v-Src Rescues Actin-based Cytoskeletal Architecture and Cell Motility and Induces Enhanced Anchorage Independence during Oncogenic Transformation of Focal Adhesion Kinase-null Fibroblasts

• Published in: The Journal of biological chemistry 2003-11, Vol.278 (48), p.47946-47959
• Main Authors: Moissoglu, Konstadinos, Gelman, Irwin H
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Alleles; Animals; Cell Adhesion; Cell Division; Cell Movement; Cell Separation; Cell Transformation, Neoplastic; Cells, Cultured; Crk protein; Cytoskeleton - metabolism; Fibroblasts - metabolism; Fibronectins - metabolism; Flow Cytometry; focal adhesion kinase; Focal Adhesion Kinase 1; Focal Adhesion Kinase 2; Focal Adhesion Protein-Tyrosine Kinases; Genes, p53 - genetics; Genes, src - genetics; growth factor receptors; MEF protein; Mice; Microscopy, Fluorescence; Oncogene Protein pp60(v-src) - physiology; Phosphorylation; Precipitin Tests; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Protein-Tyrosine Kinases - physiology; RNA Interference; Signal Transduction; Temperature; Time Factors; Transfection; Tumor Suppressor Protein p53 - metabolism; Tyrosine - chemistry; Tyrosine - metabolism; v-Src gene; Wound Healing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M302720200

The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr 397 , a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Srcinduced pathways, especially as they relate to oncogenic transformation, remain unclear. Here, we investigate the role of FAK in v-Src-induced oncogenic transformation by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK–/– mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induced abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK–/– MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescued the haptotactic, linear directional, and invasive motility defects of FAK–/– cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there was an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK–/– MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK–/–[ts72v-Src] clones exhibited 7–10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK–/–[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Srcinduced anchorage independence. Our data also indicate that the enhanced Pyk2 level found in FAK–/– MEF plays no role in v-Src-induced anchorage independence. Overall, our data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways.