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.     

PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes

PINCH1, an adaptor protein composed of five LIM domains, mediates protein-protein interactions and functions as a component of the integrin-integrin-linked kinase (ILK) complex. The integrin-ILK signaling complex plays a pivotal role in cell motility, proliferation, and survival during embryonic development of many animal species. To elucidate the physiological function of PINCH1 in mouse embryonic development, we have deleted the mouse PINCH1 gene by homologous recombination. Mice heterozygous for PINCH1 are viable and indistinguishable from wild-type littermates. However, no viable homozygous offspring were observed from PINCH1+/- intercrosses. Histological analysis of homozygous mutant embryos revealed that they had a disorganized egg cylinder by E5.5, which degenerated by E6.5. Furthermore, E5.5 PINCH1-/- embryos exhibited decreased cell proliferation and excessive cell death. We have also generated and analyzed mice in which PINCH1 has been specifically deleted in ventricular cardiomyocytes. These mice exhibit no basal phenotype, with respect to mouse survival, cardiac histology, or cardiac function as measured by echocardiography. Altogether, these data indicate that PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes.     

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.     

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.     

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.     

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.     

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.     

Oracle, a novel PDZ-LIM domain protein expressed in heart and skeletal muscle

In order to identify novel genes enriched in adult heart, we performed a subtractive hybridization for genes expressed in mouse heart but not in skeletal muscle. We identified two alternative splicing variants of a novel PDZ-LIM domain protein, which we named Oracle. Both variants contain a PDZ domain at the amino-terminus and three LIM domains at the carboxy-terminus. Highest homology of Oracle was found with the human and rat enigma proteins in the PDZ domain (62 and 61%, respectively) and in the LIM domains (60 and 69%, respectively). By Northern hybridization analysis, we showed that expression is highest in adult mouse heart, low in skeletal muscle and undetectable in other adult mouse tissues. In situ hybridization in mouse embryos confirmed and extended these data by showing high expression of Oracle mRNA in atrial and ventricular myocardial cells from E8.5. From E9.5 low expression of Oracle mRNA was detectable in myotomes. These data suggest a role for Oracle in the early development and function of heart and skeletal muscle.     

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.     

A critical role of the PINCH-integrin-linked kinase interaction in the regulation of cell shape change and migration.

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.     

Chromosomal assignment and expression pattern of the murine Lasp-1 gene

The human Lasp-1 (LIM and SH3 protein) gene was previously identified by differential screening of a breast cancer-derived metastatic lymph node cDNA library. It was located on the q12–q21 region of human chromosome 17 and was shown to be amplified and overexpressed in 12% of breast tumours. Lasp-1 defines a new LIM-protein subfamily, as it associates a C-terminal Src homology 3 (SH3) domain to a N-terminal LIM motif. In this study, the isolation and characterization of the cDNA encoding the mouse Lasp-1 protein are described, and it is shown to be highly conserved with its human counterpart. In addition to the LIM and SH3 domains, both human and mouse Lasp-1 contain an actin-binding domain. The mouse gene was mapped by in situ hybridization to the 11C–11D region of chromosome 11. Northern blot analysis shows that this gene is expressed from 7.5 to 17.5 days post-coitum of mouse embryogenesis and in almost all adult tissues.     

Gene expression patterns of the ALP family during zebrafish development

The actinin-associated LIM protein (ALP) genes belong to the PDZ/LIM protein family which is characterized by the presence of both a PDZ and a LIM domain. The ALP subfamily in mammals has four members: ALP, Elfin, Mystique and RIL. In this study, we have annotated and cloned the zebrafish ALP gene family and identified a zebrafish-specific fifth member of the family, the alp-like gene. We compared the zebrafish sequences to their human and mouse orthologues. A phylogenetic analysis based on the amino acid sequences showed the overall high degree of conservation within the family. We describe here the expression patterns for all five ALP family genes during zebrafish development. Whole mount in situ hybridization results revealed common and distinct expression patterns for the five genes. With the exception of elfin, all genes were expressed as maternal RNAs at early developmental stages. Gene expression for all of them appeared regulated and localized in specific regions at the eight different developmental stages studied. Expression for all five genes was observed in the central nervous system (CNS), which led us to further investigate brain-specific expression in sections of embryos at 2 days of development. In summary, we identified the zebrafish orthologues of the ALP family and determined their gene expression patterns during zebrafish embryogenesis. Finally, we compare our results to the limited expression data available for this gene family during mammalian development.     

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.     

Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

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. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that 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, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was 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, we provide evidence 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.     

VITO-1, a novel vestigial related protein is predominantly expressed in the skeletal muscle lineage

In order to identify novel genes expressed in skeletal muscle we performed a subtractive hybridization for genes expressed in human skeletal muscle but not in other tissues. We identified a novel scalloped interaction domain (SID) containing protein in humans and in the mouse, which we named VITO-1. Highest homology of VITO-1 was found with the Drosophila vestigial and the human TONDU proteins in the SID (54 and 40%, respectively). Using whole-mount hybridzation and Northern blot analysis, we showed that VITO-1 is expressed in the somitic myotome from E8.75 mouse embryos onwards and later on in skeletal muscle but not in the heart. Additional expression domains during development were detected in the pharyngeal pouches and clefts starting at E8.0 as well as in the cranial pharynx and in Rathkes pouch. By Northern blot analysis, we found VITO-1 to be up-regulated in C2C12 myotubes although some expression can be detected in proliferating C2C12 myoblasts. No expression was spotted in other adult mouse tissues. Likewise, expression of human Vito-1 during fetal and adult human development was found exclusively in skeletal muscle preferentially in fast skeletal muscles. These data suggest a role of VITO-1 for the development of skeletal muscles and of pharyngeal clefts/Rathkes' pouch derived structures.