Figure 2. MRTF is phosphorylated at multiple sites.

(A) Schematic representation of MRTF-A, with S/T-P phosphorylation sites substituted with alanine in MRTF26A indicated in yellow. For details of phosphorylations, see Table 1 and Figure 2—figure supplement 1. (B) Phosphorylation is largely abolished in the MRTF26A mutant. Cells expressing wildtype MRTF-A or MRTF26A (left panel) or MRTF26A-123-1A (right panel), with or without activated mDia1, were stimulated with 15% FCS or 2 μM CD as indicated, and phosphorylation monitored by SDS-PAGE and immunoblotting. See Figure 2—figure supplement 2. (C) Cells expressing wild-type MRTF-A or MRTF-A 26A were stimulated with 15% FCS for 30 min. MRTF-A localisation was assessed by immunofluorescence (N, predominantly nuclear; N/C, pancellular; C, predominantly cytoplasmic), in two independent experiments. (D) Cells depleted of endogenous MRTFs were transfected with the MRTF-A responsive 3DA-luc SRF reporter and increasing amounts of wild-type MRTF-A or MRTF-A 26A (3, 6, 12, 25, 50, 100 ng). After treatment with 15% FCS or 2 μM CD, cell extracts were assessed for luciferase activity. Two independent experiments, data presented as mean ± half-range. (E) Cells were transfected with 3DA-luc and MRTF-A derivatives as in (D), and luciferase activity measured 24 hr later. Three independent experiments, data presented as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.15460.005

Figure 2—figure supplement 1. Location of MRTF-A phosphorylation sites.

The sequence of mouse MRTF-A(fl) is shown, with the initiation codon for MRTF-A(met) highlighted in green. RPEL motifs are underlined, the ERK- and SRF-binding sequences highlighted in blue, and the bipartite NLS highlighted in grey. Residues identified as phosphorylated in our analysis are in red; those substituted by alanine in MRTF-A26A are highlighted. Residues identified as phosphorylated in high-throughput analyses of mouse and human MRTF-A in the Phosphositeplus database are highlighted in yellow (human-only overlined) (http://www.phosphosite.org/proteinAction.action?id=15910&showAllSites=true/), and those in the Phosida database in grey (see http://141.61.102.18/phosida/index.aspx). Residues S544, T545 and S549, identified as putative ERK phosphorylation sites (Muehlich et al., 2008) and mutated to alanine in our mutant STS/A, are highlighted in purple. Sites S211, S216, S222, S225, S231, S406, S408, T409, S412, S414, S541, T542, T545, S549, S553, S605 and S662 have also been detected in MEFs by others (Hsu et al., 2011; Wu et al., 2012; Yu et al., 2011). Sites S541, T545, S549 and S553 correspond to a putative primed GSK3 phosphorylation site conserved in myocardin, which apparently negatively regulates its activity by promoting degration (Badorff et al., 2005; Xie et al., 2009). Sites S98, T545 and S549 have also been detected in EGF-stimulated HeLa cells (Olsen et al., 2006).

DOI: http://dx.doi.org/10.7554/eLife.15460.006

Figure 2—figure supplement 2. Detection of MRTF-A phosphorylation with phospho-specific antibodies.

(A) Cells were transfected with Flag-MRTF-A wild-type or mutant E3, in which sites including S98, S231, S248, S605, S663, S708, S744, S775, S785, S867, T879, S897 and T899 were substituted by alanine were stimulated with 15% serum or 2 μM cytochalasin D for the indicated times. Phosphorylation was detected by immunoblotting with the indicated phosphospecific antibodies. (B) Cells were transfected as in (A) and stimulated with 15% FCS, 2 μM cytochalasin D or 100 ng/ml TPA, in the presence of 10 μM U0126 as indicated before analysis with the indicated antibodies. (C) Cells were transfected as in (A) and stimulated with 100 ng/ml TPA, in the presence of 10 μM U0126 as indicated, before analysis with the indicated antibodies. (D) Cells transfected as in A, and analysed with anti-pS33 antiserum in the presence of unphosphorylated S33 peptide (SAAPSPQSEC).

DOI: http://dx.doi.org/10.7554/eLife.15460.007