Wu L, Bernard-Trifilo JA, Lim Y, Lim ST, Mitra SK, Uryu S et al.. Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility. Oncogene 27: 1439-1448

Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA.
Oncogene (Impact Factor: 8.46). 03/2008; 27(10):1439-48. DOI: 10.1038/sj.onc.1210770
Source: PubMed


Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that alpha4 and alpha5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either alpha4beta1- or alpha5beta1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that alpha5beta1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for alpha5beta1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, alpha4beta1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatase-alpha overexpression inhibited alpha4beta1-stimulated NB motility and Src activation consistent with alpha4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In alpha4 shRNA-expressing NB cells, alpha4beta1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated alpha4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that alpha4beta1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during alpha5beta1-mediated NB migration and support the evaluation of inhibitors to alpha4, Src and FAK in the control of NB tumor progression.

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Available from: Dwayne G Stupack, Mar 11, 2014
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    • "We found FAK inhibitor and c-Src pretreatment or transfection with c-Src siRNA potently abolishing CCN1-mediated OSM expression, portending FAK-dependent c-Src signal pathway involved in CCN1-induced OSM expression. FAK-dependent c-Src activation is implicated in cell motility, cell cycle, or inhibited neuroblastoma progression [28], [52]. Studies showed this pathway regulating human epithelial cell survival and anoikis [53]. "
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    ABSTRACT: Inflammatory response and articular destruction are common symptoms of osteoarthritis. Cysteine-rich 61 (CCN1 or Cyr61), a secreted protein from the CCN family, is associated with the extracellular matrix involved in many cellular activities like growth and differentiation. Yet the mechanism of CCN1 interacting with arthritic inflammatory response is unclear. This study finds CCN1 increasing expression of oncostatin m (OSM) in human osteoblastic cells. Pretreatment of αvβ3 monoclonal antibody and inhibitors of focal adhesion kinase (FAK), c-Src, phosphatidylinositol 3-kinase (PI3K), and NF-κB inhibited CCN1-induced OSM expression in osteoblastic cells. Stimulation of cells with CCN1 increased phosphorylation of FAK, c-Src, PI3K, and NF-κB via αvβ3 receptor; CCN1 treatment of osteoblasts increased NF-κB-luciferase activity and p65 binding to NF-κB element on OSM promoter. Results indicate CCN1 heightening OSM expression via αvβ3 receptor, FAK, c-Src, PI3K, and NF-κB signal pathway in osteoblastic cells, suggesting CCN1 as a novel target in arthritis treatment.
    PLoS ONE 09/2014; 9(9):e106632. DOI:10.1371/journal.pone.0106632 · 3.23 Impact Factor
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    • "Previous studies provided evidence that Src, a member of the Src-family-kinases (SFKs), is often associated with ephrin- B ligands and mediates reverse signaling (Palmer et al., 2002; Zimmer et al., 2011). In addition, FAK and Src often act in a complex in which FAK becomes autophosphorylated at Tyr397 and then binds and activates Src (Mitra et al., 2005; Wu et al., 2008). We found that the levels of phosphorylated Src and FAK (pSrc and pFAK) are regulated in striatal and cortical neurons differentially. "
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    ABSTRACT: During embryonic development the preoptic area (POA) gives rise to two populations of neurons which are generated at the same time, cortical interneurons and striatal cells. POA-derived cortical interneurons take a superficial path and avoid the developing striatum (Str) when they migrate to their target region. We found that EphB1, which is expressed in the striatal anlage, prevents cortical interneurons from entering the Str via ephrin-B3 reverse signaling. In contrast, for striatal neurons which also express ephrin-B3, EphB1 acts as a stop signal. This dual role of EphB1 is due to differences in ephrin-B3 reverse signaling cascades. For striatal neurons, binding of EphB1 to ephrin-B3 reduces endogenously high levels of pSrc and pFAK, which then causes the cells to stop migration. In contrast, in cortical interneurons EphB1-ephrin-B3 reverse signaling leads to phosphorylation of Src and focal adhesion kinase (FAK) which then mediates repulsion. Consistent with these in vitro findings, in an ephrin-B3 knockout mouse line, we discovered misrouted cortical interneurons in the Str and an over-migration of striatal neurons in their target region. Thus, EphB1/ephrin-B3 reverse signaling has a different impact on two sets of neurons which are generated at the same time and place: it can act as a repulsive cue for migrating neurons or it can terminate neuronal migration, a novel role of the Eph/ephrin system.
    Frontiers in Cellular Neuroscience 07/2014; 8. DOI:10.3389/fncel.2014.00185 · 4.29 Impact Factor
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    • "Beads were washed three times in cell lysis buffer and twice in kinase buffer (20 mM Tris HCl, pH 7.5, 200 mM NaCl, 0.5 mM Na 3 VO 4 , 25 mM MgCl 2 , 5 mM MnCl 2 , 1 mM EDTA, and 5 mM -mercaptoethanol). Kinase assays were initiated by 200 µM ATP addition with 2 µg GST fusion protein, 250 ng of purified recombinant FAK domain, or 50 ng of purified full-length His-tagged c-Src from baculovirus (Wu et al., 2008) in the presence of 0.5 µM PF-271 or DMSO for 15 min at 32°C. Reactions were stopped by the addition of SDS sample buffer for immunoblotting. "
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    ABSTRACT: Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.
    The Journal of Cell Biology 01/2014; 204(2):247-63. DOI:10.1083/jcb.201307067 · 9.83 Impact Factor
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