Figure 3.

S104 and S106 are phosphorylated by Erk2 in vitro. (A) Purified GST-ERα was incubated with a panel of purified kinases, as indicated, according to manufacturer's instructions, followed by immunoblotting with phospho-specific antibodies (α-PS104, α-PS106, and α-PS118) or α-ER. The filled arrows indicate the position of GST-ERα, and the open arrow indicates a non-specific product seen with α-PS106 antisera. (B) Purified GST-ERα was incubated with increasing amounts of purified Erk2 MAPK (0, 5, 10, 20, 50, and 100 ng) in the absence of ligand (NL) or in the presence of E₂ (10 nM), followed by immunoblotting as before. (C) Purified GST, or wild-type GSαT-ERα-ΔLBD (ERα-ΔLBD) or GST-ERα-ΔLBD in which S104, S106, and/or S118 had been substituted by alanine (A), as indicated, were incubated with Erk2 in the presence of ³²P-γATP, followed by SDS-PAGE and autoradiography of the dried gel. Immunoblotting a duplicate gel with α-ER was used to determine the relative levels of each mutant. The bar chart shows quantification of each ³²P signal relative to the respective total GST-ERα-ΔLBD level.