Much is known about the signaling mechanisms that regulate focal adhesions, although at this time we know much more about the mechanisms that promote focal adhesion formation upon integrin activation and clustering and much less about the mechanisms that disrupt them. In particular, SFKs are critically involved in regulating focal adhesion formation and signaling downstream of integrin receptors. Activated Src localizes to focal adhesions, and this is an event that is required for cell spreading on fibronectin (16
). Fibroblasts deficient in Src kinases have focal contacts but reduced tyrosine phosphorylation at focal adhesions and defective cell adhesion to matrix (7
). Upon integrin clustering, FAK is recruited to the cytoplasmic tails (c-tails) of β-integrins, where it undergoes autophosphorylation at Y397 followed by binding and conformational activation of Src and reciprocal full activation of FAK by Src through phosphorylation of several of its tyrosine residues (30
). Src can also be directly activated by its interaction with β integrins (1
). The FAK-Src complex phosphorylates a number of substrates, including paxillin and p130Cas
, further solidifying and expanding the focal adhesion complex and ultimately linking it with the actin cytoskeleton (4
In this study, we describe an effector of SFKs that negatively regulates focal adhesion formation, thereby mediating an antiadhesive function. When phosphorylated by SFKs, Trask appears in complexes containing β1 integrin, interfering with integrin clustering and preventing the mechanical and signaling events that link the intracellular cytoskeleton with the ECM. Future studies will focus on determining the molecular mechanisms through which p-Trask prevents integrin clustering. It is now well established that the activities of the integrin heterodimer are regulated by intracellular proteins interacting with the β integrin cytoplasmic tail. In particular, proteins that can induce the separation of the intracellular tails of the α and β integrins promote an extended integrin conformation in the integrin extracellular domains (ECDs) with high affinity for ligand binding (2
). The binding of several intracellular proteins, including talin, kindlins, Dok1, tensin, and Numb, is mediated through the integrin NPxY motif (9
). While the c-tail interactions described to date have provided great insight into how intracellular proteins can regulate the affinity state of the integrin heterodimer, much less is known about inside-out signaling mechanisms that may regulate integrin clustering. p-Trask does not regulate the affinity state of the integrin heterodimer, as we have shown using assays that measure monovalent ligand binding and integrin conformation. Rather, it inhibits integrin clustering and establishment of focal adhesions. Future studies will seek to more specifically determine how Trask phosphorylation by SFKs inhibits integrin clustering. One possibility is that phosphorylation of Trask by SFKs promotes the SFK phosphorylation of the β integrin c-tail, promoting or disrupting integrin c-tail interactions that are important in clustering. In preliminary attempts thus far, we have not been able to identify p-Trask-induced tyrosine phosphorylation of β1 integrin, but the interactions of the β integrin c-tail are known to be regulated through tyrosine phosphorylation. In particular, the integrin NPxY motif binds some proteins specifically when it is phosphorylated and other proteins preferentially in its unphosphorylated state (29
). The tyrosine kinases that phosphorylate these sites have not been well established. v-Src has been shown to phosphorylate the NpxY motif within the β integrin c-tail, disrupting certain interactions and partly explaining the adhesion defects in v-src
-transformed cells (36
). However, this appears to be one of many promiscuous functions of v-src
, and an analogous physiological role for c-Src or other SFKs has not been described. It remains possible that this phosphorylation activity of cellular SFKs is induced specifically in the unanchored state, facilitated by the appearance of p-Trask. The inhibitory effects of p-Trask on integrin clustering and binding activity are almost surely mediated through the phosphorylation of its intracellular tyrosines and not through any functions attributed to its ECD, since Trask truncation mutants lacking the entire ECD inhibit cell adhesion when overexpressed and phosphorylated identically to what is seen with full-length Trask (unpublished data).
Of particular interest is the fact that the inhibitory activities of p-Trask and focal adhesion signaling are reciprocal in nature, as each of these two signaling mechanisms turns off the other, and they function in a mutually exclusive fashion. The SFK phosphorylation of Trask, whether induced physiologically by loss of anchorage or experimentally by Trask overexpression, inactivates focal adhesion signaling. The reverse also holds true, as the activation of integrin signaling, whether induced physiologically by cell adhesion to matrix or experimentally by fibronectin-induced activation of integrin receptors, promotes the dephosphorylation of Trask. It is apparent from all our experimental model systems and from all observed physiological states that Trask phosphorylation and focal adhesion signaling inactivate each other. Therefore, integrin signaling and Trask signaling inhibit each other and constitute mutually exclusive and opposing signaling programs, defining a switch that determines the cell anchorage state. Although there are five tyrosines within the intracellular domain of Trask, in extensive tyrosine mutation studies, we have found that the phosphorylation of Trask occurs as an all-or-none event consistent with its function as a switch (unpublished data). The mechanisms that mediate the dephosphorylation of Trask almost surely involve the regulation of specific protein tyrosine phosphatases (PTPs). It is likely that adhesion promotes the activation of a PTP or the interaction of a PTP with Trask, thereby shifting the phosphorylation state equilibrium toward the dephosphorylation of Trask.
Interest in Trask (also known as CDCP1) has grown because of a growing body of evidence suggesting that it may have cellular functions that are particularly important in tumor progression. A number of studies have documented increased expression of Trask/CDCP1 in some cancers of the lung, kidney, and pancreas, with a poorer prognosis conferred by its elevated expression (3
). Some studies have suggested that Trask/CDCP1 promotes anoikis resistance in cancer cells (45
). This may be a cell-specific finding, since anoikis resistance was not affected when Trask was knocked down in MDA-468 breast cancer cells (data not shown). Other studies suggest that Trask/CDCP1 may be more important for tumor metastasis. Consistent with this, a number of experimental studies using overexpression, knockdown, or monoclonal antibody targeting approaches show that the functions of Trask/CDCP1 are important in tumor invasion and metastasis (12
The link between Trask/CDCP1 and cell adhesion has been reported by others as well. Brown et al. reported that Trask/CDCP1 undergoes tyrosine phosphorylation upon loss of adhesion, but they reported that the phosphorylation event is specifically linked with the proteolytic cleavage of the Trask/CDCP1 ECD and does not occur with EDTA-induced detachment (8
). However, this study was done without the benefit of anti-Trask/CDCP1 antibodies, and the data were only indirectly inferred from the cross-reactive activities of an anti-p-FAK antibody. Our current data obtained using specific anti-Trask/CDCP1 antibody reagents as well as myc-tagged Trask/CDCP1 constructs now clearly show that the phosphorylation of Trask/CDCP1 is tightly linked with the state of anchorage and is not a consequence of proteolytic cleavage (Fig. , , , and ). In panels of the cell types studied, we found that the relative expression levels of the 85- and 140-kDa forms of Trask vary considerably among different cell types but that both forms undergo phosphorylation when anchorage is lost. Both examples were seen in this study. MDA-468 cells express predominantly the 85-kDa form of Trask, which undergoes phosphorylation upon detachment (Fig. ), mouse keratinocytes express predominantly the 140-kDa form of Trask, which undergoes phosphorylation upon scraping (Fig. ), and MCF10A cells express a mixture of the 85- and 140-kDa forms of Trask, both of which undergo phosphorylation upon detachment (Fig. ). The evidence is now clear that the phosphorylation of Trask/CDCP1 is not a consequence of the proteolytic cleavage of its ECD.
It should be noted that overexpressed Trask does not follow the same cleavage ratio as endogenous Trask. Although MDA-468 cells express predominantly the 85-kDa form of Trask, its overexpression by transfection produces both forms. This is likely due to the fact that the overexpression exceeds the cellular capacity for full cleavage. However, both forms are phosphorylated and both forms are found in integrin immune complexes when expressed (Fig. ). Therefore, Trask cleavage does not appear to play a role in the regulation of integrins. The adhesion functions of Trask appear to be entirely mediated through the tyrosine phosphorylation of its intracellular domain.