Subunit in Human Skeletal Muscle Cells - The Journal of Biological ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 24, Issue of June 11, pp. 25211–25218, 2004 Printed in U.S.A.

ERK1/2 Mediates Insulin Stimulation of Na,K-ATPase by Phosphorylation of the ␣-Subunit in Human Skeletal Muscle Cells* Received for publication, February 26, 2004, and in revised form, April 2, 2004 Published, JBC Papers in Press, April 6, 2004, DOI 10.1074/jbc.M402152200

Lubna Al-Khalili‡§, Olga Kotova‡§, Hiroki Tsuchida‡, Ingrid Ehre´n¶, Eric Fe´raille储, Anna Krook‡**, and Alexander V. Chibalin‡ ‡‡ From the ‡Section of Integrative Physiology, Department of Surgical Sciences, the ¶Section of Urology, Department of Surgical Sciences, and the **Department of Physiology and Pharmacology, Karolinska Institutet, SE-171 77 Stockholm, Sweden and the 储Division de Ne´phrologie, Hoˆpital Cantonal Universitaire, Gene`ve CH-1211, Switzerland

Insulin stimulates Naⴙ,Kⴙ-ATPase activity and induces translocation of Naⴙ,Kⴙ-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Naⴙ,Kⴙ-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Naⴙ,KⴙATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Naⴙ,Kⴙ-ATPase ␣-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive 86Rbⴙ uptake and [3H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Naⴙ,KⴙATPase ␣1- and ␣2-subunits. Insulin-stimulated Naⴙ,KⴙATPase activation, phosphorylation, and translocation of ␣-subunits to the plasma membrane were abolished by 20 ␮M PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nM wortmannin) and protein kinase C (10 ␮M GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of ␣1and ␣2-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate ␣-subunit of purified human Naⴙ,Kⴙ-ATPase in vitro. In conclusion, insulin stimulates Naⴙ,Kⴙ-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the ␣-subunits by ERK1/2 mitogenactivated protein kinase. Na⫹,K⫹-ATPase is a plasma membrane cation pump that is essential for maintenance of intracellular and extracellular sodium and potassium concentrations, cell volume, osmotic balance, and electrochemical gradients (1, 2). The regulation of Na,K-ATPase can be achieved by multiple mechanisms, including changes in intrinsic activity, subcellular distribution, and cellular abundance (3–5). The catalytic subunit of the Na⫹,K⫹ATPase is a substrate for protein kinases (2, 3, 5), and the * This work was supported by grants from the Swedish Research Council, the Swedish Heart and Lung Foundation, the Novo-Nordisk Foundation, and the Swedish Society of Medicine and Creative Peptides Sweden AB. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors have contributed equally to this work. ‡‡ To whom correspondence should be addressed: Dept. of Surgical Sciences, Section of Integrative Physiology, Karolinska Institutet, von Eulers va¨g 4, 4 tr, SE-171 77 Stockholm, Sweden. Tel.: 46-8-524-87584; Fax: 46-8-335-436; E-mail: [email protected]. This paper is available on line at http://www.jbc.org

pump phosphorylation is an important molecular mechanism for the short term control of its activity in response to hormonal stimulation (3). Skeletal muscle contains one of the largest pools of Na⫹,K⫹-ATPase in the body (6), expressing ␣- (␣1 and ␣2) and ␤- (␤1 and ␤2) subunits (4, 7). The ␣1-subunit isoform is mainly located in the sarcolemma, whereas the ␣2-subunit isoform is found both in the sarcolemma and diffusely distributed in the muscle fibers while preferentially located along the t-tubules (4, 8). Insulin plays a major role in mediating muscle K⫹-uptake to control the plasma K⫹ concentration (9) via regulation of Na⫹,K⫹-ATPase activity (4). Physiologically diverse roles for the ␣1- and ␣2-subunits have been highlighted using animal models whereby the isoform expression in skeletal muscle has been genetically altered (10). In skeletal muscle membrane fractions isolated by differential centrifugation, insulin increases ␣2-subunit abundance in the plasma membrane fraction, with no change in ␣1-distribution (11). Based on this evidence, the ␣-subunits were hypothesized to serve specific functions: the ␣1-subunit isoform was proposed to have a “housekeeping” function in maintaining basic ion transport, and the ␣2-isoform was thought to be hormonally regulated. However, using alternative techniques for monitoring protein trafficking, the ␣1-subunit has been shown to undergo a similar hormone-sensitive translocation to the plasma membrane in various tissues. Insulin promotes translocation of an exofacially epitope-tagged rat Na⫹,K⫹-ATPase ␣1-subunit to the plasma membrane in HEK-293 cells (12). In addition both cAMP and aldosterone induce translocation of ␣1-isoform in rat cortical collecting duct cells (13). Consistent with these findings, we revealed that insulin induces not only Na⫹,K⫹ATPase ␣2- but also ␣1-subunit translocation to the cell surface in skeletal muscle (14). Thus, a common mechanism of insulin regulation of Na⫹,K⫹-ATPase distribution, irrespective of structural difference between the ␣1- and ␣2-subunits, should exist. The mechanisms for regulation of Na⫹,K⫹-ATPase activity are believed to be similar between humans and rodents, however species differences (5, 15) in the structure of Na⫹,K⫹ATPase must be considered, especially in regard to the role of PKC.1 Historically, rodent Na⫹,K⫹-ATPase has been studied, and results have been extrapolated to humans. PKC has been heavily implicated in insulin-induced stimulation of Na⫹,K⫹-

1 The abbreviations used are: PKC, protein kinase C; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; HSMC, human skeletal muscle cell; MEK, ERK kinase; PI, phosphatidylinositol; MAP, mitogen-activated protein; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; NHE1, Na⫹/H⫹-exchanger 1.

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Human Na,K-ATPase Regulation by ERK1/2 Phosphorylation

ATPase (14, 16, 17). PKC-mediated phosphorylation of Ser23, plays an important role in regulation of Na⫹,K⫹-ATPase activity and membrane trafficking (18 –20). However, the human ␣-subunit lacks Ser23, highlighting important species differences in the regulation of the pump. In vitro phosphorylation of glutathione S-transferase fusion proteins containing N-terminal of Na⫹,K⫹-ATPase ␣-subunit indicate that rat ␣2 and human ␣1 are poor substrates for PKC (15). Nevertheless, insulin stimulates ␣-subunit phosphorylation on serine, threonine, and tyrosine residues (16, 21). These observations implicate a role for unidentified serine/threonine and tyrosine kinases in insulin activation of Na⫹,K⫹-ATPase in humans. We hypothesized that one of these unidentified kinases may be ERK1/2 MAP kinase. Several recent reports implicate MAP kinase in the regulation of Na⫹,K⫹-ATPase. Activation of the ERK1/2 signaling pathway leads to an increase in synthesis of Na⫹,K⫹-ATPase subunits (22, 23) and short term stimulation of Na⫹,K⫹-ATPase activity (23). Because ERK1/2 is activated in insulin-sensitive tissues, including skeletal muscle (24), we explored whether ERK1/2 is involved in the regulation of Na⫹,K⫹-ATPase in response to insulin in human skeletal muscle cells. Moreover, we determined whether ␣1- and ␣2-subunits are direct targets for ERK1/2 MAP kinase. MATERIALS AND METHODS

Antibodies and Reagents—Specific anti-␣1-subunit monoclonal (25) and anti-␣2-subunit monoclonal (26) antibodies were obtained from Drs. M. Caplan (Yale University, New Haven, CT) and K. Sweadner (Massachusetts Central Hospital, Boston, MA). Immunoprecipitation of the total Na⫹,K⫹-ATPase ␣-subunit was performed using the polyclonal antibody, anti-NK1, raised against purified rat kidney holoenzyme (16). The antibodies against a phospho-Thr-Pro motif and the anti-phospho-ERK1/2 (P-Thr202/Tyr204) were from Cell Signaling (Beverly, MA). Recombinant ERK1 and ERK2 kinases, recombinant PKC␨, and MEK1/2 inhibitor U0126 were from Upstate Cell Signaling Solutions (Charlottesville, VA). Kinase inhibitors PD98059, GF109203X, wortmannin, and purified rat brain PKC were from Calbiochem. Streptavidin-agarose beads and EZlink Sulfo-NHS-SS-biotin were from Pierce. Cell culture media and reagents were obtained from Invitrogen. Human insulin (Actrapid) was from Novo Nordisk AS (Copenhagen, Denmark). Me2SO (Calbiochem) was used as a solvent for the protein kinase and phosphatase inhibitors. All other reagents were of analytical grade (Sigma). In Silico Screen of Possible Phosphorylation Sites in Na⫹,K⫹-ATPase ␣-Subunit—The protein sequences of ␣1- and ␣2-subunits of Na⫹,K⫹ATPase of human, rat, mouse, and chicken origin were analyzed by motif-based profile scanning programs Scansite 2.0 (27, 28) (available at scansite.mit.edu) and PhosphoBase2.0 (29, 30) (www.cbs.dtu.dk/ databases/PhosphoBase). The statistical stringency criteria for predicted phosphorylation sites were set at high, medium, and low level according to the program’s user recommendations. Subject Characteristics—Skeletal muscle biopsies (rectus abdominus) were obtained with the informed consent of the donors during scheduled abdominal surgery. Subjects (3 male and 3 female) had no known metabolic disorders. Mean age was 54.5 ⫾ 6.5 years (body mass index of 26 ⫾ 1.5 kg ⫻ m⫺2 and fasting blood glucose of 5.2 ⫾ 0.3 mM). The Ethical Committee at the Karolinska Institute approved the study protocols. Cell Culture—Human skeletal muscle satellite cells were isolated from muscle biopsies and cultured as previously described (14). The experiments were performed on passages 3– 4. To initiate differentiation into myotubes, Hams/F-10 media with 20% FBS was removed from cells, and DMEM containing 1% PeSt (100 units/ml penicillin, 100 mg/ml streptomycin (Invitrogen)) and 4% FBS was added for 48 h. Medium was again changed to DMEM containing 1% PeSt and 2% FBS. Fusion and multinucleation of the cells was observed at day 3 after initiation of the differentiation protocol. Myoblasts were grown on 100-mm Petri dishes. At days 0 –14 after differentiation, myotubes were serum-starved 16 h before use to reduce the basal level of insulin- and cytokine-dependent kinase activity. Human renal tubular cells were cultured from the unaffected outer cortex of renal tissue obtained from nondiabetic patients undergoing elective nephrectomy for renal cell carcinoma, as previously described (31). Cells from the second and third passages were used for purification of Na⫹,K⫹-ATPase. Cell Incubation—For the Na⫹,K⫹-ATPase activity assay, 6-day-differentiated myotubes were preincubated for 30 min either with 0.2%

Me2SO or 20 ␮M PD98059 (MEK1 inhibitor), 1 ␮M or 10 ␮M GF109203X (PKC inhibitor), 100 nM wortmannin (PI 3-kinase inhibitor). After preexposure to Me2SO/inhibitors, cells were stimulated with insulin (100 nM) for 20 min. After treatment cells were washed twice with ice-cold PBS, and harvested by scraping cells into ice-cold lysis buffer A (20 mM Tris, pH 8.0, 135 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 10 mM Na4P2O7, 0.5 mM Na3VO4, 10 mM NaF, 1 ␮M okadaic acid, 1% Triton X-100, 10% v/v glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin). Cells were lysed by repeated pipetting, and lysates were agitated for 60 min at 4 °C and subjected to centrifugation (12,000 ⫻ g for 10 min at 4 °C). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). Lysates were kept at ⫺80 °C before subsequent Western blot analysis or immunoprecipitation with appropriate antibodies. Measurement of Ouabain-sensitive 86Rb⫹ Uptake—Na⫹,K⫹-ATPase transport activity was measured as ouabain-sensitive uptake of 86Rb⫹, under conditions of initial rate, as previously described (13). Myotubes (day 6) were grown on 6-well dishes (Costar, Cambridge, MA) and were preincubated in serum-free DMEM without or with ouabain (0.2 mM) and kinase inhibitors for 30 min at 37 °C. Thereafter, myotubes were incubated in the presence or absence of insulin (20 min) and/or inhibitors. Na⫹,K⫹-ATPase transport activity was determined after the addition of 50 ␮l of medium containing tracer amounts of 86RbCl (100 nCi/sample, Amersham Biosciences) for 5 min. Incubation was stopped by cooling on ice, and dishes were washed three times with an ice-cold washing solution containing 150 mM choline chloride, 1.2 mM MgSO4, 1.2 mM CaCl2, 2 mM BaCl2, and 5 mM HEPES, pH 7.4. Cells were lysed in 750 ␮l of lysis buffer A, and the radioactivity was measured by liquid scintillation. Protein content was determined in parallel using the BCA assay (Pierce). Ouabain-sensitive 86Rb⫹ uptake was calculated as the difference between the mean values measured in triplicate samples incubated without or with 0.2 mM ouabain. Data are expressed as percentage of control. Basal ouabain-sensitive 86Rb⫹ uptake was 3.1 ⫾ 0.3 pmol of Rb per microgram of protein per minute. [3H]Ouabain Binding—Measurement of ouabain binding sites on cell surface of differentiated myotubes was performed with [3H]ouabain, as previously described (32). Myotubes, grown on 6-well dishes (Costar, Cambridge, MA) were preincubated in serum-free DMEM in the presence or absence of insulin (20 min) and/or inhibitors. The myotubes were washed and incubated with [3H]ouabain-binding buffer (OBB, 20 mM HEPES, pH 7.4, 0.25 ␮M [3H]ouabain (specific activity: 16.5 Ci/ mmol, Amersham Biosciences), 120 mM NaCl, 5 mM KCl, 0.05 mM CaCl2, 1 mM MgCl2, 4 mM NaH2PO4, and 5 mM glucose). After 15 min of incubation in [3H]ouabain, plates were washed with [3H]ouabain-free OBB, cells were lysed in 750 ␮l of lysis buffer A, and the radioactivity was measured by liquid scintillation. Protein content was determined in parallel using the BCA assay (Pierce). Unlabeled ouabain (1 mM) was used as background control for nonspecific binding. Specific binding was calculated by subtracting the background control from [3H]ouabain. Data are expressed as percentage of control. Cell Surface Biotinylation—Myotubes (6-day) were preincubated in PBS in the absence or presence of insulin (20 min) and/or inhibitors and thereafter were exposed to EZ-link Sulfo-NHS-SS-biotin (Pierce) at a final concentration of 1.5 mg/ml in PBS at 4 °C for 60 min with gentle shaking. Cell surface biotinylation was performed as described (14). After biotinylation, cells were harvested and lysed in ice-cold buffer A as described above, and cell lysates were subjected to streptavidin precipitation. After streptavidin precipitation, samples were analyzed by SDS-PAGE with subsequent Western blot with appropriate antibodies. Metabolic Labeling of Myotubes with 32Pi—32Pi metabolic labeling was performed (16) to investigate in vivo phosphorylation of ␣-subunits of Na⫹,K⫹-ATPase. Myotubes (day 6), growing on 100-mm dishes, were incubated for 3 h at 37 °C in serum-free DMEM containing 32Pi (1 mCi/ml). Insulin and/or inhibitors were added during the last 20 or 50 min of incubation time, as described above. Incubation was terminated by cooling on ice. Myotubes were lysed in buffer A, and ␣-subunits were immunoprecipitated with polyclonal anti-NK1 rabbit antibodies. The bead pellets were mixed with 60 ␮l of Laemmli buffer (62.5 mM TrisHCl, 2% SDS, 10% glycerol, and 10 mM dithiothreitol), separated by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA). Phosphoproteins were analyzed using Bio-Imaging Analyzer BAS-1800II (Fuji Photo Film Co., Ltd., Japan), and quantification was performed using the Image Gauge software, version 3.4 (Fuji Photo Film Co., Ltd., Japan). In each experiment, the amount of radioactivity incorporated into the ␣-subunit was corrected for the amount of the protein detected by the Western blot analysis. The quantitative data is reported as percent of basal. Immunoprecipitation—Myotubes were lysed in 0.5 ml of ice-cold lysis

Human Na,K-ATPase Regulation by ERK1/2 Phosphorylation buffer A. Insoluble material was removed by centrifugation (12,000 ⫻ g for 10 min at 4 °C). Aliquots of supernatant (300 ␮g of protein) were immunoprecipitated overnight at 4 °C with 50 ␮l of polyclonal anti-NK1 rabbit antibodies or with 30 ␮l the anti-phospho-Thr-Pro mouse IgM. Immunoprecipitates were collected on protein A-Sepharose (Amersham Biosciences) or protein L-agarose (Sigma) beads, respectively. Beads were washed four times in lysis buffer A; twice in 0.1 M Tris (pH 8.0) and 0.5 M LiCl; once in 10 mM Tris (pH 7.6), 0.15 M NaCl, and 1 mM EDTA; and once in 20 mM HEPES, 5 mM MgCl2, and 1 mM dithiothreitol. Pellets were resuspended in Laemmli sample buffer. Western Blot Analysis—Aliquots of cell lysate (30 ␮g of protein) or immunoprecipitates were re-suspended in Laemmli sample buffer. Proteins were then separated by SDS-PAGE, transferred to PVDF membranes (Millipore, MA), blocked with 7.5% nonfat milk, washed with TBST (10 mM Tris HCl, 100 mM NaCl, 0.02% Tween 20), and incubated with appropriate primary antibodies overnight at 4 °C. Membranes were washed with TBST and incubated with an appropriate secondary antibody. Proteins were visualized by enhanced chemiluminescence and quantified by densitometry. Phosphoamino Acid Analysis—The phosphorylated ␣-subunit was immunoprecipitated, resolved by SDS-PAGE, and transferred to PVDF membranes, and the 32P-labled Na⫹,K⫹-ATPase ␣-subunits were identified on the membrane by using a Bio-Imaging Analyzer BAS-1800II and excised. Thereafter, the phosphorylated ␣-subunit was hydrolyzed in 6 M HCl and analyzed by two-dimensional high voltage electrophoresis on cellulose thin layer plates. Phosphoamino acid analysis was performed as described previously (33). Phosphoamino acids, on thinlayer electrophoresis plates, were analyzed using the Bio-Imaging Analyzer BAS-1800II. Na⫹,K⫹-ATPase Purification—Human renal tubular cells were grown until confluence, trypsinized, and harvested by centrifugation (160 ⫻ g for 10 min). Cells were washed three times by ice-cold PBS. The final cell pellet was resuspended in 10 –15 volumes of imidazolesucrose buffer (25 mM imidazole, 1 mM EDTA, 250 mM sucrose, pH 7.2) and homogenized by 20 strokes in glass-glass homogenizer on ice. The homogenate was centrifuged (9,000 ⫻ g for 10 min at 4 °C), and the pellet after this centrifugation are rehomogenized and centrifuged again (9,000 ⫻ g for 10 min at 4 °C). The two supernatants were combined and centrifuged (190,000 ⫻ g for 45 min at 4 °C). The pellet of crude membranes after this centrifugation was resuspended in the imidazole-sucrose buffer and was used as starting material for Na⫹,K⫹ATPase purification as described (34). Rat Na⫹,K⫹-ATPase holoenzyme was purified from rat kidney cortex as described (34). Quality of purification was verified by SDS-PAGE (7.5% gel) following Coomassie Blue staining. Na⫹,K⫹-ATPase activity has previously been determined under Vmax conditions (19). In Vitro Phosphorylation of Na⫹,K⫹-ATPase ␣-Subunit by ERK or PKC—For in vitro phosphorylation, 5 ␮g of Na⫹,K⫹-ATPase protein preparation was resuspended in 40 ␮l of 1.5⫻ phosphorylation media (final concentrations: 20 mM HEPES-Tris, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 5 mM NaF, 1 mM ␤-glycerophosphate, 200 ␮M ouabain, 0.5 mM dithiothreitol, 1.5 mM CaCl2, in the absence or presence of 0.5% Triton X-100), followed by addition of 0.1 ␮g of PKC (Calbiochem) or 0.1 ␮g of ERK1 and 0.1 ␮g of ERK2 (Upstate, Charlottesville, VA). The phosphorylation reaction for both kinases was initiated by addition of 10 ␮l of ATP (final concentration of 0.1 mM containing 3 ␮Ci/pmol [32P]ATP, Amersham Biosciences) and allowed to proceed for 1 h at 30 °C. In the case of the Na⫹,K⫹-ATPase activity measurement, the phosphorylation reaction was performed in phosphorylation media without NaF and ouabain; the final concentration of ATP was 0.5 mM. The total volume of phosphorylation sample was 60 ␮l. Reactions were terminated by the addition of 20 ␮l of 4⫻ Laemmli sample buffer, or 12-␮l aliquots were immediately used for determination of Na⫹,K⫹-ATPase activity. Samples for SDS-PAGE were incubated for 20 min at 56 °C, thereafter iodoacetamide was added to final concentration of 20 mM. The phosphorylated samples were resolved by SDS-PAGE and transferred to PVDF membranes, and the 32P-labled Na⫹,K⫹-ATPase ␣-subunits were identified by using a Bio-Imaging Analyzer BAS-1800II. Thereafter, PVDF membranes were subjected to Western blotting or to phosphoamino acid analysis. Statistics—Data are presented as mean ⫾ S.E. Comparisons between groups were performed using Student’s t test. For multiple comparisons, one-way analysis of variance with Sheffe’s correction was used. Significance was established at p ⬍ 0.05.

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RESULTS

In Silico Screen of Possible Phosphorylation Sites in Na⫹,K⫹ATPase ␣-Subunit—To determine whether Na⫹,K⫹-ATPase ␣-subunit is a potential target for protein kinases, activated by insulin, a computer-based screen for possible phosphorylation sequence motifs was performed. A phosphorylation motif scan of human ␣1-subunit was performed using a Scansite 2.0 program at high stringency level. This analysis revealed three possible phosphorylation sites: Ser491 as a possible site for calmodulin-dependent kinase II, Ser943 as possible site for cAMP-dependent protein kinase (PKA), and Thr81 as a possible site for ERK. The high stringency level scan of human ␣2subunit protein sequence suggested Thr414 as a possible site for Akt kinase, Ser936 (a homologue for Ser943 in ␣1-subunit) as a possible site for PKA, and Thr79 as a possible site for ERK. We chose to examine ERK1/2 as a candidate kinase for insulinregulation of Na⫹,K⫹-ATPase ␣-subunits, because (a) both ␣1and ␣2-subunit cell surface abundance is regulated by insulin and (b) ERK1/2 is known to be activated by insulin. The sequence scans of ␣1- and ␣2-subunits performed at a medium and low stringency level predicted Thr85, Thr86, Thr226, Ser228, Thr282, and Thr788, as possible ERK phosphorylation sites in the human ␣1-subunit, and homologue amino acid residues Thr83, Thr84, Thr224, Ser226, and Thr781 as possible ERK phosphorylation sites in the human ␣2-subunit, respectively. The prediction of ERK phosphorylation sites was independently confirmed by a different phosphorylation site prediction program (PhosphoBase2.0). Notably, the predicted ERK phosphorylation sites are conserved between human, rat, mouse, and chicken. Effect of Kinase Inhibitors on Insulin Stimulation of Ouabain-sensitive 86Rb⫹ Uptake and [3H]Ouabain Binding—Incubation of myotubes with insulin increased ouabain-sensitive 86 Rb⫹ uptake by 48% (p ⬍ 0.05). This effect was completely prevented by 20 ␮M PD98059, a specific MEK1/2 inhibitor, 10 ␮M GF109203X, a PKC inhibitor, or 100 nM wortmannin, a PI 3-kinase inhibitor. Notably a lower concentration of GF109203X (1 ␮M) as reported by Martiny-Baron et al. (35) known to inhibit the conventional and novel, but not atypical PKC isoforms did not alter insulin stimulation of ouabainsensitive 86Rb⫹ uptake (Fig. 1A). Results for [3H]ouabain binding, an assay designed to indicate changes in the number of cell surface active pump units, were comparable with the ion transport activity data (Fig. 1B). Kinase inhibitors alone did not significantly affect either basal ouabain-sensitive 86Rb⫹-uptake or [3H]ouabain binding (data not shown). It should be mentioned that U0126 and PD98059, two structurally unrelated MEK1/2 inhibitors, both prevented the insulin-stimulation of 86Rb⫹ uptake and [3H]ouabain binding (data not shown). Thus, insulin stimulates Na⫹,K⫹-ATPase activity and cell surface abundance via PI 3-kinase-, and PKC-, and ERK MAP kinase-dependent pathways. Effect of Kinase Inhibitors on Insulin-stimulated ERK Phosphorylation—Our results showing that MEK1/2 inhibitors prevented the stimulation of Na⫹,K⫹-ATPase by insulin suggested that ERK activation participates in this process. We therefore assessed insulin activation of ERK1/2 in HSMCs. For this purpose we measured ERK1/2 phosphorylation in the absence or presence of insulin and kinase inhibitors. As expected, insulin stimulation of HSMCs increased ERK1/2 phosphorylation level by 2.8-fold (Fig. 2). This effect was completely inhibited in the presence of 20 ␮M PD98059. Insulin action in ERK1/2 was partly or completely blocked in the presence of 100 nM wortmannin or 10 ␮M GF109203X, respectively (Fig. 2). In contrast, 1 ␮M GF109203X was without effect on insulin-stimulated ERK1/2 phosphorylation. Based on these observations, we con-

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FIG. 1. Effect of insulin and kinase inhibitors on ouabain sensitive 86Rbⴙ uptake (A) and [3H]ouabain binding (B) in HSMCs. Human differentiated myotubes were incubated with 100 nM insulin for 20 min in the absence or presence of 100 nM wortmannin, 1 or 10 ␮M GF109203X, or 20 ␮M PD98059, as indicated. Assays were performed as described under “Materials and Methods.” Results are means ⫾ S.E. for six independent experiments performed in triplicates. *, p ⬍ 0.05 versus control.

FIG. 2. Effect of insulin and kinase inhibitors on ERK phosphorylation in HSMCs. Human differentiated myotubes were incubated with 100 nM insulin for 20 min in absence or presence of 100 nM wortmannin, 1 or 10 ␮M GF109203X, or 20 ␮M PD98059, as described for Fig. 1. Results are means ⫾ S.E. for six independent experiments. *, p ⬍ 0.05 versus basal. A representative Western blot image is shown in the upper panel.

clude that the insulin action on the sodium pump can be mediated through an ERK1/2 MAP kinase-dependent mechanism. Na⫹,K⫹-ATPase Isoform Protein Expression from Myoblasts to Myotubes—Protein expression of ␣1 and ␣2 Na⫹,K⫹-ATPase isoforms was determined during differentiation of muscle cells in culture (Fig. 3, A and B, respectively). A significant decrease in ␣1 Na⫹,K⫹-ATPase was observed, in parallel with an increase in ␣2 Na⫹,K⫹-ATPase during myotubule differentiation. This response resembles the pattern of Na⫹,K⫹-ATPase expression in skeletal muscle during development (36). Therefore, HSMCs represents a suitable model to study the isoformspecific regulation of Na⫹,K⫹-ATPase. Cell Surface Biotinylation—The [3H]ouabain binding and ouabain-sensitive 86Rb⫹ uptake assays does not distinguish whether

FIG. 3. Protein expression of Naⴙ,Kⴙ-ATPase ␣1-subunit (A) and ␣2-subunit (B) in human skeletal muscle cells at different days of differentiation to myotubes. Representative immunoblots (upper panel) and quantitative data from six experiments (mean ⫾ S.E.) (lower panel) are shown. *, p ⬍ 0.05 versus day 0.

the MAP kinase signaling pathway regulate the two Na⫹,K⫹ATPase ␣-subunits isoforms differently. To study the isoform specificity of insulin action on Na⫹,K⫹-ATPase membrane translocation a surface biotinylation and subsequent streptavidin precipitation were employed. Insulin induced translocation of both ␣1- and ␣2-subunits to cell surface (14). Incubation of differentiated HSMCs with 100 nM insulin for 20 min increased cell surface expression of the ␣1- and ␣2-subunit (as a percentage of basal: 55% increase for ␣1 and 93% increase for ␣2, respectively, p ⬍ 0.01, Fig. 4, A and B). Importantly, the magnitude of the insulin response was greater for the ␣2-subunit. In agreement with 86Rb⫹ uptake and [3H]ouabain binding experiments (see Fig. 1), insulin-induced increase in Na⫹,K⫹-ATPase ␣-subunit cell surface expression was abolished by 20 ␮M PD98059, 10 ␮M GF109203X, or 100 nM wortmannin (Fig. 4, A and B). Thus, insulin-dependent increase in the cell surface expression of Na⫹,K⫹-ATPase isoforms relies on activation of ERK1/2 MAP kinase in human skeletal muscle cells. Phosphorylation of Na⫹,K⫹-ATPase ␣-Subunits in HSMCs in Response to Insulin—To determine whether insulin promotes phosphorylation of Na⫹,K⫹-ATPase in HSMCs, myotubes were metabolically labeled with 32Pi. Thereafter, cells were incubated with insulin for 20 min in the absence or presence of kinase inhibitors. Isoform-specific anti-␣1- and anti-␣2-monoclonal antibodies were unable to immunoprecipitate the pump subunits. Thus we used a NK1 rabbit antibody previously reported to precipitate both the ␣1- and the ␣2-subunit of Na⫹,K⫹-ATPase (16). Background phosphorylation of Na⫹,K⫹ATPase ␣-subunit was observed after 3-h incubation of HSMCs in phosphorylation media. Insulin increased phosphorylation of Na⫹,K⫹-ATPase ␣-subunits by 2.5-fold (Fig. 5A), whereas phosphorylation of the Na⫹,K⫹-ATPase ␤-subunit was not detected (data not shown). Insulin-stimulated Na⫹,K⫹-ATPase ␣-subunit phosphorylation was inhibited by 20 ␮M PD98059, 10 ␮M GF109203X, or 100 nM wortmannin. To further assess the potential role of ERK1/2-mediated Na⫹,K⫹-ATPase ␣-subunit phosphorylation, we analyzed the effect of insulin and MEK1/2 inhibitors on phosphoamino acid composition of 32P-labeled ␣-subunits. Under basal conditions, the pump ␣-subunit was


FIG. 4. Translocation of Naⴙ,Kⴙ-ATPase ␣-subunits to the cell surface in the absence or presence of insulin and PI 3-kinase, PKC, or MEK1 inhibitors. Human differentiated myotubes were incubated with 100 nM insulin and in the absence or presence of 100 nM wortmannin, 1 or 10 ␮M GF109203X, or 20 ␮M PD98059. Cell surface Na⫹,K⫹-ATPase abundance was determined by biotinylation with EZlink Sulfo-NHS-SS-biotin and streptavidin-precipitation as described under “Materials and Methods.” Na⫹,K⫹-ATPase ␣1-subunit (A) and ␣2-subunit (B) representative Western blots (upper panel) and quantitative data from six experiments (mean ⫾ S.E.) (lower panel) are shown. *, p ⬍ 0.01 versus basal.

primarily phosphorylated on serine and slightly on threonine residues (Fig. 5B). Insulin stimulation increased phosphorylation of serine and threonine and induced phosphorylation on tyrosine residues. In the presence of the MEK1/2 inhibitor PD98059, the insulin-induced phosphorylation of serine and threonine residues was markedly decreased (Fig. 5B). Interestingly, the phosphorylation of tyrosine residues was unaffected by PD98059. These data suggest that ERK1/2 MAP kinase is involved in Na⫹,K⫹-ATPase ␣-subunit phosphorylation on threonine and serine residues. In contrast, insulin-induced tyrosine phosphorylation of Na⫹,K⫹-ATPase ␣-subunit is MAP kinase-independent. Na⫹,K⫹-ATPase ␣-Subunit Is Phosphorylated by Specific -Thr-Pro- Motif Kinase in Response to Insulin—To determine whether ERK1/2 is able to directly phosphorylate Na⫹,K⫹ATPase ␣-subunit and whether this phosphorylation is isoform-specific, we utilized a phospho-threonine-proline motifspecific antibody. The -Thr-Pro- and -Ser-Pro- phosphorylation motifs are known to be primarily substrates for ERK (37, 38). Insulin stimulation resulted in increased phosphorylation of -Thr-Pro- motif measured by Western blot after immunopre-

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FIG. 5. Effect of insulin and kinase inhibitors on the phosphorylation state of Naⴙ,Kⴙ-ATPase ␣-subunit from human skeletal muscle cells. A, myotubes were metabolically labeled with 32Pi as described under “Materials and Methods” and incubated with 100 nM insulin for 30 min, in the absence or presence of 100 nM wortmannin, 1 or 10 ␮M GF109203X, or 20 ␮M PD98059. Myotubes were lysed, and equal amounts of protein (300 ␮g) were immunoprecipitated with antiNK1 antibody. A representative autoradiogram is shown in the upper panel, and quantitative data from four experiments (mean ⫾ S.E.) are shown in the lower panel. *, p ⬍ 0.05 versus basal. B, representative autoradiograms of phosphoamino acids from immunoprecipitated ␣-subunit from untreated (Basal) cells, in response to 100 nM insulin without or with 20 ␮M PD98059. Circles represent the positions of unlabeled phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr). The additional spots represent the site of sample application (ori) and nonhydrolyzed peptides.

cipitation of the total cellular pool of Na⫹,K⫹-ATPase ␣-subunit with NK1 antibodies (Fig. 6A). Phosphorylation was abolished in the presence of the MEK1 inhibitor PD98059. Because differentiated human myotubules express ␣1- and ␣2-isoforms of Na⫹,K⫹-ATPase (Fig. 3), we assessed whether insulin selectively increases phosphorylation of the -Thr-Pro- motif in these isoforms. Cell lysates were subjected to immunoprecipitation with phospho-threonine-proline-specific antibody, followed by Western blot analysis with specific anti-␣1-subunit (Fig. 6B) and ␣2-subunit (Fig. 6C) antibodies. Insulin increased the amounts of both immunoprecipitated ␣1- and ␣2-subunits of human Na⫹,K⫹-ATPase, indicating increased phosphorylation of the -Thr-Pro- motif in both isoforms. This phosphorylation was completely inhibited by pretreating cells with PD98059. Thus, our data suggest that both ␣1- and ␣2-subunits could be directly phosphorylated by ERK1/2 in intact HSMCs. In Vitro Phosphorylation of Na⫹,K⫹-ATPase by PKC and ERK—To further demonstrate that Na⫹,K⫹-ATPase ␣-subunit is a potential substrate for ERK1/2 and that PKC does not mediate the insulin-induced phosphorylation of the human ␣-subunit, we performed in vitro PKC and ERK1/2 phosphorylation of Na⫹,K⫹-ATPase purified from primary human kidney tubular cells and rat kidney cortex. Both Na⫹,K⫹-ATPase preparations did not contain protein kinase activity. Kidney Na⫹,K⫹-ATPase contains exclusively the ␣1-subunit isoform

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FIG. 6. Effect of insulin on phosphorylation of the threonineproline motif in Naⴙ,Kⴙ-ATPase ␣-subunit in intact human skeletal muscle cells. Human differentiated myotubes were incubated with or without 100 nM insulin in the presence or absence of 20 ␮M PD98059. Myotubes were lysed, and equal amounts (350 ␮g) of protein were immunoprecipitated with either anti-NK1 antibody (A) or anti-pThr-Pro-antibody (B and C). The representative blots of phosphorylation of Na⫹,K⫹-ATPase total ␣ (A), ␣1-subunit (B), and ␣2-subunit (C) Na⫹,K⫹-ATPase from five independent experiments are shown.

(39). The ␣-subunit of the Na⫹,K⫹-ATPase isolated from human kidney was a very poor substrate for purified rat brain PKC, consistent with the lack of the crucial PKC phosphorylation site Ser23 in the human ␣-subunit. In contrast, the ␣-subunit isolated from rat kidney was efficiently phosphorylated by PKC (Fig 7A). Additional in vitro phosphorylation experiments with recombinant human PKC␨ (Upstate, Charlottesville, VA) gave similar results (data not shown). In contrast to PKC, recombinant ERK1 or ERK2 (or a mixture of these kinases), equipotently phosphorylated the ␣-subunits from both human and rat Na⫹,K⫹-ATPase. However, ERK-mediated phosphorylation did not significantly affect Na⫹,K⫹-ATPase activity measured under Vmax condition (control, 13.4 ⫾ 1.3 ␮mol of Pi/mg/min versus phosphorylation, 14.4 ⫾ 1.6 ␮mol of Pi/mg/ min). Addition of 0.5% (v/v) Triton X-100 enhanced ␣-subunit phosphorylation (Fig. 7B), however solubilization of membrane-bound Na⫹,K⫹-ATPase preparation by detergent was not essential, unlike in the cases of PKA (40, 41) and protein kinase G (42) ␣-subunit phosphorylation. Interestingly, according to a sodium pump structure model (43), many of the predicted ERK phosphorylation sites are located in close proximity to the membrane: the Thr788 site is located in the M5 transmembrane fragment and, therefore, unlikely to be phosphorylated in the absence of detergent. Thus, the increased phosphorylation in the presence of detergent may be explained by increased availability of ERK phosphorylation sites by solubilization of lipid in the preparation of Na⫹,K⫹-ATPase. To determine whether ERK1/2 phosphorylation in vitro occurs on the same Thr-Pro sequence motifs as observed following activation of the PD98059-sensitive kinase in intact myotubules (Fig. 6), we subjected the in vitro phosphorylated Na⫹,K⫹-ATPase ␣-subunit from human kidney to Western blot analysis with the phospho-threonine-proline motif-specific antibody. ERK1/2 phosphorylated the -Thr-Pro- motif in vitro (Fig. 7B). A low level phosphorylation of this motif was detectable even in Na⫹,K⫹-ATPase preparations, which were not submitted to phosphorylation by ERK. This observation suggests that the -Thr-Pro- motif phosphorylation should be rela-

FIG. 7. Phosphorylation of Naⴙ,Kⴙ-ATPase ␣-subunit by PKC and ERK1/2 in vitro. Human and rat kidney Na⫹,K⫹-ATPase preparations were purified and phosphorylated by PKC and ERK1/2 in vitro, as described under “Materials and Methods.” A, comparative phosphorylation of human and rat Na⫹,K⫹-ATPase ␣-subunit by PKC and ERK1/2 in the absence of Triton X-100. Na⫹,K⫹-ATPase ␣-subunit content was assessed by Coomassie Blue staining (A, upper panel), and phosphorylation of Na⫹,K⫹-ATPase ␣-subunit was measured by autoradiography (A, lower panel) as indicated. B, purified human kidney Na⫹,K⫹-ATPase was incubated without or with ERK1/2 in the absence or presence of 0.5% Triton X-100. Na⫹,K⫹-ATPase ␣-subunit content was determined by Ponceau S staining (B, upper panel), and phosphorylation of Na⫹,K⫹-ATPase ␣-subunit was measured by autoradiography (B, middle panel) or by Western blot analysis with anti-p-Thr-Proantibody (lower panel). C, representative autoradiogram of phosphoamino acids of purified ␣-subunit phosphorylated in vitro by ERK1/2. Circles represent the positions of phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr). The additional spots represent the site of sample application (ori) and nonhydrolyzed peptides. Representative results from five independent experiments are shown.

tively stable and invulnerable to protein phosphatase activity during multiple steps of Na⫹,K⫹-ATPase purification. Alternatively, the phospho-threonine-proline motif-specific antibody may nonspecifically cross-react with a relatively large amount (⬃2 ␮g) of purified Na⫹,K⫹-ATPase ␣-subunit. Phosphoamino acid analysis of Na⫹,K⫹-ATPase ␣-subunit in vitro phosphorylated by ERK revealed that the enzyme was primarily phosphorylated on threonine residues. However, a substantial amount of phosphoserine was also detected, whereas phosphotyrosine was undetected. Thus, results from in vitro phosphorylation analysis suggests that the ␣-subunit of Na⫹,K⫹-ATPase is a good substrate for the MAP kinase ERK1/2. DISCUSSION

Insulin regulation of Na⫹,K⫹-ATPase in tissues of human origin is poorly understood. Cultured human skeletal muscle cells (HSMCs) express many components of the known insulin signaling pathway (44). Because cultured HSMCs also express multiple isoforms of Na⫹,K⫹-ATPase, they provide a good model for studying differential regulation of human Na⫹,K⫹ATPase isoforms, whose functional differences in specific cell types remain poorly understood. Results of the present study show that insulin stimulates Na⫹,K⫹-ATPase activity and the pump translocation to plasma membrane in HSMCs via phosphorylation of the ␣-subunits by ERK1/2 MAP kinase.

Human Na,K-ATPase Regulation by ERK1/2 Phosphorylation Several studies implicate a role for both PI 3-kinase and PKC in insulin-mediated activation of Na⫹,K⫹-ATPase (12, 14, 17). We have previously reported that inhibition of PI 3-kinase and atypical PKC prevents insulin-induced translocation of ␣1- and ␣2-subunits to the plasma membrane in differentiated HSMCs (14). Results of the present study confirm this observation and show that the PI 3-kinase inhibitor wortmannin (partially) and the PKC inhibitor GF109203X (completely), blocked insulinstimulated ERK1/2 phosphorylation. Although a nonspecific effect of these inhibitors on MEK1/2 cannot be excluded, this finding is in agreement with a growing number of reports suggesting that PI 3-kinase, through atypical PKCs, is involved in ERK activation (45– 47). Importantly, GF109203X, used at a concentration that inhibits only conventional and novel PKCs, had no effect on insulin-stimulated ERK1/2 phosphorylation. Thus, ERK1/2 is most likely downstream from atypical PKCs, and furthermore, inhibition of insulin-mediated Na⫹,K⫹ATPase stimulation by both PI 3-kinase and PKC inhibitors can be explained by reduced ERK1/2 signaling. In support of our hypothesis, a recent report suggests ERK is involved in sodium pump activation by angiotensin II in vascular smooth muscle cells (23). In addition to modest tyrosine phosphorylation, insulin stimulation significantly increases serine and, most notably, threonine phosphorylation of the Na⫹,K⫹-ATPase ␣-subunit in tissues of rat origin, including skeletal muscle (16, 21). Our results show that insulin induces phosphorylation of Na⫹,K⫹ATPase ␣-subunits in HSMCs. Using phosphoamino acid analysis, we showed that in human cells this phosphorylation also occurs on Tyr, Thr, and Ser residues. Computer-based phosphorylation motif screening of the Na⫹,K⫹-ATPase ␣-subunit identified potential ERK1/2 phosphorylation sites that are evolutionary conserved and present in ␣1- and ␣2-subunit isoforms, which are expressed in HSMCs. The Thr81 in the ␣1subunit and Thr79 in the ␣2-subunit exhibit the highest probability for ERK phosphorylation, in comparison with all known ERK phosphorylation sites, thus making these sites highly probable ERK targets in vivo. The following lines of experimental evidence support our hypothesis that ERK1/2 mediates insulin-stimulated phosphorylation of Na⫹,K⫹ATPase ␣-subunits in HSMCs: 1) MEK1/2 inhibition prevented insulin-induced Na⫹,K⫹-ATPase ␣-subunit Thr phosphorylation; 2) the specific ERK target sequence -Thr-Pro- of ␣1- and ␣2-subunits was phosphorylated in response to insulin; and 3) ␣-subunits of purified human and rat Na⫹,K⫹-ATPase were good substrates for ERK1 and -2. In insulin-stimulated HSMCs pre-exposed to the MEK1/2 inhibitor PD98059, Thr phosphorylation was nearly lost and Ser phosphorylation was markedly reduced, whereas Tyr phosphorylation was not altered. Thus ERK1/2 activation does not account for insulin-mediated phosphorylation of the ␣-subunits on Tyr. Phosphorylation of the Na⫹,K⫹-ATPase ␣1-subunit at Tyr-10 is required for the insulin-induced stimulation of its activity in kidney proximal tubule cells (21). However, in kidney cells, insulin stimulates Na⫹,K⫹-ATPase without affecting the pump membrane distribution (3). The physiological relevance of Na⫹,K⫹-ATPase phosphorylation on Tyr in human skeletal muscle remains to be explored. In HSMCs, insulin stimulated ouabain-sensitive 86Rb⫹ uptake, a measure of cation transport activity of the Na⫹,K⫹ATPase, and [3H]ouabain binding, a measure of the number of active Na⫹,K⫹-ATPase units at the cell surface, to a similar extent. This finding provides evidence to suggest that insulin acts mainly through an increased number of plasma membrane sodium pumps. This conclusion is supported by cell surface biotinylation experiments and by the absence of effect of in

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vitro ERK phosphorylation on ATP-hydrolyzing activity of Na⫹,K⫹-ATPase. The molecular mechanism linking ERK1/2-dependent phosphorylation of Na⫹,K⫹-ATPase ␣-subunits with increased pump surface content remains unclear. Recently, ERK-dependent phosphorylation of the Na⫹,K⫹-ATPase ␣1-subunit at nonconsensus site Ser16 has been implicated in endocytosis of the pump in response to parathyroid hormone in opossum kidney cells (48). However, in that study the possible ERK-dependent Thr phosphorylation and the balance between possible ERKdependent stimulatory and inhibitory signals were not determined. In contrast to the parathyroid hormone effect on Na⫹,K⫹-ATPase activity in opossum kidney cells, in HSMCs, insulin stimulates Na⫹,K⫹-ATPase cation transport activity and the pump cell surface abundance. Predicted ERK phosphorylation sites in Na⫹,K⫹-ATPase ␣-subunits Thr81, Thr85, and Thr86 are located in the poly-proline-rich motif LTPPPTTPE. This amino acid sequence has been shown to be involved in regulation of receptor-mediated endocytosis of Na⫹,K⫹-ATPase (49). Thus a possible explanation could be that insulin-stimulated phosphorylation of these threonine residues by ERK1/2 arrests the formation of an endocytic complex consisting in Na⫹,K⫹-ATPase, adaptor protein 2, and clathrin, thereby preventing Na⫹,K⫹-ATPase endocytosis and leading to increased plasma membrane ␣-subunit abundance due to constitutive exocytosis. The precise identification of the Na⫹,K⫹-ATPase-␣subunits amino acid residues phosphorylated by ERK1/2 in response to insulin and a further investigation of their role in trafficking of the sodium pump remain to be determined. Despite the similarities between the two ␣-subunits, the increase in ␣2-subunit cell surface expression in response to insulin is 2-fold greater, as compared with the ␣1-subunit. However, the structure and location of the proline-rich domain and predicted ERK phosphorylation sites are identical in ␣1and ␣2-subunits. Interestingly, the ␣2-subunit contains Thr414, a potential site for Akt phosphorylation, and Akt kinase is strongly activated by insulin in our HSMC model (44). Whether a specific ␣2-subunit phosphorylation by Akt occurs and whether this phosphorylation would be important for ␣2-subunit trafficking remain to be elucidated. Conversely, insulininduced activation of ERK1/2 in HSMCs leads to activation of the downstream kinase p90rsk (50), and it is prevented by MEK1 inhibitors (51). Phosphorylation of Na⫹/H⫹-exchanger 1 (NHE1) by p90rsk leads to its activation and thereby increases sodium influx (52). However, insulin stimulation of Na⫹,K⫹ATPase in HSMCs was insensitive to amiloride (data not shown), an inhibitor of NHE1, indicating that increased sodium influx through NHE1 is not involved in activation of the sodium pump. MAP kinase-dependent sodium pump activation by angiotensin II is also insensitive to amiloride in vascular smooth muscle cells (23). Nevertheless, we cannot exclude that p90rsk-mediated phosphorylation of Na⫹,K⫹-ATPase participates in the effect of insulin. Indeed, the p90rsk phosphorylation motif is similar to that for Akt and PKA (30, 51). PKA activation leads to Na⫹,K⫹-ATPase translocation from an endosomal compartment to the basolateral membrane in renal epithelial cells (13). Thus, p90rsk could mimic PKA in terms of Na⫹,K⫹-ATPase activation. The ERK1/2-mediated phosphorylation and regulation of Na⫹,K⫹-ATPase proposed here is not limited to insulin responses. For example, muscle contraction and physical exercise are both extremely potent activators of ERK1/2 (24). Moreover, acute exercise leads to increases in Na⫹,K⫹ATPase activity and translocation of both ␣1- and ␣2-subunits to the plasma membrane in skeletal muscle (53, 54). The signal-transmitting functions of Na⫹,K⫹-ATPase, as a oua-

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bain receptor, also places ERK1/2 in a central position along this newly discovered signaling pathway (55). Whether ERK1/2 mediates phosphorylation of Na⫹,K⫹-ATPase under these conditions remains to be elucidated. In conclusion, insulin stimulates Na⫹,K⫹-ATPase activity in human differentiated myotubules via translocation of ␣1- and ␣2-subunits to the plasma membrane by a PI 3-kinase-, PKC-, and ERK1/2-dependent mechanism. This activation is linked to ERK1/2-dependent Na⫹,K⫹-ATPase ␣-subunit phosphorylation on the -Thr-Pro- motif. Moreover, human Na⫹,K⫹-ATPase ␣-subunit is a good substrate for ERK1/2 in vitro. Our findings indicate that ERK1/2-dependent Na⫹,K⫹-ATPase ␣-subunit phosphorylation is a triggering signal for the Na⫹,K⫹-ATPase stimulation in human skeletal muscle. Taken together, our findings suggest that ERK1/2 is essential for insulin-stimulated Na⫹,K⫹-ATPase activation; in a broader perspective, ERK1/2 may serve as a universal trigger of the sodium pump activation in different tissues. Acknowledgments—We thank Dr. Michael Caplan and Dr. Kathleen Sweadner for the kind gift of anti-␣1- and anti-␣2-subunit antibodies. We especially thank Dr. Juleen R. Zierath and Dr. Ka¨ thi Geering for helpful discussions and critical reading of the manu¨ stman for script. We also thank Dr. Marina Kovalenko and Dr. Arne O their help with phosphoamino acids analysis. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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