Tyrosine Phosphorylation Of pp125FAK Research Articles

Effects of anesthetic agents on focal adhesion kinase (pp125FAK) tyrosine phosphorylation in rat hippocampal slices.

Tyrosine protein kinase proteins exert a prominent control on signaling pathways and may couple rapid events, such as action potential and neurotransmitter release, to long-lasting changes in synaptic strength and survival. Whether anesthetics modulate tyrosine kinase activity remains unknown. The aim of the current study was therefore to examine the effects of intravenous and volatile anesthetics on the phosphorylation of focal adhesion kinase (ppFAK), a functionally important nonreceptor tyrosine kinase, in the rat hippocampus. Phosphorylation of ppFAK was examined in hippocampal slices by immunoblotting with both antiphosphotyrosine and specific anti-ppFAK antibodies. Experiments were performed in the absence (control) or presence of various concentrations of pharmacologic or anesthetic agents or both. Clinically relevant concentrations of thiopental, propofol, etomidate, isoflurane, sevoflurane, and desflurane induced a concentration-related increase in tyrosine phosphorylation. In contrast, ketamine (up to 100 microm) and the nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane, 25 microm) did not significantly affect ppFAK phosphorylation. The anesthetic-induced increase in ppFAK phosphorylation was blocked by GF 109203X, RO 318220, and chelerythrin (100 microm), three structurally distinct inhibitors of protein kinase C and U 73122 (50 microm), an inhibitor of phospholipase C. The propofol- and isoflurane-induced increase in ppFAK phosphorylation was reversible and showed nonadditivity of effects with phorbol 12-myristate 13-acetate (an activator of protein kinase C, 0.1 microm). In contrast, ketamine (up to 100 microm), MK801 (10 microm, an N-methyl-d-aspartate receptor antagonist), bicuculline (10 microm, a gamma-aminobutyric acid type A receptor antagonist), and dantrolene (30 microm, an inhibitor of the ryanodine receptor) were ineffective in blocking anesthetic-induced activation of tyrosine phosphorylation. Except for ketamine, anesthetic agents markedly increase tyrosine phosphorylation of ppFAK in the rat hippocampus, most likely via the phospholipase C-protein kinase C pathway, whereas the nonimmobilizer F6 does not. These results suggest that ppFAK represents a target for anesthetic action in the brain.

Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in the production of constitutively active GPIIb-IIIa (αIIbβ3) integrin complexes

AbstractThe major platelet integrin αIIbβ3, also known as the platelet glycoprotein (GP) IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for fibrinogen and von Willebrand factor. In addition to its physiologic role, GPIIb-IIIa also bears a number of clinically important alloantigenic determinants. Previous studies have shown that disruption of the long-range Cys5-Cys435 disulfide bond of the β3 subunit results in the production of isoforms that bind some, but not all, anti-PlA1 alloantibodies, suggesting that mutations in this so-called long-range disulfide bond can alter the conformation of GPIIIa. The purpose of this study was to examine the effects of either the Cys5Ala or Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that both Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were expressed normally on the cell surface when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa bound well-characterized, conformationally sensitive ligand-induced binding site (LIBS) antibodies, and were capable of constitutively binding the fibrinogen-mimetic monoclonal antibodies Pl-55 and PAC-1, as well as soluble fibrinogen. Both GPIIb-Ala5IIIa– and GPIIb-Ala435IIIa–transfected CHO cells also bound more avidly to immobilized fibrinogen and were capable of mediating the tyrosine phosphorylation of pp125FAK on cell adhesion. These data are consistent with the notion that these regions of GPIIIa participate in the conformational change associated with receptor activation. Additionally, these studies may provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation.

Involvement of pp125 FAK and p60 SRC in the signaling through FcγRII–FcγRIII in murine macrophages

Cross-linking the FcγRs can activate a wide variety of biological responses in macrophages. Receptor stimulation induces activation of protein tyrosine kinase cascades that result in phagocytosis, a process known to involve cytoskeletal rearrangements. Therefore, an involvement of non-receptor tyrosine kinases such as pp125 FAK, in FcγR signaling is likely. Using the murine macrophage cell line J774, we demonstrate that FcγRII–RIII cross-linking induces a time- and dose-dependent increase in tyrosine phosphorylation of the focal adhesion kinase pp125 FAK that correlates with an increase in its catalytic activity. Interestingly enough, pp125 FAK activation results in its association both to the FcγRII–III and to p60 Src. The results presented here define a novel-signaling pathway likely to be important in low affinity FcγRII–III mediated phagocytosis.

Redistribution of glycolipid raft domain components induces insulin-mimetic signaling in rat adipocytes.

Caveolae and caveolin-containing detergent-insoluble glycolipid-enriched rafts (DIG) have been implicated to function as plasma membrane microcompartments or domains for the preassembly of signaling complexes, keeping them in the basal inactive state. So far, only limited in vivo evidence is available for the regulation of the interaction between caveolae-DIG and signaling components in response to extracellular stimuli. Here, we demonstrate that in isolated rat adipocytes, synthetic intracellular caveolin binding domain (CBD) peptide derived from caveolin-associated pp59(Lyn) (10 to 100 microM) or exogenous phosphoinositolglycan derived from glycosyl-phosphatidylinositol (GPI) membrane protein anchor (PIG; 1 to 10 microM) triggers the concentration-dependent release of caveolar components and the GPI-anchored protein Gce1, as well as the nonreceptor tyrosine kinases pp59(Lyn) and pp125(Fak), from interaction with caveolin (up to 45 to 85%). This dissociation, which parallels redistribution of the components from DIG to non-DIG areas of the adipocyte plasma membrane (up to 30 to 75%), is accompanied by tyrosine phosphorylation and activation of pp59(Lyn) and pp125(Fak) (up to 8- and 11-fold) but not of the insulin receptor. This correlates well to increased tyrosine phosphorylation of caveolin and the insulin receptor substrate protein 1 (up to 6- and 15-fold), as well as elevated phosphatidylinositol-3' kinase activity and glucose transport (to up to 7- and 13-fold). Insulin-mimetic signaling by both CBD peptide and PIG as well as redistribution induced by CBD peptide, but not by PIG, was blocked by synthetic intracellular caveolin scaffolding domain (CSD) peptide. These data suggest that in adipocytes a subset of signaling components is concentrated at caveolae-DIG via the interaction between their CBD and the CSD of caveolin. These inhibitory interactions are relieved by PIG. Thus, caveolae-DIG may operate as signalosomes for insulin-independent positive cross talk to metabolic insulin signaling downstream of the insulin receptor based on redistribution and accompanying activation of nonreceptor tyrosine kinases.

Diphenyleneiodonium Prevents Reactive Oxygen Species Generation, Tyrosine Phosphorylation, and Histamine Release in RBL-2H3 Mast Cells

Mast cells play a central role in immediate allergic reactions mediated by immunoglobulin E. It has recently been reported that mast cells generate intracellular reactive oxygen species (ROS) in response to stimulation with divergent physiologically relevant stimulants. However, the physiological role of ROS is poorly understood. Here we demonstrate that mast cell model rat basophilic leukemia (RBL-2H3) cells generate ROS in response to antigen and the calcium-ionophore A23187 via activation of diphenyleneiodonuim (DPI)-sensitive enzyme and that blockade of ROS generation by DPI suppresses histamine release induced by either stimulant. Increased tyrosine phosphorylation of pp125FAK and a 77-kDa protein coprecipitating specifically with the kinase occurred in parallel with the secretion, and blockade of ROS generation by DPI also suppressed the tyrosine phosphorylation of both proteins. These findings suggest that ROS generated by a flavoenzyme-dependent mechanism may be involved in histamine release through the pp125FAK pathway.

Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo.

Cross talk of pp125(FAK) and pp59(Lyn) non-receptor tyrosine kinases to insulin-mimetic signaling in adipocytes.

Signaling molecules downstream from the insulin receptor, such as the insulin receptor substrate protein 1 (IRS-1), are also activated by other receptor tyrosine kinases. Here we demonstrate that the non-receptor tyrosine kinases, focal adhesion kinase pp125(FAK) and Src-class kinase pp59(Lyn), after insulin-independent activation by phosphoinositolglycans (PIG), can cross talk to metabolic insulin signaling in rat and 3T3-L1 adipocytes. Introduction by electroporation of neutralizing antibodies against pp59(Lyn) and pp125(FAK) into isolated rat adipocytes blocked IRS-1 tyrosine phosphorylation in response to PIG but not insulin. Introduction of peptides encompassing either the major autophosphorylation site of pp125(FAK), tyrosine 397, or its regulatory loop with the twin tyrosines 576 and 577 inhibited PIG-induced IRS-1 tyrosine phosphorylation and glucose transport. PIG-induced pp59(Lyn) kinase activation and pp125(FAK) tyrosine phosphorylation were impaired by the former and latter peptide, respectively. Up-regulation of pp125(FAK) by integrin clustering diminished PIG-induced IRS-1 tyrosine phosphorylation and glucose transport in nonadherent but not adherent adipocytes. In conclusion, PIG induced IRS-1 tyrosine phosphorylation by causing (integrin antagonized) recruitment of IRS-1 and pp59(Lyn) to the common signaling platform molecule pp125(FAK), where cross talk of PIG-like structures and extracellular matrix proteins to metabolic insulin signaling may converge, possibly for the integration of the demands of glucose metabolism and cell architecture.

Immunolocalization of Clathrin During Electron-Dense Granule Secretion in Entamoeba histolytica

Entamoeba histolytica is characterized by the capacity of trophozoites to destroy human tissues. One of the major components in human tissues is collagen. Consequently, collagenase associated with electron-dense granules (EDG) from E. histolytica has been considered an important factor in the pathogenicity of this parasite (1). EDG secretion from E. histolytica is induced by incubation of trophozoites with collagen type I and Ca 2 1 . This induction is achieved through an increase of adhesion and tyrosine phosphorylation and activation of pp125 FAK and p42 MAPK , which results in cytoskeleton organization (2). It is known that some molecules, such as calmodulin, participate in the regulation of EDG secretion, and that the cytoskeleton is important in this biological process. However, the role of other molecules such as clathrin that participate in packaging of secretory granules in regulated secretion is unknown (3). In addition, this protein is the major coat protein of coated pits and vesicles that mediate the selective internalization and transport of receptors in mammalian cells (3). The gene of the heavy chain of this protein in trophozoites of E. histolytica has already been reported (EDZP0173). In this article, we present some evidence of the possible participation of clathrin in EDG secretion of E. histolytica.

Platelet glycoprotein IIb/IIIa receptors and Glanzmann's thrombasthenia.

Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, a system designed to act quickly and effectively to arrest hemorrhage. This system is also triggered by pathogenic events, such as the rupture of an atherosclerotic plaque, which can lead to thrombotic vaso-occlusion, ischemia, and infarction. Platelets are a major contributor to these damaging and life-threatening thrombotic phenomena because of their adhesive properties, which result in the release of soluble mediators, platelet aggregation, and enhancement of thrombin generation.1 The platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is a key component in the pathway to platelet aggregation; consequently, this receptor has become the target for therapeutic intervention. A paradigm of this antiplatelet treatment modality is found naturally in the inherited disorder Glanzmann’s thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding but only rarely demonstrate spontaneous central nervous system hemorrhage,2 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann’s thrombasthenia result in a functional deficiency of GPIIb/IIIa receptors,2 3 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. The molecular characterization of mutations causing Glanzmann’s thrombasthenia has provided a wealth of information on structure-function relations of the GPIIb/IIIa receptor. This review will briefly summarize those mutations that affect ligand-binding domains and receptor activation and present them in the context of predicted structures. More comprehensive coverage can be found in reviews discussing the structure and function of the GPIIb/IIIa receptor complex4 5 and the clinical and molecular basis of Glanzmann’s thrombasthenia.2 3 Platelets are the first line of defense in preventing blood loss from injured blood vessels via recognition and adhesion to components of the subendothelial matrix. This event is followed by formation of a platelet …

H 2O 2 activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation

Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H 2O 2 concentrations on platelet adhesion to immobilized fibrinogen and on pp72 syk and pp125 FAK tyrosine phosphorylation. Our results demonstrate that: (1) H 2O 2 does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H 2O 2 does not affect pp72 syk phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H 2O 2 reduces, in a dose-dependent fashion, pp125 FAK phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H 2O 2 near to physiological values (10–12 μM) are able to strengthen the subthreshold activation of pp125 FAK induced by epinephrine in apyrase-treated platelets; (5) H 2O 2 doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H 2O 2 to modulate pp125 FAK phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.

Rab5 GTPase regulates adenovirus endocytosis.

Adenovirus interaction with alphav integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca(2+)](c)-mediated signaling or for tyrosine phosphorylation of pp125(FAK), p130(CAS), and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins αIIbβ3 and αVβ3

AbstractInside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, αIIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of αIIbβ3. One clone (AM-1) expressed constitutively active αIIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiαIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated αVβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated αIIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to αIIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Clustering of Integrin αIIb-β3Differently Regulates Tyrosine Phosphorylation of pp72syk, PLCγ2 and pp125FAKin Concanavalin A-stimulated Platelets

Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.

Angiotensin and Endothelin

Changes in intracellular pH (pHi) can produce marked effects on cardiac function, and, therefore, it is important that the cell possess mechanisms by which pHi is regulated, especially after intracellular acidosis associated with myocardial ischemia. The Na+/H+ exchanger (NHE) and the Na+/HCO3− symport represent the 2 major pathways by which alkalinization occurs in cardiac cells. The NHE not only regulates pHi but also cell volume and intracellular signaling in response to a variety of stimuli. The NHE in mammalian myocardium1 has received considerable attention in the past decade, because it has been linked to cardiac growth and reperfusion injury.2 3 The NHE is activated by mechanical stretch in cultured cardiac myocytes and is thought to be a primary factor in influencing the anabolic state of the ventricular myocardium in response to pressure overload.4 5 Activation of the NHE occurs via phosphorylation of the cytoplasmic domain, which appears to be mediated by protein kinase C.6 Mechanical stretch of neonatal cardiac myocytes has also been associated with activation of second messengers, such as inositol triphosphate, protein kinase C, Raf-1 kinase, and mitogen-activated protein (MAP) kinase, all of which can contribute to reexpression of a number of fetal genes associated with cardiac hypertrophy, including β-myosin heavy chain, skeletal α-actin, and atrial natriuretic peptide.7 In studies using neonatal rat cardiac myocytes cultured on elastic membranes, stretch-mediated increases in MAP kinase activity and protein synthesis were attenuated by treatment of cells with the NHE antagonist Hoe 694,2 8 suggesting that the NHE has an important role in mediating growth in the pressure-overloaded myocardium. Deformation of the cardiac myocyte as it occurs in vivo, or in vitro under experimental or pathological conditions, presents a mechanical stress for cellular structures. This …

Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function.

The transendothelial movement of solutes is a dynamic process controlled by a complex interaction between the cytoskeleton and adhesion proteins. The aim of this study was to examine whether protein tyrosine phosphorylation is involved in the regulation of endothelial barrier function. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused coronary venules. Tyrosine phosphatase inhibitors, including phenylarsine oxide and sodium orthovanadate, dose and time dependently increased basal Pa. Western blot analysis of cultured coronary venular endothelial cells revealed that inhibition of tyrosine phosphatase induced an increase in phosphotyrosine content in a number of proteins, including bands at 65-70 and 120-130 kDa, which were identified as paxillin and focal adhesion kinase (pp125FAK), respectively. The time course and dose responsiveness of protein tyrosine phosphorylation were tightly correlated with those of increases in Pa. Furthermore, stimulation of endothelial cells with histamine or phorbol myristate acetate (PMA) enhanced tyrosine phosphorylation of paxillin and pp125FAK, which was blocked by the tyrosine kinase inhibitor damnacanthal. Correspondingly, the increases in venular permeability elicited by histamine and PMA were abolished in damnacanthal-treated venules. Taken together, the data suggest a possible involvement of protein tyrosine phosphorylation in the control of endothelial barrier function. Paxillin and its associated focal adhesion proteins may play a specific role in agonist-induced hyperpermeability responses in the endothelium of exchange vessels.

Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone.

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

Oxidative stress induces tyrosine phosphorylation and membrane translocation of PP60C-SRC and PP125FAK in Caco-2 cell monolayers

Oxidative stress induces tyrosine phosphorylation and membrane translocation of PP60C-SRC and PP125FAK in Caco-2 cell monolayers

Maturation of cell-substratum focal adhesions induced by depolymerization of microtubules is mediated by increased cortical tension.

Dynamics of alterations of focal adhesions (FA) induced by a microtubule-depolymerizing drug, colcemid, was examined in several types of fibroblastic cells. Evolution of individual FA in cultured cells was monitored by interference-reflection microscopy (IRM); at the end of the monitoring period (3 hours) the cells were fixed and immunofluorescence microscopy of the same FA was performed with an antibody against vinculin. Control and colcemid-treated cells remained non-motile and did not show lamellipodial activity at the edges. During the incubation, formation of new FA or disappearance of pre-existing FA did not occur in either colcemid-treated or control cultures. However, FA in colcemid-treated cells significantly increased in size in the course of a 3 hour incubation. The growth of FA was centripetal and sometimes was accompanied by the fusion of several adjacent FA. Immunofluorescence examination showed that colcemid-induced growth of FA was accompanied by accumulation of several proteins specific for these structures including vinculin, talin, paxillin and pp125FAK kinase. Immunoblotting with anti-vinculin antibody showed that incubation with colcemid considerably increased the amount of vinculin associated with the ventral membranes due to its partial redistribution from a soluble pool into the growing adhesions. A substantial increase in tyrosine phosphorylation of pp125FAK was also observed in colcemid-treated cells. In cells plated on elastic silicone rubber films, colcemid induced formation of wrinkles in the films and these wrinkles relaxed after treatment with cytochalasin D. These results confirm that microtubule depolymerization increases traction transmitted to the substratum by the actin cortex and shows that an increase in cortical tension accompanies maturation of FA. Taken together, these data show that short-term incubation with colcemid does not affect the formation of initial FA. In contrast, microtubule depolymerization considerably stimulates the maturation FA, manifested by their centripetal growth. Maturation is proposed to be mediated by increased cortical tension, which is caused by microtubule depolymerization.

The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility.

The Src family of protein tyrosine kinases is involved in transducing signals at sites of cellular adhesion. In particular, the v-Src oncoprotein resides in cellular focal adhesions, where it induces tyrosine phosphorylation of pp125FAK and focal adhesion loss during transformation. v-Src is translocated to cellular focal adhesions by an actin-dependent process. Here we have used mutant v-Src proteins that are temperature-dependent for translocation, but with secondary mutations that render them constitutively kinase-inactive or myristylation-defective, to show that neither v-Src kinase activity nor a myristyl group are required to induce association of v-Src with actin stress fibres and redistribution to sites of focal adhesions at the stress fibre termini. Moreover, switching the constitutively kinase-inactive or myristylation-defective temperature-sensitive v-Src proteins to the permissive temperature resulted in concomitant association with tyrosine-phosphorylated focal adhesion kinase (pp125FAK) and redistribution of both to focal adhesions. However, both catalytic activity and myristylation-mediated membrane association are required to induce dissociation of pp125FAK from v-Src, later degradation of pp125FAK and focal adhesion turnover during transformation and cell motility. These observations provide strong evidence that the role of the tyrosine kinase activity of the Src family at sites of cellular focal adhesions is to regulate the turnover of these structures during cell motility.