FIG. 6.

SRC-3 methylation dissociates CARM1 from the coactivator complex. (A) HEK293T cells were transfected with the indicated Flag-SRC-3 constructs. Following immunoprecipitation (IP) with anti-Flag-M2 (α-Flag) affinity gel, the protein samples were separated on a 4 to 15% SDS-PAGE gel. Endogenous CARM1 associated with Flag-SRC-3 was examined by Western blotting (upper panel). The same membrane was also used to probe for SRC-3 (lower panel). (B) R1171 is a bona fide methylation site on SRC-3. Two peptides, one with an asymmetric dimethylated arginine at R1171 and the other one without any methyl group, were incubated with recombinant SRC-3 protein in the CARM1-mediated methylation assay. Triangles indicate three different peptide concentrations used (1.5 μM, 15 μM, and 150 μM). (C) Peptide with unmodified R1171 competes with SRC-3 for binding CARM1. Peptide P1 or P2 (150 μM) was incubated together with recombinant Flag-SRC-3 and CARM1 for 1 h at 30°C. SRC-3 protein was immunoprecipitated with anti-Flag-M2 affinity gel, and the presence of associated CARM1 was determined by Western blotting. (D) The methylation region of SRC-3 overlaps with the CARM1 docking region. Wild-type (WT) or methylation site-mutated SRC-3 proteins were incubated with GST or GST-CARM1 for 2 h at 4°C. Glutathione beads were used to pull down GST control and GST-CARM1 proteins. Associated SRC-3 proteins were determined by Western blot analysis. (E) A similar experiment was performed as for panel A, except that ERα was also transfected. The levels of p300 and ERα were determined by Western blotting as well.