Protein interaction screening for the ankyrin repeats and suppressor of cytokine signaling (SOCS) box (ASB) family identify Asb11 as a novel endoplasmic reticulum resident ubiquitin ligase

Abstract

The ankyrin and SOCS (suppressor of cytokine signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting 18 members in humans, the identity of the physiological targets of the Asb proteins remains largely unexplored. To increase our understanding of the function of ASB proteins, we conducted a family-wide SILAC (stable isotope labeling by amino acids in cell culture)-based protein/protein interaction analysis. This investigation led to the identification of novel as well as known ASB-associated proteins like Cullin 5 and Elongins B/C. We observed that several proteins can be bound by more than one Asb protein. The additional exploration of this phenomenon demonstrated that ASB-Cullin 5 complexes can oligomerize and provides evidence that Cullin 5 forms heterodimeric complexes with the Cullin 4a-DDB1 complex. We also demonstrated that ASB11 is a novel endoplasmic reticulum-associated ubiquitin ligase with the ability to interact and promote the ubiquitination of Ribophorin 1, an integral protein of the oligosaccharyltransferase (OST) glycosylation complex. Moreover, expression of ASB11 can increase Ribophorin 1 protein turnover in vivo. In summary, we provide a comprehensive protein/protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases.

Keywords: Endoplasmic Reticulum (ER); Glycation; Mass Spectrometry (MS); Proteomics; Ubiquitin Ligase; Ubiquitylation.

FIGURE 1.

Protein interaction analysis of the ASB family of ubiquitin ligases. A, schematic representation of the methodology used for ASB interactome analysis. The open reading frame of ASBs 1–18 were inserted into the pcDNA4/TO backbone downstream and in-frame of an SFS epitope. SILAC-based proteomic analysis of the ASB family interactome was conducted as depicted. In brief, U2OS cells were metabolically labeled with light, medium, and heavy versions of arginine and lysine amino acids. Each experiment contains control cells transfected with empty vector (light-labeled cells), one Asb in medium-labeled cells, and another Asb in heavy-labeled cells. To expand the repertoire of putative prey, SILAC-labeled, but untransfected HuH7, HEK293T, and HeLa extracts were added to the transfected U2OS extract before SFS-tagged fusion proteins were immunoprecipitated (IP) with anti-FLAG antibody. The different immunoprecipitates were pooled and separated on SDS-PAGE and stained to visualize proteins. Bands were excised and tryptin-digested, and the peptides were analyzed by LC-MS/MS. B, network representation of the ASB protein family interactome. Proteins aligned into the inner circle share at least one interacting partner whereas proteins outside the circle only interact with one of the ASB proteins (dark orange). The known core components of the ECS ubiquitin ligase complex, Cul5, TCEB2, TCEB1, and CAND1 and their interaction partners are depicted in green.

FIGURE 2.

Asb proteins assemble in noncanonical E3 complexes. A and B, agarose-bound His₆-Asb3 (A) or His₆-Asb7 (B) recombinant proteins were mixed with U2OS cell extract containing exogenous levels of Myc-Asb6 (A) or FLAG-Asb4 (B), and the interaction was analyzed by Western blotting with anti-Myc and anti-FLAG antibodies. C, U2OS cells were transiently transfected with SFS-Asb3 and Myc-Asb6 or Myc control vector. All cells were co-transfected with Elongins B/C. Cycloheximide was added at t = 0, and cells were harvested at the indicated time points. Protein levels were analyzed with anti-FLAG, anti-Myc, and anti-β-actin antibodies. D, U2OS cells were transiently transfected with a construct encoding SFS-Asb4 or the SFS control vector. SFS-tagged fusion proteins were immunoprecipitated (IP) with anti-FLAG antibody, and associated proteins were visualized by Western blotting with anti-DDB1, anti-Cul4a, and anti-FLAG antibodies. E, U2OS cells were transiently transfected with GFP-Cullin 4a and SFS-Asb4 or the SFS control vector, as indicated. SFS fusion proteins were immunoprecipitated, and associated proteins were analyzed by Western blotting with the indicated antibodies. WCE, whole cell extract. F, U2OS cells were transiently transfected with GFP-Cullin 4a and either SFS-Asb4 or SFS control vector as indicated. Cycloheximide was added at t = 0, and cells were harvested at various time points. Protein levels were analyzed by Western blotting with the indicated antibodies. GAPDH served as a loading control.

FIGURE 3.

Asb11 and Ribophorin 1 interact in human cells. A, agarose-bound His₆-Asb11 was incubated with U2OS protein extracts containing exogenous levels of Myc-Ribophorin 1. Bound proteins were analyzed by immunoblotting with anti-His and anti-Myc antibodies. B, as in A except that extracts from untransfected U2OS cells were used to analyze binding of Asb11 to endogenously expressed Ribophorin 1. The presence of Ribophorin was analyzed by Western blotting. C, U2OS cells expressing FLAG-Asb11 stained with anti-FLAG (green), anti-PDI (red), and DAPI to visualize the nucleus (blue) were examined by confocal microscopy. Co-localization of PDI and Ribophorin 1 was observed. D, cell fractionation of U2OS cells expressing FLAG-Asb11α by differential centrifugation. The expression of Asb11 (FLAG), ER marker PDI, nuclear protein laminin A/C, mitochondrial protein Prohibitin and cytosolic protein β-actin were monitored in whole cell extracts (WCE) and in nuclear-depleted (PN), microsomal, mitochondria-depleted (PM) and rough ER (ER) fractions. E, HuH7 cells were transiently transfected with EGFP-Asb11α or Cherry-Asb11β Coverslips were stained with anti-Ribophorin 1 and DAPI (blue) and examined by confocal microscopy. F, Duolink assay to measure Asb11 and Ribophorin 1 interaction in situ. U2OS cells transiently transfected with an expression vector encoding a FLAG-tagged version of Asb11α or left untreated were grown on glass coverslips. Anti-FLAG and anti-Ribophorin 1 (center) or Cullin 5 (right) antibodies were applied to the FLAG-Asb11-transfected cells in the assay or to untransfected cells as a negative control. Bright spots indicate positive interactions.

FIGURE 4.

Ribophorin 1 binding is dependent on ankyrin repeat 4 in Asb11. A, a series of deletion mutants of Asb11α were generated, and the constructs were tested in co-immunoprecipitation assays with Myc-Ribophorin 1. B, U2OS cells were transiently transfected with expression vectors encoding Asb11α mutants deleted for the ankyrin repeats or the SOCS box as indicated in A. Cells were co-transfected with Myc-Ribophorin 1 or empty Myc vector, serving as control. Immunoprecipitates (IP) were resolved on SDS-PAGE and proteins visualized by Western blotting (WB) with the indicated antibodies.

FIGURE 5.

Asb11α has intrinsic ubiquitination activity in vitro and in vivo and ubiquitinates Ribophorin 1. A, U2OS cells were transiently transfected with SFS-Asb11α or left untreated. SFS-tagged proteins were immunoprecipitated with anti-FLAG antibody and subjected to in vitro ubiquitination with E1 and E2 enzymes, HA-ubiquitin and ATP (0.8 mm) alone or in combination with an ATP reconstitution system (0.8 mm ATP, 0.8 mm MgCl₂, 25 mm creatine phosphate, and 0.03 ng of creatine kinase). The reactions were analyzed on SDS-PAGE and immunoblotted with anti-HA and anti-FLAG antibodies. B, U2OS cells were transiently transfected with constructs encoding the SFS-Asb11α and β isoforms or empty control vector. MG132 was added 5 h prior to harvesting the cells in lysis buffer. Proteins were separated on SDS-PAGE and immunoblotted with anti-ubiquitin antibody to examine the amount of high molecular mass ubiquitin-protein conjugates. GAPDH served as a loading control. C, U2OS cells were transiently transfected with expression constructs encoding Asb11α, β, or the Asb11α-ΔSOCS mutant proteins fused to a FLAG epitope. Aliquots from whole cell extracts were assayed by immunoblotting (WB) with anti-Myc and anti-β-actin antibodies. FLAG-tagged proteins were immunoprecipitated (IP) with anti-FLAG antibody and were subsequently nedd8ylated in vitro. The reactions were used in an ubiquitination assay with the addition of E1, E2, HA-ubiquitin, and ATP. The proteins from the ubiquitination reaction were eluted from the beads with 3×FLAG peptide and immunoprecipitated with anti-Myc antibody (9E10). The α-Myc immunoprecipitates were analyzed with anti-Myc or anti-HA antibodies to assess Ribophorin 1 ubiquitination. D, U2OS cells were transiently transfected with SFS-Asb11α or empty vector control. Cells were harvested in lysis buffer, and endogenous Ribophorin 1 levels were analyzed by Western blotting. E, U2OS cells transformed with an SFS-Asb11α encoding tetracycline-inducible construct were grown with or without doxycycline to induce or repress the transgene, respectively. Cycloheximide was added at t = 0, and the amounts of Ribophorin 1, Asb11, and GAPDH were analyzed by Western blotting. GAPDH served as a loading control.