Fig 2: Protein phosphorylation profiling. A. Top panel, Protein phosphorylation profiling of p38a (T180/Y182), ERK1/2 (T202/Y204), Akt 1/2/3 (S473), CREB (S133), ß-catenin protein, p53 (S15) and c-Jun (S63) in HT1080-scr, shPTK7, PTK7, sPTK7, cPTK7/622-1070, cPTK7/726-1070, Chz, shMT1-Chz and shMT1 cells. The spots were scanned and digitized. Pixel density of the spots is shown by the graphs in the bottom panel. B. A representative array of the HT1080 cell sample. The spots (boxed) of p38a (T180/Y182), ERK1/2 (T202/Y204), Akt 1/2/3 (S473), CREB (S133), ß-catenin protein, p53 (S15) and c-Jun (S63) are numbered. C. Western blotting of phosphorylated and total cell c-Jun, CREB and ATF1 in HT1080, shPTK7, PTK7, sPTK7, cPTK7/622-1070, cPTK7/726-1070, Chz, shMT1-Chz and shMT1 cells.

Fig 3: Genome-wide transcriptional profiling. A. Heat map and the hierarchical clustering of the expression data (log2 of expression level) of the differentially expressed genes in PTK7, sPTK7, Chz, cPTK7/726-1070, cPTK7/622-1070 and shPTK7 cells relative to the parental HT1080 cells. Red and green correspond to the high and the low expression levels, respectively (color bar inset). B. The number of differentially expressed genes in the samples. Numbers show the number of the up-regulated (red) and the down-regulated (green) genes relative to HT1080 cells. C. Heat map and the hierarchical clustering of the expression data (log2 of expression level) of the differentially expressed, cell motility-related genes in the cells (selected using the IPA software) relative to HT1080 cells (Additional file 2: Table S2). Red and green correspond to the high and the low expression levels, respectively (color bar inset). D. Expression level validation of the selected up-regulated genes by immunoblotting. Top two panels, PTK7 was analyzed using the PTK7 antibody in the total cell lysates (total) and the cell surface-biotinylated cell samples (surface). Bottom panel, The FLAG- and V5-tagged recombinant PTK7 constructs were analyzed using the FLAG and V5 antibodies (total cell lysate samples), respectively. CDH11, cadherin-11; SPP1, osteopontin; IL1B, interleukin 1ß; PDPN, podoplanin; MAGEC1, melanoma antigen family C1. The numbers above the protein bands show the fold-difference of the differentially expressed genes as detected by transcriptional profiling. Tubulin, loading control.

Fig 4: PTK7 signaling. The pathways and the key proteins are predicted based on the results of our genome-wide transcriptional and protein phosphorylation profiling. Our data suggest that the stabilized full-length membrane PTK7 up-regulates the Akt and c-Jun pathways, and down-regulates the p53 pathways leading to the cell survival signaling. Full-length PTK7 also suppresses ERK and CREB-mediated gene expression and cell motility. The proteolysis of PTK7 ectodomain (Ig1-Ig7) by MT1-MMP and ADAMs generates the soluble sPTK7 form. The subsequent cleavage of the C-terminal membrane PTK7 fragment by ?-secretase releases the intracellular domain (cPTK7). The soluble sPTK7 species binds the full-length membrane PTK7 and activates the RhoA [21] and p38a pathways, which regulate the actin cytoskeleton and the expression of the migratory genes, including IL1B. The intracellular PTK7 domain fragment, cPTK7, enters the cell nucleus and induces the RAS/p90RSK/CREB signaling resulting in the CREB and ATF1 phosphorylation at Ser133 and Ser63, respectively, and the expression of the pro-migratory genes, including cadherin-11 (CDH11). We hypothesize that the ratio of the full-length membrane PTK7 to the N-terminal and the C-terminal PTK7 proteolytic fragments acts as a switch, which establishes the downstream signaling pathways that control cell survival and motility.

Fig 5: PTK7 constructs: 1–1070 PTK7, 1–694 sPTK7, cPTK7/622-1070 and cPTK7/726-1070. S, signal peptide, Ig, immunoglobulin-like domain, TM, transmembrane domain, JM, juxtamembrane region, KINASE, the catalytically inert kinase domain. The amino acid residue numbering is shown below the construct structure. MT1-MMP, ADAM and ?-secretase cleavage sites are indicated by arrows. The Chz mutation (Ala-Asn-Pro, underlined) results in an additional MT1-MMP cleavage site (Pro-Lys-Pro?Leu622; boxed). FLAG and V5-HIS are the C-terminal tags in the recombinant constructs.