Site-Specific Ubiquitination Determines Lysosomal Sorting and Signal Attenuation of the Granulocyte Colony-Stimulating Factor Receptor

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.     

G-CSFR ubiquitination critically regulates myeloid cell survival and proliferation

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis. Mutations in the G-CSFR in patients with severe congenital neutropenia (SCN) transforming to acute myelogenous leukemia (AML) have been shown to induce hypersensitivity and enhanced growth responses to G-CSF. Recent studies have demonstrated the importance of the ubiquitin/proteasome system in the initiation of negative signaling by the G-CSFR. To further investigate the role of ubiquitination in regulating G-CSFR signaling, we generated a mutant form of the G-CSFR (K762R/G-CSFR) which abrogates the attachment of ubiquitin to the lysine residue at position 762 of the G-CSFR that is deleted in the Delta716 G-CSFR form isolated from patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination of the G-CSFR was impaired in cells expressing the mutant K762R/G-CSFR compared to cells transfected with the WT G-CSFR. Cells stably transfected with the K762R/G-CSFR displayed a higher proliferation rate, increased sensitivity to G-CSF, and enhanced survival following cytokine depletion, similar to previously published data with the Delta716 G-CSFR mutant. Activation of the signaling molecules Stat5 and Akt were also increased in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged after G-CSF withdrawal. These results indicate that ubiquitination is required for regulation of G-CSFR-mediated proliferation and cell survival. Mutations that disrupt G-CSFR ubiquitination at lysine 762 induce aberrant receptor signaling and hyperproliferative responses to G-CSF, which may contribute to leukemic transformation.     

Suppressor of cytokine signaling-3 is recruited to the activated granulocyte-colony stimulating factor receptor and modulates its signal transduction

G-CSF is a polypeptide growth factor used in treatment following chemotherapy. G-CSF regulates granulopoiesis and acts on its target cells by inducing homodimerization of the G-CSFR, thereby activating intracellular signaling cascades. The G-CSFR encompasses four tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Suppressor of cytokine signaling 3 (SOCS-3), also referred to as cytokine-inducible Src homolgy 2-containing protein 3, is a member of a recently discovered family of feedback inhibitors that have been shown to inhibit the Janus kinase/STAT pathway. In this study, we demonstrate that human SOCS-3 is rapidly induced by G-CSF in polymorphonuclear neutrophils as well as in the myeloid precursor cell line U937 and that SOCS-3 negatively regulates G-CSFR-mediated STAT activation. Most importantly, we show that SOCS-3 is recruited to the G-CSFR in a phosphorylation-dependent manner and we identify phosphotyrosine (pY)729 as the major recruitment site for SOCS-3. Furthermore, we demonstrate that SOCS-3 directly binds to this pY motif. Surface plasmon resonance analysis reveals a dissociation constant (K(D)) for this interaction of around 2.8 microM. These findings strongly suggest that the recruitment of SOCS-3 to pY729 is important for the modulation of G-CSFR-mediated signal transduction by SOCS-3.     

Regulation of the differentiation of WEHI-3B D+ leukemia cells by granulocyte colony-stimulating factor receptor

To investigate the role of the G-CSF receptor (G-CSFR) in mediating the action of G-CSF, WEHI-3B D+ murine myelomonocytic leukemia cells were transfected with a plasmid containing the murine G-CSFR gene. Overexpression of G-CSFR in transfected clones was demonstrated by northern blotting, binding of [125I]rhG-CSF and cross-linking experiments. A high level of expression of the G-CSFR did not promote or suppress cellular proliferation or initiate differentiation; however, exposure of transfected cells to G-CSF in suspension culture caused a large percentage of the population to enter a differentiation pathway, as determined by two markers of the mature state, the ability of cells to reduce nitroblue tetrazolium (NBT) and to express the differentiation antigen Mac-1 (CD11b) on the cell surface. Thus, upon treatment with 10 ng/ml of G-CSF, 60% or more of transfected cells exhibited NBT positivity; whereas, in contrast, nontransfected cells exhibited only 6% NBT positivity in response to G-CSF. An eightfold increase in Mac-1 expression over that of the parental line was also observed in transfected cells exposed to G-CSF. The growth rate of the transfected clones was decreased by exposure to G-CSF, presumably due to terminal differentiation. The findings suggest that the predominant function of G-CSF and its receptor in WEHI-3B D+ cells is to mediate differentiation and that the level of the G-CSFR portion of the signal transduction mechanism in this malignant cell line is important for a response to the maturation inducing function of the cytokine.     

Loss of SHIP and CIS recruitment to the granulocyte colony-stimulating factor receptor contribute to hyperproliferative responses in severe congenital neutropenia/acute myelogenous leukemia

Mutations in the G-CSF receptor (G-CSFR) in patients with severe congenital neutropenia (SCN) are postulated to contribute to transformation to acute myelogenous leukemia (AML). These mutations result in defective receptor internalization and sustained cellular activation, suggesting a loss of negative signaling by the G-CSFR. In this paper we investigated the roles of SHIP and cytokine-inducible Src homology 2 protein (CIS) in down-modulating G-CSFR signals and demonstrate that loss of their recruitment as a consequence of receptor mutations leads to aberrant signaling. We show that SHIP binds to phosphopeptides corresponding to Tyr744 and Tyr764 in the G-CSFR and that Tyr764 is required for in vivo phosphorylation of SHIP and the formation of SHIP/Shc complexes. Cells expressing a G-CSFR form lacking Tyr764 exhibited hypersensitivity to G-CSF and enhanced proliferation, but to a lesser degree than observed with the most common mutant G-CSFR form in patients with SCN/AML, prompting us to investigate whether suppressor of cytokine signaling proteins also down-modulate G-CSFR signals. G-CSF was found to induce the expression of CIS and of CIS bound to phosphopeptides corresponding to Tyr729 and Tyr744 of the G-CSFR. The expression of CIS was prolonged in cells with the SCN/AML mutant G-CSFR lacking Tyr729 and Tyr744, which also correlated with increased G-CSFR expression. These findings suggest that SHIP and CIS interact with distal phosphotyrosine residues in the G-CSFR to negatively regulate G-CSFR signaling by limiting proliferation and modulating surface expression of the G-CSFR, respectively. Novel therapeutic approaches targeting inhibitory pathways that limit G-CSFR signaling may have promise in the treatment of patients with SCN/AML.     

The chemoattractants, IL-8 and formyl-methionyl-leucyl-phenylalanine, regulate granulocyte colony-stimulating factor signaling by inducing suppressor of cytokine signaling-1 expression

Suppressors of cytokine signaling (SOCS) are encoded by immediate early genes known to inhibit cytokine responses in a classical feedback loop. SOCS gene expression has been shown to be induced by many cytokines, growth factors, and innate immune stimuli, such as LPS. In this paper, we report that the chemoattractants, IL-8 and fMLP, up-regulate SOCS1 mRNA in human myeloid cells, primary human neutrophils, PBMCs, and dendritic cells. fMLP rapidly up-regulates SOCS1, whereas the induction of SOCS1 upon IL-8 treatment is delayed. IL-8 and fMLP did not signal via Jak/STATs in primary human macrophages, thus implicating the induction of SOCS by other intracellular pathways. As chemoattractant-induced SOCS1 expression in neutrophils may play an important role in regulating the subsequent response to growth promoting cytokines like G-CSF, we investigated the effect of chemoattractant-induced SOCS1 on cytokine signal transduction. We show that pretreatment of primary human neutrophils with fMLP or IL-8 blocks G-CSF-mediated STAT3 activation. This study provides evidence for cross-talk between chemoattractant and cytokine signal transduction pathways involving SOCS proteins, suggesting that these chemotactic factors may desensitize neutrophils to G-CSF via rapid induction of SOCS1 expression.     

SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130

Interleukin-6 is involved in the regulation of many biological activities such as gene expression, cell proliferation, and differentiation. The control of the termination of cytokine signaling is as important as the regulation of initiation of signal transduction pathways. Three families of proteins involved in the down-regulation of cytokine signaling have been described recently: (i) SH2 domain-containing protein-tyrosine phosphatases (SHP), (ii) suppressors of cytokine signaling (SOCS), and (iii) protein inhibitors of activated STATs (PIAS). We have analyzed the interplay of two inhibitors in the signal transduction pathway of interleukin-6 and demonstrate that the tyrosine phosphatase SHP2 and SOCS3 do not act independently but are functionally linked. The activation of one inhibitor modulates the activity of the other; Inhibition of SHP2 activation leads to increased SOCS3-mRNA levels, whereas increased expression of SOCS3 results in a reduction of SHP2 phosphorylation after activation of the interleukin-6 signal transduction pathway. Furthermore, we show that tyrosine 759 in gp130 is essential for both SHP2 and SOCS3 but not for SOCS1 to exert their inhibitory activities on interleukin-6 signal transduction. Besides SHP2, SOCS3 also interacts with the Tyr(P)-759 peptide of gp130. Taken together, our results suggest differences in the function of SOCS1 and SOCS3 and a link between SHP2 and SOCS3.     

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.     

Oncostatin M receptor-mediated signal transduction is negatively regulated by SOCS3 through a receptor tyrosine-independent mechanism

Down-regulation of interleukin (IL)-6-type cytokine signaling has been shown to occur, among other mechanisms, via induction of the feedback inhibitor SOCS3 (suppressor of cytokine signaling 3). Binding of SOCS3 to the phosphorylated Tyr(759) in the cytoplasmic region of gp130, the common signal transducing receptor chain of all IL-6-type cytokines, is necessary for inhibition of Janus kinase-mediated signaling. In the present study, we analyzed the effect of SOCS3 on signal transduction by the proinflammatory cytokine oncostatin M (OSM), which signals through a receptor complex of gp130 and the OSM receptor (OSMR). OSM leads to a much stronger and prolonged induction of SOCS3 in HepG2 hepatoma cells and murine embryonal fibroblasts (MEF) compared with IL-6. A negative effect of SOCS3 on OSM signaling was confirmed using MEF cells lacking SOCS3. We can show that the OSMR-mediated signaling is inhibited by SOCS3 to a similar extent as previously described for gp130. However, the inhibition occurs independent of tyrosine motifs within the OSMR. Instead, SOCS3 interacts directly with JAK1 in a stimulation-dependent manner, a mechanism so far only known for SOCS1.     

Distinct region of the granulocyte colony-stimulating factor receptor mediates proliferative signaling through activation of Janus kinase 2 and p44/42 mitogen-activated protein kinase.

The granulocyte colony-stimulating factor receptor (G-CSFR) regulates the proliferation, differentiation and survival of neutrophilic progenitor cells. In these studies, we introduced mutant G-CSFRs with cytoplasmic domains truncated approximately every 30 amino acids from the C-terminus into interleukin-3 (IL-3)-dependent myeloid LGM-1 cells. The G-CSFR membrane proximal region containing the Box 2 homology sequence was determined to be critical for proliferative signaling, as well as for activation of Janus kinase (JAK2) and p44/42 mitogen-activated protein kinase (MAPK) following G-CSF stimulation. In the presence of increasing concentrations of JAK2 or p44/42 MAPK inhibitors, LGM-1 cells expressing the full-length G-CSFR exhibited a decreased capacity to proliferate in response to G-CSF. These results demonstrate that JAK2 and p44/42 MAPK activation is involved in proliferative signaling through the G-CSFR membrane proximal region containing the Box 2 homology sequence.     

Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.     

A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.     

The cytokine-inducible Scr homology domain-containing protein negatively regulates signaling by promoting apoptosis in erythroid progenitor cells

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines, but its role in proliferation, differentiation, and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells, we generated green fluorescent protein (GFP)-tagged versions of wild type CIS, a mutant harboring an inactivated SH2 domain (CIS R107K), and a mutant with a deletion of the SOCS Box (CISDeltaBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence, both are essential for the growth inhibitory effect of CIS, whereas the CIS SOCS box is dispensable. Accordingly, the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly, apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus, CIS negatively regulates signaling at two levels, apoptosis and proliferation, and thereby sets a threshold for signal transduction.     

Distinct signal transduction through the tyrosine-containing domains of the granulocyte colony-stimulating factor receptor.

The receptor for granulocyte colony-stimulating factor (G-CSFR) is a hemopoietic growth factor receptor, which mediates proliferation and differentiation signals. The cytoplasmic region of G-CSFR carries four tyrosine residues in its C-terminal half. We constructed mutant receptors in which each tyrosine residue of G-CSFR was mutated to phenylalanine. Two mutant receptors (Tyr703 and Tyr728) neither transduced the growth-inhibitory signal nor induced the neutrophil-specific myeloperoxidase (MPO) gene. The Tyr703 mutant did not induce morphological changes in cells, whereas transformants expressing the Tyr728 mutant adhered to plates with a macrophage-like morphology upon G-CSF stimulation. Mutation of the most distal tyrosine residue (Tyr763) abolished the ability of G-CSFR to stimulate the tyrosine phosphorylation of a cellular protein with an M(r) of 54 kDa. These results indicated that the regions around the three tyrosine residues of G-CSFR play essential and distinct roles in signal transduction.     

Suppressors of cytokine signaling 4 and 5 regulate epidermal growth factor receptor signaling

Suppressors of cytokine signaling (SOCS) are Src homology-2-containing proteins originally identified as negative regulators of cytokine signaling. Accumulating evidence indicates a role for SOCS proteins in the regulation of additional signaling pathways including receptor tyrosine kinases. Notably, SOCS36E, the Drosophila ortholog of mammalian SOCS5, was recently implicated as a negative regulator of the Drosophila ortholog of EGFR. In this study, we aimed at characterizing the role of SOCS5 in the negative regulation of EGFR. Here we show that the expression of SOCS5 and its closest homolog SOCS4 is elevated in cells following treatment with EGF, similar to several negative feedback regulators of EGFR whose expression is up-regulated upon receptor activation. The expression of SOCS5 led to a marked reduction in EGFR expression levels by promoting EGFR degradation. The reduction in EGFR levels and EGF-induced signaling in SOCS5-expressing cells requires both the Src homology-2 and SOCS box domains of SOCS5. Interestingly, EGFR is degraded by SOCS5 prior to EGF treatment in a ligand- and c-Cbl-independent manner. SOCS5 can associate with EGFR and can also bind the ElonginBC protein complex via its SOCS box, which may recruit an E3 ubiquitin ligase to promote EGFR degradation. Thus, we have characterized a novel function for SOCS5 in regulating EGFR and discuss its potential role in controlling EGFR homeostasis.