FIGURE 1.

Stimulation of cullin neddylation by hDCNL1. A, Coomassie staining analysis of purified hDCNL1 in full-length or truncated forms. B and C, activation of CUL1 neddylation by hDCNL1 in vitro. The effects of GST-hDCNL1 on neddylation in vitro were measured using either Nedd8 (panel B) or ³²P-Nedd8 (panel C) as described under “Experimental Procedures.” The reaction products are visualized by immunoblot analysis (panel B) or autoradiogram (panel C). The quantification is presented graphically. D, hDCNL1-mediated activation of neddylation is sensitive to salt. The neddylation reaction was carried out as in panel C. The effect of salt (0.3 or 0.5 m NaCl) on the reaction is shown by autoradiogram and graph. E, stimulation of CUL1 neddylation by hDCNL1 in vivo. HEK293 cells were co-transfected with vectors expressing Flag-CUL1 and GST-hDCNL1 or GST-Gus. At 48 h post-transfection, cells were harvested and extracts (500 μg) were immunoprecipitated and analyzed by immunoblot analysis to detect V5-Nedd8 conjugates, Flag-CUL1 or its neddylated forms. F-CUL1-N8, Flag-CUL1-Nedd8. F-CUL1, Flag-CUL1. H, IgG heavy chain. L, IgG light chain. F, hDCNL1 stimulates in vivo neddylation of CUL3. The effect of V5-hDCNL1 on the neddylation of Myc-CUL3 was examined by transfection experiment as described for panel E. M-CUL3-N8, Myc-CUL3-Nedd8. M-CUL3, Myc-CUL1.