Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains

Abstract

The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.

FIG. 1.

Functional domains of human Cdc34. A schematic representation of domains within human Cdc34 which are responsible for catalysis, multi-Ub chain assembly, and interactions with ROC1-CUL1 for the efficient synthesis of Ub polymers is shown. Mutants made and analyzed in this study are indicated on the left, with their positions marked.

FIG. 2.

Ligation of Ub to IκBα by SCF^βTrCP2 requires human Cdc34 S95 and E108/E112. The ubiquitination reaction was carried out as described in Materials and Methods, with titration of SCF^βTrCP2 (A) and comparison of wild-type Cdc34 with Cdc34^E108A/E112A (B) or Cdc34^S95D (C) in the poly- and monoubiquitination of IκBα by SCF^βTrCP2. The reaction products are indicated, with the number of Ub moieties attached to the substrate marked in the middle.

FIG. 3.

Human Cdc34 D102/D103 and D143/M147/R149/K150/E153 are required for polyubiquitination of IκBα by SCF^βTrCP2. Reactions were carried out as described in Materials and Methods. The effects of alanine substitutions at human Cdc34 D102/D103 or D143/M147/R149/K150/E153 were analyzed by E2 titration experiments with wild-type Ub (A) or Ub^K48R (B) and by time course analyses (C and D) with 1.5 μM of E2. The reaction products are indicated, with the number of Ub moieties attached to the substrate marked in the middle.

FIG. 4.

Inhibitory effects of Cdc34^N85Q, Cdc34^Y87A, and Cdc34^S138A in the polyubiquitination of IκBα by SCF^βTrCP2. The reactions were carried out as described in Materials and Methods. The effects of mutations at human Cdc34 N85, Y87, and S138 were analyzed by E2 titration with wild-type Ub (A) or Ub^K48R (B) and by kinetic experiments (C and D) with 1.5 μM of E2. The reaction products are indicated, with the number of Ub moieties attached to the substrate marked in the middle.

FIG. 5.

Effects of human Cdc34 mutants in Ub-Ub ligation, as measured by multiple-turnover di-Ub synthesis assay. Time course experiments were carried out to measure the ligation of ³²P-Ub^K48R to bUb, as described in Materials and Methods, with 4 μM of wild-type human Cdc34 or mutant Cdc34, as specified. Where indicated, DTT was added (0.1 M) after the incubation to disrupt the thiol-ester. The reaction products are indicated.

FIG. 6.

Effects of human Cdc34 mutants in Ub-Ub ligation, as measured by a single-round pulse-chase assay. Human Cdc34 or mutant Cdc34 (4 μM), as specified, was charged with ³²P-Ub^K48R and chased with either Ub⁷⁴ (A and B) or bUb (C), using a protocol described in detail in Materials and Methods. The reaction products are indicated. Formation of di-Ub by wild-type or mutant Cdc34 was quantitated and expressed graphically.

FIG. 7.

Effects of E2 dimerization in reversing the catalytic defects of Cdc34^N85Q, Cdc34^S95D, and Cdc34^E108A/E112A. (A) GST fusion restored the ability of Cdc34^S95D and Cdc34^E108A/E112A, but not Cdc34^N85Q, to catalyze di-Ub synthesis. The di-Ub synthesis reaction was measured by the multiple-turnover assay as described in Materials and Methods, with increasing amounts of wild-type or mutant GST-Cdc34 as indicated. The reaction mix was incubated at 37°C for 60 min. Formation of di-Ub by wild-type or mutant GST-Cdc34 was quantitated and expressed graphically. (B) GST-Cdc34^N85Q forms thiol-ester conjugates with Ub. A Ub conjugation assay was performed as described in Materials and Methods. Where indicated, DTT was added (0.1 M) after the incubation to disrupt the thiol-ester. (C) Dimerization of FKBP-Cdc34^E108A/E112A in the presence of AP20187. Native gel electrophoresis was performed with purified FKBP-Cdc34 and FKBP-Cdc34^E108A/E112A (1 μg) isolated from infected High Five cells treated with or without AP20187. (D) FKBP-based dimerization stimulates the ability of Cdc34^E108A/E112A to synthesize di-Ub. The di-Ub synthesis reaction was measured by the multiple-turnover assay as described in Materials and Methods, with increasing amounts of the monomeric or dimeric (AP20187-bound) form of FKBP-Cdc34^E108A/E112A. The reaction mix was incubated at 37°C for 60 min. Formation of di-Ub by monomeric or dimeric FKBP-Cdc34^E108A/E112A was quantitated and expressed graphically.