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Akt Substrate of 160 Kd Regulates Na ,K -Atpase Trafficking in Response

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Akt Substrate of 160 Kd Regulates Na ,K -Atpase Trafficking in Response BASIC RESEARCH www.jasn.org Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia † ‡ † Daiane S. Alves,* Gunilla Thulin, Johannes Loffing, Michael Kashgarian, and Michael J. Caplan* Departments of *Cellular and Molecular Physiology and †Pathology, Yale University School of Medicine, New Haven, Connecticut; and ‡Institute of Anatomy, University of Zurich, Zurich, Switzerland ABSTRACT Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na+,K+-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na+,K+-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na+, K+-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na+,K+-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion–induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na+,K+-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na+,K+-ATPase to the energy state of renal epithelial cells. J Am Soc Nephrol 26: 2765–2776, 2015. doi: 10.1681/ASN.2013101040 The Na,K-ATPase, or sodium pump, creates the transport protein whose activity is governed through driving force for solute and fluid transport in most stimulus-induced trafficking between the plasma tissues. The energy released through the hydrolysis of membrane and intracellular compartments.8 GLUT4 onemoleculeofATPisusedbytheenzymetodrive is delivered to the cell surface from intracellular stor- exportof three Na+ ions and the import of two K+ ions, age vesicles in response to stimuli that favor increased and this transport is essential for the maintenance of glucose uptake, including insulin and muscle contrac- cellular electrochemical gradients. The Na,K-ATPase tion. AS160, or TBC1D4, is a Rab GTPase-activating is restricted in its distribution to the basolateral domain protein (GAP) that participates in regulating the trans- of the plasma membrane in most polarized epithelial location of GLUT4 to the plasma membrane.9,10 cells.1 Many cell types appear to contain two function- ally separable pools of Na,K-ATPase. In addition to the principal pool at the plasma membrane, a population Received October 3, 2013. Accepted January 6, 2015. of Na,K-ATPase is also frequently associated with the Published online ahead of print. Publication date available at 2–4 membranes of intracellular compartments. Physio- www.jasn.org. logic stimuli can promote Na,K-ATPase endocytosis Correspondence: Dr. Michael J. Caplan, Department of Cellular or translocation from the intracellular pool to the and Molecular Physiology, Yale University School of Medicine, plasma membrane.2,5–7 PO Box 208026, New Haven, CT 06520-8026. Email: michael. The glucose transporter 4 (GLUT4) of muscle and [email protected] fat cells is one of the best-studied examples of a Copyright © 2015 by the American Society of Nephrology J Am Soc Nephrol 26: 2765–2776, 2015 ISSN : 1046-6673/2611-2765 2765 BASIC RESEARCH www.jasn.org AS160 is phosphorylated by Akt on at least six amino acid res- Na,K-ATPase in a stable clonal cell line in which AS160 is ro- idues after insulin stimulation9 and this phosphorylation inhibits bustly knocked down by small hairpin RNA (shRNA) (AS160 the Rab-GAP activity of AS160. Because the cell surface accumu- KD).23 Wild-type (WT) Madin-Darby Canine Kidney (MDCK) lation of GLUT4 is dependent upon the GTP-bound state of Rab cells or AS160 KD MDCK cells were subjected or not to energy 8, Rab 10, and Rab 14,11 inhibition of AS160’s Rab-GAP activity depletion. Energy depletion was achieved by incubating cells in a leads to a redistribution of GLUT4 from intracellular storage glucose-free medium containing antimycin A and 2-deoxy-glucose compartments to the plasma membrane. Consistent with this (AA/DG). A mAb directed against the Na,K-ATPase a-subunit was model, the small interfering RNA–induced knockdown (KD) of used to detect the distribution of the sodium pump. Figure 1 AS160 expression increases the basal levels of GLUT4 at the cell depicts the Na,K-ATPase localization in untreated WT and surface and concomitantly reduces the size of the intracellular AS160 KD cells (2). After energy depletion treatment (AA/DG), pool.12,13 AS160 may also play a similar role in regulating the the Na,K-ATPase was dramatically redistributed. Abundant distribution and hence the activity of the epithelial sodium chan- Na,K-ATPase was detected in association with intracellular struc- nel in response to aldosterone stimulation.14 tures in the cytoplasm in the WT cells. The intracellular accumu- Energy depletion in renal epithelial cells results in a re- lation of the Na,K-ATPase in response to energy depletion was distribution of the cell surface Na,K-ATPase, resulting in its substantially reduced in the AS160 KD cell line, suggesting that accumulation in intracellular compartments.15 Energy depriva- AS160 plays an obligate role in sodium pump accumulation in tion induced by renal ischemia can lead to AKI, which is a com- intracellular compartments after energy depletion. mon condition associated with very significant morbidity and In order to confirm the results obtained by immunofluores- mortality.16–18 AKI is associated with detachment of the sodium cence andto quantify the extent ofNa,K-ATPaseaccumulationin pump from the plasma membrane’s subcortical cytoskeleton and cytoplasmiccompartmentsinresponsetoenergydepletioninthe with the loss of cell polarity and resultant impairment of renal MDCK WTand AS160 KD cell lines, a surface biotinylation assay function.7,19–21 Recovery from ischemic renal injury involves was performed. The basolateral surfaces of MDCK cells were restitution of cellular polarity and return of the sodium pump labeled with Sulfo-NHS-SS biotin, after which cells were sub- from intracellular compartments to the plasma membrane.22 jected to energy depletion (AA/DG). As a control, biotinylated Recently, we reported that AS160 interacts directly with the cells were maintained in regular media during the treatment (2). Na,K-ATPase a-subunit and modulates the sodium pump’ssub- Todetermine the total amount of plasma membrane Na,K-ATPase cellular distribution.23 In this study, we investigated the role of that was labeled, aliquots of cells were lysed immediately after AS160 in mediating the accumulation of the Na,K-ATPase in the biotinylation process. After the energy depletion treatment intracellular compartments after energy depletion in cultured or control incubation interval, the biotin that remained exposed at renal epithelial cells. Our data indicate that AS160 is essential the plasma membrane was stripped through an incubation with for sodium pump accumulation in cytoplasmic compartments the membrane-impermeable reducing agent 2-mercaptoethane in response to ATP depletion and that the redistribution of the sulfonate sodium (MesNa), allowing us to detect only the pool of Na,K-ATPase in response to energy depletion is mediated by internalized Na,K-ATPase that was protected from exposure to changes in the phosphorylation state of AS160. Our studies uti- the MesNa reagent. Cell lysates were incubated with streptavidin lizing AS160 knockout (KO) mice further suggest that AS160 beads and samples were subjected to SDS-PAGE and Western plays a role in governing the redistribution of the Na,K-ATPase blotting. Figure 1B shows the resulting Western blot, and Figure in distal segments of the renal tubule that is induced by renal 1C shows the quantification of the relative levels of biotinylated ischemia in vivo. Na,K-ATPase detected under each condition. Similar levels of energy depletion were achieved in both the WTand AS160 KD cells, as evidenced by the comparable increase in the level of the RESULTS phosphorylated form of the energy-sensing kinase adenosine monophosphate-stimulated protein kinase (AMPK) that is AS160 Mediates the Intracellular Accumulation of Na, detected in both cell types. As expected, upon ATP depletion, K-ATPase That Is Induced by Energy Depletion the levels of intracellular Na,K-ATPase were significantly AS160 plays an important role in regulating the GLUT4 protein’s higher in the MDCK WT cells than in the AS160 KD cell intracellular retention and its translocation to the plasma mem- line. These results are consistent with the results observed brane in response to insulin or muscle contraction.24,25 Previ- in the immunofluorescence experiments and suggest that ously, we characterized the interaction between AS160 and Na, AS160 is an essential component of the machinery that mediates K-ATPase and found that AS160 interacts with the cytoplasmic the intracellular accumulation of Na,K-ATPase in response to NP domain of the a-subunit of the sodium pump.23 Na, energy depletion. K-ATPase is internalized and retained in intracellular compart- ments in response to energy depletion and renal ischemia.7,15 The AS160 and Na,K-ATPase Interaction Is Not To investigate whether AS160 plays a role in this Na,K-ATPase Affected by Energy Depletion redistribution after energy depletion, we examined the effects We previously showed that AS160 interacts directly with the of energy depletion on the subcellular distribution of the Na,K-ATPase in MDCK cells.23 To analyze whether the 2766 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2765–2776, 2015 www.jasn.org BASIC RESEARCH Figure 1.
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