An Adjacent Pair of Human NUDT Genes on Chromosome X Are Preferentially Expressed in Testis and Encode Two New Isoforms of Diphosphoinositol Polyphosphate Phosphohydrolase
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m205476200
ISSN1083-351X
AutoresKiyoshi Hidaka, James J. Caffrey, Len Hua, Tong Zhang, John R. Falck, Gabrielle Nickel, Laura Carrel, Larry D. Barnes, Stephen B. Shears,
Tópico(s)RNA Research and Splicing
ResumoCombinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3α and 3β, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the α form is replaced by Arg-89 in the β form, making the latter ∼2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (kcat/Km = 0.2 × 105m−1s−1) compared with diphosphoinositol polyphosphates (kcat/Km = 2–40 × 105m−1 s−1). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40–50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. ThehDIPP3 genes (NUDT10 encodes hDIPP3α;NUDT11 encodes hDIPP3β) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover. Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3α and 3β, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the α form is replaced by Arg-89 in the β form, making the latter ∼2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (kcat/Km = 0.2 × 105m−1s−1) compared with diphosphoinositol polyphosphates (kcat/Km = 2–40 × 105m−1 s−1). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40–50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. ThehDIPP3 genes (NUDT10 encodes hDIPP3α;NUDT11 encodes hDIPP3β) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover. diphosphoinositol polyphosphate phosphohydrolases diadenosine 5′,5‴-P1,P6-hexaphosphate digoxigenin human diphosphoinositol polyphosphate phosphohydrolase (NUDT, (nucleoside diphosphate attached to a moietyX)-type motif) diphosphoinositol polyphosphate synthase dithiothreitol diphosphoinositol pentakisphosphate bis-diphosphoinositol tetrakisphosphate inositol hexakisphosphate nucleotide, NTS, non-translated sequence green fluorescent protein 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 4-morpholineethanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid high pressure liquid chromatography Hydrolases containing the GX5EX7REUXEEXGU 1U represents a hydrophobic residue. "Nudix-type" motif comprise a protein superfamily whose members have been proposed to act as "surveillance enzymes" (1Abel K. Anderson R.A. Shears S.B. J. Cell Sci. 2002; 114: 2207-2208Google Scholar, 2Xu W.L. Shen J.Y. Dunn C.A. Desai S. Bessman M.J. Mol. Microbiol. 2001; 39: 286-290Crossref PubMed Scopus (58) Google Scholar) that function both to eliminate potentially toxic metabolites from the cell as well as to regulate concentrations and availability of substrates, cofactors, and signaling molecules (3Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Almost all of the substrates for these hydrolases are nucleotide diphosphates. The unique exception is provided by a subgroup of phosphohydrolases (DIPPs)2 that preferentially attack diphosphoinositol polyphosphates (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar). The diphosphoinositol polyphosphates provide the most extreme example of the degree to which phosphate groups can be clustered in high density around the inositol ring. These compounds are formed when specific monoester phosphate groups on Ins(1,3,4,5,6)P5 and InsP6 are converted to diphosphates, by enzymes known as diphosphoinositol polyphosphate synthases (DINS). For example, InsP6 is phosphorylated to PP-InsP5 and [PP]2-InsP4, which contain either one or two diphosphate groups, respectively (6Menniti F.S. Miller R.N. Putney J.W., Jr. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar, 7Stephens L.R. Radenberg T. Thiel U. Vogel G. Khoo K.-H. Dell A. Jackson T.R. Hawkins P.T. Mayr G.W. J. Biol. Chem. 1993; 268: 4009-4015Abstract Full Text PDF PubMed Google Scholar). The DIPPs rapidly degrade the diphosphates back to their monoester precursors. This phosphate release relieves some of the severity of the electrostatic and steric constraints imposed upon these polyphosphorylated molecules, and so a substantial and physiologically purposeful free-energy change ensues. Indeed, evidence has accumulated that this DINS- and DIPP-catalyzed substrate cycling contributes to the control of vesicle trafficking. Initially, this hypothesis arose from observations we made several years ago (8Ali N. Duden R. Bembenek M.E. Shears S.B. Biochem. J. 1995; 310: 279-284Crossref PubMed Scopus (47) Google Scholar, 9Fleischer B. Xie J. Mayrleitner M. Shears S.B. Palmer D.J. Fleischer S. J. Biol. Chem. 1994; 269: 17826-17832Abstract Full Text PDF PubMed Google Scholar, 10Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) showing that the diphosphoinositol polyphosphates bind with high affinity to clathrin-dependent and clathrin-independent adaptor proteins. More recently, disruption of theKCS1 gene in yeast, which encodes DINS activity (11Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), was shown to impede vacuolar morphogenesis in a manner that likely represents a vesicle trafficking defect (12Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Dubois E. Scherens B. Vierendeels F., Ho, M.W.Y. Messenguy F. Shears S.B. J. Biol. Chem. 2002; 277: 23755-23763Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Several additional functions have been ascribed to diphosphoinositol polyphosphates. For example, in yeast, the turnover of diphosphoinositol polyphosphates plays roles in homologous recombination (14Saiardi A. Nagata E. Luo H.R. Snowman A.M. Snyder S.H. J. Biol. Chem. 2001; 276: 39179-39185Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the maintenance of cell wall integrity (13Dubois E. Scherens B. Vierendeels F., Ho, M.W.Y. Messenguy F. Shears S.B. J. Biol. Chem. 2002; 277: 23755-23763Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and in mediating cellular responses to environmental stress (13Dubois E. Scherens B. Vierendeels F., Ho, M.W.Y. Messenguy F. Shears S.B. J. Biol. Chem. 2002; 277: 23755-23763Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The g5R gene in the African swine fever virus was recently found to encode an active diphosphoinositol polyphosphate phosphohydrolase, suggesting a role for these compounds in viral morphogenesis (15Cartwright J.L. Safrany S.T. Dixon L.K. Darzynkiewicz E. Stepinski J. Burke R. McLennan A.G. J. Virol. 2002; 76: 1415-1421Crossref PubMed Scopus (35) Google Scholar). Finally, genetic manipulation of the turnover of diphosphoinositol polyphosphates in ovarian carcinoma cells affects apoptotic processes (16Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). To rationalize how such diverse biological processes can all be regulated by diphosphoinositol polyphosphates, it has been proposed that these compounds are ligands that modify the functions of certain proteins (11Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 17Voglmaier S.M. Bembenek M.E. Kaplin A.I. Dormán G. Olszewski J.D. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4305-4310Crossref PubMed Scopus (131) Google Scholar). In such an event, polyphosphate turnover can be envisaged to comprise a molecular switch (11Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 17Voglmaier S.M. Bembenek M.E. Kaplin A.I. Dormán G. Olszewski J.D. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4305-4310Crossref PubMed Scopus (131) Google Scholar, 18Shears S.B. Cell. Signal. 2001; 13: 151-158Crossref PubMed Scopus (165) Google Scholar), akin to G-proteins that function as a binary switch between two interconvertible GTP-bound active and GDP-bound inactive states. The rate of turnover of diphosphoinositol polyphosphates may be regulated in a combinatorial manner by cell- and developmental-specific expression of the various isoforms of DIPP and DINS. For example, three distinct DINS enzymes are known to phosphorylate InsP6 to PP-InsP5, and these proteins differ in both catalytic activity and subcellular localization (11Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 14Saiardi A. Nagata E. Luo H.R. Snowman A.M. Snyder S.H. J. Biol. Chem. 2001; 276: 39179-39185Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). As for the phosphohydrolases, we have described previously (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar) three human isoforms of DIPP, which we named type 1, type 2α, and type 2β. These three DIPP isoforms each have differing catalytic efficiencies, and their expression is apparently regulated by a variety of molecular processes (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). For example, the two type 2 proteins are transcribed from a single gene by a specialized form of alternate mRNA splicing known as intron boundary skidding (19Caffrey J.J. Shears S.B. Gene (Amst.). 2001; 269: 53-60Crossref PubMed Scopus (9) Google Scholar). Moreover, both hDIPP2α and hDIPP2β are translated from an array of mRNA transcripts of differing lengths which may have distinct turnover rates (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We now add to the range of enzymes regulating diphosphoinositol polyphosphate turnover with the molecular and biochemical characterization of a new pair of hDIPP isoforms, type 3α and type 3β. All DNA probes used in this study were generated with a PCR DIG Probe Synthesis kit (Roche Molecular Biochemicals) using the indicated PCR primers and were labeled with digoxigenin-dUTP. Cycling conditions are as follows: 94 °C/5 min, followed by 30 cycles of 94 °C/30 s, 55 °C/30 s, 72 °C/2 min; 72 °C/7 min. Reactions were subsequently held at 4 °C until analysis. Probe P1 was generated using primers (5′-GAAGTTCAAGCCCAACC-3′ (sense) and 5′-CAAGGCATTATTATCCGG-3′ (antisense)) that amplify the DNA sequence encoding amino acids 3–165 of the hDIPP2α protein (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Four master membranes containing the arrayed Human Universal cDNA Library (Stratagene, La Jolla, CA) were probed overnight as described below. The blots were washed twice for 15 min each with 0.5× SSC, 0.1% SDS at 50 °C, and hybridizing signals were detected by chemiluminescence. On these blots, each primary signal represents a pool of 96 cDNA clones; secondary blots consisting of each of these 96 cDNAs individually arrayed were probed as described below, and positive clones were then purchased from Stratagene as pure DNA stocks. PCR cycle sequencing of both strands of each cDNA clone was performed with AmpliTaq DNA Polymerase FS and dRhodamine chemistry (Applied Biosystems Inc., Foster City, CA) using universal and clone-specific primers. All clones analyzed, except one, encoded hDIPP2 open reading frames. The exception, a 2020-bp cDNA, contained an open reading frame that we designate as hDIPP3α. The cDNA clones are in the cloning vector pT7T3-18U, and each clone contains a 5′ poly(C) and a 3′ poly(A) tract, on the sense strand, as a result of the cloning strategy used to generate the library. The 5′ end of the hDIPP3α clone is reported, therefore, as the first non-C nucleotide sequenced. A single nucleotide mutation of hDIPP3α was performed with the QuickChange Site-directed Mutagenesis kit (Stratagene) with mutation oligonucleotides: 5′-CAGAACCAGGACCGCAAGCACAGAACGTACG-3′ (sense) and 5′-CGTTCTGTGCTTGCGGTCCTGGTTCTGTTCG-3′ (antisense). This results in a cDNA that codes for hDIPP3β, in which Arg-89 replaces Pro-89 in hDIPP3α. The coding region of hDIPP3α cDNA, subcloned into pBluescript II SK−, was used as a template for this reaction. The sequence of the mutated cDNA was confirmed using T3 and T7 primers, with the Big-Dye terminator cycle sequencing kit (Applied Biosystems, Inc.) and a Prism 377 genetic analyzer (Applied Biosystems, Inc). Earlier studies (20Brown C.J. Carrel L. Willard H.F. Am. J. Hum. Genet. 1997; 60: 1333-1343Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 21Carrel L. Cottle A.A. Goglin K.C. Willard H.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14440-14444Crossref PubMed Scopus (305) Google Scholar) describe the panel of mouse/human somatic cell hybrids containing either active (Xa) or inactive (Xi) chromosome X. Total RNA was isolated and reverse-transcribed (20Brown C.J. Carrel L. Willard H.F. Am. J. Hum. Genet. 1997; 60: 1333-1343Abstract Full Text PDF PubMed Scopus (94) Google Scholar), and X-linked gene expression was examined using cDNA corresponding to either 250 ng of total RNA for a positive control transcript (SHGC-32313) that escapes inactivation (21Carrel L. Cottle A.A. Goglin K.C. Willard H.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14440-14444Crossref PubMed Scopus (305) Google Scholar) or 250 ng for hDIPP3α or 50 ng for hDIPP3β. The hDIPP3α transcript was amplified using as primers 5′-CAAAGCCAGGCACAGTGTTA-3′ (sense) and 5′-GGGGCATGGTCAAATTTAGG-3′ (antisense) for 40 cycles at an annealing temperature of 58 °C. The hDIPP3β transcript was amplified using as primers 5′-TTGCCTCTCTCACTGATCCA-3′ (sense) and 5′-GCAGCCTCTTTCCTAAATGC-3′ (antisense) for 30 cycles with a 55 °C annealing temperature. Northern, dot, and arrayed library blots were pre-hybridized in DIG Easy Hyb solution (Roche Molecular Biochemicals) for 30 min, hybridized at 37 (DNA blots) or 50 °C (RNA blots) overnight with 50–100 ng of labeled probe/ml DIG Easy Hyb, and washed twice for 15 min each to the indicated stringency (see below). Hybridizing signals were labeled with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Molecular Biochemicals) for 30 min at room temperature and detected by chemiluminescence using CDP-STAR (Roche Molecular Biochemicals). Human multiple tissue expression array dot blots (CLONTECH) were hybridized with a probe (P4) that was generated with the following primers: 5′-GCGAGGACGAGGTCCTGT-3′ (sense) and 5′-GCTCATCTGTGCTTCACA (antisense). Probe P4 is specific for the hDIPP3α coding region plus ∼100 bp of the 3′-untranslated region. The blot was hybridized as described above, washed twice for 15 min each to a stringency of 0.5× SSC, 0.1% SDS at 50 °C, and processed for chemiluminescent detection of signal as described above. Northern analysis was also performed in an identical manner with a Human Multiple Tissue Northern blot (CLONTECH, Palo Alto, CA), using a hDIPP3α coding region probe (P2), generated using the following primers: 5′-GCGAGGACGAGGTCCTGT-3′ (sense) and 5′-GGGGCCATGGAGTTTCCA-3′ (antisense). This blot was washed twice for 15 min each to a stringency of 0.1× SSC, 0.1% SDS at 50 °C, and then chemiluminescent detection was performed as described above. The blot was subsequently stripped with boiling 1% SDS and re-probed with a human β-actin probe (P3) under the same hybridization and wash conditions. P3 was generated from a cDNA of human β-actin (CLONTECH) using the following primers 5-ATCGCCGCGCTCGTCGTCG-3′ (sense) and 5′-CACCGGAGTCCATCACG-3′ (antisense). Recombinant hDIPP1 and hDIPP2 proteins were prepared as described previously (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar). The entire coding regions of hDIPP3α and hDIPP3β, prepared as described above, were amplified by PCR using 5′-GATCGTCGACATGAAGTGCAAACCCAACCAG-3′ (sense) and 5′-GATCGAGCTCCGGGATCGCTATCTGGCGAGG-3′ (antisense) primers containingSalI and SacI restriction sites, respectively. The resultant PCR product was digested with these enzymes, agarose gel-purified, and ligated into the cognate sites of vector pIVEX-2.3MCS (Roche Molecular Biochemicals), and the construct was completely sequenced to verify its structure. The recombinant, poly(His)-tagged hDIPP3α and hDIPP3β proteins were synthesized in a 1-ml volume of the Rapid Translation System RTS 500 (Roche Molecular Biochemicals), according to the manufacturer's protocol. The extract was subsequently bound, at 4 °C for 3 h, to 1.5 ml of TALON metal affinity resin (CLONTECH) in 5 ml of Buffer A (50 mmphosphate buffer (pH 7.0), 300 mm NaCl, 2 mmDTT, and a protease inhibitor mixture (Roche Molecular Biochemicals product number 1-697-498)). The resin was washed three times with 15 ml of Buffer A, and bound hDIPP3 protein was then eluted with 1-ml aliquots of Buffer A plus 150 mm imidazole, by gravity flow. Most of the protein eluted in the second 1-ml fraction, which was dialyzed twice against 500 ml of Buffer A, and then the final enzyme preparations were stored at −70 °C. The peptide NGNSMAPSSPDSDP, representing the C-terminal 14 amino acid residues of hDIPP3, was synthesized with an N-terminal C residue and conjugated to keyhole limpet hemocyanin protein (SigmaGenosys, The Woodlands, TX). This conjugated peptide was used to raise antisera in New Zealand White rabbits (SigmaGenosys) following standard protocols. The anti-hDIPP2 anti-peptide antibodies were prepared as described previously (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The anti-hDIPP1 polyclonal antibodies against recombinant poly (His)-hDIPP1 hDIPP1 (5Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar) were raised in the rabbit. All serum samples were stored in aliquots at −80 °C. Recombinant hDIPP1 and hDIPP2α were prepared as described previously (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar). Human Hs1.Tes and Hs181.Tes cell lines (ATCC) were lysed by vortexing in extraction buffer containing 50 mm β-glycerophosphate (pH 8.2), 250 mm sucrose, 100 mm NaCl, 50 mm NaF, 1 mm EDTA, 10 μm EGTA, 1 mm DTT, 4 mm CHAPS, 250 μm4-(2-aminoethyl)benzenesulfonyl fluoride, 10 mmtrans-epoxysuccinyl-l-leucylamido(4-guanidino)-butane, 1 μg/ml pepstatin, 1 μg/ml leupeptin. Samples were subsequently centrifuged at 18,000 × g for 30 min at 4 °C, and the supernatants were stored at −70 °C. Mouse testes were frozen in liquid nitrogen, pulverized in a TissueMizer for 10 s, and extracted in the above buffer. Next, 200-μg aliquots of protein were separated on 4–12% polyacrylamide NuPAGE Bis-Tris gels (Invitrogen) and transferred to nitrocellulose filters, and then the filters were rinsed in Tris-buffered saline/Tween 20 (0.1%, v/v). Filters were then treated with blocking buffer (5% (w/v) nonfat dry milk in Tris-buffered saline/Tween) for 1 h at room temperature and then incubated with anti-peptide antiserum in more blocking buffer. Blots were washed with Tris-buffered saline/Tween and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (AmershamBiosciences) for 30 min. Unbound secondary antibody was washed off with Tris-buffered saline/Tween, and antibody-antigen complexes were detected by chemiluminescence using SuperSignal reagent (Pierce). PP-[3H]InsP5 (specific activity ∼20 Ci/mmol) was prepared as described previously (22Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Non-radiolabeled PP-InsP5 (with the diphosphate group added to the 5-carbon) was prepared as described previously (23Albert C. Safrany S.T. Bembenek M.E. Reddy K.M. Reddy K.K. Falck J.R. Bro¨ker M. Shears S.B. Mayr G.W. Biochem. J. 1997; 327: 553-560Crossref PubMed Scopus (73) Google Scholar). [PP]2-[3H]InsP4 was prepared as follows. Three rat brains were homogenized in 2 volumes of buffer containing 20 mm HEPES (pH 6.8), 2 mm CHAPS, 1 mm EDTA, 1 mm EGTA, 1 mm DTT plus a protease inhibitor mixture (Roche Molecular Biochemicals). A 100,000 × g supernatant was prepared, 100-μl aliquots of which were each incubated for 90 min at 37 °C with 0.5 μCi of [3H]InsP6 (specific activity ∼20 Ci/mmol; PerkinElmer Life Sciences) in 500 μl buffer containing 0.75 mm EGTA, 1.5 mm EDTA, 9 mmMgSO4, 7.5 mm ATP, 10 mm NaF, 20 mm phosphocreatine, 1 mm DTT, 20 mmHEPES (pH 6.8), 4 mm CHAPS, 20 Sigma units/ml creatine phosphokinase, and 4 μg of human DINS type 1. The reaction mixture was preincubated for 60 min at 37 °C prior to the addition of the rat brain supernatant to ensure phosphorylation of [3H]InsP6 to PP-[3H]InsP5. The resultant [PP]2-[3H]InsP4 was purified by HPLC and desalted (22Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Ap6A was synthesized from ATP as described previously (24Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar). [3H]Ap6A was custom-labeled with tritium by catalytic exchange by Moravek Biochemicals (Brea, CA). [3H]Ap6A had a radiochemical purity of 94% and was stored as a 50% ethanol solution at −20 °C. [3H]dGTP was purchased from PerkinElmer Life Sciences. To study hydrolysis of diphosphoinositol polyphosphates, purified recombinant hDIPP protein was incubated at 37 °C in 100–450 μl of buffer containing 50 mm KCl, 25 mm HEPES (pH 7.2 with KOH), 2 mm DTT, 2 mm MgSO4, 1 mm EDTA, 0.05 mg/ml bovine serum albumin, plus ∼1000 dpm of [PP]2-[3H]InsP4 or 2000 dpm of PP-[3H]InsP5. In order to determineKm and kcat values for PP-InsP5 hydrolysis, non-radioactive substrate was added to a final concentration across the range 10–1500 nm. For [PP]2-InsP4 hydrolysis, the specificity constant (kcat/Km) was determined from the first-order rate constant, with the assumption that substrate concentration (∼0.2 nm) was below 5% of itsKm value (25Crompton I.E. Waley S.G. Biochem. J. 1986; 239: 221-224Crossref PubMed Scopus (19) Google Scholar). Assays were quenched by placing the tubes on ice, followed immediately by the addition of 0.25 volume of ice-cold 250 mm EDTA (pH 7.0). Subsequently, protein was precipitated by the addition of 0.2 volume ice-cold 2 mperchloric acid (+0.1 mg/ml InsP6). After 15 min, samples were centrifuged, and the perchloric acid was precipitated by addition of 0.35 volume of 1 m K2CO3 + 5 mm EDTA. Samples were again centrifuged, and then the resultant supernatants were analyzed by HPLC on a Partisphere SAX column as described previously (12Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Recombinant hDIPP protein was also separately incubated with [3H]Ap6A in 50 mm HEPES-NaOH (pH 7.6), 1 mm MnCl2, 2 mm DTT, and 100 μg of bovine serum albumin/ml in a final volume of 100 μl for 30 min at 37 °C. The reaction was stopped and the products separated from residual substrate by boronate chromatography as described previously (26Barnes L.D. Robinson A.K. Mumford C.H. Garrison P.N. Anal. Biochem. 1985; 144: 296-304Crossref PubMed Scopus (37) Google Scholar), and the products were detected by liquid scintillation counting. HEK293 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium, 10% fetal bovine serum with 5% CO2, and cells were radiolabeled with 25 μCi/ml [3H]inositol (ARC, St. Louis, MO) for 5 days. On the 3rd day, cells (in a 6-well plate) were transfected with 5 μg of plasmid DNA encoding either GFP, GFP-hDIPP3α, or GFP-hDIPP3β, using LipofectAMINE 2000 (Invitrogen), according to the manufacturer's recommended protocol. The cDNA for the GFP-DIPP fusion constructs was prepared by subcloning the coding region of hDIPP3α and hDIPP3β into the phGFP105C1 vector (27Yamasaki M. Hashiguchi N. Tsukamoto T. Osumi T. Bioimages. 1998; 6: 1-7Google Scholar). Approximately 80% of the cells was estimated to express GFP. Cells were quenched with 1 ml of 0.6m perchloric acid supplemented with 0.1 mg/ml InsP6. Lysates were neutralized (28Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar) and analyzed by HPLC on a Partisphere SAX column (Krackler Scientific, NC) eluted with a gradient generated by mixing Buffer A (1 mmNa2EDTA) and Buffer B (1 mm Na2EDTA + 1.3 m (NH4)2HPO4 (pH 3.85), with H3PO4) as follows: 0–10 min, 0% B; 10–30 min, 0–45% B; 30–90 min, 45–100% B; 90–105 min, 100% B. One-ml fractions were collected, and the radiolabeled inositol phosphates were determined by liquid scintillation counting. Aliquots of non-radiolabeled HEK293 cells were also transfected with plasmid DNA encoding either GFP or the GFP-hDIPP3 fusion constructs as described above. Cells were lysed with 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA and 1% Triton X-100, followed by centrifugation at 12,000 × g for 20 min. Aliquots of the supernatant were analyzed by SDS-PAGE on 10% Bis-Tris NuPage gels with MES buffer; GFP-fusion proteins were detected by anti-GFP antibody (Invitrogen) or by anti-hDIPP3 antisera. The sequence alignments described in this study were obtained with Peptool version 2 and Genetool version 1 (Biotools Inc., Alberta, Canada). We have previously cloned hDIPP1 and hDIPP2 cDNAs (4Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar). In the current study we first searched for novel hDIPP isoforms by screening an arrayed human cDNA library with a 492-bp probe spanning the coding region of hDIPP2α (P1, see under "Experimental Procedures"). A novel 2020-bp cDNA was identified that encodes a 164-amino acid residue protein (Fig.1A). In the alignments shown in Fig. 1A, the sequence of this new protein is 90% identical to residues 2–165 in hDIPP2 and 74% identical to residues 2–164 in hDIPP1. We therefore propose that our new protein represents a novel DIPP isoform, which we have named type 3α (Fig. 1). This hDIPP3α protein has particularly strong conservation of a 33-residue active site found in hDIPP1 and hDIPP2 (Fig. 1A),i.e. the Nudix core domain (3Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full
Referência(s)