Disruption of Ca2+-dependent cell-matrix adhesion enhances c-Src kinase activity, but causes dissociation of the c-Src/FAK complex and dephosphorylation of tyrosine-577 of FAK in carcinoma cells

The nonreceptor tyrosine kinase c-Src is activated in most invasive cancers. Activated c-Src binds to FAK in the focal adhesion complex, resulting in the activation of the c-Src/FAK signaling cascade, which regulates cytoskeletal functions. However, the mechanisms by which c-Src/FAK signaling is regulated during conditions of anchorage-independent growth, a hallmark of tumor progression, are not clearly known. Here, an in vivo approach to measure c-Src activity was studied using phospho-specific antibodies against phosphorylated Y418 of c-Src (Src[pY418]), an autophosphorylation site of c-Src, and phosphorylated Y577 of FAK (FAK[pY577]), a known substrate of c-Src. Using genetic and pharmacological approaches to modulate c-Src activity, we showed that the levels of Src[pY418] and FAK[pY577], and the formation of a c-Src/FAK[pY577] complex correlated with the activation state of c-Src in adherent cells. Interestingly, both the in vivo level of Src[pY418] and in vitro c-Src kinase activity were increased in carcinoma cells following disruption of Ca(2+)-dependent cell-matrix adhesion. In contrast, the level of FAK[pY577] and its association with c-Src were reduced in suspended cells. The amount of FAK[pY577] in suspended cells was recovered following attachment of rounded cells to fibronectin-coated polystyrene beads, indicating that cell spreading was not required for phosphorylation of FAK. Moreover, cells expressing activated c-Src showed sustained Src[Y418] phosphorylation, but required Ca(2+)-dependent cell adhesion for phosphorylation of FAK[Y577] and association of c-Src with FAK[pY577]. These findings indicate an important role of integrin-based cell-matrix adhesion in regulating c-Src/FAK signaling under decreased anchorage conditions.     

Cytoskeletal reorganization dependence of signaling by the gonadotropin-releasing hormone receptor

Activation of classical G protein-coupled receptors (GPCRs) like the mammalian gonadotropin-releasing hormone receptor (GnRHR) typically stimulates heterotrimeric G protein molecules that subsequently activate downstream effectors. Receptor activation of heterotrimeric G protein pathways primarily controls intermediary cell metabolism by elevation or diminution of soluble cytoplasmic second messenger molecules. We have demonstrated here that stimulation of the GnRHR also results in a dramatic change in both cell adhesion and superstructural morphology. Gonadotropin-releasing hormone (GnRH) receptor activation rapidly increases the capacity of HEK293 cells expressing the GnRHR to remain matrix-adherent in the face of fluid insults. Coinciding with this profound elevation in matrix adherence, we demonstrated a GnRH-induced alteration in both cell morphology and the de novo generation of polymerized actin structures. GnRH induction of cytoskeletal remodeling was correlated with significant increases in the tyrosine phosphorylation status of a series of cytoskeletal associated proteins, e.g. focal adhesion kinase (FAK), c-Src, and microtubule-associated protein kinase (MAPK or ERK1/2). The activation of the distal downstream effector ERK1/2 was demonstrated to be sensitive to the disrupters of cytoskeletal rearrangement, cytochalasin D and latrunculin B. In addition to the sensitivity of ERKs to cytoskeletal integrity, GnRH-induced FAK and c-Src kinase activation were sensitive to these agents and the fibronectin-integrin antagonistic RGDS peptide. Activation of ERK was dependent on its protein-protein assembly with FAK and c-Src at focal adhesion complexes. Induction of the cell remodeling event leading to this signaling complex assembly occurred primarily via GnRHR activation of the monomeric G protein Rac but not RhoA. These findings demonstrated a clear divergence of GnRHR signaling via the Rac monomeric G protein focal adhesion signaling complex assembly and cytoskeletal remodeling independent of the classical heterotrimeric G protein-controlled phospholipase C-beta pathway.     

Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.     

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.     

Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215

To elucidate the molecular mechanisms by which human epidermal growth factor receptor/heregulin (HER2/HRG) influence the migratory potential of breast cancer cells, we have used phospho-specific antibodies against c-Src kinase and focal adhesion kinase (FAK). This study establishes that HER2/HRG signaling selectively upregulates Tyr phosphorylation of c-Src at Tyr-215 located within the SH2 domain, increases c-Src kinase activity and selectively upregulates Tyr phosphorylation of FAK at Tyr-861. HER2-overexpressing tumors showed increased levels of c-Src phosphorylation at Tyr-215. These findings suggest that HER2/HRG influence metastasis of breast cancer cells through a novel signaling pathway involving phosphorylation of FAK tyrosine 861 via activation of c-Src tyrosine 215.     

CEACAM6 cross-linking induces caveolin-1-dependent, Src-mediated focal adhesion kinase phosphorylation in BxPC3 pancreatic adenocarcinoma cells

Despite lacking transmembrane or intracellular domains, glycosylphosphatidylinositol-anchored proteins can modulate intracellular signaling events, in many cases through aggregation within membrane "lipid raft" microdomains. CEACAM6 is a glycosylphosphatidylinositol-linked cell surface protein of importance in the anchorage-independent survival and metastasis of pancreatic adenocarcinoma cells. We examined the effects of antibody-mediated cross-linking of CEACAM6 on intracellular signaling events and anchorage-independent survival of the CEACAM6-overexpressing pancreatic ductal adenocarcinoma cell line, BxPC3. CEACAM6 cross-linking increased c-Src activation and induced tyrosine phosphorylation of p125(FAK) focal adhesion kinase. Focal adhesion kinase phosphorylation was dependent on c-Src kinase activation, for which caveolin-1 was required. CEACAM6 cross-linking induced a significant increase in cellular resistance to anoikis. These observations represent the first characterization of the mechanism through which this important cell surface oncoprotein influences intracellular signaling events and hence malignant cellular behavior.     

Integrin activation and focal complex formation in cardiac hypertrophy

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.     

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

c-Src mediates mitogenic signals and associates with cytoskeletal proteins upon vascular endothelial growth factor stimulation in Kaposi's sarcoma cells

Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi's sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.     

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.     

Serine phosphorylation of cortactin controls focal adhesion kinase activity and cell scattering induced by Helicobacter pylori

Cell migration and invasion require the coordinated regulation of cytoskeletal architectural changes by signaling factors, including the actin-binding protein cortactin. Bacterial and viral pathogens subvert these signaling factors to promote their uptake, spread and dissemination. We show that the gastric pathogen Helicobacter pylori (Hp) targets cortactin by two independent processes leading to its tyrosine dephosphorylation and serine phosphorylation to regulate cell scattering and elongation. The phosphorylation status of cortactin dictates its subcellular localization and signaling partners. Upon infection, cortactin was found to interact with and stimulate the kinase activity of focal adhesion kinase (FAK). This interaction required the SH3 domain and phosphorylation of cortactin at serine 405 and a proline-rich sequence in FAK. Using Hp as a model, this study unravels a previously unrecognized FAK activation pathway. We propose that Hp targets cortactin to protect the gastric epithelium from excessive cell lifting and ensure sustained infection in the stomach.     

Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.     

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.     

Inhibition of osteoclast function by adenovirus expressing antisense protein-tyrosine kinase 2

Osteoclast activation is initiated by adhesion to bone, cytoskeletal rearrangement, formation of the sealing zone, and formation of the polarized ruffled membrane. Previous findings suggest that protein-tyrosine kinase 2 (PYK2), a cytoplasmic kinase related to focal adhesion kinase, participates in these events. This study examines the role of PYK2 in adhesion-mediated signaling and osteoclast function, using PYK2 antisense. We produced a recombinant adenovirus containing a 300-base pair reversed 5'-coding region of PYK2 and used full-length PYK2 as a control. Murine osteoclast-like cells or their mononuclear precursors were generated in a co-culture of bone marrow and osteoblasts. Infection with antisense adenovirus significantly reduced the expression of endogenous PYK2 protein relative to uninfected cells or to cells infected with sense PYK2 and caused: 1) a reduction in osteoclast formation in vitro; 2) inhibition of cell spreading and of actin ring formation in osteoclasts plated on glass or bone and of attachment and spreading of osteoclast precursors plated on vitronectin; 3) inhibition of bone resorption in vitro; 4) marked reduction in p130(Cas) tyrosine phosphorylation; and 5) no change in alpha(v)beta(3) integrin expression or c-Src tyrosine phosphorylation. Taken together, these findings support the hypothesis that PYK2 plays a central role in the adhesion-dependent cytoskeletal organization and sealing zone formation required for osteoclastic bone resorption.     

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.     

Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation.

Fluid shear stress (flow) modulates endothelial cell function via specific intracellular signaling events. Previously we showed that flow activated ERK1/2 in an integrin-dependent manner (Takahashi, M., and Berk, B. C. (1996) J. Clin. Invest. 98, 2623-2631). p130 Crk-associated substrate (Cas), a putative c-Src substrate, was originally identified as a highly phosphorylated protein that is localized to focal adhesions and acts as an adapter protein. Recent reports have shown that Cas is important in cardiovascular development and actin filament assembly. Flow (shear stress = 12 dynes/cm(2)) stimulated Cas tyrosine phosphorylation within 1 min in human umbilical vein endothelial cells. Phosphorylation peaked at 5 min (3.5 +/- 0.7-fold) and was sustained to 20 min. Tyrosine phosphorylation of Cas was functionally important because flow stimulated association of Cas with Crk in a time- and force-dependent manner. Flow-mediated activation of c-Src, phosphorylation of Cas, and association of Cas with Crk were all inhibited by calcium chelation and pretreatment with the Src family-specific tyrosine kinase inhibitor PP1. To determine the role of c-Src in flow-stimulated phosphorylation of Cas, we transduced cells with adenovirus encoding kinase-inactive Src. Expression of kinase-inactive Src prevented flow-induced Cas tyrosine phosphorylation but not ERK1/2 activation. Calcium-dependent activation of c-Src and tyrosine phosphorylation of Cas defines a new flow-stimulated signal pathway, different from ERK1/2 activation. This pathway may be involved in focal adhesion remodeling and actin filament assembly.     

Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis

The neural cell adhesion molecule (NCAM) has been reported to stimulate neuritogenesis either via nonreceptor tyrosine kinases or fibroblast growth factor (FGF) receptor. Here we show that lipid raft association of NCAM is crucial for activation of the nonreceptor tyrosine kinase pathway and induction of neurite outgrowth. Transfection of hippocampal neurons of NCAM-deficient mice revealed that of the three major NCAM isoforms only NCAM140 can act as a homophilic receptor that induces neurite outgrowth. Disruption of NCAM140 raft association either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the tyrosine focal adhesion kinase and extracellular signal-regulated kinase 1/2, completely blocking neurite outgrowth. Likewise, NCAM-triggered neurite outgrowth is also completely blocked by a specific FGF receptor inhibitor, indicating that cosignaling via raft-associated kinases and FGF receptor is essential for neuritogenesis.