MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seedcontaining miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitinbinding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of IntroductionMiRNAs are transcribed as long primary transcripts that are processed by RNaseIII endonucleases DROSHA and DICER into single-stranded RNAs of ϳ 22nt. 1 The nucleotides 2-7 at the 5Ј-end of miRNAs, referred to as the miRNA seed region, are important for miRNA target recognition. 2 MiRNAs regulate gene expression by pairing with the seed complementary sequences in the 3Ј untranslated region (UTR) of mRNAs. Most mammalian miRNAs both repress translation and enhance decay of their target transcript. 3,4 MiRNAs containing homologous seeds such as, for example, the Let-7 family of miRNAs, are believed to regulate the same targets. 2 Involvement of miRNAs in hematopoiesis is strongly suggested by the position of miRNA genes near translocation breakpoints and by their presence in loci targeted for deletion in human leukemias. 5 Furthermore, expression profiling data suggest a major role for miRNAs in regulation of hematopoietic cell commitment, proliferation, apoptosis, survival, and differentiation. [6][7][8][9] The importance of miRNAs during hematopoiesis has been shown by disruption of miRNA biogenesis in mice. For instance, Dicer-deleted hematopoietic stem cells are unable to reconstitute the hematopoietic system. 10 Further, conditional deletion of Dicer in T and B cells results in strong reduction of lymphocytes and diminished cell survival and functions. [11][12][13] Argonaute-2 knock-out in hematopoiesis results in impaired differentiation of B-lymphocytes and erythroid cells. 14,15 MiRNAs can be expressed in a cell type or tissue specific manner. For instance, miR-223 and miR-142 are almost exclusively expressed in hematopoietic cells. 16 MiR-223 is transcriptionally controlled by CCAAT/enhancer-binding protein ␣ (CEBPA) and suppresses the myeloid transcription factor MEF2C, a major regulator of progenitor cell proliferation and granulocyte specific functions. 17,18 In addition, specific miRNAs control cellular processes important for proliferation, survival, cytokine production and cell lineage decisions of developing T and B cells. 8,12 In hematopoietic stem cells, sustained expression of miR-155 causes a myeloproliferative disorder in mice. 19 Furthermore, forced miR-29a in hematopoietic precursors induces aberrant self-renewal and acute myeloid leukemia by still unidentified mechan...

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 Cterminus 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-β-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.

Following activation by their cognate ligands, cytokine receptors undergo intracellular routing towards lysosomes where they are degraded. Cytokine receptor signaling does not terminate at the plasma membrane, but continues throughout the endocytotic pathway. The modes of internalization and intracellular trafficking of specific receptors, the level of recycling towards the plasma membrane, the type of protein modifications (phosphorylation, ubiquitination) and the enzymes involved in these processes are remarkably diverse. This heterogeneity may contribute to the fine-tuning of signal amplitudes and duration from different receptors. The colony-stimulating factor 3 receptor (CSF3R) is unique for its balanced signaling output, first leading to proliferation of myeloid progenitors, followed by a cell cycle arrest and granulocytic differentiation. The mechanisms associated with CSF3R signal modulation, involving receptor lysine ubiquitination and redox-controlled phosphatase activities, are to a large extent confined to the signaling endosome. Interactions between signaling endosomes and the endoplasmic reticulum play a key role in this process. Here, we review the mechanisms of intracellular routing of CSF3R, their consequences for myeloid blood cell development and their implications for myeloid diseases.

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.

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.