Inhibition of focal adhesion kinase expression correlates with changes in the cytoskeleton but not apoptosis in primary cultures of chick embryo cells.

Cell adhesion to the extracellular matrix through integrin receptors can activate signaling cascades within the cell. Focal adhesion kinase (FAK) is a protein tyrosine kinase activated by integrin adhesion. The role of FAK within the cell is not clear, although evidence suggests roles in cell motility or the regulation of adhesion-dependent cell survival. We have treated primary cultures of chick embryo cells with antisense oligonucleotides to FAK to reduce the level of FAK protein expression. Levels of the related protein, proline-rich tyrosine kinase 2 (Pyk2) and the FAK substrate paxillin, were unaffected by the addition of oligonucleotides, whereas FAK expression was reduced by 70%. Levels of apoptotic cell death did not significantly increase after the addition of oligonucleotides. However, there was a change in the distribution of focal adhesion sites from a uniformly distributed pattern to a mainly peripheral pattern. This was accompanied by a loss of stress fibers and an increase in the peripheral actin cytoskeleton, as the cells became rounded. These results suggest that in these early embryonic cells, FAK expression regulates the arrangement of focal adhesions and the cytoskeleton that result in a motile phenotype, but that FAK does not appear to regulate apoptosis.

Comparison between the in vivo and in vitro expression of three adhesion-signaling proteins in embryonic cells.

We have examined the relationship between the in vivo and in vitro expression of three adhesion-signaling proteins (FAK, PYK2 and Paxillin), using cells of the early chick embryo, where pure cell populations may be isolated and cultured, and in which epithelial-to-mesenchymal transformation is occurring. Focal Adhesion Kinase (FAK) and Proline-rich Tyrosine Kinase-2 (PYK2) are related in molecular structure, and may have some overlapping functions in signal transduction associated with cell-substratum adhesion. Paxillin, a cytoskeletal protein, is also localized to focal adhesions. We show that the immunocytochemical detection of these molecules in vivo does not reflect their in vitro localization. Focal Adhesion Kinase showed a developmentally regulated localization to the cytoplasm, but not to sites of adhesion, in epithelial cells in vivo, while Paxillin was associated with migrating mesoderm cells. Proline-rich Tyrosine Kinase-2 was undetectable in vivo. The level of expression of these molecules was compared under in vivo and in vitro conditions. While the expression of Focal Adhesion Kinase showed a tissue-specific regulation of expression with the change to in vitro conditions, Proline-rich Tyrosine Kinase-2 showed a more uniform and less tissue-specific up-regulation. Levels of Paxillin expression also showed an increase with this change in conditions. We conclude that despite the structural and functional relationships between these three molecules in the developing embryo, the expression and localization of each is independently regulated. We suggest that this provides these cells with the adaptability that they require in order to respond to the changing extracellular environment in the early embryo, and to undergo epithelial-to-mesenchymal transformation.

Potential roles for focal adhesion kinase in development.

Focal adhesion kinase (pp125FAK or FAK) is a protein tyrosine kinase which is associated with intracellular signalling cascades which are initiated when the integrin family of cell adhesion molecules engage extracellular matrix molecules. In cultured cells, this molecule is physically associated with focal adhesions, which are well-defined regions of intimate cell-to-substratum adhesion. In this location, it interacts with other proteins of the focal adhesion to activate intracellular signalling events associated with cell adhesion. The in vitro expression of FAK and its level of phosphorylation appear to be related to several physiological phenomena, including cell spreading, cell differentiation, cell locomotion and cell death. Because these phenomena are all of critical importance during morphogenesis, and because FAK is expressed in embryonic cells, evidence has been accumulating to indicate that FAK may be an important modulator of developmental processes. In this review, this evidence is surveyed together with evidence from analogous situations, such as tumour cell migration and invasiveness. Although evidence suggesting a role for FAK in morphogenesis is accumulating, current uncertainties regarding its cytoplasmic location and its molecular interactions in vivo make it difficult to reach definitive conclusions regarding the significance of its contributions to developmental processes.

Cellular phenotype transformation during early embryogenesis: a role for focal adhesion kinase?

We have used the gastrulating chick embryo as a model for studying the potential role of focal adhesion kinase (FAK) in phenotypic transformation. In the gastrulating embryo, there is a well-defined epithelial to mesenchymal transformation as the upper epithelial epiblast layer of cells ingresses at the primitive streak to form the invasive mesenchymal mesoderm layer and the epithelioid endoderm layer. Immunolocalization showed that FAK was expressed primarily in the apical cytoplasm of the epiblast layer, together with some regions of the mesoderm and endoderm. Hensen's node and the primitive streak, where the transformation occurs, showed very low immunoreactivity. Levels of FAK in these individual tissues were quantified by densitometric analysis of Western blots, and FAK activation was quantified by stripping these blots and reprobing for phosphotyrosine. Immunoprecipitation indicated that the phosphotyrosine bands corresponded with the FAK bands on the blots. Although the blots confirmed that FAK was highly expressed in the epiblast, the level of FAK activation was highest in the endoderm, despite relatively low expression of the protein, Similar quantitative blotting was carried out using cells from each of the three layers cultured on different substrata. The results indicated that cells cultures on fibronectin, laminin, and Matrigel expressed differing levels of FAK, with differing levels of tyrosine phosphorylation, depending on the cell type and the substratum. We conclude that FAK is developmentally regulated during gastrulation, and that this regulation could be influenced by the changing substratum encountered by the differentiating cells during this process. However, the apical localization of FAK in much of the epiblast appears to preclude a consistent focal contact-like association of this molecule with integrins in vivo, and we therefore suggest that in the embryo, FAK may be involved in integrin-mediated signalling pathways without physical association with cell-substratum contacts.