PTK6 Regulates Survival Figure 6. Overexpression of PTK6 enhances anchorage-independent survival by regulating IGF-1R phosphorylation. A) PTK6 overexpression increases phosphorylation of IGF-1R. (Left) MCF-10A cells overexpressing a constitutively active (CA) or kinase-inactive (KD) PTK6 were grown in suspension cultures for 48 hours in the presence of IGF-1 (100ng/ml). Lysates were prepared and probed using RPPA. The values represent the average normalized expression values derived from triplicate samples. Error bars indicate standard deviation. (Right) Lysates prepared from suspension cultures of MCF-10A cells overexpressing vector control, MF-PTK6 or kinase-inactive MF-PTK6 (KD-MFPTK6) were probed with the indicated antisera. B) Lysates prepared from suspension cultures of MCF-10A cells overexpressing vector control, MF-PTK6 or wild type Akt in the presence of IGF-1 (100ng/ml) were probed with the indicated antisera. C) PTK6 co-precipitates with IGF-1R and IRS-1 adapter protein. Lysates of MCF- 10A cells overexpressing IGF-1R and wild type PTK6 were immunoprecipitated with the indicated antisera and immunoblotted with anti-PTK6 antibody. D) Anchorage-independent survival stimulated by PTK6 overexpression is suppressed by IGF-1R kinase inhibition. MCF-10A cells overexpressing vector control or MF-PTK6 were grown in suspension cultures containing IGF-1 (100ng/ml) for 48 hours in the absence or presence of BMS-536924 at the indicated concentrations. Cell death was assessed as described in Figure 1. The fold change in cell death represents the average of three independent experiments. Error bars indicate standard deviation. doi:10.1371/journal.pone.0011729.g006 (Figure 6A). By western analyses, an increase in IGF-1R phosphorylation was observed in MCF-10A cells overexpressing a catalytically active, membrane-targeted PTK6 (MF-PTK6) (Figure 6A). Increased levels of total IGF-1R accompanied the enhanced phosphorylation, whereas downregulation of PTK6 affected phosphorylation independent of receptor levels. Overex- pression of kinase-inactive PTK6 (KD-MFPTK6) also slightly enhanced IGFR phosphorylation, although not to the level observed with MF-PTK6. No change in total receptor level was observed with overexpression of KD-MFPTK6. PTK6 overex- pression does not have a general effect on membrane-associated receptor tyrosine kinases in suspension, as levels of EGFR, previously reported to activate PTK6, were unchanged (Figure 6B). Moreover, the enhanced IGF-1R phosphorylation is unlikely to be simply secondary to enhanced viability of MCF-10A cells in suspension cultures as overexpression of Akt, which potently inhibits anoikis, did not result in similar changes in IGF- 1R levels or phosphorylation (Figure 6B). Previous studies have shown that PTK6 is able to form complexes with EGF receptor family members and PTK6 couples these receptors to downstream signaling pathways [28]. PTK6 could also regulate IGF-1R signaling by physical association with the receptor complex, as PTK6 was previously shown to associate with IRS-4 in HEK-293 cells [29]. In MCF-10A cells overex- pressing IGF-1R and PTK6, PTK6 co-precipitated with IGF-1R (Figure 6C). PTK6 was also detected in immune complexes containing IRS-1, suggesting that PTK6 may regulate IGF-1R by associating with the receptor:adapter complex. To determine whether IGF-1R kinase activity is critical for the suppression of anoikis induced by PTK6 overexpression, we assessed the effect of BMS-536924, an IGF-1R/insulin receptor inhibitor, on the anchorage-independent survival of MCF-10A cells overexpressing MF-PTK6. Cells were cultured in suspension in the presence of sub-saturating doses of the inhibitor, which reversed the enhanced survival induced by PTK6 (Figure 6D and Figure S4). Collectively, these results provide evidence for PTK6’s PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11729