Volume 18, Issue 6, Pages 699-709 (June 2005) Ubiquitylation of RAG-2 by Skp2-SCF Links Destruction of the V(D)J Recombinase to the Cell Cycle  Hao Jiang, Fu-Chung Chang, Ashley E. Ross, Jihyun Lee, Keiichi Nakayama, Keiko Nakayama, Stephen Desiderio  Molecular Cell  Volume 18, Issue 6, Pages 699-709 (June 2005) DOI: 10.1016/j.molcel.2005.05.011 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Regulated Destruction of RAG-2 In Vitro (A) Radiolabeled, in vitro-translated p27, GST-R2CT89 (CT89), or GST-R2CT89(T490A) (CT89 [T490A]) were incubated with HeLa S phase extract at 30°C. At indicated times products were resolved by SDS-PAGE and visualized with a phosphorimager. (B) Radiolabeled RAG-2, RAG-2(T490A), GST-R2CT89, or GST-R2CT89(T490A) were incubated with the S phase HeLa extract under conditions supporting degradation. At indicated times, products were analyzed as in (A). (C) The percentage of intact protein remaining at each time point in (B) is plotted against time. Open squares, RAG-2(T490A); open circles, GST-R2CT89(T490A); closed squares, RAG-2; closed circles, GST-R2CT89. (D) Radiolabeled proteins were incubated individually with the HeLa S phase cell extract. The percent of input protein remaining after 90 min was determined. Solid bars, species known (Li et al., 1996) to exhibit low accumulation in vivo; open bars, species that exhibit hyperaccumulation (Li et al., 1996) in vivo. (E) Cell cycle regulation of in vitro degradation activity. HeLa cells were treated with lovastatin (Lov) or HU and then released from cell cycle blockade. Treated and untreated (Un) cells were stained with propidium iodide and analyzed by fluorescence cytometry (left). Degradation of GST-R2CT89 or GST-R2CT89(T490A) was assayed in cell-free extracts from each population (right). Molecular Cell 2005 18, 699-709DOI: (10.1016/j.molcel.2005.05.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Degradation of RAG-2 by the Ubiquitin-Proteasome Pathway (A) Radiolabeled GST-R2CT89 was incubated with the HeLa S phase extract; MG132 (100 μM), methyl-ubiquitin (Me-Ub; 1 μg/μl), or ubiquitin(K48R) (Ub[K48R]; 1 μg/μl) was added as indicated. Products were assayed after 90 min at 30°C. Arrows indicate input protein. (B) Lower mobility products represent ubiquitylated derivatives. Radiolabeled GST-R2CT89 was incubated with the HeLa S phase extract or buffer in the presence of histidine-tagged ubiquitin (His-Ub) or Me-Ub. After 1 hr at 30°C under ubiquitylation assay conditions, reactions were adsorbed to nickel. After washing, bound protein was fractionated by SDS-PAGE. Lanes 1–3, total product; lanes 4–6, bound product. (C) Left, in vitro ubiquitylation. GST, GST-R2CT89, GST-R2CT89(T490A), or GST-R2CT89(499/508A) was incubated with HeLa S phase extract or buffer under ubiquitylation conditions. Me-Ub or ubiquitin was added where indicated. Right, in vivo accumulation. HeLa cells were transfected as indicated. After 48 hr, expression was assessed by immunoblotting for GST. (D) Kinetics of ubiquitylation in vitro. GST-R2CT89 or GST-R2CT89(T490A) was incubated with the HeLa S phase extract and Me-Ub under ubiquitylation conditions. Products were assayed at indicated times. (E) RAG-2 hyperaccumulates in S phase cells upon proteasome inhibition. HeLa cells were transfected with an empty vector (V) or with plasmids encoding RAG-2 (wt) or RAG-2(T490A) (T490A). Cells were synchronized in S phase, treated with 50 μM lactacystin or DMSO for 1 hr, and harvested for cell cycle analysis (left) or protein assays (right). Asyn, asynchronous cells; Syn + DMSO, synchronized cells treated with DMSO; Syn + Lac, synchronized cells treated with lactacystin. Right, RAG-2 (top) and actin (bottom) were detected by immunoblotting; +, synchronized, lactacystin-treated; −, DMSO-treated. (F) Differential ubiquitylation of RAG-2 and RAG-2(T490A) in vivo. Cells (293) were transfected with plasmids encoding HA-tagged ubiquitin (HA-Ub), MBP-RAG-2-myc-His, and MBP-RAG-2(T490A)-myc-His, as indicated. At 22 hr after transfection, cells were treated for 2 hr with LLnL (160 μM) and lysed under denaturing conditions as described (Treier et al., 1994). Nickel precipitates were fractionated by SDS-PAGE, and ubiquitylated species were detected by immunoblotting for HA (top). Arrow indicates the position of unmodified MBP-RAG-2-myc-His. The blot was reprobed with an anti-myc antibody (bottom). Molecular Cell 2005 18, 699-709DOI: (10.1016/j.molcel.2005.05.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 CyclinA/Cdk2 Activity Is Limiting for RAG-2 Degradation In Vitro (A) Inhibition of RAG-2 degradation by p21. Radiolabeled GST-R2CT89 was assayed for degradation in the HeLa S phase extract as above, in the presence of 1.25, 2.5, 5, 10 or 20 ng purified, recombinant wild-type (lanes 3–7), or truncated (lanes 8–12) p21. Reactions carried out for 0 and 90 min in the absence of p21 were analyzed in parallel (lanes 1 and 2). (B) HeLa S phase extract was immunodepleted with anti-Cdk2 (lane 2), anti-cyclin A (lane 3), or anti-Cdc2 (lane 5) antibody, or with control IgG (lanes 1 and 4). After immunodepletion, extracts were assayed for the presence of cyclin A, Cdk2, or Cdc2 by immunoblotting. (C) The immunodepleted extracts analyzed in (B) were tested for ubiquitylation of GST-R2CT89 in the absence (lanes 2, 3, 4, 5, and 7) or presence (lanes 6 and 8) of recombinant cyclin A/Cdk2. Molecular Cell 2005 18, 699-709DOI: (10.1016/j.molcel.2005.05.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Targeted Ubquitylation of RAG-2 by Skp2 In Vitro (A) Ubiquitylation of GST-R2CT89 by anion exchange fractions of the HeLa S phase extract. Lane 1, buffer alone; lane 2, prefractionation extract; lanes 3–5, flowthrough, 300 mM and 600 mM KCl fractions; lane 6–8, as in lanes 3–5 except for addition of purified E1, Cdc34, and Ubc5C. (B) Enhancement of degradation by Cdc34. GST-R2CT89 was incubated with buffer alone (lane 2), the 300 mM KCl fraction (lane 2), or the same fraction supplemented with E1, Cdc34 and Ubc5C (lane 3), Ubc5C (lane 4), or Cdc34 (lane 5). (C) Fractions were assayed for Cul1, Skp1, and Skp2 by immunoblotting. Lane 1, prefractionation extract; lane 2, flowthrough; lanes 3–6, 100, 200, 300, and 400 mM KCl fractions, respectively. (D) HeLa S phase extract was mock depleted using control IgG (mock) or depleted (dp) with an anti-Skp2 antibody (top), anti-cdc27 antibody (bottom), or anti-β-TRCP antibody (middle) and assayed by immunoblotting. (E) Mock-depleted (lanes 2 and 8), Skp2-depleted (lanes 3–7), Cdc27-depleted (lane 9), or β-TRCP-depleted (lane 10) extract was tested for ubiquitylation (top) and degradation (bottom) of GST-R2CT89 in the absence (lanes 2, 3, 9, and 10) or presence of recombinant cyclin A/Cdk2 (lane 4), Skp1/Skp2 (lane 5), cyclinA/Cdk2, and Skp1/Skp2 (lane 6) or cyclinA/Cdk2 and Skp1 (lane 7). A reaction containing buffer alone (lane 1) was assayed in parallel. (F and G) Specific association of Skp2 with the RAG-2 degradation signal. (F) HeLa S phase extract (360 μg) was incubated with uncoupled beads (lane 2) or with beads coupled to peptides corresponding to residues 479–515 of RAG-2. Bound proteins were fractionated by SDS-PAGE, and F box proteins were detected by immunoblotting. Input extract (30 μg) was assayed in parallel (lane 1). (G) Lysates of 293 cells transfected with cDNAs for Skp2, FBXL9, FBXL2, or FBXL12 were incubated with peptide T490(Y) or P-T490(Y). Bound protein was detected by immunoblotting against a c-myc tag. Molecular Cell 2005 18, 699-709DOI: (10.1016/j.molcel.2005.05.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Reconstitution of RAG-2 Ubiquitylation with Purified Proteins (A) Underrepresentation of cyclinA and Skp2 in G1 phase HeLa cell extracts. Lane 1, G1 phase extract; lane 2, S phase extract. Indicated proteins (cyclin A, Cdk2, Skp2, Skp1) were detected by immunoblotting. (B) Skp2 and cyclinA are limiting for ubiquitylation and degradation of RAG-2 in G1 extracts. An HeLa cell G1 phase extract was tested for degradation of GST-R2CT89 (top), degradation of full-length RAG2 (middle), or ubiquitylation of GST-R2CT89 (bottom). Reactions were carried out with addition of recombinant proteins as indicated above. Control reactions containing buffer alone (lane 1) or HeLa S phase extract (lane 2) were assayed in parallel. (C) Complete reconstitution of ubiquitylation with purified proteins. The substrate GST-R2CT89 was incubated under ubiquitylation conditions with recombinant, purified cyclin A/Cdk2, Skp2, Cul1/Rbx1, and Cks1 as indicated above. Lane 1, no proteins added. Molecular Cell 2005 18, 699-709DOI: (10.1016/j.molcel.2005.05.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Skp2-Dependent Ubiquitylation of RAG-2 by a Pro-B Cell S Phase Extract (A) Ubiquitylation of GST-R2CT89, GST-R2CT89(T490A), or p27 was assayed in reactions containing Me-Ub and 63-12 or HeLa S phase extract, as indicated. Products were detected after 1 hr at 30°C. (B) The 63-12 S phase extract was immunodepleted with control IgG or anti-Skp2 antibody as indicated above, and depleted extracts were assayed for Skp2 by immunoblotting. (C) The immunodepleted extracts in (B) were assayed for ubiquitylation of GST-R2CT89 in the absence (lanes 2 and 3) or presence (lane 4) of purified Skp2. Substrate was incubated with buffer alone in parallel (lane 1). (D) Specific precipitation of Skp2 from the 63-12 S phase extract by a bead bound, RAG-2 phosphopeptide as described in Figure 4F. Molecular Cell 2005 18, 699-709DOI: (10.1016/j.molcel.2005.05.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Skp2 Regulates RAG-2 Degradation In Vivo (A) Association of Skp2 with RAG-2. Lysates of 293 cells cotransfected with Skp2ΔF together with MBP (lane 1), MBP-CT89 (lane 2), MBP-CT89(T490A) (lane 3), or MBP-CT89(499/508A) (lane 4) were adsorbed to amylose beads. Skp2 was assayed in cell lysates by immunoblotting with an anti-Skp2 antibody (top). Bound protein was assayed by immunoblotting for Skp2 (middle) or MBP (bottom). (B) HeLa (above) or 293 (below) cells were transfected with plasmids encoding RAG-2, wild-type Skp2, and Skp2ΔF as indicated. Transfection efficiency was >75% as assessed by GFP cotransfection. After 48 hr, RAG-2 and actin were detected by immunoblotting. The G1 fraction of each cell population at the time of assay is indicated. (C) Cells (293) were transfected with a control (closed diamonds) or Skp2 (open squares) siRNA, together with plasmids encoding MBP and MBP-CT89, and subjected to a pulse-chase assay. The amount of radiolabeled MBP-CT89 at various times during the chase period was plotted as percent remaining versus time. (D) Thymocytes from Skp2−/− or Skp2+/− mice were sorted by DNA content, and RAG-2 was assayed in the G1 and S/G2/M populations by immunoblotting (top). Between 1.5 × 106 and 2 × 106 cell equivalents were loaded in each lane. RAG-1 (bottom) is shown for reference. (E) Cells (293) were transfected with siRNA specific for Skp2 (lane 1), p27 (lane 2), Skp2 and p27 (lane 3), or control (lane 4) siRNA, together with plasmids encoding MBP and MBP-CT89. At 48 hr after transfection, cell lysates were assayed for MBP (top), Skp2 (middle), or p27 (bottom). Asterisk indicates a crossreactive species. (F) A model for regulation of V(D)J recombination in the cell cycle. For explanation, see text. Molecular Cell 2005 18, 699-709DOI: (10.1016/j.molcel.2005.05.011) Copyright © 2005 Elsevier Inc. Terms and Conditions