Produção Nacional
Revisado por pares
Ceramide Is a Potent Activator of Plasma Membrane Ca2+-ATPase from Kidney Proximal Tubule Cells with Protein Kinase A as an Intermediate
2007; Elsevier BV; Volume: 282; Issue: 34 Linguagem: Inglês
10.1074/jbc.m701669200
ISSN1083-351X
AutoresLindsey M.P. Cabral, Mira Wengert, Alexandre A.A. da Ressurreição, Pedro H.P. Feres-Elias, Fernando Almeida, Adalberto Vieyra, Celso Caruso‐Neves, Marcelo Einicker‐Lamas,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe kidney proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca2+-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca2+-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca2+ levels. In this paper we show that nanomolar concentrations of ceramide (Cer50 = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca2+-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca2+-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca2+-ATPase activity and PKA-mediated phosphorylation of the Ca2+ pump molecule. Ceramide also stimulates phosphorylation of renal Ca2+-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca2+ pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca2+-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells. The kidney proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca2+-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca2+-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca2+ levels. In this paper we show that nanomolar concentrations of ceramide (Cer50 = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca2+-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca2+-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca2+-ATPase activity and PKA-mediated phosphorylation of the Ca2+ pump molecule. Ceramide also stimulates phosphorylation of renal Ca2+-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca2+ pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca2+-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells. Important molecular transport processes take place across the epithelium of kidney proximal tubules. The basolateral membranes (BLM) 4The abbreviations used are: BLMbasolateral membrane(s)CerceramideC1Pceramide-1-phosphateCHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonatePKAprotein kinase APKCprotein kinase CPKAiprotein kinase A α-catalytic subunit inhibitorPMAphorbol 12-myristate 13-acetate of kidney proximal tubules cells contain different active transporters, or ion pumps, such as the very abundant Na+K+-ATPase, which is considered to be the molecular machinery responsible for Na+ reabsorption (1Féraille E. Doucet A. Physiol. Rev. 2001; 81: 345-417Crossref PubMed Scopus (433) Google Scholar). Other ion pumps are not so numerous, but some, such as the plasma membrane calcium pump, play important roles in the fine regulation of intracellular ion concentrations. Our group has recently shown that Ca2+-ATPase is exclusively located and active in caveolin-cholesterol-rich membrane microdomains or lipid rafts in the kidney BLM (2Tortelote G.G. Valverde R.H. Lemos T. Guilherme A. Einicker-Lamas M. Vieyra A. FEBS Lett. 2004; 576: 31-35Crossref PubMed Scopus (24) Google Scholar). These membranes have also been shown to house different cell signaling systems that are initiated by the activation of either different lipid kinases, with further generation of bioactive molecules (3Einicker-Lamas M. Wenceslau L.D. Bernardo R.R. Nogaroli L. Guilherme A. Oliveira M.M. Vieyra A. J. Biochem. 2003; 134: 529-536Crossref PubMed Scopus (14) Google Scholar, 4Nogaroli L. Silva O.F. Bonilha T.A. Moreno P.A.M. Bernando R.R. Vieyra A. Einicker-Lamas M. Int. J. Biochem. Cell Biol. 2005; 37: 79-90Crossref PubMed Scopus (9) Google Scholar), or protein kinases associated with the BLM (5Coka-Guevara S. Markus R.P. Caruso-Neves C. Lopes A.G. Vieyra A. Eur. J. Biochem. 1999; 263: 71-78Crossref PubMed Scopus (29) Google Scholar, 6Caruso-Neves C. Rangel L.B.A. Vives D. Vieyra A. Coka-Guevara S. Lopes A.G. Biochim. Biophys. Acta. 2000; 1468: 107-114Crossref PubMed Scopus (28) Google Scholar, 7Assunção-Miranda I. Guilherme A.L. Reis-Silva C. Costa-Sarmento G. Oliveira M.M. Vieyra A. Regul. Pept. 2005; 127: 151-157Crossref PubMed Scopus (18) Google Scholar, 8Rangel L.B. Caruso-Neves C. Lara L.S. Lopes A.G. Biochim. Biophys. Acta. 2001; 1564: 310-316Crossref Scopus (33) Google Scholar). basolateral membrane(s) ceramide ceramide-1-phosphate (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate protein kinase A protein kinase C protein kinase A α-catalytic subunit inhibitor phorbol 12-myristate 13-acetate The location of the BLM Ca2+-ATPase in caveolin-cholesterol-rich domains adds ceramides to the emerging potential regulatory network in these membranes, because those microdomains are also rich in sphingolipids (9Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar). Rafts are thought to be present in the outer leaflet of the cell membrane, where sphingomyelin, the precursor of ceramide (Cer) in a pathway catalyzed by sphingomyelinases, appears to be predominantly located. An important concept is that the assembly of the outer leaflet lipid rafts would alter the inner leaflet, thus enabling the different steps required for signal transduction to be coordinated (for review, see Ref. 10Bollinger C.R. Teichgräber V. Gulbins E. Biochim. Biophys. Acta. 2005; 1746: 284-294Crossref PubMed Scopus (276) Google Scholar). Many studies during the past decade have shown that smaller lipid rafts are merged into large membrane domains when sphingomyelin is hydrolyzed and Cer is generated (Refs. 11Liu P. Anderson R.G. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar and 12Nurminen T.A. Holopainen J.M. Zhao H. Kimmunen P.K. J. Am. Chem. Soc. 2002; 124: 12129-12134Crossref PubMed Scopus (88) Google Scholar; see also Ref. 10Bollinger C.R. Teichgräber V. Gulbins E. Biochim. Biophys. Acta. 2005; 1746: 284-294Crossref PubMed Scopus (276) Google Scholar for review). The generation of Cer molecules and their self-association leads to dramatic changes in plasma membranes with further formation of small Cer-rich microdomains, which are able to fuse spontaneously to others resulting in large domains called platforms (12Nurminen T.A. Holopainen J.M. Zhao H. Kimmunen P.K. J. Am. Chem. Soc. 2002; 124: 12129-12134Crossref PubMed Scopus (88) Google Scholar, 13Holopainen J.M. Subramanian M. Kimmunen P.K. Biochemistry. 1998; 37: 17562-17570Crossref PubMed Scopus (241) Google Scholar, 14Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (731) Google Scholar). Therefore, Cer-enriched microdomains seem to play an important role in facilitating and amplifying signaling processes, via different types of cell surface receptors, resulting in clusters of receptors and other cell signaling machinery that facilitate the effective transduction of different signals (10Bollinger C.R. Teichgräber V. Gulbins E. Biochim. Biophys. Acta. 2005; 1746: 284-294Crossref PubMed Scopus (276) Google Scholar). The importance of Cer in different cell processes is not only related to its physicochemical properties. It can be also considered a cell signaling molecule with different roles in different subcellular compartments (for review, see Ref. 15van Blitterswijk W.J. van der Luit A.H. Veldman R.J. Verheij M. Borst J. Biochem. J. 2003; 369: 199-211Crossref PubMed Scopus (391) Google Scholar). Classically, Cer generation occurs either by the activation of sphingomyelin or via de novo synthesis (16Bose R. Verheij M. Haimowitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (786) Google Scholar, 17Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). In view of its structural analogy with diacylglycerol, it has been suggested that Cer fulfills a second messenger function by binding directly to different intracellular targets (18Venkataraman K. Futerman A.H. Trends Cell Biol. 2000; 10: 408-412Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 19Huwiler A. Kolter T. Pfeilshifter J. Sandhoff K. Biochim. Biophys. Acta. 2000; 1485: 63-99Crossref PubMed Scopus (383) Google Scholar). Some of these targets have already been studied, such as different sets of protein kinases and protein phosphatases (20Ruvolo P.P. Pharmacol. Res. 2003; 47: 383-392Crossref PubMed Scopus (300) Google Scholar). Although some studies report atypical PKC, principally PKCζ, as the primary ceramide-activated PKC (20Ruvolo P.P. Pharmacol. Res. 2003; 47: 383-392Crossref PubMed Scopus (300) Google Scholar, 21Bourbon N.A. Yun J. Kester M. J. Biol. Chem. 2000; 275: 35617-35623Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), there are some reports of a direct association of Cer with PKCα and PKCδ in kidney cells (22Huwiler A. Fabbro J. Pfeilshifter J. Biochemistry. 1998; 37: 14556-14562Crossref PubMed Scopus (107) Google Scholar, 23Colombaioni L. Garcia-Gil M. Brain Res. Rev. 2004; 46: 328-355Crossref PubMed Scopus (113) Google Scholar). More than 10 years ago it was shown that Cer could also be phosphorylated by a ceramide kinase, resulting in another bioactive sphingolipid, ceramide-1-phosphate (C1P), which has a broad spectrum of cellular targets (23Colombaioni L. Garcia-Gil M. Brain Res. Rev. 2004; 46: 328-355Crossref PubMed Scopus (113) Google Scholar, 24Futerman A. Hannun Y.A. EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (541) Google Scholar, 25Baumruker T. Bornancin F. Billich A. Immunol. Lett. 2005; 96: 175-185Crossref PubMed Scopus (79) Google Scholar) including ion transporters (26Törnquist K. Blom T. Shariatmadari R. Pasternack M. Biochem. J. 2004; 380: 661-668Crossref PubMed Scopus (40) Google Scholar). In view of the preferential formation of Cer in the lipid rafts and because the Ca2+-ATPase is exclusively located in cholesterol-caveolin-rich lipid rafts in the BLM (2Tortelote G.G. Valverde R.H. Lemos T. Guilherme A. Einicker-Lamas M. Vieyra A. FEBS Lett. 2004; 576: 31-35Crossref PubMed Scopus (24) Google Scholar), we decided to investigate the effects of Cer and C1P on the kidney proximal tubule BLM Ca2+-ATPase and on the cell signaling cascade that could be involved. Materials—Buffers, bovine serum albumin, Tris-buffered saline, CHAPS, and protease inhibitors were obtained from Sigma. Percoll was from GE Healthcare. Distilled water, deionized using the Milli-Q system of resins (Millipore Corp., Marlborough, MA), was used to prepare all solutions. 32Pi was obtained from IPEN (São Paulo, Brazil). [γ-32P]ATP was prepared as described by Maia et al. (27Maia J.C.C. Gomes S.L. Juliani M.H. Morel C.M. Genes of Parasites: A Laboratory Manual. Editora Fundação Oswaldo Cruz, Rio de Janeiro, Brazil1983: 146-167Google Scholar). Ceramide (from bovine brain), C1P, histone H8, protein A-agarose from Staphylococcus aureus in saline suspension, the PKA α-catalytic subunit inhibitor 5-22 peptide (PKAi), and the PKC inhibitor calphostin C were purchased from Sigma. The PKA α-catalytic subunit (from bovine heart) was purchased from Calbiochem (La Jolla, CA). All of the other reagents were of the highest purity available. The antibody against plasma membrane Ca2+-ATPase (clone 5f10) was from Affinity Bioreagents (Golden, CO); the antibody (C-16) against isoform 1 of Ca2+-ATPase (PMCA1) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The phospho-Ser/Thr PKA substrate polyclonal antibody was from Cell Signaling Technology (Beverly, MA). Nitrocellulose membranes (Hybond) and the ECLTM system were from Amersham Biosciences. X-OmatTM diagnostic film was from Eastman Kodak Co. Pig kidneys were obtained from a slaughterhouse under the supervision of licensed veterinarians. Isolation of Basolateral Membranes—The kidneys were rapidly removed after the animals had been killed and transported in a chilled solution containing 250 mm sucrose, 10 mm Hepes-Tris (pH 7.6), 2 mm EDTA, 1 mm phenylmethanesulfonyl fluoride, and 0.15 mg/ml of soybean trypsin inhibitor. The external portion of the cortex (cortex corticis) was carefully removed, and purified BLM derived from kidney proximal tubules were prepared using the Percoll gradient method (28Boumendil-Podevin E.F. Podevin R.A. Biochim. Biophys. Acta. 1983; 735: 86-94Crossref PubMed Scopus (84) Google Scholar). Controls for contamination with other membranes were carried out as previously described (5Coka-Guevara S. Markus R.P. Caruso-Neves C. Lopes A.G. Vieyra A. Eur. J. Biochem. 1999; 263: 71-78Crossref PubMed Scopus (29) Google Scholar, 29Vieyra A. Nachbin L. Dios-Abad E. Goldfeld M. Meyer-Fernandes J.R. de Moraes L. J. Biol. Chem. 1986; 261: 4247-4255Abstract Full Text PDF PubMed Google Scholar). The specific activity of the basolateral membrane marker Na+K+-ATPase (260.8 ± 1.6 nmol/mg per min) was enriched 5-fold over the initial kidney cortex homogenate. The membranes were stored in 250 mm sucrose in liquid N2, which preserved marker and Ca2+-ATPase activities for at least 5 months. Protein Determination—A 10-μl aliquot (in triplicate) was used for protein assays, which were performed by the Folin phenol method described by Lowry et al. (30Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with the addition of 5% SDS in the samples, using bovine serum albumin as standard. Determination of Plasma Membrane Ca2+-ATPase Activity—The membranes (0.2 mg/ml) were preincubated at 37 °C for 10 min in a medium (1 ml) containing 50 mm 1,3-bis(tris(hydroxymethyl)methylamino)propane buffer (pH 7.4), 5 mm MgCl2, 10 mm NaN3, 0.5 mm ouabain, 20 μm free Ca2+ (0.2 mm EGTA, 238.3 μm CaCl2), and the concentrations of Cer or C1P indicated in the figures or figure legends. After 10 min of sonication (240 W, 25 kHz, 24–25 °C; Unique Sonifier Cleaner, Indaiatuba, São Paulo, Brazil), the samples were supplemented with 120 mm KCl and then with 5 mm ATP. Where indicated, 10 nm PKAi (the 5–22 inhibitor peptide of PKA) or 10 nm calphostin C (the inhibitor of PKC) were added to the preincubation medium. The reaction was stopped after 20 min by adding 1.5 ml of activated charcoal in 0.1 n HCl to each tube. After centrifugation in a clinical centrifuge (2,000 rpm), aliquots of the supernatants (0.5 ml) were transferred to new glass tubes to measure the amount of Pi released using the colorimetric method of Taussky and Shorr (31Taussky H.H. Shorr E. J. Biol. Chem. 1952; 202: 675-682Abstract Full Text PDF Google Scholar). Ca2+-ATPase activity was calculated as the difference between the activities measured without and with 2 mm EGTA. The free Ca2+ concentration was calculated using a computer program that took into account the different species involved in the equilibrium between EGTA, Ca2+, the different ATP forms, Mg2+,H+, and K+ (32Sorenson M.M. Coelho H.S. Reuben J.P. J. Membr. Biol. 1986; 90: 219-230Crossref PubMed Scopus (72) Google Scholar). SDS-PAGE and Immunoblotting—Electrophoresis of BLM proteins, followed by immunodetection of Ca2+-ATPase by the 5f10 or PMCA1 antibodies and of phospho-Ser/Thr residues (in the sequence recognized by PKA), was carried out as described recently (2Tortelote G.G. Valverde R.H. Lemos T. Guilherme A. Einicker-Lamas M. Vieyra A. FEBS Lett. 2004; 576: 31-35Crossref PubMed Scopus (24) Google Scholar, 33Valverde R.H. Tortelote G.G. Lemos T. Mintz E. Vieyra A. J. Biol. Chem. 2005; 280: 30611-30618Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Immunoprecipitation of Ca2+-ATPase—The BLM Ca2+ pump was immunoprecipitated using the following procedure. The BLM of kidney proximal tubules (5 mg/ml) were first incubated for 10 min in the absence or presence of 5, 20, and 50 nm Cer, before the addition of the phosphorylation medium (see below) supplemented with 10 mm NaF. After 20 min at 37 °C, the reaction was arrested with 1% CHAPS, and the tubes were incubated at room temperature for 30 min to allow the membranes to be solubilized. In the meantime, protein A-agarose was mixed with the 5f10 antibody (equal volumes of each original stock) and gently stirred for 20 min before the addition of an equal volume of bovine serum albumin (1 mg/ml) in 0.01% CHAPS. This mixture was then added to the solubilized membranes and incubated overnight at 4 °C with gentle agitation. The immunoprecipitate and the supernatant were separated by centrifugation at 1,000 × g for 4 min (4 °C). The immunoprecipitated Ca2+-ATPase samples were washed three times with cold Tris-buffered saline, solubilized in Laemmli buffer for SDS-PAGE, electrophoresed in parallel with a supernatant sample, transferred to nitrocellulose membranes, and probed with the 5f10, PMCA1 or phospho-Ser/Thr PKA antibodies. Determination of Protein Kinase A Activity—PKA activity was determined using histone H8 as a substrate for potential kinases present in BLM. Briefly, the [γ-32P]phosphoryl group of radiolabeled ATP was transferred to the histone in an incubation medium consisting of 4 mm MgCl2, 20 mm Hepes-Tris (pH 7.0), histone H8 (1.5 mg/ml), and BLM preparation (0.5 mg/ml) in a final volume of 0.1 ml. The reaction was initiated by adding 10 μm [γ-32P]ATP (7–10 μCi/μmol). After 10 min at 37 °C, the reaction was stopped by adding 0.1 ml trichloroacetic acid (40% w/v), and the tubes were immediately placed in an ice bath. The content of each tube was filtered through 0.45-μm Millipore filters, which were then washed with 20% trichloroacetic acid and phosphate buffer (2 mm, pH 7.0) to remove the unused [γ-32P]ATP. The radioactivity incorporated into the histone was determined by liquid scintillation counting (Tri-Carb 2100; Packard, Downers Grove, IL), and the PKA activity was measured as the difference between radioactivity incorporated in the presence and absence of 10 nm M PKAi, the 5-22 PKA inhibitor peptide. A control with 100 nm cAMP confirmed the association of PKA activity with the BLM. To investigate a possible direct, cAMP-independent effect of Cer on PKA, the same assay was carried out using purified PKA α-catalytic subunit instead of BLM. Determination of Protein Kinase C Activity—We used the same method described for the determination of PKA activity, but PKC activity was measured as the difference between tubes incubated in the absence and presence of 10 nm calphostin C (PKC inhibitor). Phorbol 12-myristate 13-acetate (PMA, 1 pm), a phorbol ester, was also used to confirm the presence of functional PKC in the BLM fractions. Regulatory Phosphorylation of Ca2+-ATPase—Phosphorylation of Ca2+-ATPase was also determined as described recently (33Valverde R.H. Tortelote G.G. Lemos T. Mintz E. Vieyra A. J. Biol. Chem. 2005; 280: 30611-30618Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) with slight modifications. The membranes were preincubated with the reaction medium employed to measure the Ca2+-ATPase activity (no ATP) in the presence of Cer (50 nm), PKAi (10 nm), and calphostin C (10 nm) in the combinations shown in the corresponding figure legends. The phosphorylation reaction in the PKA assays was started by adding 5 mm cold ATP. After 20 min the reaction (0.2 ml; 100 μg of BLM protein) was stopped by adding 50 μl of sample buffer (2.3 g of dithiothreitol, 3 g of SDS, 12 ml of 1.0 m Tris, pH 6.8, 15 ml of 10% glycerol in a 50-ml final volume in water), and 0.1 ml of the solubilized samples were applied to each slot for SDS-PAGE (10% acrylamide) as described above. The gel proteins were transferred to a nitrocellulose membrane, and phospho-Ser/Thr residues were recognized with the PKA substrate polyclonal antibody as described above. Phosphorylation of Ca2+-ATPase by PKC was assayed in the same medium employed for the PKA assay, except that [γ-32P]ATP (0.5–1 μCi/μmol) was used as follows. After electrophoresis and transfer, the nitrocellulose membranes were exposed overnight to a phosphor screen and analyzed using a PhosphorImager Storm 860 (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA) to measure the intensity of the 140-kDa [32P]phosphorylated bands recognized by the 5f10 antibody. Phosphorylation of Ca2+-ATPase by the BLM-resident PKC was quantified by the difference in band intensities in the absence and presence of 10 nm calphostin C. Data Analysis—The means were compared by one-way analysis of variance, taking into account the treatment of experimental groups. The differences were evaluated using the multiple comparative Bonferroni test. In all cases the n values correspond to the results obtained from different BLM preparations, except for the purified PKA α-catalytic subunit assay. Linear regression analysis with errors in both variables was used to study the extent to which stimulation of Ca2+-ATPase activity in the presence of Cer correlates with Cer-induced PKA-mediated phosphorylation of the Ca2+ pump. Fig. 1 (open circles) shows that increasing concentrations of Cer in the nanomolar range (pA ≈ 8.5) stimulate Ca2+-ATPase activity up to ∼50% over control. We also tested C1P, the phosphorylated derivative of Cer, which was ineffective in modulating Ca2+-ATPase activity (filled circles). The observed effect of Cer could be a consequence of structural changes in the membrane similar to those observed upon sphingomyelinase activation and Cer release (12Nurminen T.A. Holopainen J.M. Zhao H. Kimmunen P.K. J. Am. Chem. Soc. 2002; 124: 12129-12134Crossref PubMed Scopus (88) Google Scholar, 13Holopainen J.M. Subramanian M. Kimmunen P.K. Biochemistry. 1998; 37: 17562-17570Crossref PubMed Scopus (241) Google Scholar, 14Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (731) Google Scholar) or to direct activation of protein kinases, which are targets for Cer in intracellular compartments. Because PKA and PKC appear to be involved in the modulation of Ca2+-ATPase from different sources (33Valverde R.H. Tortelote G.G. Lemos T. Mintz E. Vieyra A. J. Biol. Chem. 2005; 280: 30611-30618Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 34Johanson J.S. Nied L.E. Haynes D.H. Biochim. Biophys. Acta. 1992; 1105: 19-28Crossref PubMed Scopus (46) Google Scholar, 35Tao J. Johanson J.S. Haynes D.H. Biochim. Biophys. Acta. 1992; 1107: 213-222Crossref PubMed Scopus (24) Google Scholar, 36Wright L.C. Chen S. Roufogalis B.D. Arch. Biochem. Biophys. 1993; 306: 277-284Crossref PubMed Scopus (48) Google Scholar, 37Dean W.L. Chen D. Brandt P.C. Vanaman T.C. J. Biol. Chem. 1997; 272: 15113-15119Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 38Bruce J.I.E. Yule D.I. Shuttleworth T.J. J. Biol. Chem. 2002; 277: 48172-48181Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) including renal tissue (5Coka-Guevara S. Markus R.P. Caruso-Neves C. Lopes A.G. Vieyra A. Eur. J. Biochem. 1999; 263: 71-78Crossref PubMed Scopus (29) Google Scholar, 7Assunção-Miranda I. Guilherme A.L. Reis-Silva C. Costa-Sarmento G. Oliveira M.M. Vieyra A. Regul. Pept. 2005; 127: 151-157Crossref PubMed Scopus (18) Google Scholar), the following experiments were conducted to investigate whether the stimulatory effect of Cer on active Ca2+ transport in kidney involves PKA or PKC, two of the renal BLM-resident kinases (6Caruso-Neves C. Rangel L.B.A. Vives D. Vieyra A. Coka-Guevara S. Lopes A.G. Biochim. Biophys. Acta. 2000; 1468: 107-114Crossref PubMed Scopus (28) Google Scholar, 7Assunção-Miranda I. Guilherme A.L. Reis-Silva C. Costa-Sarmento G. Oliveira M.M. Vieyra A. Regul. Pept. 2005; 127: 151-157Crossref PubMed Scopus (18) Google Scholar). The observation (Fig. 2) that Ca2+-ATPase is no longer stimulated by Cer when peptide 5-22 (PKAi), the highly specific inhibitor of PKA, is added simultaneously supports the hypothesis that nanomolar Cer activates this type of protein kinase in kidney membranes and that stimulation of the Ca2+ pump is a consequence of triggering this signaling cascade. The ability of Cer to activate cAMP-dependent protein kinase was therefore tested. Fig. 3 shows that Cer induces a 3-fold increase in PKA, saturating in the same concentration range as the effect on Ca2+-ATPase activity. This effect mimics that found with cAMP (hatched column in Fig. 3). However, because the effects of Cer and cAMP are additive (black column), it is clear that they both activate PKA but in different ways. The experiments presented in Fig. 4 show that Cer, in the concentration that promotes half-maximal activation of Ca2+-ATPase, strongly stimulates phosphorylation catalyzed by the purified PKA α-catalytic subunit, supporting the view that Cer activates PKA by a mechanism independent of cAMP.FIGURE 3Ceramide activates PKA in basolateral membranes: additive effect of cAMP. PKAi-inhibited kinase activity was determined in the absence or presence of Cer and cAMP in the combinations shown on the abscissa,as described under "Experimental Procedures." PKA activity was measured as the difference between total phosphorylation and that measured in the presence of 10 nm PKA inhibitor (5-22 peptide). The data are the means ± S.E. of at least six experiments performed in triplicate with different kidney membrane preparations. *, statistical difference with respect to the control without additions (p < 0.01); #, statistical difference with respect to the assays carried out without additions, with Cer alone or with cAMP alone (p < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Ceramide stimulates the α-catalytic subunit of PKA in a direct, cAMP-independent manner. Phosphorylation of histone in the presence of the PKA α-catalytic subunit (25 units/ml) was assayed in the absence or presence of Cer and PKA inhibitor (5-22 peptide) in the combinations shown on the abscissa, as described under "Experimental Procedures." The data (control and 5 nm Cer) are the means ± S.E. of four determinations. *, statistical difference with respect to the control without Cer (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Activation of PKA by Cer is associated with subsequent phosphorylation of Ca2+-ATPase, as demonstrated in Figs. 5, 6, and 8. Fig. 5 (A and C) shows that addition of 50 nm Cer promotes a PKAi-sensitive increase in the phosphorylation of a 140-kDa band detected by the specific phospho-Ser/Thr PKA antibody. The same 140-kDa band was shown to be Ca2+-ATPase by the specific antibody 5f10 (Fig. 5B). The fact that 10 nm of the PKA inhibitor completely abolishes stimulation of both Ca2+-ATPase and phosphorylation of the pump molecule in the presence of 50 nm Cer (compare Fig. 5 with Fig. 2) reinforces the view that activation of Ca2+ pumping activity requires phosphorylation.FIGURE 6Plasma membrane Ca2+-ATPase is the 140-kDa target for ceramide-stimulated, PKA-mediated phosphorylation. Basolateral membranes were phosphorylated with cold ATP in the absence or presence of the Cer concentrations shown on the abscissa, and Ca2+-ATPase was immunoprecipitated with the monoclonal antibody 5f10, as described under "Experimental Procedures." After electrophoresis of the immunoprecipitated sediments (IP) and supernatants (Sup), immunodetection of Ca2+-ATPase and of a specific phospho-Ser/Thr PKA substrate in Ca2+-ATPase was carried out on the same nitrocellulose membrane, using the 5f10 and phospho-Ser/Thr PKA substrate antibodies, respectively. A, immunodetection of phospho-Ser/Thr at 140 kDa (electrophoresis of sediments after immunoprecipitation of Ca2+-ATPase). B, immunodetection of Ca2+-ATPase in each representative blotting shown in A (electrophoresis of sediments). C, lack of Ca2+-ATPase signal in the supernatants after immunoprecipitation. D, densitometric representation of the phosphorylation patterns, calculated by the ratio between band intensity from A and the corresponding band intensity from B in the same nitrocellulose membrane. The figure shows densitometric calculations (means ± S.E.) of at least four experiments carried out with different kidney membrane preparations. *, statistical difference with respect to the control without Cer (p < 0.05).View Large Image Figure ViewerDownload Hi-res image
Referência(s)