Functional analysis of Asb-1 using genetic modification in mice

The Asbs are a family of ankyrin repeat proteins that, along with four other protein families, contain a C-terminal SOCS box motif, which was first identified in the suppressor of cytokine signaling (SOCS) proteins. While it is clear that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological roles of the other SOCS box-containing families are unknown. We have investigated Asb-1 function by generating mice that lack this protein, as well as mice that overexpress full-length or truncated Asb-1 in a wide range of tissues. Although Asb-1 is expressed in multiple organs, including the hematopoietic compartment in wild-type mice, Asb-1(-/-) mice develop normally and exhibit no anomalies of mature blood cells or their progenitors. While most organs in these mice appear normal, the testes of Asb-1(-/-) mice display a diminution of spermatogenesis with less complete filling of seminiferous tubules. In contrast, the widespread overexpression of Asb-1 in the mouse has no apparent deleterious effects.     

The SOCS box-adapting proteins for ubiquitination and proteasomal degradation

The suppressor of cytokine signalling (SOCS) box was first identified in the SH2-containing SOCS box family (cytokine-inducible SH2-containing protein, SOCS1-7) and is a 40-amino acid motif, which functions to recruit an E3 ubiquitin ligase complex consisting of the adapter proteins elongins B and C, Rbx2 and the scaffold protein Cullin5. The SOCS box is found in a diverse array of intracellular signalling molecules, many of which contain different protein interaction domains such as SPRY and WD40 domains, leucine and ankyrin repeats or other functional domains such as GTPases. In general, the SOCS box-containing proteins are thought to act as substrate-recognition modules to mediate the polyubiquitination and subsequent degradation of substrate proteins by the 26S proteasome.     

The many faces of the SOCS box

The suppressors of cytokine signalling (SOCS) box is a structural domain found at the C-terminus of over 70 human proteins. It is usually coupled to a protein interaction module such as an SH2 domain in case of SOCS proteins, a family of modulators of cytokine signaling. The SOCS box participates in the formation of E3 ligase complexes, marking activated cytokine receptor complexes for proteasomal degradation. A similar mechanism was recently uncovered for controlling SOCS activity itself, since SOCS2 was found to enhance the turnover of other SOCS proteins. The SOCS box can also add unique features to individual SOCS proteins: it can function as an adaptor domain as was demonstrated for SOCS3, or as a modulator of substrate binding in case of CIS. In this review we discuss these multiple roles of the SOCS box, which emerges as a versatile module controlling cytokine signaling via multiple mechanisms.     

ATRA-regulated Asb-2 gene induced in differentiation of HL-60 leukemia cells

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C-terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N-terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL-60 cells, and demonstrated that ASB-2, one of the ASB proteins, was rapidly induced by all-trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb-2 gene. We showed that RARalpha, one of the RARs, binds to the RARE/RXRE in the Asb-2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB-2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.     

Suppressors of cytokine signalling (SOCS) in the immune system

The suppressors of cytokine signalling (SOCS) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine responses, including those that regulate the immune system. The SOCS proteins seem to regulate signal transduction by combining direct inhibitory interactions with cytokine receptors and signalling proteins with a generic mechanism of targeting associated proteins for degradation. Evidence is emerging for the involvement of SOCS proteins in diseases of the human immune system, which raises the possibility that therapeutic strategies that are based on the manipulation of SOCS activity might be of clinical benefit.     

Ankyrin repeat and suppressors of cytokine signaling box protein asb-9 targets creatine kinase B for degradation

The suppressors of cytokine signaling (SOCS) proteins inhibit cytokine action by direct interaction with Janus kinases or activated cytokine receptors. In addition to the N-terminal and Src homology 2 domains that mediate these interactions, SOCS proteins contain a C-terminal SOCS box. DNA data base searches have identified a number of other protein families that possess a SOCS box, of which the ankyrin repeat and SOCS box-containing (Asb) proteins constitute the largest. Although it is known that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological and biochemical functions of the Asbs are largely undefined. Using a proteomics approach, we demonstrate that creatine kinase B (CKB) interacts with Asb-9 in a specific, SOCS box-independent manner. This interaction increases the polyubiquitylation of CKB and decreases total CKB levels within the cell. The targeting of CKB for degradation by Asb-9 was primarily SOCS box-dependent and suggests that Asb-9 acts as a specific ubiquitin ligase regulating levels of this evolutionarily conserved enzyme.     

ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function

Background  

The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9).     

SOCS proteins, cytokine signalling and immune regulation

Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling pathways. Studies have shown that SOCS proteins are key physiological regulators of both innate and adaptive immunity. These molecules positively and negatively regulate macrophage and dendritic-cell activation and are essential for T-cell development and differentiation. Evidence is also emerging of the involvement of SOCS proteins in diseases of the immune system. In this Review we bring together data from recent studies on SOCS proteins and their role in immunity, and propose a cohesive model of how cytokine signalling regulates immune-cell function.     

The SOCS Box Encodes a Hierarchy of Affinities for Cullin5: Implications for Ubiquitin Ligase Formation and Cytokine Signalling Suppression

The SOCS (suppressors of cytokine signalling) family of proteins inhibits the cytokine-induced signalling cascade in part by promoting the ubiquitination of signalling intermediates that are then targeted for proteasomal degradation. This activity relies upon an interaction between the SOCS box domain, the adapter complex elonginBC and a member of the Cullin family, the scaffold protein of an E3 ubiquitin ligase. In this study, we dissected this interaction in vitro using purified components. We found that all eight SOCS proteins bound Cullin5 but required prior recruitment of elonginBC. Neither SOCS nor elonginBC bound Cullin5 when in isolation. Interestingly, the affinity of each SOCS-elonginBC complex for Cullin5 varied by 2 orders of magnitude across the SOCS family. Unexpectedly, the most potent suppressors of signalling, SOCS-1 and SOCS-3, bound most weakly to the E3 ligase scaffold, with affinities 100- and 10-fold lower, respectively, than the rest of the family. The remaining six SOCS proteins all bound Cullin5 with high affinity (K_d of ∼ 10 nM) due to a slower off-rate and hence a longer half-life of the complex. This difference in affinity may reflect a difference in mode of action as only SOCS-1 and SOCS-3 have been shown to suppress signalling using both SOCS box-dependent and SOCS box-independent mechanisms. This is not the case with the other six SOCS proteins, and our data imply the existence of two distinct subclasses of SOCS proteins with a high affinity for Cullin5, the E3 ligase scaffold, possibly reflecting complete dependence upon ubiquitination for suppression of cytokine signalling.     

The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase

Suppressor of cytokine signalling 3 (SOCS3) is responsible for regulating the cellular response to a variety of cytokines, including interleukin 6 and leukaemia inhibitory factor. Identification of the SOCS box domain led to the hypothesis that SOCS3 can associate with functional E3 ubiquitin ligases and thereby induce the degradation of bound signalling proteins. This model relies upon an interaction between the SOCS box, elonginBC and a cullin protein that forms the E3 ligase scaffold. We have investigated this interaction in vitro using purified components and show that SOCS3 binds to elonginBC and cullin5 with high affinity. The SOCS3-elonginBC interaction was further characterised by determining the solution structure of the SOCS box-elonginBC ternary complex and by deletion and alanine scanning mutagenesis of the SOCS box. These studies revealed that conformational flexibility is a key feature of the SOCS-elonginBC interaction. In particular, the SOCS box is disordered in isolation and only becomes structured upon elonginBC association. The interaction depends upon the first 12 residues of the SOCS box domain and particularly on a deeply buried, conserved leucine. The SOCS box, when bound to elonginBC, binds tightly to cullin5 with 100 nM affinity. Domains upstream of the SOCS box are not required for elonginBC or cullin5 association, indicating that the SOCS box acts as an independent binding domain capable of recruiting elonginBC and cullin5 to promote E3 ligase formation.     

The SOCS box: a tale of destruction and degradation

Although initially identified in the suppressor of cytokine signaling (SOCS) family of proteins, the C-terminal SOCS box has now been identified in more than 40 proteins in nine different families. Growing evidence suggests that the SOCS box, similar to the F-box, acts as a bridge between specific substrate-binding domains and the more generic proteins that comprise a large family of E3 ubiquitin protein ligases. In this way, SOCS proteins regulate protein turnover by targeting proteins for polyubiquitination and, therefore, for proteasome-mediated degradation.     

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.     

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.     

Analysis of leptin signalling in hematopoietic cells using an adapted MAPPIT strategy

The adipocyte-secreted hormone leptin participates in the regulation of hematopoiesis and enhances proliferation of hematopoietic cells. We used an adaptation of the MAPPIT mammalian two-hybrid method to study leptin signalling in a hematopoietic setting. We confirmed the known interactions of suppressor of cytokine signalling 3 (SOCS3) and STAT5 with the Y985 and Y1077 motifs of the leptin receptor, respectively. We also provide evidence for novel interactions at the Y1077 motif, including phospholipase C gamma and several members of the SOCS protein family, further underscoring the important role of the Y1077 motif in leptin signalling.     

The N-terminal domains of SOCS proteins: a conserved region in the disordered N-termini of SOCS4 and 5

Suppressors of cytokine signaling (SOCS) proteins function as negative regulators of cytokine signaling and are involved in fine tuning the immune response. The structure and role of the SH2 domains and C‐terminal SOCS box motifs of the SOCS proteins are well characterized, but the long N‐terminal domains of SOCS4–7 remain poorly understood. Here, we present bioinformatic analyses of the N‐terminal domains of the mammalian SOCS proteins, which indicate that these domains of SOCS4, 5, 6, and 7 are largely disordered. We have also identified a conserved region of about 70 residues in the N‐terminal domains of SOCS4 and 5 that is predicted to be more ordered than the surrounding sequence. The conservation of this region can be traced as far back as lower vertebrates. As conserved regions with increased structural propensity that are located within long disordered regions often contain molecular recognition motifs, we expressed the N‐terminal conserved region of mouse SOCS4 for further analysis. This region, mSOCS4_86–155, has been characterized by circular dichroism and nuclear magnetic resonance spectroscopy, both of which indicate that it is predominantly unstructured in aqueous solution, although it becomes helical in the presence of trifluoroethanol. The high degree of sequence conservation of this region across different species and between SOCS4 and SOCS5 nonetheless implies that it has an important functional role, and presumably this region adopts a more ordered conformation in complex with its partners. The recombinant protein will be a valuable tool in identifying these partners and defining the structures of these complexes. Proteins 2011. © 2012 Wiley Periodicals, Inc.     

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.     

SOCS-6 binds to insulin receptor substrate 4, and mice lacking the SOCS-6 gene exhibit mild growth retardation

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.     

SPRY Domain-Containing SOCS Box Protein 2: Crystal Structure and Residues Critical for Protein Binding

The four mammalian SPRY (a sequence repeat in dual-specificity kinase splA and ryanodine receptors) domain-containing suppressor of cytokine signalling (SOCS) box proteins (SSB-1 to -4) are characterised by a C-terminal SOCS box and a central SPRY domain. The latter is a protein interaction module found in over 1600 proteins, with more than 70 encoded in the human genome. Here we report the crystal structure of the SPRY domain of murine SSB-2 and compare it with the SSB-2 solution structure and crystal structures of other B30.2/SPRY domain-containing family proteins. The structure is a bent β-sandwich, consisting of two seven-stranded β-sheets wrapped around a long loop that extends from the centre strands of the inner or concave β-sheet; it closely matches those of GUSTAVUS and SSB-4. The structure is also similar to those of two recently determined Neuralized homology repeat (NHR) domains (also known as NEUZ domains), with detailed comparisons, suggesting that the NEUZ/NHR domains form a subclass of SPRY domains. The binding site on SSB-2 for the prostate apoptosis response-4 (Par-4) protein has been mapped in finer detail using mutational analyses. Moreover, SSB-1 was shown to have a Par-4 binding surface similar to that identified for SSB-2. Structural perturbations of SSB-2 induced by mutations affecting its interaction with Par-4 and/or c-Met have been characterised by NMR. These comparisons, in conjunction with previously published dynamics data from NMR relaxation studies and coarse-grained dynamics simulation using normal mode analysis, further refine our understanding of the structural basis for protein recognition of SPRY domain-containing proteins.     

Suppressor of cytokine signalling (SOCS) 1 and 3 enhance cell adhesion and inhibit migration towards the chemokine eotaxin/CCL11

Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role in responses to chemokines remains poorly understood. We report that cells expressing SOCS1 and 3 exhibit enhanced adhesion and reduced migration towards the chemokine CCL11. Focal adhesion kinase (FAK) and the GTPase RhoA, control cell adhesion and migration and we show the presence of SOCS1 or 3 regulates expression and tyrosine phosphorylation of FAK, while also enhancing activation of RhoA. Our novel findings suggest that SOCS1 and 3 may control chemotaxis and adhesion by significantly enhancing both FAK and RhoA activity, thus localizing immune cells to the site of allergic inflammation.