Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells.
Na/K-ATPase in renal epithelium is expressed at the basolateral surface and thus is critical for vectorial solute transport. One potential mode of regulation of Na/K-ATPase involves the intracellular effector protein kinase C (PKC). In kidney cell lines, activation of PKC by the phorbol ester phorbol 12,13-dibutyrate (PDBu) (1 microM) inhibited Na/K-ATPase transport activity in OK cells (Vmax decreased 42%; p < 0.02), but not in LLC-PK1 cells. By immunoblot, both cell types expressed detectable levels of PKC alpha and PKC sigma. In response to PDBu, PKC alpha translocated from the cytosol to the membrane fractions of both cell lines. Phorbol ester treatment increased incorporation of 32PO4 in multiple substrates in both cell types, but a approximately 109-kDa substrate with neutral pI was detected only in the OK cell. Anti-LEAVE, directed against a highly conserved sequence in the H4-H5 loop of all known alpha isoforms of Na/K-ATPase, recognized a approximately 109-kDa membrane protein from both cell lines. Anti-LEAVE also identified a protein that comigrated with the large phosphoprotein which was only present in OK cells. Following 32PO4 loading and PDBu treatment, anti-LEAVE immunoprecipitated a approximately 109-kDa phosphoprotein in OK but not LLC-PK1 cells. These data support the notion that PKC is capable of phosphorylating the alpha subunit and inhibiting Na/K-ATPase transport activity in intact renal cells. Furthermore, they suggest that some forms of Na/K-ATPase in the kidney are not susceptible to PKC phosphorylation and that this heterogeneity may contribute to response diversity.
Division of Nephrology, Duke University Medical Center, Durham, North Carolina 27710.
J P
JP
Middleton
W A
WA
Khan
G
G
Collinsworth
Y A
YA
Hannun
R M
RM
Medford
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D009893:Opossums; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine
D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase