FIG 3.

MDM2 mediates polyubiquitylation of DDX24 in vitro. (A) In vitro ubiquitylation of DDX24 by MDM2. Recombinant human MDM2 was assayed for ubiquitylation activity with GST-FLAG-DDX24 as the substrate in the absence (–) or presence (+) of the indicated reaction mixture components. Each reaction was stopped after 2 h at 37°C by the addition of SDS sample buffer, and the mixture was subjected to immunoblot analysis with anti-FLAG. The positions of unmodified GST-FLAG-DDX24 and of GST-FLAG-DDX24 conjugated with ubiquitin (Ub) are indicated. (B) In vitro ubiquitylation assays with a panel of representative E2s. Recombinant human MDM2 was assayed for ubiquitylation activity in the presence of E1 (UBE1), ubiquitin, and a panel of E2s and with GST-FLAG-p53 (left) or GST-FLAG-DDX24 (right) as the substrate. Each reaction mixture was subjected to immunoblot analysis with antibodies to FLAG (left) or to DDX24 (right). −, no E2. (C) MDM2 mediates polyubiquitylation of DDX24 in vitro. Recombinant human MDM2 was assayed for ubiquitylation activity in the presence of E1 (UBE1), E2 (UBE2D), and either WT or methylated (Me) ubiquitin and with GST-FLAG-p53 (left) or GST-FLAG-DDX24 (right) as the substrate. Each reaction mixture was subjected to immunoblot analysis with anti-FLAG. The positions of unmodified, monoubiquitylated (MonoUb), and polyubiquitylated (PolyUb) substrates are indicated. (D) MDM2 and UBE2D mediate DDX24 polyubiquitylation with all single-lysine-substitution mutants of ubiquitin. Recombinant human MDM2 was assayed for ubiquitylation activity in the presence of E1 (UBE1), E2 (UBE2D), and either WT ubiquitin, ubiquitin mutants (in which the lysine residue at position 6, 11, 27, 29, 33, 48, or 63 was replaced with arginine), or methylated (Me) ubiquitin and with GST-FLAG-p53 (left) or GST-FLAG-DDX24 (right) as the substrate. Each reaction mixture was analyzed as described for panel B.