Identification of p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130^Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130^Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130^Cas and its subsequent binding to several SH2 domains, which depended on both the p130^Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130^Cas further increased cell migration on FN and coexpression of the p130^Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130^Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130^Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.     

Expression of a phosphorylated p130Cas substrate domain attenuates the phosphatidylinositol 3‐kinase/Akt survival pathway in tamoxifen resistant breast cancer cells

Elevated expression of p130^Cas/BCAR1 (breast cancer anti estrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. Specifically, p130^Cas signaling has been associated with antiestrogen resistance, for which the mechanism is currently unknown. TAM‐R cells, which were established by long‐term exposure of estrogen (E₂)‐dependent MCF‐7 cells to tamoxifen, displayed elevated levels of total and activated p130^Cas. Here we have investigated the effects of p130^Cas inhibition on growth factor signaling in tamoxifen resistance. To inhibit p130^Cas, a phosphorylated substrate domain of p130^Cas, that acts as a dominant‐negative (DN) p130^Cas molecule by blocking signal transduction downstream of the p130^Cas substrate domain, as well as knockdown by siRNA was employed. Interference with p130^Cas signaling/expression induced morphological changes, which were consistent with a more epithelial‐like phenotype. The phenotypic reversion was accompanied by reduced migration, attenuation of the ERK and phosphatidylinositol 3‐kinase/Akt pathways, and induction of apoptosis. Apoptosis was accompanied by downregulation of the expression of the anti‐apoptotic protein Bcl‐2. Importantly, these changes re‐sensitized TAM‐R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest that targeting the product of the BCAR1 gene by a peptide which mimics the phosphorylated substrate domain may provide a new molecular avenue for treatment of antiestrogen resistant breast cancers. J. Cell. Biochem. 107: 364–375, 2009. © 2009 Wiley‐Liss, Inc.     

The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34^Arc, and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.     

Transformation Suppression by Protein Tyrosine Phosphatase 1B Requires a Functional SH3 Ligand

We have recently shown that protein tyrosine phosphatase 1B (PTP1B) associates with the docking protein p130^Cas in 3Y1 rat fibroblasts. This interaction is mediated by a proline-rich sequence on PTP1B and the SH3 domain on p130^Cas. Expression of wild-type PTP1B (WT-PTP1B), but not a catalytically competent, proline-to-alanine point mutant that cannot bind p130^Cas (PA-PTP1B), causes substantial tyrosine dephosphorylation of p130^Cas (F. Liu, D. E. Hill, and J. Chernoff, J. Biol. Chem. 271:31290–31295, 1996). Here we demonstrate that WT-, but not PA-PTP1B, inhibits transformation of rat 3Y1 fibroblasts by v-crk, -src, and -ras, but not by v-raf. These effects on transformation correlate with the phosphorylation status of p130^Cas and two proteins that are associated with p130^Cas, Paxillin and Fak. Expression of WT-PTP1B reduces formation of p130^Cas-Crk complexes and inhibits mitogen-activated protein kinase activation by Src and Crk. These data show that transformation suppression by PTP1B requires a functional SH3 ligand and suggest that p130^Cas may represent an important physiological target of PTP1B in cells.     

BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas^−/− murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance.     

Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)

The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.     

Differential Effect of the Focal Adhesion Kinase Y397F Mutant on v-Src-Stimulated Cell Invasion and Tumor Growth

Summary Upon cell adhesion to extracellular matrix proteins, focal adhesion kinase (FAK) rapidly undergoes autophosphorylation on its Tyr-397 which consequently serves as a binding site for the Src homology 2 domains of the Src family protein kinases and several other intracellular signaling molecules. In this study, we have attempted to examine the effect of the FAK Y397F mutant on v-Src-stimulated cell transformation by establishing an inducible expression of the Y397F mutant in v-Src-transformed FAK-null (FAK^−/−) mouse embryo fibroblasts. We found that the FAK Y397F mutant had both positive and negative effects on v-Src-stimulated cell transformation; it promoted v-Src-stimulated invasion, but on the other hand it inhibited the v-Src-stimulated anchorage-independent cell growth in vitro and tumor formation in vivo . The positive effect of the Y397F mutant on v-Src-stimulated invasion was correlated with an increased expression of matrix metalloproteinase-2, both of which were inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin or a dominant negative mutant of AKT, suggesting a critical role for the phosphatidylinositol 3-kinase/AKT pathway in both events. However, the expression of the Y397F mutant rendered v-Src-transformed FAK^−/− cells susceptible to anoikis, correlated with suppression on v-Src-stimulated activation of ERK and AKT. In addition, under anoikis stress, the induction of the Y397F mutant in v-Src-transformed FAK^−/− cells selectively led to a decrease in the level of p130^Cas, but not other focal adhesion proteins such as talin, vinculin, and paxillin. These results suggest that FAK may increase the susceptibility of v-Src-transformed cells to anoikis by modulating the level of p130^Cas.     

Regulation of focal adhesion and cell migration by ANKRD28-DOCK180 interaction

DOCK180 is an atypical guanine nucleotide exchange factor of Rac1 identified originally as one of the two major proteins bound to the SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130^Cas, and recruits the Crk-p130^Cas complex to focal adhesions. Recently, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and found that ANKRD28, a protein with twenty-six ankyrin domain-repeats, interacts with the SH3 domain of DOCK180. Knockdown of ANKRD28 reduced the migration velocity and altered the distribution of focal adhesion proteins such as Crk, paxillin, and p130^Cas. On the other hand, the expression of ANKRD28, p130^Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130^Cas, which paralleled the induction of multiple long cellular processes. Depletion of ELMO, another protein bound to the SH3 domain of DOCK180, also retarded cell migration, but its expression together with p130^Cas, Crk, and DOCK180 induced extensive lamellipodial protrusion around the entire circumference without 130^Cas hyperphosphorylation. These data suggest the dual modes of DOCK180-Rac regulation for cell migration.     

A p130 Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

Background  

The adaptor protein p130 ^Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.     

Naphtho[1,2-b]furan-4,5-dione inhibits MDA-MB-231 cell migration and invasion by suppressing Src-mediated signaling pathways

Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been demonstrated to display anti-cancer activity. Breast cancer is a highly malignant carcinoma and most deaths of breast cancer are caused by metastasis. In this study, we showed that NFD blocked migration and invasion of MDA-MB-231 breast cancer cells without affecting apoptosis or growth arrest. NFD caused significant block of Src kinase activity in MDA-MB-231 cells. Moreover, NFD treatment was correlated with reduced phosphorylation of FAK at Tyr 576/577, 861 and 925 sites, p130^Cas at Tyr 410, and paxillin at Tyr 118. NFD also suppressed the activation of phosphatidylinositol 3-kinase/Akt. Consistent with inhibition of these signaling pathways and invasion, NFD reduced the expression of matrix metalloproteinase-9. Furthermore, Src antagonist PP2 caused a significant decrease in the phosphorylation of FAK, p130^Cas, paxillin, and PI3K/Akt. Our findings provide evidences that NFD inhibits Src-mediated signaling pathways involved in controlling breast cancer migration and invasion, suggesting that it has a therapeutic potential in breast cancer treatment.     

Ankyrin repeat domain 28 (ANKRD28), a novel binding partner of DOCK180, promotes cell migration by regulating focal adhesion formation

DOCK180 is a guanine exchange factor of Rac1 originally identified as a protein bound to an SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130^Cas, and recruits the Crk-p130^Cas complex to focal adhesions. To understand the role of DOCK180 in cell adhesion and migration, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and identified ANKRD28, a protein that contains twenty-six ankyrin domain repeats. Knockdown of ANKRD28 by RNA interference reduced the velocity of migration of HeLa cells, suggesting that this protein plays a physiologic role in the DOCK180-Rac1 signaling pathway. Furthermore, knockdown of ANKRD28 was found to alter the distribution of focal adhesion proteins such as Crk, paxillin, and p130^Cas. On the other hand, expression of ANKRD28, p130^Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130^Cas, and impaired detachment of the cell membrane during migration. Consequently, cells expressing ANKRD28 exhibited multiple long cellular processes. ANKRD28 associated with DOCK180 in an SH3-dependent manner and competed with ELMO, another protein bound to the SH3 domain of DOCK180. In striking contrast to ANKRD28, overexpression of ELMO induced extensive lamellipodial protrusion around the entire circumference. These data suggest that ANKRD28 specifies the localization and the activity of the DOCK180-Rac1 pathway.     

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130^Cas on Tyr-410 and an increase in the abundance of the Crk·p130^Cas complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130^Cas and disrupts the Crk·p130^Cas complex. We further determined that suppression of expression of Cdc42, Ack1, p130^Cas, or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk·p130^Cas complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.Rho GTPases are a subfamily of the Ras superfamily of small signaling molecules that are widely expressed in mammalian cells (1). RhoA, Cdc42, and Rac1 are the most extensively studied members of the Rho GTPase family, and each plays a prominent and discrete role in cell migration (2, 3). Cdc42 promotes the formation of filopodia and is required to establish cell polarity (3–5); Rac1 promotes the formation of lamellipodia at the leading edge of motile cells (6), and RhoA promotes the formation of stress fibers which generate the traction forces needed to retract the cell tail and move the cell body beyond the leading edge (7, 8). Consistent with this important role in cell motility, RhoA, Cdc42, and Rac1 are often overexpressed in human tumors including breast, lung, and colon (9), and overexpression of constitutively active RhoA, Cdc42, or Rac1 increases cell migration and invasion (2, 10, 11).The spatiotemporal regulation of Rho GTPase activity is tightly controlled by three classes of proteins. Rho-specific guanine nucleotide exchange factors (RhoGEFs)2 activate Rho proteins by facilitating the exchange of GDP for GTP; Rho GTPase-activating proteins (RhoGAPs) stimulate the intrinsic rate of hydrolysis of Rho proteins, thus converting them into their inactive state; Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) compete with RhoGEFs for binding to GDP-bound Rho proteins and sequester Rho in the inactive state (12).Dbs was identified in the screen for proteins whose overexpression cause malignant growth in murine fibroblasts (13, 14). The full-length Dbs protein (proto-Dbs) is a RhoGEF family member which contains multiple recognizable domains (Fig. 1A) including a Sec14-like domain, spectrin-like repeats, a RhoGEF domain (includes a DH and PH domain), and an SH3 domain (13). The original oncogenic version of Dbs that was identified (amino acid residues 525–1097; designated onco-Dbs) contains the RhoGEF domain alone. When expressed in murine fibroblasts, the transforming and catalytic activity of Dbs is subject to autoinhibition that is mediated by the NH₂-terminal Sec14 domain (15). Although the endogenous function of Dbs is not known, recent studies suggest that Dbs and the Rac-specific exchange factor Tiam1 regulate neurotrophin-stimulated cell migration in Schwann cells through activation of Cdc42 and Rac1, respectively (16, 17).Open in a separate windowFIGURE 1.Onco-Dbs and proto-Dbs induce cell migration in tumor-derived breast epithelial cells. A, domain structure of the onco-Dbs and proto-Dbs proteins (Sec14 = Sec14-like domain; Spec = Spectrin-like repeats; DH = Dbl homology domain; PH = pleckstrin homology domain; SH3 = Src homology 3 domain). B, stable expression of HA-epitope-tagged onco-Dbs (M_r = 65) and proto-Dbs (M_r = 129 kDa) was confirmed by Western blot using an anti-HA antibody. Three independent sets of cell lines were generated. C, T47D cells stably expressing vector (Vec), onco-Dbs, or proto-Dbs were compared in a transwell motility assay on filters pre-coated with collagen I. The motility of cells stably expressing onco-Dbs or proto-Dbs is expressed relative to that of cells stably expressing vector. Data are represented as the mean ± S.D. of three independent experiments performed in triplicate. D, T47D cells stably expressing vector, onco-Dbs, or proto-Dbs were cultured to monolayer on dishes coated with poly-l-lysine or collagen I, as indicated. Cells were serum-starved overnight, and then the surface of the plate was scraped. Migration of cells at the wound edge was monitored and photographed at 18 h. Representative images are shown. E, growth curves of T47D cells stably expressing vector, onco-Dbs, or proto-Dbs. Cells were cultured in triplicate on poly-l-lysine (filled symbols) or on dishes pre-coated with collagen I (open symbols) and counted on the indicated days. Data shown are representative of three independent experiments.Conversion of Rho proteins to their active GTP-bound state allows them to interact with effector signaling molecules. Ack1 is a nonreceptor-tyrosine kinase that binds to active Cdc42 but not Rac1 or RhoA (18, 19). Activated Ack1 is overexpressed in primary tumors and cancer cell lines and has been implicated in cancer metastasis (20). Recent studies have identified a signaling complex that regulates the motility of human breast epithelial cells that contains Cdc42, Ack1, p130^Cas, and Crk (21). Ack1 and p130^Cas interact through their respective SH3 domains, and Ack1 phosphorylates p130^Cas in a collagen I-dependent manner. p130^Cas was first identified as a hyperphosphorylated adapter protein in cells transformed by v-Src and v-Crk (22, 23). Further studies showed that p130^Cas is associated with both cellular Src and Crk in a tyrosine phosphorylation-dependent manner (24, 25). Focal adhesion kinase (FAK) binds to the NH₂ terminus of p130^Cas and phosphorylates the COOH terminus in a region that is involved in p130^Cas binding to Src (26). The binding of Crk to p130^Cas recruits binding partners to the SH3 domain of Crk, including C3G and DOCK180, which activate Rap1 and Rac1, respectively (27–31). Thus, formation of the Crk·p130^Cas complex is considered to be a molecular switch that can induce cell migration by activating Rac1 (32).Here we show that both proto-Dbs and onco-Dbs increase cell migration in human breast adenocarcinoma cells in a collagen I-dependent manner. Increased motility is dependent upon the activation of Rac1 and Cdc42 and is mediated by the assembly of Crk·p130^Cas complexes. Suppression of endogenous Dbs expression in human tumor-derived breast epithelial cells limits cell motility, suggesting that Dbs may be a critical regulator of cell behavior in breast cancer.     

The 1.1 A resolution crystal structure of the p130cas SH3 domain and ramifications for ligand selectivity

The Crk-associated tyrosine kinase substrate p130cas (CAS) is a docking protein containing an SH3 domain near its N terminus, followed by a short proline-rich segment, a large central substrate domain composed of 15 repeats of the four amino acid sequence YxxP, a serine-rich region and a carboxy-terminal domain, which possesses consensus binding sites for the SH2 and SH3 domains of Src (YDYV and RPLPSPP, respectively). The SH3 domain of CAS mediates its interaction with several proteins involved in signaling pathways such as focal adhesion kinase (FAK), tyrosine phosphatases PTP1B and PTP-PEST, and the guanine nucleotide exchange factor C3G. As a homolog of the corresponding Src docking domain, the CAS SH3 domain binds to proline-rich sequences (PxxP) of its interacting partners that can adopt a polyproline type II helix. We have determined a high-resolution X-ray structure of the recombinant human CAS SH3 domain. The domain, residues 1-69, crystallized in two related space groups, P2(1) and C222(1), that provided diffraction data to 1.1 A and 2.1 A, respectively. The crystal structure shows, in addition to the conserved SH3 domain architecture, the way in which the CAS characteristic amino acids form an atypically charged ligand-binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by the CAS SH3 domain. The structure enables modelling of the docking interactions to its ligands, for example from focal adhesion kinase, and supports structure-based drug design of inhibitors of the CAS-FAK interaction.     

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor α1‐PDX (α1‐antitrypsin Portland variant), leading to the cell‐surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen‐activated protein kinase). This outside‐in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal‐adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3‐kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up‐regulation of β1 integrins and to their location in larger focal‐adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.     

Investigation of Binding Phenomenon of NSP3 and p130Cas Mutants and Their Effect on Cell Signalling

Members of the novel SH2-containing protein (NSP3) and Crk-associated substrate (p130Cas) protein families form a multi-domain signalling platforms that mediate cell signalling process. We analysed the damaging consequences of three mutations, each from NSP3 (NSP3^L469R, NSP3^L623E, NSP3^R627E) and p130Cas (p130Cas^F794R, p130Cas^L787E, p130Cas^D797R) protein with respect to their native biological partners. Mutations depicted notable loss in interaction affinity towards their corresponding biological partners. NSP3^L469R and p130Cas^D797R mutations were predicted as most prominent in docking analysis. Molecular dynamics (MD) studies were conducted to evaluate structural consequences of most prominent mutation in NSP3 and p130Cas obtained from the docking analysis. MD analysis confirmed that mutation in NSP3^L469R and p130Cas^D797R showed significant structural deviation, changes in conformations and increased flexibility, which in turn affected the binding affinity with their biological partners. Moreover, the root mean square fluctuation has indicated a rise in fluctuation of residues involved in moderate interaction acquired between the NSP3 and p130Cas. It has significantly affected the binding interaction in mutant complexes. The results obtained in this work present a detailed overview of molecular mechanisms involved in the loss of cell signalling associated with NSP3 and p130Cas protein.     

BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130^Cas (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.     

PAF-Stimulated Protein Tyrosine Phosphorylation in Hippocampus: Involvement of NO Synthase

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat hippocampal slices. PAF caused an increase in the tyrosine phosphorylation of two phosphoproteins, which we identified by immunoprecipitation assays as the focal adhesion kinase p125^FAK and crk-associated substrate p130^Cas. The PAF effect was time- and dose-dependent. In addition, the involvement of PAF receptor was demonstrated by using PCA-4248, a specific receptor antagonist. When NO synthase was inhibited by N^G-monomethyl-L-arginine (L-NMA), PAF-stimulated protein tyrosine phosphorylation was inhibited. In conclusion, our results indicate that PAF increased the tyrosine phosphorylation of both p125^FAK and p130^Cas proteins by the production of NO in hippocampus, suggesting that PAF may play a role in the functioning of this cerebral area.     

Tumor Necrosis Factor-α (TNFα)-induced Ceramide Generation via Ceramide Synthases Regulates Loss of Focal Adhesion Kinase (FAK) and Programmed Cell Death

Ceramide synthases (CerS1–CerS6), which catalyze the N-acylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide in both the de novo and the salvage or recycling pathway of ceramide generation, have been implicated in the control of programmed cell death. However, the regulation of the de novo pathway compared with the salvage pathway is not fully understood. In the current study, we have found that late accumulation of multiple ceramide and dihydroceramide species in MCF-7 cells treated with TNFα occurred by up-regulation of both pathways of ceramide synthesis. Nevertheless, fumonisin B1 but not myriocin was able to protect from TNFα-induced cell death, suggesting that ceramide synthase activity is crucial for the progression of cell death and that the pool of ceramide involved derives from the salvage pathway rather than de novo biosynthesis. Furthermore, compared with control cells, TNFα-treated cells exhibited reduced focal adhesion kinase and subsequent plasma membrane permeabilization, which was blocked exclusively by fumonisin B1. In addition, exogenously added C6-ceramide mimicked the effects of TNFα that lead to cell death, which were inhibited by fumonisin B1. Knockdown of individual ceramide synthases identified CerS6 and its product C16-ceramide as the ceramide synthase isoform essential for the regulation of cell death. In summary, our data suggest a novel role for CerS6/C16-ceramide as an upstream effector of the loss of focal adhesion protein and plasma membrane permeabilization, via the activation of caspase-7, and identify the salvage pathway as the critical mechanism of ceramide generation that controls cell death.