Nck1 is upstream of eIF2-α phosphorylation in the cell death induced by the combination of OSU-03012 and lapatinib. A: BT474 cells (5 x 105) were co-transfected with the Ser51Ala eIF2-α mutant and GFP-Nck1. Cells were allowed to incubate for 24 h to induce ectopic expression then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested for western blotting as described previously (lower panel), or plated into 6-well plates to assay for clonogenic capacity as described previously (upper graph). Graphs represent total colony number. * denotes a p-value of <0.05. B: BT474 (upper panel) and MDA-MB-231 (lower panel) cells were treated with either vehicle (Veh) or OSU-03012 and lapatinib in combination (combo). PP1 was immunoprecipitated and the resulting membranes were immunoblotted with eIF2α and PP1. C: Our data indicate that Nck1 and PP1, which are originally in a complex with eIF2 are dissociated after treatment with the combination of OSU-03012 and lapatinib. This event frees eIF2-α to become phosphorylated by one of many upstream kinases such as PERK, leading to ER stress and eventual cell death.