Cell-extracellular matrix adhesion is an important determinant of cell morphology. We show here that migfilin, a LIM-containing protein, localizes to cell-matrix adhesions, associates with actin filaments, and is essential for cell shape modulation. Migfilin interacts with the cell-matrix adhesion protein Mig-2 (mitogen inducible gene-2), a mammalian homolog of UNC-112, and the actin binding protein filamin through its C- and N-terminal domains, respectively. Loss of Mig-2 or migfilin impairs cell shape modulation. Mig-2 recruits migfilin to cell-matrix adhesions, while the interaction with filamin mediates the association of migfilin with actin filaments. Migfilin therefore functions as an important scaffold at cell-matrix adhesions. Together, Mig-2, migfilin and filamin define a connection between cell matrix adhesions and the actin cytoskeleton and participate in the orchestration of actin assembly and cell shape modulation.

Summary Integrin-linked kinase (ILK) plays a pivotal role in connecting transmembrane receptor integrin to the actin cytoskeleton and thereby regulating diverse cell adhesion-dependent processes. The kinase domain (KD) of ILK is indispensable for its function, but the underlying molecular basis remains enigmatic. Here we present the crystal structure of the ILK KD bound to its cytoskeletal regulator, the C-terminal calponin homology domain of α-parvin. While maintaining a canonical kinase fold, the ILK KD displays a striking pseudo-active site conformation. We show that rather than performing the kinase function, this conformation specifically recognizes α-parvin for promoting effective assembly of ILK into focal adhesions. The α-parvin-bound ILK KD can simultaneously engage integrin β cytoplasmic tails. These results thus define ILK as a distinct pseudokinase that mechanically couples integrin and α-parvin for mediating cell adhesion. They also highlight functional diversity of the kinase fold and its “active” site in mediating many biological processes.

PINCH-1 is a widely expressed focal adhesion protein that forms a ternary complex with integrin-linked kinase (ILK) and CH-ILKBP/actopaxin/␣-parvin (abbreviated as ␣-parvin herein). We have used RNA interference, a powerful approach of reverse genetics, to investigate the functions of PINCH-1 and ILK in human cells. We report here the following.

Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with ␤1 and ␤3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using ␣IIb␤3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells.Cell-extracellular matrix (ECM) 3 adhesion is a fundamental process that is mediated by transmembrane receptors such as integrins (1-6). The interactions of integrins with ECM ligands can be controlled by integrin activation via "inside-out" signaling. Talin, a FERM (Band 4.1 (four point one)/ezrin/radixin/ moesin) domain-containing focal adhesion (FA) protein, can play a key role in this process (for recent reviews, see Refs. 7-10). Binding of the talin FERM domain to the ␤ integrin cytoplasmic domains results in separation of the ␣ and ␤ integrin cytoplasmic tails and consequently in an increase in integrin extracellular ligand-binding affinity (i.e. integrin activation) (11-13). Integrin extracellular ligand-binding affinity plays an important role in control of initial cell-ECM adhesion. Additionally, integrin-mediated cell-ECM adhesion can be enhanced through interactions with cytoskeletal proteins, a process that has been termed cytoskeletal strengthening (14 -16). The physical basis underlying the cytoskeletal strengthening of cell-ECM adhesion has been well described (16). However, the molecular interactions that mediate this process remain to be defined.MIG-2 (mitogen-inducible gene-2, also known as kindlin-2) is a widely expressed and evolutionarily conserved cytoplasmic protein (17-21). Genetic studies have shown that Caenorhabditis elegans UNC-112, a homolog of MIG-2, is required for attachment of body-wall muscle cells to the hypodermis (17,19). Loss of UNC-112 in C. elegans results in an embryonic lethal Pat (paralyzed, arrested elongation at two-fold) phenotype resembling that of ␣ or ␤ integrin loss (17, 19). In mammalian organisms, MIG-2 has been detected in many cell types, including fibroblasts, muscle cells, endothelial cells, and epithelial cells (20,22). In these cells, it concentrates at FAs. MIG-2 interacts with migfilin (20), a filamin-and VASP (vasodilatorstimulated p...

PINCH, integrin-linked kinase (ILK) and calponin homology-containing ILK-binding protein (CH-ILKBP) form a ternary complex that plays crucial roles at cell-extracellular matrix adhesion sites. To understand the mechanism underlying the complex formation and recruitment to cell-adhesion sites we have undertaken a combined structural, mutational and cell biological analysis. Three-dimensional structure-based point mutations identified specific PINCH and ILK sites that mediate the complex formation. Analyses of the binding defective point mutants revealed that the assembly of the PINCH-ILK-CH-ILKBP complex is essential for their localization to cell-extracellular matrix adhesion sites. The formation of the PINCH-ILK-CH-ILKBP complex precedes integrin-mediated cell adhesion and spreading. Furthermore, inhibition of protein kinase C, but not that of actin polymerization, inhibited the PINCH-ILK-CH-ILKBP complex formation, suggesting that the PINCH-ILK-CH-ILKBP complex likely serves as a downstream effector of protein kinase C in the cellular control of focal adhesion assembly. Finally,we provide evidence that the formation of the PINCH-ILK-CH-ILKBP complex,while necessary, is not sufficient for ILK localization to cell-extracellular matrix adhesion sites. These results provide new insights into the molecular mechanism underlying the assembly and regulation of cell-matrix adhesion structures.

The signals that control skeletogenesis are incompletely understood. Here we show that deleting Kindlin-2 in Prx1-expressing mesenchymal progenitors in mice causes neonatal lethality, chondrodysplasia, and loss of the skull vault. Kindlin-2 ablation reduces chondrocyte density by decreasing cell proliferation and increasing apoptosis, and disrupts column formation, thus impairing the formation of the primary ossification center and causing severe limb shortening. Remarkably, Kindlin-2 localizes to not only focal adhesions, but also to the nuclei of chondrocytes. Loss of Kindlin-2 reduces, while overexpression of Kindlin-2 increases, Sox9 expression. Furthermore, overexpression of Sox9 restores the defects in chondrogenic differentiation induced by Kindlin-2 deletion in vitro. Additionally, Kindlin-2 ablation inhibits TGF-β1-induced Smad2 phosphorylation and chondrocyte differentiation. Finally, deleting Kindlin-2 in chondrocytes directly impairs chondrocyte functions, resulting in progressive dwarfism and kyphosis in mice. These studies uncover a previously unrecognized function for Kindlin-2 and a mechanism for regulation of the chondrocyte differentiation program and chondrogenesis.

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 ILKbinding 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.Cell-extracellular matrix interactions are critically involved in the embryonic development and many physiological and pathological processes including injury repair, inflammation and metastasis. Upon adhesion to extracellular matrix, cells recruit a highly selective group of membrane and cytoplasmic proteins to the cell-extracellular matrix contact sites, where they connect the extracellular matrix to the actin cytoskeleton and regulate cell shape change, migration, and signal transduction (1-6). Many mammalian adherent cell types grown in culture form morphologically and molecularly distinct cell-matrix adhesion structures, among which the best characterized are focal adhesions (2, 3) and fibrillar adhesions (or extracellular matrix contacts) (5, 7-11). Focal adhesions represent firm substrate attachment sites that typically are arrowheadshaped and contain clusters of integrins and cytoskeletal and signaling molecules including talin, viculin, focal adhesion kinase, and paxillin. Fibrillar adhesions form between fibrillar matrix such as fibronectin fibrils and actin cytoskeleton. Fibrillar adhesions are more elongated (typical axial ratio, Ͼ7) and are rich in integrins, fibronectin, and tensin, but they are deficient in paxillin and several other components of focal adhesions (5, 10, 11). ILK 1 is a common component of both focal adhesions (12-15) and fibrillar adhesions (16). Recent studies have suggested that ILK plays a crucial role in the assembly and functions of the cell-matrix adhesion structures (17)(18)(19).PINCH is a widely expressed and evolutionarily conserved protein comprising...

Cell metabolism is strongly influenced by mechano-environment. We show here that a fraction of kindlin-2 localizes to mitochondria and interacts with pyrroline-5-carboxylate reductase 1 (PYCR1), a key enzyme for proline synthesis. Extracellular matrix (ECM) stiffening promotes kindlin-2 translocation into mitochondria and its interaction with PYCR1, resulting in elevation of PYCR1 level and consequent increase of proline synthesis and cell proliferation. Depletion of kindlin-2 reduces PYCR1 level, increases reactive oxygen species (ROS) production and apoptosis, and abolishes ECM stiffening-induced increase of proline synthesis and cell proliferation. In vivo, both kindlin-2 and PYCR1 levels are markedly increased in lung adenocarcinoma. Ablation of kindlin-2 in lung adenocarcinoma substantially reduces PYCR1 and proline levels, and diminishes fibrosis in vivo, resulting in marked inhibition of tumor growth and reduction of mortality rate. Our findings reveal a mechanoresponsive kindlin-2-PYCR1 complex that links mechano-environment to proline metabolism and signaling, and suggest a strategy to inhibit tumor growth.