Abstract
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.
Highlights
Deletion of the focal adhesion kinase (FAK) gene leads to early embryonic lethality, indicating a critical requirement for FAK in developmental processes [9]
Pyk2 overexpression induces some of the cytoskeletal rearrangements and the epithelioid morphology found in FAKϪ/Ϫ mouse embryo fibroblasts (MEF) (12, 14 –16), and at least one study suggests that Pyk2 can partially rescue activation of extracellular signal-regulated kinase (ERK)-2 in FAKϪ/Ϫ MEF
FAK Is Dispensable for Morphological Transformation and Cytoskeletal Rearrangements Induced by v-Src—To evaluate the role of FAK in v-Src-induced transformation, FAKϪ/Ϫ and FAKϩ/ϩ MEF were transduced with ecotropic retrovirus encoding ts72v-Src, whose tyrosine kinase is active enzymatically at 35 °C (PT) yet inactive at 39.5 °C (NPT)
Summary
Deletion of the FAK gene leads to early embryonic lethality, indicating a critical requirement for FAK in developmental processes [9]. It has been difficult to establish a causative role for these tyrosine phosphorylation events in specific parameters of v-Src-induced oncogenesis such as anchorage-, contact-, and mitogen-independence, metabolic changes, cytoskeletal reorganization, decreases in the number of adhesive sites, and increased motility [27]. It is assumed that FAK plays a positive role in oncogenesis based on its increased expression and activity in many metastatic tumors [32,33,34,35,36], it is unclear whether specific aspects of v-Src-induced oncogenic transformation require FAK To address this issue, we used retroviruses to transduce a temperature-sensitive v-Src allele (ts72v-Src) into otherwise genetically matched FAKϩ/ϩ and FAKϪ/Ϫ mouse embryo fibroblasts. This phenomenon was attenuated by FAK re-expression but was unaffected by either ts72v-Src or Pyk levels
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