Blocking the salt-inducible kinase 1 network prevents the increases in cell sodium transport caused by a hypertension-linked mutation in human alpha-adducin.

OBJECTIVES: Because a newly described salt-inducible kinase 1 (SIK1) network is responsible for increases in active cell sodium transport in response to elevated intracellular sodium, we hypothesized that this network could mediate the effects of the mutant (hypertensive) form of alpha-adducin on Na,K-ATPase activity. METHODS: Studies were performed in normotensive and hypertensive Milan rats and in a cell line of proximal tubule origin expressing transiently variants of alpha-adducin (human G460W/S586C; rat F316Y) that are associated with elevated blood pressure and result in increased Na,K-ATPase activity. Na,K-ATPase activity was determined as ouabain-sensitive rubidium transport. RESULTS: SIK1 activity (T182 phosphorylation) was significantly elevated in renal proximal tubule cells from Milan hypertensive rats (carrying a alpha-adducin mutation) when compared with normotensive controls. Similarly, SIK1 activity (T182 phosphorylation) was elevated in a normal renal proximal tubule cell line when transfected with the alpha-adducin variant carrying the human hypertensive mutation. Blocking the SIK1 network using negative mutants as well as different stages of its activation pathway prevented the effects induced by the hypertensive alpha-adducin. CONCLUSION: The SIK1 network may constitute an alternative target by which agents can modulate active sodium transport in renal epithelia and avoid the increases in systemic blood pressure that are associated with genetic mutations in the human alpha-adducin molecule.

SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process.

In mammalian cells, active sodium transport and its derived functions (e.g., plasma membrane potential) are dictated by the activity of the Na(+),K(+)-ATPase (NK), whose regulation is essential for maintaining cell volume and composition, as well as other vital cell functions. Here we report the existence of a salt-inducible kinase-1 (SIK1) that associates constitutively with the NK regulatory complex and is responsible for increases in its catalytic activity following small elevations in intracellular sodium concentrations. Increases in intracellular sodium are paralleled by elevations in intracellular calcium through the reversible Na(+)/Ca(2+) exchanger, leading to the activation of SIK1 (Thr-322 phosphorylation) by a calcium calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK alpha-subunit and an increase in its catalytic activity. A protein phosphatase 2A/phosphatase methylesterase-1 (PME-1) complex, which constitutively associates with the NK alpha-subunit, is activated by SIK1 through phosphorylation of PME-1 and its dissociation from the complex. These observations illustrate the existence of a distinct intracellular signaling network, with SIK1 at its core, which is triggered by a monovalent cation (Na(+)) and links sodium permeability to its active transport.

Phosphorylation of adaptor protein-2 mu2 is essential for Na+,K+-ATPase endocytosis in response to either G protein-coupled receptor or reactive oxygen species.

Activation of G protein-coupled receptor by dopamine and hypoxia-generated reactive oxygen species promote Na+,K+-ATPase endocytosis. This effect is clathrin dependent and involves the activation of protein kinase C (PKC)-zeta and phosphorylation of the Na+,K+-ATPase alpha-subunit. Because the incorporation of cargo into clathrin vesicles requires association with adaptor proteins, we studied whether phosphorylation of adaptor protein (AP)-2 plays a role in its binding to the Na+,K+-ATPase alpha-subunit and thereby in its endocytosis. Dopamine induces a time-dependent phosphorylation of the AP-2 mu2 subunit. Using specific inhibitors and dominant-negative mutants, we establish that this effect was mediated by activation of the adaptor associated kinase 1/PKC-zeta isoform. Expression of the AP-2 mu2 bearing a mutation in its phosphorylation site (T156A) prevented Na+,K+-ATPase endocytosis and changes in activity induced by dopamine. Similarly, in lung alveolar epithelial cells, hypoxia-induced endocytosis of Na+,K+-ATPase requires the binding of AP-2 to the tyrosine-based motif (Tyr-537) located in the Na+,K+-ATPase alpha-subunit, and this effect requires phosphorylation of the AP-2 mu2 subunit. We conclude that phosphorylation of AP-2 mu2 subunit is essential for Na+,K+-ATPase endocytosis in response to a variety of signals, such as dopamine or reactive oxygen species.

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.

Hypertension-linked mutation in the adducin alpha-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+,K+-ATPase trafficking in response to GPCR signals and intracellular sodium.

Alpha-adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na+,K+-ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na+,K+-ATPase activity, and their Na+,K+-ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na+,K+-ATPase because of adaptin-mu2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na+,K+-ATPase activity, mu2-subunit hyperphosphorylation, and impaired Na+,K+-ATPase endocytosis. Thus, increased renal Na+,K+-ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na+,K+-ATPase endocytosis in response to natriuretic signals.

Isoform-specific regulation of Na+,K+-ATPase endocytosis and recruitment to the plasma membrane.

The Na(+),K(+)-ATPase traffics between the plasma membrane and intracellular compartments in response to acute changes in membrane receptor activation. These effects are accomplished by a time-dependent interaction of the Na(+),K(+)-ATPase alpha-subunit with specific intracellular signaling molecules either at the plasma membrane (endocytosis) or at the endosome's membranes (recruitment). Most of these studies have been performed in rat renal epithelial cells in which only the alpha(1) isoenzyme is present. Studies in neurons from the neostriatum in which all three alpha-subunit isoforms are present indicate that neurotransmitter-dependent regulation of Na(+),K(+)-ATPase activity displays isoform specificity and also suggest a more complex organization of the intracellular signaling networks controlling Na(+),K(+)-ATPase traffic in mammalian cells.

Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na(+),K(+)-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase. Dopamine inhibited Na(+),K(+)-ATPase activity in OK cells and in those overexpressing wild type dynamin-2 but not in cells expressing a dominant-negative mutant. Dephosphorylation of dynamin is important for its assembly. Dopamine increased protein phosphatase 2A activity and dephosphorylated dynamin-2. In cells expressing a dominant-negative mutant of protein phosphatase 2A, dopamine failed to dephosphorylate dynamin-2 and to reduce Na(+),K(+)-ATPase activity. Dynamin-2 is phosphorylated at Ser(848), and expression of the S848A mutant significantly blocked the inhibitory effect of dopamine. These results demonstrate a distinct signaling network originating from the dopamine receptor that regulates the state of dynamin-2 phosphorylation and that promotes its location (by interaction with phosphatidylinositol 3-kinase) at the site of Na(+),K(+)-ATPase endocytosis.

Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta.

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 microM DA or a dopaminergic D(1) agonist, fenoldopam, but not with the dopaminergic D(2) agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase alpha1 and beta1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-delta, or isozyme-specific inhibitor peptides for PKC-delta or PKC-epsilon inhibited the DA-mediated increase in Na,K-ATPase. PKC-delta and PKC-epsilon, but not PKC-alpha or -beta, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-delta- and PKC-epsilon-specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-delta and PKC-epsilon.

Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis.

In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.