Figure 5

Constitutive phosphorylation of YB-1 due to K-RAS mutation depends on erbB1 signaling. (A) Cells were transiently transfecting with the pEGFP-C1 control vector (con. vector) or pEGFP/K-RAS^V12 (K-RAS ^V12) as described in Materials and methods. Forty-eight hours after transfection, cells were treated with 5 μM erlotinib (Erl.), 10 μM LY294002 (LY), 20 μM PD98059 (PD) or a combination of PD98059 and LY294002 (PD/LY) for 2 hours. Control cells were treated with dimethyl sulfoxide. Protein samples were isolated, and expression levels of green fluorescent protein and K-RAS, as well as the phosphorylation status of Akt, ERK1/2 and YB-1, were assessed by Western blot analysis. The blots were stripped and incubated with antibodies against K-Ras, Akt, ERK1/2 and YB-1. The densitometric values represent the ratios of P-YB-1/YB-1 and P-YB-1/actin normalized to 1 in control vector-transfected cells. The effect of indicated inhibitors on K-RAS^V12-induced YB-1 phosphorylation was investigated in at least two independent experiments and representative Western blots are shown. (B) SKBr3 cells were transfected with nontargeting small interfering RNA (siRNA) or siRNA against K-RAS as described in Materials and methods. Four days after transfection the cells were irradiated with 4 Gy ionizing radiation or treated with 100 ng/ml epidermal growth factor (EGF). At the indicated time points after irradiation and 30 minutes after EGF treatment, protein samples were isolated and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The levels of K-Ras, P-YB-1 and P-ERK1/2 were detected by Western blot analysis. The blots were stripped and incubated with ERK1/2 and YB-1 antibodies. Actin was used as a loading control. A representative Western blot of two independent experiments is shown.