The PDZ protein MPP2 interacts with c-Src in epithelial cells

c-Src is a non-receptor tyrosine kinase involved in regulating cell proliferation, cell migration and cell invasion and is tightly controlled by reversible phosphorylation on regulatory sites and through protein-protein interactions. The interaction of c-Src with PDZ proteins was recently identified as novel mechanism to restrict c-Src function. The objective of this study was to identify and characterise PDZ proteins that interact with c-Src to control its activity. By PDZ domain array screen, we identified the interaction of c-Src with the PDZ protein Membrane Protein Palmitoylated 2 (MPP2), a member of the Membrane-Associated Guanylate Kinase (MAGUK) family, to which also the Discs large (Dlg) tumour suppressor protein belongs. The function of MPP2 has not been established and the functional significance of the MPP2 c-Src interaction is not known. We found that in non-transformed breast epithelial MCF-10A cells, endogenous MPP2 associated with the cytoskeleton in filamentous structures, which partially co-localised with microtubules and c-Src. MPP2 and c-Src interacted in cells, where c-Src kinase activity promoted increased interaction of c-Src with MPP2. We furthermore found that MPP2 was able to negatively regulate c-Src kinase activity in cells, suggesting that the functional significance of the MPP2-c-Src interaction is to restrict Src activity. Consequently, the c-Src-dependent disorganisation of the cortical actin cytoskeleton of epithelial cells expressing c-Src was suppressed by MPP2. In conclusion we demonstrate here that MPP2 interacts with c-Src in cells to control c-Src activity and morphological function.     

Progesterone enhances vascular endothelial cell migration via activation of focal adhesion kinase

The mechanisms of progesterone on endothelial cell motility are poorly investigated. Previously we showed that progesterone stimulated endothelial cell migration via the activation of actin-binding protein moesin, leading to actin cytoskeleton remodelling and the formation of cell membrane structures required for cell movement. In this study, we investigated the effects of progesterone on the formation of focal adhesion complexes, which provide anchoring sites for cell movement. In cultured human umbilical endothelial cells, progesterone enhanced focal adhesion kinase (FAK) phosphorylation at Tyr(397) in a dose- and time-dependent manner. Several signalling inhibitors interfered with progesterone-induced FAK activation, including progesterone receptor (PR) antagonist ORG 31710, specific c-Src kinase inhibitor PP2, phosphatidylinosital-3 kinase (PI3K) inhibitor wortmannin as well as ρ-associated kinase (ROCK-2) inhibitor Y27632. It suggested that PR, c-Src, PI3K and ROCK-2 are implicated in this action. In line with this, we found that progesterone rapidly promoted c-Src/PI3K/Akt activity, which activated the small GTPase RhoA/ρ-associated kinase (ROCK-2) complex, resulting in FAK phosphorylation. In the presence of progesterone, endothelial cells displayed enhanced horizontal migration, which was reversed by small interfering RNAs abrogating FAK expression. In conclusion, progesterone promotes endothelial cell movement via the rapid regulation of FAK. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.     

Regulation of c-Src by binding to the PDZ domain of AF-6

c-Src is a tightly regulated non-receptor tyrosine kinase. We describe the C-terminus of c-Src as a ligand for a PDZ (postsynaptic density 95, PSD-95; discs large, Dlg; zonula occludens-1, ZO-1) domain. The C-terminal residue Leu of c-Src is essential for binding to a PDZ domain. Mutation of this residue does not affect the intrinsic kinase activity in vitro, but interferes with c-Src regulation in cells. As a candidate PDZ protein, we analysed AF-6, a junctional adhesion protein. The AF-6 PDZ domain restricts the number of c-Src substrates, whereas knockdown of AF-6 has the opposite effect. Binding of c-Src to the AF-6 PDZ domain interferes with phosphorylation of c-Src at Tyr527 by the C-terminal kinase, and reduces c-Src autophosphorylation at Tyr416, resulting in a moderately activated c-Src kinase. Unphosphorylated Tyr527 allows binding of c-Src to AF-6. This can be overcome by overexpression of CSK or strong activation of c-Src. c-Src is recruited by AF-6 to cell-cell contact sites, suggesting that c-Src is regulated by a PDZ protein in special cellular locations. We identified a novel type of c-Src regulation by interaction with a PDZ protein.     

PYK2 autophosphorylation,but not kinase activity,is necessary for adhesion-induced association with c-Src,osteoclast spreading,and bone resorption

Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts. Previous studies have shown that ligation of alpha(v)beta(3) integrin in osteoclasts induces c-Src-dependent tyrosine phosphorylation and PYK2 activation, leading to cytoskeletal rearrangement, migration, and polarization of these cells. In this study, we examined the role of PYK2 kinase activity and its major autophosphorylation site in adhesion-dependent signaling and cytoskeletal organization during osteoclast spreading and migration. By infecting pre-fusion osteoclasts using recombinant adenovirus expressing PYK2 and its mutants, we demonstrated that mutation at the autophosphorylation site (Y402F) abolishes PYK2 association with c-Src and reduces significantly phosphorylation at tyrosines 579/580 and 881 resulting in inhibition of osteoclast spreading and bone resorption. Overexpression of the kinase-dead PYK2(K475A) mutant had no effect on cell spreading, interaction with c-Src, or the phosphorylation level of Tyr-402, Tyr-579/580, and Tyr-881 relative to PYK2(wt)-expressing cells. Taken together these findings suggest that Tyr-402 is the major docking site for c-Src and can be phosphorylated by another tyrosine kinase in osteoclasts but not in HEK293 cells. Interestingly, both PYK2(Y402F) and PYK2(K457A) translocate normally to podosomes and have no effect on macrophage colony-stimulating factor-induced osteoclast migration. Whereas PYK2(Y402F) dominant negatively blocks osteoclast spreading and bone resorption, PYK2(K457A) may function in part as an adaptor by initially recruiting c-Src to the adhesion complex, which appears to activate PYK2 by phosphorylating additional tyrosines in its regulatory and C-terminal domains. We thus concluded that phosphorylation at Tyr-402 in PYK2 is essential in the regulation of adhesion-dependent cytoskeletal organization in osteoclasts.     

Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527.

We have characterized the mechanism by which the subcellular distribution of c-Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c-Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c-Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c-Src inside the cell. The first 251 amino acids of c-Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c-Src in focal adhesions resides in the amino-terminal half. Point mutations and deletions in the first 251 amino acids of c-Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino-terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non-catalytic portion of c-Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c-Src may regulate focal adhesions and cellular adhesion by a kinase-independent mechanism.     

Overexpression of c-Src enhances cell-matrix adhesion and cell migration in PDGF-stimulated NIH3T3 fibroblasts

c-Src is normally associated with the plasma membrane, but upon activation by tyrosine kinase receptors it translocates to the cytoskeleton. Activation of c-Src alters its conformation and induces the association of c-Src with cytoskeletal proteins. c-Src is implicated in tyrosine phosphorylation of cytoskeletal proteins, which might affect the cytoskeletal architecture. Rearrangements of the cytoskeleton affect cell-matrix adhesion and cell migration. In this study NIH3T3 fibroblasts, that overexpress c-Src, were used to analyze the effect of c-Src on both cell-matrix adhesion and cell migration. Upon PDGF stimulation translocation of c-Src to the cytoskeleton was detected. PDGF treatment also increased cell-matrix adhesion and cell migration. The cell line with the highest c-Src expression showed the largest increases in both phenomena. These findings suggest that translocation of c-Src to the cytoskeleton results in enhanced cell-matrix adhesion and cell migration.     

Close homolog of L1 is an enhancer of integrin-mediated cell migration

Close homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules expressed by subpopulations of neurons and glia in the central and peripheral nervous system. It promotes neurite outgrowth and neuronal survival in vitro. This study describes a novel function for CHL1 in potentiating integrin-dependent cell migration toward extracellular matrix proteins. Expression of CHL1 in HEK293 cells stimulated their haptotactic migration toward collagen I, fibronectin, laminin, and vitronectin substrates in Transwell assays. CHL1-potentiated cell migration to collagen I was dependent on alpha1beta1 and alpha2beta1 integrins, as shown with function blocking antibodies. Potentiated migration relied on the early integrin signaling intermediates c-Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Enhancement of migration was disrupted by mutation of a potential integrin interaction motif Asp-Gly-Glu-Ala (DGEA) in the sixth immunoglobulin domain of CHL1, suggesting that CHL1 functionally interacts with beta1 integrins through this domain. CHL1 was shown to associate with beta1 integrins on the cell surface by antibody-induced co-capping. Through a cytoplasmic domain sequence containing a conserved tyrosine residue (Phe-Ile-Gly-Ala-Tyr), CHL1 recruited the actin cytoskeletal adapter protein ankyrin to the plasma membrane, and this sequence was necessary for promoting integrin-dependent migration to extracellular matrix proteins. These results support a role for CHL1 in integrin-dependent cell migration that may be physiologically important in regulating cell migration in nerve regeneration and cortical development.     

Co-operative effect of c-Src and ezrin in deregulation of cell-cell contacts and scattering of mammary carcinoma cells

The non-receptor tyrosine kinase c-Src is activated in many human cancer types, and induces deregulation of cadherin-based cell-cell contacts and actin cytoskeleton. Because ezrin, a protein which cross-links the plasma membrane with the actin cytoskeleton, is often over-expressed in human cancers, and participates in cell adhesion, motility, and cell scattering, we therefore investigated whether c-Src co-operates with ezrin in regulating cell-cell contacts in a murine mammary carcinoma cell line, SP1. SP1 cells over-expressing wild type ezrin, or an activated c-Src mutant, formed loose aggregates which scattered spontaneously when plated on plastic. When wild type ezrin and activated c-Src were co-expressed, scattering was increased, cell-cell contacts disrupted, and cell aggregation prevented. Pre-treatment with the c-Src family kinase inhibitor PP2 partially restored aggregation of cells expressing activated c-Src and wild type ezrin, indicating that c-Src family kinases act co-operatively with ezrin in regulating cell-cell contacts. Furthermore, expression of a truncated NH2-terminal domain of ezrin, which has dominant negative function, blocked the cell scattering effect of activated c-Src and promoted formation of cohesive cell-cell contacts. Together, these results suggest co-operativity between c-Src and ezrin in deregulation of cell-cell contacts and enhancing scattering of mammary carcinoma cells.     

Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells

Invadopodia are filopodia-like projections possessing protease activity that participate in tumor cell invasion. We demonstrate that co-localization of cortactin and phosphotyrosine identifies a subset of cortactin puncta termed "invadopodial complexes" that we find to be closely associated with the plasma membrane at active sites of focal degradation of the extracellular matrix in MDA-MB-231 breast cancer cells. Manipulation of c-Src activity in cells by transfection with kinase activated c-Src(527) or kinase inactive c-Src(295) results in a dramatic increase or decrease, respectively, in the number of these structures associated with changes in the number of sites of active matrix degradation. Overexpression of kinase-inactive c-Src(295) does not prevent localization of cortactin at the membrane; however, co-localized phosphotyrosine staining is decreased. Thus, elevated phosphotyrosine at invadopodial complexes is specifically associated with the proteolytic activity of invadopodia. Further, invadopodial complexes are spatially, morphologically and compositionally distinct from focal adhesions as determined by localization of focal adhesion kinase (FAK), which is not present in invadopodial complexes. Expression of kinase-inactive c-Src(295) blocks invadopodia activity, but does not block filopodia formation. Thus, invadopodia, but not filopodia, are highly correlated with matrix invasion, and sites of invadopodial activity can be identified by the formation of invadopodial complexes.     

CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration

In this study we have demonstrated that both CD44 (the hyaluronan (HA) receptor) and c-Src kinase are expressed in human ovarian tumor cells (SK-OV-3.ipl cell line), and that these two proteins are physically associated as a complex in vivo. Using a recombinant cytoplasmic domain of CD44 and an in vitro binding assay, we have detected a specific interaction between CD44 and c-Src kinase. Furthermore, the binding of HA to SK-OV-3.ipl cells promotes c-Src kinase recruitment to CD44 and stimulates c-Src kinase activity, which, in turn, increases tyrosine phosphorylation of the cytoskeletal protein, cortactin. Subsequently, tyrosine phosphorylation of cortactin attenuates its ability to cross-link filamentous actin in vitro. In addition, transfection of SK-OV-3.ipl cells with a dominant active form of c-Src (Y527F)cDNA promotes CD44 and c-Src association with cortactin in membrane projections, and stimulates HA-dependent/CD44-specific ovarian tumor cell migration. Finally, overexpression of a dominant-negative mutant of c-Src kinase (K295R) in SK-OV-3.ipl cells impairs the tumor cell-specific phenotype. Taken together, these findings strongly suggest that CD44 interaction with c-Src kinase plays a pivotal role in initiating cortactin-regulated cytoskeleton function and HA-dependent tumor cell migration, which may be required for human ovarian cancer progression.     

Disruption of cell-substrate adhesion activates the protein tyrosine kinase pp60(c-src)

Treatment of confluent chicken embryo fibroblasts (CEFs) with trypsin results in a dose- and time-dependent increase in c-Src protein tyrosine kinase (PTK) activity. A similar, but less marked, increase in c-Src PTK activity occurs upon incubation of CEFs in calcium-free phosphate-buffered saline, which also causes a decrease in cell-substrate adhesion. The increase in c-Src PTK activity following disruption of cell-substrate adhesion correlates with a decrease in the phosphorylation of c-Src at the regulatory site, Tyr527. The phosphotyrosine phosphatase inhibitor phenylarsine oxide blocks the increase in c-Src PTK activity seen following treatment with trypsin and the morphological changes associated with the disruption of cell-substrate adhesion. In contrast, disruption of cell-substrate adhesion causes a decrease in FAK PTK activity that rapidly returns to control levels when the cells are plated on fibronection-coated dishes. Treatment of cells with cytochalasin D, which disrupts actin filaments but not cell-substrate adhesion, causes only a slight increase in c-Src PTK activity. Thus, these studies demonstrate a ligand-independent mechanism for the activation of c-Src that is consistent with its role in both cell adhesion and cell motility. Furthermore, these data suggest that similar to adhesion, loss of adhesion is not a passive process but can activate specific signaling pathways that may have significant effects on cellular function.     

Potential molecular mechanism for c-Src kinase-mediated regulation of intestinal cell migration

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.     

c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY(418))-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY(418)-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α(2)β(1)-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.     

Integrin-mediated RON growth factor receptor phosphorylation requires tyrosine kinase activity of both the receptor and c-Src

Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation.     

Disruption of Cell–Substrate Adhesion Activates the Protein Tyrosine Kinase pp60

Treatment of confluent chicken embryo fibroblasts (CEFs) with trypsin results in a dose- and time-dependent increase in c-Src protein tyrosine kinase (PTK) activity. A similar, but less marked, increase in c-Src PTK activity occurs upon incubation of CEFs in calcium-free phosphate-buffered saline, which also causes a decrease in cell–substrate adhesion. The increase in c-Src PTK activity following disruption of cell–substrate adhesion correlates with a decrease in the phosphorylation of c-Src at the regulatory site, Tyr527. The phosphotyrosine phosphatase inhibitor phenylarsine oxide blocks the increase in c-Src PTK activity seen following treatment with trypsin and the morphological changes associated with the disruption of cell–substrate adhesion. In contrast, disruption of cell–substrate adhesion causes a decrease in FAK PTK activity that rapidly returns to control levels when the cells are plated on fibronection-coated dishes. Treatment of cells with cytochalasin D, which disrupts actin filaments but not cell–substrate adhesion, causes only a slight increase in c-Src PTK activity. Thus, these studies demonstrate a ligand-independent mechanism for the activation of c-Src that is consistent with its role in both cell adhesion and cell motility. Furthermore, these data suggest that similar to adhesion, loss of adhesion is not a passive process but can activate specific signaling pathways that may have significant effects on cellular function.     

Tyrosine phosphatase epsilonM stimulates migration and survival of porcine aortic endothelial cells by activating c-Src

The cell growth, survival, and migration of vascular endothelial cells (ECs) are positively regulated by several protein tyrosine kinase receptors. Therefore, protein tyrosine phosphatases (PTPs) must also be important for these processes. The present study found that transmembranal PTPepsilonM, but not cytoplasmic PTPepsilonC, is expressed in porcine ECs and in rat smooth muscle cells, both of which were prepared from the aorta. The overexpression of wild-type PTPepsilonM promoted cell survival and migration in porcine aortic ECs even in medium without and with 1% serum, respectively. A catalytically inactive, substrate-trapping mutant of PTPepsilonM, respectively, did not affect and conversely suppressed cell survival and migration. Interestingly, the forced expression of wild-type PTPepsilonC reduced cell viability in contrast to PTPepsilonM in ECs lacking endogenous PTPepsilonC, indicating the biological significance of selective expression of PTPepsilon isoforms in the vasculature. PTPepsilonM activated c-Src kinase probably by directly dephosphorylating phospho-Tyr527, a negative regulatory site of c-Src. The increases in cell survival and migration induced by overexpressed PTPepsilonM were suppressed by the c-Src inhibitor SU6656. Considering the behaviors of vascular ECs in the pathogenesis of atherosclerosis, these data suggest that PTPepsilonM negatively regulates the development of this disease by activating c-Src.     

Involvement of a protein kinase C and protein phosphatases in adhesion of CD4+ T cells to and detachment from extracellular matrix proteins.

For immune surveillance and function to be effective, T lymphocytes constantly recirculate via lymph and blood between lymphoid organs and body tissues. To enable efficient cell movement and migration, cell adhesion to components of the basement membrane and the extracellular matrix (ECM) must be a rapid and transitory process. Whether phosphorylation and dephosphorylation of cellular proteins are involved in this phenomena was explored by monitoring the adhesion of T cells to immobilized ECM proteins. A short exposure of 51Cr-labeled human CD4+ T cells to phorbol esters in vitro induced a rapid beta 1-integrin-mediated adhesion to both fibronectin and laminin, as determined by inhibition with anti-integrin antibodies. Adhesion was reversible; detachment from the immobilized ECM ligands occurred between 20 and 120 min without further intervention. This T cell adhesion was regulated by the activation of protein kinase C because (a) staurosporine and H-7 inhibitors of protein kinase C suppressed T cell adhesion, and (b) PMA-induced down-regulation of intracellular levels of protein kinase C was associated with the abrogation of the T cell adhesiveness to fibronectin and laminin. Furthermore, inhibition of protein phosphatases activity by okadaic acid delayed the detachment of the T cells from fibronectin or laminin. Thus, we suggest that T cell-ECM interactions such as adhesion and detachment are regulated, respectively, by protein kinase C and protein phosphatases.     

The transmembrane adaptor Cbp/PAG1 controls the malignant potential of human non-small cell lung cancers that have c-src upregulation

The tyrosine kinase c-Src is upregulated in various human cancers, although the precise regulatory mechanism underlying this upregulation is unclear. We previously reported that a transmembrane adaptor Csk-binding protein (Cbp; PAG1) plays an important role in controlling the cell transformation that is induced by the activation of c-Src. To elucidate the in vivo role of Cbp, we examined the function of Cbp in lung cancer cell lines and tissues. In this study, we found that Cbp was markedly downregulated in human non-small cell lung cancer (NSCLC) cells. The ectopic expression of Cbp suppressed the anchorage-independent growth of the NSCLC cell lines (A549 and Lu99) that had upregulated c-Src, whereas the Cbp expression had little effect on other NSCLC cell lines (PC9 and Lu65) that express normal levels of c-Src. The expression of Cbp suppressed the kinase activity of c-Src in A549 cells by recruiting c-Src and its negative regulator, C-terminal Src kinase (Csk), to lipid rafts. The treatment with Src inhibitors, such as PP2, dasatinib, and saracatinib, also suppressed the growth of A549 cells. Furthermore, Cbp expression attenuated the ability of A549 cells to form tumors in nude mice, invade in vitro, and metastasize in vivo. In addition, we found a significant inverse correlation between the level of Cbp expression and the extent of lymph node metastasis in human lung cancers. These results indicate that Cbp is required for the Csk-mediated inactivation of c-Src and may control the promotion of malignancy in NSCLC tumors that are characterized by c-Src upregulation.     

Synergistic promotion of c-Src activation and cell migration by Cas and AND-34/BCAR3

The adapter molecule p130Cas (Cas) plays a role in cellular processes such as proliferation, survival, cell adhesion, and migration. The ability of Cas to promote migration has been shown to be dependent upon its carboxyl terminus, which contains a bipartite binding site for the protein tyrosine kinase c-Src (Src). The association between Src and Cas enhances Src kinase activity, and like Cas, Src plays an important role in cell proliferation and migration. In this study, we show that Src and Cas function cooperatively to promote cell migration in a manner that depends upon kinase-active Src. Another carboxyl-terminal binding partner of Cas, AND-34/BCAR3 (AND-34), functions synergistically with Cas to enhance Src activation and cell migration. The carboxyl-terminal guanine nucleotide exchange factor domain of AND-34, as well as the activity of its putative target Rap1, contribute to these events. A mechanism through which AND-34 may regulate Cas-dependent cell migration is suggested by the finding that Cas becomes redistributed from focal adhesions to lamellipodia located at the leading edge of AND-34 overexpressing cells. These data thus provide insight into how Cas and AND-34 may function together to stimulate Src signaling pathways and promote cell migration.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.