Involvement of Cell-Cell Interactions in the Rapid Stimulation of Cas Tyrosine Phosphorylation and Src Kinase Activity by Transforming Growth Factor-β1

Abstract

Transforming growth factor-␤ (TGF-␤) regulates a wide range of physiological and pathological cellular processes, including cell migration, mesenchymal transition, extracellular matrix synthesis, and cell death. Cas (Crk-associated substrate, 130 kDa), an adaptor protein localized at focal adhesions and stress fibers, is also known to have important functions in cell migration and the induction of immediate-early gene expression. Here, we report that a rapid and transient tyrosine phosphorylation of Cas is induced by TGF-␤1 and that E-cadherin-mediated cell-cell interaction and the Src kinase pathway are involved in this early TGF-␤ signaling. The addition of TGF-␤1 to epithelial cells rapidly induced tyrosine phosphorylation of Cas and promoted the formation of complexes between focal adhesion molecules. Cas phosphorylation required the integrity of the actin cytoskeleton but was not dependent on cell adhesion, implying that Cas-dependent signaling may be distinct from integrin signaling. TGF-␤1 also stimulated Src kinase activity, and specific inhibitors of Src completely blocked the induction of Cas phosphorylation by TGF-␤1. The Cas phosphorylation and Src kinase activation seen in our results were induced in an epithelial phenotype-specific manner. Stable transfection of E-cadherin to L929 cells and L cells as well as E-cadherin blocking assay revealed that E-cadherin-mediated cellcell interactions were essential for both Cas phosphorylation and Src kinase activation. Taken together, our data suggest that rapid Cas phosphorylation and Src kinase activation may play a novel role in TGF-␤ signal transduction. Transforming growth factor-␤ (TGF-␤)1 regulates a wide range of physiological and pathological cellular processes, including differentiation, immune response, inflammation, extracellular matrix synthesis, angiogenesis, and wound healing in humans (1-3). In epithelial and endothelial cells, TGF-␤ strongly inhibits cell growth (4, 5) and, in fibroblasts, acts in both growth stimulatory and growth inhibitory manners depending on the stage of differentiation and culture conditions (6, 7). TGF-␤ exhibits a tumor suppressor activity, and components of its signaling pathway are frequently mutated or silenced in colon and pancreatic cancers (8). However, accumulating data indicate that TGF-␤ can positively affect tumorigenesis and contribute to the progression and invasiveness of tumors (9 -11). This tumor-activating activity of TGF-␤ is associated with its ability to induce an epithelial to mesenchymal transition and stimulate cell migration (12). The epithelial to mesenchymal transition induced by TGF-␤ results in the disruption of the polarized morphology of epithelial cells, the formation of stress fibers, and an enhancement of cell migration (11, 13-15).The cell migration and reorganization of the actin cytoskeleton induced by many extracellular factors are accompanied by dramatic changes in the tyrosine phosphorylation of several signaling proteins localized at the focal adhesion plaques (16,17). The rapid...

Ligand release-independent transactivation of epidermal growth factor receptor by transforming growth factor-β involves multiple signaling pathways

“…EGFR transactivation then induces c-Fos/c-Jun expression via the ERK signaling cascade. E-cadherin-mediated cell-cell interactions are also required for this event, similar with the effect of TGF-b on c-Src activation in epithelial cells, previously reported by us (Kim and Joo, 2002).…”

“…Cell-cell interactions via E-cadherin involve extracellular binding and intracellular anchorage to the actin-based cytoskeleton (Takeichi, 1991). Moreover, we showed previously that E-cadherin expression and cell-cell interaction mediated by it are important for TGF-b-induced c-Src activation (Kim and Joo, 2002). Hence, we investigated the effects of E-cadherin expression and cell-cell interactions mediated by it on EGFR transactivation.…”

“…We demonstrated previously that TGF-b induces rapid and transient activation of c-Src in HaCaT cells (Kim and Joo, 2002), implicating increased tyrosine phosphorylation of multiple proteins. To investigate whether TGF-b1 induces tyrosine phosphorylation of multiple proteins in HaCaT cells, serum-starved cells were stimulated with 4 ng/ml of TGF-b1 for various periods, lysed and analysed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with anti-phosphotyrosine monoclonal antibody (mAb) (PY20).…”

Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

“…Accumulating data indicate that these pathways have been implicated in a number of phenomena associated with tumor promotion (6). The Src kinase pathway is involved in this early TGF-␤ signaling (3,7). The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-␤ receptors (8).…”

“…One of the factors regulating the metastatic process is considered to be transforming growth factor-␤ (TGF-␤), which is a multifunctional cytokine that elicits numerous cellular effects pertinent to the metastatic process (1). TGF-␤ regulates a wide range of physiological and pathological cellular processes, including cell growth, differentiation, invasion, migration, mesenchymal transition, extracellular matrix synthesis, and cell death in many cell types including ovarian cancer cells (3). Recent data demonstrated that TGF-␤ specifically stimulates up-regulation of uPA mRNA and protein in certain types of neoplastic cells (1).…”