TY - JOUR
T1 - Phosphorylation of the catalyic α-subunit constitutes a triggering signal for Na+,K+-ATPase endocytosis
AU - Chibalin, Alexander V.
AU - Pedemonte, Carlos H.
AU - Katz, Adrian I.
AU - Féraille, Eric
AU - Berggren, Per Olof
AU - Bertorello, Alejandro M.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1998/4/10
Y1 - 1998/4/10
N2 - 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 α- and β-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 α-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 α- subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal 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 α-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the α-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent α-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the α-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.
AB - 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 α- and β-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 α-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 α- subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal 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 α-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the α-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent α-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the α-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.
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U2 - 10.1074/jbc.273.15.8814
DO - 10.1074/jbc.273.15.8814
M3 - Article
C2 - 9535860
AN - SCOPUS:0032502713
VL - 273
SP - 8814
EP - 8819
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -