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Hearts of this ILK rely on TNNI3K, a MAPKKK that regulated TNNI3.
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Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.
How intracellular cytoskeletal and signaling proteins connect and communicate with the extracellular matrix (ECM) is a fundamental question in cell biology. Recent biochemical, cell biological, and genetic studies have revealed important roles of cytoplasmic integrin-linked kinase (ILK) and its interactive proteins in these processes. Cell adhesion to ECM is an important process that controls cell shape change, migration, proliferation, survival, and differentiation. Upon adhesion to ECM, integrins and a selective group of cytoskeletal and signaling proteins are recruited to cell matrix contact sites where they link the actin cytoskeleton to the ECM and mediate signal transduction between the intracellular and extracellular compartments. In this review, we discuss the molecular activities and cellular functions of ILK, a protein that is emerging as a key component of the cell-ECM adhesion structures.
Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling.
Interaction of cells with the extracellular matrix (ECM) results in the regulation of cell growth, differentiation and migration by coordinated signal transduction through integrins and growth-factor receptors. Integrins achieve signalling by interacting with intracellular effectors that couple integrins and growth-factor receptors to downstream components. One well-studied effector is focal-adhesion kinase (FAK), but recently another protein kinase, integrin-linked kinase (ILK), has been identified as a receptor-proximal effector of integrin and growth-factor signalling. ILK appears to interact with and be influenced by a number of different signalling pathways, and this provides new routes for integrin-mediated signalling. This article discusses ILK structure and function and recent genetic and biochemical evidence about the role of ILK in signal transduction.
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.
Integrin-linked kinase and PINCH: partners in regulation of cell-extracellular matrix interaction and signal transduction.
Integrin-linked kinase (ILK) is a focal adhesion serine/threonine protein kinase that is emerging as a key signaling protein functioning at one of the early convergence points of integrin- and growth factor-signaling pathways. ILK binds to PINCH through the N-terminal ankyrin (ANK) repeat domain and the PINCH binding is crucial for focal adhesion localization of ILK. The ILK-PINCH interaction also connects ILK to Nck-2, an SH2-SH3-containing adaptor protein that interacts with components of growth factor and small GTPase signaling pathways. The kinase activity of ILK is regulated by both cell adhesion and growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner. ILK phosphorylates downstream targets such as protein kinase B (PKB, also known as Akt) and glycogen synthase kinase 3 (GSK-3) and regulates their activities. Overexpression of ILK in epithelial cells leads to striking morphological changes mimicking epithelial-mesenchymal transition, including upregulation of integrin-mediated fibronectin matrix assembly and downregulation of cell-cell adhesions. Furthermore, ILK regulates nuclear translocation of (beta)-catenin and gene expression, and promotes cell cycle progression and tumor formation. Recent genetic studies in Drosophila melanogaster and Caenorhabditis elegans have shown that lack of expression of ILK or PINCH results in phenotypes resembling those of integrin-null mutants, which demonstrates that ILK and PINCH are indispensable for integrin function during embryonic development.
Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties.
Integrin-linked kinase (ILK) is an ankyrin repeat-containing Ser/Thr kinase that interacts with the cytoplasmic domains of beta(1) and beta(3) integrins. ILK is widely expressed in tissues throughout the body, and, as might be expected, appears to mediate a diversity of functions relating to its role in coupling integrins and growth factor receptors to downstream signaling pathways. Through its downstream targets protein kinase B/Akt and glycogen synthase kinase-3beta, ILK appears to be involved in several oncogenesis-related events, including suppression of apoptosis and promotion of cell survival, as well as cell migration and invasion. Over-expression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression. Inoculation of nude mice with ILK over-expressing cells leads to tumor formation. Furthermore, increased ILK expression and activity have been correlated with malignancy in several human tumor types, including breast, prostate, brain, and colon carcinomas. Based on these findings, ILK represents an excellent therapeutic target for the prevention of tumor progression. Here, we provide an overview of the physical and biochemical properties of ILK, and present data describing the impact of small-molecule ILK inhibitors on several ILK-mediated cellular functions.
Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats.
Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that 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, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was 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, was 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, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed 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.
Molecular dissection of actopaxin-integrin-linked kinase-Paxillin interactions and their role in subcellular localization.
Paxillin is a focal adhesion adapter protein involved in integrin signaling. We have recently reported that the paxillin LD1 motif acts as a binding interface for both the actin-binding protein actopaxin and the serine/threonine integrin-linked kinase (ILK). In this report we demonstrate the direct association between actopaxin and ILK and dissect the role of the respective interactions in their subcellular localization. Co-immunoprecipitation experiments were employed to map the binding sites on ILK and actopaxin. ILK binds to the CH2 domain of actopaxin. However, an actopaxin CH2 domain mutant defective for paxillin binding (paxillin binding subdomain mutant) retains the capacity to bind ILK, indicating that paxillin and ILK binding sites on actopaxin are distinct. Actopaxin binds to the C terminus of ILK. Despite the direct binding between actopaxin and ILK, mutation analysis confirmed a primary role for paxillin in their localization to focal adhesions. Interestingly, an ILK mutant (E359K) that was previously reported to act as dominant negative for ILK function was unable to bind actopaxin or paxillin and failed to localize to focal adhesions. This mutant also exhibited in vitro kinase activity comparable with wild-type ILK. Taken together, these data suggest that normal ILK signaling is dependent on efficient localization involving multiple protein interactions.
Characterization of normal spermiation and spermiation failure induced by hormone suppression in adult rats.
At the end of spermatogenesis, elongated spermatids are released from supporting Sertoli cells via the process termed spermiation. Previous studies have shown that spermiation failure occurs after hormone suppression, in which spermatids are retained instead of releasing. However, the molecular mechanisms involved in spermiation and spermiation failure are largely unknown. The aims of the present study were, first, to characterize the ultrastructural events associated with normal spermiation and spermiation failure using light and electron microscopy and, second, to investigate the localization of cell adhesion-associated (beta1-integrin and cadherins) and junction-associated molecules (integrin-associated kinase [ILK], beta-catenin, and espin) during these processes. Four adult Sprague-Dawley rats received testosterone and estradiol implants and FSH antibody (2 mg kg-1 day-1) for 7 days to suppress testicular testosterone and FSH and to induce spermiation failure. Four rats treated with saline were used as controls. After testosterone and FSH suppression, spermiation at the ultrastructural level appeared to be normal until the final disengagement of the spermatids from Sertoli cells (stage VIII), at which stage a large number of retained spermatids were noted. Immunohistochemical localization of espin showed that during spermiation, removal of the ectoplasmic specialization (ES) occurred 30 h before spermatid disengagement, suggesting that non-ES junctions mediate the spermatid-Sertoli cell interaction before and during disengagement. beta1-Integrin and beta-catenin remained associated with spermatids after ES removal and until disengagement; however, ILK was removed along with the ES. Though detectable, N-cadherin was not associated with the spermatid-Sertoli cell junction. After testosterone and FSH suppression, beta1-integrin, but not N-cadherin or beta-catenin, remained associated with spermatids that failed to spermiate. In conclusion, hormone suppression-induced spermiation failure is caused by defects in the disengagement of spermatids from the Sertoli cell, and this process likely is mediated by beta1-integrin in an ILK-independent mechanism.
Integrin-linked kinase (ILK): a "hot" therapeutic target.
Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy.
The distribution and regulation of integrin-linked kinase in normal and diabetic kidneys.
Alteration in cell adhesion and extracellular matrix deposition is a hallmark of diabetic glomerulosclerosis. Integrin-linked kinase (ILK) is a recently identified integrin cytoplasmic-binding protein that has been implicated in the regulation of cell adhesion and extracellular matrix deposition. To begin to investigate whether ILK is involved in the pathogenesis of diabetic glomerulosclerosis, we have analyzed the distribution and regulation of ILK in normal and diabetic kidneys as well as in isolated mesangial cells. We have found that ILK is normally expressed at high concentration in visceral epithelial cells. In diabetic glomeruli, ILK expression in the mesangium is dramatically increased. The increase in ILK level is associated with diffuse mesangial expansion. In glomeruli where advanced nodular sclerosis and global sclerosis were dominant, ILK level was reduced, suggesting that the increase in ILK expression likely associates with relatively early glomerulosclerosis. Additionally, we have found that exposure of mesangial cells to high concentrations of glucose significantly increased the ILK level. Finally, we show that ILK localizes to regions of cell membranes that are in close contact with mesangial fibronectin matrix. These results suggest that ILK is likely involved in mesangial matrix expansion in response to hyperglycemia in the pathogenesis of diabetic glomerulosclerosis.
Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.
Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.
Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes.
Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. In order to evaluate the role of PINCH in integrin-mediated cellular events, we have tested directly the in vivo function of PINCH in Drosophila melanogaster. We demonstrate that the steamer duck (stck) alleles that were first identified in a screen for potential integrin effectors represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with betaPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, we show that integrins are required for proper localization of PINCH at the myotendinous junction. The integrin-linked kinase, ILK, is also essential for integrin function. We demonstrate that Drosophila PINCH and ILK are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, ILK localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of ILK. Our results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion.
Integrin-linked kinase expression increases with prostate tumor grade.
PURPOSE: Integrin-linked kinase (ILK) overexpression can suppress anoikis, promote anchorage-independent cell cycle progression, and induce tumorigenesis and invasion. Inhibition of ILK in prostatic adenocarcinoma (CaP) cells elicits cell cycle arrest and induces apoptosis. Furthermore, ILK expression increases with androgen-independent progression of human CaP cell lines, suggesting that increased ILK expression may be associated with CaP progression. EXPERIMENTAL DESIGN: To assess whether ILK expression may be related to CaP development and/or progression, we have evaluated ILK expression by immunohistochemistry in 100 human prostate tissues. RESULTS: We show that ILK expression increases significantly with CaP progression. ILK immunostaining is specifically increased in high-grade, primary human CaP relative to adjacent benign prostatic hyperplasia (P < 0.001), benign prostatic hyperplasia from patients without cancer (P < 0.002), and low-grade CaP (P = 0.003). ILK overexpression is specifically associated with the increased proliferative index (P = 0.001) that typifies CaP progression. Strikingly, intense uniform ILK immunostaining was inversely related to 5-year patient survival (P = 0.004). CONCLUSIONS: ILK expression increases dramatically with CaP progression. ILK expression is also specifically related to the disproportionately increased proliferative index that contributes to the net gain of CaP cells during progression. Finally, enhanced ILK expression is inversely related to 5-year patient survival. These data therefore implicate increased ILK expression in prostate tumor progression.
Identification and characterization of a mouse protein kinase that is highly homologous to human integrin-linked kinase.
Integrin-linked kinase (ILK) is a recently identified human protein kinase that has been implicated in integrin-mediated signal transduction and tumorigenesis. We have identified a mouse molecule that is highly homologous to human ILK. The mouse ILK homologue protein is readily recognized by antibodies raised against the human ILK protein, and the gene encoding the mouse ILK homologue is widely expressed in mouse tissues. The mouse ILK homologue gene has been mapped to chromosome 7E1 band. A second locus in the mouse chromosome 9E1-3 region has also been detected with a mouse ILK homologue cDNA probe by fluorescence in situ hybridization, suggesting the possible existence of an ILK pseudo-gene or a family of ILK genes in the mouse.
Phosphorylation of the beta1 integrin cytoplasmic domain: toward an understanding of function and mechanism.
As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the beta1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of beta1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in beta1 phosphorylation and selective loss of beta1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with beta1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with beta1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with beta1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with beta1. A possible role for regulated beta1 phosphorylation in cell migration is discussed.
alpha(v) integrins regulate cell proliferation through integrin-linked kinase (ILK) in ovarian cancer cells.
Integrins regulate both adhesion and signaling processes involved in proliferation and survival. alpha(v)beta(3) and alpha(v)beta(5) integrins have been shown to mediate cell adhesion and migration. Here we used human ovarian cancer cell lines (IGROV1, SKOV-3) that express alpha(v)beta(3) and alpha(v)beta(5) to study their role in cell proliferation and the signaling pathways involved. We found that alpha(v) integrins regulate cell proliferation through activation of integrin-linked kinase (ILK). An anti-alpha(v)-blocking antibody specifically inhibits the growth of IGROV1 and SKOV-3. The inhibition of cell proliferation involves alpha(v)beta(3) in IGROV1 cells, and both alpha(v)beta(3) and alpha(v)beta(5) in SKOV-3 cells. The reduced growth rate induced by alpha(v) integrin blockade is linked in both cell lines to G1/S cell cycle arrest. alpha(v) integrin blockade by neutralizing antibody as well as cyclic-RGD peptide caused an inhibition of ILK activity and phosphorylation of PKB/Akt on serine-473 but not on threonine-308, and was accompanied by an increase in p27(Kip1) expression. Overexpression of wild-type ILK rescued the phosphorylation of PKB/Akt on serine-473 in cells treated with anti-alpha(v) antibody. Inhibition of ILK by a pharmacological inhibitor results in inhibition of cell proliferation, PKB/Akt phosphorylation and increase of p27(Kip1). These results demonstrate that alpha(v) integrins regulate ovarian cancer cell proliferation through ILK.