Phosphorylation of Trask by Src kinases inhibits integrin clustering and functions in exclusion with focal adhesion signaling.

Src family kinases (SFKs) are a family of nonreceptor protein tyrosine kinases with a domain structure consisting of a highly conserved kinase domain as well as an SH2 domain and an SH3 domain, a C-terminal negative-regulatory tyrosine residue, and an N-terminal myristoylation site. Three members of the family, Src, Yes, and Fyn, are ubiquitously expressed, while the expression of the other members is largely restricted to specific hematopoietic cell lineages. SFKs participate in numerous cellular pathways in association with growth factor receptors, G protein-coupled receptors, steroid hormones, STAT transcription factors, and integrin receptors (14, 17, 34). The role of SFKs in regulating cell adhesion signaling at sites of adhesion to the extracellular matrix (ECM) is particularly well established. Upon integrin engagement with the ECM and clustering of integrins at sites of cell adhesion to matrix, macromolecular complexes are assembled in association with the intracellular tails of activated integrins (30, 40, 48). Within these focal adhesions, focal adhesion kinase (FAK) is activated by autophosphorylation at tyrosine 397, creating a binding site for the Src SH2 domain (30, 37). Upon binding to FAK, Src is activated and phosphorylates a number of additional tyrosine residues on FAK, creating additional binding sites for SFKs and other proteins. Activated Src also phosphorylates a number of additional cytoskeletal proteins, including paxillin and p130Cas and proteins involved in regulating the RhoA, Rac1, and Cdc42 GTPases (23). These events function to stabilize focal adhesions, generating a force-induced mechanical link with the actin cytoskeleton, and regulate the surrounding membrane dynamics. SFKs are required for proper establishment of focal adhesions, as fibroblasts deficient in Src kinases have significantly reduced tyrosine phosphorylation at focal contacts and defective cell adhesion to matrix (7, 26, 47). Although this loss-of-function model supports the current molecular models of focal adhesion establishment, the conclusions are not reciprocated by gain-of-function experiments. The constitutively activated v-src oncogene product interacts with focal contacts, phosphorylating target proteins within them (20, 33). 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 (11). Therefore, the evidence suggests that SFKs are capable of promoting both adhesive and antiadhesive functions, and most current models reconcile this by proposing that SFKs function in focal adhesion turnover (15). The mechanisms that mediate the antiadhesive functions of SFKs are less well understood. Some evidence suggests that SFKs can mediate focal adhesion disassembly through a RhoA- and mDia1-mediated pathway or through a calpain-mediated pathway (18, 51). In this paper, we describe a novel mechanism by which SFKs can negatively regulate focal adhesion assembly. We have been studying a novel substrate of SFKs named Trask. Trask is a recently described 140-kDa transmembrane protein with little homology to known families of proteins. It has a large extracellular region containing CUB domains and a smaller intracellular region containing five tyrosines (5). Trask is widely expressed in epithelial cells and tissues as a variable blend of 140-kDa and 85-kDa forms, the latter due to proteolytic cleavage of its distal extracellular region by serine proteases, including the membrane bound MT-SP1 (5, 42). Trask is phosphorylated in vitro by SFKs, including Src and Yes, and is also phosphorylated by SFKs in cells, and its phosphorylation can be inhibited by all classes of SFK inhibitors (5). The phosphorylation of Trask is exclusively dependent on SFKs, since it fails to undergo phosphorylation in Src/Yes/Fyn knockout cells (SYF cells) unless transfected with an SFK member (50). Trask has also been independently identified as a cancer-associated gene by other groups. In a microarray analysis of colon cancers, it was identified as a transcript with increased expression in tumors compared with that in adjacent normal tissues and was named CDCP1 (39). In another line of study, a subtractive immunization screen designed to identify antibodies against more metastatic variants of HEp-3 carcinoma cells identified a surface protein that was named SIMA135, which is identical to Trask/CDCP1 (22). The suggestion that Trask/CDCP1 is important in cancer progression has been further supported by correlative studies of human tumors, although the data are mixed and the nature of this association and the cellular role of Trask/CDCP1 in cancer is a matter of ongoing interest and investigation. In an extensive analysis of Trask expression and phosphorylation in human tissues, we found that Trask is widely expressed in most epithelial tissues; however, the SFK phosphorylation of Trask is restricted to physiological circumstances of detachment, such as in mitotically detached cells in the colonic crypts (42). However, in a large survey of human tumor sections, we found that Trask is phosphorylated in many epithelial cancers at all stages, including preinvasive cancers such as tubular adenomas, but not in their normal-tissue counterparts (50). In other studies, the elevated expression of Trask/CDCP1 has been linked with poorer prognosis in cancers of the lung, kidney, and pancreas (3, 24, 31) but with better prognosis in endometrioid cancer (28). The phosphorylation of Trask is linked with cell adhesion such that Trask is phosphorylated almost instantly when epithelial cells detach from matrix and is dephosphorylated when cells readhere to matrix (42). In the current study, we mechanistically studied the link between Trask phosphorylation and cell adhesion through loss-of-function and gain-of-function studies, looking at cell adhesiveness, at the affinity, binding, and clustering of integrins, and at focal adhesion assembly and signaling. We report that the SFK phosphorylation of Trask inhibits cell adhesion through the inhibition of integrin binding activity. This is mediated through the inhibition of clustering, but not regulation of affinity state, and consequent inhibition of focal adhesion assembly and signaling. We also found that the SFK phosphorylation of Trask functions in opposition to and in exclusion with focal adhesion signaling. These two signaling pathways oppose and inactivate each other, defining a switch that regulates cell anchorage state.