Suppressor Of Cytokine Signaling Box Research Articles

ASB3 promotes hepatocellular carcinoma progression by mediating DR5 ubiquitination in TRAIL resistance.

Ankyrin-repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis invitro and invivo. Liver-specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)-induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a DR5-dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

WSB1, a Hypoxia-Inducible E3 Ligase, Promotes Myofibroblast Accumulation and Attenuates Alveolar Epithelial Regeneration in Mouse Lung Fibrosis

Idiopathic pulmonary fibrosis is a progressive interstitial lung disease for which there is no curative therapy available. Repetitive alveolar epithelial injury repair, myofibroblast accumulation, and excessive collagen deposition are key pathologic features of idiopathic pulmonary fibrosis, eventually leading to cellular hypoxia and respiratory failure. The precise mechanism driving this complex maladaptive process remains inadequately understood. WD repeat and suppressor of cytokine signaling box containing 1 (WSB1) is an E3 ubiquitin ligase, the expression of which is associated strongly with hypoxia, and forms a positive feedback loop with hypoxia-inducible factor 1α (HIF-1α) under anoxic condition. This study explored the expression, cellular distribution, and function of WSB1 in bleomycin (BLM)-induced mouse lung injury and fibrosis. WSB1 expression was highly induced by BLM injury and correlated with the progression of lung fibrosis. Significantly, conditional deletion of Wsb1 in adult mice ameliorated BLM-induced pulmonary fibrosis. Phenotypically, Wsb1-deficient mice showed reduced lipofibroblast to myofibroblast transition, but enhanced alveolar type 2 proliferation and differentiation into alveolar type 1 after BLM injury. Proteomic analysis of mouse lung tissues identified caveolin 2 as a potential downstream target of WSB1, contributing to BLM-induced epithelial injury repair and fibrosis. These findings unravel a vital role for WSB1 induction in lung injury repair, thus highlighting it as a potential therapeutic target for pulmonary fibrosis.

A Rab-bit hole: Rab40 GTPases as new regulators of the actin cytoskeleton and cell migration.

The regulation of machinery involved in cell migration is vital to the maintenance of proper organism function. When migration is dysregulated, a variety of phenotypes ranging from developmental disorders to cancer metastasis can occur. One of the primary structures involved in cell migration is the actin cytoskeleton. Actin assembly and disassembly form a variety of dynamic structures which provide the pushing and contractile forces necessary for cells to properly migrate. As such, actin dynamics are tightly regulated. Classically, the Rho family of GTPases are considered the major regulators of the actin cytoskeleton during cell migration. Together, this family establishes polarity in the migrating cell by stimulating the formation of various actin structures in specific cellular locations. However, while the Rho GTPases are acknowledged as the core machinery regulating actin dynamics and cell migration, a variety of other proteins have become established as modulators of actin structures and cell migration. One such group of proteins is the Rab40 family of GTPases, an evolutionarily and functionally unique family of Rabs. Rab40 originated as a single protein in the bilaterians and, through multiple duplication events, expanded to a four-protein family in higher primates. Furthermore, unlike other members of the Rab family, Rab40 proteins contain a C-terminally located suppressor of cytokine signaling (SOCS) box domain. Through the SOCS box, Rab40 proteins interact with Cullin5 to form an E3 ubiquitin ligase complex. As a member of this complex, Rab40 ubiquitinates its effectors, controlling their degradation, localization, and activation. Because substrates of the Rab40/Cullin5 complex can play a role in regulating actin structures and cell migration, the Rab40 family of proteins has recently emerged as unique modulators of cell migration machinery.

An unconventional role of an ASB family protein in NF-κB activation and inflammatory response during microbial infection and colitis

Nuclear factor κB (NF-κB)-mediated signaling pathway plays a crucial role in the regulation of inflammatory process, innate and adaptive immune responses. The hyperactivation of inflammatory response causes host cell death, tissue damage, and autoinflammatory disorders, such as sepsis and inflammatory bowel disease. However, how these processes are precisely controlled is still poorly understood. In this study, we demonstrated that ankyrin repeat and suppressor of cytokine signaling box containing 1 (ASB1) is involved in the positive regulation of inflammatory responses by enhancing the stability of TAB2 and its downstream signaling pathways, including NF-κB and mitogen-activated protein kinase pathways. Mechanistically, unlike other members of the ASB family that induce ubiquitination-mediated degradation of their target proteins, ASB1 associates with TAB2 to inhibit K48-linked polyubiquitination and thereby promote the stability of TAB2 upon stimulation of cytokines and lipopolysaccharide (LPS), which indicates that ASB1 plays a noncanonical role to further stabilize the target protein rather than induce its degradation. The deficiency of Asb1 protects mice from Salmonella typhimurium- or LPS-induced septic shock and increases the survival of mice. Moreover, Asb1-deficient mice exhibited less severe colitis and intestinal inflammation induced by dextran sodium sulfate. Given the crucial role of ASB proteins in inflammatory signaling pathways, our study offers insights into the immune regulation in pathogen infection and inflammatory disorders with therapeutic implications.

The Suppressor of Cytokine Signaling-6 Gene and Its Expression during Wound Healing and Infection in the Triangle Sail Mussel Hyriopsis cumingii

Suppressors of cytokine signaling (SOCS) family members can inhibit multiple biological functions of cytokines, hormones, and growth factors. In this study, a SOCS6 gene homolog from triangle sail mussel Hyriopsis cumingii (HcSOCS6) was cloned and characterized. The full-length cDNA of HcSOCS6 was 1,823 bp and encoded 458 amino acid residues. No introns were found in the genomic sequence. The deduced HcSOCS6 protein possesses highly conserved functional domains, including the Src homology 2 domain and the SOCS box. Phylogenetic analysis showed that HcSOCS6 was clustered with other molluscan genes. Transcripts of HcSOCS6 were constitutively expressed in all examined tissues, at high levels in the hepatopancreas and gills and low levels in hemocytes. Transcription of HcSOCS6 was significantly upregulated in hemocytes and the hepatopancreas after injecting with lipopolysaccharide, but it was only upregulated in hemocytes after Aeromonas hydrophila stimulation. The expression of HcSOCS6 in mantle significantly increased during wound healing, especially on the third day. These results suggested that HcSOCS6 was involved in the immune response and wound healing in the triangle sail mussel.

Identification of crucial miRNAs and lncRNAs for ossification of ligamentum flavum.

The present study aimed to screen crucial micro (mi)RNAs and long non-coding (lnc)RNAs involved in the development of ossification of ligamentum flavum (OLF) based on the miRNA-mRNA and lncRNA-miRNA-mRNA competing endogenous (ce)RNA regulatory network analyses, which are rarely reported. The differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs) and differentially expressed miRNAs (DEMs) between 4 OLF and 4 healthy controls were identified using two microarray datasets GSE106253 and GSE106256 collected from the Gene Expression Omnibus database. A protein-protein interaction (PPI) network was constructed, followed by calculation of topological characteristics and sub-module analysis in order to obtain hub DEGs. The miRNA-mRNA and lncRNA-miRNA networks that were established based on their interaction pairs, obtained from miRwalk and starBase databases, respectively, were integrated to form the ceRNA network. The underlying functions of mRNAs were predicted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The present study screened 828 DEGs, 119 DELs and 81 DEMs between OLF and controls. PPI network and module analyses identified interleukin (IL)10, adenylate cyclase (ADCY)5, suppressor of cytokine signaling (SOCS)3, G protein subunit gamma (GNG) 4, collagen type II α 1 chain (COL2A1) and collagen type XIII α 1 chain (COL13A1) as hub genes. The miRNA-mRNA network analysis demonstrated IL10 could be regulated by miR-210-3p, while COL13A1 and COL2A1 could be modulated by miR-329-3p and miR-222-5p, respectively. lncRNA-miRNA-mRNA ceRNA network analysis identified that small nucleolar RNA host gene 16-hsa-miR-196a-5p-SOCS3, ankyrin repeat and SOCS box containing 16-AS1-hsa-miR-379-5p-GNG4, nuclear enriched abundant transcript 1-has-miR-181b-5p-ADCY5, rhophilin 1-AS1-hsa-miR-299-3p-WNT7B interaction axes may be crucial. DAVID analysis predicted IL10, ADCY5, GNG4 and SOCS3 were involved in ‘adaptive immune response’, ‘Chemokine signaling pathway’ and ‘regulation of apoptosis’ processes, while COL2A1, COL13A1 and WNT7B may be ossification related. In conclusion, the identification of these crucial miRNAs and lncRNAs may be conducive for explaining the pathogenesis of OLF and provide certain natural, endogenous and nontoxic drug targets for the treatment of OLF.

Asb2α-Filamin A Axis Is Essential for Actin Cytoskeleton Remodeling During Heart Development.

Heart development involves differentiation of cardiac progenitors and assembly of the contractile sarcomere apparatus of cardiomyocytes. However, little is known about the mechanisms that regulate actin cytoskeleton remodeling during cardiac cell differentiation. The Asb2α (Ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) CRL5 (cullin 5 RING E3 ubiquitin ligase) triggers polyubiquitylation and subsequent degradation by the proteasome of FLNs (filamins). Here, we investigate the role of Asb2α in heart development and its mechanisms of action. Using Asb2 knockout embryos, we show that Asb2 is an essential gene, critical to heart morphogenesis and function, although its loss does not interfere with the overall patterning of the embryonic heart tube. We show that the Asb2α E3 ubiquitin ligase controls Flna stability in immature cardiomyocytes. Importantly, Asb2α-mediated degradation of the actin-binding protein Flna marks a previously unrecognized intermediate step in cardiac cell differentiation characterized by cell shape changes and actin cytoskeleton remodeling. We further establish that in the absence of Asb2α, myofibrils are disorganized and that heartbeats are inefficient, leading to embryonic lethality in mice. These findings identify Asb2α as an unsuspected key regulator of cardiac cell differentiation and shed light on the molecular and cellular mechanisms determining the onset of myocardial cell architecture and its link with early cardiac function. Although Flna is known to play roles in cytoskeleton organization and to be required for heart function, this study now reveals that its degradation mediated by Asb2α ensures essential functions in differentiating cardiac progenitors.

Suppressor of cytokine signaling 2 (SOCS2) negatively regulates the expression of antimicrobial peptides by affecting the Stat transcriptional activity in shrimp Marsupenaeus japonicus.

The suppressor of cytokine signaling (SOCS) family is a kind of negative regulators in the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in mammals and Drosophila. In kuruma shrimp, Marsupenaeus japonicus, SOCS2 is identified and its expression can be stimulated by peptidoglycan and polycytidylic acid. However, if SOCS2 participates in regulating Jak/Stat pathway in shrimp still needs further study. In this study, SOCS2 with Src homology 2 domain and SOCS box was identified in kuruma shrimp, M. japonicus. SOCS2 existed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine, the expression of SOCS2 was upregulated significantly in the hemocytes and intestine of shrimp challenged with Vibrio anguillarum at 6h. To analyze SOCS2 function in shrimp immunity, bacterial clearance and survival rate were analyzed after knockdown of SOCS2 in shrimp challenged with V.anguillarum. Results showed that bacterial clearance increased, and the survival rate improved significantly comparing with controls. The SOCS2 was expressed in Escherichia coli and the recombinant SOCS2 was injected into shrimp, and Stat phosphorylation and translocation were analyzed. The result showed that "overexpression" of SOCS2 declined Stat phosphorylation level and inhibited Stat translocation into the nucleus. After knockdown of SOCS2 in shrimp prior to V.anguillarum infection, the expression level of antimicrobial peptides, including anti-lipopolysaccharide factors C1, C2 and D1, and Crustin I was upregulated significantly, and the expression of the AMPs was declined after recombinant SOCS2 injection. The SOCS2 expression was also decreased in Stat-knockdown shrimp challenged by V.anguillarum at 6 and 12h. Therefore, SOCS2 negatively regulates the AMP expression by inhibiting Stat phosphorylation and translocation into nucleus in shrimp, meanwhile, SOCS2 expression was also regulated by Jak/Stat pathway.

Molecular characterization and immune response to lipopolysaccharide (LPS) of the suppressor of cytokine signaling (SOCS)-1, 2 and 3 genes in Nile tilapia (Oreochromis niloticus).

Suppressor of cytokine signaling (SOCS) proteins are inverse feedback regulators of cytokine and hormone signaling mediated by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway that are involved in immunity, growth and development of organisms. In the present study, three SOCS genes, SOCS-1, SOCS-2 and SOCS-3, were identified in an economically important fish, Nile tilapia (Oreochromis niloticus) referred to as NtSOCS-1, NtSOCS-2 and NtSOCS-3. Multiple alignments showed that, the three SOCS molecules share highly conserved functional domains, including the SRC homology 2 (SH2) domain, the extended SH2 subdomain (ESS) and the SOCS box with others vertebrate counterparts. Phylogenetic analysis indicated that NtSOCS-1, 2 and 3 belong to the SOCS type II subfamily. Whereas NtSOCS-1 and 3 showed close evolutionary relationship with Perciformes, NtSOCS-2 was more related to Salmoniformes. Tissue specific expression results showed that, NtSOCS-1, 2 and 3 were constitutively expressed in all nine tissues examined. NtSOCS-1 and 3 were highly expressed in immune-related tissues, such as gills, foregut and head kidney. However, NtSOCS-2 was superlatively expressed in liver, brain and heart. In vivo, NtSOCS-1 and 3 mRNA levels were up-regulated after lipopolysaccharide (LPS) challenge while NtSOCS-2 was down-regulated. In vitro, LPS stimulation increased NtSOCS-3 mRNA expression, however it inhibited the transcription of NtSOCS-1 and 2. Collectively, our findings suggest that, the NtSOCS-1 and 3 might play significant role(s) in innate immune response, while NtSOCS-2 may be more involved in metabolic regulation.

SOCS6 is a selective suppressor of receptor tyrosine kinase signaling.

The suppressors of cytokine signaling (SOCS) are well-known negative regulators of cytokine receptor signaling. SOCS6 is one of eight members of the SOCS family of proteins. Similar to other SOCS proteins, SOCS6 consists of an uncharacterized extended N-terminal region followed by an SH2 domain and a SOCS box. Unlike other SOCS proteins, SOCS6 is mainly involved in negative regulation of receptor tyrosine kinase signaling. SOCS6 is widely expressed in many tissues and is found to be downregulated in many cancers including colorectal cancer, gastric cancer, lung cancer, ovarian cancer, stomach cancer, thyroid cancer, hepatocellular carcinoma, and pancreatic cancer. SOCS6 is involved in negative regulation of receptor signaling by increasing degradation mediated by ubiquitination of receptors or substrate proteins and induces apoptosis by targeting mitochondrial proteins. Therefore, SOCS6 turns out as an important regulator of survival signaling and its activity is required for controlling receptor tyrosine kinase signaling.

SOCS proteins in regulation of receptor tyrosine kinase signaling.

Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment. The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also evident that RTKs can sometimes bypass SOCS regulation and SOCS proteins can even potentiate RTKs-mediated mitogenic signaling. Thus, apart from negative regulation of receptor signaling, SOCS proteins may also influence signaling in other ways.

Disruption of RAB40AL function leads to Martin–Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder

Background and aimMartin–Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the...

Abstract 2206: SOCS1 and SOCS3 attenuate Lck-mediated cellular transformation through distinct mechanisms

Abstract The suppressor of cytokine signaling (SOCS) proteins are negative feedback inhibitors of Janus kinase (JAK) – signal transducer and activator of transcription (STAT) signaling in response to cytokine stimulation. Among eight SOCS family members, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2-containing protein) are best characterized both structurally and functionally. We showed previously that CIS, SOCS1 and SOCS3 were not expressed in Lck-transformed cells (Cell Signal 18:851; Int J Oncol 36:1201). It suggests that defective negative feedback control may contribute to constitutive JAK-STAT5 activation in Lck-mediated oncogenesis (J Immunol 159:5206; Mol Cancer Res 4:39). These findings also raise the possibility that SOCS proteins may function as tumor suppressors. Indeed, by enforced SOCS1 and SOCS3 expression, we were able to reduce Lck-induced cellular transformation (Int J Oncol 36:1201). In this report, we further examine the molecular mechanisms underlying SOCS tumor-suppressing activity. Through comparative studies of SOCS1, SOCS2, SOCS3 and CIS, we demonstrate here two distinct properties of SOCS1 and SOCS3 toward oncogenic Lck kinase. First, SOCS1 has the highest affinity in binding to oncogenic Lck kinase. We identified the positive regulatory phospho-tyrosine 394 residue (Y394) in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These data suggest that SOCS1 may inhibit Lck kinase activity in a manner similar to JAK kinases. Second, only SOCS3 becomes phosphorylated in the presence of oncogenic Lck kinase. We identified the phosphorylation sites as Y204 and Y221 located in the C-terminal SOCS box of SOCS3. We further analyzed the contribution of SOCS3 tyrosine phosphorylation on its tumor suppressor activity. Preliminary studies suggested that SOCS3 phosphorylation on Y204 and Y221 might play an important role in blocking cell proliferation and cell cycle progression of Lck-transformed cells. SOCS3 tyrosine phosphorylation can lead to altered protein stability and crosstalk with other signal transduction pathways, such as MAPK signaling. Experiments are in progress to further define the molecular details of SOCS3 phosphorylation involved in tumor suppression. All together, these results provide important mechanistic insights into the distinct roles of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases. This work is supported in part by NIH grant CA107210. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2206. doi:10.1158/1538-7445.AM2011-2206

Molecular Insight into the Conformational Dynamics of the Elongin BC Complex and Its Interaction with HIV-1 Vif

The human immunodeficiency virus type 1 virion infectivity factor (Vif) inhibits the innate viral immunity afforded by the APOBEC3 family of cytidine deaminases. Vif targets the APOBEC3 family for poly-ubiquitination and subsequent proteasomal degradation by linking the Elongin-BC-dependent ubiquitin ligase complex with the APOBEC3 proteins. The interaction between Vif and the heterodimeric Elongin BC complex, which is mediated by Vif's viral suppressor of cytokine signaling box, is essential for Vif function. The biophysical consequences of the full-length Vif:Elongin BC interaction have not been extensively reported. In this study, hydrogen exchange mass spectrometry was used to dissect the Vif:Elongin BC interaction. Elongin C was found to be highly dynamic in the Elongin BC complex while Elongin B was much more stable. Recombinant full-length Vif interacted with the Elongin BC complex in vitro with a Kd of 1.9 μM and resulted in observable changes in deuterium uptake in both Elongin C and B. Upon binding to Elongin BC, no significant global conformational changes were detected in Vif by hydrogen exchange mass spectrometry, but a short fragment of Vif that consisted of the viral suppressor of cytokine signaling box showed decreased deuterium incorporation upon Elongin BC incubation, suggesting that this region folds upon binding.

Abstract 4013: Functional analysis of Bcr-Abl-dependent phosphorylation of SOCSs

Abstract Previous studies have demonstrated that constitutive activation of JAK/STAT (Janus tyrosine kinase/signal transducers and activators of transcription) signaling is required for efficient transformation by the Abelson murine leukemia virus (A-MuLV), which results from the expression of the v-Abl oncogene. Although the activation of JAK/STAT signaling pathway in cells expressing BCR-ABL still remains controversial, there is increasing evidence that deregulation of the JAK or STAT activity plays an important role in BCR-ABL malignant transformation. The suppressors of cytokine signaling (SOCS) are inhibitors of Jak kinases and as such are critically involved in a negative feedback loop of Jak/STAT signal transduction pathway. The mechanism by which Abl oncogene bypasses SOCS negative regulation remains poorly defined. Here, we found that co-expression of BCR-ABL with some SOCS proteins resulted in phosphorylation of SOCSs. Moreover, we observed that BCR-ABL-dependent phosphorylation of SOCSs altered SOCS function at least at two levels. One involves alteration of direct interaction of SOCSs with JAKs, which maintains JAK kinase activity. The other level involves inhibition of interaction of SOCS box with the Elongin BC complex, which disrupted the formation of E3 ubiquitin ligase complex that mediates proteosomal degradation of JAK kinases. Together, these results suggest that the BCR-ABL may regulate the inhibitory function of SOCSs through mediating phosphorylation of SOCS proteins. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4013.

Abstract 1208: Suppressor of cytokine signaling 6 (SOCS6) is targeted to mitochondria and regulates mitochondrial morphology and function

Abstract Mitochondria are highly motile organelles that constantly undergo fission and fusion. Impairment of mitochondrial dynamics is associated with mitochondrial dysfunction and frequently linked to the pathogenesis of neurodegenerative diseases and cancer. We have previously shown that loss-of-function of suppressor of cytokine signaling (SOCS) 6 is a frequent event in human gastric cancer and ectopic expression of SOCS6 sensitized cells to UV-induced apoptosis. In this study, we have identified SOCS6 as a novel mitochondrial protein that regulates mitochondrial dynamics. By confocal microscopy, we showed that ectopically expressed SOCS6 is predominantly localized in mitochondria, and that the carboxyl-terminal SOCS box is important for its mitochondrial localization. We further showed that localization of SOCS6 in mitochondria is accompanied with increased mitochondrial fragmentation with attenuated membrane potential, both of which can be reversed by coexpression of mitochondrial fusion proteins Mfn1 or Mfn2. Our data further suggested that SOCS6 interferes with mitochondrial dynamics by interrupting the fusion process. Firstly, SOCS6 expression severely impaired the dynamics and mobility of mitochondria in live cells as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Secondly, SOCS6 expression significantly hampered intermitochondrial transfer and fusion in the somatic hybrid produced by PEG-induced fusion. Furthermore, elimination of the expression of endogenous SOCS6 by RNA interference technique led to elongated mitochondria, and conferred cells increased resistance to UV-induced apoptosis. Taken together, our data suggest that SOCS6 is a mitochondrial targeting protein involved in the regulation of mitochondrial dynamics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1208.

A Label-free Quantitative Proteomics Strategy to Identify E3 Ubiquitin Ligase Substrates Targeted to Proteasome Degradation

The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.

The E3 ubiquitin ligase specificity subunit ASB2β is a novel regulator of muscle differentiation that targets filamin B to proteasomal degradation

Ubiquitin-mediated protein degradation is the main mechanism for controlled proteolysis, which is crucial for muscle development and maintenance. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 gene (ASB2) encodes the specificity subunit of an E3 ubiquitin ligase complex involved in differentiation of hematopoietic cells. Here, we provide the first evidence that a novel ASB2 isoform, ASB2beta, is important for muscle differentiation. ASB2beta is expressed in muscle cells during embryogenesis and in adult tissues. ASB2beta is part of an active E3 ubiquitin ligase complex and targets the actin-binding protein filamin B (FLNb) for proteasomal degradation. Thus, ASB2beta regulates FLNb functions by controlling its degradation. Knockdown of endogenous ASB2beta by shRNAs during induced differentiation of C2C12 cells delayed FLNb degradation as well as myoblast fusion and expression of muscle contractile proteins. Finally, knockdown of FLNb in ASB2beta knockdown cells restores myogenic differentiation. Altogether, our results suggest that ASB2beta is involved in muscle differentiation through the targeting of FLNb to destruction by the proteasome.

ASB2 targets filamins A and B to proteasomal degradation

AbstractThe ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and filamin degradation; conversely, ASB2 expression in leukemia cells induces filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both filamin A and filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of filamins for degradation.

Elongin B/C Recruitment Regulates Substrate Binding by CIS

SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins.