Dissociation of spectrin-ankyrin complex as a basis for lossof Na-K-ATPase pola - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 28 下一页

返回列表

查看: 604907\|回复: 273	go Dissociation of spectrin-ankyrin complex as a basis for lossof Na-K-ATPase pola [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：RobertWoroniecki, Jean R.Ferdinand, JonS.Morrow,  PrasadDevarajan,作者单位：1 Division of Pediatric Nephrology, Albert Einstein Collegeof Medicine, New York, New York 10467; Division of Pathology,Yale University School of Medicine, New Haven, Connecticut 06520; and Department of Nephrology, Cincinnati Children‘sHospital Medical Center, Cincinnati, Ohio 45229-3039


      $ q9 z. g+ J+ _. U3 D9 R) a8 T

         2 ~! O. e7 {9 G/ q  v0 B
         - s, x* e7 ?  x3 M  B4 E" o
         5 o  I; g6 ]7 O


      ; y0 p& m' g5 F: G- S. B4 F" F0 k

      # w" X: T( o: }% Z/ [5 {9 t7 L( J
      【摘要】
   The polarizeddistribution of Na-K-ATPase at the basolateral membranes of renaltubule epithelial cells is maintained via a tethering interaction withthe underlying spectrin-ankyrin cytoskeleton. In this study, we haveexplored the mechanism underlying the loss of Na-K-ATPase polarityafter ischemic injury in Madin-Darby canine kidney (MDCK)cells, utilizing a novel antibody raised against a recently describedkidney-specific isoform of ankyrin. In control MDCK cells, ankyrin wascolocalized with Na-K-ATPase at the basolateral membrane. ATP depletionresulted in a duration-dependent mislocation of Na-K-ATPase andankyrin throughout the cytoplasm. Colocalization studies showed apartial overlap between the distribution of ankyrin and Na-K-ATPase atall periods after ATP depletion. By immunoprecipitation withanti-ankyrin antibody, the mislocated Na-K-ATPase remained bound toankyrin at all time points after ATP depletion. However, theinteraction between ankyrin and spectrin was markedly diminished within3 h of ATP depletion and was completely lost after 6 h. Insolution binding assays using a fusion peptide of glutathione S -transferase with the ankyrin binding domain ofNa-K-ATPase, a complex with ankyrin was detected at all time pointsafter ATP depletion, but spectrin was lost from the complex in aduration-dependent manner. The loss of spectrin binding was notattributable to spectrin degradation but was associated withhyperphosphorylation of ankyrin. The results suggest that adissociation of the membrane-cytoskeleton complex at thespectrin-ankyrin interface may contribute to the loss of Na-K-ATPasepolarity after ischemic injury and reaffirm a critical adapterrole for ankyrin in the normal maintenance of Na-K-ATPase polarity.
      【关键词】 phosphorylation MadinDarby canine kidney adenosine‘triphosphatase depletion
   INTRODUCTION! ?4 Z# h, |+ ~/ ?; o- a+ x

TETHERING INTERACTIONS BETWEEN integral membrane proteins and the underlyingspectrin-based cytoskeleton play key roles in several cellularactivities, including the establishment and maintenance of orderedmembrane domains ( 10 ). Ankyrins are a family of conserved proteins that have emerged as adapter molecules mediating such linkages, because they possess binding sites for a variety of integralmembrane proteins as well as for spectrin ( 3-5, 8-10 ). A particularly well-characterized example is thelinkage between ankyrin and -Na-K-ATPase, mediated primarily byresidues within ankyrin's repeats domain and the second cytoplasmicdomain of -Na-K-ATPase ( 11, 37, 40 ). This interactionis especially critical to the cells lining the kidney tubules, whichvectorially transport ions and nutrients via mechanisms that aredependent on the polarized basolateral colocalization ofNa-K-ATPase, ankyrin, and spectrin ( 29, 31, 36 ). One ofthe major consequences of acute ischemic injury to renal tubulecells is the disruption of polarity, with a coordinate mislocation ofNa-K-ATPase, ankyrin, and spectrin to alternate cellular sites( 28, 33, 36 ). This has been demonstrated both in vitro( 24-27 ) and in human biopsy samples ( 1 ),with important implications for the abnormal handling of sodium andglucose by the postischemic kidney ( 21, 24, 35 ).However, the molecular basis for the loss of Na-K-ATPase polarity afterischemic renal injury remains incompletely understood.
  \0 }( _! b2 e# A
We have recently cloned and characterized a novel renal isoform ofankyrin, termed AnkG190, based on the predicted size of the polypeptide( 37 ). Others and we have shown that AnkG190 is the majorisoform associated with the basolateral domain of renal epithelialcells ( 30, 37 ). In this study, we have developed ananti-peptide polyclonal antibody directed to the uniqueNH 2 -terminal sequences of AnkG190. We demonstrate thatAnkG190 interacts with -Na-K-ATPase at the lateral domain ofMadin-Darby canine kidney (MDCK) cells. ATP depletion results in aduration-dependent loss of -Na-K-ATPase polarity. Using a series ofcomplementary assays, we show that ankyrin remains bound to -Na-K-ATPase after ATP depletion but that the interaction betweenankyrin and spectrin is lost. We suggest that a dissociation of themembrane-cytoskeleton complex at the spectrin-ankyrin interface,possibly mediated by a posttranslational mechanism involvingserine/threonine and tyrosine phosphorylation of ankyrin, maycontribute to the loss of Na-K-ATPase polarity that followsischemic injury.0 N7 C6 S% M/ \$ G0 {( F

MATERIALS AND METHODS( g( @1 I; M- e2 c* w2 c
& O2 w4 S7 z% ^, u# P
Cell culture and ATP depletion. MDCK type II cells, an established polarized renal tubule epithelialcell line obtained from American Type Culture Collection (Rockville,MD), were passaged in complete DMEM with 10% fetal bovine serum(GIBCO, Gaithersburg, MD) and analyzed within 1 day of reachingconfluence. Cells were grown on six-well tissue culture-treated polystyrene plates (Costar, Cambridge, MA) except for microscopy, forwhich they were grown on coverslips placed within the wells. In aseparate set of experiments, cells were grown on Transwell filters(Costar) to ensure complete polarization. ATP depletion was used as awell-established model of reversible ischemic injury to MDCKcells ( 2, 6, 15, 26, 38 ). Briefly, confluent cells werewashed with PBS and incubated for varying time periods in glucose-freeDMEM (GIBCO) in the presence of 1 µM antimycin A (Sigma, St. Louis,MO) as an inhibitor of oxidative phosphorylation ( 15 ). Wehave previously shown that MDCK cells subjected to this protocolundergo partial but reversible ATP depletion ( 15 ). After~8 h, a subset of cells thus stressed initiate the process ofapoptosis, but the majority remain adherent and viable( 15 ).

Preparation of polyclonal antibodies to AnkG190. Affinity-purified polyclonal antibodies were generated (QualityControlled Biochemicals, Hopkinton, MA) in two rabbits after injectionof a glutaraldehyde-conjugated synthetic peptide containing residues15-31 of AnkG190. These residues were chosen because they areunique to AnkG190 ( 37 ). Antibody titers of hyperimmune sera were monitored by ELISA using the BSA-coupled peptide.

Microscopy. Immunofluorescence microscopy was performed at room temperature aspreviously described ( 12, 13, 29 ). Briefly, 4-µm sections of paraffin-embedded rat kidney or MDCK cells at confluence orafter varying periods of ATP depletion were fixed with acetone for 15 min, blocked in goat serum for 30 min, incubated in primary antibody in2% BSA containing 10% goat serum for 60 min, washed, incubated insecondary antibodies conjugated to Cy2 or Cy3 (Amersham, ArlingtonHeights, IL), and visualized with a microscope (Olympus AX70, LakeSuccess, NY) equipped for epilumination. The antibodies used were thepolyclonal to AnkG190 at 1:200 dilution and a monoclonal to -Na-K-ATPase at 1:1,000 (Upstate Biotechnology, Lake Placid, NY).
1 H& [7 x3 E! b7 h2 g9 W
Immunoprecipitations. Immunoprecipitations were performed as previously described ( 13, 29 ). Briefly, cells in six-well plates were lysed for 20 min at4°C in 2 ml of IP buffer [10 mM Tris · HCl, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 0.5% deoxycholate, 1% Nonidet P-40, and 1×Complete protease inhibitor (Roche Applied Science, Indianapolis, IN)]. In a separate set of experiments, cells were extracted in situwith a low-salt buffer (10 mM PIPES, pH 6.8, 50 mM NaCl, 300 mMsucrose, 0.5% Triton X-100, and 1× Complete protease inhibitor) toisolate a cytosolic fraction, as previously described( 11-13, 37, 40 ). The lysates were centrifuged for 1 min at 10,000 g and precleared by a 60-min incubation at4°C with 25 µl of preimmune serum and 200 µl of a 50% proteinA-Sepharose solution (Upstate Biotechnology). The cleared supernate wasincubated with 20 µl of the polyclonal AnkG190 antibody for 4 hat 4°C and for an additional 2 h with 200 µl of a 50% proteinA-Sepharose solution. The lysates were centrifuged for 1 min at 10,000 g, washed three times with IP buffer, and the pellet wassubjected to SDS-PAGE and Western analysis. The antibodies used werethe polyclonal to AnkG190 at 1:200 dilution, a monoclonal to -Na-K-ATPase at 1:5,000 (Upstate Biotechnology), a polyclonal to II spectrin (10D) at 1:200 dilution, and a monoclonal to tubulin at1:10,000 (Sigma).& Z  o. l$ f/ v1 h2 b4 K

Ankyrin binding assay. Solution binding assays were performed using glutathione S -transferase (GST) fusion peptides as previously described( 11, 40 ). The minimal ankyrin binding (MAB) domain of -Na-K-ATPase lies within residues 142-166, and the preparationand purification of recombinant peptides representing this region havebeen previously detailed ( 11, 40 ). A construct encodingfor MAB was expressed in bacteria as a GST fusion using the pGEXprokaryotic expression system (Pharmacia, Piscataway, NJ) and purifiedusing glutathione-agarose ( 11, 40 ). GST alone wasexpressed as a control peptide. Proteins were analyzed by SDS-PAGEfollowed by staining with Coomassie blue and were quantified by aBradford assay (Bio-Rad, Hercules, CA). Each fusion protein (GST orGST-MAB, 50 µg at 1 mg/ml) was conjugated to 50 µl of a 50% slurryof glutathione-agarose for 1 h at 4°C with gentle rotation andincubated at 4°C overnight with 1 ml of a cytoskeletal fraction (300 µg of total protein) from confluent MDCK cells extracted in situ,using a buffer containing 10 mM PIPES, 500 mM NaCl, 300 mM sucrose, 3 mM MgCl 2, 0.5% Triton X-100, and 1× Complete (Roche)protease inhibitors ( 11, 40 ). The beads were pelleted andwashed twice with PBS, and aliquots were analyzed by SDS-PAGE followedby Western blotting and enhanced chemiluminescence (Amersham) withantibodies as above.

Ankyrin phosphorylation assays. The phosphorylation status of ankyrin was examined usingprotocols as recommended by the manufacturer (UpstateBiotechnology). Briefly, cells were preincubated for 60 min withprotease inhibitor [1× Complete protease inhibitor (Roche)] and acocktail of phosphatase inhibitors including 1 mM sodium vanadate(Calbiochem), 10 mM okadaic acid (GIBCO), and 1 nM calyculin A (GIBCO).Cells were then extracted in situ with high-salt lysis buffer (500 mMNaCl, 1% Nonidet P-40, 50 mM Tris, pH 8.0), and lysates werecentrifuged at 14,000 rpm for 10 min at 4°C. The clear supernates (1 ml) were incubated overnight at 4°C with either 20 µl ofpreconjugated anti-phosphotyrosine agarose (Upstate Biotechnology) orwith a combination of 20 µl of anti-phosphoserine/threonine antibody and 20 µl of IgG-agarose (Upstate Biotechnology). Bound complexes were recovered by centrifugation, washed with 1× PBS, and subjected toWestern analysis with antibody to AnkG190 as described above.8 [4 t' g9 ^: _
' J& ^9 H6 `" C7 }5 C) G  M
RESULTS( C, J0 Q3 ~/ t: q, d, ~
7 I' Q! N3 p) v0 `7 |" d
Ankyrin is polarized to the basolateral membrane of kidney tubulesand MDCK cells. We generated polyclonal antibodies directed to the uniqueNH 2 -terminal sequences of AnkG190. The antibodyspecifically recognized an immunoreactive peptide at 190 kDa in ratkidney lysates and a 210-kDa peptide in MDCK cell lysates, whereas thepreimmune serum was devoid of cross-reactivity (Fig. 1 ). By immunofluorescence microscopy, AnkG190 was localized predominantly to the basolateral domains of kidney tubule cells (not shown) and cultured MDCK cells (Fig. 1 ), whereas no staining was observed with the preimmune serum(not shown). Double-staining of MDCK cells revealed a colocalization ofAnkG190 with -Na-K-ATPase along the basolateral membrane (Fig. 2 G ).0 p0 H/ p! u6 L5 s7 O

Fig. 1. Ankyrin is polarized to the basolateral membrane ofMadin-Darby canine kidney (MDCK) cells. A : Western blot ofMDCK cells or rat kidney lysates with preimmune serum (Pre) orpolyclonal AnkG190 antibody (Ank). Left : molecular massmarkers. B : immunofluorescence microscopy of MDCK cells withAnkG190 antibody. Results were reproducible in 3 separate experiments.Bar = 10 µm.
/ O2 f) u" @+ Y7 l3 X  e
Fig. 2. ATP depletion results in a duration-dependent loss of polarizeddistribution of Na-K-ATPase and ankyrin. MDCK cells, untreated( left ) or ATP depleted for 3 ( middle ) or 6 h( right ), were double-stained with antibodies to ankyrin(red, A - C ) and -Na-K-ATPase (green, D - F ). G - I : mergedimages. Results are representative of 3 experiments. Bar = 10 µm.
9 S( \5 Y$ i) z& h) T5 @
ATP depletion results in a duration-dependent loss of polarizeddistribution of Na-K-ATPase and ankyrin. The well-established ATP depletion model was used to study the behaviorof ankyrin and Na-K-ATPase after ischemic injury in MDCK cells.Within 3 h of ATP depletion, there was a partial loss ofbasolateral membrane staining of AnkG190 (Fig. 2 B ) and -Na-K-ATPase (Fig. 2 E ), with both proteins beginningto assume a punctate cytoplasmic distribution characteristic ofinternalized Na-K-ATPase. By 6 h of ATP depletion, the cytoplasmicmislocation of both AnkG190 (Fig. 2 C ) and -Na-K-ATPase(Fig. 2 F ) was complete, and no staining of either proteinwas visible at the basolateral membrane. Another uniform observationwas that the abundance of immunoreactive ankyrin was increased after6 h of ATP depletion (Fig. 2, C vs. A ). This is consistent with our previous findings of enhanced ankyrin expression in a rat model of renal ischemia ( 34 ).

Ankyrin remains bound to Na-K-ATPase after ATP depletion. Because a tethering interaction with ankyrin is crucial to the normalbasolateral distribution of Na-K-ATPase, it was of interest todetermine the behavior of ankyrin in cells with mislocated Na-K-ATPaseafter ATP depletion. Double-immunostaining revealed that AnkG190remained partially colocalized with -Na-K-ATPase at all time periodsafter ATP depletion (Fig. 2, H and I ).8 T5 T4 Q3 {+ |; U
( m0 t4 c$ z% H, H
The finding that ankyrin continues to interact with Na-K-ATPase afterATP depletion was confirmed using two additional complementary assays.First, analysis of the polyclonal AnkG190 antibody immunoprecipitates demonstrated that control cells contained functional complexes ofAnkG190, -Na-K-ATPase, and II-spectrin (Fig. 3 A ). Conversely, in cellsdepleted of ATP, -Na-K-ATPase continued to immunoprecipitate withankyrin, whereas spectrin was lost from the complex. The loss ofspectrin from the complex was duration dependent (Fig. 3 A )and could not be attributed to its degradation, because the 240-kDaimmunoreactive spectrin band remained intact in whole cell lysates atall time periods examined (Fig. 3 B ). In a separate set ofexperiments, with cells grown on Transwell filters to optimize thegeneration and maintenance of cell polarity, identical results wereobtained (not shown). In another set of experiments, the cells wereextracted with a low-salt buffer to isolate a purely cytosolicfraction. Ankyrin in this cytosolic fraction continued to associatewith -Na-K-ATPase after 6 h of ATP depletion but not withspectrin (not shown).! d) w( Q- s* V* d1 E/ }( |

Fig. 3. Ankyrin remains bound to Na-K-ATPase after ATPdepletion-immunoprecipitation assays. A : MDCK cells,untreated (Con) or ATP depleted for various periods as shown, wereanalyzed for complexes with ankyrin. The negative control was beadsalone (Beads). Equal aliquots of cell lysates were probed withanti-tubulin antibody before precipitation to verify equal loading ofsamples. B : Western blot of cell lysates at various timeperiods as indicated, with anti-spectrin antibody. Left :molecular mass (in kDa). Results are representative of 3 experiments.

In the second assay, the in vitro interaction of ankyrin fromATP-depleted cell lysates with a GST fusion protein containing theminimal ankyrin binding domain of -Na-K-ATPase (GST-MAB) wasevaluated. In control cells, this peptide bound strongly to anankyrin-spectrin complex (Fig. 4 ). AfterATP depletion, GST-MAB continued to avidly bind ankyrin, but spectrinwas progressively lost from the complex as a function of the durationof ATP depletion (Fig. 4 ). The negative control, GST alone, did notinteract with ankyrin or spectrin.
  M3 c2 B9 k" [& g0 {7 f8 \: Y
Fig. 4. Ankyrin remains bound to Na-K-ATPase after ATPdepletion-solution binding assays. Top : Coomassieblue-stained gel of the recombinant peptides glutathione S -transferase (GST) alone [negative control (Con)] andGST-minimal ankyrin binding (MAB) construct. Middle and bottom : Western blots with antibodies to ankyrin andspectrin, respectively. Left: molecular mass markers (in kDa). Resultsare representative of 2 experiments.
$ a. K% D2 m, ]" `4 a
An additional observation gleaned from Figs. 3 and 4 pertains to theenhanced ankyrin expression after ATP depletion. A significant increasein the abundance of immunoreactive ankyrin was noted after 6-12 hof ATP depletion, consistent with the enhanced ankyrin immunofluorescence observed in Fig. 2 and with our previous in vivoobservations ( 34 ). Densitometric analysis of multipleblots revealed that ankyrin protein abundance increased by 2.5- ± (SD) 0.5-fold at 6 and 12 h of ATP depletion.9 w; D5 B; \& N+ D( \8 Y) b
! W" e6 g" s$ Q# @( f6 q1 n
Ankyrin undergoes phosphorylation after ATP depletion. Because membrane-cytoskeletal interactions in general, and theinteractions between spectrin and ankyrin in particular, are oftenregulated by phosphorylation ( 7, 10, 22 ), it was ofinterest to determine whether the phosphorylation status of ankyrin changed after ATP depletion. This was measured byevaluating the amount of ankyrin that was present inanti-phosphotyrosine or anti-phosphoserine/threonine precipitates.Surprisingly, despite the diminished ATP content of these cells,increases in both tyrosine and serine/threonine phosphorylation onankyrin were detected, as shown in Fig. 5. Enhanced phosphorylation was detectedas early as 3 h after the start of ATP depletion and persistedthrough 6 h of ATP depletion. These changes correlatedwith the rate of spectrin loss from the ankyrin-Na-K-ATPase complex.
. M' P; P8 K. V
Fig. 5. Ankyrin is phosphorylated after ATP depletion. Westernblot with ankyrin antibody of MDCK cell lysates after various periodsof ATP depletion and immunoprecipitation with anti-phosphotyrosine oranti-phosphoserine/threonine. Equal aliquots of cell lysates wereprobed with anti-tubulin antibody before precipitation to verify equalloading of samples.

DISCUSSION

We have developed a polyclonal antibody directed to the uniqueNH 2 -terminal sequences of AnkG190 and demonstrated thatAnkG190 interacts with -Na-K-ATPase at the lateral domain ofpolarized MDCK cells. On ATP depletion, there is a duration-dependentloss of Na-K-ATPase and ankyrin polarity. Using a series ofcomplementary assays, we have demonstrated that ankyrin remains boundto Na-K-ATPase after ATP depletion but that the interaction betweenankyrin and spectrin is lost. We suggest that a dissociation ofthe membrane-cytoskeleton complex at the spectrin-ankyrin interface maycontribute to the loss of Na-K-ATPase polarity and contribute to itsinternalization after ATP depletion.' E6 C2 b& o/ G+ m/ b+ V1 n

Ischemic renal injury remains a leading cause of acute renalfailure, and despite important technical advances in treatment theassociated mortality and morbidity remain dismally high ( 28, 33, 36 ). An improved understanding of the pathophysiology from acellular and molecular standpoint may facilitate the development ofnovel therapeutic interventions. Although the pathogenesis of acuterenal failure is clearly multifactorial, persistent afferent arteriolarvasoconstriction is considered an important factor and has beenpostulated to occur secondarily to the loss of proximal tubule cellpolarity ( 36 ). Previous studies have established thatischemic injury modeled by ATP depletion results in adisruption of polarized basolateral membrane distribution ofNa-K-ATPase, ankyrin, and spectrin in cultured proximal tubule cells( 24-27 ), leading to impaired sodium reabsorption( 24, 35 ). Loss of Na-K-ATPase polarity and increaseddelivery of filtered sodium to the macula densa have also beendocumented in human cadaveric kidneys afterischemia-reperfusion injury ( 1, 21 ). Itis reasonable to hypothesize that the ensuing activation oftubuloglomerular feedback contributes to the persistentvasoconstriction characteristic of ischemic acute renalfailure. However, the molecular basis for the loss of Na-K-ATPasepolarity after ischemic renal injury remains incompletely understood.
+ b! V) @& O# c3 z; p
Several lines of evidence gleaned from the present study suggest that adissociation of the membrane-cytoskeleton complex at thespectrin-ankyrin interface may be responsible for the loss ofNa-K-ATPase polarity after ATP depletion. First, the internalized andmislocalized -Na-K-ATPase remains substantially colocalized withankyrin. Second, ankyrin remains complexed with -Na-K-ATPase inimmunoprecipitates, whereas spectrin is lost from the complex inproportion to the duration of ATP depletion. Third, the isolated ankyrin binding domain of Na-K-ATPase continues to interact in vitrowith ankyrin after ATP depletion whereas spectrin does not. Fourth, II spectrin remains intact and does not undergo degradation afterATP depletion. Collectively, these results support earlier studiesdemonstrating the lack of association between ankyrin and spectrinafter ATP depletion in LLC-PK 1 cells ( 26 ) anddemonstrate the persistence of a crucial ankyrin-Na-K-ATPase linkage.Others have similarly shown that in large measure, both spectrin and ankyrin remain intact after ischemic injury to MDCK cells( 14 )." \6 I+ F7 n6 R, S% c) x( ?
8 s1 Q; N  C* }8 a  R6 E/ `! b* g7 E
The phosphorylation state of ankyrin is an important determinant of itsinteraction with spectrin ( 7, 16-19, 22, 23 ). Unphosphorylated ankyrin preferentially binds to the fully functional tetrameric spectrin unit rather than to dimers with a 10-fold greateraffinity; this preferential binding is abolished by phosphorylation. Although the sites and physiological significance of phosphorylation remain unclear, an attractive mechanism by which ATP depletion mightinfluence spectrin-ankyrin interactions would be by altering thephosphorylation status of ankyrin. Our findings indicate that ATPdepletion results paradoxically in a marked increase in both tyrosineand serine/threonine phosphorylation of ankyrin, suggesting a possiblebiochemical mechanism underlying the loss of association betweenspectrin and ankyrin. While the role of phosphorylation in ankyrin isunknown, it has been shown that hyperphosphorylation of ankyrin onserine/threonine in avian red blood cells by casein kinase IIsuppresses its ability to bind spectrin ( 16, 18 ). Caseinkinase II is also known to phosphorylate spectrin ( 39 ). Normally, ankyrin and spectrin undergo a futile cycle of rapid phosphorylation and dephosphorylation, reflecting dynamic control ofmembrane and cytoskeletal organization. During a period of ATPdepletion, this cycle is presumably interrupted or unbalanced. Themechanisms that account for changes in kinase/phosphatase activityunder these conditions remain unexplored. Another factor that mayinfluence the spectrin-ankyrin interaction after ATP depletion includeschanges in the oligomeric status of spectrin per se. In erythrocytes,the affinity of spectrin for ankyrin is cooperatively linked to itsoligomeric state, with ankyrin favoring spectrin oligomers andtetramers over spectrin dimers ( 7, 19 ). Becausespectrin's oligomerization state can be influenced by a variety offactors including phosphorylation, calcium, calmodulin, and proteolysis( 20, 23 ), these factors may also contribute, underconditions of renal tubule cell injury, to the deconstruction of thecortical spectrin-ankyrin skeleton.+ J, G; {3 z6 e" j8 f- x  B
, k: M  e* x/ b, X  \
Finally, an additional observation offered by this study is the findingthat immunodetectable ankyrin is upregulated after ATP depletion inMDCK cells, consistent with our previous findings in a rat model ofrenal ischemia ( 34 ). Although the molecular mechanisms and significance of this change are unclear, it is intriguing to speculate that the overexpressed ankyrin, via its continued ability to interact with Na-K-ATPase, may play a role in therestoration of cell polarity during recovery from ischemia, lending emerging evidence for the notion that ankyrin-spectrin bindingis a necessary cofactor for the appropriate membrane delivery andsorting of a subset of proteins that includes Na-K-ATPase ( 13 ), anion exchanger ( 17 ), CD45( 32 ), and others ( 5 ).. |3 B! W3 P) A+ s% f
6 \3 L! |: j. E+ y. b8 D: \, j; Y
In summary, the present study suggests that a dissociation of themembrane-cytoskeleton complex at the spectrin-ankyrin interface maycontribute to the loss of Na-K-ATPase surface display and polarityafter ATP depletion. It will be important in future studies to examinethe phosphorylation status of ankyrin after ischemic injury inanimal models to further elucidate the possible mechanisms underlyingthe selective loss of spectrin binding and preferential retention ofthe interaction between ankyrin and Na-K-ATPase.4 y+ R3 N7 Z7 \

ACKNOWLEDGEMENTS8 T/ @: X) Q+ i; Y! O
# }! M) |4 x6 }7 O2 R; |' I, ]/ L
This work was supported by National Institute of Diabetes andDigestive and Kidney Diseases Grants DK-53289 (to P. Devarajan), DK-07110 (to R. Woroniecki), and DK-43812 (to J. S. Morrow).. [, g: f( e; a
      【参考文献】- c8 H8 T; S9 V2 w. ~1 \
1. Alejandro, VSJ,Nelson WJ,Huie P,Sibley RK,Dafoe D,Kuo P,Scandling JD, Jr,andMyers BD. Postischemic injury, delayed function and Na,K-ATPase distribution in the transplanted kidney. Kidney Int 48:1308-1315,1995  .; x7 F. `- x7 m9 v2 a
. l- G3 r7 e4 Q. y

0 p  j6 i( e% A7 E
2. Bacallao, R,Garfinkel A,Monke S,Zampighi G,andMandel LJ. ATP depletion: a novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton. J Cell Sci 107:3301-3313,1994 .

3. Beck, KA,andNelson WJ. The spectrin-based membrane skeleton as a membrane protein-sorting machine. Am J Physiol Cell Physiol 270:C1263-C1270,1996 ., x( E2 f3 w- p9 c

6 W. S  V1 q( `! P
4. Bennett, V,andBaines AJ. Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol Rev 81:1353-1392,2001 .: d  s" I8 a  Z; \
! ]0 ^$ h5 x$ r: Q) h$ f5 D

3 _6 t0 o) @3 Z" {0 L6 s
5. Bennett, V,andChen L. Ankyrins and cellular targeting of diverse membrane proteins to physiological sites. Curr Opin Cell Biol 13:61-67,2001  .
+ _# Y( \  o  r5 t2 F& E0 C

6. Bush, KT,George SK,Zhang PL,andNigam SK. Pretreatment with inducers of ER molecular chaperones protects epithelial cells subjected to ATP depletion. Am J Physiol Renal Physiol 277:F211-F218,1999 .0 K; k7 n8 a  q' r
' L7 _2 t/ p( i7 `; B5 ]3 u: x

7. Cianci, CD,Giorgi M,andMorrow JS. Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3. J Cell Biochem 37:301-315,1988  .* s; V& U- r- z' y4 X9 T2 B
5 Z& i- W2 n1 x/ l, U

8. De Matteis, MA,andMorrow JS. The role of ankyrin and spectrin in membrane transport and domain formation. Curr Opin Cell Biol 10:542-549,1998  .

; c0 O1 N0 H7 ?$ {
9. De Matteis, MA,andMorrow JS. Spectrin tethers and mesh in the biosynthetic pathway. J Cell Sci 113:2331-2343,2000 .- n  n2 c3 v: i! B' z. }. N

* P* W% r( o- ]) E; j" C/ j

10. Devarajan, P,andMorrow JS. The spectrin cytoskeleton and organization of polarized epithelial cell membranes. Curr Top Membr Transp 43:97-128,1996.
3 ~5 I( a. H# F. S4 ^. ^
0 |, {; X( F9 T2 b! F: R

11. Devarajan, P,Scaramuzzino DA,andMorrow JS. Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase subunit. Proc Natl Acad Sci USA 91:2965-2969,1994 .2 e8 d2 m% _* ^# O0 [
3 l+ C* }( p. Z
  z3 L0 S& j% b6 `/ R

12. Devarajan, P,Stabach PR,Mann AS,Ardito T,Kashgarian M,andMorrow JS. Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds I spectrin and associates with the Golgi apparatus. J Cell Biol 133:819-830,1996 .5 b  ]$ z6 j3 a

4 v& d* C; g: H/ ]( F. v: b

13. Devarajan, P,Stabach PR,De Matteis MA,andMorrow JS. Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in MDCK cells. Proc Natl Acad Sci USA 94:10711-10716,1997 .

' B7 L9 H" v7 [$ r. z6 b

14. Doctor, RB,Bennett V,andMandel LJ. Degradation of spectrin and ankyrin in the ischemic rat kidney. Am J Physiol Cell Physiol 264:C1003-C1013,1993 .
" _9 j: s/ e' H) y3 _6 ?& C' j

15. Feldenberg, LR,Thevananther S,Del Rio M,De Leon M,andDevarajan P. Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells. Am J Physiol Renal Physiol 276:F837-F846,1999 .
( i; q, v% o0 i4 F% q

+ l- D' d% F, n/ o" E9 l& }, x
16. Ghosh, S,andCox JV. Dynamics of ankyrin-containing complexes in chicken embryonic erythroid cells: role of phosphorylation. Mol Biol Cell 12:3864-3874,2001 .
! M3 }; Q: ]2 B( C7 h9 u

17. Ghosh, S,Cox KH,andCox JV. Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during recycling to the Golgi. Mol Biol Cell 10:455-469,1999 .
* L6 ~' E4 C! G$ T. V. l

8 C  l* Z& D; v6 q6 F
18. Ghosh, S,Dorsey FC,andCox JV. CK2 constitutively associates with and phosphorylates chicken erythroid ankyrin and regulates its ability to bind to spectrin. J Cell Sci 115:4107-4115,2002 .

19. Giorgi, M,Cianci CD,Gallagher PG,andMorrow JS. Spectrin oligomerization is cooperatively coupled to membrane assembly: a linkage targeted by many hereditary hemolytic anemias? Exp Mol Pathol 70:215-230,2001  .
& H; L. N5 D" J' m8 n/ E9 w% m0 J

% Z9 X  H2 C( w% Q: J1 _4 J2 W' y
20. Harris, AS,andMorrow JS. Calmodulin and calcium-dependent protease 1 coordinately regulate the interaction of fodrin with actin. Proc Natl Acad Sci USA 87:3009-3013,1990 .! u. |- I2 y  l3 a0 C

7 W6 c+ _* M6 ]

21. Kwon, O,Corrigan G,Myers BD,Sibley R,Scandling JD,Dafoe D,Alfrey E,andNelson WJ. Sodium reabsorption and distribution of Na,K-ATPase during postischemic injury to the renal allograft. Kidney Int 55:973-975,1999.) T% d  U5 m+ f9 P4 e8 Q5 ^

6 S/ O& ^8 `% s
- U* d3 H' k0 B  S
22. Lu, PW,Soong CJ,andTao M. Phosphorylation of ankyrin decreases its affinity for spectrin tetramer. J Biol Chem 260:14958-14964,1985 .
5 W; E+ H) W& I+ a
# P$ @& x1 I$ f2 m0 \2 A  T6 T

23. Manno, S,Takakuwa Y,Nagao K,andMohandas N. Modulation of erythrocyte membrane mechanical function by beta-spectrin phosphorylation and dephosphorylation. J Biol Chem 270:5659-5665,1995 .

. i7 n. M+ f: Y5 v- v

24. Molitoris, BA. Na,K-ATPase that redistributes to the apical membrane during ATP depletion remains functional. Am J Physiol Renal Fluid Electrolyte Physiol 265:F693-F697,1993 ., i$ U0 a4 \- e

1 X( O' Y& c/ k  ^8 x5 f( a% y. L
' T' ^, W! t; Z, J$ s
25. Molitoris, BA,Dahl R,andGeerdes A. Cytoskeletal disruption and apical redistribution of proximal tubule Na,K-ATPase during ischemia. Am J Physiol Renal Fluid Electrolyte Physiol 263:F488-F495,1992 .% |- `+ i% O4 q, f6 P/ L
% V' C: u4 m# c

2 s9 k7 H; k; E  T* F
26. Molitoris, BA,Dahl R,andHosford M. Cellular ATP depletion induces disruption of the spectrin cytoskeletal network. Am J Physiol Renal Fluid Electrolyte Physiol 271:F790-F798,1996 .

27. Molitoris, BA,Geerdes A,andMcIntosh JR. Dissociation and redistribution of Na,K-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion. J Clin Invest 88:462-469,1991  ./ k! z3 a: g# c* i, L" o* M. u4 i
* y6 l4 G5 T/ a& J3 j3 y( ^3 M
) X+ q1 }0 k. z/ f( C( A1 \. C. N9 @
) e# r- _/ S/ [, O; y4 T/ N/ x
28. Molitoris, BA,Weinberg JM,Venkatachalam MA,Zager RA,Nath KA,andGoligorsky MS. Acute renal failure. II. Experimental models of acute renal failure: imperfect but indispensable. Am J Physiol Renal Physiol 278:F1-F12,2000 .
& U9 U7 z: `+ @
( X6 N# I7 z3 ^2 g' D

29. Morrow, JS,Cianci C,Ardito T,Mann A,andKashgarian MT. Ankyrin links fodrin to alpha Na/K ATPase in Madin-Darby canine kidney cells and in renal tubule cells. J Cell Biol 108:455-465,1989 .4 o7 }4 i& B* S. K' l4 x
& p- v1 [) ?" B8 Q7 `% {
8 m' ]* c6 t8 W5 x5 y6 A
/ U% F+ ]+ _; Y) e: n
30. Peters, LL,John KM,Lu FM,Eicher EM,Higgins A,Tialamas M,Turtzo LC,Otsuka AJ,andLux SE. Ank3 (epithelial ankyrin), a widely distributed new member of the ankyrin gene family and the major ankyrin in kidney, is expressed in alternatively spliced forms, including forms that lack the repeat domain. J Cell Biol 130:313-330,1995 .
, q6 Y* ?% h/ j

31. Piepenhagen, PA,andNelson WJ. Biogenesis of polarized epithelial cells during kidney development in situ: roles of E-cadherin-mediated cell-cell adhesion and membrane cytoskeleton organization. Mol Biol Cell 11:3161-3177,1998.8 {/ @3 Y3 Z8 B$ v% @
8 @' `1 H* }* L" o/ ?) a- |
! f& G: Y7 u% [- Z8 t" a
4 q* S$ M; ?# @5 b. _
32. Pradhan, D,andMorrow JS. The spectrin-ankyrin skeleton controls CD45 surface display and interleukin-2 production. Immunity 17:303-315,2002  .% {5 E: _4 E% Z
$ `3 W4 `* }( F$ p, X6 O

33. Sheridan, AM,andBonventre JV. Cell biology and molecular mechanisms of injury in ischemic acute renal failure. Curr Opin Nephrol Hypertens 9:427-434,2000  .0 \) J1 a! L% _" A

34. Siegel, NJ,Van Why S,andDevarajan P. Renal cell injury: metabolic and structural alterations. Pediatr Res 36:129-136,1994  .7 H- s+ I: g+ J3 L* ]8 T
& B' b3 h: h6 p' K) @' J) r* [  S
- y  A1 v9 t  ^# [. i! I$ k
9 [; J2 D7 B4 @$ A+ S$ `1 G! |
35. Spiegel, DM,Wilson PD,andMolitoris BA. Epithelial polarity following ischemia: a requirement for normal cell function. Am J Physiol Renal Fluid Electrolyte Physiol 256:F430-F436,1989 .0 J0 u) R7 t4 K" f( p  m

. Z% s2 _4 i# y- R, \

36. Sutton, TA,andMolitoris BA. Mechanisms of cellular injury in ischemic acute renal failure. Semin Nephrol 18:490-497,1998  .( \) K; @& f' \3 i9 |9 R5 {
; Q) g. ^1 P$ U; z2 k+ @: `3 {
( v+ x: ^' k( ?) h1 N2 u

37. Thevananther, S,Kolli AH,andDevarajan P. Identification of a novel ankyrin isoform (AnkG190) in kidney and lung that associates with the plasma membrane and binds -Na,K-ATPase. J Biol Chem 273:23952-23958,1998 ." V- A- F$ |* n
/ R/ t* ^6 G4 ?0 v. c
+ k) o$ @6 {/ U: Q  q/ {: I, x3 m
. _# Q  C7 c: `
38. Tsumakoto, T,andNigam SK. Tight-junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly. J Biol Chem 272:16133-16139,1997 .

39. Wei, T,andTao M. Human erythrocyte casein kinase II: characterization and phosphorylation of membrane cytoskeletal proteins. Arch Biochem Biophys 307:206-216,1993  ." B7 c+ M. T6 s* w# _8 l8 V8 h( y

  I% S6 m) m1 {+ e0 ?, ]
40. Zhang, Z,Devarajan P,Dorfman AL,andMorrow JS. Structure of the ankyrin-binding domain of -Na,K-ATPase. J Biol Chem 273:18681-18684,1998 .

收藏0 分享0 顶0 踩0