Substrate-recognition component of a SCF-like ECS (Elongin-Cullin-SOCS-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins. Recognizes at least two forms of creatine kinase, CKB and CKMT1A.
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). RESULTS: We found that a variant of ASB9 that lacks the SOCS box (ASB9DeltaSOCS) was naturally detected in human cell lines but not in peripheral blood mononuclear cells or normal hepatocytes. We also identified ubiquitous mitochondrial creatine kinase (uMtCK) as a new target of ASB9 in human embryonic kidney 293 (HEK293) cells. The ankyrin repeat domains of ASB9 can associate with the substrate binding site of uMtCK in a SOCS box-independent manner. The overexpression of ASB9, but not ASB9DeltaSOCS, induces ubiquitination of uMtCK. ASB9 and ASB9DeltaSOCS can interact and colocalise with uMtCK in the mitochondria. However, only expression of ASB9 induced abnormal mitochondrial structure and a decrease of mitochondrial membrane potential. Furthermore, the creatine kinase activities and cell growth were significantly reduced by ASB9 but not by ASB9DeltaSOCS. CONCLUSIONS: ASB9 interacts with the creatine kinase system and negatively regulates cell growth. The differential expression and function of ASB9 and ASB9DeltaSOCS may be a key factor in the growth of human cell lines and primary cells.
Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9) is a specific substrate-recognition subunit of an elongin C-cullin-SOCS box E3 ubiquitin ligase complex. It recognizes its substrate, brain type creatine kinase (CKB), using the ankyrin repeat domain; and facilitates the polyubiquitination of CKB to mediate proteasomal degradation through the SOCS box domain. HASB9-2 is an isoform of hASB9 that contains one ankyrin repeat domain. In this study, the crystal structure of hASB9-2 is shown at 2.2-Å resolution using molecular replacement. Overall, hASB9-2 forms a slightly curved arch with a characteristic L-shaped cross-section. Amino acid substitution analysis based on docking experiments revealed that His103 and Phe107 in hASB9-2 are essential for binding to CKB. Analysis of truncation mutants demonstrated that the first six ankyrin repeats along with the N-terminal region of hASB9-2 contribute to the interaction with CKB.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
Several attempts have been made to systematically map protein-protein interaction, or 'interactome', networks. However, it remains difficult to assess the quality and coverage of existing data sets. Here we describe a framework that uses an empirically-based approach to rigorously dissect quality parameters of currently available human interactome maps. Our results indicate that high-throughput yeast two-hybrid (HT-Y2H) interactions for human proteins are more precise than literature-curated interactions supported by a single publication, suggesting that HT-Y2H is suitable to map a significant portion of the human interactome. We estimate that the human interactome contains approximately 130,000 binary interactions, most of which remain to be mapped. Similar to estimates of DNA sequence data quality and genome size early in the Human Genome Project, estimates of protein interaction data quality and interactome size are crucial to establish the magnitude of the task of comprehensive human interactome mapping and to elucidate a path toward this goal.
Evidence
2:
Inferred from Physical InteractionIntAct
Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
The process in which a signal is passed on to downstream components within the cell, which become activated themselves to further propagate the signal and finally trigger a change in the function or state of the cell.
Any process that activates or increases the frequency, rate or extent of the chemical reactions and pathways resulting in the breakdown of a protein by the destruction of the native, active configuration, with or without the hydrolysis of peptide bonds.
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.
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.
Protein involved in ubiquitin-like modifier processing, activation, conjugation or deconjugation such as Ubl-activating enzymes (E1s), Ubl-conjugating enzymes (E2s), Ubl-protein ligases (E3s), some thiol proteases (Ubiquitin carboxyl-terminal hydrolases (UCH), Ubiquitin- specific processing proteases (UBP) and ubiquitin-like proteases) and the ubiquitin-like modifier proteins. Besides signaling proteolysis, ubiquitination for example can be a signal for trafficking, kinase activation and other nonproteolytic fates.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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