-ECM adhesion, and its alteration leads to dynamic cell shape change, migration, and survival (2). The molecular details of such connection, however, are highly complex, involving a large protein complex network called focal adhesions (FAs) (3, 4).Integrin-linked kinase (ILK) is a 50-kDa FA protein that contains an N-terminal ankyrin repeat domain (ARD), a middle pleckstrin homology domain, and a C-terminal kinase domain. Originally discovered as an integrin β cytoplasmic tail-binding protein (5), ILK has been established as a major regulator that controls the complex FA assembly and transmits many cell adhesive signals between integrins and actin (6-8). Soon after the discovery of ILK, Tu et al. (9) identified an ILK binding partner called PINCH that contains five LIM domains. Extensive studies have shown that the PINCH binding to ILK is essential for triggering the FA assembly and for relaying diverse mechanical and biochemical signals between ECM and the actin cytoskeleton (9-11). Consistent with the importance of the ILK/PINCH association in almost all cellular behavior and fate, ablation of either ILK (12) or PINCH in mice is embryonically lethal (13, 14). PINCH also has a highly homologous isoform called PINCH-2. However, although complementary to PINCH in many cellular behaviors (for reviews, see Refs. 8 and 15), PINCH-2 appears to be involved at the later stage of development (16), and thus its ablation in mice is not embryonically lethal (17). At the clinical level, dysregulation of the ILK/PINCH interaction has been implicated in the development of numerous human disorders such as cancer (6, 18) and heart diseases (19, 20). A Phase I clinical trial is ongoing on a drug called thymosin β-4 (RegeneRx) that appears to specifically target ILK/PINCH for treating myocardial infarction, a major heart failure disorder (19).Despite the cellular, physiological, and pathological importance of the ILK/PINCH interaction, the structural basis for how exactly PINCH binds to ILK has not been well understood. Previous biochemical/structural analyses have indicated that ILK utilizes its N-terminal ARD to recognize the LIM1 domain of PINCH, and such binding may promote the targeting of PINCH to FAs (9, 21). However, the precise atomic basis for such targeting process is elusive. No structure of any ARD·LIM complex has been reported. Using a combination of NMR-based techniques, we have solved the solution structure of the ILK ARD·PINCH LIM1 complex that revealed an interface that is distinct from other ARD and LIM bound to non-ARD/LIM domains. Structure-based mutation of a hot spot in PINCH LIM1, which is not conserved in other LIM domains, abolished the PINCH binding to ILK and its localization to FAs. These results not only reveal a unique LIM/ARD recognition mode but also provide a definitive functional basis for how PINCH is recruited by ILK to focal adhesion site, a major step toward the dynamic cell adhesion and migration processes.     

Molecular dissection of PINCH-1 reveals a mechanism of coupling and uncoupling of cell shape modulation and survival

How cells couple and uncouple regulation of cellular processes such as shape change and survival is an important question in molecular cell biology. PINCH-1, a widely expressed protein consisting of five LIM domains and a C-terminal tail, is an essential focal adhesion protein with multiple functions including regulation of the integrin-linked kinase (ILK) level, cell shape, and survival signaling. We show here that the LIM1-mediated interaction with ILK regulates all these three processes. By contrast, the LIM4-mediated interaction with Nck-2, which regulates cell morphology and migration, is not required for the control of the ILK level and survival. Remarkably, a short 15-residue tail C-terminal to LIM5 is required for both cell shape modulation and survival, albeit it is not required for the control of the ILK level. The C-terminal tail not only regulates PINCH-1 localization to focal adhesions but also functions after it localizes there. These findings suggest that PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but yet distinct domain interactions.     

UNC-97/PINCH is involved in the assembly of integrin cell adhesion complexes in Caenorhabditis elegans body wall muscle

UNC-97/PINCH is an evolutionarily conserved protein that contains five LIM domains and is located at cell-extracellular matrix attachment sites known as cell adhesion complexes. To understand the role of UNC-97/PINCH in cell adhesion, we undertook a combined genetic and cell biological approach to identify the steps required to assemble cell adhesion complexes in Caenorhabditis elegans. First, we have generated a complete loss of function mutation in the unc-97 coding region. unc-97 null mutants arrest development during embryogenesis and reveal that the myofilament lattice and its attachment structures, which include PAT-4/ILK (integrin-linked kinase) and integrin fail to assemble into properly organized arrays. Although in the absence of UNC-97/PINCH, PAT-4/ILK and integrin fail to organize normally, they are capable of colocalizing together at the muscle cell membrane. Alternatively, in integrin and pat-4 mutants, UNC-97/PINCH fails to localize to the muscle cell membrane and instead is found diffusely throughout the muscle cell cytoplasm. In agreement with mammalian studies, we show that LIM domain 1 of UNC-97/PINCH is required for its interaction with PAT-4/ILK in yeast two-hybrid assays. Additionally, we find, by LIM domain deletion analysis, that LIM1 is required for the localization of UNC-97/PINCH to cell adhesion complexes. Our results provide evidence that UNC-97/PINCH is required for the development of C. elegans and is required for the formation of integrin based adhesion structures.     

Oligomerizing potential of a focal adhesion LIM protein Hic-5 organizing a nuclear-cytoplasmic shuttling complex

Hic-5 is a focal adhesion LIM protein serving as a scaffold in integrin signaling. The protein comprises four LD domains in its N-terminal half and four LIM domains in its C-terminal half with a nuclear export signal in LD3 and is shuttled between the cytoplasmic and nuclear compartments. In this study, immunoprecipitation and in vitro cross-linking experiments showed that Hic-5 homo-oligomerized through its most C-terminal LIM domain, LIM4. Strikingly, paxillin, the protein most homologous to Hic-5, did not show this capability. Gel filtration analysis also revealed that Hic-5 differs from paxillin in that it has multiple forms in the cellular environment, and Hic-5 but not paxillin was capable of hetero-oligomerization with a LIM-only protein, PINCH, another molecular scaffold at focal adhesions. The fourth LIM domain of Hic-5 and the fifth LIM domain region of PINCH constituted the interface for the interaction. The complex included integrin-linked kinase, a binding partner of PINCH, which also interacted with Hic-5 through the region encompassing the pleckstrin homology-like domain and LIM domains of Hic-5. Of note, Hic-5 marginally affected the subcellular distribution of PINCH but directed its shuttling between the cytoplasmic and nuclear compartments in the presence of integrin-linked kinase. Uncoupling of the two signaling platforms of Hic-5 and PINCH through interference with the hetero-oligomerization resulted in impairment of cellular growth. Hic-5 is, thus, a molecular scaffold with the potential to dock with another scaffold through the LIM domain, organizing a mobile supramolecular unit and coordinating the adhesion signal with cellular activities in the two compartments.     

Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption

Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.     

Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility

Cell dynamics mediated through cell-extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine-aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)-PEST. PTP-PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP-PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2-3 inhibited cell adhesion and spreading while LIM3-4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.     

The focal adhesion and nuclear targeting capacity of the LIM-containing lipoma-preferred partner (LPP) protein

Targeting of proteins to a particular cellular compartment is a critical determinant for proper functioning. LPP (LIM-containing lipoma-preferred partner) is a LIM domain protein that is localized at sites of cell adhesion and transiently in the nucleus. In various benign and malignant tumors, LPP is present in a mutant form, which permanently localizes the LIM domains in the nucleus. Here, we have investigated which regions in LPP target the protein to its subcellular locations. We found that the LIM domains are the main focal adhesion targeting elements and that the proline-rich region of LPP, which harbors binding sites for alpha-actinin and vasodilator-stimulated phosphoprotein (VASP), has a weak targeting capacity. All of the LIM domains of LPP cooperate in order to provide robust targeting to focal adhesions, and the linker between LIM domains 1 and 2 plays a pivotal role in this targeting. When overexpressed in the cytoplasm of cells, the LIM domains of LPP can deplete endogenous LPP and vinculin from focal adhesions. The proline-rich region of LPP contains targeting sites for focal adhesions and stress fibers that are distinct from the alpha-actinin and VASP binding sites, and the LPP LIM domains are dispensable for targeting LPP to the nucleus. Our studies have defined novel functional domains in the LPP protein.     

Targeting of zyxin to sites of actin membrane interaction and to the nucleus

The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for alpha-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains.     

The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.     

Purification and SAXS Analysis of the Integrin Linked Kinase,PINCH, Parvin (IPP) Heterotrimeric Complex

The heterotrimeric protein complex containing the integrin linked kinase (ILK), parvin, and PINCH proteins, termed the IPP complex, is an essential component of focal adhesions, where it interacts with many proteins to mediate signaling from integrin adhesion receptors. Here we conduct a biochemical and structural analysis of the minimal IPP complex, comprising full-length human ILK, the LIM1 domain of PINCH1, and the CH2 domain of α-parvin. We provide a detailed purification protocol for IPP and show that the purified IPP complex is stable and monodisperse in solution. Using small-angle X-ray scattering (SAXS), we also conduct the first structural characterization of IPP, which reveals an elongated shape with dimensions 120×60×40 Å. Flexibility analysis using the ensemble optimization method (EOM) is consistent with an IPP complex structure with limited flexibility, raising the possibility that inter-domain interactions exist. However, our studies suggest that the inter-domain linker in ILK is accessible and we detect no inter-domain contacts by gel filtration analysis. This study provides a structural foundation to understand the conformational restraints that govern the IPP complex.     

A new focal adhesion protein that interacts with integrin-linked kinase and regulates cell adhesion and spreading

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 NH(2)-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.     

Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes

Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. In order to evaluate the role of PINCH in integrin-mediated cellular events, we have tested directly the in vivo function of PINCH in Drosophila melanogaster. We demonstrate that the steamer duck (stck) alleles that were first identified in a screen for potential integrin effectors represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with betaPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, we show that integrins are required for proper localization of PINCH at the myotendinous junction. The integrin-linked kinase, ILK, is also essential for integrin function. We demonstrate that Drosophila PINCH and ILK are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, ILK localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of ILK. Our results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion.     

Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, alpha-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, alpha-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation.     

Tandem zyxin LIM sequences do not enhance force sensitive accumulation

The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano-signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing protein candidate. Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force-bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity.     

PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.