Regulation of AMP Deaminase by Phosphoinositides
1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês
10.1074/jbc.274.36.25701
ISSN1083-351X
AutoresBrian Sims, Donna K. Mahnke-Zizelman, Adam A. Profit, Glenn D. Prestwich, Richard L. Sabina, Anne B. Theibert,
Tópico(s)Biochemical and Molecular Research
ResumoAMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a Ki of 110 nm. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)Xn(R/K)X KK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo. AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a Ki of 110 nm. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)Xn(R/K)X KK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo. Phosphoinositides and inositol polyphosphates (referred to collectively as inositides) are components of many pathways in eukaryotic cells, functioning in second messenger cascades, acting as regulators of many proteins, and operating as membrane localization signals (1Toker A. Cantley L. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1225) Google Scholar, 2Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (245) Google Scholar, 3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar). Numerous protein and lipid kinases, adaptor proteins, ion channels, phospholipases, modulators of small GTPases, and actin-binding proteins are regulated by inositides (1Toker A. Cantley L. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1225) Google Scholar, 2Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (245) Google Scholar, 3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar). To identify novel targets for inositides, our laboratories and others have used purification schemes employing affinity resins that contain tethered inositol polyphosphate head groups (3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar, 4Prestwich G.D. Acc. Chem. Res. 1996; 29: 503-513Crossref Scopus (107) Google Scholar, 5Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar, 6Voglmaier S.M. Keen J.H. Murphy J. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar, 7Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 8Tanaka K. Imajoh-Ohmi S. Sawada T. Shirae R. Hashimoto Y. Iwasaki S. Kaibuchi K. Kanaho Y. Shirai T. Terada Y. Kimura K. Nagata S. Fukui Y. Eur. J. Biochem. 1997; 245: 512-519Crossref PubMed Scopus (82) Google Scholar, 9Kanematsu T. Misumi Y. Watanabe Y. Ozaki S. Koga T. Iwanage S. Ikehara Y. Hirata M. Biochem. J. 1996; 313: 319-325Crossref PubMed Scopus (90) Google Scholar). These affinity purifications were successful in the identification of inositide binding in the clathrin adaptor/assembly protein AP-2 (6Voglmaier S.M. Keen J.H. Murphy J. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar), centaurin α (7Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), a centaurin α orthologue (8Tanaka K. Imajoh-Ohmi S. Sawada T. Shirae R. Hashimoto Y. Iwasaki S. Kaibuchi K. Kanaho Y. Shirai T. Terada Y. Kimura K. Nagata S. Fukui Y. Eur. J. Biochem. 1997; 245: 512-519Crossref PubMed Scopus (82) Google Scholar), and a phospholipase C (PLC)1-related protein (9Kanematsu T. Misumi Y. Watanabe Y. Ozaki S. Koga T. Iwanage S. Ikehara Y. Hirata M. Biochem. J. 1996; 313: 319-325Crossref PubMed Scopus (90) Google Scholar). In addition to AP-2 and centaurin α, we isolated several other proteins from rat brain, one of which was approximately 80 kDa (5Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar). Published reports show that inositol polyphosphates modulate the activity of the enzyme AMP deaminase (EC 3.5.4.6) (AMPD) (10Yoshino M. Kawamura Y. Fujisawa K. Ogasawara N. J. Biochem. (Tokyo). 1976; 80: 309-313Crossref PubMed Scopus (13) Google Scholar, 11Spychala J. Biochem. Biophys. Res. Commun. 1987; 148: 106-111Crossref PubMed Scopus (4) Google Scholar), an enzyme family whose endogenous, purified subunit molecular masses are between 66 and 88 kDa. Therefore, we hypothesized that AMPD could be the 80-kDa protein isolated using the inositide affinity resin. AMPD is a diverse and highly regulated enzyme located at a branchpoint in the adenine nucleotide catabolic pathway and is important in regulating nucleotide pools. AMPD is also a component of the purine nucleotide cycle, an energy-generating pathway reportedly operative in many animal tissues (reviewed in Ref. 12Van Waarde A. Biol. Rev. 1988; 63: 259-298Crossref PubMed Google Scholar). The AMPD1 gene encodes human isoform M and rat isoform A (13Sabina R.L. Morisaki T. Clarke P. Eddy R. Shows T.B. Morton C.C. Holmes EW. J. Biol. Chem. 1990; 265: 9423-9433Abstract Full Text PDF PubMed Google Scholar); the AMPD2 gene encodes the human isoform L and rat isoform B (14Bausch-Jurken M.T. Mahnke-Zizelman D.K. Morisaki T. Sabina R.L. J. Biol. Chem. 1992; 267: 22407-22413Abstract Full Text PDF PubMed Google Scholar, 15Morisaki T. Sabina R.L. Holmes E.W. J. Biol. Chem. 1990; 265: 11482-11486Abstract Full Text PDF PubMed Google Scholar); and the AMPD3 gene encodes the human isoform E and the rat isoform C (16Mahnke-Zizelman D.K. Sabina R.L. J. Biol. Chem. 1992; 267: 20866-20877Abstract Full Text PDF PubMed Google Scholar, 17Mahnke-Zizelman D.K. D'Cunha J. Wojnar J. Brogley M.A. Sabina R.L. Biochem. J. 1997; 326: 521-529Crossref PubMed Scopus (18) Google Scholar). A single AMPD gene has also been identified in yeast (18Meyer S.L. Kvalnes-Krick K.L. Schramm V.L. Biochemistry. 1989; 28: 8734-8743Crossref PubMed Scopus (29) Google Scholar). All human AMPD isoforms contain similar C-terminal regions and substantially divergent N-terminal domains. The AMPD1 isoform is found almost exclusively in skeletal muscle, whereas the AMPD2 and AMPD3 isoforms are widely expressed in many tissues and cells (19Ogasawara N. Goto H. Yamada Y. Watanabe T. Eur. J. Biochem. 1978; 87: 297-304Crossref PubMed Scopus (72) Google Scholar, 20Ogasawara N. Goto H. Yamada Y. Watanabe T. Asano T. Biochim. Biophys. Acta. 1982; 714: 298-306Crossref PubMed Scopus (96) Google Scholar), including mammalian brain (21Ogasawara N. Goto H. Watanabe T. FEBS Lett. 1975; 58: 245-248Crossref PubMed Scopus (22) Google Scholar,22Sims B Powers R.E. Sabina R.L. Theibert A.B. Neurobiol. Aging. 1998; 19: 385-391Crossref PubMed Scopus (25) Google Scholar). AMPD activity is highly regulated through interactions with other proteins (23Ashby B. Frieden C. J. Biol. Chem. 1978; 253: 8728-8735Abstract Full Text PDF PubMed Google Scholar, 24Rundell K.W. Tullson P.C. 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In particular, nonskeletal muscle AMPD activities are regulated by fatty acids, fatty acyl coenzyme As, phosphatidic acid, and phosphatidylcholine (PC) with Ki values ranging from 10 to 100 μm (33Skladanowski A. Kaletha K. Zydowo M. Int. J. Biochem. 1978; 9: 43-45Crossref PubMed Scopus (9) Google Scholar, 34Yoshino M. Miyajima E. Tsushima K. J. Biol. Chem. 1979; 254: 1521-1525Abstract Full Text PDF PubMed Google Scholar, 35Yoshino M. Miyajima E. Tsushima K. FEBS Lett. 1976; 72: 143-146Crossref PubMed Scopus (17) Google Scholar, 36Tanfani F. Kossowska E. Purzycka-Preis J. Zydowo M.M. Wozniak M. Tartaglini E. Bertoli E. Biochem. J. 1993; 291: 921-926Crossref PubMed Scopus (10) Google Scholar, 37Raffin J.P. Purzycka-Preis J. Prus E. Wozniak M. Zydowo M. Comp. Biochem. Physiol. B: Comp. Biochem. 1985; 80: 685-692Crossref PubMed Scopus (3) Google Scholar, 38Prus E. Zydowo M. Int. J. Biochem. 1983; 15: 1169-1173Crossref PubMed Scopus (5) Google Scholar, 39Wozniak M. Kossowska E. Purzycka-Preis J. Zydowo M. Biochem. J. 1988; 255: 977-981Crossref PubMed Scopus (7) Google Scholar). The high concentrations required for these modulatory effects suggest that these lipid interactions may be low affinity or nonspecific. However, the interaction with lipids may prove to be physiologically relevant. Whereas hydropathy analysis suggests that AMPD isoforms do not contain putative transmembrane spanning domains, the AMPD3 enzyme can associate with erythrocyte membrane fractions (40Pipoly G.M. Nathens G.R. Chang D. Deuel T.F. J. Clin Invest. 1979; 63: 1066-1076Crossref PubMed Scopus (12) Google Scholar, 41Rao N. Hara L. Askari A. Biochim. Biophys. Acta. 1968; 151: 651-654Crossref PubMed Scopus (25) Google Scholar). In this report, we provide several independent lines of evidence that the specific, high affinity interaction between the AMPD3 isoform and phosphoinositides constitutes an important mechanism for enzyme regulation and localization. Aminopropyl-InsP4 affigel, (d,l)-1-O-[125I][N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]-myo-inositiol 3,4,5-trisphosphate (ASA-InsP4), and 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-myo-inositol 3,4,5-trisphosphate (BZDC-InsP4) were synthesized as described (3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar, 4Prestwich G.D. Acc. Chem. Res. 1996; 29: 503-513Crossref Scopus (107) Google Scholar, 5Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar). PC, PS, and PE were from Avanti Polar Lipids (Alabaster, AL). PtdIns(4,5)P2, PtdIns(4)P, and PtdIns were from Sigma. PtdIns(3,4,5)P3, PtdIns(3, 4)P2, and PtdIns(3)P were from Matreya Inc. (Pleasant Gap, PA) or were gifts from Echelon Research Laboratories Inc. (Salt Lake City, UT). All other reagents were from Sigma. The purification was performed as described (3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar, 5Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar, 6Voglmaier S.M. Keen J.H. Murphy J. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar, 7Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) with the following minor modifications. Rat brain supernatant or CHAPS extracted membranes were incubated with heparin-agarose (1 ml of resin/rat brain) overnight, washed with 250 mm NaCl in Prep buffer (PB; 25 mm Tris, pH 7.7, 1 mm EDTA, 1 mmEGTA, 1 mm β-mercaptoethanol, 250 mg/ml CBZ-phenylalanine, 100 mg/ml phenylmethylsulfonyl fluoride, 5 mg/liter each chymostatin, antipain, and pepstatin, 10 mg/liter aprotinin, and 10 mg/liter leupeptin), and eluted with 1.5 m NaCl in PB for 1 h. Heparin-agarose eluates were concentrated using Centriprep10 (Amicon Corp., Beverely, MA) to a final volume of 1–2 ml/10–15 rat brains, diluted with 50 mm Tris, pH 7.4, 1 mm EDTA, and loaded on an aminopropyl-InsP4column on an fast performance liquid chromatography (Amersham Pharmacia Biotech; column dimensions, 10 × 3 cm) at a rate of 0.2 ml/min. The column was washed with 10 ml of 0.2 m NaCl and eluted with a NaCl gradient of 0.2–2 m NaCl. Wild type and N-terminally truncated (ΔM90) human AMPD3 recombinant enzymes were produced in Sf9 (Spodoptera frugiperda) insect cells using a baculoviral expression system and partially purified by phosphocellulose chromatography as described previously (42Mahnke-Zizelman D.K. Tullson P.C. Sabina R.L. J. Biol. Chem. 1998; 273: 35118-35125Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). AMPD activity was determined using a phenol/hypochlorite reaction (11Spychala J. Biochem. Biophys. Res. Commun. 1987; 148: 106-111Crossref PubMed Scopus (4) Google Scholar). The reaction contained 25 mm sodium citrate, pH 6.0, 50 mm potassium chloride, 10 mm AMP, and samples were incubated at 37 °C for 10 min, followed by addition of 2.5 ml of 100 mmphenol, 200 mm sodium nitroprusside in H2O, 2.5 ml of 125 mm sodium hydroxide, 200 mm dibasic sodium phosphate, 0.1% sodium hypochlorite. Absorbance was measured at 625 nm. Absolute ammonia was determined with ammonium sulfate. For kinetic determinations, AMPD activity was assayed using a high pressure liquid chromatography method to separate substrate (AMP) from product (IMP) as described previously (42Mahnke-Zizelman D.K. Tullson P.C. Sabina R.L. J. Biol. Chem. 1998; 273: 35118-35125Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Photolabeling of aminopropyl-InsP4 purified fractions using [125I]ASA-InsP4 (see Fig. 1) was performed as described in Refs. 3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar, 5Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar, and 7Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar. For experiments described in Fig. 2, human AMPD3 recombinant protein (2 μg) was incubated with [3H]BZDC-InsP4 or [3H]BZDC-PtdIns(4,5)P2 in 25 mmTris-HCl, pH 7.4, 1 mm EDTA, and 1 mm potassium phosphate and exposed to UV light (366 nm) for 60 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis. Gels were fluorographed using Entensify (NEN Life Science Products), dried, and exposed to Hyperfilm (Amersham Pharmacia Biotech) at −70 °C for 14 days.Figure 2Photoaffinity labeling of human AMPD3 recombinant protein with inositide-based probes. Photolabeling was performed with [3H]BZDC-Ins(1,3,4,5)P4(A–C) or [3H]BZDC-PtdIns(4,5)P2(D). The Total lane indicates the labeling in the presence of photolabel only. Competition including various unlabeled inositides or phospholipids is shown. Representative autorads are presented; photolabeling was performed in three independent experiments with comparable results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Samples (2–15 μg) were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (Schleicher & Schuell), and probed with a rabbit polyclonal antibody to the recombinant AMPD3 isoform (42Mahnke-Zizelman D.K. Tullson P.C. Sabina R.L. J. Biol. Chem. 1998; 273: 35118-35125Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were detected using 3,3′-diaminobenzidine. Immunoblot data were quantified by densitometry using a Bio-Rad model GS-670 Imaging Densitometer. Binding of AMPD to unilamellar mixed micelles was performed as described in Ref. 43Bromann P.A. Boetticher E.E. Lomasney J.W. J. Biol. Chem. 1997; 272: 16240-16246Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar. 400 μg of PE was added to various concentrations of other phospholipids and dried under N2. The lipids were resuspended in 1 ml of 180 mm sucrose, pelleted at 10,000 × g for 15 min, and redissolved in 1 ml of 50 mm HEPES, 1 mm EDTA, and 1 mm EGTA. Human AMPD3 recombinant protein (2 μg) was added to the micelles for 30 min at 25 °C, and centrifuged at 400,000 × g for 40 min. The pellets were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with the anti-AMPD3 antibody. PC12 cells were cultured on 10-cm culture plates and grown to confluency in cell culture media (RPMI, fetal bovine serum, horse serum, penicillin/streptomycin, andl-glutamine). Cells were collected by trituration, centrifuged at 1000 × g for 5 min, and lysed by sonication, and the supernatants and membranes were separated by centrifugation at 12,000 × g for 5 min. Phosphoinositide levels were determined as described in Ref. 44Li Y. Lombardini J.B. Brain Res. 1991; 553: 89-96Crossref PubMed Scopus (38) Google Scholar. PC12 cells were preincubated with 10 mm taurine for 2 h, and then 30 μCi of [γ-32P]ATP was added to the culture media and incubated at 37 °C for 3 h. Lipids were extracted with 1 ml of chloroform-methanol (1:2 v/v) and 0.25 ml of 10 mm EDTA, pH 7.4, and centrifuged at 1000 × g for 5 min. The organic layer was removed, washed with 0.125 ml of 2.4 mHCl and 1 ml of CH3OH-H20 (1:1 v/v) and centrifuged for 5 min. The organic layer was removed, dried under N2, and dissolved in chloroform-methanol (2:1 v/v). Lipids were separated using Whatman (Maidstone, UK) silica gel plates with chloroform/methanol/4 m NH4OH (90:65:20 v/v/v). Using an aminopropyl-InsP4 resin to purify inositide-binding proteins from rat brain, we previously identified a protein at approximately 80 kDa that eluted in fractions containing the clathrin adaptor/assembly protein AP-2 (3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar, 5Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar, 6Voglmaier S.M. Keen J.H. Murphy J. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar, 45Profit A.A. Chen J. Gu Q.-M. Chaudhary A. Prasad K. Lafer E.M. Prestwich G.D. Arch. Biochem. Biophys. 1998; 357: 85-94Crossref PubMed Scopus (10) Google Scholar). Published reports have shown that AMPD, an enzyme family whose native purified subunit molecular masses are between 66–88 kDa, is regulated by inositol polyphosphates (10Yoshino M. Kawamura Y. Fujisawa K. Ogasawara N. J. Biochem. (Tokyo). 1976; 80: 309-313Crossref PubMed Scopus (13) Google Scholar, 11Spychala J. Biochem. Biophys. Res. Commun. 1987; 148: 106-111Crossref PubMed Scopus (4) Google Scholar). Therefore we tested whether AMPD activity was enriched in fractions containing the 80-kDa protein eluted from the aminopropyl-InsP4 resin (Fig.1 A). All of the AMPD activity bound to the resin, and a symmetrical peak of AMPD activity was eluted in the fractions containing AP-2 and the 80-kDa protein. Similar to AP-2, the 80-kDa protein in the peak activity fractions incorporated an [125I]ASA-InsP4 photoaffinity label (Fig.1 B, odd fractions). Photolabeling was specific because including 30 μm unlabeled Ins(1,3,4,5)P4 displaced the label (Fig. 1 B, even fractions). By batch purification, the aminopropyl-InsP4 resin gave a 71-fold purification with a specific activity of 917.1 nmol/min/mg and an 86% yield. Immunoblot analysis of batch eluted aminopropyl-InsP4 eluate with an anti-AMPD3 serum (Fig. 1 C) indicated a single strongly immunoreactive band at approximately 80 kDa. Because both AMPD2 and AMPD3 are expressed in brain (21Ogasawara N. Goto H. Watanabe T. FEBS Lett. 1975; 58: 245-248Crossref PubMed Scopus (22) Google Scholar, 22Sims B Powers R.E. Sabina R.L. Theibert A.B. Neurobiol. Aging. 1998; 19: 385-391Crossref PubMed Scopus (25) Google Scholar) and all the AMPD activity was recovered in a single peak, the AMPD2 and AMPD3 isoforms appear to behave similarly in this purification. Thus, comparable with other high affinity inositide-binding proteins, endogenous rat brain AMPD is effectively purified using an inositide affinity resin (3Theibert A.B. Prestwich G.D. Jackson T. Hammonds-Odie L. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 117-150Google Scholar, 4Prestwich G.D. Acc. Chem. Res. 1996; 29: 503-513Crossref Scopus (107) Google Scholar, 5Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar, 6Voglmaier S.M. Keen J.H. Murphy J. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar, 7Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 8Tanaka K. Imajoh-Ohmi S. Sawada T. Shirae R. Hashimoto Y. Iwasaki S. Kaibuchi K. Kanaho Y. Shirai T. Terada Y. Kimura K. Nagata S. Fukui Y. Eur. J. Biochem. 1997; 245: 512-519Crossref PubMed Scopus (82) Google Scholar, 9Kanematsu T. Misumi Y. Watanabe Y. Ozaki S. Koga T. Iwanage S. Ikehara Y. Hirata M. Biochem. J. 1996; 313: 319-325Crossref PubMed Scopus (90) Google Scholar, 45Profit A.A. Chen J. Gu Q.-M. Chaudhary A. Prasad K. Lafer E.M. Prestwich G.D. Arch. Biochem. Biophys. 1998; 357: 85-94Crossref PubMed Scopus (10) Google Scholar,46Prestwich G.D. Chaudhary A. Chen J. Feng L. Mehrotra B. Peng J. Bruzik K. Advances in Phosphoinositides. American Chemical Society, Washington, D.C.1998: 24-37Google Scholar). Recent advances in recombinant expression of human AMPD cDNAs have provided larger quantities of higher purity enzymes than can be obtained from endogenous sources (42Mahnke-Zizelman D.K. Tullson P.C. Sabina R.L. J. Biol. Chem. 1998; 273: 35118-35125Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Therefore, the human AMPD3 recombinant enzyme was used to determine the specificity and affinity of inositide binding in AMPD. AMPD was specifically labeled using a high efficiency [3H]BZDC-Ins(1,3,4,5)P4 photoprobe (Fig.2, A–C, Total). A variety of inositides and phospholipids were tested for displacement of the photolabel. PtdIns(4,5)P2 and PtdIns(4)P were the most potent inhibitors of photolabeling, with 50% displacement of the label (IC50) effected by addition of 250 nm (Fig. 2, A and B). PtdIns(3,4,5)P3 was weaker, with an IC50 between 1–2 μm (Fig.2 B). Ins(1,3,4,5)P4 and inositol (1,2,3,4,5,6)-hexakisphosphate were substantially less effective and displaced the photolabel only when added at 10 μm (Fig.2 B). Phosphatidylinositol (PI) and PS displaced the photolabel when added at concentrations greater than 10 μm (Fig. 2 C), whereas PE or PC did not displace the label even at 30 μm. AMPD could also be photolabeled with a [3H]BZDC-PtdIns(4,5)P2probe (Fig. 2 D). Lower concentrations of PtdIns(4,5)P2 were required to effect displacement in this label (IC50 = 100 nm) presumably because lower concentrations of the [3H]BZDC-PtdIns(4,5)P2label were used. These data show that the human AMPD3 recombinant enzyme binds phosphoinositides with high affinity, with both the inositol head group and glycerolipid moieties essential for high affinity binding. To ensure that differences in lipid accessibility or micellar size were not the basis for the apparent specificity differences observed, we examined the interaction of AMPD3 recombinant protein with unilamellar mixed lipid micelles. Binding of AMPD to mixed micelles that contained PE as the core lipid and increasing concentrations of either PtdIns(4,5)P2, PS, PI, or PC, is shown in Fig.3. A fraction of the human AMPD3 recombinant enzyme consistently associated with the PE micelle pellet, presumably through a low affinity interaction with PE that is present in high concentrations in the micelles. However, addition of PtdIns(4,5)P2 to the PE micelles produced a substantial increase in the binding of AMPD to the micelles. The increase in AMPD associated with the lipid micelle was concentration-dependent and saturable with half-maximal association observed at approximately 200 nm. In contrast, addition of PS, PI, or PC to the PE micelles did not lead to any additional increase in association of AMPD compared with PE alone, indicating that the enhanced binding was selective for PtdIns(4,5)P2. These data demonstrate that the human AMPD3 recombinant enzyme recognizes PtdIns(4,5)P2 in a mixed micelle with an affinity and selectivity similar to that determined in the photoaffinity labeling. We next examined the effect of phosphoinositides on AMPD catalytic activity. Phosphoinositides potently inhibit human AMPD3 recombinant activity in a dose-dependent manner (Fig.4). PtdIns(4,5)P2 was the most effective inhibitor of enzyme activity, with an IC50of approximately 100 nm (Fig. 4 A, open symbols). The only other phospholipids that inhibited activity in the submicromolar range were phosphoinositides (Fig. 4, B and C). PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, and PtdIns(3,4,5) were between 1.5- and 3-fold less potent than PtdIns(4,5)P2 but did not effect complete inhibition even at 3 μm. Other phospholipids, such as PI, PC, and PS, produced either no or only slight (<20%) inhibition when the concentration was increased to 3 μm (Fig. 4, C and D). Inositol-1,4,5-trisphosphate, Ins(1,3,4,5)P4, and inositol (1,2,3,4,5,6)-hexakisphosphate had no effect on activity, even at 30 μm (Fig.4 D). Endogenous rat brain AMPD is also potently inhibited by phosphoinositides (Fig. 4 A, closed symbols). Phosphoinositides were approximate
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