Integrin linked kinase


Identification of Integrin-linked kinase

The interaction of cells with the extracellular matrix regulates cell shape, motility, growth, survival, differentiation and gene expression, through integrin-mediated signal transduction. A two-hybrid screen was performed to isolate genes encoding proteins that interact with the beta 1-integrin cytoplasmic domain. The most frequently isolated complementary DNA encodes a new, 59K serine/threonine protein kinase, containing four ankyrin-like repeats. This integrin-linked kinase (ILK) phosphorylates a beta 1-integrin cytoplasmic domain peptide in vitro and coimmunoprecipitates with beta 1 in lysates of mammalian cells. Endogenous ILK kinase activity is reduced in response to fibronectin. Overexpression of p59ILK disrupts epithelial cell architecture and inhibits adhesion to integrin substrates, while inducing anchorage-independent growth. It is proposed that ILK is a receptor-proximal protein kinase regulating integrin-mediated signal transduction (Hannigan, 1996).

Integrin-linked kinase protein interactions

PINCH (Drosophila homolog: Steamer duck) 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 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).

Paxillin is a focal adhesion adapter protein involved in integrin signaling. Paxillin LD motifs bind several focal adhesion proteins including the focal adhesion kinase, vinculin, the Arf-GTPase-activating protein paxillin-kinase linker, and the newly identified actin-binding protein actopaxin. Microsequencing of peptides derived from a 50-kDa paxillin LD1 motif-binding protein reveal 100% identity with integrin-linked kinase (ILK)-1, a serine/threonine kinase that has been implicated in integrin, growth factor, and Wnt signaling pathways. Cloning of ILK from rat smooth muscle cells generates a cDNA that exhibits 99.6% identity at the amino acid level with human ILK-1. A monoclonal antibody raised against a region of the carboxyl terminus of ILK, which is identical in rat and human ILK-1 protein, recognizes a 50-kDa protein in all cultured cells and tissues examined. ILK binds directly to the paxillin LD1 motif in vitro. Co-immunoprecipitation from fibroblasts confirmed that the association between paxillin and ILK occurs in vivo in both adherent cells and cells in suspension. Immunofluorescence microscopy of fibroblasts demonstrates that endogenous ILK as well as transfected green fluorescent protein-ILK co-localizes with paxillin in focal adhesions. Analysis of the deduced amino acid sequence of ILK has identified a paxillin-binding subdomain in the carboxyl terminus of ILK. In contrast to wild-type ILK, paxillin-binding subdomain mutants of ILK are unable to bind to the paxillin LD1 motif in vitro and fail to localize to focal adhesions. Thus, paxillin binding is necessary for efficient focal adhesion targeting of ILK and may therefore impact the role of ILK in integrin-mediated signal transduction events (Nikolopoulos, 2001).

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP is independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Conditional expression of ILKAP in HEK 293 cells results in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but does not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, does not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, are differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant, ILKAP strongly inhibits insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but does not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signaling. Consistent with this, active ILKAP selectively inhibits transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. It is proposed that ILKAP regulates ILK1 activity, targeting ILK1 signaling of Wnt pathway components via modulation of GSK3beta phosphorylation (Leung-Hagesteijn, 2001).

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. In-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry show that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB-serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt (Persad, 2001).

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).

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. A novel ILK-binding protein, affixin, has been identified that consists of two tandem calponin homology domains. In CHO cells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion (Yamaji, 2001).

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; see Drosophila Parvin). 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).

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).

Thymosin beta4 forms a functional complex with PINCH and integrin-linked kinase

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).

Rac is a downstream target of PINCH-1, ILK, and parvin

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 interacts with Hic-5: Hic-5 directs PINCH shuttling between the cytoplasmic and nuclear compartments in the presence of integrin-linked kinase

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).

Integrin-linked kinase and cell signaling

Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D- and E-dependent kinases, the latter via the up-regulation of cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27, respectively. This adhesion-dependent regulation of cell cycle is thought to be mediated by integrins. Stable transfection and overexpression of the integrin-linked kinase, which interacts with the beta1 and beta3 integrin cytoplasmic domains, induces anchorage-independent cell cycle progression but not serum-independent growth of rat intestinal epithelial cells (IEC18). ILK overexpression results in increased expression of cyclin D1, activation of Cdk4 and cyclin E-associated kinases, and hyperphosphorylation of the retinoblastoma protein. In addition, ILK overexpression results in the expression of p21 and p27 Cdk inhibitors with altered electrophoretic mobilities, with the p27 from ILK-overexpressing cells having reduced inhibitory activity. The transfer of serum-exposed IEC18 cells from adherent cultures to suspension cultures results in a rapid down-regulation of expression of cyclin D1 and cyclin A proteins as well as in retinoblastoma protein dephosphorylation. In marked contrast, transfer of ILK-overexpressing cells from adherent to suspension cultures results in continued high levels of expression of cyclin D1 and cyclin A proteins, and a substantial proportion of the retinoblastoma protein remains in a hyperphosphorylated state. These results indicate that, when overexpressed, ILK induces signaling pathways resulting in the stimulation of G1/S cyclin-Cdk activities, which are normally regulated by cell adhesion and integrin engagement (Radeva, 1997).

Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine-threonine protein kinase capable of interacting with the cytoplasmic domains of integrin beta1, beta2, and beta3 subunits. Overexpression of ILK in epithelial cells disrupts cell-extracellular matrix as well as cell-cell interactions; suppresses suspension-induced apoptosis (also called Anoikis), and stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of beta-catenin, where the latter associates with a T cell factor/lymphocyte enhancer-binding factor 1 (TCF/LEF-1) to form an activated transcription factor. ILK activity is rapidly, but transiently, stimulated upon attachment of cells to fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore, phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK in vitro, and in addition, membrane targetted constitutively active Pi(3)K activates ILK in vivo. ILK is an upstream effector of the Pi(3)K-dependent regulation of both protein kinase B (PKB/AKT) and glycogen synthase kinase 3 (GSK-3). Specifically, ILK can directly phosphorylate GSK-3 in vitro and when stably, or transiently, overexpressed in cells can inhibit GSK-3 activity, whereas the overexpression of kinase-deficient ILK enhances GSK-3 activity. In addition, kinase-active ILK can phosphorylate PKB/AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of PKB/AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB/AKT activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor mediated, activation of PKB/AKT, and inhibition of GSK-3 (Delcommenne, 1998).

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine/threonine protein kinase that can interact directly with the cytoplasmic domains of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell-cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. Modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of E-cadherin expression, translocation of beta-catenin to the nucleus, formation of a complex between beta-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1/beta-catenin complex. LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-ras or v-src oncogenes do not result in the activation of LEF-1/beta-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1/beta-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cell-matrix interactions and cell-cell adhesion as well as components of the Wnt signaling pathway (Novak, 1998).

Loss of functional adenomatous polyposis coli (APC) protein results in the stabilization of cytosolic beta-catenin and activation of genes that are responsive to Lef/Tcf family transcription factors. An independent cell adhesion and integrin linked kinase (ILK)-dependent pathway can also activate beta-catenin/LEF mediated gene transcription and downregulate E-cadherin expression. In addition, ILK activity and expression are elevated in adenomatous polyposis and colon carcinomas. To examine the role of this pathway in the background of APC mutations, ILK activity was inhibited in APC-/- human colon carcinoma cell lines. In all cases, inhibition of ILK results in substantial inhibition of TCF mediated gene transcription and inhibition of transcription and expression of the TCF regulated gene, cyclin D1. Inhibition of ILK results in decreased nuclear beta-catenin expression, and in the inhibition of phosphorylation of GSK-3 and stimulation of its activity, leading to accelerated degradation of beta-catenin. In addition, inhibition of ILK suppresses cell growth in culture as well as growth of human colon carcinoma cells in SCID mice. Strikingly, inhibition of ILK also results in the transcriptional stimulation of E-cadherin expression and correlates with the inhibition of gene transcription of snail, a repressor of E-cadherin gene expression. Overexpression of ILK causes a stimulation of expression of snail, but snail expression was found not to be regulated by beta-catenin/Tcf. These data demonstrate that ILK can regulate beta-catenin/TCF and snail transcription factors by distinct pathways. It is proposed that inhibition of ILK may be a useful strategy in the control of progression of colon as well as other carcinomas (Tan, 2001).

Cell attachment and the assembly of cytoskeletal and signaling complexes downstream of integrins are intimately linked and coordinated. Although many intracellular proteins have been implicated in these processes, a new paradigm is emerging from biochemical and genetic studies that implicates integrin-linked kinase (ILK) and its interacting proteins, such as CH-ILKBP (alpha-parvin), paxillin, and PINCH in coupling integrins to the actin cytoskeleton and signaling complexes. Genetic studies in Drosophila, Caenorhabditis elegans, and mice point to an essential role of ILK as an adaptor protein in mediating integrin-dependent cell attachment and cytoskeletal organization. This study demonstrates that inhibiting ILK kinase activity, or expression, results in the inhibition of cell attachment, cell migration, F-actin organization, and the specific cytoskeletal localization of CH-ILKBP and paxillin in human cells. The kinase activity of ILK is elevated in the cytoskeletal fraction and the interaction of CH-ILKBP with ILK within the cytoskeleton stimulates ILK activity and downstream signaling to PKB/Akt and GSK-3. Interestingly, the interaction of CH-ILKBP with ILK is regulated by the Pi3 kinase pathway, because inhibition of Pi3 kinase activity by pharmacological inhibitors, or by the tumor suppressor PTEN, inhibits this interaction as well as cell attachment and signaling. These data demonstrate that the kinase and adaptor properties of ILK function together, in a Pi3 kinase-dependent manner, to regulate integrin-mediated cell attachment and signal transduction (Attwell, 2003).

Integrin-linked kinase is an adaptor with essential functions during mouse development

The development of multicellular organisms requires integrin-mediated interactions between cells and their extracellular environment. Integrin binding to extracellular matrix catalyses assembly of multiprotein complexes, which transduce mechanical and chemical signals that regulate many aspects of cell physiology. Integrin-linked kinase (Ilk) is a multifunctional protein that binds beta-integrin cytoplasmic domains and regulates actin dynamics by recruiting actin binding focal adhesion proteins such as alpha- and beta-parvin that are related to the alpha-actinin superfamily. Unlike other members of the alpha-actinin superfamily, which are large multidomain proteins, alpha-parvin lacks a rod domain or any other C-terminal structural modules and therefore represents the smallest known protein of the superfamily. Ilk has also been shown to possess serine/threonine kinase activity and to phosphorylate signalling proteins such as Akt1 and glycogen synthase kinase 3beta (Gsk3beta) in mammalian cells; however, these functions have been shown by genetic studies not to occur in flies and worms. This study shows that mice carrying point mutations in the proposed autophosphorylation site of the putative kinase domain and in the pleckstrin homology domain are normal. In contrast, mice with point mutations in the conserved lysine residue of the potential ATP-binding site of the kinase domain, which mediates Ilk binding to alpha-parvin, die owing to renal agenesis. Similar renal defects occur in alpha-parvin-null mice. Thus, evidence is provided that the kinase activity of Ilk is dispensable for mammalian development; however, an interaction between Ilk and alpha-parvin is critical for kidney development (Lange, 2009).

Spatial coordination of actin polymerization and ILK-Akt2 activity during endothelial cell migration

Eukaryotic cell migration proceeds by cycles of protrusion, adhesion, and contraction, regulated by actin polymerization, focal adhesion assembly, and matrix degradation. However, mechanisms coordinating these processes remain largely unknown. This study shows that local regulation of thymosin-beta4 (Tbeta4) binding to actin monomer (G-actin) coordinates actin polymerization with metalloproteinase synthesis to promote endothelial cell motility. In particular and quite unexpectedly, FRET analysis reveals diminished interaction between Tbeta4 and G-actin at the cell leading edge despite their colocalization there. Profilin-dependent dissociation of G-actin-Tbeta4 complexes simultaneously liberates actin for filament assembly and facilitates Tbeta4 binding to integrin-linked kinase (ILK) in the lamellipodia. Tbeta4-ILK complexes then recruit and activate Akt2, resulting in matrix metalloproteinase-2 production. Thus, the actin-Tbeta4 complex constitutes a latent coordinating center for cell migratory behavior, allowing profilin to initiate a cascade of events at the leading edge that couples actin polymerization to matrix degradation (Fan, 2009).

ILK phosphorylates PKB/Akt and binds PINCH, paxillin, and parvin, all important components regulating cell migration. ILK is critical for EC migration and angiogenesis. The mechanism by which Tβ4 stimulates MMP expression is unclear. Because Tβ4 coimmunoprecipitates with ILK-PINCH, activation of Akt in the ILK-Akt complex could be responsible for MMP expression and increased cell migration. These studies show an interaction of Tβ4 with the kinase domain of ILK, and that formation of the ternary Tβ4–ILK-Akt2 complex increases Akt2 phosphorylation with a consequent increase in MMP-2 expression and cell migration. These Tβ4-mediated processes are regulated by G-actin binding to Tβ4: dissociation of actin–Tβ4 complex promotes Tβ4-ILK interaction and MMP-2 expression. Thus, these results provide a molecular mechanism for Tβ4-promoted protease synthesis and cell migration (Fan, 2009).

The Akt family consists of three isoforms: Akt1, Akt2, and Akt3. The role of Akt2 in cell migration is controversial: Akt2 knockout elevates Rac and Pak1 activities to increase fibroblast cell motility, but knockdown studies indicate that Tβ4-inducible Akt2 activation induces protease synthesis to facilitate EC migration. Consistent with these findings, transient knockdown or overexpression studies show a stimulatory role of Akt2 in cell migration and invasion in multiple cell types. Thus, Akt2 may have dual roles in induction of MMP and inhibition of migratory signaling, including Rac. Akt1 is critical for actin polymerization and cell motility, particularly for EC migration and in vivo angiogenesis. The data show that Tβ4 stimulates stronger ILK-mediated activation of Akt2 than that of Akt1. It is speculated that weaker Akt1 induction results in a tighter localization of active Akt1 to the leading edge, enhancing local actin polymerization (Fan, 2009).

Integrin-linked kinase controls vascular wall formation by negatively regulating Rho/ROCK-mediated vascular smooth muscle cell contraction

Vascular smooth muscle cells (VSMCs) form contractile layers around larger blood vessels in a process that is essential for the formation of a fully functional vasculature. This study shows that integrin-linked kinase (ILK) is required for the formation of a unitary layer of aligned VSMCs around arterioles and the regulation of blood vessel constriction in mice. In the absence of ILK, activated Rho/ROCK signaling induces the elevated phosphorylation of myosin light chain leading to abnormally enhanced VSMC contraction in vitro and in vivo. These findings identify ILK as a key component regulating vascular wall formation by negatively modulating VSMC contractility (Kogata, 2009).

It has been reported that the major β integrin subunit β1 contributes to vascular smooth muscle function (Abraham, 2008). Gene inactivation of β1 integrin in mice impairs VSMC spreading and differentiation, and increases VSMC proliferation leading postnatal lethality (Abraham, 2008). This study reports that ILK plays a fundamental role in developing vascular wall assembly that is distinct from that of β1 integrin. Mice lacking ILK expression in vascular wall cells fail to assemble their VSMCs into a unitary layer, which results in defective vascular remodeling and embryonic lethality. Moreover, this study shows that loss of ILK causes increased VSMC contractility due to elevated myosin light-chain phosphorylation through activation of Rho/ROCK signaling (Kogata, 2009).

Integrin-linked kinase and differentiation

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20%-30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the beta-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of beta(1) and beta(3) integrin subunits and promotes anchorage-independent growth. ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter is abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide -54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3beta (GSK-3beta) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduces ILK induction of cyclin D1 expression. ILK induces CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induces cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increases both ILK activity and cyclin D1 levels. It is concluded that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells (D'Amico, 2000).

Myogenic differentiation is a highly orchestrated, multistep process that is coordinately regulated by growth factors and cell adhesion. Integrin-linked kinase (ILK), an intracellular integrin- and PINCH-binding serine/threonine protein kinase, is an important regulator of myogenic differentiation. ILK is abundantly expressed in C2C12 myoblasts, both before and after induction of terminal myogenic differentiation. However, a noticeable amount of ILK in the Triton X-100-soluble cellular fractions is significantly reduced during terminal myogenic differentiation, suggesting that ILK is involved in cellular control of myogenic differentiation. To further investigate this, the wild-type and mutant forms of ILK were overexpressed in C2C12 myoblasts. Overexpression of ILK in the myoblasts inhibits the expression of myogenic proteins (myogenin, MyoD, and myosin heavy chain) and the subsequent formation of multinucleated myotubes. Furthermore, mutations that eliminate either the PINCH-binding or the kinase activity of ILK abolishes its ability to inhibit myogenic protein expression and allows myotube formation. Although overexpression of the ILK mutants is permissive for the initiation of terminal myogenic differentiation, the myotubes derived from myoblasts overexpressing the ILK mutants frequently exhibit an abnormal morphology (giant myotubes containing clustered nuclei), suggesting that ILK functions not only in the initial decision making process, but also in later stages (fusion or maintaining myotube integrity) of myogenic differentiation. Overexpression of ILK, but not that of the PINCH-binding defective or the kinase-deficient ILK mutants, prevents inactivation of MAP kinase, which is obligatory for the initiation of myogenic differentiation. Finally, inhibition of MAP kinase activation reverses the ILK-induced suppression of myogenic protein expression. Thus, ILK likely influences the initial decision making process of myogenic differentiation by regulation of MAP kinase activation (Huang, 2000).

Integrin-linked kinase localizes to the centrosome and regulates mitotic spindle organization

Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffold protein with well defined roles in focal adhesions in integrin-mediated cell adhesion, spreading, migration, and signaling. Using mass spectrometry-based proteomic approaches, centrosomal and mitotic spindle proteins were identified as interactors of ILK. alpha- and beta-tubulin, ch-TOG (XMAP215), and RUVBL1 (Pontin 52) associate with ILK and colocalize with it to mitotic centrosomes. Inhibition of ILK activity or expression induces profound apoptosis-independent defects in the organization of the mitotic spindle and DNA segregation. ILK fails to localize to the centrosomes of abnormal spindles in RUVBL1-depleted cells. Additionally, depletion of ILK expression or inhibition of its activity inhibits Aurora A-TACC3/ch-TOG interactions, which are essential for spindle pole organization and mitosis. These data demonstrate a critical and unexpected function for ILK in the organization of centrosomal protein complexes during mitotic spindle assembly and DNA segregation (Fielding, 2008).

Integrin-linked kinase and cell growth, cell survival, and tumorigenesis

Signals generated by the interaction of beta1 integrins with laminin in the basement membrane contribute to mammary epithelial cell morphogenesis and differentiation. The integrin-linked kinase (ILK) is one of the signaling moieties that associates with the cytoplasmic domain of beta1 integrin subunits with some specificity. Forced expression of a dominant negative, kinase-dead form of ILK subtly alters mouse mammary epithelial cell morphogenesis but it does not prevent differentiative milk protein expression. In contrast, forced overexpression of wild-type ILK strongly inhibits both morphogenesis and differentiation. Overexpression of wild-type ILK also causes the cells to lose the cell-cell adhesion molecule E-cadherin: they become invasive, reorganize cortical actin into cytoplasmic stress fibers, and switch from an epithelial cytokeratin to a mesenchymal vimentin intermediate filament phenotype. Forced expression of E-cadherin in the latter mesenchymal cells rescues epithelial cytokeratin expression and it partially restores the ability of the cells to differentiate and undergo morphogenesis. These data demonstrate that ILK, which responds to interactions between cells and the extracellular matrix, induces a mesenchymal transformation in mammary epithelial cells, at least in part, by disrupting cell-cell junctions (Somasiri, 2001).

Fibronectin (Fn) matrix plays important roles in many biological processes including morphogenesis and tumorigenesis. Recent studies have demonstrated a critical role of integrin cytoplasmic domains in regulating Fn matrix assembly, implying that intracellular integrin-binding proteins may be involved in controlling extracellular Fn matrix assembly. Overexpression of integrin-linked kinase (ILK), a newly identified serine/threonine kinase that binds to the integrin beta1 cytoplasmic domain, dramatically stimulates Fn matrix assembly in epithelial cells. The integrin-linked kinase activity is involved in transducing signals leading to the up-regulation of Fn matrix assembly, since overexpression of a kinase-inactive ILK mutant fails to enhance the matrix assembly. Moreover, the increase in Fn matrix assembly induced by ILK overexpression is accompanied by a substantial reduction in the cellular E-cadherin. ILK-overexpressing epithelial cells readily form tumors in nude mice, despite forming an extensive Fn matrix. These results identify ILK as an important regulator of pericellular Fn matrix assembly, and suggest a novel critical role of this integrin-linked kinase in cell growth, cell survival, and tumorigenesis (Wu, 1998).

Integrin-linked kinase modulates longevity and thermotolerance in C. elegans through neuronal control of HSF-1

Integrin-signaling complexes play important roles in cytoskeletal organization and cell adhesion in many species. Components of the integrin-signaling complex have been linked to aging in both Caenorhabditis elegans and Drosophila, but the mechanisms underlying this function are unknown. This study investigated the role of Integrin-linked kinase (ILK), a key component of the integrin-signaling complex, in lifespan determination. Genetic reduction of ILK in both C. elegans and Drosophila increases resistance to heat stress, and leads to lifespan extension in C. elegans without majorly affecting cytoskeletal integrity. In C. elegans, longevity and thermotolerance induced by ILK depletion is mediated by the heat-shock factor-1 (HSF-1), a major transcriptional regulator of the heat-shock response (HSR). Reduction of ILK levels increases hsf-1 transcription and activation, and leads to enhanced expression of a subset of genes with roles in the HSR. Moreover, induction of HSR-related genes, longevity, and thermotolerance caused by ILK reduction required the thermosensory neuron AFD and interneuron AIY, which are known to play a critical role in the canonical HSR. Notably, ILK was expressed in neighboring neurons, but not in AFD or AIY, implying that ILK reduction initiates cell non-autonomous signaling through thermosensory neurons to elicit a non-canonical HSR. These results thus identify HSF-1 as a novel effector of the organismal response to reduced ILK levels, and show that ILK inhibition regulates HSF-1 in a cell non-autonomous fashion to enhance stress resistance and lifespan in C. elegans (Kumsta, 2013).

Integrin linked kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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