ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005


The Latest on Twitter

Kidney Week

Abstract: FR-PO607

14-3-3 Epsilon Regulates NPT2a Activity in the Renal Proximal Tubule

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic


  • Gagnon, Kenneth, University of Louisville, Louisville, Kentucky, United States
  • Barati, Michelle T., University of Louisville, Louisville, Kentucky, United States
  • Kitterman, Kathleen, University of Louisville, Louisville, Kentucky, United States
  • Bushau, Adrienne M., University of Louisville, Louisville, Kentucky, United States
  • Clark, Barbara, University of Louisville, Louisville, Kentucky, United States
  • Lederer, Eleanor D., University of Louisville; Robley Rex VA Medical Center, Louisville, Kentucky, United States

14-3-3 is an adapter protein implicated in the regulation of a large spectrum of signaling pathways. We have demonstrated an increased association between the Na-phosphate cotransporter type 2a (NPT2a) and 14-3-3 epsilon in the absence of the Na-H Exchanger Regulator Factor 1 (NHERF1) in proximal tubule cells. We hypothesize that 14-3-3 epsilon associates with a phosphorylated residue in the carboxy-terminal PDZ-1 binding motif of NPT2a and inhibits NHERF1-mediated Npt2a forward trafficking to the apical membrane.


We generated cDNA constructs modifying the threonine residue in the NPT2a PDZ-1 binding motif “TRL” to either mimic phosphorylation (T635D, T635E) or prevent phosphorylation (T635A). We combined these wild type and mutant NPT2a cDNAs with NHERF1 FL or 14-3-3 epsilon FL in IRES-containing bicistronic mammalian expression vectors. We then transiently transfected these constructs in NHERF1-deficient opossum kidney (OKH) cells and assessed membrane expression with confocal fluorescent microscopy and sodium-dependent phosphate cotransport activity with 32P phosphate uptake assays.


NPT2a FL and NHERF1 FL exhibited membrane co-localization. Phosphomimic NPT2a (DRL) appears as cytosolic punctate regions with no NHERF1 FL co-localization. Phosphomimic NPT2a (ERL) and non-phosphomimic NPT2a (ARL) exhibit both reduced membrane localization (compared to NPT2a FL) and cytosolic punctate regions but neither co-localized with NHERF1 FL. 32P uptake assays corroborated the confocal immunofluorescence with a 7-fold increase in phosphate uptake in OKH cells expressing NPT2a FL and NHERF1 FL versus water-transfected OKH cells. NPT2a ERL and NPT2a ARL with NHERF1 had a 2-fold increase in phosphate uptake versus water-transfected OKH cells. NPT2a FL and 14-3-3 epsilon FL do not exhibit cytosolic co-localization. NPT2a phosphomimics (DRL & ERL) both exhibit reduced membrane localization and cytosolic punctate expression, but no co-localization with 14-3-3 epsilon. Non-phosphomimic NPT2a (ARL) exhibits no membrane expression or co-localization with 14-3-3 epsilon FL.


These studies demonstrate that the phosphorylation state of T635 in the PDZ binding motif of NPT2a is a determinant in the molecular switch between NHERF1 and 14-3-3 epsilon regulation of the trafficking and functional activity of the cotransporter.


  • Veterans Affairs Support