Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II

Abstract

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-alpha by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.

FIG. 1.

ASB3 expression and protein-binding patterns. (A) A human adult tissue Northern blot (BD Biosciences Clontech, Palo Alto, Calif.) was probed with a ³²P-labeled probe generated from ASB3 cDNA. (B) With different cell lines, as indicated, RT-PCR was performed to examine asb3 (top) and β-actin (bottom) mRNA expression. (C) Whole extracts (1.5 mg) prepared from 293 cells transfected with c-Myc-ASB3 were incubated with an antibody array (300 antibodies immobilized on a PVDF membrane) overnight at 4°C. After being washed, the array was blotted with anti-c-Myc-HRP for 4 h followed by ECL detection. Anti-c-Myc-HRP-positive signals were detected at the spots printed with antibodies against c-Myc (C11), FAST (E2), HEF-1 (E16), Survivin (L10), TNF-R2 (L18), Toso (L19), and VHR (M13).

FIG. 2.

TNF-R2 recruits ASB3 in cells. (A) c-Myc-ASB3 was transiently transfected into 293 cells, followed by treatment with or without TNF-α for 30 min. Whole extracts prepared from these cells were immunoprecipitated with anti-TNF-R2 followed by Western blot analysis with anti-c-Myc or anti-TNF-R2. (B) 293 cells were transfected with TNF-R2 alone (1 μg pcDNA3 empty vector and 1 μg TNF-R2) or with TNF-R2 and c-Myc-ASB3 (1 μg TNF-R2 and 1 μg c-Myc-ASB3). Forty-eight hours after transfection, cell extracts were prepared for the same immunoprecipitation/Western blot analysis as that described for panel A. As an immunoprecipitation control, immunoglobulin G (normal mouse IgG) was incubated with the extracts prepared from 293 cells expressing TNF-R2 and c-Myc-ASB3. (C) 293 cells were transfected with TNF-R1 alone or with TNF-R1 and c-Myc-ASB3 under similar conditions to those used for panel B. Anti-TNF-R1 precipitates were analyzed for ASB3 binding under similar conditions to those used for panel B. (D) 293 cells were transfected with TNF-R2 (0.1 μg), c-Myc-ASB3 (1 μg), and either empty vector or mTNF-α (0.5 μg). After 48 h, the cells were either left untreated or treated with 10 μM MG132, followed by protein extraction and immunoprecipitation as described for panel A.

FIG. 3.

ANK repeats of ASB3 and the C terminus of TNF-R2 are responsible for their interaction. (A) Schematic illustration of TNF-R2 deletion mutants. TM, transmembrane domain. (B) ASB3 (1 μg) was transfected into 293 cells with full-length (FL) TNF-R2 (1 μg) or a deletion mutant, as indicated. After transfection for 48 h, whole-cell extracts were prepared and immunoprecipitated with anti-c-Myc, followed by anti-TNF-R2 or anti-c-Myc blotting as indicated. TNF-R2 expression levels are shown in the bottom panel. (C) 293 cells were cotransfected with ASB3 (1 μg) and TRAF2 (1 μg), with or without TNF-R2, as indicated. Whole-cell extracts were prepared and immunoprecipitated with anti-c-Myc, followed by Western blot analysis with anti-TRAF2 or anti-c-Myc. (D) Schematic illustration of c-Myc-ASB3 with ANK deletion mutations. (E) 293 cells were cotransfected with full-length ASB3 (1 μg) or an ANK deletion mutant and with TNF-R2 (1 μg) or an empty vector (1 μg). Forty-eight hours after transfection, whole-cell extracts were prepared for immunoprecipitation with anti-TNF-R2, followed by Western blot analysis with anti-c-Myc or anti-TNF-R2. The ASB3 expression input (anti-c-Myc) is shown in the bottom panel. Asterisks mark anti-c-Myc signals.

FIG. 4.

ASB3 recruits Elongin-C. (A) Alignment of SOCS boxes of ASB family members and SOCS family members from the human genome database. CE-ASB is a putative ASB identified from the Caenorhabilitis elegans genome database (accession no. U39995). The α-helices (cyan shading) and β-strands (yellow shading) were predicted with the 3D-PSSM web tool, version 2.5.6 (http://www.sbg.bio.ic.ac.uk/∼3dpssm/). Tyrosine residues of α4-helix, the leucine and cysteine residues responsible for the Elongin-C (14) interaction, and the two conserved arginine residues of the α2-helix are shown with red letters. (B) 293 cells were cotransfected with ASB3 or SOCS-1 and Elongin-C and Elongin-B. Whole-cell extracts were prepared for immunoprecipitation with control IgG or anti-c-Myc, followed by Western blot analysis with anti-Elongin-C (top) or anti-c-Myc (middle). The bottom panel shows the expression of Elongin-C.

FIG. 5.

ASB3 promotes TNF-R2 ubiquitination. (A) 293 cells were transfected with an empty vector (1 μg), TNF-R2 (1 μg), c-Myc-ASB3 (1 μg), and HA-ubiquitin (0.5 μg) as indicated. Twenty-four hours after transfection, whole-cell extracts were prepared and subjected to immunoprecipitation with anti-TNF-R2, followed by Western blot analysis with anti-HA. (B) 293 cells were cotransfected with full-length TNF-R2 and with an empty vector or ASB3 and HA-ubiquitin, as indicated. TNF-R2 ubiquitination was analyzed as described for panel A except that cells were pretreated with MG132 (25 μM) for 30 min prior to cell harvest. TNF-R2 levels are shown in the bottom panel. (C) 293 cells were cotransfected with full-length (FL) TNF-R2 or TNF-R2Δ424 and with ASB3 and HA-ubiquitin, as indicated. TNF-R2 ubiquitination was analyzed as described for panel A. TNF-R2 levels are shown in the bottom panel. (D) 293 cells were transfected with TNF-R2 as described for panel A and then left untreated or treated with TNF-α for 30 min. The cells were then lysed for immunoprecipitation with anti-TNF-R2. The immunoprecipitates were eluted from the beads by boiling in the presence of 0.5% SDS for 5 min, followed by reimmunoprecipitation (re-IP) with anti-TNF-R2. After separation by SDS-PAGE, the proteins were transferred to a PVDF membrane, followed by blotting with anti-HA (top) and anti-TNF-R2 (bottom). (E) In vitro ubiquitination assay. Immunoprecipitated TNF-R2 and/or immunoprecipitated c-Myc-ASB3 was incubated in a control reaction mixture with only Ub and ATP (lane 1). For lanes 2, 3, and 4, immunoprecipitated TNF-R2 and/or immunoprecipitated c-Myc-ASB3 was incubated in a complete reaction mixture containing E1 (50 μM), E2 (1 μM), Ub, and ATP. For lane 5, Ub was replaced with an equal amount of methyl-Ub, whereas for lane 6, no Ub was added. For lane 7, normal mouse IgG plus goat IgG precipitates were incubated in a complete reaction mixture. The reactions were separated by SDS-PAGE, transferred to a PVDF membrane, and blotted with anti-ubiquitin (top). The same membrane was reblotted with anti-TNF-R2 (middle) or anti-c-Myc (bottom).

FIG. 6.

TNF-R2 C terminus bears multiple lysine sites for ubiquitination. (A) Sequence alignment of TNF-R2 C termini of human, bovine, mouse, and rat proteins. (B) 293 cells were cotransfected with wild-type TNF-R2 or various TNF-R2 mutants with Lys→Arg substitutions and with HA-ubiquitin. Twenty-four hours after transfection, whole-cell extracts were prepared for immunoprecipitation with anti-TNF-R2, followed by Western blot analysis with anti-HA (top panel). Anti-TNF-R2 immunoprecipitates were probed with anti-c-Myc (middle panel), and immunoprecipitated TNF-R2 levels are shown in the bottom panel.

FIG. 7.

ASB3 induces TNF-R2 proteasome-dependent degradation. (A) 293 cells were cotransfected with TNF-R2 (1 μg) and 1.5 μg c-Myc-ASB3 or c-Myc-ASB3ΔSB for 48 h, followed by a CHX (25 μg/ml) chase for the indicated times. Whole-cell extracts were immunoblotted with anti-TNF-R2 (top panel), anti-c-Myc (middle panel), and anti-TRAF2 (low panel). (B) 293 cells were cotransfected with TNF-R2 and the empty pcDNA vector (EV), c-Myc-ASB3, or c-Myc-ASB3ΔSB, followed by a CHX chase in the presence or absence of MG132 (10 μM) or NH₄Cl (15 mM). TNF-R2 and ASB3 proteins were detected as described for panel A. (C) 293 cells were cotransfected with wild-type TNF-R2 or TNF-R2 lysine mutants and with either an empty vector or ASB3 and ubiquitin for 48 h, followed by a CHX chase (25 μg/ml) for an additional 5 h. Cells were either left untreated or treated with TNF-α (10 ng/ml) for 30 min. TNF-R2 and ASB3 protein levels were immunoblotted with anti-TNF-R2 or anti-c-Myc as described above. The right panel shows the TNF-R2 protein levels prior to the CHX chase. (D) Inhibition of ASB3 and Elongin-C expression by ASB3 and Elongin-C siRNAs. 293 cells were transfected with a siRNA directed against green fluorescent protein (GFP; control siRNA), ASB3, Elongin-C, or both ASB3 and Elongin-C. The levels of TNF-R2 proteins (top) and β-tubulin (bottom) were analyzed by immunoblotting with anti-TNF-R2 and anti-β-tubulin, respectively. Elongin-C, asb3, and β-actin mRNA levels were measured by RT-PCR.

FIG. 8.

ASB3 inhibits JNK activation by TNF-R2. (A) 293 cells were cotransfected with TNF-R2 and HA-JNK1 and with pcDNA3, ASB3, or ASB3ΔSB. Forty-eight hours after transfection, whole-cell extracts were prepared and immunoprecipitated with anti-HA (JNK1). Immunoprecipitated HA-JNK1 was analyzed in an in vitro kinase assay with GST-c-Jun as the substrate. The bottom panel shows immunoprecipitated HA-JNK1 blotted with anti-HA. (B) 293 cells were cotransfected with HA-JNK1 and with TRAF-2 and ASB3 (or ASB3ΔSB). The immunoprecipitated HA-JNK1 was analyzed in an in vitro kinase assay with GST-c-Jun as the substrate. The expression levels of TRAF2 in the cell extracts (anti-TRAF2 blotting) are shown in the bottom panel. (C and D) 293 cells were cotransfected with c-Jun-Gal4- and Gal4-responsive luciferase reporters, Renilla luciferase reporter constructs, and pcDNA, mTNF-α, TNF-R2, c-Myc-ASB3, or c-Myc-ASB3ΔSB, as indicated. After 24 h, whole-cell extracts were prepared and split into half for immunoblotting with the indicated antibodies (D) and for a dual-luciferase assay (C).

FIG. 9.

ASB3 knockout potentiates TNF-α-induced apoptosis in 4E3 cells. (A) 4E3 and Jurkat cells were treated with TNF-α (10 ng/ml) for the indicated times. Whole-cell extracts were subjected to Western blot analysis with anti-IκBα (top) or anti-β-tubulin (bottom). (B) 4E3 and Jurkat cells were treated as described for panel A. Whole-cell extracts were examined by Western blot analysis with specific antibodies against phospho-JNK (Cell Signaling, Beverly, Mass.), JNK (Pharmingen, San Diego, Calif.), and TRAF2 (Santa Cruz Biotechnology Inc.). (C) Whole-cell extracts were prepared from 4E3 cells following TNF-α treatment for the indicated times. The protein levels of TNF-R2 (top panel) and β-tubulin (bottom panel) were analyzed by Western blot analysis with anti-TNF-R2 and anti-β-tubulin. (D) 4E3 cells, transfected with either control (ctl) siRNA (GFP sequence) or ASB3 siRNA for 48 h, were treated with TNF-α for 15 min. Whole extracts prepared from these cells were subjected to Western blot analysis with anti-TNF-R2. RT-PCRs were performed with the RNAs prepared from these cells to evaluate asb3 expression or β-actin expression. (E) 4E3 cells were transfected with either GFP siRNA (ctl) or ASB3 siRNA for 48 h. Whole-cell extracts prepared from these cells were subjected to Western blot analysis with anti-TNF-R2 and anti-β-tubulin. RT-PCRs were performed with RNAs prepared from these cells to evaluate asb3 expression or β-actin expression. 4E3 cells were transfected with a Renilla luciferase reporter construct (pRLTK; Promega) and either control siRNA or ASB3 siRNA for 48 h. Each group was split in two, with one group maintained in medium and the other treated with TNF-α. At the indicated times, the percentage of specific cytotoxicity was determined by measuring the loss of Renilla luciferase activity. The means and standard deviations of three independent experiments are shown.