Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice.
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ABSTRACT:
SUBMITTER: Bulus N
PROVIDER: S-EPMC7949147 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val(386) and Thr(387) were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and ?-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins.
| S-EPMC3696662 | biostudies-literature
Project description:Integrin-linked kinase (ILK) is one of the few evolutionarily conserved focal adhesion proteins involved in diverse cell adhesion-dependent physiological and pathological responses. Despite more than a decade of studies and extensive literature, the kinase function of ILK is controversial. ILK contains a highly degraded kinase active site but it has been argued that ILK may be an unusual manganese (Mn)-dependent serine-threonine kinase that targets specific substrates such as glycogen synthase kinase-3? (GSK-3?). In this study, we have tackled this issue by a systematic bottom-up biochemical, proteomic, structural, and thermodynamic analysis of ILK. We show that recombinant ILK from either bacteria or mammalian cells exhibits no kinase activity on GSK-3? in the presence of either Mn(2+) or the conventional kinase co-factor Mg(2+). A comprehensive and unbiased whole cell-based kinase assay using entire mammalian CG-4 and C2C12 cell lysate did not identify any specific ILK substrates. High resolution crystallographic structure analysis further confirmed that the Mn-bound ILK adopts the same pseudo active site conformation as that of the Mg-bound ILK. More importantly, thermodynamic analysis revealed that the K220M mutation, previously thought to inactivate ILK by disrupting ATP binding, significantly impairs the structural integrity and stability of ILK, which provides a new basis for understanding how this mutation caused renal agenesis, a failure of fetal kidney development. Collectively, our data provide strong evidence that ILK lacks intrinsic kinase function. It is a bona fide pseudokinase that likely evolved from an ancestral catalytic counterpart to act as a distinct scaffold to mediate protein-protein interactions during focal adhesion assembly and many other cellular events.
| S-EPMC3122243 | biostudies-literature
Project description:ILK is essential for proper development of hair follicles, and for epidermal integrity and repair after injury. To better understand the pathways modulated by ILK in the epidermis, we compared the transcriptomes of ILK-deficient and -expressing epidermis using microarray analyses. Ilktm1Star (with floxed Ilk alleles) and Tg(KRT14-cre)1Amc/J mice were bred, and the resulting mice were bred again with Ilktm1Star mice, to generate animals heterozygos for the KRT14-cre transgene and either heterozygous (ILK-expressing) or homozygous (ILK-deficient) for the floxed Ilk alleles. The epidermis of 3 day-old animals was harvested and used to prepare RNA for the microarrays. The animals used were littermates. RNA from the epidermis of five ILK-deficient and five ILK-expressing mice were used.
2012-01-18 | E-GEOD-35129 | biostudies-arrayexpress
Project description:ILK is essential for proper development of hair follicles, and for epidermal integrity and repair after injury. To better understand the pathways modulated by ILK in the epidermis, we compared the transcriptomes of ILK-deficient and -expressing epidermis using microarray analyses.
2012-01-18 | GSE35129 | GEO
Project description:Integrin-linked kinase (ILK) is a multifunctional molecular actor in cell-matrix interactions, cell adhesion, and anchorage-dependent cell growth. It combines functions of a signal transductor and a scaffold protein through its interaction with integrins, then facilitating further protein recruitment within the ILK-PINCH-Parvin complex. ILK is involved in crucial cellular processes including proliferation, survival, differentiation, migration, invasion, and angiogenesis, which reflects on systemic changes in the kidney, heart, muscle, skin, and vascular system, also during the embryonal development. Dysfunction of ILK underlies the pathogenesis of various diseases, including the pro-oncogenic activity in tumorigenesis. ILK localizes mostly to the cell membrane and remains an important component of focal adhesion. We do know much about ILK but a lot still remains either uncovered or unclear. Although it was initially classified as a serine/threonine-protein kinase, its catalytical activity is now questioned due to structural and functional issues, leaving the exact molecular mechanism of signal transduction by ILK unsolved. While it is known that the three isoforms of ILK vary in length, the presence of crucial domains, and modification sites, most of the research tends to focus on the main isoform of this protein while the issue of functional differences of ILK2 and ILK3 still awaits clarification. The activity of ILK is regulated on the transcriptional, protein, and post-transcriptional levels. The crucial role of phosphorylation and ubiquitylation has been investigated, but the functions of the vast majority of modifications are still unknown. In the light of all those open issues, here we present an extensive literature survey covering a wide spectrum of latest findings as well as a past-to-present view on controversies regarding ILK, finishing with pointing out some open questions to be resolved by further research.
| S-EPMC8799556 | biostudies-literature
Project description:Integrin-linked kinase (ILK) is a serine/threonine kinase that has been linked to human and experimental heart failure, but its role in the heart is not fully understood.To define the role of cardiomyocyte ILK, we generated cardiac-specific ILK knockout mice using ?-myosin heavy chain-driven Cre expression. Cardiac-specific ILK knockout mice spontaneously developed lethal dilated cardiomyopathy and heart failure with an early increase in apoptosis, fibrosis, and cardiac inflammation. To identify downstream effectors, we used deep sequence analysis of gene expression to compare comprehensive transcriptional profiles of cardiac-specific ILK knockout and wild-type hearts from 10-day-old mice before the development of cardiac dysfunction. Approximately 2×10(6) cDNA clones from each genotype were sequenced, corresponding to 33 274 independent transcripts. A total of 93 genes were altered, using nominal thresholds of >1.4-fold change and P<0.001. The most highly upregulated gene was osteopontin (47-fold increase; P=9.6×10(-45)), an inflammatory chemokine implicated in heart failure pathophysiology. ILK also regulated osteopontin expression in cardiomyocytes in vitro. Importantly, blocking antibodies to osteopontin mitigated but did not fully rescue the functional decline in cardiac-specific ILK knockout mice.Cardiomyocyte-specific ILK deletion leads to a lethal cardiomyopathy characterized by cardiomyocyte death, fibrosis, and inflammation. Comprehensive profiling identifies ILK-dependent transcriptional effects and implicates osteopontin as a contributor to these phenotypes.
| S-EPMC3950354 | biostudies-literature
Project description:Arrhythmogenic cardiomyopathy is a genetic heart muscle disorder characterized by fibro-fatty replacement of cardiomyocytes leading to life-threatening ventricular arrhythmias, heart failure, and sudden cardiac death. Mutations in genes encoding cardiac junctional proteins are known to cause about half of cases, while remaining genetic causes are unknown. Using exome sequencing, we identified 2 missense variants (p.H33N and p.H77Y) that were predicted to be damaging in the integrin-linked kinase (ILK) gene in 2 unrelated families. The p.H33N variant was found to be de novo. ILK links integrins and the actin cytoskeleton, and is essential for the maintenance of normal cardiac function. Both of the new variants are located in the ILK ankyrin repeat domain, which binds to the first LIM domain of the adaptor proteins PINCH1 and PINCH2. In silico binding studies proposed that the human variants disrupt the ILK-PINCH complex. Recombinant mutant ILK expressed in H9c2 rat myoblast cells shows aberrant prominent cytoplasmic localization compared to the wild-type. Expression of human wild-type and mutant ILK under the control of the cardiac-specific cmlc2 promotor in zebrafish shows that p.H77Y and p.P70L, a variant previously reported in a dilated cardiomyopathy family, cause cardiac dysfunction and death by about 2-3 weeks of age. Our findings provide genetic and functional evidence that ILK is a cardiomyopathy disease gene and highlight its relevance for diagnosis and genetic counseling of inherited cardiomyopathies.
| S-EPMC7412573 | biostudies-literature
Project description:Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.
| S-EPMC3998073 | biostudies-literature
Project description:Integrin-linked kinase (ILK) is a distinct intracellular adaptor essential for integrin-mediated cell-extracellular matrix adhesion, cell spreading, and migration. Acting as a major docking platform in focal adhesions, ILK engages many proteins to dynamically link integrins with the cytoskeleton, but the underlying mechanism remains elusive. Here, we have characterized the interaction of ILK with kindlin-2, a key regulator for integrin bidirectional signaling. We show that human kindlin-2 binds to human ILK with high affinity. Using systematic mapping approaches, we have identified a major ILK binding site involving a 20-residue fragment (residues 339-358) in kindlin-2. NMR-based analysis reveals a helical conformation of this fragment that utilizes its leucine-rich surface to recognize the ILK pseudokinase domain in a mode that is distinct from another ILK pseudokinase domain binding protein, ?-parvin. Structure-based mutational experiments further demonstrate that the kindlin-2 binding to ILK is crucial for the kindlin-2 localization to focal adhesions and cell spreading (integrin outside-in signaling) but dispensable for the kindlin-2-mediated integrin activation (integrin inside-out signaling). These data define a specific mode of the kindlin-2/ILK interaction with mechanistic implications as to how it spatiotemporally mediates integrin signaling and cell adhesion.
| S-EPMC4192489 | biostudies-literature
Project description:The entry into epithelial cells and the prevention of primary immune responses are a prerequisite for a successful colonization and subsequent infection of the human host by Streptococcus pyogenes (group A streptococci, GAS). Here, we demonstrate that interaction of GAS with plasminogen promotes an integrin-mediated internalization of the bacteria into keratinocytes, which is independent from the serine protease activity of potentially generated plasmin. ?(1)?(1)- and ?(5)?(1)-integrins were identified as the major keratinocyte receptors involved in this process. Inhibition of integrin-linked kinase (ILK) expression by siRNA silencing or blocking of PI3K and Akt with specific inhibitors, reduced the GAS M49-plasminogen/plasmin-mediated invasion of keratinocytes. In addition, blocking of actin polymerization significantly reduced GAS internalization into keratinocytes. Altogether, these results provide a first model of plasminogen-mediated GAS invasion into keratinocytes. Furthermore, we demonstrate that plasminogen binding protects the bacteria against macrophage killing.
| S-EPMC3122219 | biostudies-literature