Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells

Tyrosine phosphorylation of membrane-associated proteins is involved at two distinct sites of contact between cells and the extracellular matrix: adhesion plaques (cell adhesion and de-adhesion) and invadopodia (invasion into the extracellular matrix). Adhesion plaques from chicken embryonic fibroblasts or from cells transformed by Rous sarcoma virus contain low levels of tyrosine-phosphorylated proteins (YPPs) which were below the level of detection in 0.5-microns thin, frozen sections. In contrast, intense localization of YPPs was observed at invadopodia of transformed cells at sites of degradation and invasion into the fibronectin-coated gelatin substratum, but not in membrane extensions free of contact with the extracellular matrix. Local extracellular matrix degradation and formation of invadopodia were blocked by genistein, an inhibitor of tyrosine-specific kinases, but cells remained attached to the substratum and retained their free-membrane extensions. Invadopodia reduced or lost YPP labeling after treatment of the cells with genistein, but adhesion plaques retained YPP labeling. The plasma membrane contact fractions of normal and transformed cells have been isolated form cells grown on gelatin cross-linked substratum using a novel fractionation scheme, and analyzed by immunoblotting. Four major YPPs (150, 130, 81, and 77 kD) characterize invadopodial membranes in contact with the matrix, and are probably responsible for the intense YPP labeling associated with invadopodia extending into sites of matrix degradation. YPP150 may be an invadopodal-specific YPP since it is approximately 3.6-fold enriched in the invasive contact fraction relative to the cell body fraction and is not observed in normal contacts. YPP130 is enriched in transformed cell contacts but may also be present in normal contacts. The two major YPPs of normal contacts (130 and 71 kD) are much lower in abundance than the major tyrosine-phosphorylated bands associated with invadopodial membranes, and likely represent major adhesion plaque YPPs. YPP150, paxillin, and tensin appear to be enriched in the cell contact fractions containing adhesion plaques and invadopodia relative to the cell body fraction, but are also present in the soluble supernate fraction. However, vinculin, talin, and alpha-actinin that are localized at invadopodia, are equally concentrated in cell bodies and cell contacts as is the membrane-adhesion receptor beta 1 integrin. Thus, tyrosine phosphorylation of the membrane-bound proteins may contribute to the cytoskeletal and plasma membrane events leading to the formation and function of invadopodia that contact and proteolytically degrade the extracellular matrix; we have identified several candidate YPPs that may participate in the regulation of these processes.     

Preferential Localization of Tyrosine-Phosphorylated Paxillin in Focal Adhesions

Focal adhesions are sites for integrin-mediated attachment of cultured cells to the extracellular matrix. Localization studies have shown that focal adhesions can be stained by antiphosphotyrosine antibodies, but the role of tyrosine-phosphorylated proteins in focal adhesions is not known. By using ventral plasma membranes prepared from chicken embryo fibroblasts spread on the substrate, we present evidence for the preferential localization of a minor pool of tyrosine-phosphorylated paxillin in focal adhesions. Ventral plasma membranes showed an enrichment in β1-integrins, and in several tyrosine-phosphorylated polypeptides, while focal adhesion proteins like vinculin and paxillin, although localized to focal adhesions in ventral plasma membranes, were not particularly enriched in these preparations compared to whole cell lysates. Biochemical and morphological analysis of ventral plasma membranes showed a dramatic increase in the level of tyrosine-phosphorylation of the pool of paxillin localized to the adhesive sites, when compared to the paxillin present in whole cell lysates. The observed preferential localization of tyrosine-phosphorylated paxillin to focal adhesions may represent a general mechanism to compartmentalize focal adhesion components from large non-phosphorylated, cytosolic pools.     

Preferential Localization of Tyrosine-Phosphorylated Paxillin in Focal Adhesions

Focal adhesions are sites for integrin-mediated attachment of cultured cells to the extracellular matrix. Localization studies have shown that focal adhesions can be stained by antiphosphotyrosine antibodies, but the role of tyrosine-phosphorylated proteins in focal adhesions is not known. By using ventral plasma membranes prepared from chicken embryo fibroblasts spread on the substrate, we present evidence for the preferential localization of a minor pool of tyrosine-phosphorylated paxillin in focal adhesions. Ventral plasma membranes showed an enrichment in β1-integrins, and in several tyrosine-phosphorylated polypeptides, while focal adhesion proteins like vinculin and paxillin, although localized to focal adhesions in ventral plasma membranes, were not particularly enriched in these preparations compared to whole cell lysates. Biochemical and morphological analysis of ventral plasma membranes showed a dramatic increase in the level of tyrosine-phosphorylation of the pool of paxillin localized to the adhesive sites, when compared to the paxillin present in whole cell lysates. The observed preferential localization of tyrosine-phosphorylated paxillin to focal adhesions may represent a general mechanism to compartmentalize focal adhesion components from large non-phosphorylated, cytosolic pools.     

Leishmania infection inhibits macrophage motility by altering F‐actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.     

Paxillin enables attachment-independent tyrosine phosphorylation of focal adhesion kinase and transformation by RAS

Paxillin and HIC5 are closely related adapter proteins that regulate cell migration and are tyrosine-phosphorylated by focal adhesion kinase (FAK). Paxillin, HIC5, and FAK tyrosine phosphorylation increase upon cell attachment and decrease upon detachment from extracellular matrix. Unexpectedly, we found that although FAK tyrosine phosphorylation in attached cells did not require paxillin, in detached fibroblasts there was remaining FAK tyrosine phosphorylation that required expression of paxillin and was not supported by HIC5. The support of attachment-independent FAK tyrosine phosphorylation required the paxillin LIM domains and suggested that paxillin might facilitate oncogenic transformation. Paxillin but not HIC5 augmented anchorage-independent cell proliferation induced by RAS. Both anchorage-independent FAK tyrosine phosphorylation and RAS-induced colony formation required multiple docking sites on paxillin, including LD4 (docking sites for FAK-Src and GIT1/2-PIX-NCK-PAK complex), LD5, and all four carboxyl-terminal LIM domains (that bind tubulin and PTP-PEST). Analysis using paxillin mutants dissociated domains of paxillin that are required for regulation of cell migration from domains that are required for anchorage-independent cell proliferation and demonstrated essential functions of the paxillin LIM domains that are not found in HIC5 LIM domains. These results highlight the role of paxillin in facilitating attachment-independent signal transduction implicated in cancer.     

Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions

This study establishes that the physical state of the extracellular matrix can regulate integrin-mediated cytoskeletal assembly and tyrosine phosphorylation to generate two distinct types of cell-matrix adhesions. In primary fibroblasts, alpha(5)beta(1) integrin associates mainly with fibronectin fibrils and forms adhesions structurally distinct from focal contacts, independent of actomyosin-mediated cell contractility. These "fibrillar adhesions" are enriched in tensin, but contain low levels of the typical focal contact components paxillin, vinculin, and tyrosine-phosphorylated proteins. However, when the fibronectin is covalently linked to the substrate, alpha(5)beta(1) integrin forms highly tyrosine-phosphorylated, "classical" focal contacts containing high levels of paxillin and vinculin. These experiments indicate that the physical state of the matrix, not just its molecular composition, is a critical factor in defining cytoskeletal organization and phosphorylation at adhesion sites. We propose that molecular organization of adhesion sites is controlled by at least two mechanisms: 1) specific integrins associate with their ligands in transmembrane complexes with appropriate cytoplasmic anchor proteins (e.g., fibronectin-alpha(5)beta(1) integrin-tensin complexes), and 2) physical properties (e.g., rigidity) of the extracellular matrix regulate local tension at adhesion sites and activate local tyrosine phosphorylation, recruiting a variety of plaque molecules to these sites. These mechanisms generate structurally and functionally distinct types of matrix adhesions in fibroblasts.     

Serine phosphorylation regulates paxillin turnover during cell migration

Background

Paxillin acts as an adaptor protein that localizes to focal adhesion. This protein is regulated during cell migration by phosphorylation on tyrosine, serine and threonine residues. Most of these phosphorylations have been implicated in the regulation of different steps of cell migration. The two major phosphorylation sites of paxillin in response to adhesion to an extracellular matrix are serines 188 and 190. However, the function of this phosphorylation event remains unknown. The purpose of this work was to determine the role of paxillin phosphorylation on residues S188 and S190 in the regulation of cell migration.

Results

We used NBT-II epithelial cells that can be induced to migrate when plated on collagen. To examine the role of paxillin serines 188/190 in cell migration, we constructed an EGFP-tagged paxillin mutant in which S188/S190 were mutated into unphosphorylatable alanine residues. We provide evidence that paxillin is regulated by proteasomal degradation following polyubiquitylation of the protein. During active cell migration on collagen, paxillin is protected from proteasome-dependent degradation. We demonstrate that phosphorylation of serines 188/190 is necessary for the protective effect of collagen. In an effort to understand the physiological relevance of paxillin protection from degradation, we show that cells expressing the paxillin S188/190A interfering mutant spread less, have reduced protrusive activity but migrate more actively.

Conclusion

Our data demonstrate for the first time that serine-regulated degradation of paxillin plays a key role in the modulation of membrane dynamics and consequently, in the control of cell motility.     

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21^WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α_vβ₃ were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.     

Integrin engagement, the actin cytoskeleton, and c-Src are required for the calcitonin-induced tyrosine phosphorylation of paxillin and HEF1, but not for calcitonin-induced Erk1/2 phosphorylation

We have previously shown that in a HEK-293 cell line that overexpresses the C1a isoform of the calcitonin receptor (C1a-HEK), calcitonin induces the tyrosine phosphorylation of the focal adhesion-associated proteins HEF1 (a p130(Cas)-like docking protein), paxillin, and focal adhesion kinase and that it also stimulates the phosphorylation and activation of Erk1 and Erk2. We report here that cell attachment to the extracellular matrix, an intact actin cytoskeleton, and c-Src are absolutely required for the calcitonin-induced phosphorylation of focal adhesion-associated proteins. In contrast to the phosphorylation of paxillin and HEF1 in cells attached to fibronectin-coated dishes, calcitonin failed to stimulate the phosphorylation of paxillin and HEF1 in suspended cells, in cells attached to poly-d-lysine-coated dishes, and in attached cells pretreated with the RGD-containing peptide GRGDS. Overexpression of wild-type c-Src increased calcitonin-induced paxillin and HEF1 phosphorylation, whereas overexpression of kinase-dead Src or Src lacking a functional SH2 domain inhibited the calcitonin-stimulated tyrosine phosphorylation of these proteins. Overexpression of Src lacking the SH3 domain did not affect the calcitonin-induced phosphorylation of paxillin and HEF1. In contrast to the regulation of paxillin and HEF1 phosphorylation, the calcitonin-induced phosphorylation of Erk1 and Erk2 did not appear to involve c-Src and was only partially dependent on cell adhesion to the extracellular matrix and an intact actin cytoskeleton. Furthermore, inhibition of Erk1 and Erk2 phosphorylation had no effect on the calcitonin-induced phosphorylation of paxillin and HEF1. Thus, in C1a-HEK cells, the calcitonin receptor is coupled to the tyrosine phosphorylation of focal adhesion-associated proteins and to Erk1/2 phosphorylation by mechanisms that are in large part independent.     

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca²⁺ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction. tissue transfection; plasmids; cytoskeleton; talin; immunofluorescence     

mRNA sequence of the Xenopus laevis paxillin gene and its expression.

Paxillin is a cytoskeletal protein found in structures of focal adhesions where cells adhere to the extracellular matrix. We isolated paxillin cDNA from the Xenopus laevis ovary. The cDNA sequence encodes a protein of 539 amino acids with four LIM and five LD motifs. 80% of the amino acids of frog paxillin are shared by human and chicken paxillins. Northern analysis showed that the frog gene is expressed in the spleen, kidney, testis and ovary. Immunocytochemistry showed that paxillin protein is accumulated in the nucleus as well as in the periphery of the cytoplasm of the A6 cell. This intriguing result shows that paxillin, which has been characterized as a cytoskeletal protein, is capable of translocating to the nucleus.     

Syndesmos, a syndecan-4 cytoplasmic domain interactor, binds to the focal adhesion adaptor proteins paxillin and Hic-5.

Syndecan-4 and integrins are the primary transmembrane receptors of focal adhesions in cells adherent to extracellular matrix molecules. Syndesmos is a cytoplasmic protein that interacts specifically with the cytoplasmic domain of syndecan-4, and it co-localizes with syndecan-4 in focal contacts. In the present study we sought possible interactors with syndesmos. We find that syndesmos interacts with the focal adhesion adaptor protein paxillin. The binding of syndesmos to paxillin is direct, and these interactions are triggered by the activation of protein kinase C. Syndesmos also binds the paxillin homolog, Hic-5. The connection of syndecan-4 with paxillin through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperative signaling of these two receptors in the assembly of focal adhesions and actin stress fibers.     

Focal adhesion kinase signaling is decreased in polyamine-depleted IEC-6 cells

Polyamines are essential to the migration ofepithelial cells in the intestinal mucosa. Cells depleted of polyaminesdo not attach as rapidly to the extracellular matrix and do not form the actin stress fibers essential for migration. Because both attachment and stress fiber formation depend on integrin signaling andthe formation of focal adhesions, we examined these and related processes in polyamine-depleted IEC-6 cells. There was general decreased tyrosine phosphorylation of focal adhesion kinase (FAK), and,specifically, decreased phosphorylation of Tyr-925, the paxillin binding site. In control cells, FAK phosphorylation was rapid afterattachment to the extracellular matrix, while attached cells depletedof polyamines had significantly delayed phosphorylation. FAK activitywas also significantly inhibited in polyamine-depleted cells as was thephosphorylation of paxillin. Polyamine-depleted cells failed to spreadnormally after attachment, and immunocytochemistry showed littlecolocalization of FAK and actin compared with controls. Focal adhesioncomplex formation was greatly reduced in the absence of polyamines.These data suggest that defective integrin signaling may, at least inpart, account for the decreased rates of attachment, actin stress fiberformation, spreading, and migration observed in polyamine-depleted cells.

     

Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1 cells

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD(416)G and DSPD(748)G) in vitro, and point mutations substituting the Asp of DVPD(416)G and DSPD(748)G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD(416)G generated a 74-kDa fragment, which was in turn cleaved at DSPD(748)G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.     

Integrin adhesions: Who’s on first? What's on second?

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

Patterning of the membrane cytoskeleton by the extracellular matrix

The extracellular matrices of different tissues contain components which affect the migration, morphology and differentiation of many types of cells. These forms of cell behavior often involve dramatic changes in cytoskeletal organization. Extracellular matrix components are recognized by specific cell surface receptors which span the membrane and interact with the actin cytoskeleton. In cultured cells, the matrix receptors are concentrated in sites of cell attachment called focal adhesions. Information that is conveyed from the extracellular matrix to the cytoskeleton may involve matrix components, cell surface receptors, as well as the proteins at the cytoplasmic face of the focal adhesion which link the receptors to the actin cytoskeleton.     

Human caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner

The signals involved in restitution during mucosal healing are poorly understood. We compared focal adhesion kinase (FAK) and paxillin protein and phosphorylation, extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 activation, as well as FAK and paxillin organization in static and migrating human intestinal Caco-2 cells on matrix proteins and anionically derivatized polystyrene dishes (tissue culture plastic). We also studied effects of FAK, ERK, and p38 blockade in a monolayer-wounding model. Compared with static cells, cells migrating across matrix proteins matrix-dependently decreased membrane/cytoskeletal FAK and paxillin and cytosolic FAK. Tyrosine phosphorylated FAK and paxillin changed proportionately to FAK and paxillin protein. Conversely, cells migrating on plastic increased FAK and paxillin protein and phosphorylation. Migration matrix-dependently activated p38 and inactivated ERK1 and ERK2. Total p38, ERK1, and ERK2 did not change. Caco-2 motility was inhibited by transfection of FRNK (the COOH-terminal region of FAK) and PD-98059, a mitogen-activated protein kinase-ERK kinase inhibitor, but not by SB-203580, a p38 inhibitor, suggesting that FAK and ERK modulate Caco-2 migration. In contrast to adhesion-induced phosphorylation, matrix may regulate motile intestinal epithelial cells by altering amounts and distribution of focal adhesion plaque proteins available for phosphorylation as well as by p38 activation and ERK inactivation. Motility across plastic differs from migration across matrix.     

Paxillin phosphorylation controls invadopodia/podosomes spatiotemporal organization

In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.     

Paxillin modulates squamous cancer cell adhesion and is important in pressure-augmented adhesion

Paxillin is an adapter protein regulating signaling and focal adhesion assembly that has been linked to malignant potential in many malignancies. Overexpression of paxillin has been noted in aggressive tumors. Integrin-mediated binding through the focal adhesion complex is important in metastatic adhesion and is upregulated by extracellular pressure in malignant colonocytes through FAK and Src activation. Neither head and neck cancers nor paxillin have been studied in this regard. We hypothesized that paxillin would play a role in modulating squamous cancer adhesion both at baseline and under conditions of increased extracellular pressure. Using SCC25 tongue squamous cancer cells stably transfected with either an empty selection vector or paxillin expression and selection vectors, we studied adhesion to collagen, paxillin, FAK, and Src expression and phosphorylation in cells maintained for 30 min under ambient or 15 mmHg increased pressure conditions. Paxillin-overexpressing cells exhibited adhesion 121 +/- 2.9% of that observed in vector-only cells (n = 6, P < 0.001) under ambient pressure. Paxillin-overexpression reduced FAK phosphorylation. Pressure stimulated adhesion to 118 +/- 2.3% (n = 6, P < 0.001) of baseline in vector-only cells, similar to its effect in the parental line, and induced paxillin, FAK, and Src phosphorylation. However, increased pressure did not stimulate adhesion or phosphorylate paxillin, FAK, or Src further in paxillin-overexpressing cells. Metastasizing squamous cancer cell adhesiveness may be increased by paxillin-overexpression or by paxillin activation by extracellular pressure during surgical manipulation or growth within a constraining compartment. Targeting paxillin in patients with malignancy and minimal tumor manipulation during surgical resection may be important therapeutic adjuncts.     

Characterization of the tyrosine kinases RAFTK/Pyk2 and FAK in nerve growth factor-induced neuronal differentiation

The related adhesion focal tyrosine kinase (RAFTK), a member of the focal adhesion kinase (FAK) family and highly expressed in brain, is a key mediator of various extracellular signals that elevate intracellular Ca(2+) concentration. We investigated RAFTK and FAK signaling upon nerve growth factor (NGF) stimulation of PC12 cells. NGF induced the tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner, whereas no change in the tyrosine phosphorylation of FAK was observed. Chemical inhibition showed that RAFTK phosphorylation was inhibited by blocking phospholipase Cgamma activity or intracellular Ca(2+). Blocking of extracellular Ca(2+) or phosphatidylinositol 3-kinase activity partially reduced the phosphorylation of RAFTK. In addition, disruption of actin polymerization abolished RAFTK phosphorylation, indicating that an intact actin-based cytoskeletal organization is required for RAFTK phosphorylation. The focal adhesion molecule paxillin was co-immunoprecipitated with RAFTK, and its tyrosine phosphorylation was increased in a Ca(2+)-dependent manner upon NGF stimulation. Confocal microscopic analysis demonstrated that RAFTK translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase (within 5 min) of NGF stimulation, whereas FAK co-localized with paxillin at "point contacts," which are the primary cell adhesion sites in neuronal cells. Significant distribution of RAFTK was observed in the neurites and growth cones of differentiated PC12 cells. Furthermore, potassium depolarization induced the tyrosine phosphorylation of both RAFTK and paxillin in an intracellular Ca(2+)-dependent manner in the differentiated PC12 cells. Taken together, these results demonstrate that RAFTK is involved in NGF-induced cytoskeletal organization and may play a role in neurite and growth cone function(s).