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SOCS Proteins: Negative Regulators of Cytokine Signaling

The Walter and Eliza Hall Institute of Medical Research and the Cooperative Research Center for Cellular Growth Factors, Royal Melbourne Hospital, Victoria, Australia

Key Words. SOCS • Cytokine • Signal transduction • JAK • STAT

Danielle L. Krebs, Ph.D., The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia. Telephone: 61-3-9345-2525; Fax: 61-3-9347-0852; e-mail:krebs{at}wehi.edu.au

	ABSTRACT

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
Cytokines regulate the growth and differentiation of cells by binding to cell-surface receptors and activating intracellular signal transduction cascades such as the JAK-STAT pathway. Cytokine signaling is negatively regulated with respect to both magnitude and duration, and it is now clear that the suppressor of cytokine signaling (SOCS) family of proteins (SOCS1-SOCS7 and CIS) contributes significantly to this process. Transcripts encoding CIS, SOCS1, SOCS2, and SOCS3 are upregulated in response to cytokine stimulation, and the corresponding SOCS proteins inhibit cytokine-induced signaling pathways. SOCS proteins therefore form part of a classical negative feedback circuit. SOCS family members modulate signaling by several mechanisms, which include inactivation of the Janus kinases (JAKs), blocking access of the signal transducers and activators of transcription (STATs) to receptor binding sites, and ubiquitination of signaling proteins and their subsequent targeting to the proteasome. Gene targeting has been used to generate mice lacking socs1, socs2, or socs3, in order to elucidate the physiological function of these SOCS family members. The analysis of socs1^–/– mice has revealed that SOCS1 plays a key role in the negative regulation of interferon- signaling and in T cell differentiation. Socs2^–/– mice are 30%-40% larger than wild-type mice, demonstrating that SOCS2 is a critical regulator of postnatal growth. Additionally, the study of embryos lacking socs3 has revealed that SOCS3 is an important regulator of fetal liver hematopoiesis. The biological role of other SOCS proteins remains to be determined.

	INTRODUCTION

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
Cytokines comprise a large family of secreted glycoproteins that regulate fundamental biological processes including hematopoiesis, immunity, wound healing, and the development of the nervous system. Cytokines relay biological information to target cells by binding to receptors on the cell surface. Although cytokine receptors lack intrinsic kinase activity, more often than not they are constitutively associated with members of the Janus kinase (JAK) family of tyrosine kinases, which includes TYK2, JAK1, JAK2, and JAK3. Cytokine binding induces receptor aggregation, resulting in the juxtaposition of JAKs and their activation via cross-phosphorylation. Activated JAKs phosphorylate multiple tyrosine residues in the cytoplasmic domain of cytokine receptors, creating recruitment sites for signaling proteins containing Src-homology 2 (SH2) or phosphotyrosine binding (PTB) domains. In this way, cytokine stimulation initiates multiple signal transduction cascades such as those involving RAS, phosphatidylinositol 3-kinase (PI3K), phospholipase C-, and the signal transducers and activators of transcription (STATs). Together, these pathways culminate in the regulation of gene expression in the nucleus, leading to target cell differentiation, proliferation, survival, apoptosis or activation [1].

The STAT family of transcription factors consists of STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. The targeted disruption of genes encoding STAT family members has revealed that STATs are necessary for many cytokine-induced biological responses. STAT proteins bind to tyrosine-phosphorylated cytokine receptors through their SH2 domains and become phosphorylated on specific C-terminal tyrosine residues by the JAKs. Phosphorylated STATs then dissociate from the receptor and form homo- or heterodimers through their SH2 domains. STAT dimers immediately enter the nucleus, where they bind to specific DNA sequences in the promoters of target genes, and thus regulate transcription [2].

Cytokine-induced signal transduction pathways must be tightly regulated to avoid the detrimental consequences of excessive stimulation. Recently, it has become clear that at least three different classes of inhibitors contribute to the negative regulation of cytokine signaling: protein tyrosine phosphatases such as SHP-1 and CD45, the protein inhibitors of activated STATs (PIAS) and the suppressor of cytokine signaling (SOCS) proteins [3, 4]. Here we review the cloning of the socs gene family, the mechanism of SOCS action and the physiological function of SOCS proteins.

	DISCOVERY OF THE SOCS FAMILY OF PROTEINS

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
Socs1 was cloned in 1997 by three groups which used different experimental approaches. Starr et al. cloned socs1 using a functional screen for inhibitors of cytokine signaling [5]. In this study, the M1 myeloid leukemic cell line was infected with a retroviral cDNA library, and M1 colonies that failed to undergo interleukin-6 (IL-6)-induced macrophage differentiation were isolated. The cDNA in one IL-6-resistant M1 colony encoded an SH2 domain-containing protein, which Starr et al. named suppressor of cytokine signaling 1 (SOCS1). Naka et al. identified SOCS1 as a protein recognized by a monoclonal antibody directed against the STAT3 SH2 domain, and named it STAT-induced STAT-inhibitor [6]. SOCS1 was also identified by Endo et al. as a protein that bound to the catalytic domain of JAK2 in a yeast two-hybrid screen [7]. Endo et al. named this protein JAK-binding protein.

Database searches using the predicted amino acid sequence of SOCS1 revealed that at least 20 proteins in mice and humans share sequence homology within a 40-residue C-terminal motif referred to as the SOCS box [5, 8]. One of these genes, cytokine-inducible SH2-containing protein (CIS), had been previously cloned as an immediate early gene induced by IL-2, IL-3, and erythropoietin (Epo) [9]. The 20 SOCS box-containing proteins were classified into five subfamilies based on the domains they contained in their central regions. Proteins containing a central SH2 domain were termed SOCS (SOCS1-SOCS7 and CIS), proteins containing WD-40 repeats were termed WSB (WSB1 and WSB2), proteins containing ankyrin repeats were termed ASB (ASB1-ASB3), and proteins containing SPRY domains were termed SSB (SSB1-SSB3). In addition, a subfamily containing a GTPase domain was identified (RAR and RAR-like) [8]. Database searches also revealed a Drosophila melanogaster ortholog of SOCS5 [10].

Interestingly, a comparison of the primary amino acid sequence of SOCS subfamily members shows that pairs of SOCS proteins are more similar to each other than to other SOCS proteins. Indeed, CIS and SOCS2, SOCS1 and SOCS3, SOCS4 and SOCS5, and SOCS6 and SOCS7 all form related pairs. Structurally, the N-terminal regions of SOCS proteins are variable in length, ranging from 50-380 amino acids [8]. The SOCS N-terminal regions contain no recognizable motifs, the exception being SOCS7 which contains a putative nuclear localization signal and multiple proline-rich regions [11]. Presently, there are three systems of nomenclature for the SOCS proteins/genes, although the SOCS nomenclature has become the most widely used (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. The alternative names and domain structure of the SOCS protein family. The kinase inhibitory region of SOCS1 and SOCS3 is indicated in red.

	SOCS MRNAS ARE INDUCED IN RESPONSE TO CYTOKINE STIMULATION

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
Transcripts encoding SOCS1, SOCS2, SOCS3, and CIS are often present in cells at low or undetectable levels, but are rapidly induced by a broad spectrum of cytokines, both in vitro and in vivo. For example, SOCS1, SOCS2, SOCS3, and CIS mRNAs are all induced in response to stimulation with IL-2, IL-3, IL-4, IL-6, interferon- (IFN-), Epo, G-CSF, GM-CSF, leukemia inhibitory factor (LIF), prolactin, and growth hormone (GH) [5, 6, 9,13-16].

In general, there is no simple relationship between a particular cytokine and the pattern of SOCS mRNAs it induces. Rather, the cytokine-induced expression of SOCS family members tends to vary with respect to the cell line or tissue being studied. For example, IFN- induces the expression of SOCS1 and SOCS3 but not SOCS2 or CIS mRNAs in the NIH-3T3 cell line, but only SOCS1 mRNA is induced by IFN- in the M1 cell line [17]. In most cases, cytokine stimulation results in the upregulation of several SOCS family members, the exception being SOCS3, which is specifically induced by some cytokines. For example, injection of either leptin or ciliary neurotrophic factor induces the expression of SOCS3 but not SOCS1, SOCS2 or CIS mRNA in the mouse hypothalamus [18-20]. Likewise, IL-10 and lipopolysaccharide specifically induce SOCS3 mRNA in monocytes and macrophages, respectively [21, 22].

	STATS MEDIATE INDUCTION OF SOCS TRANSCRIPTION

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
Clearly, SOCS expression is tightly regulated at the transcriptional level. It is now apparent that the STAT family of transcription factors contributes significantly to the transcriptional upregulation of the cis, socs1 and socs3 genes. The promoter of the cis gene contains four STAT5-binding sites, all of which are required for Epo-dependent activation of the cis promoter in reporter assays [23]. In addition, the IL-3-induced expression of CIS mRNA is inhibited in cells expressing a dominant negative version of STAT5, and CIS expression in mouse ovary is abolished in mice lacking both STAT5a and STAT5b [24, 25].

Expression of the socs1 gene is also regulated by the STATs. The socs1 promoter contains putative STAT1-, STAT3- and STAT6-binding sites, and a dominant negative version of STAT3 inhibits the IL-6- or LIF-induced expression of SOCS1 mRNA [6, 26]. Recently, Saito et al. [26] reported that the IFN--induced expression of SOCS1 mRNA is eliminated in fibroblasts lacking STAT1. Somewhat surprisingly, STAT1 was found to act indirectly by inducing the expression of the interferon regulatory factor-1 (IRF-1) transcription factor, which in turn stimulates transcription of the socs1 gene [26].

Expression of the socs3 gene is also STAT-regulated. The socs3 promoter contains a STAT1/STAT3 binding element which is necessary and sufficient for LIF-dependent activation of the socs3 promoter in reporter assays. In addition, the LIF-induced expression of SOCS3 mRNA is inhibited in AtT-20 cells expressing a dominant negative version of STAT3 [27]. There is evidence that STAT5b also regulates socs3 expression. Indeed, the GH-induced expression of socs3 mRNA in liver is downregulated in mice lacking STAT5b, and STAT5b can bind to the STAT1/STAT3 element in the socs3 promoter [16, 28]. It is likely that socs2 expression is also regulated by the STATs; however, this remains to be investigated.

As previously discussed, SOCS mRNAs are induced by cytokines, and the corresponding SOCS proteins can extinguish the signaling pathways that stimulated their production. SOCS proteins therefore act in part of a classical negative feedback loop (Fig. 2). For example, the expression of CIS is induced by STAT5 in response to stimulation with Epo, and CIS inhibits Epo-induced STAT5 activation [23]. In some cases, the induction of SOCS proteins by one cytokine can inhibit signaling by another. For example, IL-6 upregulates SOCS1 expression in CD4⁺ T cells. This inhibits IFN- signaling, thereby preventing CD4⁺ Th1 cell differentiation [29]. Thus, SOCS proteins might mediate crosstalk between cytokine receptors.

View larger version (27K): [in this window] [in a new window]   Figure 2. The molecular mechanism by which SOCS proteins negatively regulate cytokine signaling. Cytokine stimulation activates the JAK-STAT pathway, leading to the induction of CIS, SOCS1, SOCS2, and/or SOCS3. These SOCS proteins then inhibit the signaling pathways that initially led to their production. SOCS proteins therefore act in part of a negative feedback loop. CIS, SOCS1, SOCS2, and SOCS3 appear to inhibit signaling by different mechanisms: SOCS1 binds to the JAKs and inhibits catalytic activity, SOCS3 binds to JAK-proximal sites on cytokine receptors and inhibits JAK activity, and CIS blocks the binding of STATs to cytokine receptors. The mechanism of SOCS2 action remains to be determined.

	OVEREXPRESSION STUDIES REVEAL THAT SOCS PROTEINS INHIBIT CYTOKINE SIGNALING

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

SOCS1 transgenic mice have also been created [34]. In these mice, socs1 is expressed under the control of the lck proximal promoter and therefore overexpressed in the T cell lineage. In primary thymocytes from these mice, the activation of STATs in response to stimulation with IFN-, IL-4, IL-6, or IL-7 is significantly reduced. Interestingly, the phenotype of SOCS1 transgenic mice is similar to that of mice lacking either the -common cytokine receptor chain or JAK3, which suggests that SOCS1 can attenuate signaling from a wide range of cytokines.

In addition to regulating the JAK-STAT pathway, SOCS proteins might have a wider range of action. Indeed, SOCS family members can bind to a variety of signaling proteins in vitro, indicating that they could modulate many signaling systems (Table 1). However, additional research is required to determine the significance of these protein interactions.

	THE PHYSIOLOGICAL FUNCTION OF SOCS PROTEINS

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
Overexpression studies reveal the potential of SOCS family members to inhibit multiple cytokine-induced signaling pathways. However, because these studies involve the ectopic expression of SOCS proteins, they could overestimate the range of SOCS action. In addition, the artificially high expression of SOCS proteins in these studies might lead to a qualitative difference in their action. This is exemplified by the finding that SOCS2 inhibits GH signaling when expressed at low level; however, higher expression levels of SOCS2 actually stimulate GH signaling [35]. It is perhaps not surprising that research using loss-of-function mouse models has provided new insight into SOCS function.

To investigate the physiological function of SOCS1, three groups have used gene targeting to disrupt the socs1 gene in mice [36-38]. Although socs1^–/– mice are normal at birth, they exhibit stunted growth and die at 3 weeks of age with a syndrome characterized by severe lymphopenia, activation of peripheral T cells, fatty degeneration and necrosis of the liver, and macrophage infiltration of major organs.

Interestingly, the neonatal defects exhibited by socs1^–/– mice appear to occur primarily as a result of unbridled IFN- signaling. Indeed, the administration of IFN- to neonatal wild-type mice induces a pathology similar to that observed in socs1^–/– mice, and socs1^–/– mice that also lack the IFN- gene do not die neonatally [38-40]. Evidence of enhanced IFN- signaling in socs1^–/– mice is suggested by observations that STAT1 is constitutively activated, and the IFN--inducible genes IRF1 and iNOS are upregulated in socs1^–/– animals [40]. Two factors likely contribute to the excessive IFN- signaling in mice lacking SOCS1. First, sensitive assays reveal an elevation in the level of serum IFN- in some, but not all of these mice [38; Hilton et al., unpublished data]. Second, socs1^–/– mice and tissues exhibit an intrinsic hypersensitivity to IFN-, as illustrated by the finding that socs1^–/– bone-marrow-derived macrophages clear a Leishmania major parasite infection in response to 100-fold less IFN- than do wild-type macrophages [40].

Because T cells are the primary source of IFN- production, it is likely that the excess IFN- is derived from the aberrantly activated T cells in socs1^–/– mice. Consistent with this, socs1^–/– mice that also lack the rag2 gene, and therefore lack functional lymphocytes, exhibit undetectable levels of IFN- and survive. Furthermore, reconstitution of the lymphoid lineage of irradiated jak3^–/– mice with socs1^–/– bone marrow recapitulates the same fatal syndrome [38].

As mentioned above, mice lacking socs1 exhibit severe lymphopenia. This was initially attributed to accelerated lymphocyte apoptosis due to upregulation of the proapoptotic protein Bax [37]. More recently, Morita et al. reported that apoptosis in socs1^–/– mice might result from excessive tumor necrosis factor- (TNF-) signaling [41]. Indeed, serum TNF- levels are elevated in socs1^–/– animals, and socs1^–/– murine embryonic fibroblasts exhibit enhanced sensitivity to TNF--induced cell death. The analysis of mice lacking SOCS1 therefore suggests that SOCS1 plays a key role in the negative regulation of signaling by IFN-. Additionally, SOCS1 likely regulates T cell differentiation to prevent the emergence of activated T cells that produce excess IFN-; a situation that may have secondary consequences such as the upregulation of proinflammatory cytokines like TNF-.

Recently, Metcalf et al. generated mice lacking the socs2 gene, in order to elucidate the function of SOCS2 in vivo [42]. Although socs2^–/– mice appear normal until 3 weeks of age, they subsequently grow 30%-40% larger than their wild-type littermates. This weight increase is not attributable to excess fat, but rather to a significant increase in visceral organ weight, carcass weight, long bone length and body length. Another abnormality exhibited by socs2^–/– mice is a thickening of the dermis, which is associated with excess collagen accumulation.

Given that socs2^–/– mice are excessively large, an attractive hypothesis is that socs2 acts to negatively regulate growth-promoting cytokines such as GH and IGF-1. The observation that socs2^–/– mice are phenotypically similar to both GH and IGF-1 transgenic mice supports this. Indeed, GH transgenic mice, as well as humans with elevated levels of GH, exhibit an increase in bone growth, and IGF-1 transgenic mice exhibit an increase in body weight and an enhancement in growth rate [43]. Furthermore, both GH and IGF-1 transgenic mice possess an increased deposition of collagen in the dermis [44, 45]. Thus, socs2^–/– mice exhibit abnormalities associated with augmented GH and IGF-1 signaling.

There is also more direct evidence of enhanced GH signaling in socs2^–/– mice. One outcome of GH signaling is the production of IGF-1, and socs2^–/– mice exhibit elevated levels of IGF-1 in the heart, lungs and spleen [42]. GH also modulates the expression of major urinary protein (MUP). MUP is a GH pulse-dependent product that is downregulated in transgenic mice that constitutively overexpress GH, and therefore lack pulsatile GH signaling. Intriguingly, MUP levels are decreased in socs2^–/– urine, suggesting that GH signaling might be disregulated in these mice [42]. SOCS2 therefore appears to play an essential physiological role in the regulation of growth, possibly due to its ability to modulate GH and/or IGF-1 signaling.

The biological function of SOCS3 has also been recently investigated by Marine et al. [30]. The authors reported that SOCS3 is expressed at low levels in adult tissues; however, it is specifically expressed in fetal liver erythroid progenitors during a stage of erythropoiesis characterized by a massive, Epo-dependent expansion of cells of the erythroid lineage. To investigate the function of SOCS3 in vivo, Marine et al. generated transgenic mice that overexpress SOCS3, as well as mice that lack the socs3 gene. Both socs3^–/– mice and SOCS3 transgenic mice die during the embryonic stage of development. Interestingly, about 75% of embryos containing the SOCS3 transgene completely lack fetal liver erythropoiesis, whereas many socs3^–/– embryos were reported to exhibit marked erythrocytosis throughout the embryo [30]. Taken together, these experiments suggest that SOCS3 may play an important role in the regulation of fetal liver erythropoiesis. Given that Epo signaling is required for erythropoiesis, it is possible that SOCS3 modulates this process by attenuating Epo signaling. The fact that SOCS3 can inhibit Epo signaling in vitro is consistent with this idea [46].

	MOLECULAR MECHANISM OF SOCS ACTION

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
SOCS family members can inhibit cytokine signaling by several different mechanisms. Many studies show that SOCS1 directly interacts with JAK1, JAK2, JAK3, and TYK2 and, in doing so, inhibits their catalytic activity [6, 7, 10, 47]. SOCS3 also inhibits JAK2 activity; however, compared with SOCS1, SOCS3 binds to JAK2 with a lower affinity and is a significantly less efficient inhibitor [15, 48-50]. Unlike SOCS1 and SOCS3, CIS and SOCS2 do not bind to, or inhibit the JAKs [15, 47].

Structure-function studies using truncated versions of SOCS1 have revealed the mechanism by which SOCS1 inhibits JAK activity. Interestingly, SOCS1 binds to phosphorylated Y1007 of JAK2, which lies within the JAK2 activation loop and regulates JAK activity [47]. The SOCS1 SH2 domain is sufficient for JAK2 binding; however, both the SH2 domain and 24 residues immediately N-terminal to the SH2 domain are required for high affinity binding and inhibition of JAK2 activity [10, 47, 51]. Within this 24-residue region, both SOCS1 and SOCS3 exhibit homology within a 12-residue sequence that is absolutely required for inhibition of JAK2 activity. This region has been termed the kinase inhibitory region (KIR) [47]. Interestingly, the KIR resembles the JAK activation loop, which in itself is a JAK substrate. Yasukawa et al. have therefore hypothesized that the KIR might inhibit JAK activity by acting as a pseudosubstrate that prevents access of legitimate substrates to the JAK catalytic pocket [47].

Another way SOCS proteins might inhibit signaling is by competing with STATs for common PTB sites within the cytoplasmic domains of cytokine receptors. Ram and Waxman have suggested that the inhibitory action of CIS occurs, in part, by this mechanism [52]. Indeed, CIS inhibits GH-induced STAT5b activation, and both CIS and STAT5b bind to an overlapping set of phosphotyrosine residues on the GH receptor [32, 52, 53]. In addition, the inhibitory action of CIS is more complete when the expression level of STAT5b is decreased, which suggests that CIS and STAT5b might compete for GH-receptor binding sites [31, 52]. Thus, CIS might inhibit GH signaling by blocking access of STAT5b to the activated GH receptor. CIS also inhibits Epo signaling and binds to the Epo receptor at Y401, which is one of the two STAT5 binding sites on this receptor [54]. This suggests that CIS might modulate Epo signaling by masking the STAT5 binding site on the Epo receptor.

The mechanism by which SOCS3 inhibits cytokine signaling appears to be intermediate between that exhibited by CIS and SOCS1. Although SOCS3 is a very poor JAK2 inhibitor, the ability of SOCS3 to inhibit GH-induced JAK2 activation increases significantly when the expression level of the GH receptor is increased [53]. Similarly, the ability of SOCS3 to inhibit IL-2-induced JAK1 activation is enhanced in the presence of the IL-2Rß chain [14]. Thus, the inhibition of JAK activity by SOCS3 is augmented when SOCS3 is bound to cytokine receptors. Interestingly, a chimeric protein containing the CIS N-terminal domain fused to the SOCS3 SH2 domain and SOCS box does not inhibit GH signaling, suggesting that the SOCS3 N-terminal domain is required for inhibition [53].

Recently, Nicholson et al. further investigated the mechanism of SOCS3 action by identifying the primary SOCS3-binding partners within the gp130 signaling pathway [55]. This was accomplished by determining the affinity between SOCS3 and various phosphopeptides derived from either the JAKs, the STATs, or the gp130 subunit of the LIF/IL-6 receptors. The authors found that SOCS3 binds with high affinity to a phosphopeptide corresponding to a region surrounding Y757 within gp130, while it exhibits weak binding to all other phosphopeptides tested. Furthermore, the inhibition of gp130 signaling by SOCS3, but not SOCS1, is significantly reduced when Y757 on gp130 is mutated to phenylalanine [55, 56]. This suggests that optimal inhibition by SOCS3 occurs when SOCS3 is bound to gp130. SOCS3 also binds to the leptin and Epo receptors, and mutation of the SOCS3 binding site on these receptors interferes with the ability of SOCS3 to inhibit leptin and Epo signaling, respectively [46, 50]. Taken together, these studies suggest that SOCS3 is brought into the vicinity of the JAKs by binding to activated cytokine receptors. Once localized at the receptor, SOCS3 likely inhibits JAK activity through its KIR.

A third line of evidence shows that SOCS proteins might act by promoting the degradation of specific signaling proteins. Zhang et al. suggest that SOCS box-containing proteins act as adapter molecules that recruit activated signaling proteins to the proteasome [58]. All SOCS proteins tested associate with a complex containing elongins B and C (elongin BC) through their SOCS box [58, 59]. In turn, elongin BC associates with cullin-2 to form part of a putative E3 ubiquitin ligase. Given that SOCS proteins contain SH2 domains that bind to tyrosine-phosphorylated signaling proteins, they could therefore act as adapters that facilitate the ubiquitination and subsequent degradation of associated signaling proteins.

This proposed function of SOCS proteins is analogous to that of the von Hippel-Lindau tumor suppressor protein (VHL), which also binds to elongin BC and cullin-2 to form an E3 ubiquitin ligase complex. Recently, VHL was shown to act as an adapter that brings the subunit of the hypoxia-inducible factor-1 transcription factor into the vicinity of the E3 ubiquitin ligase complex, thus targeting it for proteasomal degradation [60]. Interestingly, elongin BC binds to a site on VHL that exhibits significant homology to the SOCS box. The similarity between VHL and SOCS family members suggests that they may have analogous functions.

In keeping with the above-mentioned model, there is evidence that SOCS and SOCS-associated proteins are ubiquitinated and degraded by the proteasome. For example, SOCS1 binds to the guanine-nucleotide exchange factor VAV in an SH2-domain-dependent manner and, when coexpressed, SOCS1 stimulates the ubiquitination and degradation of VAV [61]. Additionally, CIS can be monoubiquitinated, and the monoubiquitinated form of CIS accumulates in the presence of proteasome inhibitors [54]. CIS is also degraded in response to GH stimulation in a proteasome-dependent manner, and the inhibition of GH signaling by CIS is blocked by proteasome inhibitors [52]. This suggests that CIS uses the proteasome to negatively regulate GH signaling, likely by targeting the GH receptor/JAK2/CIS complex for degradation.

	CONCLUSIONS

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
Since the discovery of the SOCS family in 1997, significant advances have been made in understanding the function of SOCS proteins. In overexpression systems, SOCS1, SOCS3, and CIS inhibit signaling by a wide range of cytokines; however, these SOCS proteins appear to have a more restricted function in vivo. Indeed, the analysis of mice lacking socs1 has revealed that SOCS1 is a critical regulator of IFN- signaling and T cell differentiation. Interestingly, the study of mice lacking socs2 shows that SOCS2 regulates postnatal growth, likely through its ability to influence the GH/IGF-1 axis. Mice lacking socs3 die in utero, and the analysis of socs3^–/– embryos has revealed that SOCS3 might play a key role in the regulation of fetal liver erythropoiesis, likely by regulating Epo signaling.

In vitro and transgenic mouse studies implicate CIS in the negative regulation of signaling by Epo, GH, prolactin, and other cytokines that signal through STAT5. Nevertheless, mice lacking the cis gene have been generated, and are phenotypically normal, perhaps due to functional compensation by other SOCS proteins [30]. It will be interesting to explore whether CIS, SOCS1, SOCS2, and SOCS3 have overlapping actions that are not revealed by mice lacking single socs genes. Another challenge will be to elucidate the function of SOCS4, SOCS5, SOCS6, and SOCS7.

Biochemical studies have provided great insight into the mechanism by which SOCS family members inhibit cytokine signaling. Both SOCS1 and SOCS3 contain a 12-residue KIR in their N-terminal domain, which is capable of inhibiting JAK catalytic activity. The KIR likely acts as a pseudosubstrate that blocks access of legitimate substrates to the JAK catalytic pocket. Interestingly, while both SOCS1 and SOCS3 inhibit the JAKs in a similar manner, they appear to be recruited to the JAKs by distinct mechanisms. SOCS1 directly binds to active JAKs, whereas SOCS3 likely binds to JAK-proximal sites on the intracellular domain of activated cytokine receptors. Unlike SOCS1 and SOCS3, neither CIS nor SOCS2 can bind to the JAKs or inhibit JAK activity. CIS likely inhibits signaling by masking STAT5 binding sites on activated cytokine receptors; however, the molecular mechanism of SOCS2 action remains to be determined. SOCS proteins also appear to inhibit signaling by recruiting the proteasomal machinery to signaling complexes, which results in the ubiquitination and subsequent degradation of neighboring proteins.

	ACKNOWLEDGMENT

Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 
The original work reported here was supported by the National Health and Medical Research Council, Canberra, the Anti-Cancer Council of Victoria, an Australian Government Cooperative Research Centres Program Grant, the National Institutes of Health, Bethesda, the J.D. and L. Harris Trust, and AMRAD Operations Pty Ltd., Melbourne.

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Top Abstract Introduction Discovery of the SOCS... SOCS mRNAs are Induced... STATs Mediate Induction of... Overexpression Studies Reveal... The Physiological Function of... Molecular Mechanism of SOCS... Conclusions References

 

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