Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130(Cas) tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1ΔC) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130(Cas) phosphorylation. Importantly, knockdown of NRP1 or p130(Cas) or expression of either NRP1ΔC or a non-tyrosine-phosphorylatable substrate domain mutant protein (p130(Cas15F)) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130(Cas) pathway in the regulation of chemotaxis.     

p130Cas: A key signalling node in health and disease

p130Cas/breast cancer anti-oestrogen resistance 1 (BCAR1) is a member of the Cas (Crk-associated substrate) family of adaptor proteins, which have emerged as key signalling nodes capable of interactions with multiple proteins, with important regulatory roles in normal and pathological cell function. The Cas family of proteins is characterised by the presence of multiple conserved motifs for protein–protein interactions, and by extensive tyrosine and serine phosphorylations. Recent studies show that p130Cas contributes to migration, cell cycle control and apoptosis. p130Cas is essential during early embryogenesis, with a critical role in cardiovascular development. Furthermore, p130Cas has been reported to be involved in the development and progression of several human cancers. p130Cas is able to perform roles in multiple processes due to its capacity to regulate a diverse array of signalling pathways, transducing signals from growth factor receptor tyrosine kinases, non-receptor tyrosine kinases, and integrins. In this review we summarise the current understanding of the structure, function, and regulation of p130Cas, and discuss the importance of p130Cas in both physiological and pathophysiological settings, with a focus on the cardiovascular system and cancer.     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration

Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.     

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.     

Sphingosine-1-phosphate induces the association of membrane-type 1 matrix metalloproteinase with p130Cas in endothelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in sphingosine-1-phosphate(S1P)-dependent migration of endothelial cells but the underlying mechanisms remain largely unknown. Herein, we show that S1P promotes the relocalization of MT1-MMP to peripheral actin-rich membrane ruffles that is coincident with its association with the adaptor protein p130Cas at the leading edge of migrating cells. Immunoprecipitation and confocal microscopy analyses suggest that this interaction required the tyrosine phosphorylation of p130Cas and also involves S1P-dependent phosphorylation of MT1-MMP within its cytoplasmic sequence. The interaction of MT1-MMP with p130Cas at the cell periphery suggests the existence of a close interplay between pericellular proteolysis and signaling pathways involved in EC migration.     

Organization of functional domains in the docking protein p130Cas

The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.     

Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism

The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.     

SH2-containing inositol 5'-phosphatase SHIP2 associates with the p130(Cas) adapter protein and regulates cellular adhesion and spreading

In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605-18609, 1998). We describe here a novel interaction between SHIP2 and the p130(Cas) adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130(Cas) association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130(Cas). The faster-migrating form of p130(Cas) was no longer recognized by antibodies to the amino terminus of p130(Cas) and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130(Cas) was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (DeltaRV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading.     

p130Cas regulates the activity of AND-34, a novel Ral, Rap1, and R-Ras guanine nucleotide exchange factor

We previously identified a novel murine protein, AND-34, with a carboxyl-terminal domain homologous to Ras family guanine nucleotide exchange factors (GEFs), which bound to the focal adhesion docking protein p130(Cas). Work by others has implicated both the human homologue of AND-34, BCAR3, and human p130(Cas), BCAR1, in the resistance of breast cancer cells to the anti-estrogen tamoxifen. Here we report that AND-34 displays GEF activity on RalA, Rap1A, and R-Ras but not Ha-Ras GTPases in cells. In contrast to several other Ral-GEFs, the Ral GEF activity of AND-34 is not augmented by constitutively active Ha-Ras(Val-12), consistent with the absence of a detectable Ras-binding domain. Efficient binding to AND-34 required both the Src-binding domain and a flanking carboxyl-terminal region of p130(Cas). The p130(Cas)-binding site mapped to a carboxyl-terminal sequence within the AND-34 GEF domain. Overexpression of p130(Cas), but not an AND-34-binding mutant of p130(Cas), inhibited the Ral GEF activity of co-transfected AND-34. This work identifies a new potential function for p130(Cas) and a new regulatory pathway involved in the control of Ral, Rap, and R-Ras GTPases that may participate in the progression of breast cancer cells to tamoxifen resistance.     

Selective inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) identifies a central role for VEGFR-2 in human aortic endothelial cell responses to VEGF

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR-family proteins consist of VEGFR-1/Flt-1, VEGFR-2/KDR/Flk-1, and VEGFR-3/Flt-4. Among these, VEGFR-2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1-3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR-specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR-2 tyrosine kinase, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), to explore the role of VEGFR-2 in endothelial cell function. Consistent with its reported effects on VEGFR-2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR-2, but not of VEGFR-1, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR-1 and VEGFR-2, but not VEGFR-3, in the absence or presence of ZM323881. Inhibition of VEGFR-2 blocked activation of extracellular regulated-kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR-1-specific ligand, placental growth factor (PIGF). Inhibition of VEGFR-2 also perturbed VEGF-induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor-2 inhibition also reversed VEGF-stimulated phosphorylation of CrkII and its Src homology 2 (SH2)-binding protein p130Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR-2 thus blocked all VEGF-induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR-2 is critical for VEGF signaling and/or that VEGFR-2 may function in a heterodimer with VEGFR-1 in human vascular endothelial cells.     

Tyrosine Y189 in the Substrate Domain of the Adhesion Docking Protein NEDD9 Is Conserved with p130Cas Y253 and Regulates NEDD9-Mediated Migration and Focal Adhesion Dynamics

The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.     

The interaction of CFLAR with p130Cas promotes cell migration

CASP8 and FADD Like Apoptosis Regulator (CFLAR) is a key anti-apoptotic regulator for resistance to apoptosis mediated by Fas and TRAIL. In addition to its anti-apoptotic function, CFLAR is also an important mediator of tumor growth. High level of CFLAR expression correlates with a more aggressive tumor. However, the mechanism of CFLAR signaling in malignant progression is not clear. Here we report a novel CFLAR-associated protein p130Cas, which is a general regulator of cell growth and cell migration. CFLAR-p130Cas association is mediated by the DED domain of CFLAR and the SD domain of p130Cas. Immunofluorescence observation showed that CFLAR had the colocalization with p130Cas at the focal adhesion of cell membrane. CFLAR overexpression promoted p130Cas phosphorylation and the formation of focal adhesion complex. Moreover, the enhancement of cell migration induced by CFLAR overexpression was obviously inhibited by p130Cas siRNA. In silico analysis on human database suggests high expressions of CFLAR or/and p130Cas are associated with poor prognosis of patients with lung cancer. Together, our results suggest a new mechanism for CFLAR involved in tumor development via association with p130Cas.     

CrkI and p130(Cas) complex regulates the migration and invasion of prostate cancer cells

Prostate cancer metastasis is often associated with poor prognosis. The molecular coupling of the adaptor protein Crk to the docking protein p130(Cas) serves as a switch that regulates cell migration in several invasive cancer cells and Ack appears to act upstream of CrkII to modulate the cell motility. However, the precise role of Ack, Crk and p130(Cas) complex in prostate cancer migration remains unknown. In this study we examined the expression of Crk and p130(Cas) in prostate cancer cell lines, and found that CrkI and p130(Cas) protein level was higher in highly invasive PC-3M and PC-3 cell lines than in moderately invasive DU-145 cells. Upon shRNA mediated knockdown of CrkI and p130(Cas) in PC-3M cells, cell migration and invasion were significantly inhibited as analyzed by wound healing assay and transwell invasion assay. Furthermore, co-immunoprecipitation assay showed that p130(Cas) interacted with CrkI in PC-3M cells and the stability of p130(Cas) and CrkI depended on each other. AckI interacted with both CrkI and p130(Cas) and the interaction of AckI with CrkI seemed to be independent of p130(Cas) . Taken together, our results demonstrate the high expression of CrkI and p130(Cas) in invasive prostate cancer cells and the important role of CrkI/p130(Cas) complex in the migration and invasion of prostate cancer cells. These data suggest that CrkI/p130(Cas) could be exploited as potential molecular therapeutic target for prostate cancer metastasis.     

P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas -/- mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.     

A role for phosphoinositides in tyrosine phosphorylation of p125 focal adhesion kinase in rat pancreatic acini

Previous studies have shown that different agonists increase tyrosine phosphorylation of the focal adhesion related proteins p125(FAK), p130(Cas), and paxillin in different cell types and that tyrosine phosphorylation depends on the integrity of the actin cytoskeleton. Because phosphoinositides are important for the maintenance of the cytoskeleton, the role of phosphoinositides in the tyrosine phosphorylation of these proteins in response to occupancy of m3 muscarinic and CCK(A) receptors has been investigated in pancreatic acini. Addition of carbachol or CCK-8 to pancreatic acini resulted in rapid increases in the tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin. Pretreatment of pancreatic acini with LY294002 or wortmannin resulted in a concentration-dependent inhibition of tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin stimulated by carbachol or CCK-8. Carbachol- or CCK-8-stimulated tyrosine phosphorylation of these proteins was not inhibited by rapamycin, PD 98059 or SB 203580, and thus it was dissociated from the activation of p70 S6 or MAP kinases. These results indicate that m3 muscarinic and CCK(A) receptor-mediated increase in p125(FAK), p130(Cas), and paxillin tyrosine phosphorylation in pancreatic acini depends on the ability of these cells to synthesise phosphoinositides.     

Suppression of ultraviolet irradiation-induced apoptosis by overexpression of focal adhesion kinase in Madin-Darby canine kidney cells.

Focal adhesion kinase (FAK) has been implicated to play a role in suppression of apoptosis. In this study, we have demonstrated that UV irradiation induced cleavage of FAK and two of its interacting proteins Src and p130(Cas) in Madin-Darby canine kidney cells, concomitant with an increase in cell death. The cleavage of these proteins upon UV irradiation was completely inhibited by ZVAD-FMK, a broad range inhibitor of caspases, and apparently delayed by Bcl2 overexpression. To examine if FAK plays a role in suppressing UV-induced apoptosis, stable Madin-Darby canine kidney cell lines overexpressing FAK were established. Our results showed that a marked (30-40%) increase in cell survival upon UV irradiation was achieved by this strategy. In our efforts to determine the mechanism by which FAK transduces survival signals to the downstream, we found that a FAK mutant deficient in binding to phosphatidylinositol 3-kinase failed to promote cell survival. Moreover, the expression of the Src homology 3 domain of p130(Cas), which competed with endogenous p130(Cas) for FAK binding, abrogated the FAK-promoted cell survival. Together, these results suggest that the integrity of FAK and its binding to phosphatidylinositol 3-kinase and p130(Cas) are required for FAK to exert its antiapoptotic function.     

NCK and PAK participate in the signaling pathway by which vascular endothelial growth factor stimulates the assembly of focal adhesions

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.     

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.