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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

Authors

  • 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
Background

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.

Methods

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.

Results

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.

Conclusion

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.

Funding

  • Veterans Affairs Support