Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains

Abstract

Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP carboxyl terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements, together with fluorescence quenching and mass spectrometry, to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labeled WW3, WW4, and, to a lesser extent, WW2 domains, and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.

Figure 1

Domain structure of human Nedd4-2 and the protein constructs used in this study. (A) Schematic representation of the Nedd4-2 domain structure showing the positions of 14-3-3 binding motifs S342, T367, and S448; the Ca²⁺ lipid binding domain is shown in gray (denoted as C2), and the WW1-4 domains are shown in yellow, teal, blue, and magenta (denoted as W1-4). The N- and C-lobes of the HECT domain are shown in raspberry and salmon, respectively. (B) The constructs used in this study are Nedd4-2^186−975 (C942) and Nedd4-2^190−581 (C209, C218, C389, C414, C508, C522, and C571). The positions of the cysteine residues used for 1,5-IAEDANS labeling are shown in red. To see this figure in color, go online.

Figure 2

Mean fluorescence lifetimes of Nedd4-2 variants and their changes upon 14-3-3η binding. The positions of AEDANS-labeled cysteine residues within Nedd4-2 domains are indicated at the bottom. C209 and C218, WW1; C389 and C414, WW2; C414 and C508, WW3; C571, WW4; C943, the C-lobe of HECT domain. Each error bar reflects the standard deviation of a single-curve data analysis.

Figure 3

WW domains of Nedd4-2 exhibit different mobilities. Fluorescence anisotropy decays of AEDANS-labeled Nedd4-2^190−581 variants. To see this figure in color, go online.

Figure 4

14-3-3η binding affects the mobility of Nedd4-2 WW domains. Fluorescence anisotropy decays of Nedd4-2 variants with AEDANS-labeled individual WW domains in the absence and presence of 14-3-3η. (A) WW1 domain variants C209 and C218, (B) WW2 domain variants C389 and C414, and (C) WW3 and WW4 domain variants C508, C522, and C571. To see this figure in color, go online.

Figure 5

14-3-3η binding affects the solvent exposure of Nedd4-2 domains, as shown by the apparent bimolecular quenching constants of AEDANS-labeled Nedd4-2 cysteine variants in the absence and presence of 14-3-3η. Each error bar reflects the standard deviation of a single-curve data analysis.

Figure 6

Limited proteolysis assay showing the protective effect of 14-3-3 to pNedd4-2^186−975. (A) Phosphorylated Nedd4-2^186−975 in the absence and presence of 14-3-3η digested with trypsin for 10, 20, and 30 min. The protease/Nedd4-2 ratio was 1:1000 (w/w). The reactions were stopped by boiling the samples with SDS/PAGE loading buffer at the times indicated before SDS-PAGE analysis. The black rectangle marks the quantified region. Unedited gel image is provided in supporting material (Fig. S15). (B) The density of the bands representing degraded Nedd4-2^186−975 in the presence and absence of 14-3-3η were quantified in ImageLab (Bio-Rad). Error bars represent the standard deviation of four independent experiments. Asterisks indicate significant differences, according to Student's t-tests comparing relative changes between samples with and without 14-3-3η at selected time points (^∗p ≤ 0.01, ^∗∗∗p ≤ 0.0001).

Figure 7

Phosphorylation followed by 14-3-3 binding regulates Nedd4-2. (A) Nedd4-2 binds to ENaC PY motifs and catalyzes its ubiquitination, reducing the rate of sodium transport and consequently the surface expression of these channels. Phosphorylation by various kinases (PKA, SGK, and Akt) triggers 14-3-3 protein binding, which sterically blocks WW domains and affects the structure of the active site, thereby preventing Nedd4-2 binding to ENaC and its ubiquitination. The 14-3-3 binding motifs S342, T367, and S448 are shown as teal circles with P. (B) Positions of AEDANS-labeled cysteine residues in the SAXS-based model of the pNedd4-2^186−975:14-3-3η complex (30). The 14-3-3η protomers are shown in pale green and pale cyan. In the HECT domain of Nedd4-2^186−975, the N-lobe is indicated in raspberry and the C-lobe in salmon. WW2, WW3, and WW4 domains are indicated in teal, blue, and magenta, respectively. Phosphorylated 14-3-3 binding motifs of Nedd4-2 are shown as orange sticks (PDB: 6ZBT and 6ZC9 (30)). The positions of the cysteine residues in the WW2, WW3, WW4, and HECT domains labeled by AEDANS are indicated as red balls. The WW1 domain is not shown. The PY motif (L⁹⁴⁸PPY⁹⁵¹) is shown in red. To see this figure in color, go online.