Mutations of Ser-23 of the alpha1 subunit of the rat Na+/K+-ATPase to negatively charged amino acid residues mimic the functional effect of PKC-mediated phosphorylation.
The Na+/K+-ATPase is a target protein for protein kinase C (PKC). The PKC-mediated phosphorylation of the rat alpha1 subunit at Ser-23 results in the inhibition of its transport function. To understand the molecular basis of the inhibition by PKC, the Ser-23 in the rat alpha1 subunit has been replaced by negatively (Asp, Glu) or positively (Lys) charged, or uncharged (Gln, Ala) residues, and the mutants were expressed in Xenopus oocytes. Ouabain-specific 86Rb uptake and pump-generated current as well as sensitivity to ouabain and to external K+ have been investigated. When Ser-23 was replaced by the negatively charged residues, transport function was inhibited, and simultaneously synthesis of the alpha subunits was enhanced. In addition, if Ser-23 was substituted by Glu, the K(I) value for inhibition of transport by ouabain was drastically increased from 46.5 microM to 1.05 mM. The data suggest that insertion of a negative charge within the N-terminus of alpha subunit of the Na+/K+-ATPase due to phosphorylation of Ser-23 plays an important role in the PKC-mediated inhibition of transport function.
Vasilets
L A
LA
Max-Planck-Institute for Biophysics, Frankfurt/Main, Germany. larisa.vasilets@medizin.uni-halle.de
Postina
R
R
Kirichenko
S N
SN
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000235:genetics*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D017931:DNA Primers; D012694:Serine; D010042:Ouabain; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium