Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct

Abstract

Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.

Tea Drinking Alleviates Diabetic Symptoms via Upregulating Renal Water Reabsorption Proteins and Downregulating Renal Gluconeogenic Enzymes in db/db Mice

“…PKC‐α knockout mice have defective urine concentration. [ 41 ] The mechanisms of action of PKC‐α in enhancing urine concentration include: 1) PKC‐α regulates urea permeability, [ 42–44 ] 2) PKC‐α upregulates AQP2 through phosphorylating cyclic adenosine binding protein, which is a transcriptional factor of AQP2, [ 21,22 ] 3) PKC‐α promotes vasopressin‐stimulated AQP2 trafficking via altering microtubule assembly, [ 23 ] 4) PKC decreases phosphorylation of AQP2 at Ser261, [ 24 ] which inversely regulates AQP2 trafficking, [ 45,46 ] and 5) PKC‐α increases phosphorylation of UT‐A1 at Ser494 [ 47 ] and enhances UT‐A1 cell surface expression. [ 25 ] In the present study, both GT and BT infusions increased renal PKC‐α and renal membrane PKC‐α in db/db mice.…”

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Interestingly, while PKA phosphorylation sites on UT-A1 have been mapped to S486, S499, and S84 (56,89,455), the phosphorylation sites regulated by EPAC appear to be different and are not currently mapped (420). A PKC-dependent, hypertonicity-stimulated UT-A1 phosphorylation at site S494 was recently identified (88). Phosphorylation of S494 was independent of the cAMP pathway as activation of PKA and EPAC was without effect on UT-A1 phosphorylation at S494.…”

Urea transport and clinical potential of urearetics

“…A recent study used mutagenesis to identify S494 as the PKC phosphorylation site (23) (Figure 2). A polyclonal antibody to phospho-S494-UT-A1 was generated and used to show that activators of PKC, phorbol dibutyrate and hypertonicity, increased UT-A1 phosphorylation at S494 while activators of either PKA or Epac did not (23). Activation of both PKA and PKC, but not PKC alone, increased the apical plasma membrane accumulation of UT-A1 (23).…”

“…A polyclonal antibody to phospho-S494-UT-A1 was generated and used to show that activators of PKC, phorbol dibutyrate and hypertonicity, increased UT-A1 phosphorylation at S494 while activators of either PKA or Epac did not (23). Activation of both PKA and PKC, but not PKC alone, increased the apical plasma membrane accumulation of UT-A1 (23). These findings suggest UT-A1 phosphorylation at S494 by PKC may increase vasopressin-stimulated urea transport by enhancing UT-A1 retention in the apical plasma membrane (23).…”

“…Activation of both PKA and PKC, but not PKC alone, increased the apical plasma membrane accumulation of UT-A1 (23). These findings suggest UT-A1 phosphorylation at S494 by PKC may increase vasopressin-stimulated urea transport by enhancing UT-A1 retention in the apical plasma membrane (23). …”