Abstract
Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor β (PDGFRβ) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRβ did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRβ lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.
Highlights
The platelet-derived growth factor (PDGF) isoforms are mitogens and chemotactic agents for smooth muscle cells, pericytes, fibroblasts and glial cells of the brain [23], regulating embryonic development, would healing, interstitial fluid pressure and blood–brain barrier [3]
We show that the ubiquitin-specific proteases USP17 and USP4 efficiently remove ubiquitination of PDGF receptor β (PDGFRβ), affecting its internalization and early-endosomal sorting, thereby controlling the timing of PDGFRβ-induced STAT3 activation, which in turn affects transcription of STAT3-inducible genes, including STAT3, colony-stimulating factor-1 (CSF1), junB, c-myc and CDKN1A, adversely affecting cell proliferation
◂Fig. 5 USP4 and USP17L22 affect the timing of activation of STAT3. a, b Induction of USP17L22 results in a shift of STAT3 activation to later time points in BJhTERT inducible cells
Summary
The platelet-derived growth factor (PDGF) isoforms are mitogens and chemotactic agents for smooth muscle cells, pericytes, fibroblasts and glial cells of the brain [23], regulating embryonic development, would healing, interstitial fluid pressure and blood–brain barrier [3]. Ubiquitination is a post-translational modification of cellular proteins that plays important roles in, e.g., proteasomal degradation of proteins, intracellular trafficking, receptor internalization and downregulation, DNA repair and cell proliferation [43]. Ubiquitination of RTKs following ligand binding and phosphorylation is a hallmark of receptormediated endocytosis and serves to mediate interactions of RTKs with the sorting machineries at the cell surface and at endosomes [15]. USP17 was found to be required for clathrin-mediated endocytosis of EGFR and was proposed to have a general role in endocytosis [26] and to affect cell migration and plasma membrane localization of H-Ras by acting on isoform 2 of Ras converting enzyme 1 (RCE1) at the endoplasmic reticulum [9, 10, 27, 38]
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