Focal Adhesion Kinase pp125and the β1 Integrin Subunit Are Constitutively Complexed in HaCaT Cells

Binding of integrins to the extracellular matrix (ECM) activates various signal transduction pathways and regulates gene expression in many cell types. Integrin-dependent cytoplasmic protein/protein interactions are necessary for activation of those signal transduction cascades. In our studies we investigated a possible association of pp125^FAK, an adhesion involved tyrosine kinase, with the integrin β1 subunit. Further we wanted to know to which extent protein tyrosine phosphorylation affects cell adhesion to the ECM and the possible β1 integrin/pp125^FAKcomplex. We were able to show that in HaCaT cells (a human keratinocyte derived cell line) the integrin β1 subunit is associated with tyrosine kinase pp125^FAK. This association was observed in ECM-adherent cells and nonadherent cells and is independent of tyrosine phosphorylation. However, cell adhesion of HaCaT cells to specific substrates requires tyrosine phosphorylation since genistein treatment that blocks phosphorylation of many cellular proteins as pp125^FAKled to a reduced substrate adhesion.     

Possible involvement of focal adhesion kinase,p125FAK,in osteoclastic bone resorption

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D₃. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115-to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115–130 kD was identified as focal adhesion kinase (p125^FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p 125^FAK antibody revealed that p125^FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125^FAK but also changed the intracellular localization of p125^FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125^FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125^FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125^FAK is critical for regulating osteoclast function.     

Echistatin inhibits pp72 and pp125 phosphorylation in fibrinogen-adherent platelets

The adhesion of ADP-stimulated platelets to immobilized fibrinogen induces the tyrosine phosphorylation of multiple proteins which include pp72^syk and pp125^FAK. The phosphorylation of these two proteins increases as function of time of platelet adhesion to fibrinogen; however, pp72^syk results strongly phosphorylated already after 15 min. whereas pp125^FAK reaches high levels of phosphorylation after 1 h of platelet adhesion. Phosphorylation of both proteins is only slightly detectable when platelets are held in suspension or when platelets are allowed to adhere to bovine serum albumin, a non-specific substrate. Echistatin, an Arg-Gly-Asp (RGD)-containing snake-venom protein, affects protein tyrosine phosphorylation promoted by platelet adhesion to fibrinogen, by causing an approximately 44% and 39% decrease of pp72^syk and pp125^FAK phosphorylation, respectively. The interaction of echistatin with fibrinogen receptor glycoprotein Ilb-Illa on platelet surface might be responsible for the block of integrin-mediated signaling cascade, including pp72^syk and pp125^FAK inactivation.     

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

 Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK^−/− mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130^CAS/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.     

Mechanical Strain Increases Protein Tyrosine Phosphorylation in Airway Smooth Muscle Cells

Mechanical stress contributes to normal structure and function of the lung as well as pathology in such diseases as bronchopulmonary dysplasia and adult respiratory distress syndrome. Stress-related increases in airway smooth muscle (ASM) quantity are reflectedin vitrowhere cultured ASM cells respond to cyclic deformational strain with increased proliferation, cell reorientation, protein production, stress fibers, and focal adhesions. To understand the mechanisms of mechanical signaling in ASM cells, we investigated whether strain increased tyrosine phosphorylation of focal adhesion-related proteins. ASM cells were grown to confluence on collagen type I and subjected to 30 min of cyclic deformation strain (2 s of 25% deformation of the substratum, 2 s relaxation) and compared at various time points with identical cells not subjected to strain for phosphotyrosine content of three focal adhesion-concentrated proteins (pp125^FAK, paxillin, and talin) by Western blotting. Strain caused a rapid increase in tyrosine phosphorylation of pp125^FAKand paxillin. Tyrosine phosphorylation decreased by 4 h in pp125^FAKafter discontinuing strain but remained elevated in paxillin at 24 h. Increases in tyrosine phosphorylation of talin were not found. In separate studies, when cells were strained in the presence of tyrosine kinase inhibitors (genistein and herbimycin A), strain-induced reorientation and elongation were inhibited. Mechanochemical signal transduction appears to mediate cell morphologic changes through quantitative and possibly qualitative changes in tyrosine phosphorylation of adhesion-related proteins.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

The modulation of cell adhesion molecule expression and intercellular junction formation in the developing avian inner ear

The cells that constitute the membranous labyrinth in the vertebrate inner ear are all derived from a single embryonic source, namely, the otocyst. The mature inner ear epithelia contain different regions with highly differentiated cells, displaying a highly specialized cytoarchitecture. The present study was designed to determine the presence of adherens-type intercellular junctions in this tissue and study the expression of cell adhesion molecules (CAMs) associated with these junctions, namely, A-CAM and L-CAM, in the developing avian inner ear epithelia. The results presented here show that throughout the early otocyst, A-CAM is coexpressed with L-CAM. The formation of asymmetries between sensory and nonsensory areas in the epithelium is accompanied by the modulation of CAMs expression and the assembly of intercellular junctional complexes. A-CAM and L-CAM display reciprocal expression patterns, the former being expressed mostly in the mosaic sensory epithelium, while L-CAM becomes conspicuous in the nonsensory areas but its expression in the sensory region is markedly reduced. Adherens-type junctions and numerous desmosomes are found in the junctional complexes of early otocyst cells. The former persist to maturity of the various inner ear epithelia, whereas desmosomes disappear from junctional complexes of hair cells but remain in the intercellular junctional complexes of all other cell types in the membranous labyrinth. Thus, adherens type intercellular junctions comprise the only defined cytoskeleton-bound junction in mature hair cells. A-CAM-positive cells are also found in the region of the acoustic ganglion in early developmental stages but not in the mature neural elements.     

Desmosomal adhesion: structural basis,molecular mechanism and regulation (Review)

Desmosomes are adhesive intercellular junctions of epithelia and cardiac muscle. They have an essential function in maintaining the integrity of tissues, which is compromised in human genetic and autoimmune disease that targets desmosomal components. Recent evidence (1) suggests new roles for the function of desmosomal adhesion in tissue morphogenesis, (2) gives new insights into the molecular mechanism of adhesion, (3) indicates that the desmosomal adhesion molecules, desmocollin and desmoglein, may contribute to the regulation of epidermal diffentiation, and (4) shows that the affinity of desmosomal adhesion is regulated by protein kinase C.     

Desmosomal adhesion: structural basis,molecular mechanism and regulation (Review)

Desmosomes are adhesive intercellular junctions of epithelia and cardiac muscle. They have an essential function in maintaining the integrity of tissues, which is compromised in human genetic and autoimmune disease that targets desmosomal components. Recent evidence (1) suggests new roles for the function of desmosomal adhesion in tissue morphogenesis, (2) gives new insights into the molecular mechanism of adhesion, (3) indicates that the desmosomal adhesion molecules, desmocollin and desmoglein, may contribute to the regulation of epidermal diffentiation, and (4) shows that the affinity of desmosomal adhesion is regulated by protein kinase C.     

Roles and modes of action of nectins in cell–cell adhesion

Nectins are Ca²⁺-independent immunoglobulin (Ig)-like cell–cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell–cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell–cell junctions and cell–cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell–cell adhesion and recruit E-cadherin to the nectin-based cell–cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

Adhesion remodeling underlying tissue morphogenesis

Cell-adhesion molecules localized at adherens junctions (AJs) maintain the polarized architecture of epithelial cells but limit their movements. The morphogenesis of a developing epithelium is associated with the control of both cell shape and cell contacts. Epithelial cells remodel their contacts, and intercellular adhesion controlled by cadherin molecules is spatially and temporally regulated. Cell shape depends, in part, on the regulation of cell adhesion between different groups of cells. Patterned epithelial cell movements such as those that occur during cell intercalation--a universal process whereby cells exchange neighbors--rely on the polarized remodeling of AJs. Recent studies show that the understanding of adhesion will benefit from studies of developing organisms in which adhesion is regulated.     

Involvement of nectin in inactivation of integrin alpha(v)beta(3) after the establishment of cell-cell adhesion

Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin alpha(v)beta(3) is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin alpha(v)beta(3) becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin alpha(v)beta(3) during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Igamma90 (PIPKIgamma90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin alpha(v)beta(3) at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIgamma is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase mu (PTPmu) effectively dephosphorylated PIPKIgamma and thus canceled the PIPKIgamma-dependent activation of integrin alpha(v)beta(3) by blocking the interaction of integrin alpha(v)beta(3) with talin. Moreover, PTPmu associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIgamma90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPmu-induced inactivation of PIPKIgamma.     

Beyond the epithelium: cadherin function in fibrous connective tissues

In fibrous connective tissues, fibroblasts are organized into syncytia, cellular networks that enable matrix remodeling and that are interconnected by intercellular adherens junctions (AJs). The AJs of fibroblasts are mediated by N-cadherin, a broadly expressed classical cadherin that is critically involved in developmental processes, wound healing and several diseases of mesenchymal tissues. In contrast to E-cadherin-dependent junctions of epithelia, the formation of AJs in fibrous connective tissues is relatively uncharacterized. Work over the last several years has documented an expanding list of molecules which function to regulate N-cadherin mediated junctions such as: Fer, PTP1B, cortactin, calcium, gelsolin, PIP5KIgamma, PIP2, and the Rho family of GTPases. We present an overview on the regulation of N-cadherin-mediated junction formation that highlights recent molecular advances in the field and rationalizes the roles of N-cadherin in connective tissue function.     

Spatial and temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during avian embryogenesis

A-CAM (adherens-junction-specific cell adhesion molecule) is a calcium-dependent adhesion molecule which is associated with intercellular adherens junctions in various tissues (Volk & Geiger, 1986, J. Cell Biol. 103, 1441-1450 and 1451-1464). In the present report, we have investigated the distribution of A-CAM during avian morphogenesis by immunofluorescence microscopy and immunoblotting. A-CAM appeared at the onset of gastrulation on developing mesodermal and endodermal cells and was then expressed on tissues derived from the three primary germ layers. During embryonic life, A-CAM was constitutively expressed in a number of tissues including the central and peripheral nervous system, myocardium, muscles, notochord, skin and lens whereas it was found transiently in many tissues ranging from the nephritic tubules and the endoderm of visceral arches to ectodermal placodes. In the adult, in addition to the nervous system, A-CAM was restricted to the skin, lens, heart and testis, and exhibited an apparent molecular weight higher than the one found in the embryo. The prevalence and cell-surface modulation of A-CAM could frequently be correlated with morphogenetic events such as mesenchyme condensation into epithelia or cell clusters (e.g. formation of the somitic epithelium, kidney tubules and peripheral ganglia), dissociation of epithelia (e.g. dissociation of the somitic epithelium and segregation of neural crest from the neural tube), separation of cell populations (e.g. fibroblasts and myotubes in the heart) and reorganizations of epithelia (e.g. neurulation). In addition, using electron microscopy, the expression of A-CAM on the surface of aggregating and separating cells could be correlated with the formation and disappearance of adherens junctions. This precisely scheduled control of A-CAM correlated with early morphogenetic events during embryogenesis suggests that this CAM could play a crucial role in these processes.     

Cdc42 antagonizes Rho1 activity at adherens junctions to limit epithelial cell apical tension

In epithelia, cells are arranged in an orderly pattern with a defined orientation and shape. Cadherin containing apical adherens junctions (AJs) and the associated actomyosin cytoskeleton likely contribute to epithelial cell shape by providing apical tension. The Rho guanosine triphosphatases are well known regulators of cell junction formation, maintenance, and function. Specifically, Rho promotes actomyosin activity and cell contractility; however, what controls and localizes this Rho activity as epithelia remodel is unresolved. Using mosaic clonal analysis in the Drosophila melanogaster pupal eye, we find that Cdc42 is critical for limiting apical cell tension by antagonizing Rho activity at AJs. Cdc42 localizes Par6–atypical protein kinase C (aPKC) to AJs, where this complex limits Rho1 activity and thus actomyosin contractility, independent of its effects on Wiskott-Aldrich syndrome protein and p21-activated kinase. Thus, in addition to its role in the establishment and maintenance of apical–basal polarity in forming epithelia, the Cdc42–Par6–aPKC polarity complex is required to limit Rho activity at AJs and thus modulate apical tension so as to shape the final epithelium.     

PAF-Stimulated Protein Tyrosine Phosphorylation in Hippocampus: Involvement of NO Synthase

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat hippocampal slices. PAF caused an increase in the tyrosine phosphorylation of two phosphoproteins, which we identified by immunoprecipitation assays as the focal adhesion kinase p125^FAK and crk-associated substrate p130^Cas. The PAF effect was time- and dose-dependent. In addition, the involvement of PAF receptor was demonstrated by using PCA-4248, a specific receptor antagonist. When NO synthase was inhibited by N^G-monomethyl-L-arginine (L-NMA), PAF-stimulated protein tyrosine phosphorylation was inhibited. In conclusion, our results indicate that PAF increased the tyrosine phosphorylation of both p125^FAK and p130^Cas proteins by the production of NO in hippocampus, suggesting that PAF may play a role in the functioning of this cerebral area.     

An Examination of Focal Adhesion Formation and Tyrosine Phosphorylation in Fibroblasts Isolated from src¯, fyn¯, and yes¯ Mice

As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125^FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59^fyn, pp60^v-src, and activated pp60^c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60^c-src p59^fyn, or pp62^c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyc¯, srn¯ and yes¯ fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60^src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59^fyn and pp62^c-yes, and particularly in those lacking pp60^c-scr. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells.