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‐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.

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.

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.

Ubiquitination of lysine (K) residues in the cytoplasmic domain of cytokine receptors plays a major role in intracellular receptor routing and control of signal duration. However, which ubiquitin (E3) ligases and whether specific lysines, eg. present in conserved motifs, are involved in ubiquitin-mediated routing is still largely unknown. We recently showed that SOCS3, which forms an Elongin/Cullin-based E3 ligase (ECSSOCS3), is involved in ligand-induced ubiquitination, lysosomal routing and degradation of the G-CSFR (Irandoust et al, EMBO J . 2007, 26:1782–93). Strikingly, ECSSOCS3-mediated mechanisms largely depended on ubiquitination of a single juxtamembrane lysine at position 632 (K632), even though 4 additional conserved lysines are present in the cytoplasmic domain of the G-CSFR. This juxtamembrane lysine located 5 amino acids upstream of the box1 region is conserved among several other cytokine receptors. We sought to unravel the configuration required for ubiquitination of K632 and for its function in attenuation of G-CSF signaling. First, we tested whether K632 is part of a domain that can function as an isolated motif. To this end, we fused the K632-containing domain (D) encompassing the juxtamembrane and box1 regions to the COOH-terminus of a lysine-less G-CSFR (K5R-D). Despite efficient ubiquitination of the lysine within the reallocated domain, K5R-D, similar to K5R, was severely hampered in lysosomal routing. This resulted in prolonged STAT5 activation and in G-CSF-induced hyperproliferation of myeloid 32D cells indicating that the juxtamembrane domain of the G-CSFR does not function as an isolated domain that can be shifted to a different cytoplasmic location. To delineate how imperative the positioning of K632 is for its function, we inserted 5 alanines immediately upstream or downstream of K632. However, these insertions did not affect G-CSFR signal duration suggesting that there is no stringent proximity of K632 to the cell membrane and no strict positioning relative to the SOCS3 recuitment site (Y729) to direct downregulation of G-CSFR signaling. To further study this flexibility in positioning we ‘walked' lysines through the juxtamembrane domain and the adjacent box 1-region by mutating amino acids at indicated positions to a lysine in an otherwise lysine-less G-CSFR. Mutants 628K and 630K were hardly ubiquitinated and, as predicted, were hampered in lysosomal routing. In contrast, lysines on position 631, 633, 634, 638 and 644 were ubiquitinated comparably to K632. Interestingly, while mutants 631K, 633K and 634K showed characteristics indistinguishable from 632K, mutants 638K and 644K displayed slightly decreased signal attenuation suggesting a gradual decline of functionality of lysines moved to box-1. Finally, moving the lysine downstream to position 672, which corresponds to the second lysine in the wt G-CSFR, resulted in complete loss of functionality. In conclusion, these results indicate that the positioning of a lysine within the juxtamembrane domain is crucial for ECSSOCS3-mediated signal downregulation and lysosomal routing of the G-CSFR, although a limited flexibility is tolerated towards the box1 region. The fact that inactive lysines are still ubiquitinated implies that interaction with effector proteins is involved in lysosomal routing rather than reduced or lost activity of E3 ligases. Given its high conservation among several cytokine receptors we are currently investigating a common role for the juxtamembrane lysine in cytokine receptor routing and signal attenuation.

Signals induced by granulocyte colony-stimulating factor (G-CSF), the major cytokine involved in neutrophil development, are tightly controlled by ligand-induced receptor internalization. Truncated G-CSF receptors (G-CSF-Rs) that fail to internalize show sustained proliferation and defective differentiation signaling. Steady-state forward routing also determines cell surface levels of cytokine receptors, but mechanisms controlling this are poorly understood. Here, we show that WD40 and suppressor of cytokine signaling (SOCS) box protein-2 (Wsb-2), an SOCS box-containing WD40 protein with currently unknown function, binds to the COOH-terminal region of G-CSF-R. Removal of this region did not affect internalization, yet resulted in increased membrane expression of G-CSF-R and enhanced proliferation signaling at the expense of differentiation induction. Conversely, Wsb-2 binding to the G-CSF-R reduced its cell surface expression and inhibited proliferation signaling. These effects depended on the SOCS box involved in ubiquitylation and on cytosolic lysines of G-CSF-R and imply a major role for ubiquitylation through the G-CSF-R C-terminus in forward routing of the receptor. Importantly, the Wsb-2 gene is commonly disrupted by virus integrations in mouse leukemia. We conclude that control of forward routing of G-CSF-R is essential for a balanced response of myeloid progenitors to G-CSF and suggest that disturbance of this balance may contribute to myeloid leukemia.

The granulocyte colony-stimulating factor receptor (G-CSF-R) induces proliferation, survival and differentiation of myeloid progenitor cells in a tightly controlled temporal fashion. These responses depend on multiple signaling mechanisms that are activated via distinct regions in the cytoplasmic domain of wild type (WT) G-CSF-R. About 20% of severe congenital neutropenia patients acquire mutations that truncate the C-terminus of G-CSF-R, which is often associated with disease progression to acute myeloid leukemia. Myeloid cells expressing these truncated G-CSF-R hyperproliferate and are hampered in differentiation in response to G-CSF. Multiple mechanisms have been linked to perturbed signaling of truncated G-CSF-R. Specifically, defective internalization has been associated with a prolonged activation status of the truncated G-CSF-R, which is e.g. reflected by strongly increased and sustained activation of STAT5. We found that internalized WT G-CSF-R are rapidly targeted to lysosomes, suggesting that lysosomal degradation is a major mechanism for signal attenuation. Lysine (K) residues in the cytoplasmic tail of transmembrane receptors are often determinants for ubiquitin-mediated sorting into multi-vesicular bodies and lysosomes. We observed that G-CSF-R is ubiquitinated and subsequently assessed the contribution of K residues in G-CSF-R routing and signaling. To this end, we generated mutant G-CSF-R-K5R, in which all five conserved cytoplasmic K residues were replaced by arginine (R). To study differential localization of internalized K5R and WT G-CSF-R, we introduced constitutively active Rab5 (Rab5-Q79L) to enlarge early endosomes. Under these conditions, internalized WT G-CSF-R colocalized extensively with endosomal microdomains containing the endosome-to-lysosome sorting protein Hrs. In contrast, internalized G-CSF-R-K5R was mostly found outside these domains. Consequently, lysosomal routing of G-CSF-R-K5R was severely impaired, which resulted in strongly increased receptor protein levels. Upon removal of G-CSF, downregulation of STAT5 activity in 32D/K5R cells was significantly delayed compared to 32D/WT cells, establishing that the lysine residues are not only crucial for receptor stability, but also for duration of signaling. Moreover, in the continuous presence of G-CSF, 32D/K5R cells showed strongly increased STAT5 activity and proliferation and reduced granulocytic differentiation compared to 32D/WT. Similar results were obtained in colony assays with g-csfr deficient primary bone marrow progenitors transduced with K5R or WT G-CSF-R. To substantiate a relationship between signal duration and receptor degradation, we inhibited lysosomal degradation by pharmacological inhibitors and by knockdown of Hrs and the related protein Tsg101 by RNA interference. As expected, receptor stability was significantly increased by these treatments. Surprisingly however, this did not influence STAT5 activity, suggesting that signal attenuation predominantly occurred in a pre-lysosomal compartment. We conclude from these data that ubiquitinated lysine residues of G-CSF-R are required to target internalized receptors for lysosomal degradation. However, they also appear to be involved in contracting an as yet unidentified inhibitor, perhaps a phosphatase, to a specific Hrs-positive subendosomal compartment, thereby switching off G-CSF-R already prior to its degradation in lysosomes.