FIGURE 4.

Maf1 phosphorylation status correlates with cellular mTOR signaling. A, MG63 cells were transfected with wild-type FLAG-Maf1 for 24 h and treated with vehicle-DMSO (Cont), 0.5 μm CCI-779, or 0.5 μm WYE-132 for an additional 3 h followed by lysis in NuPAGE-LDS buffer. For calf intestinal alkaline phosphatase (CIAP) treatment, total lysates from vehicle-treated cells were prepared in a buffer lacking phosphatase inhibitors (Phos Inh.) and treated for 1 h at 37 °C. Lysates were probed with FLAG or β-actin antibodies. B, indicated cell lines were transiently transfected FLAG-Maf1 expression vector. 24 h post-transfection cells were treated with 0.5 μm CCI-779, 0.5 μm WYE-132, 5 μg/ml U0126, 10 μg/ml α-amanitin, 100 ng/ml taxol, or DMSO-control for 3 h, as described in Fig. 2. Total cellular lysates were subjected to immunoblotting with antibodies for FLAG, phospho-S6K1, phospho-AKT, total AKT, phospho-ERK (extracellular signal-regulated kinase), and β-actin. Dephosphorylation of the Maf1 was monitored by its shift in migration on SDS-PAGE. The arrowheads indicate migration of the phosphorylated versus hypophosphorylated form of Maf1. C, HEK293 cells were subjected to amino acid withdrawal (−AA) for 2 h and then incubated with amino acids (+AA) for 1 h with or without WYE-132. Lysates were immunoblotted with FLAG, P-S6K1, total 4E-BP1, or β-actin antibodies.