Figure 5.

Activation of Tyr phosphorylation by NDP-MSH. A, Serum-deprived HBL cells were challenged with NDP-MSH for the times shown and Tyr phosphorylation was detected by Western blot. B, Failure of cAMP to induce Tyr phosphorylation in melanoma cells. HBL cells were incubated with NDP-MSH or FSK and probed for pTyr by Western blot. C, RTK inhibition completely blocks ERK activation by NDP-MSH. Control, untransfected HBL cells were preincubated with increasing concentrations of AG1478 for 45 min and then treated with NDP-MSH. Cell extracts were analyzed for ERK phosphorylation. As a control for specificity, cells transiently transfected with empty vector or the NRAS constitutively active mutant Q61R, as indicated, were incubated with a fixed 50 μm concentration of AG1478 for 1 h, and pERK was detected by Western blot. D, NDP-MSH-induced Tyr phosphorylation is not mediated by the EGFR. HBL cells transfected with empty vector or the Q61R NRAS mutant were pretreated or not with the specific EGFR inhibitor PD153035 (0.1 μm, 45 min) and then challenged with 10⁻⁷ m NDP-MSH for the times shown. pERK levels were estimated by Western blot. Identical results were obtained in two independent experiments.