Fig. 2.
MCV sT induces cellular 4E-BP1 hyperphosphorylation. (A) MCV sT induces 4E-BP1T37/T46 and 4E-BP1S65/S101 phosphorylation. 4E-BP1 phosphospecies are named α through δ according to molecular mass. Higher molecular mass isoforms, particularly β–δ, were increased following sT expression in 293 cells and include authentic phosphorylation sites as detected by phospho-specific antibodies. A 2D gel fractionation of these same lysates (pH 3–6 isoelectric focusing/SDS/PAGE) is aligned to the 1D gel. Arrowheads indicate new 4E-BP1 isoelectric focusing spots after sT expression detected by p4E-BP1T37/T46, p4E-BP1S65/S101, and total 4E-BP1 antibodies. (B) MCV sT-induced 4E-BP1 phosphorylation is partially resistant to mTOR inhibition. The 293 cells were transfected with MCV sT, sTmLSD, or empty vector expression plasmids for 48 h; treated with the mTOR inhibitor PP242; and harvested at indicated time points. MCV sT depends on an intact LSD region to maintain PP242-resistant 4E-BP1 phosphorylation. Representative results are shown from three independent experiments.