Rorsman C, Tsioumpekou M, Heldin CH, Lennartsson J
J. Biol. Chem. 291 (22) 11608-11618 [2016-05-27; online 2016-04-05]
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.