Abstract
Myocardin-related transcription factor A (MRTF-A) is a known actin-regulated transcriptional coactivator of serum response factor (SRF). Stimulation of actin polymerization activates MRTF-A by releasing it from G-actin and thus allowing it to bind to and activate SRF. Here, we compared protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2 in two independent phosphoproteomic approaches using anisomycin-treated MEF cells and LPS-stimulated mouse macrophages, respectively. Two MRTF-A sites, Ser351 (corresponding to Ser312 in human) and Ser371 (Ser333 in human), showed significantly stronger phosphorylation (12-fold and 6-fold increase) in the cells expressing MK2. MRTF-A is phosphorylated at these sites in a stress-, but not in a mitogen-induced manner, and p38MAPK/MK2 catalytic activities are indispensable for this phosphorylation. MK2-mediated phosphorylation of MRTF-A at Ser312 and Ser333 was further confirmed in an in vitro kinase assay and using the phospho-protein kinase-D (PKD)-consensus motif antibody (anti-LXRXXpS/pT), the p38MAPK inhibitor BIRB-796, MK2/3-deficient cells and MRTF-A phospho-site mutants. Unexpectedly, dimerization, subcellular localization and translocation, interaction with actin, SRF or SMAD3 and transactivating potential of MRTF-A seem to be unaffected by manipulating the p38MAPK/MK2-dependent phosphorylations. Hence, MRTF-A is stress-dependently phosphorylated by MK2 at Ser312 and Ser333 with so far undetected functional and physiological consequences.
Highlights
It should be noted that the sites S351 and S371 identified in mouse correspond to S312 and S333 in human myocardin-related transcription factors (MRTFs)-A, respectively
To analyse whether MRTF-A overexpression interferes with stress-induced immediate early genes (IEGs) expression, we used MK2/3 DKO mouse embryonic fibroblasts (MEF) transduced with the retroviral TetOn human MRTF-A (hMRTF-A) and rescued or not with MK2
MRTF-A is a transcriptional coactivator of SRF linking actin dynamics with transcription of SRF-dependent genes encoding contractile and cytoskeletal proteins[5,13,26]
Summary
We monitored the p38MAPK-dependence of MRTF-A phosphorylation in HeLa cells transfected with an expression construct for Flag-tagged hMRTF-A. MRTF-A detection by the PKD motif antibodies was strongly increased after anisomycin treatment and almost completely suppressed by application of the p38 inhibitor BIRB796 indicating that MRTF-A is phosphorylated upon stress in a p38-dependent manner (Fig. 2A).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
