Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes

  1. Fangfei Qu
  2. Damaris N Lorenzo
  3. Samantha J King
  4. Rebecca Brooks
  5. James E Bear
  6. Vann Bennett  Is a corresponding author
  1. Duke University Medical Center, United States
  2. The University of North Carolina at Chapel Hil, United States

Abstract

Endosomal membrane trafficking requires coordination between phosphoinositide lipids, Rab GTPases, and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport. Here we report that Ankyrin-B (AnkB), through integrating all three systems, functions as a critical node in the protein circuitry underlying polarized recycling of α5β1-integrin in mouse embryonic fibroblasts, which enables persistent fibroblast migration along fibronectin gradients. AnkB associates with phosphatidylinositol 3-phosphate (PI3P)-positive organelles in fibroblasts and binds dynactin to promote their long-range motility. We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like (RabGAP1L) and recruits it to PI3P-positive organelles, where RabGAP1L inactivates Rab22A, and promotes polarized trafficking to the leading edge of migrating fibroblasts. We further determine that α5β1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling. Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5β1-integrin and likely of other specialized endocytic cargos.

Article and author information

Author details

  1. Fangfei Qu

    Department of Cell Biology, Duke University Medical Center, Durham, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Damaris N Lorenzo

    Department of Cell Biology, Duke University Medical Center, Durham, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Samantha J King

    UNC Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hil, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Rebecca Brooks

    UNC Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hil, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. James E Bear

    UNC Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hil, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Vann Bennett

    Department of Cell Biology, Duke University Medical Center, Durham, United States
    For correspondence
    vann.bennett@duke.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2695-7209

Funding

Howard Hughes Medical Institute

  • Fangfei Qu
  • Damaris N Lorenzo
  • Vann Bennett

National Institutes of Health (National Institute of Health grant GM110155)

  • Samantha J King
  • Rebecca Brooks
  • James E Bear

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed strictly following the guide for the laboratory animal care and use at Duke University Medical Center. All of the animals were handled according to approved Institutional Animal Care and Use Committee (IACUC) protocol (# A149-15-05) of Duke University.

Reviewing Editor

  1. Johanna Ivaska, University of Turku, Finland

Publication history

  1. Received: August 6, 2016
  2. Accepted: October 7, 2016
  3. Accepted Manuscript published: October 8, 2016 (version 1)
  4. Version of Record published: November 1, 2016 (version 2)

Copyright

© 2016, Qu 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

  • 2,337
    Page views
  • 631
    Downloads
  • 17
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Fangfei Qu
  2. Damaris N Lorenzo
  3. Samantha J King
  4. Rebecca Brooks
  5. James E Bear
  6. Vann Bennett
(2016)
Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes
eLife 5:e20417.
https://doi.org/10.7554/eLife.20417

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Morgane Boone et al.
    Research Advance Updated

    In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Florian Bleffert et al.
    Research Article Updated

    Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.