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The Ubiquitin Ligases c-Cbl and Cbl-b Negatively Regulate Platelet-derived Growth Factor (PDGF) BB-induced Chemotaxis by Affecting PDGF Receptor beta (PDGFR beta) Internalization and Signaling
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 22, p. 11608-11618Article in journal (Refereed) Published
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Text
Abstract [en]

Protein ubiquitination controls protein stability and subcellular localization of tyrosine kinase receptors, hence affecting signaling both quantitatively and qualitatively. In this report, we demonstrate that, after ligand stimulation, the PDGF beta receptor (PDGFR beta) becomes ubiquitinated in a manner requiring both the c-Cbl and Cbl-b ubiquitin ligases. Simultaneous depletion of c-Cbl and Cbl-b resulted in reduced ligand-induced PDGFR beta clearance from the cell surface because of reduced endocytosis of the receptor. Cbl-b formed a complex with c-Cbl, as well as with the PDGFR beta, in response to PDGF-BB stimulation. We were unable to find a direct interaction between the receptor and c-Cbl, raising the possibility that Cbl-b is necessary for c-Cbl to interact with PDGFR beta. Phosphorylated Tyr-1021 in PDGFR beta was the primary interaction site for Cbl-b, with some contribution from Tyr-1009. Depletion of c-Cbl and Cbl-b led to an increased ligand-induced tyrosine phosphorylation of the receptor. Several tyrosine residues with elevated phosphorylation (i.e. Tyr-579, Tyr-581, Tyr-1009, and Tyr-1021) have previously been shown to interact with Src kinases and PLC gamma. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLC gamma phosphorylation were enhanced, whereas activation of other pathways, such as Erk1/2 MAP kinase and Akt, were not affected. In addition, Stat3 phosphorylation, which has been connected to Src activity, was also elevated in cells lacking c-Cbl and Cbl-b. Functionally, we found that cells depleted of c-Cbl and Cbl-b were more prone to migrate toward PDGF-BB, whereas no reproducible effect on cell proliferation could be observed. In conclusion, internalization as well as signaling via PDGFR beta are controlled by ubiquitination.

Place, publisher, year, edition, pages
2016. Vol. 291, no 22, p. 11608-11618
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-299504DOI: 10.1074/jbc.M115.705814ISI: 000377264800013PubMedID: 27048651OAI: oai:DiVA.org:uu-299504DiVA, id: diva2:949688
Funder
Swedish Cancer SocietyAvailable from: 2016-07-22 Created: 2016-07-22 Last updated: 2019-02-03Bibliographically approved
In thesis
1. Studies of PDGF receptor signaling in vitro and in vivo
Open this publication in new window or tab >>Studies of PDGF receptor signaling in vitro and in vivo
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelet-derived growth factor receptor (PDGFR) signaling is essential for proliferation, migration and survival of cells of mesenchymal origin; however, its deregulation has been associated with various diseases, including cancer. The aim of this thesis was to clarify the molecular mechanisms of PDGFR signaling regulation. We have studied PDGFR downregulation, identified the E3 ligases and deubiquitinases (DUBs) acting on the receptor, characterized the role of the downstream effector Erk5, as well as elucidated the role of PDGFRβ isoform in tumor growth and angiogenesis.

As Erk5 activation has been associated with tumorigenesis, it is important to delineate the pathway from activated PDGFR to Erk5. Here, we demonstrate not only a complex mechanism for PDGF-induced Erk5 activation that involves Mek5, Mekk2, Mek1/2, PI3K and classical PKCs, but also a novel function for Erk5 by showing that PDGF-BB affects BMP-Smad signaling in an Erk5 pathway-dependent manner, indicating a crosstalk between tyrosine kinase receptor and serine/threonine receptor signaling.

By investigating PDGFRβ downregulation, we demonstrated that ubiquitination of PDGFRβ, mediated by Cbl-b and c-Cbl, is essential for the receptor internalization, signaling, as well as downstream biological responses. Additionally, as ubiquitination is a reversible post-translational modification, we identified USP4 as one of the DUBs acting on PDGFRβ and discovered that USP4 interacts with PDGFRβ, removing both K48- and K63-linked polyubiquitin chains, and increases its stability, in both normal and cancer cells.

Although several studies have highlighted the therapeutic benefit of PDGFR inhibition in cancer treatment, all available PDGFR kinase inhibitors have secondary targets; consequently, the details underlying the importance of PDGFR in tumorigenesis remain unknown. By targeting specifically PDGFRβ in the stroma of various tumor models, we showed that specific inhibition of PDGFRβ signaling suppresses growth of tumors with high levels of PDGF-BB, whereas the multi-target kinase inhibitor imatinib has less effect, indicating the significance of selective targeting of PDGFRβ.

Our data provide new insights into the molecular events underlying PDGFRβ signaling and downregulation, highlight its importance as cancer therapeutic target and lead the way for further discoveries.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 67
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1538
Keywords
PDGF, signaling, cancer, Erk5, kinase, Cbl, ubiquitination, deubiquitinase, pericytes, tumor growth, angiogenesis, low molecular weight inhibitors
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-376248 (URN)978-91-513-0571-4 (ISBN)
Public defence
2019-03-22, B42, Biomedical Center, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-02-27 Created: 2019-02-03 Last updated: 2019-03-18

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Rorsman, CharlotteTsioumpekou, MariaHeldin, Carl-HenrikLennartsson, Johan

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