C. elegans Unc-97 constitutes a novel component of muscular adherens junctions. Unc-97 and homologues from several other species define the PINCH family, a family of LIM proteins whose modular composition of five LIM domains implicates them as potential adapter molecules. unc-97 expression is restricted to tissue types that attach to the hypodermis, specifically body wall muscles, vulval muscles, and mechanosensory neurons. In body wall muscles, the UNC-97 protein colocalizes with the ß-integrin PAT-3 to the focal adhesion-like attachment sites of muscles. Partial and complete loss-of-function studies demonstrate that Unc-97 affects the structural integrity of the integrin containing muscle adherens junctions and contributes to the mechanosensory functions of touch neurons. The expression of a Drosophila homologue of unc-97 in two integrin containing cell types, muscles, and muscle-attached epidermal cells, suggests that unc-97 function in adherens junction assembly and stability has been conserved across phylogeny. In addition to its localization to adherens junctions Unc-97 can also be detected in the nucleus, suggesting multiple functions for this LIM domain protein (Hobert, 1999).
The X-linked unc-97 gene is defined by a single recessive allele, su110. Adult animals homozygous for the su110 mutation are limp, egg laying-defective, and show movement defects that vary from slow moving to paralyzed animals. This probably reflects the degree of disorganization of the vulval and body wall muscles. unc-97 mutants have shallow, easily disrupted muscle sarcomeres. Using polarized light microscopy it has also been found that mutant muscle cells are fragile, but if animals are handled carefully intact sarcomeres can be observed. When handled gently, unc-97(su110) muscle largely resembles wild-type muscle. Sarcomere dimensions and overall organization are similar between unc-97(su110) and wild-type muscle, but the dense bodies in su110 animals are never as clearly defined as in wild-type animals. The combination of coverslip pressure and slight rolling of an unc-97(su110) animal will also lead to the collapse of the sarcomere structure. Under similar conditions sarcomeres within wild-type muscle cells do not collapse. Anchorage of the sarcomere complex to the plasma membrane is therefore weak and easily disrupted in unc-97(su110) animals (Hobert, 1999).
The lability of the sarcomeres as observed by polarized light microscopy prompted an analysis of the structural integrity of the muscle attachment structures in more detail. To this end, the subcellular localization of two defined components of these muscle-hypodermal adherens junctions was determined. Immunhistochemistry with a ß-integrin/ PAT-3 monoclonal antibody revealed that the dense bodies do not for the most part show up as rows of discrete spots in unc-97 mutants, but instead appear primarily as diffuse stripes running parallel to the M line. Similarly, vinculin, which normally localizes exclusively to dense bodies, shows a diffuse stripe-like appearance in unc-97 mutants; the diffuse stripes do not always run in parallel, but occasionally fuse to one another. Due to the presence of M lines in the anti-PAT-3-stained animals, the fusion of these stripes can more easily be observed using anti-vinculin stained antibodies. These observations are consistent with UNC-97 playing a role in determining the structural integrity of adherens junctions, perhaps through clustering integrin or other dense body-associated proteins (Hobert, 1999).
The C. elegans proteoglycan perlecan, encoded by the unc-52 gene, localizes to the basement membrane underlying the muscle. Staining with anti-UNC-52 antibodies reveals staining at periodicities corresponding to the sites of the dense bodies and M-line structures. In unc-97(su110) mutant animals, the periodicity of the staining is abrogated; instead, UNC-52 appears more diffuse. The effect of the intracellular UNC-97 protein on the localization of the extracellular UNC-52 protein demonstrates that the correct localization of extracellular matrix components depends on the structural integrity of the muscular integrin complexes (Hobert, 1999).
To monitor the expression pattern of Unc-97, the transcriptional regulatory regions of the Unc-97 gene were fused to GFP and its expression was analyzed in transgenic C. elegans. A translational fusion protein, encoding all exons and introns of Unc-97 reveals a similar set of expressing cells. In both larvae and adult animals, Unc-97 is expressed in two different cell types, muscles and neurons. Strong expression is detectable in body wall muscle cells and vulval muscle cells, whereas no expression can be detected in pharyngeal muscles, intestinal muscle, or the anal depressor muscle. Weak expression can be observed in the anal sphincter muscle. In the nervous system, Unc-97 is expressed in the six mechanosensory receptor neurons, ALML/R, PLML/R, AVM, and PVM, that are responsible for sensing light touch. Intriguingly, a common theme of all the Unc-97-expressing cell types is their attachment via the extracellular matrix to the hypodermis. Unc-97 expression was examined at earlier developmental stages to determine its onset of expression in development. In embryos, Unc-97 expression can first be observed at mid-embryogenesis at ~300 min of development (Hobert, 1999).
The structural components that anchor myofibers to the extracellular matrix, such as integrin, vinculin, talin, and alpha-actinin are remarkably conserved in C. elegans and resemble the protein composition of adherens junctions in other systems, such as focal adhesions in tissue culture cells. It is thus likely that the fundamental mechanisms required to assemble these structure are conserved as well. Based on functional analysis of Unc-97 and the presence of highly related homologous molecules in flies and vertebrates, it is suggested that members of the PINCH family of LIM-only proteins are a critical component of muscle attachment and adherens junction assemblies across phylogeny. Indeed, the mouse Unc-97 homologue PINCH-1 has recently been shown to colocalize with ß1-integrins at focal adhesions. This interaction appears to be mediated by the ankyrin-repeat containing serine/threonine kinase Ilk, which by direct association with both ß1-integrins (Hannagan, 1996) and the mouse Unc-97 homologue PINCH-1 (Wu, C., personal communication to Hobert, 1999) appears to serve as a bridging molecule (Hobert, 1999).
Cellular attachment sites contain several LIM domain proteins, such as zyxin, paxillin, cysteine-rich proteins (CRP proteins: see Drosophila Mlp60A and Mlp84B) and others. In muscles, the CRP3/MLP and ALP proteins localize to Z-discs, attachment points of myofibers in vertebrates. Drosophila CRP homologues also localize to muscle attachment sites. However, the functional requirement for any LIM containing protein at these subcellular sites has until now only been reported for the CRP3/MLP protein, whose loss-of-function causes disorganization of cardiac myofibers. A similar requirement is demonstrated in this study for the LIM-only protein Unc-97 in body wall muscles in C. elegans. Since the C. elegans genome contains a gene highly related to the CRP proteins (T04C9.4), it is speculated that other LIM proteins besides Unc-97 will also be involved in adherens junction assembly (Hobert, 1999).
The su110 allele indicates that Unc-97 has an important role in maintaining sarcomere organization in growing and adult animals. Moreover, the RNAi (probable loss-of-function) experiment indicates an important regulatory role for Unc-97 during embryonic muscle development. However, it is unclear so far as to whether the embryonic defects caused by Unc-97 RNA interference reflect a requirement for Unc-97 in the initial stages of assembly of the adherens junctions or reflects a requirement for Unc-97 in stabilizing adherens junctions after they are formed to resist the mechanical stress they are exposed to upon elongation of the animal. Since Unc-97 expression clearly coincides with the onset of expression of adherens junction components and their assembly into these structures, a model is favored in which Unc-97 is involved in the initial assembly of the adherens junction components and keeps residing in these structures to ensure their stability (Hobert, 1999).
Although it is unclear how Unc-97 cooperates with ß-integrins and other focal adhesion components such as vinculin to determine adherens junction integrity, the modular organization of Unc-97 into five LIM domains implicates Unc-97 in binding to multiple proteins. Unc-97 might be a scaffold or adapter protein onto which various different components assemble to form a functional muscle attachment site. Since defective myofibrils cause as a secondary consequence the destabilization of muscle attachment sites, it is for example possible that Unc-97 serves as an anchor between myofibrils and membrane-anchored integrin components. In this model, dense body defects in Unc-97(su110) mutant animals would not arise from direct defects in the dense body structure per se, but would represent a secondary consequence of the myofibril disorganization (Hobert, 1999).
Analysis of Unc-97 localization in a living, nonfixed animal, using a rescuing, i.e., functionally intact Unc-97:: GFP reporter gene, lends support for an authentic in vivo dual subcellular localization of this LIM protein. However, at this point it is unclear whether the nuclear localization of Unc-97 is indeed functionally significant. In contrast to the localization of Unc-97 to adherens junctions, which correspond to the site of action of Unc-97 as inferred from the Unc-97 mutant phenotype, no such clear correlation exists to a possible nuclear function of Unc-97. However, the demonstration of a genetic interaction of Unc-97 with the LIM homeodomain transcription factor mec-3 could be explained on the basis of a physical interaction between these proteins and could thus reflect a functional requirement for Unc-97 in the nucleus. LIM- LIM interaction have been previously described and the direct interaction of Unc-97 with MEC-3 could affect the transcriptional activity of the MEC-3 transcription factor. Alternatively, it is also entirely possible that the genetic interaction of Unc-97 and mec-3 reflects an independent requirement for these genes in mechanosensory processes (Hobert, 1999).
Although further experiments will need to address the physiological significance of Unc-97 in the nucleus, there are several attractive hypotheses regarding a potential nuclear function of Unc-97. Unc-97 could be directly involved in gene regulatory events by interacting with specific transcription factors. The vertebrate LIM-only proteins CRP3/MLP and SLIM1/KyoT have been directly implicated in gene regulation via interaction with the transcription factors MyoD and RBP-J, respectively, whereas the LIM-only protein LMO2 assembles higher order transcriptional activation complexes by bridging other transcription factors that are directly involved in DNA binding . Alternatively, but not necessarily mutually exclusive, Unc-97 could represent a structural component of specific subnuclear domains. The localization of Unc-97 to discrete dots in muscle nuclei supports this hypothesis, although the nature of these dots is entirely unclear. Subnuclear domains of different types, such as speckles, coiled bodies, gems, and Kr bodies/nuclear domains have been described in various systems. Factors localizing to these domains are involved in distinct nuclear processes such transcriptional silencing and RNA processing (Hobert, 1999 and references therein).
Lastly, in regard to the dual subcellular localization of Unc-97 it is also tempting to speculate that Unc-97 transmits a signal from attachment sites to the nucleus. Although the question whether Unc-97 dynamically shuttles between attachment sites and the nucleus has not been addressed, focal adhesion-nuclear shuttling has been recently demonstrated for the LIM protein zyxin. The mouse Unc-97 homologue PINCH-1 has been shown to interact specifically with, and might serve as a substrate of, the integrin-linked kinase Ilk (Tu, 1999), a serine-threonine kinase implicated in integrin signal transduction. This observation might point to a potential role for Unc-97 in integrin-mediated signal transduction (Hobert, 1999).
Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion-like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of beta-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). It has been shown that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines (Miller, 2006).
The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent OFT septation are critical events during vertebrate heart development. Four modified differential display (DD) screens were performed in the chick embryo to identify genes that may be involved in CNC and heart development. Full-length sequence of one of the DD clones has been obtained and identified as chick PINCH-1. This particularly interesting new cysteine-histidine-rich protein contains five protein-binding LIM domains (five double zinc fingers), a nuclear localization signal, and a nuclear export signal, allowing it to participate in integrin and growth factor signaling and possibly act as a transcription factor. Chick PINCH-1 is expressed in neural crest cells, both in the neural fold and cardiac OFT, and is also expressed in mesoderm derived-structures, including the myocardium, during avian embryogenesis. The normal expression pattern and overexpression in neural crest cell explants suggest that PINCH-1 may be a regulator of neural crest cell adhesion and migration (Martinsen, 2006).
Autoantibody eluted from aged human red blood cells was used to immunoscreen a human fetal liver expression library and led to the isolation of a cDNA encoding a novel 35.8 kDa protein with five LIM domains. An autoepitope homologous to the "senescent cell antigen" on the red blood cell membrane anion exchange protein is present in the first zinc finger of the third LIM domain. The gene for this novel protein is highly conserved in vertebrates, and its 4.6 kb mRNA is widely expressed in human tissues. Recombinant autoantigens such as the one reported here have potential applications in vitro for the purification, identification and quantitation of autoantibodies, and in vivo for the removal of autoantibodies, increasing red blood cell lifespan and reducing the need for transfusion (Rearden, 1994).
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 (Drosophila homolog; Dreadlocks). A new isoform of PINCH is described that is called PINCH2. PINCH has therefore been renamed 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, differential expression pattern of the mRNAs is observed. 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. PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes (Braun, 2003).
The distribution and abundance of PINCH in patients with breast, prostate, lung, colon, and skin carcinomas has been . Immunostaining for PINCH is increased in the cytoplasm of fibroblastoid cells in areas of the tumor-associated stroma in all carcinomatous tissues evaluated. The most intense stromal immunostaining for PINCH was noted at invasive edges, particularly in breast carcinomatous tissue. Immunoblotting of lysates from normal breast and breast carcinomatous tissue confirmed that PINCH protein expression is markedly increased in breast carcinomatous tissues. It is concluded that PINCH is up-regulated in tumor-associated stromal cells, particularly at invasive edges, and may be a marker for stroma manifesting the ability to facilitate invasion. Because of this and because PINCH functions as a "molecular switch" in signal transduction, PINCH may be a new target for drug discovery aimed at the tumor-associated stroma (Wang-Rodriguez, 2003).
PINCH2 belongs, together with PINCH1, to a family of focal adhesion proteins, the members of which are composed of five LIM domains. PINCH1 and PINCH2 interact, through their first LIM domain, with integrin-linked kinase and thereby link integrins with several signal transduction pathways. Despite their high similarity, PINCH1 and PINCH2 exert distinct functions during cell spreading and cell survival. To investigate the function of PINCH2 in vivo, PINCH2 was deleted in the mouse using the loxP/Cre system. In contrast to PINCH1-deficient mice, which die at the peri-implantation stage, PINCH2-null mice are viable, fertile and show no overt phenotype. Histological analysis of tissues that express high levels of PINCH2 such as bladder and kidney revealed no apparent abnormalities, but showed a significant upregulation of PINCH1, suggesting that the two PINCH proteins may have, at least in part, overlapping function in vivo. To further test this possibility, PINCH1-null mouse embryonic fibroblasts, which express neither PINCH1 nor PINCH2, were established. It was found that in fibroblasts with a PINCH1/2-null background, PINCH2 is able to rescue the spreading and adhesion defects of mutant fibroblasts to the same extent as PINCH1. Furthermore, the LIM1 domain only of either PINCH1 or PINCH2 can prevent ILK degradation despite their failure to localize to focal adhesions. Altogether these results suggest that PINCH1 and PINCH2 share overlapping functions and operate dependently and independently of their subcellular localization (Stanchi, 2005).
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. 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 (Xu, 2005).
Integrin-linked kinase (Ilk) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. Ilk is a constituent of cell-matrix focal adhesions. Ilk Is recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, Ilk Is absent in E-cadherin-mediated cell-cell adherens junctions. PINCH is a protein consisting of five LIM domains, and has been identified as an Ilk binding protein. The Ilk-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of Ilk and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The Ilk ANK repeats domain, which is capable of interacting with PINCH in vitro, can also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex is markedly reduced in the absence of the C-terminal domain of Ilk. The PINCH binding defective ANK1 deletion Ilk mutant, unlike the wild-type Ilk, is unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of Ilk. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of Ilk, since an Ilk mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site fails to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where Ilk functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating Ilk cellular function (Li, 1999).
Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. ILK is a constituent of cell-matrix focal adhesions. ILK is recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK is absent in E-cadherin-mediated cell-cell adherens junctions. PINCH, a protein consisting of five LIM domains, has been identified as an ILK binding protein. ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeat domain, which is capable of interacting with PINCH in vitro, can also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex is markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, is unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, since an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site fails to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function (Lynch, 1999).
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. 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. These proteins interact 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. PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and studies 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 (Tu, 1999).
Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. A new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP) has been identified and characterized. 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. CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolishes 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 impairs the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibits 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 (Tu, 2001).
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. The PINCH LIM1 domain NMR structure has been solved and its binding to ILK has been characterized. 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 demonstrate a 1:1 complex between PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift mapping experiment has 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 indicates that the LIM motif might have a highly variable mode in recognizing various target proteins (Velyvis, 2001).
Integrin-linked kinase (Ilk) is a multidomain protein that plays important roles at cell-extracellular matrix (ECM) adhesion sites. A new LIM-domain containing protein (termed as PINCH-2) is described 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 shows that PINCH-2 localizes to both cell-ECM contact sites and the nucleus. Deletion of the first LIM (LIM1) domain of PINCH-2 abolishes 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, is 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 inhibits the PINCH-1-Ilk interaction and reduces 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 (Zhang, 2002a).
PINCH, integrin-linked kinase (Ilk) and calponin homology-containing Ilk-binding protein (CH-IlkBP) form a ternary complex that plays crucial roles at cell-extracellular matrix adhesion sites. To understand the mechanism underlying the complex formation and recruitment to cell-adhesion sites a combined structural, mutational and cell biological analysis was undertaken. Three-dimensional structure-based point mutations identified specific PINCH and Ilk sites that mediate the complex formation. Analyses of the binding defective point mutants revealed that the assembly of the PINCH-Ilk-CH-IlkBP complex is essential for their localization to cell-extracellular matrix adhesion sites. The formation of the PINCH-Ilk-CH-IlkBP complex precedes integrin-mediated cell adhesion and spreading. Furthermore, inhibition of protein kinase C, but not that of actin polymerization, inhibits the PINCH-Ilk-CH-IlkBP complex formation, suggesting that the PINCH-Ilk-CH-IlkBP complex likely serves as a downstream effector of protein kinase C in the cellular control of focal adhesion assembly. Finally, evidence is provided that the formation of the PINCH-Ilk-CH-IlkBP complex, while necessary, is not sufficient for Ilk localization to cell-extracellular matrix adhesion sites. These results provide new insights into the molecular mechanism underlying the assembly and regulation of cell-matrix adhesion structures (Zhang, 2002b).
PINCH co-localizes with Ilk in both focal adhesions and fibrillar adhesions. The molecular basis underlying the targeting of PINCH to the cell-matrix contact sites and the functional significance of the PINCH-Ilk interaction have been investigated. 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, retard cell spreading, and reduce cell motility. These results suggest that PINCH, through its interaction with Ilk, is crucially involved in the regulation of cell shape change and motility (Zhang, 2002c).
PINCH-1 is a widely expressed focal adhesion protein that forms a ternary complex with integrin-linked kinase (ILK) and CH-ILKBP/actopaxin/alpha-parvin (abbreviated as alpha-parvin herein). RNAi was used to investigate the functions of PINCH-1 and ILK in human cells. PINCH-1 and ILK, but not alpha-parvin, are shown to be essential for prompt cell spreading and motility. PINCH-1 and ILK, like alpha-parvin, are crucial for cell survival. Also, PINCH-1 and ILK are required for optimal activating phosphorylation of PKB/Akt, an important signaling intermediate of the survival pathway. Whereas depletion of ILK reduces Ser473 phosphorylation but not Thr308 phosphorylation of PKB/Akt, depletion of PINCH-1 reduces both the Ser473 and Thr308 phosphorylation of PKB/Akt. PINCH-1 and ILK function in the survival pathway not only upstream but also downstream (or in parallel) of protein kinase B (PKB)/Akt. This study also shows that, PINCH-1, ILK and to a less extent alpha-parvin are mutually dependent in maintenance of their protein, but not mRNA, levels. The coordinated down-regulation of PINCH-1, ILK, and alpha-parvin proteins is mediated at least in part by proteasomes. Finally, increased expression of PINCH-2, an ILK-binding protein that is structurally related to PINCH-1, prevented the down-regulation of ILK and alpha-parvin induced by the loss of PINCH-1 but failed to restore the survival signaling or cell shape modulation. These results provide new insights into the functions of PINCH proteins in regulation of ILK and alpha-parvin and control of cell behavior (Fukuda, 2003).
Many of the protein-protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. A widely expressed, Nck-related adaptor protein termed Nck-2 has been characterized. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-beta, and IRS-1. The association of Nck-2 with EGF receptors is regulated by EGF stimulation and involves largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributes to the complex formation. The association of Nck-2 with PDGF receptor-beta is dependent on PDGF activation and is mediated solely by the SH2 domain of Nck-2. Additionally, a stable association has been detected between Nck-2 and IRS-1 that is mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, evidence is provided indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain-containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways (Tu, 1998).
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. The LIM1 domain of PINCH functions in focal adhesions by binding specifically to integrin-linked kinase. Using NMR spectroscopy, it has been shown 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 disrupts LIM-SH3 interaction and substantially impairs 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 (Velyvis, 2003).
Weak protein-protein interactions (PPIs) are critical determinants of many biological processes. However, in contrast to a large growing number of well-characterized, strong PPIs, the weak PPIs are poorly explored. Genome wide, there exist few 3D structures of weak PPIs with, and none with. This study reports the NMR structure of an extremely weak focal adhesion complex between Nck-2 SH3 domain and PINCH-1 LIM4 domain. The structure exhibits a remarkably small and polar interface with distinct binding modes for both SH3 and LIM domains. Such an interface suggests a transient Nck-2/PINCH-1 association process that may trigger rapid focal adhesion turnover during integrin signaling. Genetic rescue experiments demonstrate that this interface is indeed involved in mediating cell shape change and migration. Together, the data provide a molecular basis for an ultraweak PPI in regulating focal adhesion dynamics during integrin signaling (Vaynberg, 2005).
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 interacts with Hic-5 through the region encompassing the pleckstrin homology-like domain and LIM domains of Hic-5. Of note, Hic-5 marginally affects the subcellular distribution of PINCH but directs 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 (Mori, 2006).
Heart disease is a leading cause of death in newborn children and in adults. Efforts to promote cardiac repair through the use of stem cells hold promise but typically involve isolation and introduction of progenitor cells. This study shows that the G-actin sequestering peptide thymosin beta4 promotes myocardial and endothelial cell migration in the embryonic heart and retains this property in postnatal cardiomyocytes. Survival of embryonic and postnatal cardiomyocytes in culture is also enhanced by thymosin beta4. Thymosin beta4 forms a functional complex with PINCH and integrin-linked kinase (ILK), resulting in activation of the survival kinase Akt (also known as protein kinase B). After coronary artery ligation in mice, thymosin beta4 treatment results in upregulation of ILK and Akt activity in the heart, enhanced early myocyte survival and improved cardiac function. These findings suggest that thymosin beta4 promotes cardiomyocyte migration, survival and repair and the pathway it regulates may be a new therapeutic target in the setting of acute myocardial damage (Bock-Marquette, 2004).
Proteins at cell-extracellular matrix adhesions (e.g. focal adhesions) are crucially involved in regulation of cell morphology and survival. CH-ILKBP/actopaxin/alpha-parvin and affixin/beta-parvin (abbreviated as alpha- and beta-parvin, respectively), two structurally closely related integrin-linked kinase (ILK)-binding focal adhesion proteins, are co-expressed in human cells. Depletion of alpha-parvin dramatically increases the level of beta-parvin, suggesting that beta-parvin is negatively regulated by alpha-parvin in human cells. Loss of PINCH-1 or ILK, to which alpha- and beta-parvin bind, significantly reduces the activation of Rac, a key signaling event that controls lamellipodium formation and cell spreading. It was surprising to find that loss of alpha-parvin, but not that of beta-parvin, markedly stimulates Rac activation and enhances lamellipodium formation. Overexpression of beta-parvin, however, is insufficient for stimulation of Rac activation or lamellipodium formation, although it is sufficient for promotion of apoptosis, another important cellular process that is regulated by PINCH-1, ILK, and alpha-parvin. In addition, the interactions of ILK with alpha- and beta-parvin are mutually exclusive. Overexpression of beta-parvin or its CH(2) fragment, but not a CH(2) deletion mutant, inhibited the ILK-alpha-parvin complex formation. Finally, evidence is provided suggesting that inhibition of the ILK-alpha-parvin complex is sufficient, although not necessary, for promotion of apoptosis. These results identify Rac as a downstream target of PINCH-1, ILK, and parvin. Furthermore, they demonstrate that alpha- and beta-parvins play distinct roles in mammalian cells and suggest that the formation of the ILK-alpha-parvin complex is crucial for protection of cells from apoptosis (Zhang, 2004).
PINCH is a double zinc finger domain (LIM)-only adapter protein that functions to recruit the integrin-linked kinase (Ilk) to sites of integrin activation. Genetic studies have shown that PINCH and Ilk are required for integrin signaling. Since integrin activation is associated with Schwann cell migration, neurite outgrowth and regeneration, this study examined PINCH in the normal peripheral nervous system and after chronic constriction injury (CCI) in adult Sprague-Dawley rats. Immunohistochemistry identified PINCH immunoreactivity in cell bodies of dorsal root ganglia (DRG) neurons, axons, satellite cells, and Schwann cells. PINCH immunostaining was localized to the membrane of uninjured DRG cell bodies consistent with its localization at a site of integrin activation. In contrast, 5 days following CCI, PINCH immunostaining was diffuse throughout the DRG cell cytoplasm. Confocal microscopy of primary and transformed Schwann cells localized PINCH in cytoplasmic, perinuclear and nuclear areas. Examination of the PINCH sequence revealed a putative leucine-rich nuclear export signal (NES) and an overlapping basic nuclear localization signal (NLS). To demonstrate nuclear export of PINCH, rabbit anti-PINCH IgG was microinjected into Schwann cell nuclei and allowed to combine with PINCH contained within the nucleus. Immunofluorescence showed that the PINCH and anti-PINCH IgG complex rapidly translocated to the cytoplasm. Treatment with leptomycin B causes nuclear accumulation of PINCH, indicating that the CRM1 pathway mediates nuclear export of PINCH. Ilk activity in Schwann cells is enhanced by platelet-derived growth factor (PDGF) and tumor necrosis factor alpha. PINCH immunoprecipitates from PDGF- and TNFalpha-stimulated Schwann cells contained several high-molecular-weight threonine-phosphorylated proteins. Taken together, these results indicate that PINCH is an abundant shuttling/signaling protein in Schwann cells and DRG neurons (Campana, 2003).
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