FIGURE 3.

Calcium activates Nedd4 E3 ubiquitin ligase activity. A, HA-tagged human Nedd4-2a, Nedd4-2b, Nedd4-2bΔC2, or rat Nedd4-1 was transfected into HEK293 cells, immunoprecipitated from the cell lysates, and used for the E3 ubiquitin ligase activity assay with or without addition of 5 mm CaCl₂. The polyubiquitin products were detected by immunoblotting with anti-ubiquitin antibody (top panels), and the Nedd4 protein amount used in the ligase assay was detected by immunoblotting with anti-HA antibody (bottom panels). B, calcium activation assayed with purified Nedd4-2. His-tagged Nedd4-2a was expressed in E. coli and purified with affinity beads. Purified his-tagged Nedd4-2a (100 ng) was used for the ubiquitin ligase activity assay in the presence of indicated concentration of CaCl₂. C and D, calcium activates immunoprecipitated Nedd4 E3 ubiquitin ligase from HEK293 cells. HA-tagged Nedd4-2a was transfected into HEK293 cells and immunoprecipitated with anti-HA antibody from cell homogenates. The E3 ubiquitin ligase activity of Nedd4-2a was assayed in presence of CaCl₂ at indicated concentrations. C, a representative immunoblotting data; D, quantification of immunoblotting data from two independent assays of calcium-induced activation of Nedd4-2a E3 ubiquitin ligase activity by Kodak EDAS290 image system. The activation fold is defined as ([UB]n/[HA-Nedd4-2a])/([[UB]n]₀/[HA-Nedd4-2a]₀), where [UB]n stands for the quantity of poly-ubiquitin products at any concentration of calcium and [HA-Nedd4-2a] for the quantity of the corresponding HA-Nedd4-2a used in the assay; [[UB]n]₀ for the quantity of poly-ubiquitin products with no calcium and [HA-Nedd4-2a]₀ for the quantity of HA-Nedd4-2a used in the assay with no calcium.