Figure 2.

MK2 and MK5 phosphorylate SRC-3-S857 in vitro. (A) Schematic diagram of SRC-3 amino acid sequence (aa). Common phosphorylation sites are indicated above the sequence and the consensus sequence of the S857 phosphorylation site is indicated below. Preferred consensus sequences for phosphorylation by MAPKKAPK (i), PKA (ii) and MAPK (iii) are shown in the lower part of the figure; Hyd indicates hydrophobic aa, X indicates any amino acid⁵⁷. (B) MK2 and MK5 phosphorylates SRC-3 at S857 in vitro. The in vitro kinase assay was performed by incubating GST-CID-SRC-3 WT (2 μg) with 0.1U of different active kinases at 30 °C for 15 min. Phosphorylation of SRC-3-S857 in GST-CID-SRC-3 WT and the total amount of GST-CID-SRC-3 WT was detected using anti-P-S857-SRC-3 and anti-GST antibodies, respectively. The full-length blots are presented in supplementary figure S8. (C,D) MK2 and MK5 phosphorylate SRC-3-S857 in a dose and time dependent manner in vitro. (C) An in vitro kinase assay was performed by incubating increasing amounts (5, 10, 20 and 40 ng) of active MK2 or active MK5 together with 2 μg GST-CID-SRC-3 WT at 30 °C for 15 min. (D) An in vitro kinase assay was performed by incubating 100 ng of active MK2 or MK5 together with 2 μg GST-CID-SRC-3 WT at 30 °C for different periods of time (5, 10, 15, 20 min). For both (C) and (D), anti-P-S857-SRC-3 antibody was used to detect SRC-3-S857 phosphorylation and an anti-GST antibody visualize the input of GST-CID-SRC-3 WT fusion protein for each reaction. The full-length blots are presented in supplementary figures S9,S10. (E) Neither MK5 nor ERK3 phosphorylate SRC-3-S857 in vivo. H1299 cells were transfected with either 20 nM scrambled siRNA, siRNA against MK5 or against ERK3. After 48 h, the cells were lysed and SRC-3 was immunoprecipitated with anti-SRC-3 antibody. The immunoprecipitate (IP) and whole cell extracts (WCE) were analyzed by Western-blotting. The full-length blots are presented in supplementary figure S11.