Figure 4. ERK-mediated phosphorylation of S98 inhibits actin binding.

(A) Sequences of MRTF-A(fl) and MRTF-A(BSAC), with RPEL1 highlighted in pink and the ERK-binding motif in blue. (B) Cells expressing Flag-MRTF-A and RafER were treated with 4OHT or vehicle and 10 μM U0126 as indicated. Lysates were analysed by immunoblot for MRTF-A pS98, MRTF-A pS33, and MRTF-A (Flag). (C) Cells expressing wild-type MRTF-A(fl) or derivatives with the indicated ERK-binding site mutations, or MRTF-A (BSAC), were analysed by immunoblotting with MRTF-A phospho-S98 antibody before and after serum stimulation. (D) Phosphorylation of recombinant MRTF-A(2-199) by ERK2 in the absence or presence of G-actin was monitored by MRTF-A phospho-S98 antibody (top) or coomassie staining (bottom). (E) S33 phosphorylation occurs independently of the ERK-binding motif. Cells expressing MRTF-A derivatives were analysed as in (C) using anti-MRTF-A phospho-S33. (F) Size exclusion chromatography of complexes formed between G-actin and recombinant MRTF-A(67–199) derivatives, or G-actin alone, colour coded. (G) Left, calibration curve; right, apparent stoichiometry of G-actin binding.

DOI: http://dx.doi.org/10.7554/eLife.15460.010

Figure 4—figure supplement 1. ERK phosphorylation of MRTF-A S98.

(A) Cells expressing Flag-MRTF-A(2–204)-PK and S98 mutants, with or without MEK^R4F, were maintained in 0.3% FCS for 20 hr, then stimulated with 100 ng/ml TPA and phosphorylation of S98 and S33 analysed by immunoblotting. (B) Immunofluorescence assay of NIH3T3 cells before and after a 15' TPA stimulation with U0126 addition as indicated. (C) ERK interacts with the MRTF-A N-terminal sequences via the ERK docking motif. GST MRTF-A (2–115) or docking site mutant derivatives were used in pulldown assays with recombinant ERK2. (D) G-actin does not detectably compete for ERK binding in the pulldown assay. Assays with GST-MRTF-A (2–199) were as in (C) except that increasing amounts of G-actin were included.

DOI: http://dx.doi.org/10.7554/eLife.15460.011

Figure 4—figure supplement 2. S98 phosphorylation impairs assembly of MRTF-A / G-actin complexes.

(A) Top, sequences of the RPEL1 containing peptides used in the fluorescence polarisation anisotropy assay, with mutation and phosphorylation sites indicated. Bottom, fluorescence anisotropy assay of G-actin binding to FAM-labeled RPEL1 peptides. Data represent mean of at least two independent experiments. (B) Size exclusion chromatography profiles of G-actin / Spacer1-RPEL2 peptide complexes or G-actin alone are shown, colour coded, with calibration at the right, and data are summarised below.

DOI: http://dx.doi.org/10.7554/eLife.15460.012