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The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

The receptors for granulocyte-macrophage colony-stimulating factor (GM- CSF) and interleukin-3 and -5 (IL-3, IL-5) share a common signaling subunit (beta c). However, in the mouse, IL-3 can also use an alternative IL-3-specific receptor beta-chain (beta IL-3). To assess the relative contributions of beta c and beta IL-3 to IL-3 receptor formation and function, mice were generated in which the beta IL-3 gene was functionally inactivated by replacement of exons 9–13 with a neomycin resistance cassette. Bone marrow cells from these mice displayed a lower affinity IL-3 receptor than normal and were hyporesponsive to IL-3, but the mice displayed no obvious hematopoietic abnormalities. The data suggested that beta c and beta IL-3 are normally coexpressed on IL-3-responsive cells and have identical qualitative signaling capacities. Receptor transmodulation studies on bone marrow cells from wild-type, beta c -/-, and beta IL-3 -/- mice showed that the previously described hierarchical pattern of transmodulation was dependent on the relative numbers of both beta IL-3 and beta c receptor chains and also provided evidence for an unexpected interaction between beta c chains and G-CSF and M-CSF receptors.

A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7–17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low- affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7–17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7–17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM- CSF receptor required for ligand binding.

Differentiation induction in murine M1 leukemia cells by interleukin 6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M (OSM) is postulated to occur via a common receptor chain, gp130. In this study, growth factor-induced differentiation of M1 cells was accompanied by a late and persistent decrease in levels of mRNA and protein for a helix-loop-helix transcription factor, the SCL gene product. To evaluate whether reduced SCL expression was instrumental in monocyte differentiation, an SCL cDNA expression vector was introduced into M1 cells to obtain cell lines in which overexpression of SCL mRNA and protein was enforced. This resulted in a reduction in cells differentiating in response to LIF and OSM but not in response to IL-6. Scatchard analysis indicated that both parental and SCL-transfected cell lines exhibited similar receptor numbers and receptor affinities for LIF, OSM, and IL-6, suggesting that the differential responsiveness was not due to selective receptor down-modulation. Thus, these data implicate SCL in monocytic differentiation and provide evidence for differential receptor signaling pathways despite utilization of a common gp130 subunit by all three receptors.

Murine receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and Multi-CSF (interleukin-3) can exist in both high- and low-affinity forms and demonstrate trans-modulation by several different ligands. In contrast the recently cloned human GM-CSF receptor and murine interleukin-3 (IL-3) receptor display only low-affinity binding. To begin to understand the molecular basis of the formation of high- and low-affinity receptors and their trans-modulation we have developed methods for the solubilization and assay of GM-CSF and interleukin-3 receptors so that their binding characteristics can be studied in cell-free solution. Both receptors displayed a single class of high-affinity binding on intact FDC-P1 cells and IL-3 receptors had unaltered binding characteristics in cells, membranes and in detergent solution. However, GM-CSF receptors were converted to a single class of low-affinity binding in detergent solution while both high- and low-affinity forms were evident in membranes. The basis of affinity conversion of GM-CSF receptors was exclusively a change in the kinetic dissociation rate of ligand. Cross-linking experiments suggested that high-affinity receptors for GM-CSF and IL-3 might consist of two different protein species and, if this is so, the data suggest that this association is more stable for IL-3 than for GM-CSF receptors.

The suppressors of cytokine signaling (SOCS) proteins are a family of SH2 domain-containing intracellular inhibitors of cytokine signal transduction that act by several different mechanisms. Recent evidence suggests that the action of the SOCS proteins may extend beyond the cytokine receptors to signaling initiated by members of the tyrosine kinase receptor family. In this study, the ability of SOCS-5 to negatively regulate signaling cascades downstream of the epidermal growth factor receptor (EGF-R) has been examined by using an EGF-responsive cell line engineered to constitutively express the EGF-R and SOCS-5 or SOCS-5 mutants. SOCS-5 associated with the EGF-R complex in an EGF-independent manner, and the mitogenic response to EGF of all SOCS-5-expressing cell lines was dramatically inhibited when compared with control cell lines. Furthermore, this effect was abrogated after deletion of the SOCS-5 SOCS box. This result suggests that the inhibition of signaling occurs through enhanced proteasomal degradation of the EGF-R through SOCS box recruitment of E3 ubiquitin ligase activity.

Leukemia inhibitory factor (LIF) has many biological actions which parallel those of IL-1, IL-6 and tumor necrosis factor-alpha, but its role in the pathogenesis of human disease is unknown. A specific radioreceptor competition assay capable of detecting LIF at concentrations above 1 ng/ml (45 pM) was developed. To identify disease states in which LIF might be involved, a cross-sectional survey of serum and body fluids from approximately 1,500 subjects with a variety of diseases was performed using the LIF radioreceptor competition assay. Serum LIF concentrations were transiently elevated (2-200 ng/ml) in six subjects with meningococcal or Gram-negative septic shock, and in a subject with idiopathic fulminant hepatic failure. Moderately elevated LIF concentrations (> 10 ng/ml) were detected in cerebrospinal fluid from subjects with bacterial meningitis, in effusions associated with pneumonia and peritonitis, and in amniotic fluid from a woman with chorioamnionitis. Low LIF concentrations (1-10 ng/ml) were present in synovial fluid from subjects with inflammatory arthritis, amniotic fluid from women in labor, and some reactive, chronic inflammatory and malignant effusions and cyst fluids, but rarely in transudates. These initial findings suggest that LIF might be involved in the pathogenesis of inflammation and septic shock.

SOCS-1 was originally identified as an inhibitor of interleukin-6 signal transduction and is a member of a family of proteins (SOCS-1 to SOCS-7 and CIS) that contain an SH2 domain and a conserved carboxyl-terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino-terminal and SH2 domains are essential for SOCS-1 and SOCS-3 to inhibit cytokine signaling. Inhibition of cytokine-dependent activation of STAT3 occurred in cells expressing either SOCS-1 or SOCS-3, but unlike SOCS-1, SOCS-3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS-1 and SOCS-3 action when over-expressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS-1−/− mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS-1−/− mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS-1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects. J. Leukoc. Biol. 66: 588–592; 1999.