Asb2 mediates Notch-induced E47 and Jak2 degradation. (A) Asb2 accelerates E2A and Jak2 degradation as efficiently as N1-IC. Left, BaF3 cells transduced with vector, HPC4-tagged Asb2 or N1-IC-expressing retroviruses were used for immunoblotting endogenous E2A, Jak2 and tubulin. Right, vector or Asb2-Flag-transduced cells were also treated with 12 μM MG-132 (+) or DMSO (−) for 2 h. Immunoblotting was performed with antibodies against E2A and Jak2, as well as Erk1 for loading control. Asb2 was detected with an antibody against its HPC4 tag. (B) Stimulation of ubiquitination of E2A and Jak2 by Asb2 in vivo. BaF3 cells transduced with vector, Asb2 and N1-IC retroviruses were treated with 5 μM MG-132 for 5 h. Whole-cell extracts were used for IP with anti-E2A and Jak2 antibodies, respectively. The precipitates were immunoblotted with a monoclonal antibody against ubiquitin. The amounts of endogenous E2A and Jak2 proteins brought down were determined by probing with anti-E2A or Jak2 antibodies. (C) Asb1 but not Asb12 induces E47 and Jak2 degradation. Left, E47 plus or minus indicated that Asb constructs were co-transfected into NIH3T3 cells. A plasmid expressing GFP was included in all transfection experiments to monitor transfection efficiency. Right, Jak2 and Epo receptor plus or minus indicated that Asb constructs were co-transfected into 293T cells and treated with 5 ng/ml of Epo for Jak2 activation for 1.5 h prior to harvest. Immunoblot analyses were performed with antibodies against the indicated proteins. Asb proteins were detected with an anti-Flag antibody whereas control was carried out with anti-GFP antibodies. (D) Notch or Asb2-induced E47 degradation is blocked by a DN mutant of Asb2 (Asb2ΔC). E47 plus or minus N1-IC or flag-tagged Asb2 were co-transfected into NIH3T3 cells along with or without flag-tagged Asb2ΔC. A plasmid expressing GFP was included in all transfection experiments to monitor transfection efficiency. Immunoblot analyses were performed with antibodies against the indicated proteins. (E) Left, Jak2 and Epo receptor plus or minus N1-IC were co-transfected into 293T cells along with or without Asb2ΔC and treated with 5 ng/ml of Epo for Jak2 activation for 1.5 h prior to harvest for immunoblotting. Right, BaF3 cells transduced with vector or Asb2ΔC-expressing retrovirus were co-cultured with OP42 stromal cells transduced with vector control or retrovirus expressing Notch ligand, DL1, for 1.5 h. Endogenous Jak2 levels were determined by immunoblotting and loading controls were performed by probing tubulin.

Interaction of Asb2 with components of Cul5-based E3 ligase complexes. (A) Asb2 competes with SOCS1 to bind to Jak2. 293T cells were co-transfected with indicated amounts of indicated constructs. IP was performed with anti-Jak2 or control IgG (Con) in the NP40 lysis buffer. The immunoprecipitates and inputs were immunoblotted with antibodies against the Flag or HPC4 tag and Jak2. (B) Asb2 competes with SOCS1 to bind to ElonginB/C. 293T cells were co-transfected with indicated amounts of indicated constructs. IP was performed with an anti-myc antibody or control IgG in the NP40 lysis buffer. The anti-Myc immunoprecipitates were subjected to immunoblotting with antibodies against the Flag or HPC4 tag or Elongin proteins. (C, D) Asb2 interacts more strongly with Jak2 than with ElonginB/C or Cul5. 293T cells were transfected with indicated constructs. IP was carried out in either the NP40 or RIPA buffer with indicated antibodies. The precipitates were analyzed by probing for the indicated proteins.

Interaction of Asb2 and Skp2 with substrates. (A) Downregulation of Skp2 diminishes the association of Asb2 with E47. Constructs expressing E47 and Asb2-Flag were co-transfected into 293T cells with or without shRNA constructs against Skp2 (S) or a random control sequence (C). The anti-Flag or control immunoprecipitates along with inputs were immunoblotted for the indicated proteins. (B) Skp2 facilitates the interaction between Asb2 and E47. Indicated constructs were co-transfected into 293T cells. IP and immunoblotting were performed as in A. (C) Asb2 and Jak2 interaction is independent of but strengthened by Skp2. Asb2-Flag and Jak2-HA were coexpressed with or without Skp2-T7 expressing or anti-Skp2 shRNA constructs in 293T cells. IP was performed with the anti-HA and control antibodies in the NP40 lysis buffer. The amounts of Asb2-Flag and Skp2-T7 co-precipitated were determined by immunoblotting with antibodies against the indicated proteins. (D) Skp2 and Jak2 interaction is enhanced by Asb2. Indicated constructs were transfected into 293T cells. Immunoprecipitates of anti-Skp2 or control antibodies obtained in RIPA buffer were probed for the indicated proteins.

Dependence of both Cul1 and Cul5 in Notch and Asb2-induced degradation of E47 and Jak2. (A) N1-IC and Asb2-induced degradation of E47 and Jak2 was attenuated by DN mutant-Cul1 (1) or Cul5 (5). E47 and GFP constructs were co-transfected with or without N1-IC or Asb2 plus or minus DN-Cul1 or DN-Cul5 into NIH3T3 cells. Jak2, GFP and EpoR constructs were co-transfected with or without N1-IC or Asb2 plus or minus DN-Cul1 or DN-Cul5 into 293T cells and treated with 5 ng/ml of Epo for 1.5 h prior to harvesting the cells. Immunoblot assays were performed with antibodies against the indicated proteins. (B) Abrogation of Asb2-induced ubiquitination of E47 and Jak2 by either DN-Cul1 or DN-Cul5. 293T cells were co-transfected with the indicated constructs. IP was carried out with anti-E47 or Jak2 antibodies after transfected cells were treated with MG-132 at a concentration of 5 μM for 5 h. The precipitates were subjected to immunoblotting with the anti-HA monoclonal antibody to visualize ubiquitinated E47 and Jak2, respectively. The amounts of E47 and Jak2 precipitated were determined by probing with anti-E47 and Jak2 antibodies. (C) Downregulation of either Cul1 or Cul5 rescues both N1-IC and Asb2-induced protein degradation. HeLa cells were co-transfected with E47 or Jak2-HPC4 plus GFP-expressing constructs with or without N1-IC or Asb2. After 24 h, the cells were then reverse transfected with 10 nM of control or siRNAs targeting Cul1 or Cul5 using Lipofectamine 2000 for 12 h and incubated for additional 24 h in culture medium. Whole-cell lysates were immunoblotted with antibodies against the indicated proteins.

Notch signaling upregulates Asb2 expression. (A) BaF3 cells were transduced with retrovirus expressing GFP or GFP plus the IC of Notch1 (N1-IC). GFP-positive cells were sorted and used for RNA isolation and quantitative real-time PCR (qRT-PCR). Levels of transcripts were normalized against that of β-actin by calculating ΔC_T. Data shows the average ± SD, which was calculated as described ⁵¹, and is a representative of three independent experiments. For immunoblotting, total lysates of the same cells were used with antibodies against Asb2 and tubulin as a loading control. (B) Lin⁻CD19⁺IgM⁻ bone marrow cells prepared by immune-magnetic depletion with antibodies against Mac-1, Ter119, CD3, CD8 and IgM were co-cultured on OP42 stromal cells transduced with vector or DL1-expressing retrovirus in the presence of cytokines (10 ng/ml SCF, 10 ng/ml Flt-3 ligand and 5 ng/ml IL-7) along with 10 μM of a GSI or DMSO for 1.5 h. The co-cultured bone marrow cells were carefully harvested and used for qRT-PCR and immunoblot assays as described in A.

Interaction of Asb2 with Cullins. (A) Whole-cell lysates of vector or Asb2-Flag-transfected 293T cells were used in IP assays with an anti-Flag antibody in a lysis buffer containing 0.5% NP40. The precipitates as well as the input controls were immunoblotted with antibodies against indicated proteins. (B) 293T cells were transfected with or without Asb2-Flag. IP was performed with anti-Cul1 or Cul5 antibodies or control IgG. The precipitates were probed for indicated proteins. (C) 293T cells were co-transfected with indicated cullin constructs, Cul2 or myc-tagged Cul3, 4 and 7, plus or minus Asb2. Cullins were pulled down with antibodies against Cul2 or the myc tag and the precipitates were analyzed for Asb2 and Cullins.

Mapping protein interacting domains of Asb2. (A) Schematic diagram of human Asb2 and its mutant constructs. Ankyrin repeats are marked as gray boxes and the black box represents the SOCS box. Numbers indicate the positions in amino acid sequence of Asb2. Internal deletions are designated by “v” shaped lines. All constructs contain a Flag tag at the C terminus. Regions responsible for Skp2, Jak2 and ElonginB/C/Cul5 binding are sketched on top of the full-length construct. (B) Interaction between Asb2 and Jak2. Jak2 was co-transfected with indicated constructs into 293T cells. IP was performed with anti-Flag or control antibodies in the RIPA buffer. The precipitates and inputs were probed with anti-Jak2 and anti-Flag antibodies. (C, D) Interaction between Asb2 and ElonginB/C or Cul5. 293T cells were co-transfected with indicated constructs. IP was carried out with anti-Flag (C) or anti-His (D) antibodies in the NP40 lysis buffer. IP with control IgG was included in each of the experiments. The precipitates were analyzed by immunoblotting with anti-Cul5, anti-Myc and anti-Flag antibodies. (E) Interaction of Asb2 with Skp2. Skp2 was co-transfected with indicated constructs. Anti-Flag or control immunoprecipitates obtained in the RIPA buffer were subjected to immunoblotting with anti-Skp2 and anti-Flag antibodies.

Asb2 and Skp2 bridge the assembly of a Cul1- and Cul5-containing heterodimeric E3 ligase complex. (A) Association of Asb2 with Cul1 depends on Skp2. Constructs expressing full-length or deletion mutant Asb2 were co-transfected into 293T cells with or without Skp2 or control shRNA constructs. The anti-Flag or control immunoprecipitates obtained in the NP40 lysis buffer were immunoblotted with anti-Cul1 and anti-Flag antibodies. (B) Skp2 interacts with Cul5 in the NP40 but not RIPA buffer. 293T cells were transfected with or without Asb2, and IP was performed with anti-Skp2 or control antibodies in the NP-40 lysis and RIPA buffer, respectively. The immunoprecipitates were probed for the indicated proteins. (C) N1-IC (N1) or Asb2 was transfected into 293T cells. IP was performed with anti-Cul1 or Cul5 antibodies or control. The precipitates were immunoblotted with anti-Nedd8 and Cul1 or Cul5 antibodies. (D) Augmentation of Cul5 and Asb2 interaction by Skp2. 293T cells were co-transfected with indicated constructs. The anti-Flag or control immunoprecipitates obtained in the NP40 buffer were subjected to immunoblotting with anti-Cul5, anti-Skp2 and anti-Flag antibodies.

Asb2-mediated dimeric cullin-based complexes. Canonical Cul1 or Cul5 monomeric E3 ligase complexes are thought to be responsible for ubiquitination of their respective substrates. Activation of Notch signaling upregulates Asb2 expression, and Asb2 displace SOCS proteins to associate with ElonginB/C and Cul5. Interaction between Asb2 and Skp2 bridges the formation a dimeric complex containing Cul1 and Cul5 and their associates. The manner in which E47 and Jak2 interact with Asb2 and Skp2 is illustrated separately. The enhancement of ubiquitination by the availability of two sets of E2 enzymes to catalyze ubiquitin ligation is illustrated by arrows.