ERK1 associates with alpha(v)beta 3 integrin and regulates cell spreading on vitronectin

Kinases that associate with integrins are likely to mediate the assembly/disassembly of cell:matrix junctions during cell migration. Here we show that ERK1 associates with alpha(v)beta(3) integrin following the addition of platelet-derived growth factor to serum-starved Swiss or NIH 3T3 fibroblasts in an interaction that is mediated by the central region of the beta(3) integrin cytodomain. alpha(v)beta(3).ERK1 association occurred prior to focal complex formation and was seen to initiate in small punctate complexes primarily in the peripheral regions of the plasma membrane. Expression of a dominant negative mutant of ERK1 (but not ERK2) significantly reduced the spreading of cells on vitronectin, whereas cell spreading on fibronectin was unaffected by inhibition of ERK1. In contrast, inhibition of ERK activation by PD98059 had no effect on the platelet-derived growth factor-regulated Rab4-dependent flux of alpha(v)beta(3) integrin from early endosomes to the plasma membrane, an event that is also necessary for cells to spread efficiently on vitronectin. We propose that alpha(v)beta(3) integrin must recycle to the plasma membrane via the Rab4 pathway and recruit active ERK1 in order to function efficiently.     

Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin alpha(v)beta(3), has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of alpha(v)beta(3) in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin alpha(v)beta(3) on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. alpha(v)beta(3) was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-alpha(v)beta(3) monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin alpha(v)beta(3), only L1 serves as a potential ligand for alpha(v)beta(3) during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and alpha(v)beta(3) antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin alpha(v)beta(3) on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.     

Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation

Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.     

Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction.     

Cytokines modulate integrin alpha(v)beta(3)-mediated human endothelial cell adhesion and calcium signaling.

Angiogenesis is a complex process regulated by the interactions of endothelial cells with cytokines and the adhesive protein matrix. The cytokines basic fibroblast growth factor (bFGF) and tumor necrosis factor-alpha (TNF-alpha) are two of the modulators of angiogenesis. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor activity, in particular, alpha(v)beta(3). In this study, we examined the ability of these angiogenic factors to modulate the adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized disintegrins (i.e., rhodostomin and arietin), which are specific in antagonizing integrin alpha(v)beta(3) in cells. As these disintegrins were immobilized as substrates, they acted as agonists to induce HUVEC adhesion in a dose- and alpha(v)beta(3)-dependent manner. In addition, adhesion also triggered a sustained increase of intracellular free calcium. Furthermore, bFGF-primed HUVECs potentiated, but TNF-alpha primed cells attenuated, about 50% adhesion events and calcium signaling triggered by immobilized disintegrin compared to naive cells, respectively. The mechanisms of modulating alpha(v)beta(3)-dependent HUVEC adhesion by cytokines may be related to changes of integrin alpha(v)beta(3) conformation, as demonstrating the antagonistic effect of Mn(2+) on decreased adhesion by TNF-alpha pretreatment, and confirmed with flow cytometric analysis probed by anti-LIBS1 mAb. However, cytokine pretreatment did not alter the expression of this integrin on the cell surface, as determined by flow cytometry. Phosphoinositide-3 kinase may be one of the signaling molecules involved in the enhanced adhesion of bFGF-primed cells.     

Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.     

Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly

Cell-extracellular matrix (cell-ECM) interactions mediated by integrin receptors are essential for providing positional and environmental information necessary for many cell functions, such as proliferation, differentiation and survival. In vitro studies on cell adhesion to randomly adsorbed molecules on substrates have been limited to sub-micrometer patches, thus preventing the detailed study of structural arrangement of integrins and their ligands. In this article, we illustrate the role of the distance between integrin ligands, namely the RGD (arginine-glycine-aspartate) sequence present in ECM proteins, in the control of cell adhesion. By using substrates, which carry cyclic RGD peptides arranged in highly defined nanopatterns, we investigated the dynamics of cell spreading and the molecular composition of adhesion sites in relation to a fixed spacing between the peptides on the surface. Our novel approach for in vitro studies on cell adhesion indicates that not only the composition, but also the spatial organization of the extracellular environment is important in regulating cell-ECM interactions.     

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling

The p38 mitogen-activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor-dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2-induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2-induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2-induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2-induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2-driven angiogenesis.     

Identification of an alpha(3)beta(1) integrin recognition sequence in thrombospondin-1.

A synthetic peptide containing amino acid residues 190-201 of thrombospondin-1 (TSP1) promoted adhesion of MDA-MB-435 breast carcinoma cells when immobilized and inhibited adhesion of the same cells to TSP1 when added in solution. Adhesion to this peptide was enhanced by a beta(1) integrin-activating antibody, Mn(2+), and insulin-like growth factor I and was inhibited by an alpha(3)beta(1) integrin function-blocking antibody. The soluble peptide inhibited adhesion of cells to the immobilized TSP1 peptide or spreading on intact TSP1 but at the same concentrations did not inhibit attachment or spreading on type IV collagen or fibronectin. Substitution of several residues in the TSP1 peptide with Ala residues abolished or diminished the inhibitory activity of the peptide in solution, but only substitution of Arg-198 completely inactivated the adhesive activity of the immobilized peptide. The essential residues for activity of the peptide as a soluble inhibitor are Asn-196, Val-197, and Arg-198, but flanking residues enhance the inhibitory activity of this core sequence, either by altering the conformation of the active sequence or by interacting with the integrin. This functional sequence is conserved in all known mammalian TSP1 sequences and in TSP1 from Xenopus laevis. The TSP1 peptide also inhibited adhesion of MDA-MB-435 cells to the laminin-1 peptide GD6, which contains a potential integrin-recognition sequence Asn-Leu-Arg and is derived from a similar position in a pentraxin module. Adhesion studies using recombinant TSP1 fragments also localized beta1 integrin-dependent adhesion to residues 175-242 of this region, which contain the active sequence.     

Distinct biological consequences of integrin alpha v beta 3-mediated melanoma cell adhesion to fibrinogen and its plasmic fragments.

Fibrinogen/fibrin and its proteolytic fragments serve as potential adhesive substrates during thrombosis, wound healing, and cancer. In this report we examined the biological response of human melanoma cells exposed to fibrinogen and its naturally occurring plasmic breakdown products that are known constituents of the tumor stroma. Plasmin treatment of fibrinogen first results in fragment X, which is characterized by removal of the COOH-terminal portion of the alpha chain including an RGD sequence (A alpha 572-575). Further digestion leads to fragment D comprising primarily an intact COOH-terminal stretch of the gamma chain containing the platelet adhesion sequence HHLGGAKQAGDV. In a sensitive adhesion assay M21 human melanoma cells utilized integrin alpha v beta 3 to attach to all three of these ligands. However, only intact fibrinogen promoted significant cell spreading, while fragment X produced minimal spreading and fragment D promoted only adhesion. These results indicate that fibrinogen contains at least two alpha v beta 3-dependent adhesive sites and these promote distinct biological responses of human melanoma cells. The differential functional properties of these ligands directly correlate to their relative binding affinity for purified alpha v beta 3 as measured in a solid-phase receptor binding assay. These results provide evidence that a single integrin can promote distinct biological signals depending on the molecular nature of the ligand binding event.     

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.     

Processing of integrin alpha(v) subunit by membrane type 1 matrix metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase.

Recently, we have shown that membrane type 1 matrix metalloproteinase (MT1-MMP) exhibits integrin convertase activity. Similar to furin-like proprotein convertases, MT1-MMP directly processes a single chain precursor of alpha(v) integrin subunit (pro-alpha(v)) into the heavy and light alpha-chains connected by a disulfide bridge. To evaluate functionality of MT1-MMP-processed integrins, we examined breast carcinoma MCF7 cells co-expressing alpha(v)beta(3) integrin with either the wild type or mutant MT1-MMP in a variety of migration and adhesion tests. Specific inhibitors of proprotein convertases and MMP were employed in our cell system to attenuate the individual pathways of pro-alpha(v) maturation. We present evidence that MT1-MMP cleavage of pro-alpha(v) in the cells did not affect RGD-ligand binding of the resulting alpha(v)beta(3) integrin but enhanced outside-in signal transduction through a focal adhesion kinase pathway. Enhanced tyrosine phosphorylation of focal adhesion kinase in cells co-expressing MT1-MMP and alpha(v)beta(3) integrin contributed to efficient adhesion and, especially, migration of cells on vitronectin, a ligand of alpha(v)beta(3) integrin. These mechanisms underscore the significance of a coordinated interplay between MT1-MMP and alpha(v)beta(3) integrin in tumor cells and identify downstream signaling pathways resulting from their interactions. Regulation of integrin maturation and functionality may be an important role of MT1-MMP in tumor cells.     

alpha v beta3 and alpha5beta1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration

Accumulating evidence suggests that integrin recycling regulates cell migration. However, the lack of reagents to selectively target the trafficking of individual heterodimers, as opposed to endocytic transport as a whole, has made it difficult to define the contribution made by particular recycling pathways to directional cell movement. We show that autophosphorylation of protein kinase D1 (PKD1) at Ser(916) is necessary for its association with alphavbeta3 integrin. Expression of PKD1(916A) or the use of mutants of beta3 that do not bind to PKD1 selectively inhibits short-loop, Rab4-dependent recycling of alphavbeta3, and this suppresses the persistence of fibroblast migration. However, we report that short-loop recycling does not directly contribute to fibroblast migration by moving alphavbeta3 to the cell front, but by antagonizing alpha5beta1 recycling, which, in turn, influences the cell's decision to migrate with persistence or to move randomly.     

Distinct ligand-binding modes for integrin alpha(v)beta(3)-mediated adhesion to fibronectin versus vitronectin.

Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including alpha(v)beta(3) can bind to multiple ligands. The binding of alpha(v)beta(3) to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the alpha(v)beta(3)-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the alpha(v)beta(3)-vitronectin bond required phosphorylation of the beta(3) cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of beta(3) controlled by Tyr-747 and Tyr-759. In contrast, alpha(v)beta(3)-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of alpha(v)beta(3).     

Overexpressed Csk tyrosine kinase is localized in focal adhesions, causes reorganization of alpha v beta 5 integrin, and interferes with HeLa cell spreading.

The C-terminal Src kinase p50csk phosphorylates Src family tyrosine kinases and down-regulates their activity in vitro. To gain insight into the cellular functions of this potentially antioncogenic enzyme, we have overexpressed the csk cDNA by using an inducible promoter in HeLa cells. Despite some differences in basal Src activity in the clones analyzed, Src activity was not significantly suppressed, while the amount of p50csk and Csk activity increased at least 10-fold during 3 days of induction. Immunofluorescence for the induced p50csk was localized in the cytoplasm and distinctly in focal adhesions, in which the amount of phosphotyrosine containing proteins was also increased. Point and deletion mutagenesis experiments showed that localization in focal adhesions was dependent on the SH2 and SH3 domains of Csk but not on its catalytic activity. Csk formed a complex with the focal adhesion protein paxillin in cells, and its SH2 domain was shown to interact with pp125FAK and paxillin in vitro. After Csk induction, the cells became spherical and more loosely attached to the culture substratum, and the alpha v beta 5 integrin complex (vitronectin receptor) of focal adhesions was redistributed to a novel type of structure consisting of punctate plaques on the ventral cell surface. These phenotypic changes occurred in several clones analyzed and were totally reversible when Csk was switched off, but they did not occur in cells overexpressing the catalytically inactive Csk R-222 mutant or luciferase. Our results thus show that a fraction of cellular Csk is targeted to focal adhesions via its SH2 and SH3 domains, probably interacting with tyrosyl-phosphorylated focal adhesion proteins. They also suggest that Csk is involved in the regulation of integrins controlling cell attachment and shape.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3.

This paper reports a detailed conformational characterization in solution by 1H-NMR in H2O and DMSO-d6 and molecular modeling simulations of cyclic peptides containing the RGDDV pharmacophore and the RGDY(Me)R pharmacophore. These two pentapeptide sequences when properly constrained in cyclic peptides are low to sub-nanomolar inhibitors of integrin alpha(v)beta3. The peptides containing the RGDDY(Me)R sequence bind potently to integrin alphaIIb3 as well. The conformations found in H2O and in DMSO-d6 solutions are valuable for the design of peptidomimetics of these two pharmacophores. The structure-activity relationships of the RGDDV and RGDY(Me)R pharmacophores within cyclic peptides are discussed. Specifically, the orientation of surface-accessible chemical features on the ligand, such as hydrophobic, positive and negative ionizable groups, which are considered to be responsible for the desired biological activity, is focused on.     

Del1 mediates VSMC adhesion, migration, and proliferation through interaction with integrin alpha(v)beta(3)

Del1 is a matrix protein transiently expressed by embryonic endothelial cells. It was recently demonstrated that vascular endothelial cells adhere and interact with Del1 through alpha(v)beta(3)- integrins, providing an autocrine angiogenic signaling pathway in this cell type. To determine whether Del1 might signal to other cell types in the vessel wall in a paracrine fashion, studies were conducted with vascular smooth muscle cells (VSMC). Del1 promoted adhesion and migration of VSMC in a dose-dependent fashion. These functions were mediated through alpha(v)beta(3)-integrins, as the vitronectin receptor inhibitory peptide containing penacillamine (PCN) arginine-glycine-aspartic acid (PCN-RGD) and an antibody specific for the alpha(v)beta(3)-integrin specifically blocked both adhesion and migration. Adhesion of VSMC to Del1 was associated with organization of actin filaments and formation of focal contacts enriched in vinculin and alpha(v)beta(3). Furthermore, Del1 supported VSMC proliferation at least in part by inhibiting these cells from undergoing apoptosis. These data, in conjunction with evidence that Del1 expression is reactivated in vascular injury, suggest that Del1 may have a paracrine role in vessel wall development and remodeling.     

P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.     

The CK2 phosphorylation of vitronectin. Promotion of cell adhesion via the alpha(v)beta 3-phosphatidylinositol 3-kinase pathway

Phosphorylation of vitronectin (Vn) by casein kinase II was previously shown to occur at Thr50 and Thr57 and to augment a major physiological function of vitronectin-cell adhesion and spreading. Here we show that this phosphorylation increases cell adhesion via the alpha(v)beta3 (not via the alpha(v)beta5 integrin), suggesting that alpha(v)beta3 differs from alpha(v)beta5 in its biorecognition profile. Although both the phospho (CK2-PVn) and non-phospho (Vn) analogs of vitronectin (simulated by mutants Vn(T50E,T57E), and Vn(T50A,T57A), respectively) trigger the alpha(v)beta3 as well as the alpha(v)beta5 integrins, and equally activate the ERK pathway, these two forms are different in their activation of the focal adhesion kinase/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway. Specifically, we show (i) that, upon exposure of cells to Vn/CK2-PVn, their PKB activation depends on the availability of the alpha(v)beta3 integrin on their surface; (ii) that upon adhesion of the beta3-transfected cells onto the CK2-PVn, the extent of PKB activation coincides with the enhanced adhesion of these cells, and (iii) that both the PKB activation and the elevation in the adhesion of these cells is PI3K-dependent. The occurrence of a cell surface receptor that specifically distinguishes between a phosphorylated and a non-phosphorylated analog of Vn, together with the fact that it preferentially activates a distinct intra-cellular signaling pathway, suggest that extra-cellular CK2 phosphorylation may play an important role in the regulation of cell adhesion and migration.