Schematic representation of the Trask mutant constructs. All constructs contain the signal peptide and transmembrane regions for proper membrane localization and an in-frame c-terminal myc tag for easy identification. The triangles indicate the naturally occurring cleavage sites after the signal peptide and at Arg368 of the extracellular domain. Abbreviations indicate the signal peptide (SP), CUB domains, transmembrane region (TM), proline-rich region (PRR), and myc tags. The extracellular region homology to CUB domains is low, and the precise region and number of CUB domains is somewhat speculative. The location of the 5 intracellular tyrosines are indicated by Y.

A) The induced constructs were immunoprecipitated by anti-myc antibodies from the engineered transfectants cell types and vector control cells and immunoblotted with pTyr antibodies. The lysates from the larger size constructs (wt, YΔF, M5) were run on lower % gels and the smaller size constructs (M4, M8) were run on higher % gels for optimal separation and identification. The bands corresponding to the various constructs are indicated by arrows. The starts indicate the immunoglobulin chains. These blots are representative of multiple experiments (4X for wt, 4X for YΔF, 2X for M5, 2X for M4, 3X for M8, and 2X for vector control). B) Anti-yes immune complexes were obtained from each of the mutant cell types and immunoblotted using anti-myc antibodies to detect the co-immunoprecipitation of the transfected constructs. The M8 mutant Trask protein appears as 3 bands likely due to the significant effect of differential phosphorylation on the gel migration of this small construct. These blots are representative of multiple experiments (5X for wt, 1X for M5, 2X for M7, 2X for M4, and 3X for M8). C) The interaction of the YΔF mutant with Yes was similarly assayed as above. The anti-myc immunoblot shows that the Yes-Trask interaction is lost in the YΔF mutant. The immunoblots were redone using anti-Trask antibodies, again confirming that the Yes-Trask interaction is lost in the YΔF mutant. The stars indicates the immunoglobulin chains. The arrows indicate the Yes protein. These blots are representative of three experiments.

A) HEK293TR cells were engineered to express the M4-Trask mutant in a tet-inducible fashion. The doxycycline inducible expression of M4-Trask is shown here for four different clones. This blot is representative of two independent experiments. B) HEK293TR/M4-Trask cells were plated in the presence or absence of 1 ug/ml doxycycline and the following day observed under phase contrast microscopy. Although not obvious in these still photographs, many of the cell clumps in the doxycycline treated arm are loosely adhered or entirely in suspension. This experiment has been performed 4 times with similar results.

A) MDA-468TR cells were engineered to inducibly overexpress the M4, M5, M7, M8, and M9 and YΔF Trask mutants in response to doxycycline treatment. For each construct four clones were studied to account for clonal variability. The dox-inducible expression of the M4, M5, M7, M8, and M9 and YΔF Trask mutants are shown by anti-myc immunoblots. The myc immunoblots for the wt-Trask over-expressing cells were previously published [7]. These induction blots are representative of two experiments done with the entire clone set, and with 4 experiments done with selected Trask-M4 and Trask-YΔF clones. B) The membrane localization of each of the induced constructs was verified following doxycycline induction by anti-myc staining of fixed cells and immunofluorescence microscopy.

A) The indicated cell types were plated in 6-well plates in the presence or absence of 1 ug/ml doxycycline and the following day imaged under phase contrast microscopy. These views are representative of multiple fields of view (shown in Figure S2) and representative of multiple independent experiments (>10X for wt, 5X for M4, 3X for M5, 3X for M7, 3X for M8, 3X for M9, and 5X for YΔF). B) The indicated cell types were treated with 1 ug/ml doxycycline or control and the following day seeded into 96-well plates in triplicate wells and the adherent cell population was quantified by crystal violet staining as described in methods. Results were normalized to non-induced controls, and averaged across the triplicate wells as well as across three independent experiments done on different days (n = 9), in different clones, with the indicated SEM. The reduced cell adhesion following dox induction is statistically significant for WT-Trask (p<0.0001), M4-Trask (p<0.0001), M5-Trask (p<0.0001), and M8-Trask (p<0.0001) cell types. C) The indicated cell types were treated with 1 ug/ml doxycycline or control overnight and the following day total cell lysates were used to determine focal adhesion signaling. FAK phosphorylation was done by anti-FAK immunoprecipitation followed by anti-phosphotyrosine immunoblotting. These blots are representative of multiple experiments (5X for wt, 5X for vector control, 2X for M4, 2X for M5, 2X for M7, 2X for M8, 2X for M9, and 5X for YΔF).

A,B) HEK293 (A) or MDA-468 (B) cells were transiently transfected with the indicated mutant constructs or wildtype or vector controls and total cell lysates were assayed as indicated. These blots are representative of multiple experiments (shown in Figure S4). C) Cell lysates from five cell lines were harvested while adherent (A) or after 2 hours of culture in suspension (S) and immunoblotted as indicated. Lanes correspond to 1) L3.3 cells, 2) SW480 cells, 3) DLD1 cells, 4) PC3 cells, and 5) MDA-468 cells. Arrows indicate the larger uncleaved and smaller cleaved forms of Trask. These blots are representative of two experiments. The MDA-468 blot is representative of >5 independent experiments.