title: The Ubiquitin Ligases c-Cbl and Cbl-b Negatively Regulate Platelet-derived Growth Factor (PDGF) BB-induced Chemotaxis by Affecting PDGF Receptor β (PDGFRβ) Internalization and Signaling.
abstract: 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 β receptor (PDGFRβ) 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 PDGFRβ 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 PDGFRβ, 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 PDGFRβ. Phosphorylated Tyr-1021 in PDGFRβ 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 PLCγ. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLCγ 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 PDGFRβ are controlled by ubiquitination.
authors.affiliation: From the Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, 75124 Uppsala, Sweden.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Rorsman
authors.affiliation: From the Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, 75124 Uppsala, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Tsioumpekou
authors.affiliation: From the Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, 75124 Uppsala, Sweden.
authors.forename: Carl-Henrik
authors.initials: CH
authors.lastname: Heldin
authors.affiliation: From the Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, 75124 Uppsala, Sweden Johan.Lennartsson@LICR.uu.se.
authors.forename: Johan
authors.initials: J
authors.lastname: Lennartsson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000077214:Becaplermin; D015153:Blotting, Western; D002478:Cells, Cultured; D002633:Chemotaxis / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis / Q000378:metabolism*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D054875:Ubiquitination; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin; D014443:Tyrosine; C492211:CBLB protein, human; D050721:Proto-Oncogene Proteins c-cbl; D020797:Receptor, Platelet-Derived Growth Factor beta; D016315:Proto-Oncogene Proteins c-abl; D019061:src-Family Kinases; D048052:Mitogen-Activated Protein Kinase 3
keywords: CBLB; E3 ubiquitin ligase; PLC; STAT3; Src; chemotaxis; protein kinase; receptor tyrosine kinase