Alternative Ubiquitin Activation/Conjugation Cascades Interact with N-End Rule Ubiquitin Ligases to Control Degradation of RGS Proteins  Peter C.W. Lee, Mathew E. Sowa, Steven P. Gygi, J. Wade Harper  Molecular Cell  Volume 43, Issue 3, Pages 392-405 (August 2011) DOI: 10.1016/j.molcel.2011.05.034 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Identification of UBR1, UBR2, and UBR3 as Targets of the USE1 Ubiquitin conjugating Enzyme (A) Scheme depicting the yeast three-hybrid screening strategy employed to identify proteins interacting with USE1. (B) Domain structures of UBR1, UBR2, and UBR3, along with the positions of the cDNA clones identified in our yeast three-hybrid screen. (C) Flow chart for proteomic analysis of 293T cells stably expressing HA-USE1. (D) Summary of proteomic data, depicting total spectral counts (TSCs), DN-scores, and Z-scores for proteins found in association with HA-USE1. Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 USE1 Interacts with UBR Proteins via the RING Domain (A) Extracts from mouse NIH 3T3 cells were subjected to immunoprecipitation with either control IgG or α-USE1 antibodies, and the presence of UBR1 and UBR2 were determined by immunoblotting. (B) Extracts from 293T cells transfected with the indicated siRNAs were employed for immunoprecipitation using α-USE1 or control IgG, and immune complexes were blotted with the indicated antibodies. (C) Extracts from 293T cells expressing HA-USE1 and FLAG-UBR2 were immunoprecipitated with α-FLAG antibodies, and immune complexes were immunoblotted with the indicated antibodies. (D) Domain structure of UBR3 and the UBR3ΔN fragment employed for proteomics is shown along with the total spectral counts (TSCs) and DN-scores for associated E2s identified by LC-MS/MS. DN-scores greater than 1 are considered significant. (E) Association of MYC-UBR3ΔN with FLAG-USE1, FLAG-UBE2A, and FLAG-UBE2B was examined by immunoprecipitation and immunoblotting after transfection of 293T cells with vectors expressing the indicated proteins. (F) Alignment of the RING domains from human UBR1, UBR2, and UBR3, and mouse UBR1 and UBR2 and comparison to the RING domain of c-CBL. The asterisks indicate the position of mutations that were made in UBR2 to generate RING domain defective proteins. (G and H) Mutations in the UBR2 RING domain abolish association with USE1 and UBE2A. The indicated FLAG-tagged mUBR2 proteins were transiently transfected with HA-USE1 and either α-FLAG (G) or α-HA (H) immune complexes isolated prior to immunoblotting. Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 UBR2 Promotes K48-Specific Ubiquitin Discharge by USE1 and UBE2A in a RING Domain-Dependent Manner (A) Scheme employed for measuring ubiquitin discharge of USE1 and UBE2A (see text for details). (B) Coomassie gel of FLAG-UBR2 isolated from 293T cells expressing FLAG-UBR2. (C) Discharge reactions performed using FLAG-UBR2, FLAG-UBR2W1170A, or control purification eluates with either charged USE1 (top panel) or charged UBE2A (lower panel). Reactions were analyzed by immunoblotting after separation on nonreducing gels. (D) A panel of ubiquitin mutants containing no Lys residues (K0) or various Lys only mutants, as well as wild-type ubiquitin (Ub), were used to charge USE1 or UBE2A. Samples were run on either reducing or nonreducing gels prior to immunoblotting to detect charged and uncharged forms of the E2. (E and F) UBR2-dependent discharge reactions were performed using USE1 or UBE2A charged with the indicated ubiquitin derivatives as described in (A) and (C). Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 UBA6-USE1 and UBA1-UBE2A/B Ubiquitin Conjugation Cascades Contribute to Degradation of a Model N-End Rule Substrate (A and B) HeLa cells expressing the model N-end rule substrate Ub-R-GFP were transfected with the indicated siRNAs (30 nM), and extracts from cells were employed for immunoblotting (A) or cells were analyzed for GFP-content by flow cytometry (B). The gray shading in the flow diagrams indicate the window employed for identifying GFP-positive cells (fluorescence intensity > 103). Where noted, cells were treated with Velcade for 3 hr prior to harvesting. The error bars in (B) represent standard error for triplicate analyses. (C and D) HeLa cells expressing the non-N-end rule substrate Ub-M-GFP were transfected with the indicated siRNAs, and extracts from cells were employed for immunoblotting (D) or cells were analyzed for GFP-content by flow cytometry (C). Where noted, cells were treated with Velcade for 3 hr prior to harvesting. The error bars in (C) represent standard error for triplicate analyses. (E and F) USE1 and UBE2 collaborate to promote turnover of the model N-end rule substrate R-GFP. HeLa/R-GFP cells were transfected with 10 or 20 nM of the indicated siRNAs, and GFP intensity was analyzed by flow cytometry 72 hr after transfection (E). Expression of USE1 and UBE2 were determined by immunoblotting (F). The error bars in (E) represent standard error for triplicate analyses. (G) Schematic diagram depicting the strategy for CRE-mediated deletion of the UBA6 gene in MEFs. The catalytic ThiF domain is shared in exons 16 and 17 and is flanked on the left by a lox site (solid triangle) and on the right by a FRT-NEO-FRT (F-N-F) cassette followed by a lox site. FLP recombinase was employed to remove the neo gene and exons 16 and 17 were removed by Adeno-CRE recombinase to produce UBA6Δ. (H and I) Loss of UBA6 in MEFs leads to a loss in cell viability. Wild-type or UBA6flox/flox cells were treated with control virus or Adeno-CRE and cell viability monitored over a 5-day period using CellTiter-Glo luminescent assay (n = 5, with normalization to WT cells at day zero) (H). Cell extracts at the indicated time points were immunoblotted for UBA6 to confirm depletion (I). (J) Loss of USE1 charging with ubiquitin in MEFs lacking UBA6. UBA6flox/flox MEFs were infected with control or Adeno-CRE virus, and after 48 hr the extent of thioester formation for UBA6 and USE1 were examined as described previously (Jin et al., 2007). (K) UBA6flox/flox MEFs were transfected with either the N-end rule substrate Ub-R-GFP or the Ub-M-GFP control and subsequently infected with Adeno-CRE for 48 hr. Where noted, Velcade was added for 2 hr before harvesting. Whole cell extracts were immunoblotted for the indicated proteins, using α-tubulin as a loading control. Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Inactivation of UBA6-USE1 and UBA1-UBE2A/B Pathways Stabilizes RGS4 and RGS5 (A–D) 293T cells stably expressing RGS4-HA (A) or RGS5-HA (B–D) were transfected with the indicated siRNAs, and after 72 hr whole cell extracts were blotted with the indicated antibodies. Where noted, Velcade was added for 2 hr prior to harvesting cells as a control for stabilization of the RGS protein. α-tubulin was used as a loading control. (C) and (D) examine accumulation of RGS5-HA with multiple siRNAs for UBA6 and USE1. (E) UBA6flox/flox MEFs were transfected with RGS4-HA or RGS5-HA and subsequently treated with Adeno-CRE (48 hr) in the presence or absence of Velcade (2 hr prior to harvesting), and whole cell extracts were blotted with α-UBA6 to visualize depletion and α-HA to detect RGS proteins. α-tubulin was used as a loading control. (F–H) 293T cells stably expressing RGS4-HA (F and H) or RGS5-HA (G) were transfected with the indicated siRNAs, and after 72 hr a cyclohexamide chase experiment was performed by immunoblotting. (I–K) 293T cells stably expressing RGS4-HA (I and K) or RGS5-HA (J) were transfected with the indicated siRNAs, and after 24 hr cells were transfected with a vector expressing an RNAi-resistant form of either wild-type (WT) or catalytically inactive (Cys to Ser mutant, CS) USE1 or UBE2A (MYC-tagged). After a further 48 hr, whole cell extracts were blotted with the indicated antibodies, with α-tubulin used as a loading control. Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 In Vitro K48-Specific Ubiquitylation of the N-End Rule RGS4 Substrate by UBR2 in Combination with Either UBA6-USE1 or UBA1-UBE2A (A) Scheme used to produce N-end rule substrates for RGS4. V-RGS4 was used as a negative control. (B) The indicated E1-E2 pairs were incubated in the presence of R-RGS4-HA or V-RGS4-HA in the presence or absence of FLAG-UBR2 (purified from 293T cells) and wild-type ubiquitin, and reaction products were examined by SDS-PAGE and immunoblotting with α-HA antibodies. (C) Ubiquitylation assays with R-RGS4-HA were performed as in (B) using either wild-type, K48, K63, or ubiquitin lacking all Lys residues (K0). Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Spatial Control of RGS Turnover by the UBA6-USE1 and UBA1-UBE2A/B Pathways (A and B) UBA6 is specifically localized in the cytoplasm. Cytoplasmic and nuclear fractions of 293T cells stably expressing either RGS4-HA or RGS5-HA were immunoblotted with α-UBA1 or α-UBA6 antibodies (A). α-tubulin and α-TBP were used as controls for cytoplasmic and nuclear compartments, respectively. HeLa cells were transfected with either control siRNA or siUBA6, and after 48 hr cells were subjected to immunoflourescence with α-UBA6 antibodies (B). DNA was stained with Hoechst 33342 to illuminate nuclei. (C) 293T cells expressing RGS4-HA (lanes 1–9) or RGS5-HA (lanes 10–18) were transfected with 30 nM siCK (Control), siUSE1, or siUBE2A/B, and after 72 hr cells were lysed (total extract) and then subjected to fractionation to isolate cytoplasmic and nuclear compartments. Equal amounts of extracts relative to starting cell numbers were separated by SDS-PAGE and immunoblotted with the indicated antibodies. α-TBP and α-tubulin were used as nuclear and cytoplasmic compartment markers, respectively. Approximately 5% of USE1 can be detected in the nucleus when 20-fold more extract is analyzed by immunoblotting (lanes 19–21). (D) Codepletion of USE1 and UBE2 stabilizes RGS5 to a greater extent than depletion of either E2 alone. 293T/RGS5-HA were transfected with 20 nM siRNAs and subjected to immunoblotting after 72 hr. (E) Depletion of UBA6 in MEFs leads to reduced levels of phosphorylated ERK1/2. UBA6flox/flox MEFs were treated with Adeno-CRE or control virus, and after 48 hr extracts were subjected to immunoblotting for ERK1/2 and phospho-ERK1/2. (F) UBA6 is required for phosphorylation of JNK in response to LPS. UBA6flox/flox MEFs were treated with CRE or control virus, and after 48 hr cells were stimulated with LPS to activate JNK1/2 phosphorylation (2 hr). Cell extracts were then subjected to immunoblotting for JNK1/2 and phospho-JNK1/2. (G) Model depicting dual ubiquitin activation and conjugation cascades for RGS proteins. The UBA6-USE1 pathway is functional in the cytoplasm, due to the specific localization of UBA6 in this compartment. The UBA1-UBE2 pathway is active in both the cytoplasm and nucleus. The finding that depletion of either USE1 or UBE2 stabilizes RGS proteins suggests that the pool of RGS proteins that are targeted by each of these pathways is distinct. Molecular Cell 2011 43, 392-405DOI: (10.1016/j.molcel.2011.05.034) Copyright © 2011 Elsevier Inc. Terms and Conditions