v-Src rescues actin-based cytoskeletal architecture and cell motility and induces enhanced anchorage independence during oncogenic transformation of focal adhesion kinase-null fibroblasts.
The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, 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 Src-induced 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 Src-induced 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.
D000199:Actins / Q000378:metabolism*; D000483:Alleles; D000818:Animals; D002448:Cell Adhesion; D002455:Cell Division; D002465:Cell Movement; D002469:Cell Separation; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016158:Genes, p53 / Q000235:genetics; D016391:Genes, src / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D015688:Oncogene Protein pp60(v-src) / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D034622:RNA Interference; D015398:Signal Transduction; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D014945:Wound Healing
D000199:Actins; D005353:Fibronectins; D016159:Tumor Suppressor Protein p53; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); C496051:Ptk2 protein, mouse; C496053:Ptk2b protein, mouse