Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2) Regulation by 14-3-3 Protein Binding at Canonical Serum and Glucocorticoid Kinase 1 (SGK1) Phosphorylation Sites

Abstract

Background: Coordinate regulation by kinases and 14-3-3 proteins regulates sodium transport through phosphorylation and inhibition of E3 ligases. Results: Phosphorylation at similar but distinct target motifs can either inhibit or stabilize E3 ligases. Conclusion: E3 ligases integrate multiple kinase inputs to regulate sodium transport and protein stability. Significance: These findings broaden our knowledge of how E3 ligases and sodium transport are regulated by phosphorylation.

Harvest and primary culture of the murine aldosterone-sensitive distal nephron

“…HEK293T cells were transiently transfected as indicated with FLAG-tagged mouse Nedd4-2 with ␣ (HA-tagged)-, ␤-, and ␥-subunits of mouse ENaC as described previously (8). Twenty-four hours after transfection, cells were harvested in 500 l lysis buffer containing protease and phosphatase inhibitors as described previously (18).…”

“…Human embryonic kidney (HEK293T) cells and mouse polarized kidney cortical collecting duct (mpkC-CD c14) cells were maintained and cultured as described previously (8). MpkCCDc14 cells were pretreated for 6 h with vehicle (100% ethanol) or 1 M aldosterone (Sigma-Aldrich) with 2 final hours of vehicle (DMSO, Sigma-Aldrich), 50 M LY294002 (EMD Millipore), or 100 nM insulin (Sigma-Aldrich).…”

Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action

“…These minor sites are believed to be more SGK1 specific (32). All 3 of these phosphorylation sites mediate binding to 14-3-3 proteins, which sequester and inactivate NEDD4-2 (30,33). Mutation of these residues effectively renders the NEDD4-2 protein resistant to inhibition by SGK1 (14,34).…”

“…Aldosterone induces the transcription of SGK1, which interferes with NEDD4-2 function by phosphorylating residues that alter PY motif binding, including a canonical "major" site at mouse serine 328 (analogous to Xenopus serine 444) (30). To test whether SGK1 can inhibit NEDD4-2-mediated regulation of WNK1, we first tested the effect of small-molecule SGK1 inhibitors on WNK1 abundance in mpkCCD c14 cells.…”

Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ

“…Refs. [15][16][17][18]. The 14-3-3 isoforms ␤ and ⑀ constitutes a heterodimer that interacts with a phosphorylated form of the E3 ligase Nedd4-2, blocking the interaction of Nedd4-2 with ENaC, reducing ENaC ubiquitylation and thereby increasing apical ENaC density and sodium transport.…”