Upon binding to FAK, Src is PJ34 hydrochloride activated 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. These events function to stabilize focal adhesions, generating a force-induced mechanical link with the actin cytoskeleton, and regulate the surrounding membrane dynamics. While our understanding of adhesion complex formation has greatly evolved in Creatinine recent years, our understanding of how adhesion complexes are disrupted remains largely primitive. The mechanisms that promote the disruption of cell adhesion are of particular interest in cancer biology, specially for the study of invasion and metastasis. The functions of the Trask ECD are largely unknown. CUB domains are extracellular protein-protein interaction modules, typically found in many proteases. Indeed Trask is stably associated with the CUB domain containing membrane protease MT-SP1 and is a proteolytic substrate of MT-SP1. Trask is cleaved by other serine proteases including trypsin and plasmin as well and this accounts for the expression of larger and smaller forms of Trask that are typically observed in different ratios in different cell types. Some investigators have suggested that cleavage of the Trask/ CDCP1 ECD results in phosphorylation of its ICD. The evidence that has promoted this suggestion is that cells treated with trypsin simultaneously undergo Trask cleavage, cell detachment, and Trask phosphorylation. Although the cleavage and phosphorylation of Trask/CDCP1 may occur simultaneously in some circumstances, the cleavage of the ECD does not appear to be required for phosphorylation of the ICD as the phosphorylation of Trask is also seen with EDTA-induced cell detachment without any increase in Trask cleavage beyond the basal state. Clearly, we need more experimental studies to explore the functional relationship between the Trask ECD and ICD, particularly as it pertains to its role in the regulation of cell adhesion. In the current study we have undertaken a structurefunction analysis of Trask in order to determine whether its antiadhesive functions reside within the ECD or ICD and whether this involves transmembrane signaling. The data reveals that the antiadhesive functions of Trask are directly mediated through the tyrosine phosphorylation of its ICD and the ECD is dispensible for the inhibition of cell adhesion when phosphorylated. The partial nature of the proteolytic cleavage is similar to that seen with tet-inducible overexpression of the wildtype Trask which we have previously shown. While there are differences in the relative expression of cleaved and uncleaved products of the wildtype and mutant Trask constructs, these could be due to the different stability of these proteins. All constructs localize to the cell membrane as shown by fluorescence microscopy of anti-myc immunostained cells following doxycycline treatment. Therefore neither the ECD or the ICD functions are required for membrane localization. The YDF mutant also localizes to the membrane indicating that phosphorylation is also not required for membrane localization. The signal peptide and transmembrane regions were preserved in all constructs, since they are known to be required for proper membrane localization of type I transmembrane proteins. The state of phosphorylation of each expressed construct was determined by anti-myc immunoprecipitation followed by phosphotyrosine immunoblotting. When overexpressed, wildtype Trask undergoes constitutive tyrosine phosphorylation.