Sandra E. Nicholson

Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL‐6 signal transduction

SOCS‐1 (suppressor of cytokine signaling‐1) is a representative of a family of negative regulators of cytokine signaling (SOCS‐1 to SOCS‐7 and CIS) characterized by a highly conserved C‐terminal SOCS box preceded by an SH2 domain. This study comprehensively examined the ability of several SOCS family members to negatively regulate the gp130 signaling pathway. SOCS‐1 and SOCS‐3 inhibited both interleukin‐6 (IL‐6)‐ and leukemia inhibitory factor (LIF)‐induced macrophage differentiation of murine monocytic leukemic M1 cells and LIF induction of a Stat3‐responsive reporter construct in 293T fibroblasts. Deletion of amino acids 51–78 in the N‐terminal region of SOCS‐1 prevented inhibition of LIF signaling. The SOCS‐1 and SOCS‐3 N‐terminal regions were functionally interchangeable, but this did not extend to other SOCS family members. Mutation of SH2 domains abrogated the ability of both SOCS‐1 and SOCS‐3 to inhibit LIF signal transduction. Unlike SOCS‐1, SOCS‐3 was unable to inhibit JAK kinase activity in vitro, suggesting that SOCS‐1 and SOCS‐3 act on the JAK–STAT pathway in different ways. Thus, although inhibition of signaling by SOCS‐1 and SOCS‐3 requires both the SH2 and N‐terminal domains, their mechanisms of action appear to be biochemically different.

Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation

Toll-like receptor (TLR) signals that initiate innate immune responses to pathogens must be tightly regulated to prevent excessive inflammatory damage to the host. The adaptor protein Mal is specifically involved in signaling via TLR2 and TLR4. We demonstrate here that after TLR2 and TLR4 stimulation Mal becomes phosphorylated by Brutons tyrosine kinase (Btk) and then interacts with SOCS-1, which results in Mal polyubiquitination and subsequent degradation. Removal of SOCS-1 regulation potentiates Mal-dependent p65 phosphorylation and transactivation of NF-κB, leading to amplified inflammatory responses. These data identify a target of SOCS-1 that regulates TLR signaling via a mechanism distinct from an autocrine cytokine response. The transient activation of Mal and subsequent SOCS-1–mediated degradation is a rapid and selective means of limiting primary innate immune response.

SOCS regulation of the JAK/STAT signalling pathway

The suppressor of cytokine signalling (SOCS) proteins were, as their name suggests, first described as inhibitors of cytokine signalling. While their actions clearly now extend to other intracellular pathways, they remain key negative regulators of cytokine and growth factor signalling. In this review we focus on the mechanics of SOCS action and the complexities of the mouse models that have underpinned our current understanding of SOCS biology.

Biology and significance of the JAK/STAT signalling pathways

Since its discovery two decades ago, the activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway by numerous cytokines and growth factors has resulted in it becoming one of the most well-studied intracellular signalling networks. The field has progressed from the identification of the individual components to high-resolution crystal structures of both JAK and STAT, and an understanding of the complexities of the molecular activation and deactivation cycle which results in a diverse, yet highly specific and regulated pattern of transcriptional responses. While there is still more to learn, we now appreciate how disruption and deregulation of this pathway can result in clinical disease and look forward to adoption of the next generation of JAK inhibitors in routine clinical treatment.

The SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo

Suppressor of Cytokine Signaling-1 (SOCS-1) is an essential physiological inhibitor of IFN-γ signaling. Mice lacking this gene die in the early postnatal period from a disease characterized by hyperresponsiveness to endogenous IFN-γ. The SOCS box is a C-terminal domain shared with over 30 other proteins that links SOCS proteins to an E3 ubiquitin ligase activity and the proteasome, but whether it contributes to inhibition of cytokine signaling is currently disputed. We have deleted only the SOCS box of the SOCS-1 gene in mice and show that such mice have an increased responsiveness to IFN-γ and slowly develop a fatal inflammatory disease. These results demonstrate that deletion of the SOCS box leads to a partial loss of function of SOCS-1.

SOCS-6 Binds to Insulin Receptor Substrate 4, and Mice Lacking the SOCS-6 Gene Exhibit Mild Growth Retardation

ABSTRACT SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6−/− mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6−/− mice weighed approximately 10% less than wild-type littermates.

The SOCS proteins: a new family of negative regulators of signal transduction.

The negative regulation of cytokine signaling, with the exception of the tyrosine phosphatases, is not widely understood. We recently identified a new family of negative regulators by retroviral expression of hematopoietic cDNA library in the monocytic leukemic cell line, M1. This was coupled with selection for cells that were no longer able to differentiate in response to interleukin‐6. From this screen, SOCS‐1 was cloned and was shown to arrest cytokine signaling by binding to and inhibiting the intrinsic enzymatic activity of the JAK family of protein tyrosine kinases. SOCS‐1 expression is induced in response to a range of cytokines and as such is thought to form part of a classic negative feedback loop. The SOCS family of proteins is linked by the presence of a conserved carboxy‐terminal domain termed the SOCS box and now encompasses five distinct protein groups on the basis of the structural elements found amino‐terminal to the SOCS box: (1) those that contain SH2 domains, (2) those that contain WD‐40 repeats, (3) ankyrin repeats, (4) SPRY domains, and (5) GTPase domains. As yet the function of the SOCS box remains unknown, but given the level of conservation within such diverse proteins, it is likely to have a broadly similar role in each. J. Leukoc. Biol. 63: 665–668; 1998.

Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction.

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.

The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development

Naïve T helper cells differentiate into Th1 and Th2 subsets, which have unique cytokine signatures, activators and transcriptional targets. The Th1/Th2 cytokine milieu is a key paradigm in lineage commitment, and IL-4 (Il4), IL-13 (Il13) and Stat6 are important mediators of Th2 development. We show here, for the first time, that this paradigm applies also to mammary epithelial cells, which undergo a switch from Th1 to Th2 cytokine production upon the induction of differentiation. Thus, the Th1 cytokines IL-12 (Il12), interferon gamma (INFγ; also known as Ifng) and Tnfα are downregulated concomitantly with the upregulation of the Th2 cytokines IL-4, IL-13 and IL-5 (Il5) as epithelial cells commit to the luminal lineage. Moreover, we show that Th2 cytokines play a crucial role in mammary gland development in vivo, because differentiation and alveolar morphogenesis are reduced in both Stat6 and IL-4/IL-13 doubly deficient mice during pregnancy. This unexpected discovery demonstrates a role for immune cell cytokines in epithelial cell fate and function, and adds an unexpected tier of complexity to the previously held paradigm that steroid and peptide hormones are the primary regulators of mammary gland development.

Suppression of cytokine signaling: the SOCS perspective.

The discovery of the Suppressor of Cytokine Signaling (SOCS) family of proteins has resulted in a significant body of research dedicated to dissecting their biological functions and the molecular mechanisms by which they achieve potent and specific inhibition of cytokine and growth factor signaling. The Australian contribution to this field has been substantial, with the initial discovery of SOCS1 by Hilton, Starr and colleagues (discovered concurrently by two other groups) and the following work, providing a new perspective on the regulation of JAK/STAT signaling. In this review, we reflect on the critical discoveries that have lead to our current understanding of how SOCS proteins function and discuss what we see as important questions for future research.