Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

Abstract

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs, and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in regulation of MRTF-A.

Article and author information

Author details

  1. Richard Panayiotou

    Signaling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Francesc Miralles

    Signaling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Rafal Pawlowski

    Signaling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Jessica Diring

    Signaling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Helen R Flynn

    Signaling and Transcription Group, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Mark Skehel

    Mass Spectrometry science technology platform, Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Richard Treisman

    Signalling and Transcription Group, Francis Crick Institute, London, United Kingdom
    For correspondence
    Richard.Treisman@Crick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

Copyright

© 2016, Panayiotou et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,850
    views
  • 711
    downloads
  • 67
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Richard Panayiotou
  2. Francesc Miralles
  3. Rafal Pawlowski
  4. Jessica Diring
  5. Helen R Flynn
  6. Mark Skehel
  7. Richard Treisman
(2016)
Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity
eLife 5:e15460.
https://doi.org/10.7554/eLife.15460

Share this article

https://doi.org/10.7554/eLife.15460

Further reading

    1. Cell Biology
    2. Computational and Systems Biology
    Sarah De Beuckeleer, Tim Van De Looverbosch ... Winnok H De Vos
    Research Article

    Induced pluripotent stem cell (iPSC) technology is revolutionizing cell biology. However, the variability between individual iPSC lines and the lack of efficient technology to comprehensively characterize iPSC-derived cell types hinder its adoption in routine preclinical screening settings. To facilitate the validation of iPSC-derived cell culture composition, we have implemented an imaging assay based on cell painting and convolutional neural networks to recognize cell types in dense and mixed cultures with high fidelity. We have benchmarked our approach using pure and mixed cultures of neuroblastoma and astrocytoma cell lines and attained a classification accuracy above 96%. Through iterative data erosion, we found that inputs containing the nuclear region of interest and its close environment, allow achieving equally high classification accuracy as inputs containing the whole cell for semi-confluent cultures and preserved prediction accuracy even in very dense cultures. We then applied this regionally restricted cell profiling approach to evaluate the differentiation status of iPSC-derived neural cultures, by determining the ratio of postmitotic neurons and neural progenitors. We found that the cell-based prediction significantly outperformed an approach in which the population-level time in culture was used as a classification criterion (96% vs 86%, respectively). In mixed iPSC-derived neuronal cultures, microglia could be unequivocally discriminated from neurons, regardless of their reactivity state, and a tiered strategy allowed for further distinguishing activated from non-activated cell states, albeit with lower accuracy. Thus, morphological single-cell profiling provides a means to quantify cell composition in complex mixed neural cultures and holds promise for use in the quality control of iPSC-derived cell culture models.

    1. Cell Biology
    Joan Chang, Adam Pickard ... Karl E Kadler
    Research Article

    Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33B secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33B led to loss of fibril rhythmicity and overabundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33B and integrin α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33B, integrin α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.