| 1. |
- Delis, Costas, et al.
(författare)
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AtHESPERIN : a novel regulator of circadian rhythms with poly(A)-degrading activity in plants
- 2016
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Ingår i: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 13:1, s. 68-82
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Tidskriftsartikel (refereegranskat)abstract
- We report the identification and characterization of a novel gene, AtHesperin (AtHESP) that codes for a deadenylase in Arabidopsis thaliana. The gene is under circadian clock-gene regulation and has similarity to the mammalian Nocturnin. AtHESP can efficiently degrade poly(A) substrates exhibiting allosteric kinetics. Size exclusion chromatography and native electrophoresis coupled with kinetic analysis support that the native enzyme is oligomeric with at least 3 binding sites. Knockdown and overexpression of AtHESP in plant lines affects the expression and rhythmicity of the clock core oscillator genes TOC1 and CCA1. This study demonstrates an evolutionary conserved poly(A)-degrading activity in plants and suggests deadenylation as a mechanism involved in the regulation of the circadian clock. A role of AtHESP in stress response in plants is also depicted.
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| 2. |
- Reyhani, Vahid, et al.
(författare)
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PDGF-BB enhances collagen gel contraction through a PI3K-PLCγ-PKC-cofilin pathway
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Cell-mediated contraction of collagenous matrices is modulated by various growth factors and cytokines, such as platelet-derived growth factor-BB (PDGF-BB). Here we used a genetic cell model to delineate defined signaling pathways that enhance collagen gel contraction downstream of ligand-stimulated platelet-derived growth factor receptor-β (PDGF-Rβ). Our data show that PDGF BB-enhanced activations of phosphatidylinositol 3'-kinase (PI3K) and phospholipase Cγ (PLCγ) were necessary for PDGF-enhanced collagen gel contraction. Importantly, other defined signaling pathways down-stream of PDGF-Rβ were, however, dispensable. The decisive roles for PI3K and PLCγ were corroborated by experiments using selective inhibitors. Furthermore, we show that de-phosphorylation and thereby activation of cofilin that is important for the turnover of actin filaments, is depended on PI3K and PLCγ down-stream of PDGF-Rβ. Moreover, inhibition of protein kinase C (PKC) by GÖ6976 and bisindolylmaleimide-II abolished cofilin de-phosphorylation, as well as PDGF-enhanced contraction. In contrast, activation of the PKC protein family by 4β-phorbol 12-myristate 13-acetate (PMA) did not accelerate collagen gel contraction although it induced long-term cofilin de-phosphorylation, showing the need of a dynamic control of cofilin de-phosphorylation for PDGF-enhanced collagen gel contraction. Taken together, our data point to the involvement of a PI3K/PLCγ-PKC-cofilin pathway in both PDGF-enhanced cofilin de-phosphorylation and PDGF-enhanced collagen gel contraction.
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| 3. |
- Rorsman, Charlotte, et al.
(författare)
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The Ubiquitin Ligases c-Cbl and Cbl-b Negatively Regulate Platelet-derived Growth Factor (PDGF) BB-induced Chemotaxis by Affecting PDGF Receptor beta (PDGFR beta) Internalization and Signaling
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:22, s. 11608-11618
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Tidskriftsartikel (refereegranskat)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 beta receptor (PDGFR beta) 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 PDGFR beta 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 PDGFR beta, 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 PDGFR beta. Phosphorylated Tyr-1021 in PDGFR beta 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 PLC gamma. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLC gamma 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 PDGFR beta are controlled by ubiquitination.
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| 4. |
- Sarri, Niki, et al.
(författare)
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Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFR beta and affect PDGF-BB-induced STAT3 signalling
- 2022
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer. - 1420-682X .- 1420-9071. ; 79:2
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Tidskriftsartikel (refereegranskat)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 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.
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| 5. |
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| 6. |
- Tsioumpekou, Maria, et al.
(författare)
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Platelet-derived growth factor (PDGF)-induced activation of Erk5 MAP-kinase is dependent on Mekk2, Mek1/2, PKC and PI3-kinase, and affects BMP signaling
- 2016
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Ingår i: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 28:9, s. 1422-1431
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Tidskriftsartikel (refereegranskat)abstract
- Platelet-derived growth factor-BB (PDGF-BB) binds to its tyrosine kinase receptors (PDGFRs) and stimulates mitogenicity and survival of cells of mesenchymal origin. Activation of PDGFRs initiates a number of downstream signaling pathways, including phosphatidyl 3'-inositol kinase (PI3-kinase), phospholipase C gamma and MAP kinase pathways. In this report, we show that Erk5 MAP kinase is activated in response to PDGF-BB in the smooth muscle cell line MOVAS in a manner dependent on Mekk2, Mek1/2, Mek5, PI3-kinase and protein kinase C (PKC). The cooperation of Mek1/2 and Mekk2 in the activation of Erk5, suggests a close co-regulation between the Erk1/2 and Erk5 MAP kinase pathways. Furthermore, we found that classical PKCs are important for Erk5 activation. In addition, we found that PKC zeta interacts with Erk5 and may exert a negative feed-back effect. We observed no nuclear accumulation of Erk5 in response to PDGF-BB stimulation, however, we identified a mechanism by which cytoplasmic Erk5 influences gene expression; Erk5 was essential for PDGF-BB-mediated Smad1/5/8 signaling by stimulating release and/or activation of bone morphogenetic protein(s) (BMPs). Thus, PDGF-BB-induced Erk5 activation involves parallel stimulatory and inhibitory pathways and promotes Smad1/5/8 signaling. (C) 2016 Published by Elsevier Inc.
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| 7. |
- Tsioumpekou, Maria, et al.
(författare)
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Specific targeting of PDGFR beta in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression
- 2020
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Ingår i: Theranostics. - : IVYSPRING INT PUBL. - 1838-7640. ; 10:3, s. 1122-1135
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Tidskriftsartikel (refereegranskat)abstract
- PDGF-BB/PDGFR beta signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFR beta signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFR beta ATP binding site that allows specific targeting of PDGFR beta using the compound 1-NaPP1. To evaluate the impact of selective PDGFR beta inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFR beta expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). Results: We found that specific impairment of PDGFR beta kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFR beta kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFR beta by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFR beta by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFR beta ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data. Conclusion: Specific targeting of PDGFR beta signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFR beta in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.
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| 8. |
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| 9. |
- Tsioumpekou, Maria
(författare)
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Studies of PDGF receptor signaling in vitro and in vivo
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Platelet-derived growth factor receptor (PDGFR) signaling is essential for proliferation, migration and survival of cells of mesenchymal origin; however, its deregulation has been associated with various diseases, including cancer. The aim of this thesis was to clarify the molecular mechanisms of PDGFR signaling regulation. We have studied PDGFR downregulation, identified the E3 ligases and deubiquitinases (DUBs) acting on the receptor, characterized the role of the downstream effector Erk5, as well as elucidated the role of PDGFRβ isoform in tumor growth and angiogenesis.As Erk5 activation has been associated with tumorigenesis, it is important to delineate the pathway from activated PDGFR to Erk5. Here, we demonstrate not only a complex mechanism for PDGF-induced Erk5 activation that involves Mek5, Mekk2, Mek1/2, PI3K and classical PKCs, but also a novel function for Erk5 by showing that PDGF-BB affects BMP-Smad signaling in an Erk5 pathway-dependent manner, indicating a crosstalk between tyrosine kinase receptor and serine/threonine receptor signaling.By investigating PDGFRβ downregulation, we demonstrated that ubiquitination of PDGFRβ, mediated by Cbl-b and c-Cbl, is essential for the receptor internalization, signaling, as well as downstream biological responses. Additionally, as ubiquitination is a reversible post-translational modification, we identified USP4 as one of the DUBs acting on PDGFRβ and discovered that USP4 interacts with PDGFRβ, removing both K48- and K63-linked polyubiquitin chains, and increases its stability, in both normal and cancer cells.Although several studies have highlighted the therapeutic benefit of PDGFR inhibition in cancer treatment, all available PDGFR kinase inhibitors have secondary targets; consequently, the details underlying the importance of PDGFR in tumorigenesis remain unknown. By targeting specifically PDGFRβ in the stroma of various tumor models, we showed that specific inhibition of PDGFRβ signaling suppresses growth of tumors with high levels of PDGF-BB, whereas the multi-target kinase inhibitor imatinib has less effect, indicating the significance of selective targeting of PDGFRβ.Our data provide new insights into the molecular events underlying PDGFRβ signaling and downregulation, highlight its importance as cancer therapeutic target and lead the way for further discoveries.
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| 10. |
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