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.
Subject(s)
Down-Regulation , Endoplasmic Reticulum/metabolism , Endosomes/metabolism , Granulocyte Colony-Stimulating Factor/metabolism , Peroxiredoxins/metabolism , Signal Transduction , 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/metabolismABSTRACT
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.
Subject(s)
Colony-Stimulating Factors/pharmacology , Endopeptidases/metabolism , Immediate-Early Proteins/metabolism , Lysosomes/metabolism , Receptors, Colony-Stimulating Factor/metabolism , Signal Transduction , 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 , UbiquitinationABSTRACT
BACKGROUND: Huntington's disease is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD or Huntington's disease gene. Although micro array studies on patient and animal tissue provide valuable information, the primary effect of mutant huntingtin will inevitably be masked by secondary processes in advanced stages of the disease. Thus, cell models are instrumental to study early, direct effects of mutant huntingtin. mRNA changes were studied in an inducible PC12 model of Huntington's disease, before and after aggregates became visible, to identify groups of genes that could play a role in the early pathology of Huntington's disease. RESULTS: Before aggregation, up-regulation of gene expression predominated, while after aggregates became visible, down-regulation and up-regulation occurred to the same extent. After aggregates became visible there was a down-regulation of dopamine biosynthesis genes accompanied by down-regulation of dopamine levels in culture, indicating the utility of this model to identify functionally relevant pathways. Furthermore, genes of the anti-oxidant Nrf2-ARE pathway were up-regulated, possibly as a protective mechanism. In parallel, we discovered alterations in genes which may result in increased oxidative stress and damage. CONCLUSION: Up-regulation of gene expression may be more important in HD pathology than previously appreciated. In addition, given the pathogenic impact of oxidative stress and neuroinflammation, the Nrf2-ARE signaling pathway constitutes a new attractive therapeutic target for HD.
