Brunton VG, Avizienyte E, Fincham VJ, Serrels B, Metcalf III CA, Sawyer TK, Frame MCIdentification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior. Cancer Res 65: 1335-1342
ABSTRACT Src tyrosine kinase expression and activity are elevated during colon cancer progression. How this contributes to the malignant phenotype is not fully understood. We show that in KM12C colon carcinoma cells, expression of kinase-deficient Src proteins (SrcMF and Src251) does not alter cell growth. Src kinase activity is required for turnover of cell-matrix adhesions and, in particular, the Src-dependent phosphorylation of focal adhesion kinase (FAK) is required for their disassembly. Surprisingly, we found that expression of SrcMF or Src251 resulted in increased tyrosine phosphorylation of FAK on Tyr(407), Tyr(576), Tyr(577), and Tyr(861), which are considered to be Src kinase substrates. This Src kinase-independent phosphorylation of FAK required an intact Src SH2 domain that mediates association of Src and FAK at peripheral adhesions. Use of a novel highly potent and selective Src kinase inhibitor AP23464 combined with experiments in Src/Fyn/Yes-deficient fibroblasts showed that increased phosphorylation of FAK in cells expressing SrcMF did not require Src-like kinases. However, specific phosphorylation on Tyr(925) of FAK was not evident in SrcMF- or Src251-expressing cells, and lack of Src kinase-dependent phosphorylation on this site was associated with impaired adhesion turnover. Our data show that Src kinase activity is required for adhesion turnover associated with cell migration in cancer cells and that, in addition to the catalytic activity, Src also acts as an adaptor to recruit other kinases that can phosphorylate key substrates including FAK. These studies have implications for tumor progression with respect to the use of Src kinase inhibitors.
- SourceAvailable from: Renato Socodato
[Show abstract] [Hide abstract]
- "Stably transfected (st.) subclones were confirmed by western blot for all constructs: shCsk st. 5 97% reduction in Csk; shSrc st. 5 89% reduction in c- Src; Src CA st. 5 76% increase in c-Src activity; Src KD st. 5 42% decrease in c-Src activity. c-Src expression was measures by western blotting using an antibody that only recognizes c-Src while c-Src activity was measured in both cell lines by western blotting using an anti-FAK pTyr, 925 which represent a direct target for c-Src among other SFKs (Brunton et al., 2005; Socodato et al., 2012). "
ABSTRACT: Microglial cells are the resident macrophages of the central nervous system (CNS). Their function is essential for neuronal tissue homeostasis. After inflammatory stimuli, microglial cells become activated changing from a resting and highly ramified cell shape to an amoeboid-like morphology. These morphological changes are associated with the release of pro-inflammatory cytokines and glutamate, as well as with high phagocytic activity. The acquisition of such phenotype has been associated with activation of cytoplasmic tyrosine kinases, including those of the Src family (SFKs). In this study, using both in vivo and in vitro inflammation models coupled to FRET-based time-lapse microscopy, lentiviruses-mediated shRNA delivery and genetic gain-of-function experiments, we demonstrate that among SFKs c-Src function is necessary and sufficient for triggering microglia pro-inflammatory signature, glutamate release, microglia-induced neuronal loss and phagocytosis. c-Src inhibition in retinal neuroinflammation experimental paradigms consisting of intravitreal injection of LPS or ischemia-reperfusion injury significantly reduced microglia activation changing their morphology to a more resting phenotype and prevented neuronal apoptosis. Our data demonstrate an essential role for c-Src in microglial cell activation.Glia 03/2015; 63(3). DOI:10.1002/glia.22767 · 6.03 Impact Factor
[Show abstract] [Hide abstract]
- "The overexpression, deregulation or mutation of c-Src associated with alterations in its activity have been observed in various cancer types, such as colon, breast, and pancreatic types  . The oncogenic potential of an increased Src activity involves the control of cell proliferation and the regulation of cytoskeletal-linked events, such as migration, spreading, and invasion    . Considerable evidences also suggest that c-Src kinase inhibition may enhance the anti-tumor efficacy of hormonal and cytotoxic agents in preclinical models   . "
ABSTRACT: The main idea of this work was to find predictive Quantitative Structure-Activity Relationships (QSAR) for a wide set of c-Src Tyrosine Kinase inhibitors, by means of resorting to a conformation-independent representation of the chemical structure. In this way, our attempt was to avoid the availability of X-ray crystallographic structural information of the target. Therefore, in a set composed of 80 pyrrolo-pyrimidine derivatives, 1179 theoretical descriptors were simultaneously analyzed through linear regression models obtained with the Replacement Method variable subset selection technique. Alternatively, the flexible (activity dependent) descriptor approach was also applied in this study. The models were validated and tested through the use of an external test set of compounds, the Leave-Group-Out Cross Validation method, Y-Randomization and Applicability Domain analysis. Our results were compared with previously published ones based on Docking Analysis and 3D-QSAR. The obtained conformation-independent approach was in good agreement with experimental observations.Chemometrics and Intelligent Laboratory Systems 05/2014; 134. DOI:10.1016/j.chemolab.2014.03.003 · 2.32 Impact Factor
[Show abstract] [Hide abstract]
- "Emerging evidence supports the important role for FAK in the endothelial cell motility response . Since in cancer cells specific Src-dependent phosphorylation of FAK Y925 is associated with the ability of cells to dynamically regulate cell adhesions , we next investigated the phosphorylation state of FAK in endothelial cells silenced for DAB2. We performed immunoprecipitation analyses of cell extracts from quiescent cells by using anti-Y925 FAK antibodies. "
ABSTRACT: Disabled-2 (DAB2) is an adaptor protein implicated in signal transduction pathways and in protein traffic regulation. Here, we show that DAB2 is highly expressed in human endothelial cells. DAB2 silencing in endothelial cells by lentiviral-mediated small hairpin RNA expression affects cell migration and differentiation into capillary-like structures while increasing cell proliferation and viability. DAB2 knockdown causes activation of the Src-FAK signal pathway, extracellular-signal regulated kinase and c-Jun NH2-terminal kinase activation, and inhibition of p38 phosphorylation. In DAB2 silenced endothelial cells, pharmacological inhibition of Src with its specific inhibitor PP2 abolishes focal adhesion kinase activation and restores differentiation of endothelial cells. These results suggest that DAB2, via Src and focal adhesion signaling, plays a role in human endothelial cell function.FEBS Letters 08/2008; 582(17):2542-8. DOI:10.1016/j.febslet.2008.06.025 · 3.34 Impact Factor