Activation of protein kinase A acutely inhibits and phosphorylates Na/H exchanger NHE-3.
In the mammalian renal proximal tubule, protein kinase A (PKA) plays an important role in mediating hormonal regulation of apical membrane Na/H exchanger activity. This exchanger is likely encoded by NHE-3. The present studies examined regulation of NHE-3 by PKA. NHE-3 was stably expressed in Na/H exchanger-deficient fibroblasts (AP-1/NHE-3 cells). PKA activation (0.1 mM 8-BrcAMP x 20 min) inhibited NHE-3 activity by 39% (P < 0.01) with no change in NHE-3 protein abundance in the plasma membrane. To define the structural requirements for PKA-mediated inhibition, full-length NHE-3 and a cytoplasmic domain-truncated mutant (NHE-3 delta cyto) were expressed in Xenopus laevis oocytes. 8-BrcAMP inhibited NHE-3 activity by 27% (P < 0.05), an effect that was blocked by 10(-7) M PKA inhibitor peptide. NHE-3 delta cyto had baseline activity similar to that of full-length NHE-3 but its activity was not regulated by 8-BrcAMP. The purified recombinant cytoplasmic domain of NHE-3 was phosphorylated in vitro by the catalytic subunit of PKA on serine residues. In AP-1/NHE-3 cells, NHE-3 was immunoprecipitated as a approximately 87-kD phosphoprotein. Addition of 0.1 mM 8-BrcAMP increased the phosphocontent of NHE-3 by threefold. In summary, acute activation of PKA inhibits NHE-3 activity, an effect that is likely mediated by phosphorylation of its cytoplasmic domain.
Moe
O W
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Department of Internal Medicine, Department of Veterans Affairs, Dallas, Texas, USA.
Amemiya
M
M
Yamaji
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D015870:Gene Expression; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D017868:Cyclic AMP-Dependent Protein Kinases