Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.     

Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation

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.     

Migfilin, a Molecular Switch in Regulation of Integrin Activation

The linkage of heterodimeric (α/β) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin β cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin β CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin β CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.Cells reside in a protein network, the extracellular matrix (ECM).⁴ Cell-ECM contact is crucial for many physiological and pathophysiological processes and is primarily mediated by heterodimeric (α/β) transmembrane receptors, the integrins (1). Integrins engage a variety of ECM proteins via their extracellular domains while connecting to the actin cytoskeleton via their small cytoplasmic tails (CTs). The ability of integrins to bind to their ligands is uniquely controlled by the integrin CTs via a process called “inside-out signaling,” i.e. upon cellular stimulation, an integrin, typically expressed in a latent state, can receive intracellular signal(s) at its CT, which transmits through the transmembrane domain to the extracellular domain thereby converting the receptor from a low to a high affinity state (integrin activation). How such long range information transfer is initiated and regulated has been the central topic of integrin/cell adhesion research over the decades (for reviews see Refs. 2-5). Structural/biochemical studies have indicated that the inside-out signaling involves the unclasping of the integrin α/β CT complex (6-9), followed by extensive rearrangement of transmembrane domain and extracellular domain (10-13). Talin, a large actin-linking protein, was found to play a key role in the unclasping process by binding to the integrin β CTs (7-8, 14). Talin activity appears to be controlled by multiple factors or pathways (15-20).Relevant to this study is the role of filamin, another major actin cross-linking protein (21-22), in integrin activation. Filamin was found to share an overlapping binding site on integrin β CTs with talin and thus suppress the talin-integrin interaction (16). Gene silencing of filamin in various cell lines to remove the filamin-integrin connection enhances integrin activation (16, 23), whereas increased filamin-integrin interaction inhibits cell migration (24), a process critically dependent on integrin activation. Together these observations support the notion that filamin binding to integrin serves as a cellular brake to control the dynamics of the integrin activation by inhibiting talin function and ECM-cytoskeleton communication. The mechanism as to how the filamin brake is turned off to promote integrin activation and cell migration is not understood.Filamin is known to contain an N-terminal actin binding domain (ABD) and a long rod-like domain of 24 immunoglobulin-like repeats, of which repeat 21 (IgFLN21) was shown to play a key role in binding to integrin β CTs and blocking the talin-integrin β CT interaction (16). Interestingly, IgFLN21 also recognizes another intracellular protein called migfilin, which has been shown to be an important regulator of integrin-mediated cytoskeletal rearrangements, cell shape change (25), and cell migration (26).In an effort to dissect the complex intermolecular interactions between migfilin, filamin, and integrin, we have undertaken a detailed structural/functional analysis. Using NMR spectroscopy, we have mapped the precise IgFLN21 binding region in migfilin, which is located at the extreme N terminus (residues 1-24) of migfilin (migfilin-N), and we solved solution structure of the IgFLN21-migfilin-N complex. To our surprise, despite little sequence homology, migfilin binds to the same region in IgFLN21 where integrin β CT binds. Detailed NMR and biochemical analyses demonstrate that the migfilin-filamin interaction is an order of magnitude higher than the integrin-filamin interaction and that the migfilin binding to filamin can competitively dissociate filamin from integrin and thus promote the talin-integrin interaction. Using multiple functional approaches, we further show that migfilin, but not its filamin binding defective mutant, significantly enhances integrin activation. These data suggest a novel regulatory pathway in which the binding of filamin to its downstream target migfilin switches off the integrin-filamin connection, thereby promoting talin binding to and activation of integrins.     

The Regulation Mechanism for the Auto-Inhibition of Binding of Human Filamin A to Integrin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Human filamins are large actin cross-linking proteins that connect integrins to the cytoskeleton. Filamin binding to the cytoplasmic tail of β integrins has been shown to prevent integrin activation in cells, which is important for controlling cell adhesion and migration. The molecular-level mechanism for filamin binding to integrin has been unclear, however, as it was recently demonstrated that filamin undergoes intramolecular auto-inhibition of integrin binding. In this study, using steered molecular dynamics simulations, we found that mechanical force applied to filamin can expose cryptic integrin binding sites. The forces required for this are considerably lower than those for filamin immunoglobulin domain unfolding. The mechanical-force-induced unfolding of filamin and exposure of integrin binding sites occur through stable intermediates where integrin binding is possible. Accordingly, our results support filamin's role as a mechanotransducer, since force-induced conformational changes allow binding of integrin and other transmembrane and intracellular proteins. This observed force-induced conformational change can also be one of possible mechanisms involved in the regulation of integrin activation.     

Structural basis of the migfilin-filamin interaction and competition with integrin beta tails

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro(5) and Pro(19) and binds to the CD face of the IgFLNa21 beta-sandwich. This interaction is similar to the previously characterized beta 7 integrin-IgFLNa21 interaction and migfilin and integrin beta tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.     

RSK2 Protein Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.     

Kindlins: essential regulators of integrin signalling and cell-matrix adhesion

Integrin-mediated cell-ECM (extracellular matrix) adhesion is a fundamental process that controls cell behaviour. For correct cell-ECM adhesion, both the ligand-binding affinity and the spatial organization of integrins must be precisely controlled; how integrins are regulated, however, is not completely understood. Kindlins constitute a family of evolutionarily conserved cytoplasmic components of cell-ECM adhesions that bind to beta-integrin cytoplasmic tails directly and cooperate with talin in integrin activation. In addition, kindlins interact with many components of cell-ECM adhesions--such as migfilin and integrin-linked kinase--to promote cytoskeletal reorganization. Loss of kindlins causes severe defects in integrin signalling, cell-ECM adhesion and cytoskeletal organization, resulting in early embryonic lethality (kindlin-2), postnatal lethality (kindlin-3) and Kindler syndrome (kindlin-1). It is therefore clear that kindlins, together with several other integrin-proximal proteins, are essential for integrin signalling and cell-ECM adhesion regulation.     

Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration

Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.     

Interaction Analyses of 14-3-3ζ, Dok1, and Phosphorylated Integrin β Cytoplasmic Tails Reveal a Bi-molecular Switch in Integrin Regulation

Integrins are hetero-dimeric (α and β subunits) type I transmembrane proteins that facilitate cell adhesion and migration. The cytoplasmic tails (CTs) of integrins interact with a plethora of intra-cellular proteins that are required for integrin bidirectional signaling. In particular, the β CTs of integrins are known to recruit a variety of cytosolic proteins that often have overlapping recognition sites. However, the chronological sequence of β CTs/cytosolic proteins interactions remains to be fully characterized. Previous studies have shown that the scaffold protein 14-3-3ζ binds to phosphorylated β CTs in activated integrins, whereas interactions of Dok-1 with phosphorylated β CTs maintained integrins in the resting state. In this study, we examined the binding interactions between 14-3-3ζ, Dok1, and phosphorylated integrin β2 and β3 CTs. We show that the scaffold protein 14-3-3ζ interacts with the phosphotyrosine binding (PTB) domain of Dok1 even in the absence of the phosphorylated integrin β CTs. The interactions were mapped onto the β-sheet region of the PTB domain of Dok1. Furthermore, we provide evidence that the 14-3-3ζ/Dok1 binary complex is able to bind to their cognate phosphorylated sequence motifs in the integrin β CTs. We demonstrate that Thr phosphorylated pTTT β2 CT or pTST β3 CT can bind to 14-3-3ζ that is in complex with the Dok1 PTB domain, whereas Ser phosphorylated β2 CT or Tyr phosphorylated β3 CT interacted with Dok1 in 14-3-3ζ/Dok1 complex. Based on these data, we propose that 14-3-3ζ/Dok1 complex could serve as a molecular switch providing novel molecular insights into the regulating integrin activation.     

Integrin regulation by vascular endothelial growth factor in human brain microvascular endothelial cells: role of alpha6beta1 integrin in angiogenesis

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of alpha(6) integrin in HBMECs. VEGF significantly increased alpha(6)beta(1) integrin expression, but not alpha(6)beta(4) integrin expression in these cells. Specific down-regulation of alpha(6) integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of alpha(6)beta(1) integrins in HBMECs. Functional blocking of alpha(6) integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of alpha(6)beta(1) integrins, which may promote VEGF-driven tumor angiogenesis in vivo.     

Functional and structural insights into ASB2alpha, a novel regulator of integrin-dependent adhesion of hematopoietic cells

By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor α oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2α) and a muscle-type (ASB2β) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2α is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2α structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2α, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of β1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2α, together with ankyrin repeats 1 to 10, is necessary for association of ASB2α with filamin A. Importantly, the ASB2α N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and β integrins. Together, these data provide new insights into the molecular mechanisms of ASB2α binding to filamin.     

SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways

The FERM-like domain-containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture-based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.     

Integrin affinity modulation in angiogenesis

Integrins, transmembrane glycoprotein receptors, play vital roles in pathological angiogenesis, but their precise regulatory functions are not completely understood and remain controversial. This study aims to assess the regulatory functions of individual beta subunits of endothelial integrins in angiogenic responses induced by vascular endothelial growth factor (VEGF). Inhibition of expression of β1, β3, or β5 integrins in endothelial cells resulted in down regulation of EC adhesion and migration on the primary ligand for the corresponding integrin receptor, while no effects on the recognition of other ligands were detected. Although inhibition of expression of each subunit substantially affected capillary growth stimulated by VEGF, the loss of β3 integrin was the most inhibitory. EC stimulation by VEGF induced formation of the high affinity (activated) state of αVβ3 in a monolayer and activated αVβ3 was co-localized with VEGF receptor-2 (VEGFR-2). Inhibition of expression of β1, β3, or β5 did not affect expression levels of VEGFR-2 in EC. However, inhibition of β3, but not β1 or β5, resulted in substantial inhibition of VEGFR-2 phosphorylation stimulated by VEGF. Exogenous stimulation of αVβ3 integrin with activating antibodies augmented VEGF-dependent phosphorylation of VEGFR-2, whereas integrin blockade suppressed this response. Most importantly, activated αVβ3 was detected on endothelial cells of tumor vasculature. Activation of αVβ3 was substantially increased in highly-vascularized tumors as compared to normal tissues. Moreover, activated αVβ3 was co-localized with VEGFR-2 on endothelial cells of proliferating blood vessels. Together, these results show the unique role of αVβ3 integrin in cross-talk with VEGFR-2 in the context of pathological angiogenesis.     

Identification and Characterization of Multiple Similar Ligand-binding Repeats in Filamin: IMPLICATION ON FILAMIN-MEDIATED RECEPTOR CLUSTERING AND CROSS-TALK*

The actin-binding protein filamin links membrane receptors to the underlying cytoskeleton. The cytoplasmic domains of these membrane receptors have been shown to bind to various filamin immunoglobulin repeats. Notably, among 24 human filamin repeats, repeat 17 was reported to specifically bind to platelet receptor glycoprotein Ibα and repeat 21 to integrins. However, a complete sequence alignment of all 24 human filamin repeats reveals that repeats 17 and 21 actually belong to a distinct filamin repeat subgroup (containing repeats 4, 9, 12, 17, 19, 21, and 23) that shares a conserved ligand-binding site. Using isothermal calorimetry and NMR analyses, we show that all repeats in this subgroup can actually bind glycoprotein Ibα, integrins, and a cytoskeleton regulator migfilin in similar manners. These data provide a new view on the ligand specificity of the filamin repeats. They also suggest a multiple ligand binding mechanism where similar repeats within a filamin monomer may promote receptor clustering or receptor cross-talking for regulation of the cytoskeleton organization and diverse filamin-mediated cellular activities.     

Vascular endothelial cells promote cortical neurite outgrowth via an integrin β3-dependent mechanism

The interaction of neurons with their non-neuronal milieu plays a crucial role in the formation of neural networks, and wide variety of cell-contact-dependent signals that promote neurite elongation have been identified. In this study, we found that vascular endothelial cells promote neurite elongation in an integrin β3-dependent manner. Vascular endothelial cells from the cerebral cortex promoted neurite elongation of cortical neurons in a cell contact-dependent manner. This effect was mediated by arginine–glycine–aspartic acid (RGD), a major recognition sequence for integrins. Pharmacological blockade of integrin β3 abolished the neurite elongation effect induced by the endothelial cells. Immunocytochemical analysis revealed that integrin β3 was expressed on cultured cortical neurons. These results demonstrate that the neurite elongation promoted by vascular endothelial cells requires integrin β3. Vascular endothelial cells may therefore play a role in the development and repair of neural networks in the central nervous system.     

α9β1 Integrin in melanoma cells can signal different adhesion states for migration and anchorage

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The α9 integrin subunit, which pairs only with β1, has specific roles in the immune system and may regulate cell migration. Melanoma cells express abundant α9β1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported α9β1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced α9β1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of α9β1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by β1 integrin activating antibody. The α9β1 integrin translocated to focal adhesions, where active β1 integrin was also detected by conformation-specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration. Endogenous α9β1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although α9β1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration.     

Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in human umbilical vein endothelial cells

CYR61 (CCN1) is an extracellular matrix-associated protein of the CCN family, which also includes CTGF (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). Purified CYR61 induces neovascularization in corneal implants, and Cyr61-null mice suffer embryonic death due to vascular defects, thus establishing that CYR61 is an important regulator of angiogenesis. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. In culture, CYR61 functions through integrin-mediated pathways to promote cell adhesion, migration, and proliferation. Here we show that CYR61 can also promote cell survival and tubule formation in human umbilical vein endothelial cells. Furthermore, we have dissected the integrin receptor requirements of CYR61 with respect to its pro-angiogenic activities. Thus, CYR61-induced cell adhesion and tubule formation occur through interaction with integrin alpha(6)beta(1) in early passage endothelial cells in which integrins have not been activated. By contrast, in endothelial cells in which integrins are activated by phorbol ester or vascular endothelial growth factor, CYR61-promoted cell adhesion, migration, survival, growth factor-induced mitogenesis, and endothelial tubule formation are all mediated through integrin alpha(v)beta(3). These findings indicate that CYR61 is an activation-dependent ligand of integrin alpha(v)beta(3) and an activation-independent ligand of integrin alpha(6)beta(1) and that these integrins differentially mediate the pro-angiogenic activities of CYR61. These findings help to define the mechanisms by which CYR61 acts as an angiogenic regulator, provide a molecular interpretation for the loss of vascular integrity and increased apoptosis of vascular cells in Cyr61-null mice, and underscore the importance of CYR61 in the development and homeostasis of the vascular system.     

Role of integrin-linked kinase in leukocyte recruitment

Chemokines modulate leukocyte integrin avidity to coordinate adhesion and subsequent transendothelial migration, although the sequential signaling pathways involved remain poorly characterized. Here we show that integrin-linked kinase (ILK), a 59-kDa serine-threonine protein kinase that interacts principally with beta(1) integrins, is highly expressed in human mononuclear cells and is activated by exposure of leukocytes to the chemokine monocyte chemoattractant protein-1. Biochemical inhibitor studies show that chemokine-triggered activation of ILK is downstream of phosphoinositide 3-kinase. In functional assays under physiologically relevant flow conditions, overexpression of wild-type ILK in human monocytic cells diminishes beta(1) integrin/vascular cell adhesion molecule-1-dependent firm adhesion to human endothelial cells. These data implicate ILK in the dynamic signaling events involved in the regulation of leukocyte integrin avidity for endothelial substrates.     

Mechanisms of integrin activation and trafficking

Integrin adhesion receptors are essential for the normal function of most multicellular organisms, and defective integrin activation or integrin signaling is associated with an array of pathological conditions. Integrins are regulated by conformational changes, clustering, and trafficking, and regulatory mechanisms differ strongly between individual integrins and between cell types. Whereas integrins in circulating blood cells are activated by an inside-out-induced conformational change that favors high-affinity ligand binding, β1-integrins in adherent cells can be activated by force or clustering. In addition, endocytosis and recycling play an important role in the regulation of integrin turnover and integrin redistribution in adherent cells, especially during dynamic processes such as cell migration and invasion. Integrin trafficking is strongly regulated by their cytoplasmic tails, and the mechanisms are now being identified.