A Cas-BCAR3 co-regulatory circuit controls lamellipodia dynamics

  1. Elizabeth M Steenkiste
  2. Jason D Berndt
  3. Carissa Pilling
  4. Christopher Simpkins
  5. Jonathan A Cooper  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States

Abstract

Integrin adhesion complexes regulate cytoskeletal dynamics during cell migration. Adhesion activates phosphorylation of integrin-associated signaling proteins, including Cas (p130Cas, BCAR1), by Src-family kinases. Cas regulates leading-edge protrusion and migration in cooperation with its binding partner, BCAR3. However, it has been unclear how Cas and BCAR3 cooperate. Here, using normal epithelial cells, we find that BCAR3 localization to integrin adhesions requires Cas. In return, Cas phosphorylation, as well as lamellipodia dynamics and cell migration, requires BCAR3. These functions require the BCAR3 SH2 domain and a specific phosphorylation site, Tyr 117, that is also required for BCAR3 downregulation by the ubiquitin-proteasome system. These findings place BCAR3 in a co-regulatory positive-feedback circuit with Cas, with BCAR3 requiring Cas for localization and Cas requiring BCAR3 for activation and downstream signaling. The use of a single phosphorylation site in BCAR3 for activation and degradation ensures reliable negative feedback by the ubiquitin-proteasome system.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files, with the exception of the raw mass spectrometry data, which have been deposited in the Dryad Digital Repository.

The following data sets were generated

Article and author information

Author details

  1. Elizabeth M Steenkiste

    Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6452-2340
  2. Jason D Berndt

    Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    No competing interests declared.
  3. Carissa Pilling

    Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    No competing interests declared.
  4. Christopher Simpkins

    Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3174-6609
  5. Jonathan A Cooper

    Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    For correspondence
    jcooper@fhcrc.org
    Competing interests
    Jonathan A Cooper, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8626-7827

Funding

National Institutes of Health (T32 GM007270)

  • Elizabeth M Steenkiste

National Institutes of Health (R01 GM109463)

  • Elizabeth M Steenkiste
  • Jason D Berndt
  • Carissa Pilling
  • Christopher Simpkins
  • Jonathan A Cooper

National Institutes of Health (P30 CA015704)

  • Elizabeth M Steenkiste
  • Jason D Berndt
  • Carissa Pilling
  • Christopher Simpkins
  • Jonathan A Cooper

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

Reviewing Editor

  1. Margaret C Frame, University of Edinburgh, United Kingdom

Version history

  1. Received: January 30, 2021
  2. Accepted: June 21, 2021
  3. Accepted Manuscript published: June 25, 2021 (version 1)
  4. Version of Record published: July 8, 2021 (version 2)

Copyright

© 2021, Steenkiste 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

  • 676
    Page views
  • 66
    Downloads
  • 1
    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. Elizabeth M Steenkiste
  2. Jason D Berndt
  3. Carissa Pilling
  4. Christopher Simpkins
  5. Jonathan A Cooper
(2021)
A Cas-BCAR3 co-regulatory circuit controls lamellipodia dynamics
eLife 10:e67078.
https://doi.org/10.7554/eLife.67078

Further reading

    1. Cell Biology
    Samarpan Maiti, Kaushik Bhattacharya ... Didier Picard
    Research Article

    Cells are exposed to a wide variety of internal and external stresses. Although many studies have focused on cellular responses to acute and severe stresses, little is known about how cellular systems adapt to sublethal chronic stresses. Using mammalian cells in culture, we discovered that they adapt to chronic mild stresses of up to two weeks, notably proteotoxic stresses such as heat, by increasing their size and translation, thereby scaling the amount of total protein. These adaptations render them more resilient to persistent and subsequent stresses. We demonstrate that Hsf1, well known for its role in acute stress responses, is required for the cell size increase, and that the molecular chaperone Hsp90 is essential for coupling the cell size increase to augmented translation. We term this translational reprogramming the ‘rewiring stress response’, and propose that this protective process of chronic stress adaptation contributes to the increase in size as cells get older, and that its failure promotes aging.

    1. Cell Biology
    2. Medicine
    Thao DV Le, Dianxin Liu ... Julio E Ayala
    Research Article Updated

    The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.