What introduces significant complexity in understanding how phosphotyrosine signaling regulates cell adhesion is the unexpected vacillating role played by SFKs. In the simplest schema to explain how phosphotyrosine signaling could operate in two modes, one would envision that certain tyrosine kinases would function to promote cell adhesion while another group of tyrosine kinases would function to oppose it. But it appears that SFKs play both roles in a decisive way. Trask appears to be uniquely phosphorylated by SFKs as evidenced by studies with Src-selective inhibitors as well as co-transfection studies in SYF cells24,29
and therefore SFKs inhibit cell adhesion through the phosphorylation of Trask. However, SFKs are also well known central players in the formation of focal adhesion complexes, in particular in complex with activated FAK and phosphorylate many substrates at focal adhesions, stabilizing them and linking them to the actin cytoskeleton.8,35–37
This apparent dichotomous role of SFKs suggests that they may embody functions more complex than workhorse functions. The traditional view of kinases is that they are regulated at the level of activity and by turning on and off, they function to transmit signals along a pathway. The fact that SFKs can phosphorylate focal adhesion targets and can phosphorylate Trask in exclusion of one another with opposing biological affects suggests that SFKs function as branch points in cell signaling with the capacity for temporal discrimination in selecting substrates.
The dichotomous role of SFKs in the regulation of both pro-adhesive and anti-adhesive signaling mechanisms also may help reconcile some of the conceptually conflicting evidence regarding the functions of SFKs. The role of SFKs in regulating focal adhesion formation and signaling downstream of integrin activation is well established. Activated Src localizes to focal adhesions, an event that is required for cell spreading on fibronectin.38
Fibroblasts deficient in SFKs have focal contacts but reduced tyrosine phosphorylation at focal adhesions and defective cell adhesion to matrix.36,38,39
But the kinetics of focal adhesion assembly are more rapid in these cells, introducing complexity in the model.36
Furthermore, the pro-adhesive role of SFKs suggested by these loss-of-function experiments are not reciprocated by gain-of-function experiments. The constitutively activated v-src
oncogene product interacts with focal contacts, phosphorylating target proteins within them.40,41
However, the activities of the v-src
product are destructive to focal adhesions and in fact v-src
transformed cells appear to have significantly reduced focal adhesions.42
To reconcile these datasets, SFKs have been proposed to function in the turnover of focal adhesions, a dynamic model that is tolerant to potentially opposing roles for SFKs.43
But the SFK effectors that could mediate the proposed anti-adhesive component of the turnover model have not been well defined. The identification of Trask as an anti-adhesive effector of SFKs now fills a conceptual gap that enables a better mechanistic model of how SFKs can promote the turnover of focal adhesions.
A caveat to this is that much of the cited studies of focal adhesions were performed in fibroblasts and Trask is abundantly expressed in epithelial cells but appears to have very low expression in mesenchymal cells.32
But there is some, albeit low expression in fibroblasts and although we can't detect the expression of Trask in 3T3 cells, we can detect pY-Trask in v-src
transformed 3T3 cells by immunoprecipitation and phosphotyrosine immunoblotting (). This may be due to the sensitivity limits of our antibodies and their weaker affinity for the mouse Trask protein and the likely much higher sensitivity and cross-reactivity of anti-phosphotyrosine antibodies with mouse Trask. The higher expression of Trask in epithelial cells may reflect the more complex nature of adhesion in epithelial cells, including the existence of both cell-matrix and cell-cell adhesions and junctions, the link with cell polarity, survival and many other functions.
Cell lysates from the indicated cell lines were immunoprecipitated with rabbit polyclonal anti-Trask antibodies and immunoblotted with mouse anti-Trask or antiphosphotyrosine monoclonal antibodies.