Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

<div><p>Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system <em>in vitro</em>. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.</p> </div

UbcH5a and UbcH6 promote TdT ubiquitylation.

<p>(A) UbcH5a enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Myc-TdT (lanes 2 to 5), and/or Flag-UbcH5a (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated Myc-TdT was detected by immunoblotting with an anti-Myc antibody. Myc-TdT and Flag-UbcH5a in the lysate were detected using an anti-Myc or anti-Flag antibody. (B) UbcH6 enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH6 (lanes 1, 2, 4 and 5) in the indicated combinations. After incubation for 24 h, the cells were treated with 10 µM MG132 for another 12 h. The cells were lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH6 in the lysate were detected using an anti-Flag or anti-Myc antibody. (C) UbcH7 does not enhance TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH7 (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH7 in the lysate were detected using an anti-Flag or anti-Myc antibody.</p

UbcH5a or UbcH6 binds to TdT <i>in vivo</i>.

<p>(A) Immunoprecipitation using Myc-TdT as the bait. 293 T cells were co-transfected with expression vectors encoding Flag-UbcH5a (lanes 1 and 3) and Myc-TdT (lanes 2 and 3). Immunoprecipitation was carried out using an anti-Myc antibody. Immunoprecipitants were subjected to SDS-PAGE and following immunoblotting using an anti-Flag or anti-Myc antibody. (B) Immunoprecipitation using Flag-TdT as the bait. 293 T cells were co-transfected with expression vectors encoding Myc-UbcH6 (lanes 1 and 3) and Flag-TdT (lanes 2 and 3), and immunoprecipitation was carried out using an anti-Flag antibody. Immunoprecipitants were separated by SDS-PAGE and immunoblotted with an anti-Flag or anti-Myc antibody. (C) Immunoprecipitation using Flag-TdT as the bait. 293 T cells were co-transfected with expression vectors encoding Myc-UbcH7 (lanes 1 and 3) and Flag-TdT (lanes 2 and 3). Immunoprecipitation using an anti-Flag antibody. Immunoprecipitants were separated by SDS-PAGE and immunoblotted with an anti-Flag or anti-Myc antibody. (D) Immunoprecipitation of endogenous TdT in Jurkat cell lysates, using an anti-TdT antibody (lane 2) or rabbit pre-immune serum (pre IS) (lane 3). Immunoprecipitants were separated by SDS-PAGE and immunoblotted with an anti-UbcH5, anti-UbcH6 or anti-TdT antibody. Each immunoprecipitation used 1 mg whole cell lysate.</p

TdT ubiquitylation <i>in vivo</i> or <i>in vitro</i>.

<p>(A) Endogenous TdT ubiquitylation in Jurkat cells. Immunoprecipitation was carried out using an anti-TdT antibody (lanes 4 and 6) or rabbit pre-immune serum (pre-IS) (lanes 3 and 5). Immunoprecipitants were subjected to SDS-PAGE and immunoblotted with anti-Ub (FK2) or anti-TdT antibody. One mg of whole cell lysate was used for each immunoprecipitation. (B) <i>In vitro</i> TdT ubiquitylation. The substrate His-TdT was incubated under ubiquitylation conditions with purified proteins. His-TdT and His-BPOZ-2 were purified from <i>E. coli</i> and the Flag-tagged proteins were purified from 293 T cells. To obtain Cul3/Rbx1 or BPOZ-2/Cul3/Rbx1 complexes, 293 T cells were co-transfected with the circled plasmids (lanes 10 and 11). After electrophoresis with a denaturing 7.5% polyacrylamide gel, ubiquitylated TdT was detected using an anti-TdT antibody. (C and D) Kinetics of TdT ubiquitylation in the absence (C) or presence (D) of the BPOZ-2/Cul3/Rbx1 complex. The incubation time after the addition of His-TdT is given above. The reaction products were separated by 7.5% SDS-PAGE and immunoblots were probed with anti-TdT antibody or anti-Flag antibody. The ratio of ubiquitylated His-TdT to unmodified His-TdT was determined with ImageJ. The band density for ubiquitylated His-TdT was defined as 100%.</p

TdT directly binds to E2.

<p>(A) UbcH5a (E2) binds to TdT <i>in vitro</i>. His-UbcH5a was incubated with GST-bound (lanes 2 and 4) or GST-TdT-bound (lanes 3 and 5) Glutathione Sepharose 4B in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of His-Ub. Proteins bound to the beads were eluted by boiling with Laemmli buffer. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-His, anti-TdT, or anti-GST antibody. (B) The pol β-like region in TdT binds to UbcH5a. His-TdT deletion mutants were incubated with GST-bound (lane 2) or GST-UbcH5a-bound (lane 3) Glutathione Sepharose 4B. Proteins bound to the beads were eluted with Laemmli buffer after boiling. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting using an anti-His antibody. (C) E3-independent ubiquitylation of either His-TdT (aa 150–509 or wt) was carried out with UBE1, UbcH5a, and Ub. Ubiquitylated proteins were detected by immunoblotting using an anti-TdT antibody. (D) TdT is poly-ubiquitylated through lysine(s) other than Lys48. TdT was ubiquitylated in a reaction mixture without Ub (lane 1) or containing UbcH5a with His-Ub wt (lane 2), His-Ub K48R (lane 3), Me-Ub (lane 4), or lysine-less (K0) Ub (lane 5). Ubiquitylated His-TdT was detected by immunoblotting using an anti-TdT antibody. (E) <i>In vitro</i> ubiquitylation of TdT mutants. E3-independent ubiquitylation was carried out for wild-type (wt) or point-mutated His-TdTs. TdT was ubiquitylated in the reaction mixture containing UBE1, UbcH5a, and Ub. His-TdT ubiquitylation was detected by immunoblotting using an anti-TdT antibody.</p

The SPA motif in UbcH5a is essential for binding to TdT and for TdT ubiquitylation.

<p>(A) Amino acid sequence alignment of E2s. The catalytic cysteine is indicated in boldface; the SPA motif is indicated by the gray background. UbcH5, UbcH6, and UbcH13, which bind to TdT, have a SPA motif in loop 7. (B) Mutation of the SPA motif in UbcH5a to AAA. The UbcH5a mutant (mtUbcH5a) was expressed in <i>E. coli</i> as a His-tagged protein and purified. His-mtUbcH5a (lane 1) and His-wtUbcH5a (lane 2) were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). (C) Reduction of UbcH5a’s binding ability by the mutated SPA motif. His-wtUbcH5a and His-mtUbcH5a were incubated with Glutathione Sepharose 4B-bound GST (lanes 3 and 4, respectively) or GST-TdT (lanes 5 and 6, respectively). Proteins bound to the beads were eluted by boiling with Laemmli buffer and immunoblotted with an anti-His or anti-GST antibody. (D) Formation of thioester adducts of wtUbcH5a and mtUbcH5a expressed in <i>E. coli</i>. The thioester reaction mixture contained Ub, UBE1, and UbcH5a as indicated. The same amount of each UbcH5a was used for CBB staining. After 5 min at RT, reactions were stopped by adding Laemmli buffer with (lanes 3 and 6) or without (lanes 1, 2, 4, and 5) β-mercaptoethanol, and proteins were separated by SDS-PAGE. UbcH5a thioester adducts were detected by immunoblotting using an anti-UbcH5 antibody. (E) TdT is not ubiquitylated by mtUbcH5a. E3-independent (lanes 1 to 6) or Cul3-dependent TdT ubiquitylation by the BPOZ-2/Cul3/Rbx1 complex (lanes 7 to 12) was carried out. TdT was incubated in a ubiquitylation reaction mixture containing E1, ubiquitin, and wtUbcH5a or mtUbcH5a. His-TdT ubiquitylation was detected by immunoblotting with an anti-TdT antibody.</p

UbcH5a or UbcH6 directly binds to TdT and E3-independently ubiquitylates TdT <i>in vitro</i>.

<p>(A) Ten E2 enzymes were subjected to SDS-PAGE and stained by CBB. (B) Binding between TdT and E2 enzymes <i>in vitro</i>. Ten purified recombinant His-E2 enzymes were incubated with GST- (lane 2), GST-TdT (lane 3) bound Glutathione Sepharose 4B. Proteins bound to the beads were eluted with Laemmli buffer after boiling. The eluates were subjected to SDS-PAGE and detected by immunoblotting using an anti-His antibody. (C) E3-independent TdT ubiquitylation was carried out by 10 E2 enzymes. The substrate His-TdT was incubated with His-Ub, His-UBE1, and His-tagged E2 as indicated (lanes 2–10). After electrophoresis in a denaturing 7.5% polyacrylamide gel, ubiquitylated TdT was detected using an anti-TdT antibody.</p

Knockdown of UbcH5, UbcH6, or UbcH7 mRNA reduces TdT ubiquitylation.

<p>(A and B) 293 T cells were transfected with a control siRNA or siRNAs targeting UbcH5a or UbcH5c using the X-tremeGENE Transfection reagent 24 h prior to the plasmid transfection. The cells were then transfected with Myc-TdT and His-Ub using the GeneJuice Transfection Reagent. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h. mRNA levels of UbcH5 isoforms were measured by real-time PCR, normalized to GAPDH, and are expressed as their ratio to cells transfected with a control siRNA (A). Ubiquitylated Myc-TdT and UbcH5 in the lysate were detected by immunoblotting with an anti-Myc, anti-UbcH5, or anti-actin antibody (B). (C) 293 T cells were transfected with a control siRNA or siRNAs targeting UbcH6 or UbcH7 using the X-tremeGENE Transfection reagent 24 h prior to the plasmid transfection. The cells were then transfected with Myc-TdT and His-Ub using the GeneJuice Transfection Reagent. After incubation for 24 h, the cells were treated with 10 µM MG132 for another 12 h. Ubiquitylated Myc-TdT, UbcH6, and UbcH7 in the lysate were detected by immunoblotting with an anti-Myc, anti-UbcH6, anti-UbcH7, or anti-actin antibody.</p