Mechanisms of modulation of PDGFRβ signaling

Sammanfattning: Platelet-derived growth factors (PDGF) constitute a family of five functional dimers that bind to two structurally related tyrosine kinase receptors i.e. PDGF receptor α and β (PDGFRα and PDGFRβ, respectively), controlling cell growth, proliferation, and migration in cells of mesenchymal origin. However, the aberrant activation of PDGF-induced intracellular signalling pathways is a frequent event in cancer. Therefore, the aim of this thesis has been to discover novel molecular mechanisms of modulation of PDGFRβ signalling. Since mitogen-activated protein (MAP) kinases are activated in PDGF signalling and their spatiotemporal activity is defined by a balance in phosphorylation and dephosphorylation events, in paper I we focused on dual-specificity MAPK phosphatases (MPKs or DUSPs).  We found MKP2/DUSP4 to be induced in response to PDGF-BB stimulation. We then demonstrated that the expression of MKP2/DUSP4 was dependent on ERK1/2 activation and on the STAT3/p53 signalling.  Endocytosis of RTKs is another mechanism that serves for signal attenuation and termination and this process can be regulated by ubiquitination or deubiquitination of cell-surface receptors. In paper II, we have identified that ubiquitin specific proteases USP4 and USP17 act as deubiquitinases (DUBs) for PDGFRβ. Both deubiquitinases impacted the timing of PDGFRβ trafficking and prolonged STAT3 activation. Consequently, high transcriptional activity of STAT3 led to the increased expression of STAT3-inducible genes c-MYC, CSF1, JUNB and CDKN1A. USP4 deletion attenuated cell proliferation in response to PDGF-BB stimulation. The family of Cbl E3 ligases is essential for ubiquitination of PDGFRβ upon ligand stimulation, followed by the receptor internalization from the cell surface and downregulation of signalling. In paper III, we have identified a new E3 ligase, i.e. tripartite motif-containing protein TRIM21, that deubiquitinates PDGFRβ and regulates its basal levels and its availability on the cell surface in a PDGF-BB independent manner. In paper IV, we described a regulatory role of the endoribonuclease Ras GTPase-activating protein-binding protein 1 (G3BP1) in PDGF signalling. G3BP1 was identified as a PDGFRβ interacting protein which also interacts with BAF155, a core component of SWI/SNF chromatin remodelling complex. G3BP1 depletion upregulated c-FOS, c-MYC and c-JUN mRNA and negatively affected STAT3 and ERK1/2 mRNA and protein levels, stalling cell proliferation. Collectively, we present new mechanisms that regulate PDGF signalling by controlling either PDGFRβ protein levels, availability on the cell surface, subcellular trafficking or activation of downstream signalling affecting regulation of cell proliferation.