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• W2093961415 abstract "Eukaryotic vacuolar-type H+-ATPases (V-ATPases) are regulated by the reversible disassembly of the active V1V0 holoenzyme into a cytosolic V1 complex and a membrane-bound V0 complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V1 subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A and is the only V-ATPase subunit that is phosphorylated by protein kinase A. Subunit C can be phosphorylated as single polypeptide as well as a part of the V1 complex but not as a part of the V1V0 holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V1 complex from which subunit C had been removed before. Using salivary glands of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via protein kinase A, we show that the membrane-permeable cAMP analog 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (8-CPT-cAMP) causes phosphorylation of subunit C in a tissue homogenate and that phosphorylation is reduced by incubation with antibodies against subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C, but this is abolished by H-89, an inhibitor of protein kinase A. These data suggest that subunit C binds to and serves as a substrate for protein kinase A and that this phosphorylation may be a regulatory switch for the formation of the active V1V0 holoenzyme. Eukaryotic vacuolar-type H+-ATPases (V-ATPases) are regulated by the reversible disassembly of the active V1V0 holoenzyme into a cytosolic V1 complex and a membrane-bound V0 complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V1 subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A and is the only V-ATPase subunit that is phosphorylated by protein kinase A. Subunit C can be phosphorylated as single polypeptide as well as a part of the V1 complex but not as a part of the V1V0 holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V1 complex from which subunit C had been removed before. Using salivary glands of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via protein kinase A, we show that the membrane-permeable cAMP analog 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (8-CPT-cAMP) causes phosphorylation of subunit C in a tissue homogenate and that phosphorylation is reduced by incubation with antibodies against subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C, but this is abolished by H-89, an inhibitor of protein kinase A. These data suggest that subunit C binds to and serves as a substrate for protein kinase A and that this phosphorylation may be a regulatory switch for the formation of the active V1V0 holoenzyme. Vacuolar type H+-ATPases (V-ATPases) 3The abbreviations used are: V-ATPase, vacuolar H+-ATPase; V1C, V1 complex saturated with subunit C; 5-HT, 5-hydroxytryptamine; PKA, protein kinase A; 8-CPT-cAMP, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate; GABA, γ-amino butyric acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 3The abbreviations used are: V-ATPase, vacuolar H+-ATPase; V1C, V1 complex saturated with subunit C; 5-HT, 5-hydroxytryptamine; PKA, protein kinase A; 8-CPT-cAMP, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate; GABA, γ-amino butyric acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. are the most versatile proton pumps, being common to all eukaryotic organisms, and are found in endomembrane systems and in the plasma membrane (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Crossref PubMed Scopus (998) Google Scholar, 2Nelson N. J. Bioenerg. Biomembr. 2003; 35: 281-289Crossref PubMed Scopus (66) Google Scholar, 3Inoue T. Wang Y. Jefferies K. Qi J. Hinton A. Forgac M. J. Bioenerg. Biomembr. 2005; 37: 393-398Crossref PubMed Scopus (43) Google Scholar). V-ATPases are multi-subunit transporters composed of a catalytic ATP-hydrolyzing V1 complex (≈550 kDa), which resides on the cytoplasmic side of the membrane, and a membrane-bound proton-translocating V0 complex (≈250 kDa). V-ATPase-dependent proton pumping is essential for cellular pH homeostasis and creates an electrochemical proton gradient that energizes secondary transport mechanisms in a wide variety of organelles and membrane systems. Acidification of organelles by V-ATPase activity is crucial to various cellular processes such as neurotransmitter uptake into synaptic vesicles, intracellular protein trafficking, and the secretion and activation of lysosomal enzymes for protein processing and degradation (4Kanner B.I. Schuldiner S. CRC Crit. Rev. Biochem. 1987; 22: 1-38Crossref PubMed Scopus (404) Google Scholar, 5Moriyama Y. Maeda M. Futai M. J. Exp. Biol. 1992; 172: 171-178Crossref PubMed Google Scholar, 6Johnson L.S. Dunn K.W. Pytowsky B. McGraw T.E. Mol. Biol. Cell. 1993; 4: 1251-1266Crossref PubMed Scopus (173) Google Scholar, 7Schoonderwoert V.T. Martens G.J. J. Membr. Biol. 2001; 182: 159-169Crossref PubMed Scopus (53) Google Scholar). Located in the plasma membrane of specialized cells, V-ATPases are involved in processes such as cation secretion, bone resorption, renal acidification, and osmoregulation (8Wieczorek H. Putzenlechner M. Zeiske W. Klein U. J. Biol. Chem. 1991; 266: 15340-15347Abstract Full Text PDF PubMed Google Scholar, 9Beyenbach K.W. News Physiol. Sci. 2001; 16: 145-151PubMed Google Scholar, 10Rein J. Zimmermann B. Hille C. Walz B. Baumann O. J. Exp. Biol. 2006; 209: 1716-1724Crossref PubMed Scopus (20) Google Scholar, 11Blair H.C. Teitelbaum S.L. Ghiselli R. Gluck S. Science. 1989; 245: 855-857Crossref PubMed Scopus (732) Google Scholar, 12Schlesinger P.H. Blair H.C. Teitelbaum S.L. Edwards J.C. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 13Gluck S.L. J. Bioenerg. Biomembr. 1992; 24: 351-359Crossref PubMed Scopus (53) Google Scholar, 14Ehrenfeld J. Klein U. J. Exp. Biol. 1997; 200: 247-256Crossref PubMed Google Scholar, 15Wilson J.M. Laurent P. Tufts B.L. Benos D.J. Donowitz M. Vogl A.W. Randall D.J. J. Exp. Biol. 2000; 203: 2279-2296Crossref PubMed Google Scholar, 16Kirschner L.B. J. Exp. Biol. 2004; 207: 1439-1452Crossref PubMed Scopus (153) Google Scholar). With respect to this diversity of function, mutations in genes encoding V-ATPase subunits obviously lead to several diseases, e.g. osteopetrosis (17Frattini A. Orchard P.J. Sobacchi C. Giliani S. Abinun M. Mattsson J.P. Keeling D.J. Andersson A.K. Wallbrandt P. Zecca L. Notarangelo L.D. Vezzoni P. Villa A. Nat. Genet. 2000; 25: 343-346Crossref PubMed Scopus (563) Google Scholar) or renal tubular acidosis (18Karet F.E. Finberg K.E. Nelson R.D. Nayir A. Mocan H. Sanjad S.A. Rodriguez-Soriano J. Santos F. Cremers C.W. Di Pietro A. Nat. Genet. 1999; 21: 84-90Crossref PubMed Scopus (580) Google Scholar). Several mechanisms have been proposed for the regulation of V-ATPase activity (3Inoue T. Wang Y. Jefferies K. Qi J. Hinton A. Forgac M. J. Bioenerg. Biomembr. 2005; 37: 393-398Crossref PubMed Scopus (43) Google Scholar). The most prominent and physiologically relevant mechanism is the reversible disassembly of the V-ATPase holoenzyme into its V1 and V0 complexes as discovered in the midgut of the tobacco hornworm Manduca sexta and in the yeast Saccharomyces cerevisiae (19Sumner J.P. Dow J.A. Earley F.G. Klein U. Jaeger D. Wieczorek H. J. Biol. Chem. 1995; 270: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 20Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar). In both systems, a nutrient drop induced by glucose deprivation in yeast or the cessation of feeding because of molting or starvation in the insect lead to the reversible disassembly of the functional V-ATPase holoenzyme into its inactive V1 and V0 complexes (21Kane P.M. FEBS Lett. 2000; 469: 137-141Crossref PubMed Scopus (64) Google Scholar, 22Wieczorek H. Grüber G. Harvey W.R. Huss M. Merzendorfer H. Zeiske W. J. Exp. Biol. 2000; 203: 127-135Crossref PubMed Google Scholar). Although more recent additional examples support the notion that the reversible disassembly/reassembly is a widely used mechanism for the regulation of V-ATPase activity (23Zimmermann B. Dames P. Walz B. Baumann O. J. Exp. Biol. 2003; 206: 1867-1876Crossref PubMed Scopus (43) Google Scholar, 24Trombetta E.S. Ebersold M. Garrett W. Pypaert M. Mellman I. Science. 2003; 299: 1400-1403Crossref PubMed Scopus (564) Google Scholar, 25Sautin Y.Y. Lu M. Gaugler A. Zhang L. Gluck S.L. Mol. Cell. Biol. 2005; 25: 575-589Crossref PubMed Scopus (185) Google Scholar), the signaling cascades that trigger the association/dissociation process remain elusive. The salivary glands of the blowfly Calliphora vicina have served as a model system for the analysis of the regulation of V-ATPase reassembly and activation. V-ATPase activity in these tubiform glands is under the control of the hormone serotonin (5-hydroxytryptamine, 5-HT). In the presence of 5-HT, the V1 complex reallocates within minutes to the apical membrane and reassociates with the V0 complex, thus leading to the active holoenzyme (23Zimmermann B. Dames P. Walz B. Baumann O. J. Exp. Biol. 2003; 206: 1867-1876Crossref PubMed Scopus (43) Google Scholar). Recent results demonstrate that a 5-HT-induced increase in intracellular cAMP induces V-ATPase reassembly (26Dames P. Zimmermann B. Schmidt R. Rein J. Voss M. Schewe B. Walz B. Baumann O. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3926-3931Crossref PubMed Scopus (61) Google Scholar) and that protein kinase A (PKA) is the downstream target of cAMP in this scenario. 4J. Rein, M. Voss, B. Walz, and O. Baumann, unpublished results. 4J. Rein, M. Voss, B. Walz, and O. Baumann, unpublished results. These results lead to the assumption that one or more subunits of the V-ATPase become phosphorylated via PKA and that this phosphorylation may act as a trigger for holoenzyme formation in the blowfly salivary gland and possibly other systems. Regarding the reversible assembly/disassembly, the V1 subunit C is unique among V-ATPase subunits because it is released to the cytosol upon dissociation of the holoenzyme into its V1 and V0 complexes (21Kane P.M. FEBS Lett. 2000; 469: 137-141Crossref PubMed Scopus (64) Google Scholar, 27Merzendorfer H. Reinecke S. Zhao X.F. Jacobmeier B. Harvey W.R. Wieczorek H. Biochim. Biophys. Acta. 2000; 25: 369-379Crossref Scopus (42) Google Scholar). Subunit C is an elongated molecule (28Drory O. Frolow F. Nelson N. EMBO Rep. 2004; 5: 1148-1152Crossref PubMed Scopus (108) Google Scholar) that appears to bridge the V1 with the V0 complex (29Inoue T. Forgac M. J. Biol. Chem. 2005; 280: 27896-27903Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Moreover, subunit C binds to actin filaments, and this interaction may be involved in stabilizing the proton pump in its assembled state (30Vitavska O. Merzendorfer H. Wieczorek H. J. Biol. Chem. 2005; 280: 1070-1076Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 31Vitavska O. Wieczorek H. Merzendorfer H. J. Biol. Chem. 2003; 278: 18499-18505Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). These properties make subunit C suited to the control of V-ATPase reassembly state and to the mediation of the relevant cellular signals. Here we test the above-mentioned hypothesis and demonstrate that V-ATPase subunit C becomes phosphorylated by PKA. Animals and Preparation—M. sexta (Lepidoptera, Sphingidae) was reared at 27 °C under long day conditions (16 h light) at the University of Osnabrück. Blowflies (C. vicina) were reared at 25 °C under a light-dark cycle of 12 h:12 h at the University of Potsdam. At 1-3 weeks after the eclosion of the flies, the abdominal portions of their salivary glands were dissected in physiological saline containing 128 mm NaCl, 10 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 2.7 mm sodium glutamate, 2.7 mm malic acid, 10 mm d-glucose, and 10 mm Tris-HCl, pH 7.2. Reagents—ProQ Diamond phosphoprotein stain was obtained from Invitrogen, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (8-CPT-cAMP) was from Biolog LSI (Bremen, Germany), and H-89 was from Axxora (Grünberg, Germany). 5-HT, protease inhibitor cocktail (catalog no. P8340) and the catalytic subunit of bovine PKA were purchased from Sigma. [γ-32P]ATP was from GE Healthcare (Munich, Germany). Antibodies—Monospecific polyclonal antibodies directed against the recombinant subunit C from M. sexta were produced in guinea pigs (serum 488-1; Ref. 27Merzendorfer H. Reinecke S. Zhao X.F. Jacobmeier B. Harvey W.R. Wieczorek H. Biochim. Biophys. Acta. 2000; 25: 369-379Crossref Scopus (42) Google Scholar). On Western blots of blowfly salivary gland homogenates, these antibodies identified a single intense band at 42 kDa (Figs. 7 and 8) corresponding approximately to the molecular mass of Manduca subunit C (27Merzendorfer H. Reinecke S. Zhao X.F. Jacobmeier B. Harvey W.R. Wieczorek H. Biochim. Biophys. Acta. 2000; 25: 369-379Crossref Scopus (42) Google Scholar). Guinea pig polyclonal antibodies against γ-amino butyric acid (GABA) were from Biotrend (Cologne, Germany), rabbit antiserum against the Drosophila PKA catalytic subunit was from Daniel Kalderon (Columbia University, New York, NY), mouse antibody E7 against β-tubulin (32Chu D.T.W. Klymkowsky M.W. Dev. Biol. 1989; 136: 104-117Crossref PubMed Scopus (128) Google Scholar) was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA), alkaline phosphatase-conjugated anti-guinea pig antibodies were from Sigma, and horseradish-peroxidase-conjugated anti-guinea pig antibodies were from Jackson ImmunoResearch (West Grove, PA).FIGURE 3Subunits of the V1 complex are not phosphorylated by PKA. The 8 μm purified V1 complex from M. sexta was incubated with or without 0.7 μm PKA catalytic subunit (PKA-C). After centrifugation in a sucrose gradient, the fractions containing the V1 complex were collected and subjected to SDS-PAGE. As a control for the phosphorylation reaction, the 6 μm recombinant subunit C from M. sexta was incubated with the PKA catalytic subunit. The gels were stained with Coomassie Blue (A) after phosphoprotein staining with ProQ Diamond (B). A molecular mass marker (M) containing two phosphorylated proteins (ovalbumin, 45 kDa; β-casein, 24 kDa) was used. The positions of the V1 subunits are marked by capital letters (in A, the asterisk denotes a decay product of subunit B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Stimulus-induced phosphorylation inC. vicinasalivary glands. A cell lysate of blowfly salivary glands was incubated with 50 μm 8-CPT-cAMP for 30 min at 37 °C. The proteins were separated by SDS-PAGE and analyzed by ProQ Diamond phosphoprotein staining. Coomassie Blue staining of the gels confirms that similar amounts of protein were loaded. An immunoblot (WB) indicates the position of subunit C. After incubation with 8-CPT-cAMP, the phospho-signal at 42 kDa is enhanced (arrowhead).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8Effect of anti-subunit-C antibodies on PKA-dependent phosphorylation.C. vicina salivary glands were homogenized on ice and centrifuged. The supernatant was divided into equal volumes and preincubated without (-) or with (+) polyclonal guinea pig antiserum against subunit C (GPαC-sub) or GABA (GPαGABA). The mixtures were supplemented with 50 μm 8-CPT-cAMP and incubated for 15 min at 37 °C. The samples were subjected to SDS-PAGE and analyzed by ProQ Diamond and afterward by Coomassie Blue staining. An immunoblot (WB) indicates the position of subunit C. A magnification of the corresponding region in the phosphoprotein image shows that the 8-CPT-cAMP-induced increase in phospho-signal at 42 kDa (arrowhead) is largely blocked by incubation with GPαC-sub but not with GPαGABA. Note that the 8-CPT-cAMP-induced increase in phospho-signal of other proteins (arrow) is not influenced by GPαC-sub.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Preparation of the V1 Complex, the V1V0 Holoenzyme and the Recombinant Subunit C—The V1 complex as well as the V1V0 holoenzyme were purified from the posterior midgut of fifth instar larvae of M. sexta according to published procedures (33Gräf R. Harvey W.R. Wieczorek H. J. Exp. Biol. 1996; 271: 20908-20913Scopus (123) Google Scholar, 34Huss M. Ingenhorst G. König S. Gaβel M. Dröse S. Zeeck A. Altendorf K. Wieczorek H. J. Biol. Chem. 2002; 277: 40544-40548Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) with the modification that all buffers contained the protein phosphatase inhibitors fluoride (20 mm) and vanadate (1 mm). V1 complex without subunit C was prepared by gel chromatography in the presence of 25% methanol as described by Vitavska et al. (31Vitavska O. Wieczorek H. Merzendorfer H. J. Biol. Chem. 2003; 278: 18499-18505Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The V-ATPase subunit C from M. sexta was expressed in Escherichia coli BL 21 cells by using the pET-16b expression system from Novagen (EMD Biosciences, Madison, WI) and was purified by nickel-nitrilotriacetic acid affinity chromatography as described previously (27Merzendorfer H. Reinecke S. Zhao X.F. Jacobmeier B. Harvey W.R. Wieczorek H. Biochim. Biophys. Acta. 2000; 25: 369-379Crossref Scopus (42) Google Scholar). Protein determination was also performed as described previously (35Schweikl H. Klein U. Schindlbeck M. Wieczorek H. J. Biol. Chem. 1989; 264: 11136-11142Abstract Full Text PDF PubMed Google Scholar). Overlay Blots with Recombinant Subunit C—Samples containing 1.5 and 6 μg of PKA catalytic subunit were transferred onto a nitrocellulose membrane via the slot-blot technique; transfer was controlled by Ponceau S staining. After being blocked for 1 h at room temperature with 3% gelatin, the membrane was incubated with 0.35 μm recombinant subunit C from M. sexta for 1 h in the presence of 1% gelatin in TNNT buffer (20 mm Tris-HCl, pH 7.5, 0.5 m NaCl, 0.02% NaN3, and 0.05% Tween 20). Nonspecifically bound subunit C was removed by washing the membrane in TNNT buffer three times for 5 min each. For labeling of bound subunit C, the membrane was incubated for 1 h with anti-C antibodies at a dilution of 1:2,000 in 1% gelatin/TNNT buffer. After three washes of 5 min each in TNNT buffer, the membrane was incubated for 1 h with alkaline-phosphatase-conjugated anti-guinea pig antibodies at a dilution of 1:30,000 in 1% gelatin/TNNT buffer. The membrane was then washed as above, and the color reaction was performed with 0.34‰ nitro blue tetrazolium, 0.18‰ 5-bromo-4-chloro-3-indolyl phosphate in 50 mm Tris-HCl, pH 9.5, 0.1 m NaCl, and 50 mm MgCl2. Another membrane without incubation with recombinant subunit C was used as a negative control. Phosphorylation Assays—For experiments with [32P]ATP, 6 μm subunit C was incubated with 1.4 μm PKA catalytic subunit in the presence of 5 μCi of [γ-32P]ATP in 50 μl of PKA buffer (20 mm Na-Hepes, pH 7.5, 0.1 m NaCl, 4 mm MgCl2, 10 mm dithiothreitol, and 2 mm ATP) for 3 h at 30 °C. The reaction was stopped by adding 0.25 volumes 5× SDS sample buffer (625 mm Tris-HCl, pH 6.8, 25% sucrose, 10% SDS, 0.025% bromophenol blue, and 10% β-mercaptoethanol). After SDS-PAGE (17.4% total acrylamide concentration [T], 0.4% cross-linker concentration [C]) and Coomassie Blue staining, the gel was exposed to a phosphoscreen and finally analyzed by a phosphorimaging device (Molecular Dynamics, Sunnyvale, CA). In nonradioactive experiments, phosphoproteins were detected by the Pro-Q Diamond phosphoprotein stain. First, 8 μm V1 complex from M. sexta was incubated with or without 0.7 μm PKA catalytic subunit in PKA buffer for 3 h at 30 °C. To separate the PKA catalytic subunit from the V1 complex, the samples were loaded onto a discontinuous sucrose gradient (34Huss M. Ingenhorst G. König S. Gaβel M. Dröse S. Zeeck A. Altendorf K. Wieczorek H. J. Biol. Chem. 2002; 277: 40544-40548Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) in 16 mm Tris-HCl, pH 8.1, 0.32 mm EDTA, 0.2 m NaCl, 9.6 mm β-mercaptoethanol and centrifuged at 4 °C for 1.5 h at 310,000 × g. The fractions containing the V1 complex without the PKA catalytic subunit were collected and concentrated by precipitation with 20% trichloroacetic acid. The pellets were resuspended, heated in SDS sample buffer for 45 s at 95 °C, and loaded onto a gel. To control the phosphorylation reaction, 6 μm recombinant subunit C from M. sexta was incubated with or without 0.7 μm PKA catalytic subunit for 1 h at 30 °C. After SDS-PAGE as above, the gels were analyzed by Pro-Q Diamond phosphoprotein stain and afterward by Coomassie Blue staining. To check whether subunit C can be phosphorylated after its reassociation with the V1 complex, the V1 complex without subunit C was incubated with a 10-fold excess of recombinant subunit C for 12 h at 4 °C. V1 complex saturated with subunit C (V1C) was collected by gel chromatography on a Superdex 200 column. Then 5 μm V1C complex was incubated with 0.5 μm catalytic subunit of PKA for 2 h at 30 °C and afterward subjected to SDS-PAGE. The gels were stained with Pro-Q Diamond to detect phosphoproteins and afterward with Coomassie Blue. Isolated blowfly salivary glands were homogenized on ice in sample buffer A (20 mm Tris-HCl, pH 7.8, 0.1 m KCl, 10 mm MgCl2, 10 mm Na2ATP, 5 mm EGTA, 1% Triton X-100), supplemented with protease inhibitor cocktail according to the manufacturerʼns instructions and centrifuged for 15 min at 20,000 × g at 4 °C to remove cell debris. The resulting supernatant was divided into pools of equal volumes, supplemented with the respective test reagent(s) (50 μm 8-CPT-cAMP, antibodies), and incubated for 15 or 30 min at 37 °C. The preparations were mixed with sample buffer B (250 mm Tris-HCl, pH 6.8, 5% SDS, 10% β-mercaptoethanol, 10% glycerol) to give a final SDS concentration of 1% and were heated for 5 min at 70 °C. After SDS-PAGE (14.4% T, 0.4% C), phosphoproteins were stained with ProQ Diamond and imaged. Subsequently, the proteins were stained with Coomassie Blue. Two-dimensional Electrophoresis—Two-dimensional electrophoresis was carried out with Mini-PROTEAN 2D and Mini-PROTEAN II cell from Bio-Rad. Isoelectric focusing was performed at 20 °C in denaturating 5% polyacrylamide tube gels (8 m urea, 2% Triton X-100, 2% Servalyt 3/10) with a pH 3-10 gradient. Before the loading of samples, the pH gradient was established by pre-electrophoresis: for 10 min at 200 V, 15 min at 300 V, and 15 min at 400 V. Isolated blowfly salivary glands were incubated at room temperature for 5 min with the respective test reagent(s) (30 nm 5-HT, 100 μm 8-CPT-cAMP, and 50 μm H-89) diluted in physiological saline; a control group was bathed in physiological saline only. Subsequently, the glands were homogenized on ice in sample buffer A and centrifuged for 30 min at 120,000 × g at 4 °C. The supernatants were mixed with sample buffer B to give a final SDS concentration of 1.4% and heated for 5 min at 70 °C. For the alkylation of cysteine residues, iodacetamide was added to give a final concentration of 6%, and the solution was incubated for 45 min at 30 °C. The samples were diluted in sample buffer C (8 m urea, 2% CHAPS, 2% Servalyt 3/10, 0.0025% bromphenol blue) to give a final SDS concentration of 0.24% to substitute SDS by CHAPS. Proteins (from an equivalent of 5 glands/assay) were separated by the following voltage profile: 10 min at 500 V, 3.5 h at 750 V, and 1 h at 1000 V. Tube gels were rinsed in equilibration buffer (62.5 mm Tris-HCl, pH 6.8, 2.3% SDS, 10% glycerol, 0.0025% bromphenol blue) just before SDS-PAGE (10.3% T, 0.3% C). The proteins were electrotransferred onto polyvinylidene difluoride membranes and probed by Western blot analysis as described previously (23Zimmermann B. Dames P. Walz B. Baumann O. J. Exp. Biol. 2003; 206: 1867-1876Crossref PubMed Scopus (43) Google Scholar). Interaction of the V-ATPase Subunit C with the Catalytic Subunit of Protein Kinase A—To test our assumption that subunit C can be phosphorylated by PKA, we initially chose the V-ATPase from midgut plasma membranes of the tobacco hornworm, M. sexta, a useful model system for the investigation of this family of proteins. First, we checked whether a direct interaction of subunit C with the catalytic subunit of PKA could be detected by overlay blotting. We used the catalytic subunit of bovine heart PKA (with more than 80% amino acid sequence identity with the respective Drosophila enzyme) as an appropriate substitute for the insect PKA. After loading the PKA catalytic subunit onto nitrocellulose and incubation with or without recombinant V-ATPase subunit C from M. sexta, strong immuno-signals developed on the membrane incubated with subunit C as compared with the control membrane (Fig. 1). Interaction of the PKA catalytic subunit with the M. sexta subunit C was also confirmed by overlay blots after SDS-PAGE (not depicted). These results support the conclusion that the V-ATPase subunit C binds to the catalytic subunit of PKA and thus might also be a physiologically relevant substrate for phosphorylation. Phosphorylation of Subunit C by Protein Kinase A—Phosphorylation was investigated by incubation of subunit C with the catalytic subunit of PKA in the presence of [γ-32P]ATP. Fig. 2 demonstrates that, after SDS-PAGE and phosphorimaging analysis, subunit C is labeled with 32P in a PKA-dependent manner. The upper band at ∼42 kDa represents subunit C with a molecular mass of 44 kDa as deduced from the cDNA sequence, whereas the lower band corresponds to the PKA catalytic subunit, which has a molecular mass of about 40 kDa and is known to become autophosphorylated (36Steinberg R.A. Cauthron R.D. Symcox M.M. Shuntoh H. Mol. Cell. Biol. 1993; 13: 2332-2341Crossref PubMed Google Scholar). Phosphorylation of subunit C does not exclude the possibility that other V-ATPase subunits are also phosphorylated, especially in view of a recent report that a WNK (with no K (lysine)) kinase from Arabidopsis phosphorylates not only subunit C but also the V1 subunits A, G, and either B or H (37Hong-Hermesdorf A. Bruex A. Grüber A. Grüber G. Schumacher K. FEBS Lett. 2006; 580: 932-939Crossref PubMed Scopus (91) Google Scholar). Therefore, we tested whether other subunits of the V1 complex could also be phosphorylated by PKA. After incubation of the V1 complex with and without the PKA catalytic subunit, sucrose density gradient centrifugation was performed, and fractions containing only the V1 complex were analyzed by SDS-PAGE. Fig. 3 shows gels after Coomassie staining and after Pro-Q Diamond phosphoprotein staining. The latter stain has, compared with the use of radiolabeled ATP, the advantage that phosphorylated proteins can be detected without prior incubation with a protein kinase and ATP. Thus, Fig. 3 also demonstrates that the recombinant subunit C, which had been expressed in E. coli, is not phosphorylated a priori and remains nonphosphorylated after incubation with ATP in the absence of the PKA catalytic subunit (Fig. 3B). In the presence of the PKA catalytic subunit, however, a strong phospho-signal could be observed at subunit C (Fig. 3B). In the V1 complex purified from M. sexta, no subunit including subunit C was found to be phosphorylated, either in the absence or in the presence of the PKA catalytic subunit (Fig. 3B). Therefore, we suggest that the V1 complex purified from the midgut cytosol does not contain phosphorylated subunits and that subunits cannot be phosphorylated by PKA as long as they are part of the complex. This also applies to subunit C, which usually occurs in substoichiometric amounts in the isolated V1 complex (22Wieczorek H. Grüber G. Harvey W.R. Huss M. Merzendorfer H. Zeiske W. J. Exp. Biol. 2000; 203: 127-135Crossref PubMed Google Scholar). However, there could be a qualitative difference between the substoichiometric subunit C that remains bound to the V1 complex purified from M. sexta midgut and the subunit C that dissociates upon enzyme disassembly. Hence we reassociated the recombinant subunit C with the V1 complex from which we had removed before all of subunit C. The resultant V1C complex was then incubated with the catalytic subunit of PKA. Indeed, subunit C in this complex could be phosphorylated by PKA (Fig. 4), thus confirming our suspicion. Thus, the free cytosolic subunit C as well as subunit C reassociated with the V1 complex both appear to be competent for phosphorylation by PKA. After dissociation of the V1V0 holoenzyme, the majority of subunit C occurs freely in the cytosol (30Vitavska O. Merzendorfer H. Wieczorek H. J. Biol. Chem. 2005; 280: 1070-1076Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, it might be expected that the activation of PKA leads predominantly to the" @default.
• W2093961415 created "2016-06-24" @default.
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• W2093961415 date "2007-11-01" @default.
• W2093961415 modified "2023-09-26" @default.
• W2093961415 title "Stimulus-induced Phosphorylation of Vacuolar H+-ATPase by Protein Kinase A" @default.
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