PTK6 Regulates Survival Figure 5. PTK6 downregulation affects IGF-1 dependent signaling pathways. A) Reverse-phase protein arrays (RPPA) reveal changes in IGF- 1-stimulated signaling with PTK6 downregulation. RPPA analysis was performed on lysates of IGF-1R cells overexpressing Bcl2 (IGF-1R/Bcl2) that were transfected with control or PTK6 siRNA oligonucleotides in suspension cultures containing IGF-1 (100ng/ml). The heatmap represents values normalized to luciferase control from transfections performed in triplicate. B) shRNA-mediated downregulation of PTK6 in MCF-7 cells decreases ligand-stimulated IGF-1R phosphorylation. MCF-7 cells expressing control or PTK6 shRNA were stimulated in suspension cultures for the indicated times with 100ng/ml IGF-1. Lysates were probed with the indicated antisera. C) DOV-13 cells cultured on adherent plates were stimulated with 100ng/ml IGF-1 for 20 minutes. Lysates were probed with the indicated antisera. D) PTK6 downregulation inhibits IGF-1- stimulated activation of Akt and Erk/MAPK. Lysates of MCF-7 cells stimulated with the indicated concentrations of IGF-1 for 60 minutes (left panel) or 15 minutes (right panel) in suspension cultures were probed with the indicated antisera. doi:10.1371/journal.pone.0011729.g005 S6, suggesting a widepspread impact of PTK6 downregulation on signaling pathways, either as a primary or secondary consequence. Interestingly, we observed that the signal intensities for phospho- IGF-1R and total IGF-1R were also decreased on the protein array, which raised the possibility that PTK6 modulates activation and/or expression of the receptor itself, thereby influencing multiple downstream signaling molecules. Western analyses of lysates derived from IGF-1 stimulated MCF-7 cells confirmed that downregulation of PTK6 resulted in decreased levels of IGF-1 receptor phosphorylation of known autophosphorylation sites (tyrosine 1131, 1135 and 1136) which are required for maximal IGF-1R kinase activity (Figure 5B). This decrease in IGF-1R phosphorylation was not associated with a decrease in IGF-1R levels. The effects of PTK6 downregulation on IGF-1 receptor phosphorylation are unlikely to be secondary to changes in viability induced by matrix deprivation as these changes were observed in IGF-1R cells co-expressing Bcl2 which are protected from anoikis induced by PTK6 downregulation. Furthermore, similar changes in IGF-1 receptor phosphorylation were observed with downregulation of PTK6 in attached DOV-13 ovarian cancer cells (Figure 5C). PTK6 downregulation-induced decreases in signaling downstream of IGF-1R (e.g. Akt, Erk) were also confirmed in MCF-7 cells acutely stimulated with IGF-1 in suspension cultures (Figure 5D). The effect of overexpressing PTK6 on IGF-1R phosphorylation was also examined (Figure 6). In the RPPA studies an enhanced signal for phospho-IGF-1R (phosphorylated at tyrosine 1131/ 1135/1136) was detected with overexpression of active, PTK6 PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11729