Although recent studies show that the 14-3-3 protein is a negative regulator of ubiquitin E3 protein ligases, the molecular mechanism remains largely unknown. We previously demonstrated that 14-3-3 specifically binds one of the E3 enzymes, Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2), which can be phosphorylated by serum glucocorticoid-inducible protein kinase 1 (SGK1); this binding protects the phosphorylated/inactive hNedd4-2 from phosphatase-catalyzed dephosphorylation [Ichimura, T., et al. (2005) J. Biol. Chem. 280, 13187-13194]. Here we report an additional mechanism of 14-3-3-mediated regulation of hNedd4-2. Using surface plasmon resonance spectrometry, we show that 14-3-3 inhibits the interaction between the WW domains of hNedd4-2 and the PY motif of the epithelial Na(+) channel, ENaC. The inhibition was dose-dependent and was dependent on SGK1-catalyzed phosphorylation of Ser468 located between the WW domains. Importantly, a mutant of hNedd4-2, which can be phosphorylated by SGK1 but cannot bind 14-3-3, reduced SGK1-mediated stimulation of the ENaC-induced current in Xenopus laevis oocytes. In addition, 14-3-3 had similar effects on hNedd4-2 that had been phosphorylated by cAMP-dependent protein kinase (PKA). Our results, together with the recent finding on 14-3-3/parkin interactions [Sato, S., et al. (2006) EMBO J. 25, 211-221], suggest that 14-3-3 suppresses ubiquitin E3 ligase activities by inhibiting the formation of the enzyme/substrate complex.