18 results on '"Roovers O"'
Search Results
2. Malignant Transformation Involving CXXC4 Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia.
- Author
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Olofsen, P.A., Fatrai, S., Strien, P.M.H. van, Obenauer, J.C., Looper, H.W.J. de, Hoogenboezem, R.M., Erpelinck-Verschueren, C.A., Vermeulen, M.P.W.M., Roovers, O., Haferlach, T, Jansen, J.H., Ghazvini, M., Bindels, E.M., Schneider, R.K., Pater, E.M. de, Touw, I.P., Olofsen, P.A., Fatrai, S., Strien, P.M.H. van, Obenauer, J.C., Looper, H.W.J. de, Hoogenboezem, R.M., Erpelinck-Verschueren, C.A., Vermeulen, M.P.W.M., Roovers, O., Haferlach, T, Jansen, J.H., Ghazvini, M., Bindels, E.M., Schneider, R.K., Pater, E.M. de, and Touw, I.P.
- Abstract
Contains fulltext : 229624.pdf (publisher's version ) (Open Access)
- Published
- 2020
3. Malignant Transformation Involving CXXC4 Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia
- Author
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Olofsen, P.A. (Patricia A.), Fatrai, S. (Szabolcs), Strien, P.M.H. (Paulette) van, Obenauer, J. (Julia), Looper, H. (Hans) de, Hoogenboezem, R.M. (Remco), Erpelinck, C.A.J. (Claudia), Vermeulen, M.P.W.M. (Michael P.W.M.), Roovers, O. (Onno), Haferlach, T. (Torsten), Jansen, J.H. (Joop), Ghazvini, M. (Mehrnaz), Bindels, E.M.J. (Eric), Schneider-Kramann, R.K.M. (Rebekka), Pater, E. (Emma) de, Touw, I.P. (Ivo), Olofsen, P.A. (Patricia A.), Fatrai, S. (Szabolcs), Strien, P.M.H. (Paulette) van, Obenauer, J. (Julia), Looper, H. (Hans) de, Hoogenboezem, R.M. (Remco), Erpelinck, C.A.J. (Claudia), Vermeulen, M.P.W.M. (Michael P.W.M.), Roovers, O. (Onno), Haferlach, T. (Torsten), Jansen, J.H. (Joop), Ghazvini, M. (Mehrnaz), Bindels, E.M.J. (Eric), Schneider-Kramann, R.K.M. (Rebekka), Pater, E. (Emma) de, and Touw, I.P. (Ivo)
- Abstract
Olofsen et al. show that acquisition of a mutation in Cxxc4 results in increased CXXC4 protein levels, reduced TET2 protein, increased inflammatory signaling, and leukemic progression of a CSF3R/RUNX1 mutant mouse model of severe congenital neutropenia (SCN).Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.
- Published
- 2020
- Full Text
- View/download PDF
4. Peroxiredoxin-controlled G-CSF signalling at the endoplasmic reticulum-early endosome interface
- Author
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Palande, K.K. (Karishma), Roovers, O. (Onno), Gits, J. (Judith), Verwijmeren, C. (Carola), Iuchi, Y. (Yoshihito), Fujii, J. (Junichi), Neel, B., Karisch, R. (Robert), Tavernier, J., Touw, I.P. (Ivo), Palande, K.K. (Karishma), Roovers, O. (Onno), Gits, J. (Judith), Verwijmeren, C. (Carola), Iuchi, Y. (Yoshihito), Fujii, J. (Junichi), Neel, B., Karisch, R. (Robert), Tavernier, J., and Touw, I.P. (Ivo)
- Abstract
Reactive oxygen species (ROS) regulate growth factor receptor signalling at least in part by inhibiting oxidation-sensitive phosphatases. An emerging concept is that ROS act locally to affect signal transduction in different subcellular compartments and that ROS levels are regulated by antioxidant proteins at the same local level. Here, we show that the ER-resident antioxidant peroxiredoxin 4 (Prdx4) interacts with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This interaction occurs when the activated G-CSFR resides in early endosomes. Prdx4 inhibits G-CSF-induced signalling and proliferation in myeloid progenitors, depending on its redox-active cysteine core. Protein tyrosine phosphatase 1b (Ptp1b) appears to be a major downstream effector controlling these responses. Conversely, Ptp1b might keep Prdx4 active by reducing its phosphorylation. These findings unveil a new signal transduction regulatory circuitry involving redox-controlled processes in the ER and activated cytokine receptors in endosomes.
- Published
- 2011
- Full Text
- View/download PDF
5. Suppressor of cytokine signaling 3 controls lysosomal routing of G-CSF receptor
- Author
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Irandoust, M. (Mahban), Aarts, L.H.J. (Bart), Roovers, O. (Onno), Gits, J. (Judith), Erkeland, S.J. (Stefan), Touw, I.P. (Ivo), Irandoust, M. (Mahban), Aarts, L.H.J. (Bart), Roovers, O. (Onno), Gits, J. (Judith), Erkeland, S.J. (Stefan), and Touw, I.P. (Ivo)
- Abstract
The hematopoietic system provides an attractive model for studying growth factor-controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony-stimulating factor receptor (G-CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G-CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G-CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G-CSFR mutants lacking K632 show a perturbed proliferation/ differentiation balance in response to G-CSF. This is the first demonstration of SOCS-mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output.
- Published
- 2007
- Full Text
- View/download PDF
6. Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics
- Author
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Aarts, L.H.J. (Bart), Roovers, O. (Onno), Ward, A.C. (Alister), Touw, I.P. (Ivo), Aarts, L.H.J. (Bart), Roovers, O. (Onno), Ward, A.C. (Alister), and Touw, I.P. (Ivo)
- Abstract
We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position -4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.
- Published
- 2004
7. Novel role of WD40 and SOCS box protein-2 in steady-state distribution of granulocyte colony-stimulating factor receptor and G-CSF-controlled proliferation and differentiation signaling
- Author
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Erkeland, S J, primary, Aarts, L H, additional, Irandoust, M, additional, Roovers, O, additional, Klomp, A, additional, Valkhof, M, additional, Gits, J, additional, Eyckerman, S, additional, Tavernier, J, additional, and Touw, I P, additional
- Published
- 2006
- Full Text
- View/download PDF
8. Truncated CSF3 receptors induce pro-inflammatory responses in severe congenital neutropenia.
- Author
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Olofsen PA, Bosch DA, de Looper HWJ, van Strien PMH, Hoogenboezem RM, Roovers O, van der Velden VHJ, Bindels EMJ, De Pater EM, and Touw IP
- Subjects
- Mice, Animals, Congenital Bone Marrow Failure Syndromes genetics, Mutation, Leukemia, Myeloid, Acute diagnosis, Myelodysplastic Syndromes genetics, Myelodysplastic Syndromes complications
- Abstract
Severe congenital neutropenia (SCN) patients are prone to develop myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML). Leukaemic progression of SCN is associated with the early acquisition of CSF3R mutations in haematopoietic progenitor cells (HPCs), which truncate the colony-stimulating factor 3 receptor (CSF3R). These mutant clones may arise years before MDS/AML becomes overt. Introduction and activation of CSF3R truncation mutants in normal HPCs causes a clonally dominant myeloproliferative state in mice treated with CSF3. Paradoxically, in SCN patients receiving CSF3 therapy, clonal dominance of CSF3R mutant clones usually occurs only after the acquisition of additional mutations shortly before frank MDS or AML is diagnosed. To seek an explanation for this discrepancy, we introduced a patient-derived CSF3R-truncating mutation in ELANE-SCN and HAX1-SCN derived and control induced pluripotent stem cells and compared the CSF3 responses of HPCs generated from these lines. In contrast to CSF3R-mutant control HPCs, CSF3R-mutant HPCs from SCN patients do not show increased proliferation but display elevated levels of inflammatory signalling. Thus, activation of the truncated CSF3R in SCN-HPCs does not evoke clonal outgrowth but causes a sustained pro-inflammatory state, which has ramifications for how these CSF3R mutants contribute to the leukaemic transformation of SCN., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)
- Published
- 2023
- Full Text
- View/download PDF
9. PML-controlled responses in severe congenital neutropenia with ELANE-misfolding mutations.
- Author
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Olofsen PA, Bosch DA, Roovers O, van Strien PMH, de Looper HWJ, Hoogenboezem RM, Barnhoorn S, Mastroberardino PG, Ghazvini M, van der Velden VHJ, Bindels EMJ, de Pater EM, and Touw IP
- Subjects
- Adaptor Proteins, Signal Transducing, Congenital Bone Marrow Failure Syndromes, Granulocyte Colony-Stimulating Factor, Humans, Mutation, Leukocyte Elastase genetics, Neutropenia congenital, Neutropenia genetics
- Abstract
Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy., (© 2021 by The American Society of Hematology.)
- Published
- 2021
- Full Text
- View/download PDF
10. Malignant Transformation Involving CXXC4 Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia.
- Author
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Olofsen PA, Fatrai S, van Strien PMH, Obenauer JC, de Looper HWJ, Hoogenboezem RM, Erpelinck-Verschueren CAJ, Vermeulen MPWM, Roovers O, Haferlach T, Jansen JH, Ghazvini M, Bindels EMJ, Schneider RK, de Pater EM, and Touw IP
- Subjects
- Animals, Cell Line, Cell Line, Tumor, Cell Transformation, Neoplastic pathology, Core Binding Factor Alpha 2 Subunit genetics, HEK293 Cells, Humans, Inflammation genetics, Inflammation pathology, K562 Cells, Mice, Neutropenia genetics, Neutropenia pathology, Signal Transduction genetics, Cell Transformation, Neoplastic genetics, Congenital Bone Marrow Failure Syndromes genetics, Congenital Bone Marrow Failure Syndromes pathology, DNA-Binding Proteins genetics, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute pathology, Mutation genetics, Neutropenia congenital, Transcription Factors genetics
- Abstract
Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor ( CSF3R ) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R / RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)
- Published
- 2020
- Full Text
- View/download PDF
11. Lentiviral Hematopoietic Stem Cell Gene Therapy Corrects Murine Pompe Disease.
- Author
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Stok M, de Boer H, Huston MW, Jacobs EH, Roovers O, Visser TP, Jahr H, Duncker DJ, van Deel ED, Reuser AJJ, van Til NP, and Wagemaker G
- Abstract
Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA ( GAAco ), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition., (© 2020 The Author(s).)
- Published
- 2020
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12. Autophagy inhibition as a potential future targeted therapy for ETV6-RUNX1-driven B-cell precursor acute lymphoblastic leukemia.
- Author
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Polak R, Bierings MB, van der Leije CS, Sanders MA, Roovers O, Marchante JRM, Boer JM, Cornelissen JJ, Pieters R, den Boer ML, and Buitenhuis M
- Subjects
- Autophagic Cell Death genetics, Child, Child, Preschool, Class III Phosphatidylinositol 3-Kinases genetics, Class III Phosphatidylinositol 3-Kinases metabolism, Core Binding Factor Alpha 2 Subunit genetics, Female, Humans, Male, Oncogene Proteins, Fusion genetics, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma genetics, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma metabolism, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma pathology, Asparaginase pharmacology, Autophagic Cell Death drug effects, Core Binding Factor Alpha 2 Subunit metabolism, Drug Delivery Systems, Oncogene Proteins, Fusion metabolism, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma drug therapy
- Abstract
Translocation t(12;21), resulting in the ETV6-RUNX1 (or TEL-AML1) fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia and is considered a first hit in leukemogenesis. A targeted therapy approach is not available for children with this subtype of leukemia. To identify the molecular mechanisms underlying ETV6-RUNX1-driven leukemia, we performed gene expression profiling of healthy hematopoietic progenitors in which we ectopically expressed ETV6-RUNX1. We reveal an ETV6-RUNX1-driven transcriptional network that induces proliferation, survival and cellular homeostasis. In addition, Vps34, an important regulator of autophagy, was found to be induced by ETV6-RUNX1 and up-regulated in ETV6-RUNX1-positive leukemic patient cells. We show that induction of Vps34 was transcriptionally regulated by ETV6-RUNX1 and correlated with high levels of autophagy. Knockdown of Vps34 in ETV6-RUNX1-positive cell lines severely reduced proliferation and survival. Inhibition of autophagy by hydroxychloroquine, a well-tolerated autophagy inhibitor, reduced cell viability in both ETV6-RUNX1-positive cell lines and primary acute lymphoblastic leukemia samples, and selectively sensitized primary ETV6-RUNX1-positive leukemia samples to L asparaginase. These findings reveal a causal relationship between ETV6-RUNX1 and autophagy, and provide pre-clinical evidence for the efficacy of autophagy inhibitors in ETV6-RUNX1-driven leukemia., (Copyright© 2019 Ferrata Storti Foundation.)
- Published
- 2019
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13. Peroxiredoxin-controlled G-CSF signalling at the endoplasmic reticulum-early endosome interface.
- Author
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Palande K, Roovers O, Gits J, Verwijmeren C, Iuchi Y, Fujii J, Neel BG, Karisch R, Tavernier J, and Touw IP
- Subjects
- Animals, Cell Line, Cell Proliferation, Endoplasmic Reticulum enzymology, Endoplasmic Reticulum genetics, Endosomes enzymology, Endosomes genetics, Granulocyte Colony-Stimulating Factor genetics, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Humans, Mice, Peroxiredoxins genetics, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Protein Tyrosine Phosphatase, Non-Receptor Type 1 genetics, Protein Tyrosine Phosphatase, Non-Receptor Type 1 metabolism, Reactive Oxygen Species, Receptors, Granulocyte Colony-Stimulating Factor genetics, Receptors, Granulocyte Colony-Stimulating Factor metabolism, Down-Regulation, Endoplasmic Reticulum metabolism, Endosomes metabolism, Granulocyte Colony-Stimulating Factor metabolism, Peroxiredoxins metabolism, Signal Transduction
- Abstract
Reactive oxygen species (ROS) regulate growth factor receptor signalling at least in part by inhibiting oxidation-sensitive phosphatases. An emerging concept is that ROS act locally to affect signal transduction in different subcellular compartments and that ROS levels are regulated by antioxidant proteins at the same local level. Here, we show that the ER-resident antioxidant peroxiredoxin 4 (Prdx4) interacts with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This interaction occurs when the activated G-CSFR resides in early endosomes. Prdx4 inhibits G-CSF-induced signalling and proliferation in myeloid progenitors, depending on its redox-active cysteine core. Protein tyrosine phosphatase 1b (Ptp1b) appears to be a major downstream effector controlling these responses. Conversely, Ptp1b might keep Prdx4 active by reducing its phosphorylation. These findings unveil a new signal transduction regulatory circuitry involving redox-controlled processes in the ER and activated cytokine receptors in endosomes.
- Published
- 2011
- Full Text
- View/download PDF
14. The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor.
- Author
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Meenhuis A, Verwijmeren C, Roovers O, and Touw IP
- Subjects
- Animals, Cells, Cultured, Endopeptidases genetics, Endosomal Sorting Complexes Required for Transport genetics, Endosomal Sorting Complexes Required for Transport metabolism, Humans, Immediate-Early Proteins genetics, Mice, Receptors, Colony-Stimulating Factor genetics, Ubiquitin Thiolesterase genetics, Ubiquitin Thiolesterase metabolism, Ubiquitination, Colony-Stimulating Factors pharmacology, Endopeptidases metabolism, Immediate-Early Proteins metabolism, Lysosomes metabolism, Receptors, Colony-Stimulating Factor metabolism, Signal Transduction
- Abstract
Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities., (© The Authors Journal compilation © 2011 Biochemical Society)
- Published
- 2011
- Full Text
- View/download PDF
15. Site-specific ubiquitination determines lysosomal sorting and signal attenuation of the granulocyte colony-stimulating factor receptor.
- Author
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Wölfler A, Irandoust M, Meenhuis A, Gits J, Roovers O, and Touw IP
- Subjects
- Amino Acid Sequence, Animals, DNA Mutational Analysis, Endocytosis physiology, HeLa Cells, Humans, Lysine metabolism, Molecular Sequence Data, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Suppressor of Cytokine Signaling 3 Protein, Suppressor of Cytokine Signaling Proteins metabolism, Ubiquitination, Lysosomes metabolism, Protein Transport physiology, Receptors, Granulocyte Colony-Stimulating Factor metabolism, Signal Transduction physiology
- Abstract
Ubiquitination of cytokine receptors controls intracellular receptor routing and signal duration, but the underlying molecular determinants are unclear. The suppressor of cytokine signaling protein SOCS3 drives lysosomal degradation of the granulocyte colony-stimulating factor receptor (G-CSFR), depending on SOCS3-mediated ubiquitination of a specific lysine located in a conserved juxtamembrane motif. Here, we show that, despite ubiquitination of other lysines, positioning of a lysine within the membrane-proximal region is indispensable for this process. Neither reallocation of the motif nor fusion of ubiquitin to the C-terminus of the G-CSFR could drive lysosomal routing. However, within this region, the lysine could be shifted 12 amino acids toward the C-terminus without losing its function, arguing against the existence of a linear sorting motif and demonstrating that positioning of the lysine relative to the SOCS3 docking site is flexible. G-CSFR ubiquitination peaked after endocytosis, was inhibited by methyl-beta-cyclodextrin as well as hyperosmotic sucrose and severely reduced in internalization-defective G-CSFR mutants, indicating that ubiquitination mainly occurs at endosomes. Apart from elucidating structural and spatio-temporal aspects of SOCS3-mediated ubiquitination, these findings have implications for the abnormal signaling function of G-CSFR mutants found in severe congenital neutropenia, a hematopoietic disorder with a high leukemia risk.
- Published
- 2009
- Full Text
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16. Janus kinases promote cell-surface expression and provoke autonomous signalling from routing-defective G-CSF receptors.
- Author
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Meenhuis A, Irandoust M, Wölfler A, Roovers O, Valkhof M, and Touw IP
- Subjects
- Animals, Cell Line, Tumor, Endocytosis, HeLa Cells, Humans, Ligands, Lysosomes metabolism, Mice, Receptors, Colony-Stimulating Factor genetics, Receptors, Colony-Stimulating Factor metabolism, Receptors, Granulocyte Colony-Stimulating Factor genetics, Transfection, Ubiquitination, Cell Membrane metabolism, Janus Kinases metabolism, Receptors, Granulocyte Colony-Stimulating Factor metabolism, Signal Transduction
- Abstract
CSF3R [G-CSF (granulocyte colony-stimulating factor) receptor] controls survival, proliferation and differentiation of myeloid progenitor cells via activation of multiple JAKs (Janus kinases). In addition to their role in phosphorylation of receptor tyrosine residues and downstream signalling substrates, JAKs have recently been implicated in controlling expression of cytokine receptors, predominantly by masking critical motifs involved in endocytosis and lysosomal targeting. In the present study, we show that increasing the levels of JAK1, JAK2 and TYK2 (tyrosine kinase 2) elevated steady-state CSF3R cell-surface expression and enhanced CSF3R protein stability in haematopoietic cells. This effect was not due to inhibition of endocytotic routing, since JAKs did not functionally interfere with the dileucine-based internalization motif or lysine-mediated lysosomal degradation of CSF3R. Rather, JAKs appeared to act on CSF3R in the biosynthetic pathway at the level of the ER (endoplasmic reticulum). Strikingly, increased JAK levels synergized with internalization- or lysosomal-routing-defective CSF3R mutants to confer growth-factor independent STAT3 (signal transducer and activator of transcription 3) activation and cell survival, providing a model for how increased JAK expression and disturbed intracellular routing of CSF3R synergize in the transformation of haematopoietic cells.
- Published
- 2009
- Full Text
- View/download PDF
17. Suppressor of cytokine signaling 3 controls lysosomal routing of G-CSF receptor.
- Author
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Irandoust MI, Aarts LH, Roovers O, Gits J, Erkeland SJ, and Touw IP
- Subjects
- Amino Acid Motifs, Animals, Cell Proliferation, Endocytosis, HeLa Cells, Humans, Ligands, Lysine metabolism, Mice, Models, Biological, Mutant Proteins metabolism, Myeloid Cells cytology, Myeloid Cells metabolism, Protein Transport, STAT5 Transcription Factor metabolism, Suppressor of Cytokine Signaling 3 Protein, Ubiquitin metabolism, Lysosomes metabolism, Receptors, Granulocyte Colony-Stimulating Factor metabolism, Suppressor of Cytokine Signaling Proteins metabolism
- Abstract
The hematopoietic system provides an attractive model for studying growth factor-controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony-stimulating factor receptor (G-CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G-CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G-CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G-CSFR mutants lacking K632 show a perturbed proliferation/differentiation balance in response to G-CSF. This is the first demonstration of SOCS-mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output.
- Published
- 2007
- Full Text
- View/download PDF
18. Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics.
- Author
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Aarts LH, Roovers O, Ward AC, and Touw IP
- Subjects
- 3T3 Cells, Animals, Binding Sites, COS Cells, Chlorocebus aethiops, Endocytosis, Genes, Reporter, HeLa Cells, Humans, Kinetics, Ligands, Mice, Mice, Knockout, Microscopy, Confocal, Mutation, Organelles metabolism, Organelles ultrastructure, Peptide Fragments chemistry, Protein Transport, Receptors, Granulocyte Colony-Stimulating Factor deficiency, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Restriction Mapping, Sequence Deletion, Transfection, Granulocyte Colony-Stimulating Factor pharmacology, Receptors, Granulocyte Colony-Stimulating Factor chemistry, Receptors, Granulocyte Colony-Stimulating Factor metabolism
- Abstract
We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position -4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.
- Published
- 2004
- Full Text
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