Focal Adhesion Targeting: The Critical Determinant of FAK Regulation and Substrate Phosphorylation

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion–targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion–dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion–targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion–dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.

Regulation of F-Actin Binding to Platelet Moesin In Vitro by Both Phosphorylation of Threonine 558 and Polyphosphatidylinositides

Activation of human platelets with thrombin transiently increases phosphorylation at 558threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or 125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with α- or β/γ-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, ∼1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin’s high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen MatricesV⃞

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including β1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both β1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only β1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-β1 and anti-α2 integrin mAbs, whereas mAbs blocking CD44, α3, α5, α6, or αv integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of β1 integrins was not restored via CD44. Because α2β1-mediated migration was neither synergized nor replaced by CD44–HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.

Biogenesis of Polarized Epithelial Cells During Kidney Development In Situ: Roles of E-Cadherin–mediated Cell–Cell Adhesion and Membrane Cytoskeleton Organization

Organization of proteins into structurally and functionally distinct plasma membrane domains is an essential characteristic of polarized epithelial cells. Based on studies with cultured kidney cells, we have hypothesized that a mechanism for restricting Na/K-ATPase to the basal-lateral membrane involves E-cadherin–mediated cell–cell adhesion and integration of Na/K-ATPase into the Triton X-100–insoluble ankyrin- and spectrin-based membrane cytoskeleton. In this study, we examined the relevance of these in vitro observations to the generation of epithelial cell polarity in vivo during mouse kidney development. Using differential detergent extraction, immunoblotting, and immunofluorescence histochemistry, we demonstrate the following. First, expression of the 220-kDa splice variant of ankyrin-3 correlates with the development of resistance to Triton X-100 extraction for Na/K-ATPase, E-cadherin, and catenins and precedes maximal accumulation of Na/K-ATPase. Second, expression of the 190-kDa slice variant of ankyrin-3 correlates with maximal accumulation of Na/K-ATPase. Third, Na/K-ATPase, ankyrin-3, and fodrin specifically colocalize at the basal-lateral plasma membrane of all epithelial cells in which they are expressed and during all stages of nephrogenesis. Fourth, the relative immunofluorescence staining intensities of Na/K-ATPase, ankyrin-3, and fodrin become more similar during development until they are essentially identical in adult kidney. Thus, renal epithelial cells in vivo regulate the accumulation of E-cadherin–mediated adherens junctions, the membrane cytoskeleton, and Na/K-ATPase through sequential protein expression and assembly on the basal-lateral membrane. These results are consistent with a mechanism in which generation and maintenance of polarized distributions of these proteins in vivo and in vitro involve cell–cell adhesion, assembly of the membrane cytoskeleton complex, and concomitant integration and retention of Na/K-ATPase in this complex.

Integrin-mediated Adhesion of Endothelial Cells Induces JAK2 and STAT5A Activation: Role in the Control of c-fos Gene Expression

Integrin-mediated adhesion induces several signaling pathways leading to regulation of gene transcription, control of cell cycle entry and survival from apoptosis. Here we investigate the involvement of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in integrin-mediated signaling. Plating primary human endothelial cells from umbilical cord and the human endothelial cell line ECV304 on matrix proteins or on antibody to β1- or αv-integrin subunits induces transient tyrosine phosphorylation of JAK2 and STAT5A. Consistent with a role for the JAK/STAT pathway in regulation of gene transcription, adhesion to matrix proteins leads to the formation of STAT5A-containing complexes with the serum-inducible element of c-fos promoter. Stable expression of a dominant negative form of STAT5A in NIH3T3 cells reduces fibronectin-induced c-fos mRNA expression, indicating the involvement of STAT5A in integrin-mediated c-fos transcription. Thus these data present a new integrin-dependent signaling mechanism involving the JAK/STAT pathway in response to cell–matrix interaction.

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60c-src or p59fyn results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60c-src or p59fyn to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60c-src is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60c-src from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60c-src to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55–65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.

Defining Extracellular Integrin α-Chain Sites That Affect Cell Adhesion and Adhesion Strengthening without Altering Soluble Ligand Binding

It was previously shown that mutations of integrin α4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in α4β1-dependent cell adhesion. Some reports have suggested that α-chain “EF-hand” sites may interact directly with ligands. However, we show here that mutations of three different α4 “EF-hand” sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on α4β1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin α4 “EF-hand-like” sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.

Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

The TEL/platelet-derived growth factor β receptor (PDGFβR) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGFβR kinase-dependent signaling pathways

The TEL/PDGFβR fusion protein is the product of the t(5;12) translocation in patients with chronic myelomonocytic leukemia. The TEL/PDGFβR is an unusual fusion of a putative transcription factor, TEL, to a receptor tyrosine kinase. The translocation fuses the amino terminus of TEL, containing the helix-loop-helix (HLH) domain, to the transmembrane and cytoplasmic domain of the PDGFβR. We hypothesized that TEL/PDGFβR self-association, mediated by the HLH domain of TEL, would lead to constitutive activation of the PDGFβR tyrosine kinase domain and cellular transformation. Analysis of in vitro-translated TEL/PDGFβR confirmed that the protein self-associated and that self-association was abrogated by deletion of 51 aa within the TEL HLH domain. In vivo, TEL/PDGFβR was detected as a 100-kDa protein that was constitutively phosphorylated on tyrosine and transformed the murine hematopoietic cell line Ba/F3 to interleukin 3 growth factor independence. Transformation of Ba/F3 cells required the HLH domain of TEL and the kinase activity of the PDGFβR portion of the fusion protein. Immunoblotting demonstrated that TEL/PDGFβR associated with multiple signaling molecules known to associate with the activated PDGFβR, including phospholipase C γ1, SHP2, and phosphoinositol-3-kinase. TEL/PDGFβR is a novel transforming protein that self-associates and activates PDGFβR-dependent signaling pathways. Oligomerization of TEL/PDGFβR that is dependent on the TEL HLH domain provides further evidence that the HLH domain, highly conserved among ETS family members, is a self-association motif.

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48–CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s−1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2–CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of ≈104 M−1 and a dissociation rate of ≥6 s−1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.

Red blood cell adhesion on a solid/liquid interface

Red blood cells (RBCs), previously fixed with glutaraldehyde, adhere to glass slides coated with fibrinogen. The RBC deposition process on the horizontal glass surface is investigated by analyzing the relative surface covered by the RBCs, as well as the variance of this surface coverage, as a function of the concentration of particles. This study is performed by optical microscopy and image analysis. A model, derived from the classical random sequential adsorption model, has been developed to account for the experimental results. This model highlights the strong influence of the hydrodynamic interactions during the deposition process.

A structural basis for integrin activation by the cytoplasmic tail of the αIIb-subunit

A key step in the activation of heterodimeric integrin adhesion receptors is the transmission of an agonist-induced cellular signal from the short α- and/or β-cytoplasmic tails to the extracellular domains of the receptor. The structural details of how the cytoplasmic tails mediate such an inside-out signaling process remain unclear. We report herein the NMR structures of a membrane-anchored cytoplasmic tail of the αIIb-subunit and of a mutant αIIb-cytoplasmic tail that renders platelet integrin αIIbβ3 constitutively active. The structure of the wild-type αIIb-cytoplasmic tail reveals a “closed” conformation where the highly conserved N-terminal membrane-proximal region forms an α-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix. The structure of the active mutant is significantly different, having an “open” conformation where the interactions between the N-terminal helix and C-terminal region are abolished. Consistent with these structural differences, the two peptides differ in function: the wild-type peptide suppressed αIIbβ3 activation, whereas the mutant peptide did not. These results provide an atomic explanation for extensive biochemical/mutational data and support a conformation-based “on/off switch” model for integrin activation.

Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: A parasite adhesion trait implicated in cerebral malaria

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated α, β, γ, δ, and ɛ. The DBL domain from the A4tres that binds ICAM-1 is DBLβ type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBLβ domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBLβ domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.