14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels.
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.
Nagaki
Kazunori
K
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
Yamamura
Hisao
H
Shimada
Shoichi
S
Saito
Taro
T
Hisanaga
Shin-ichi
S
Taoka
Masato
M
Isobe
Toshiaki
T
Ichimura
Tohru
T
D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D048948:14-3-3 Proteins; D017931:DNA Primers; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005982:Glutathione Transferase; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D015222:Sodium Channels; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus