Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct.
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.
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia mabloun@emory.edu.
Mitsi A
MA
Blount
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Penelope
P
Cipriani
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Sara K
SK
Redd
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Ronald J
RJ
Ordas
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Lauren N
LN
Black
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Diane L
DL
Gumina
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Carol A
CA
Hoban
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia.
Janet D
JD
Klein
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia.
Jeff M
http://orcid.org/0000-0001-9822-0607
JM
Sands
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D006982:Hypertonic Solutions / Q000494:pharmacology; D007679:Kidney Medulla / Q000187:drug effects / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000201:enzymology*; D018374:LLC-PK1 Cells; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009995:Osmosis; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D017207:Rats, Sprague-Dawley; D012694:Serine; D013552:Swine; D014162:Transfection; D000096982:Urea Transporters
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020536:Enzyme Activators; D006982:Hypertonic Solutions; D026901:Membrane Transport Proteins; D051571:Protein Kinase C-alpha; D047428:Protein Kinase Inhibitors; D012694:Serine; D000096982:Urea Transporters
PKC; UT-A1; cAMP; urea transport; vasopressin