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RAS GTPase-activating protein-binding protein 1 (G3BP1) is a component of the RAS signaling pathway and a phosphorylation-dependent RNA/DNA endoribonuclease that links signal transduction and RNA metabolism. We identified G3BP1 as a nuclear interactor of platelet-derived growth factor receptor-β, forming a complex with BAF155, a component of the SWI/SNF chromatin remodeling complex, as well as with the transcription factor STAT3. Depletion of G3BP1 in human primary fibroblasts AG1523 reduced PDGF-BB-induced activation of STAT3 while increasing mRNA levels of FOS, MYC, and CCND1, which encode proteins involved in growth stimulation. Both STAT1 and cyclin D1 mRNA and protein levels were elevated upon G3BP1 knockdown, identifying G3BP1 as a negative regulator of STAT1 and cyclin D1 expression. G3BP1 depletion did not abolish PDGF-BB-induced proliferation of human primary fibroblasts. Thus, G3BP1 interacts with the SWI/SNF chromatin remodeling complex in the nucleus and regulates cell growth pathways by modulating STAT signaling and transcription of growth-associated genes.

The ability to analyze whether DNA contains lesions is essential in identifying mutagenic substances. Currently, the detection of single-stranded DNA breaks (SSBs) lacks precision. To address this limitation, we develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq), which enables the mapping of SSBs. The method requires a highly error-prone DNA polymerase, so we engineer a chimeric DNA polymerase, Sloppymerase, capable of replicating DNA in the absence of one nucleotide. Following the omission of a specific nucleotide (e.g., dATP) from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions where deoxyadenosine should occur. This mismatch pattern, coupled with the retention of sequence information flanking these sites, ensures that the identified hits are bona fide SSBs. STEEL-seq is compatible with a variety of sequencing technologies, as demonstrated using Sanger, Illumina, PacBio, and Nanopore systems. Using STEEL-seq, we determine the SSB/base pair frequency in the human genome to range between 0.7 and 3.8 x 10-6 with an enrichment in active promoter regions.

The active form of vitamin D, 1,25-dihydroxyvitamin D₃, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D₃. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D₃-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D₃-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D₃. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D₃. 1,25-Dihydroxyvitamin D₃ increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D₃-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D₃-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D₃ levels.

This study explored the signaling interplay between the vitamin D receptor (VDR) and receptor tyrosine kinases (RTKs). Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB promotes cell proliferation in normal and cancer cells. At the same time, the active form of vitamin D (1,25(OH)₂-vitamin D₃) inhibits proliferation in some cells. Although EGF receptors (EGFR) and PDGF receptors (PDGFR) activate similar downstream pathways, we found that they interact with VDR signaling in distinct ways. We confirmed that 1,25(OH)₂-vitamin D₃ induces CYP24A1 gene expression in U2OS, T98G, and U251 cells. We found this to be potentiated when combined with EGF. In contrast, PDGF-BB did not impact 1,25(OH)₂-vitamin D₃-induced CYP24A1 expression in U2OS cells. The increase in CYP24A1 expression due to the combined action of EGF and 1,25(OH)₂-vitamin D₃ was dependent on AKT and ERK1/2 activation. Another VDR-responsive gene, CYP27B1, was unaffected by the addition of EGF, suggesting that EGF may have gene-specific effects on VDR signaling. While PDGF-BB did not influence CYP24A1 expression, 1,25(OH)₂-vitamin D₃ significantly influenced PDGF-BB-induced receptor phosphorylation and cell proliferation. In summary, we found that EGF, but not PDGF-BB, influenced the expression of the VDR-dependent gene CYP24A1, while 1,25(OH)₂-vitamin D₃ had an inhibitory effect on PDGFR signaling and proliferation. These findings highlight unique crosstalk between 1,25(OH)₂-vitamin D₃ signaling and EGF or PDGF-BB.

This study investigates the role of Rho GTPases, specifically Cdc42, Rac1, and RhoA, in platelet-derived growth factor receptors (PDGFRα and PDGFRβ) signaling. Signal transducer and activator of transcription (STAT) proteins, essential for cellular processes such as proliferation and immune response, are activated downstream of PDGFRs. Dysregulation of these pathways is linked to various diseases, including cancer. The current study examines the effects of Rho GTPase depletion on PDGFR phosphorylation, STAT protein stability, and downstream signaling. Results indicate that depletion of Cdc42, Rac1, or RhoA impairs PDGFR phosphorylation and reduces STAT1 and STAT3 signaling, without significantly affecting AKT and ERK1/2 pathways. The findings highlight the critical regulatory roles of Rho GTPases in PDGFR-mediated STAT signaling.

Platelet-derived growth factor (PDGF)-induced signalling via PDGF receptor β (PDGFRβ) leads to activation of downstream signalling pathways which regulate multiple cellular responses. It is unclear how PDGFRβ is degraded; both lysosomal and proteasomal degradation have been suggested. In this study, we have characterised the proteolytic cleavage of ligand-activated PDGFRβ, which results in two fragments: a larger fragment containing the extracellular domain, the transmembrane segment, and a part of the intracellular juxtamembrane region with a molecular mass of ∼130 kDa, and an intracellular ∼70 kDa fragment released into the cytoplasm. The proteolytic processing did not take place without internalisation of PDGFRβ. In addition, chelation of intracellular Ca²⁺ inhibited proteolytic processing. Inhibition of the proteasome affected signal transduction by increasing the phosphorylation of PDGFRβ, PLCγ, and STAT3 while reducing it on Erk1/2 and not affecting Akt. The proteolytic cleavage was observed in fibroblasts or cells that had undergone epithelial-mesenchymal transition.

The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.

Background

The platelet-derived growth factor (PDGF) family of ligands exerts their cellular effects by binding to α- and β-tyrosine kinase receptors (PDGFRα and PDGFRβ, respectively). SUMOylation is an important posttranslational modification (PTM) which regulates protein stability, localization, activation and protein interactions. A mass spectrometry screen has demonstrated SUMOylation of PDGFRα. However, the functional role of SUMOylation of PDGFRα has remained unknown.

Results

In the present study, we validated that PDGFRα is SUMOylated on lysine residue 917 as was previously reported using a mass spectrometry approach. Mutation of lysine residue 917 to arginine (K917R) in PDGFRα substantially decreased SUMOylation, indicating that this amino acid residue is a major SUMOylation site. Whereas no difference in the stability of wild-type and mutant receptor was observed, the K917R mutant PDGFRα was less ubiquitinated than wild-type PDGFRα. The internalization and trafficking of the receptor to early and late endosomes were not affected by the mutation, neither was the localization of the PDGFRα to Golgi. However, the K917R mutant PDGFRα showed delayed activation of PLC-γ and enhanced activation of STAT3. Functional assays showed that the mutation of K917 of PDGFRα decreased cell proliferation in response to PDGF-BB stimulation.

Conclusions

SUMOylation of PDGFRα decreases ubiquitination of the receptor and affects ligand-induced signaling and cell proliferation.

Activation of platelet-derived growth factor (PDGF) receptors α and β (PDGFRα and PDGFRβ) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRβ in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRβ. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRβ in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRβ in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRβ, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRβ, thus suggesting that ubiquitination of PDGFRβ by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.

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 beta (PDGFR beta) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFR beta 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 PDFGR beta lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.