No abstract

Dopamine inhibits Na؉ ,K ؉ -ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the ␣ subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat ␣ 1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat ␣ 1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na ؉ ,K ؉ -ATPase activity and increased ␣ subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type ␣ 1 subunit or the S11A ␣ 1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A ␣ 1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na ؉ ,K ؉ -ATPase activity, although it did not alter the increased ␣ subunit phosphorylation induced by this agonist.We conclude that dopamine-induced inhibition of Na ؉ ,K ؉ -ATPase activity in rat renal tubule cells requires endocytosis of the ␣ subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

Inhibition of Na؉ ,K ؉ -ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na ؉ ,K ؉ -ATPase activity in response to dopamine is associated with endocytosis of its ␣-and ␤-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na ؉ ,K ؉ -ATPase and inhibition of its activity were accompanied by phosphorylation of the ␣-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the ␣-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent ␣-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the ␣-subunit with a concomitant decrease in Na ؉ ,K ؉ -ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent ␣-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser 11 and Ser 18 ). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the ␣-subunit is essential for Na ؉ ,K ؉ -ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

Endocytosis of Na + ,K + -ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I A phosphoinositide-3 kinase (PI3K-I A ) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I A , through its p85α subunit-SH3 domain, binds to a proline-rich region in the Na + ,K + -ATPase catalytic α subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na + ,K + -ATPase α subunit and results in increased PI3K-I A activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na + ,K + -ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I A and its activation during Na + ,K + -ATPase endocytosis in response to G protein-coupled receptor signals.