Figure 3
From: ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity
ERK2 directly phosphorylates residue serine 74 and serine 77 in Bmf. (a) Alignment of Bmf protein sequences surrounding serine 74 and serine 77 among different species. Conserved serine 74 and serine 77 residues are shaded. The amino-acid positions relative to the start codon are shown in parentheses. (b) WM793TR cell lines expressing wild type, S74A, S77A, S74A/S77A (AA), S74D, S77D, S74D/S77D (DD) HA-Bmf were induced with with or without 100 ng/ml doxycycline for 7 h. Cells lysates were analyzed by western blotting using antibodies against the HA-tag and actin. (c) 1205LuTR/HA-Bmf (WT, S77A and AA) cells and A375TR/HA-Bmf (WT, S77A and AA) cells were treated with or without 100 ng/ml doxycycline for 7 h. Cell lysates were analyzed as in b. (d) WM793TR/HA Bmf cells were treated with 100 ng/ml doxycycline for 1.5 h and 1 μM okadaic acid was added for another 30 min. Cells were then lysed for western blot analysis. Indicated are the percentages of Bmf that is slow migrating, as determined by quantitation of band intensity. (e) WM793TR/HA-Bmf S74A and WM793TR/HA-Bmf S77A cells were transfected with control, ERK1, ERK2 and p38α siRNA. Seventy-two hours post transfection, cells were treated with 100 ng/ml doxycycline and with or without 10 μM U0126 for 7 h before lysis for western blot analysis on indicated proteins. (f) Activated ERK2 was incubated with bacterially expressed GST-Bmf WT, GST-Bmf S74A, GST-Bmf S77A and GST-Bmf AA in the presence of [γ-32P]ATP. Phosphorylation of GST-Bmf variants was examined by SDS-PAGE followed by autoradiography. Coomassie staining of the same gel is shown below
