Regulation of Embryonic Cell Adhesion by the Prion Protein

Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca⁺²-independent homophilic cell adhesion and signaling; and (2) modulates Ca⁺²-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.     

Regulation of the Cell Cycle by Focal Adhesion Kinase

In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant ΔC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in ΔC14. Biochemical analyses indicated that FAK mutant ΔC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKΔC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that ΔC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.     

Regulation of Monocyte Adhesion and Migration by Nox4

We showed that metabolic disorders promote thiol oxidative stress in monocytes, priming monocytes for accelerated chemokine-induced recruitment, and accumulation at sites of vascular injury and the progression of atherosclerosis. The aim of this study was to identify both the source of reactive oxygen species (ROS) responsible for thiol oxidation in primed and dysfunctional monocytes and the molecular mechanisms through which ROS accelerate the migration and recruitment of monocyte-derived macrophages. We found that Nox4, a recently identified NADPH oxidase in monocytes and macrophages, localized to focal adhesions and the actin cytoskeleton, and associated with phospho-FAK, paxillin, and actin, implicating Nox4 in the regulation of monocyte adhesion and migration. We also identified Nox4 as a new, metabolic stress-inducible source of ROS that controls actin S-glutathionylation and turnover in monocytes and macrophages, providing a novel mechanistic link between Nox4-derived H₂O₂ and monocyte adhesion and migration. Actin associated with Nox4 was S-glutathionylated, and Nox4 association with actin was enhanced in metabolically-stressed monocytes. Metabolic stress induced Nox4 and accelerated monocyte adhesion and chemotaxis in a Nox4-dependent mechanism. In conclusion, our data suggest that monocytic Nox4 is a central regulator of actin dynamics, and induction of Nox4 is the rate-limiting step in metabolic stress-induced monocyte priming and dysfunction associated with accelerated atherosclerosis and the progression of atherosclerotic plaques.     

Allosteric Regulation of E-Cadherin Adhesion

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120^ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120^ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120^ctn dephosphorylation.     

Allosteric Regulation of Focal Adhesion Kinase by PIP2 and ATP

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains—the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation.     

Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.Cyclic AMP (cAMP) is a second messenger that relays a wide range of hormone responses. The discovery of Epac as a direct effector of cAMP (15, 29) has triggered the elucidation of many cAMP-regulated processes that could not be explained by the previously known effectors protein kinase A (PKA) and cyclic nucleotide-regulated ion channels (21). Both Epac family members, Epac1 and Epac2, act as guanine nucleotide exchange factors (GEFs) for the small G proteins Rap1 and Rap2. Thereby, Epac functions in processes such as exocytosis (28, 48, 59), cell-cell junction formation (13, 20, 30, 53, 64), and cell-extracellular matrix (ECM) adhesion (55). Adhesion to the ECM induced by Epac1 and Rap is mediated by actin-linked integrin molecules and is implicated in diverse biological processes such as homing of endothelial progenitor cells to ischemic tissue (9), remodeling of the vasculature (10, 36), and transendothelial migration of leukocytes (37, 60).Epac1 and Epac2 are multidomain proteins containing a C-terminal catalytic region, which consists of a CDC25 homology domain responsible for GEF activity, a Ras exchange motif (REM), which stabilizes the CDC25 homology domain, and a Ras association (RA) domain. In the autoinhibited state, the catalytic site is sterically covered by the N-terminal regulatory region, which harbors a DEP (Dishevelled, Egl-10, and pleckstrin) domain and one or two cyclic nucleotide-binding domains in Epac1 and Epac2, respectively. As demonstrated by the crystal structures of both active and inactive Epac2, autoinhibition is released by a conformational change induced by the binding of cAMP (56, 57).After its production at the plasma membrane (PM) by adenylate cylases, cAMP becomes compartmentalized due to local degradation by spatially restricted phosphodiesterases (1). Further compartmentalization of cAMP signaling is established by the confined targeting of the cAMP effector proteins. Numerous adaptor proteins that target PKA to distinct subcellular locations and mediate the assembly of large signaling complexes have been identified (3). Similarly, cAMP-Epac signaling appears to be spatially regulated by diverse anchoring mechanisms, which may reflect the many different functions assigned to Epac. For instance, the DNA damage-responsive kinase DNA-PK is regulated by nuclear Epac1 (26), whereas membrane recruitment by activated Ras is essential for the role of Epac2 in neurite outgrowth (34, 35). Recently, we reported that Epac1 translocates to the PM upon the binding of cAMP and that this translocation contributes to Rap-mediated cell-ECM adhesion (51). Although the anchor at the PM remains elusive, it has become clear that the cAMP-dependent translocation of Epac1 involves its DEP domain (amino acids 50 to 148) and requires the cAMP-induced conformation.In this study, we reveal an additional targeting mechanism of Epac1 by showing that its N terminus interacts with members of the ezrin-radixin-moesin (ERM) family. ERM proteins show high sequence similarity and function as scaffolding proteins that link the actin cytoskeleton to the PM (18, 42, 47). Inactive ERM proteins reside in the cytoplasm in an autoinhibited state maintained by an intramolecular interaction between the N-terminal FERM (4.1 protein, ezrin, radixin, moesin) domain and the C-terminal actin binding domain (ABD). This autoinhibition is released by binding to phosphatidylinositol-4,5-bisphosphate (PIP₂) and threonine phosphorylation of the ABD, which induce the open conformation of the protein (reviewed in reference 8). Several kinases have been implicated in phosphorylation of this threonine in the ABD, including protein kinase C α (PKC α), PKC θ, NIK, Mst4, and the Rho effector ROCK (2, 40, 46, 50, 61). Active ERM proteins directly link the actin cytoskeleton to the PM and allow the recruitment of multiple signaling proteins. In this manner, ERM proteins function in numerous processes, such as the formation of microvilli, adherens junction stabilization, and leukocyte polarization (12, 18, 42, 47). Here, we demonstrate that ERM proteins also function as PM anchors for Epac1. The underlying interaction is mediated by the N terminus (residues 1 to 49) of Epac1 and is independent of its conformational state. Instead, the interaction is regulated at the level of the ERM proteins, which bind Epac1 when they are in their active, open conformation. G protein-coupled receptor (GPCR)-mediated signaling that results in activation of ERM proteins increases binding of Epac1 and results in a clustered localization of Epac1 at the PM. Together with DEP domain-mediated PM translocation, ERM proteins control cell adhesion mediated by Epac1. In conclusion, our data show that ERM proteins mediate PM recruitment of Epac1 and couple Epac1 activity to integrin-mediated cell adhesion.     

Regulation of Adhesion and Migration in the Germinal Center Microenvironment

T cell dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell homing to the GC and interaction with FDC critically depend on integrin-mediated adhesion. We have recently indentified the c-met-encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signalling pathway regulating B cell adhesion (van der Voort et al., 1997, J. Exp. Med. 185, 2121–2131). The c-Met protein is expressed on B cells localized in the dark zone of the GC (centroblasts) and is induced by CD40 plus BCR ligation. Stimulation of c-Met with HGF/SF. which is produced at high levels by tonsillar stromal cells and FDC, leads to receptor phosphorylation and to enhanced integrin-mediated adhesion of B cells to both VCAM-l and fibronectin. Interestingly, these responses to HGF/SF are promoted by heparan-sulfate proteoglycan forms of CD44 (CD44-HS). Like c-Met, CD44-HS is induced on B cells by CD40 ligation. It efficiently binds HGF/SF and strongly promotes signalling through c-Met. We conclude that integrin regulation during antigen specific B cell differentiation involves cross-talk between the HGF/SF-c-Met pathway and CD44-HS.     

Cell-Cell Recognition in Saccharomyces cerevisiae: Regulation of Mating-Specific Adhesion

Mating-specific adhesion between haploid yeast cells of opposite mating type (a and alpha) was studied by using a quantitative agar plate assay. Washed a and alpha cells that had not previously been exposed to their respective opposite mating type ("naive" cells) adhered relatively weakly. In water, only 5 to 10% of the a cells stuck tightly enough to alpha cells to give rise subsequently to diploid clones on the assay plates. Under optimum conditions (pH 6 to 7, at least 0.1 M Nacl or 0.01 M Mg(2+)), there was about 20% adhesion. Nevertheless, this weak binding defined a mating type-specific interaction because, even under optimum conditions, the homologous interactions (a with a and alpha with alpha) yielded only 3 to 5% cohesion. In contrast to these results, washed cells that had been preincubated in the cell-free culture medium of their opposite mating type ("preconditioned" cells) adhered quite strongly. The degree of adhesion between preconditioned cells (40 to 50%) was essentially unaffected by extremes of ionic strength, pH, and temperature and by the absence of divalent cation. This strong interaction was also mating type specific since cohesion between preconditioned cells of like mating type was only about 5%. The increase in agglutinability was obtained if only the a cells were preconditioned and could be induced by highly purified preparations of natural or synthetically prepared alpha-factor, an oligopeptide pheromone released by the alpha cells. The appearance of increased adhesiveness was blocked by an inhibitor of RNA synthesis and by an inhibitor of protein synthesis, but not by an inhibitor of polysaccharide synthesis. Adhesion between preconditioned cells could be inhibited by pretreatment with functionally univalent succinylated concanavalin A or with extracts from preconditioned cells of the opposite mating type. These results confirm in a quantitative manner that the recognition between conjugating cells of S. cerevisiae is a developmentally regulated event that is under the control of the mating type locus.     

Role of Cell Surface O-Linked Oligosaccharides in Adhesion of HL60 Cells to Fibronectin: Regulation of Integrin-Dependent Cell Adhesion by O-Linked Oligosaccharide Elongation

The adhesion of the myelogenous leukemia cell line, HL60, to fibronectin and its fragments, heparin binding fragment (40 kDa) and cell attachment fragment (120 kDa), was enhanced by culturing with benzyl-α-GalNAc (BZαGalNAc). Enhancement of cell adhesion to fibronectin was also observed on treatment of HL60 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA). However, an additive effect of BZαGalNAc and TPA treatments was not observed. The expression of VLA4 and VLA5 did not change during treatment with BZαGalNAc or TPA. Cell adhesion to fibronectin before and after treatment with BZαGalNAc or TPA was inhibited by anti-VLA4 and anti-VLA5 monoclonal antibodies. Staining of the cells with Helix pomatia lectin demonstrated that culturing of the cells with BZαGalNAc blocked elongation of O-linked oligosaccharides on the cell surface and led to accumulation of GalNAc-O-Ser/Thr. Labeling of cell surface carbohydrates with [³H]-glucosamine followed by treatment with TPA revealed that O-glycosylated glycoproteins including CD43 were released from the cell surface during this treatment. These findings indicate that integrin-dependent cell adhesion, particularly VLA4- or VLA5-dependent cell adhesion, of HL60 cells is prevented with the extension of O-linked oligosaccharides and recovers with the disappearance of O-linked oligosaccharides from the cell surface.     

Visualizing and Manipulating Focal Adhesion Kinase Regulation in Live Cells

Focal Adhesion Kinase (FAK) is essential for cell migration and plays an important role in tumor metastasis. However, the complex intermolecular and intramolecular interactions that regulate FAK activity at the focal adhesion remain unresolved. We have engineered a toolbox of FRET sensors that retain all of the individual FAK domains but modulate a key intramolecular regulatory interaction between the band 4.1/ezrin/radixin/moesin (FERM) and kinase domains of FAK. We demonstrate systematic control and quantitative measurement of the FERM-kinase interaction at focal adhesions, which in turn allows us to control cell migration. Using these sensors, we find that Tyr-397 phosphorylation, rather than kinase activity of FAK, is the key determinant of cell migration. Our sensors directly demonstrate, for the first time, a pH-dependent change in a protein-protein interaction at a macromolecular structure in live cells. The FERM-kinase interaction at focal adhesions is enhanced at acidic pH, with a concomitant decrease in Tyr-397 phosphorylation, providing a potential mechanism for enhanced migration of cancer cells.     

Regulation of Homotypic Cell-Cell Adhesion by Branched N-Glycosylation of N-cadherin Extracellular EC2 and EC3 Domains

The effects of altering N-cadherin N-glycosylation on several cadherin-mediated cellular behaviors were investigated using small interfering RNA and site-directed mutagenesis. In HT1080 fibrosarcoma cells, small interfering RNA-directed knockdown of N-acetylglucosaminyltransferase V (GnT-V), a glycosyltransferase up-regulated by oncogene signaling, caused decreased expression of N-linked β(1,6)-branched glycans expressed on N-cadherin, resulting in enhanced N-cadherin-mediated cell-cell adhesion, but had no effect on N-cadherin expression on the cell surface. This effect on adhesion was accompanied by decreased cell migration and invasion, opposite of the effects observed when GnT-V was overexpressed in these cells (Guo, H. B., Lee, I., Kamar, M., and Pierce, M. (2003) J. Biol. Chem. 278, 52412–52424). A detailed study using site-directed mutagenesis demonstrated that three of the eight putative N-glycosylation sites in the N-cadherin sequence showed N-glycan expression. Moreover, all three of these sites, located in the extracellular domains EC2 and EC3, were shown by leucoagglutinating phytohemagglutinin binding to express at least some β(1,6)-branched glycans, products of GnT-V activity. Deletion of these sites had no effect on cadherin levels on the cell surface but led to increased stabilization of cell-cell contacts, cell-cell adhesion- mediated intracellular signaling, and reduced cell migration. We show for the first time that these deletions had little effect on formation of the N-cadherin-catenin complex but instead resulted in increased N-cadherin cis-dimerization. Branched N-glycan expression at three sites in the EC2 and -3 domains regulates N-cadherin-mediated cell-cell contact formation, outside-in signaling, and cell migration and is probably a significant contributor to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling.     

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release

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Regulation of Focal Adhesion Dynamics and Cell Motility by the EB2 and Hax1 Protein Complex

Cell migration is a fundamental cellular process requiring integrated activities of the cytoskeleton, membrane, and cell/extracellular matrix adhesions. Many cytoskeletal activities rely on microtubule filaments. It has been speculated that microtubules can serve as tracks to deliver proteins essential for focal adhesion turnover. Three microtubule end-binding proteins (EB1, EB2, and EB3) in mammalian cells can track the plus ends of growing microtubules. EB1 and EB3 together can regulate microtubule dynamics by promoting microtubule growth and suppressing catastrophe, whereas, in contrast, EB2 does not play a direct role in microtubule dynamic instability, and little is known about the cellular function of EB2. By quantitative proteomics, we identified mammalian HCLS1-associated protein X-1 (HAX1) as an EB2-specific interacting protein. Knockdown of HAX1 and EB2 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration in vitro and in vivo. Our results further demonstrate that cell motility and focal adhesion turnover require interaction between Hax1 and EB2. Together, our findings provide new insights for this critical cellular process, suggesting that EB2 association with Hax1 plays a significant role in focal adhesion turnover and epidermal migration.     

Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.     

Cell Adhesion Strength Is Controlled by Intermolecular Spacing of Adhesion Receptors

Spatial patterning of biochemical cues on the micro- and nanometer scale controls numerous cellular processes such as spreading, adhesion, migration, and proliferation. Using force microscopy we show that the lateral spacing of individual integrin receptor-ligand bonds determines the strength of cell adhesion. For spacings ≥90 nm, focal contact formation was inhibited and the detachment forces as well as the stiffness of the cell body were significantly decreased compared to spacings ≤50 nm. Analyzing cell detachment at the subcellular level revealed that rupture forces of focal contacts increase with loading rate as predicted by a theoretical model for adhesion clusters. Furthermore, we show that the weak link between the intra- and extracellular space is at the intracellular side of a focal contact. Our results show that cells can amplify small differences in adhesive cues to large differences in cell adhesion strength.     

CD44 in Cancer Progression: Adhesion, Migration and Growth Regulation

It is well established that the large array of functions that a tumour cell has to fulfil to settle as a metastasis in a distant organ requires cooperative activities between the tumour and the surrounding tissue and that several classes of molecules are involved, such as cell-cell and cell-matrix adhesion molecules and matrix degrading enzymes, to name only a few. Furthermore, metastasis formation requires concerted activities between tumour cells and surrounding cells as well as matrix elements and possibly concerted activities between individual molecules of the tumour cell itself. Adhesion molecules have originally been thought to be essential for the formation of multicellular organisms and to tether cells to the extracellular matrix or to neighbouring cells. CD44 transmembrane glycoproteins belong to the families of adhesion molecules and have originally been described to mediate lymphocyte homing to peripheral lymphoid tissues. It was soon recognized that the molecules, under selective conditions, may suffice to initiate metastatic spread of tumour cells. The question remained as to how a single adhesion molecule can fulfil that task. This review outlines that adhesion is by no means a passive task. Rather, ligand binding, as exemplified for CD44 and other similar adhesion molecules, initiates a cascade of events that can be started by adherence to the extracellular matrix. This leads to activation of the molecule itself, binding to additional ligands, such as growth factors and matrix degrading enzymes, complex formation with additional transmembrane molecules and association with cytoskeletal elements and signal transducing molecules. Thus, through the interplay of CD44 with its ligands and associating molecules CD44 modulates adhesiveness, motility, matrix degradation, proliferation and cell survival, features that together may well allow a tumour cell to proceed through all steps of the metastatic cascade.     

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.     

Dynamic Regulation of a Cell Adhesion Protein Complex Including CADM1 by Combinatorial Analysis of FRAP with Exponential Curve-Fitting

Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation.     

Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

Background

Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Principal Findings

Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.

Conclusions

Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement.