Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction

We have investigated a role for the amino-terminal FERM-like domain of the focal adhesion kinase (FAK) as a negative regulator of its own activity and phosphorylation state. Deletion of the first 375 amino acids from the amino terminus of FAK increases its catalytic activity in vitro, its phosphorylation when expressed in mammalian cells, and the phosphorylation of a FAK substrate, paxillin. Deletion mutants are phosphorylated in suspension, suggesting that they are no longer regulated by adhesion. The amino terminus of FAK can interact with the kinase domain of FAK in vitro and in vivo, suggesting that it might act as an autoinhibitor of FAK activity. The amino terminus of FAK can act in trans to inhibit FAK phosphorylation when expressed in mammalian cells or to directly inhibit FAK activity in vitro. Expression of the amino terminus of FAK inhibits cell cycle progression in CHO cells, consistent with its inhibition of FAK phosphorylation and function in trans. A glutathione S-transferase fusion protein containing the cytoplasmic tail of the beta1 integrin stimulates FAK activity in vitro, suggesting that FAK could be regulated by molecular interactions with the amino terminus. Based on these and previous data, we propose a working model for activation of FAK in cell adhesion.     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.     

Spatial and temporal regulation of focal adhesion kinase activity in living cells

Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.     

The focal adhesion kinase amino-terminal domain localises to nuclei and intercellular junctions in HEK 293 and MDCK cells independently of tyrosine 397 and the carboxy-terminal domain

The function and intracellular localisation of the non-catalytic NH(2)-terminal region of focal adhesion kinase (FAK) are unclear. We investigated the targetting of the FAK NH(2)-terminal domain in HEK 293 and epithelial MDCK cells. Exogenous expression of a variety of GFP-fused and epitope-tagged NH(2) terminal domain constructs either including or lacking the major Tyr 397 autophosphorylation and Src-binding site targeted to nuclei and cell-cell junctions in HEK 293 cells and co-localised at junctions with occludin, and beta1 integrin subunits at junctions. Mutation of Tyr 397 also had no effect on localisation of the NH(2)-terminal domain. In contrast, constructs encoding either the kinase or focal adhesion targeting (FAT) domains but lacking the NH(2)-terminal region failed to localise to intercellular junctions or nuclei. The NH(2)-terminal domain was not associated with beta1 integrin subunits as indicated by co-immunoprecipitation experiments, but did co-localise with cortical actin filaments. The NH(2)-terminal domain also targetted to nuclei and intercellular junctions in MDCK cells, whereas full-length FAK localised only to focal adhesions in these cells. These results indicate that the FAK NH(2)-terminal domain targets to epithelial intercellular junctions and nuclei and suggest novel functions for FAK NH(2)-terminal domain fragments independent of Y397, kinase, and FAT domains.     

In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.     

How focal adhesion kinase achieves regulation by linking ligand binding, localization and action

Focal adhesion kinase (FAK) has an astonishing number of ligands and functions, which enable it to contribute to embryonic development and human health. FAK can promote different effects in similar cellular environments or similar effects in different cellular environments. Recent advances in structural and cellular analysis of FAK are starting to reveal the interrelationships between the conformations, localizations, interactions, and functions of FAK. This review focuses on our emerging understanding of how the structural framework of FAK mechanistically allows it to integrate manifold stimuli into environment-specific functions.     

Resealing of endothelial junctions by focal adhesion kinase

Endothelial cell (EC) junctions determine vascular barrier properties and are subject to transient opening to allow liquid flux from blood to tissue. Although EC junctions open in the presence of permeability-enhancing factors, including oxidants, the mechanisms by which they reseal remain inadequately understood. To model opening and resealing of EC junctions in the presence of an oxidant, we quantified changes in H(2)O(2)-induced transendothelial resistance (TER) in monolayers of rat lung microvascular EC. During a 30-min exposure, H(2)O(2) (100 microM) decreased TER for an initial approximately 10 min, indicating junctional opening. Subsequently, despite continuous presence of H(2)O(2), TER recovered to baseline, indicating the activation of junctional resealing mechanisms. These bimodal TER transients matched the time course of loss and then gain of E-cadherin at EC junctions. The timing of the TER decrease matched the onset of focal adhesion formation, while F-actin increase at the cell periphery occurred with a time course that complemented the recovery of peripheral E-cadherin. In monolayers expressing a focal adhesion kinase (FAK) mutant (del-FAK) that inhibits FAK activity, the initial H(2)O(2)-induced junctional opening was present, although the subsequent junctional recovery was blocked. Expression of transfected E-cadherin was evident at the cell periphery of wild-type but not del-FAK-expressing EC. E-cadherin overexpression in del-FAK-expressing EC failed to effect major rescue of the junctional resealing response. These findings indicate that in oxidant-induced EC junction opening, FAK plays a critical role in remodeling the adherens junction to reseal the barrier.     

Stretch-induced phosphorylation of focal adhesion kinase in endothelial cells: role of mitochondrial oxidants

Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2'7'-dichlorofluorescin (DCFH), increased 152 +/- 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 microM) or rotenone (2 microM) attenuated this increase, whereas L-nitroarginine (100 microM), allopurinol (100 microM), or apocynin (30 microM) had no effect. The antioxidants ebselen (5 microM) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 microM) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 microM) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretch-induced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or G?-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.     

RNA interference elucidates the role of focal adhesion kinase in HLA class I-mediated focal adhesion complex formation and proliferation in human endothelial cells

Ligation of class I molecules by anti-HLA Ab stimulates an intracellular signaling cascade resulting in endothelial cell (EC) survival and proliferation, and has been implicated in the process of chronic allograft rejection and transplant-associated vasculopathy. In this study, we used small interfering RNA blockade of focal adhesion kinase (FAK) protein to determine its role in class I-mediated organization of the actin cytoskeleton, cell survival, and cell proliferation in primary cultures of human aortic EC. Knockdown of FAK appreciably inhibited class I-mediated phosphorylation of Src at Tyr(418), p85 PI3K, and Akt at both Thr(308) and Ser(473) sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try(118) and blocked class I-induced paxillin assembly into focal contacts. FAK small interfering RNA completely abrogated class I-mediated formation of actin stress fibers. Interestingly, FAK knockdown did not modify fibroblast growth factor receptor expression induced by class I ligation. However, FAK knockdown blocked HLA class I-stimulated cell cycle proliferation in the presence and absence of basic fibroblast growth factor. This study shows that FAK plays a critical role in class I-induced cell proliferation, cell survival, and focal adhesion assembly in EC and may promote the development of transplant-associated vasculopathy.     

Crystal structure of the FERM domain of focal adhesion kinase

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.     

Cell density regulates tyrosine phosphorylation and localization of focal adhesion kinase

We have investigated tyrosine phosphorylation of cellular proteins at different cell densities. A tyrosine-phosphorylated protein of 120 kDa was detected when cells were plated sparsely. The phosphorylation level of the protein gradually declined as the cells were plated at higher densities or when the sparsely plated cells approached confluence. This density-dependent phosphorylation was also associated with cell attachment since it disappeared when the cells were detached from plates or when the cells were cultured in suspension. Immunoblotting and immunoprecipitation analyses with specific antibodies revealed that the 120-kDa protein corresponded to the focal adhesion kinase (FAK) and the protein level of FAK was not altered at different cell densities. In vitro kinase assays demonstrated that the kinase activity of FAK decreased with increasing cell densities in parallel with its dephosphorylation. Cell density also affects localization of FAK associated with rearrangement of actin stress fibers. At low cell densities, FAK and actin stress fiber are distributed around the periphery of cells while they are dispersed over the ventral surface in high-density cells. Finally, the density-regulated tyrosine phosphorylation and localization of FAK appeared to be mediated by an insoluble factor produced by high-density cells.     

Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation

We have previously described regulation of focal adhesion kinase (FAK) by its amino-terminal FERM-like domain through an autoinhibitory interaction with its kinase domain (Cooper, L. A., Shen, T. L., and Guan, J. L. (2003) Mol. Cell. Biol. 23, 8030-8041). Here we show that the first two subdomains of the FERM-like domain are independently capable of inhibiting phosphorylation of FAK in trans. We characterized several point mutations within the first subdomain of the FERM-like domain and find that mutation of Lys-38 to alanine results in a FAK mutant that is strongly hyperphosphorylated when expressed in mammalian cells, and promotes increased phosphorylation of the FAK substrate paxillin. A second mutation of Lys-78 to alanine results in a FAK mutant that is underphosphorylated, but can be activated by extracellular matrix stimuli. Like deletion of the amino terminus itself the K38A mutation is phosphorylated in suspension. The Delta375 truncation mutant of FAK is strongly phosphorylated both when Tyr-397 is mutated to phenylalanine, and in the presence of the Src inhibitor, PP2, suggesting that removal of the amino terminus can render FAK Src independent. This is in contrast to the K38A mutant that is not phosphorylated in the Y397F background, and which shows decreased phosphorylation in the presence of the Src inhibitor PP2, suggesting that regulation of FAK by Src is a secondary step in its activation. The K38A mutation weakens the interaction between the amino terminus of FAK and its own kinase domain, and disrupts the ability of the amino terminus to inhibit the phosphorylation of FAK in trans. The K38A mutation of FAK also increases the ability of FAK to promote cell cycle progression and cell migration, suggesting that hyperphosphorylation of this mutant can positively affect FAK function in cells. Together, these data strongly suggest a role for the first FAK subdomain of the FERM domain in its normal regulation and function in the cell.     

Focal adhesion kinase regulation of N-WASP subcellular localization and function

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr(256). We found that phosphorylation of Tyr(256) affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr(256) of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr(256) plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.     

Paxillin binding is not the sole determinant of focal adhesion localization or dominant-negative activity of focal adhesion kinase/focal adhesion kinase-related nonkinase

The carboxy-terminal 150 residues of the focal adhesion kinase (FAK) comprise the focal adhesion-targeting sequence, which is responsible for its subcellular localization. The mechanism of focal adhesion targeting has not been fully elucidated. We describe a mutational analysis of the focal adhesion-targeting sequence of FAK to further examine the mechanism of focal adhesion targeting and explore additional functions encoded by the carboxy-terminus of FAK. The results demonstrate that paxillin binding is dispensable for focal adhesion targeting of FAK. Cell adhesion-dependent tyrosine phosphorylation strictly correlated with the ability of mutants to target to focal adhesions. Focal adhesion targeting was also a requirement for maximal FAK-dependent tyrosine phosphorylation of paxillin and FAK-related nonkinase (FRNK)-dependent inhibition of endogenous FAK function. However, there were additional requirements for these latter functions because we identified mutants that target to focal adhesions, yet are defective for the induction of paxillin phosphorylation or the dominant-negative function of FRNK. Furthermore, the paxillin-binding activity of FRNK mutants did not correlate with their ability to inhibit FAK, suggesting that FRNK has other targets in addition to paxillin.     

The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) is critical for recruitment of FAK to focal adhesions and contains tyrosine 926, which, when phosphorylated, binds the SH2 domain of Grb2. Structural studies have shown that the FAT domain is a four-helix bundle that exists as a monomer and a dimer due to domain swapping of helix 1. Here, we report the NMR solution structure of the avian FAT domain, which is similar in overall structure to the X-ray crystal structures of monomeric forms of the FAT domain, except that loop 1 is longer and less structured in solution. Residues in this region undergo temperature-dependent exchange broadening and sample aberrant phi and psi angles, which suggests that this region samples multiple conformations. We have also identified a mutant that dimerizes approximately 8 fold more than WT FAT domain and exhibits increased phosphorylation of tyrosine 926 both in vitro and in vivo.     

Identification of an amino-terminal substrate-binding domain in the Yersinia tyrosine phosphatase that is required for efficient recognition of focal adhesion targets

YopH is a protein tyrosine phosphatase (PTP) that is delivered into host mammalian cells via a type III secretion pathway in pathogenic Yersinia species. Although YopH is a highly active PTP, it preferentially targets a subset of tyrosine-phosphorylated proteins in host cells, including p130^Cas. Previous in vitro studies have indicated that the carboxy-terminal PTP domain contributes specificity to the interaction of YopH with substrates. However, it is not known if the PTP domain is sufficient for substrate recognition by YopH. Here, we have identified paxillin as an additional substrate of YopH in HeLa cells. In addition, we have identified a domain in the amino-terminal region of YopH that binds to both p130^Cas and paxillin and is required for the efficient recognition of substrates by the wild-type enzyme. This 'substrate-binding' domain exhibits a ligand specificity that is similar to that of the Crk Src homology 2 (SH2) domain, and it binds substrates directly in a phosphotyrosine-dependent manner. The substrate-binding domain of YopH may represent a novel type of protein–protein interaction module, as it lacks significant sequence similarity with any known SH2 or phosphotyrosine-binding (PTB) domain.     

The structural basis of localization and signaling by the focal adhesion targeting domain

The localization of focal adhesion kinase (FAK) to sites of integrin clustering initiates downstream signaling. The C-terminal focal adhesion targeting (FAT) domain causes this localization by interacting with talin and paxillin. FAT also mediates signaling through Grb2 via phosphorylated Y925. We report two crystal structures of the FAT domain. Large rearrangements of the structure are indicated to allow phosphorylation of Y925 and subsequent interaction with Grb2. Sequence homology and structural compatibility suggest a FAT-like fold for the C-terminal domains of CAS, Efs/Sin, and HEF1. A structure-based alignment including these proteins and the vinculin tail domain reveals a conserved region that could play a role in focal adhesion targeting. Previously postulated "paxillin binding subdomains" may contribute to structural integrity rather than directly to paxillin binding.     

Phosphorylation-dependent regulation of unique nuclear and nucleolar localization signals of LIM kinase 2 in endothelial cells

LIM kinases (LIMKs) regulate actin dynamics through cofilin phosphorylation and also have a function in the nucleus. Recently we have shown that LIMK2 shuttles between cytoplasm and nucleus in endothelial cells and that nuclear import is inhibited by protein kinase C-mediated phosphorylation of Ser-283. Here we aimed to identify the structural features of LIMK2 responsible for nuclear import. We found that the kinase domain of LIMK2 is localized exclusively in the nucleus and, in contrast to the kinase domain of LIMK1, it accumulated in the nucleolus. Through site-directed mutagenesis, we identified the basic amino acid-rich motif KKRTLRKNDRKKR (amino acids 491-503) as the functional nuclear and nucleolar localization signal of LIMK2. After fusing this motif to enhanced green fluorescent protein, the fusion protein localized exclusively in the nucleus and nucleolus. Mutagenesis studies showed that phosphorylation of Thr-494, a putative protein kinase C phosphorylation site identified within the nuclear localization signal, inhibits nuclear import of the enhanced green fluorescent protein-PDZ kinase domain of LIMK2. After inhibiting nuclear export with leptomycin B, phosphorylation of either Ser-283 or Thr-494 reduced the nuclear import of LIMK2. Phosphorylation of both Ser-283 and Thr-494 sites inhibited nuclear import completely. Our findings identify a unique basic amino acid-rich motif (amino acids 491-503) in LIMK2 which is not present in LIMK1 that serves to target the protein not only to the nucleus but also to the nucleolus. Phosphorylation of Thr-494 within this motif negatively regulates nuclear import of LIMK2.     

Reactive oxygen species activate focal adhesion kinase, paxillin and P130CAS tyrosine phosphorylation in endothelial cells

Reactive oxygen species (ROS), particularly hydroxyl radical (HO), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 μM) and herbimycin A (0.9 μM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120–130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125^FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 μM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.