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The Urea Transporter UT‐A1 is Directly Phosphorylated by PKC

Hypertonicity increases urea transport, as well as phosphorylation and membrane accumulation of UT‐A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Recent work from our laboratory demonstrated that hypertonicity stimulates urea transport through PKC‐mediated phosphorylation. This finding led us to speculate that PKC directly phosphorylates UT‐A1. Using in silico analysis, we determined that UT‐A1 has eight sites that may be directly phosphorylated by PKC: S23, S79, T447, S494, T545, T549, S554, and S910. In order to test these sites, site‐directed mutagenesis was used to replace either Ser or Thr with an Ala in order to conserve the molecular volume of the amino acid side chain at that position while ablating the phosphate acceptor site. These mutant constructs were transiently transfected into LLC‐PK1 cells. Cells were successfully transfected with either wild type UT‐A1 (WT) or one of the eight single‐site mutations were metabolically labeled with [32P]orthophosphate, then treated with either vehicle or phorboldibutyrate (PDBu), a known PKC activator. Immunoprecipitated UT‐A1 from each sample was analyzed for total phosphorylation by autoradiography. Of the eight sites, we determined that one, S494, is directly phosphorylated by PKC. We conclude that PKC phosphorylates UT‐A1 at S494. Support: NIDDK.