Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.

Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with interferon-gamma (IFN-gamma). Conversely, IL-6-induced STAT activation is normal in Socs1-deficient cells, whereas STAT1 activation induced by IFN-gamma is prolonged. Microarray analysis shows that the pattern of gene expression induced by IL-6 in Socs3-deficient livers mimics that induced by IFN-gamma. Our data indicate that SOCS3 and SOCS1 have reciprocal functions in IL-6 and IFN-gamma regulation and imply that SOCS3 has a role in preventing IFN-gamma-like responses in cells stimulated by IL-6.

Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1-/- mice were shown to exhibit excessive responses typical of those induced by interferon gamma (IFNgamma), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNgamma-dependent capacity to kill L. major parasites. The complex disease in SOCS1-/- mice was prevented by administration of anti-IFNgamma antibodies and did not occur in SOCS1-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential for resistance to a variety of infections, the potential toxic action of IFNgamma, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNgamma action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses.

Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba͞F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

The four members of the recently identified suppressor of cytokines signaling family (SOCS-1, SOCS-2, SOCS-3, and CIS, where CIS is cytokine-inducible SH2-containing protein) appear, by various means, to negatively regulate cytokine signal transduction. Structurally, the SOCS proteins are composed of an N-terminal region of variable length and amino acid composition, a central SH2 domain, and a previously unrecognized C-terminal motif that we have called the SOCS box. By using the SOCS box amino acid sequence consensus, we have searched DNA databases and have identified a further 16 proteins that contain this motif. These proteins fall into five classes based on the protein motifs found N-terminal of the SOCS box. In addition to four new SOCS proteins (SOCS-4 to SOCS-7) containing an SH2 domain and a SOCS box, we describe three new families of proteins that contain either WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3) or ankyrin repeats (ASB-1 to -3) N-terminal of the SOCS box. In addition, we show that a class of small GTPases also contains a SOCS box. The expression of representative members of each class of proteins differs markedly, as does the regulation of expression by cytokines. The function of the WSB, SSB, and ASB protein families remains to be determined.Cytokines act by binding to and inducing dimerization of members of the hemopoietin receptor family expressed on the surface of responsive cells (1). Although the cytoplasmic proteins that then transduce the signal are relatively welldefined and include the Janus kinase (JAK) family of kinases and signal transducers and activators of transcription (STAT) transcription factors (2, 3), the proteins involved in limiting signal transduction are not well characterized.The four known members of the suppressor of cytokine signaling (SOCS) family (CIS, SOCS-1͞SSI-1͞JA B-1, SOCS-2, and SOCS-3, where CIS is cytokine-inducible SH2-containing protein) represent a family of negative regulators of cytokine signal transduction (4-9). The SOCS proteins appear to form part of a classical negative feedback loop that regulates cytokine signal transduction. Transcription of each of the SOCS genes occurs rapidly in vitro and in vivo in response to cytokines, and once produced, the various members of the SOCS family appear to inhibit signaling in different ways. For SOCS-1, inhibition of signal transduction appears to occur by binding to and inhibiting the catalytic activity of members of the JAK family of cytoplasmic kinases (4-6), while CIS appears to act by competing with signaling molecules such as the STATs for binding to phosphorylated receptor cytoplasmic domains (7, 9).The SOCS proteins share structural similarities. Each has an N-terminal region of variable length and highly variable amino acid sequence, a central SH2 domain, and a striking region of C-terminal homology that we designated the SOCS box (4). Given the sequence similarity evident in the SOCS box of the four SOCS proteins and its conserved position at the C terminus of each protein, ...

Suppressor of cytokine signalling-2 (SOCS-2) is a member of the suppressor of cytokine signalling family, a group of related proteins implicated in the negative regulation of cytokine action through inhibition of the Janus kinase (JAK) signal transducers and activators of transcription (STAT) signal-transduction pathway. Here we use mice unable to express SOCS-2 to examine its function in vivo. SOCS-2(-/-) mice grew significantly larger than their wild-type littermates. Increased body weight became evident after weaning and was associated with significantly increased long bone lengths and the proportionate enlargement of most organs. Characteristics of deregulated growth hormone and insulin-like growth factor-I (IGF-I) signalling, including decreased production of major urinary protein, increased local IGF-I production, and collagen accumulation in the dermis, were observed in SOCS-2-deficient mice, indicating that SOCS-2 may have an essential negative regulatory role in the growth hormone/IGF-I 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.