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• W2059778621 abstract "A novel serine/threonine protein phosphatase (PPase) designated PP7 was identified from cDNA produced from human retina RNA. PP7 has a molecular mass of ∼75 kDa, and the deduced amino acid sequence of PP7 contains a phosphatase catalytic core domain that possesses all of the invariant motifs of the PP1, PP2A, PP2B, PP4, PP5, and PP6 gene family. However, PP7 has unique N- and C-terminal regions and shares <35% identity with the other known PPases. The unique C-terminal region of PP7 contains multiple Ca2+ binding sites (i.e. EF-hand motifs). This region of PP7 is similar to the Drosophila retinal degeneration C gene product (rdgC), and PP7 and rdgC share 42.1% identity. Unlike the other known PPases, the expression of PP7 is not ubiquitous; PP7 was only detected in retina and retinal-derived Y-79 retinoblastoma cells. Expression of recombinant human PP7 in baculovirus-infected SF21 insect cells produces an active soluble enzyme that is capable of utilizing phosphohistone and p-nitrophenyl phosphate as substrates. The activity of recombinant PP7 is dependent on Mg2+ and is activated by calcium (IC50 ≅ 250 μm). PP7 is not affected by calmodulin and is insensitive to inhibition by okadaic acid, microcystin-LR, calyculin A, and cantharidin. A novel serine/threonine protein phosphatase (PPase) designated PP7 was identified from cDNA produced from human retina RNA. PP7 has a molecular mass of ∼75 kDa, and the deduced amino acid sequence of PP7 contains a phosphatase catalytic core domain that possesses all of the invariant motifs of the PP1, PP2A, PP2B, PP4, PP5, and PP6 gene family. However, PP7 has unique N- and C-terminal regions and shares <35% identity with the other known PPases. The unique C-terminal region of PP7 contains multiple Ca2+ binding sites (i.e. EF-hand motifs). This region of PP7 is similar to the Drosophila retinal degeneration C gene product (rdgC), and PP7 and rdgC share 42.1% identity. Unlike the other known PPases, the expression of PP7 is not ubiquitous; PP7 was only detected in retina and retinal-derived Y-79 retinoblastoma cells. Expression of recombinant human PP7 in baculovirus-infected SF21 insect cells produces an active soluble enzyme that is capable of utilizing phosphohistone and p-nitrophenyl phosphate as substrates. The activity of recombinant PP7 is dependent on Mg2+ and is activated by calcium (IC50 ≅ 250 μm). PP7 is not affected by calmodulin and is insensitive to inhibition by okadaic acid, microcystin-LR, calyculin A, and cantharidin. In eukaryotes, the reversible phosphorylation of proteins catalyzed by protein kinases and protein phosphatases determines the biological activities of many proteins and is recognized as a major mechanism controlling cellular processes as diverse as cell cycle progression, metabolism, gene expression, and phototransduction. Traditionally, the protein phosphatases that catalyze the dephosphorylation of serine and threonine residues (PPases) 1The abbreviations used are: PPase, Ser/Thr, protein phosphatase; PCR, polymerase chain reaction; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pNPP,p-nitrophenyl phosphate; RP, retinitis pigmentosa; microcystin-LR, microcystin variant leucine-arginine. 1The abbreviations used are: PPase, Ser/Thr, protein phosphatase; PCR, polymerase chain reaction; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pNPP,p-nitrophenyl phosphate; RP, retinitis pigmentosa; microcystin-LR, microcystin variant leucine-arginine. have been classified into four subtypes (PP1, PP2A, PP2B, and PP2C) based on (a) their biochemical characteristics, (b) their sensitivities to specific inhibitors, and (c) a limited amount of substrate specificity that can be demonstrated in vitro (for review see Refs. 1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar and 2Shenolikar S. Nairn A.C. Adv. Second Messenger Phosphoprotein Res. 1991; 23: 1-119PubMed Google Scholar). More recent studies indicate that the primary amino acid sequences of PP1, PP2A, and PP2B are related, whereas PP2C is structurally distinct and belongs to a completely different gene family (3Cohen P.T.W. Brewis N.D. Hughes V. Mann D.J. FEBS Lett. 1990; 268: 355-359Crossref PubMed Scopus (189) Google Scholar, 4Walter G. Mumby M. Biochim. Biophys. Acta. 1993; 1155: 207-226PubMed Google Scholar). In addition, multiple isoforms of all four PPases have been cloned. In mammals, three isoforms of PP1, which demonstrate >90% identity (5Song Q. Khanna K.K. Lu H. Lavin M.F. Gene. 1993; 129: 291-295Crossref PubMed Scopus (20) Google Scholar, 6Barker H.M. Craig S.P. Spurr N.K. Cohen P.T. Biochim. Biophys. Acta. 1993; 1178: 228-233Crossref PubMed Scopus (59) Google Scholar, 7Barker H.M. Brewis N.D. Street A.J. Spurr N.K. Cohen P.T. Biochim. Biophys. Acta. 1994; 1220: 212-218Crossref PubMed Scopus (66) Google Scholar), two isoforms of PP2A with >97% identity (8Green D.D Yang S.I. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4880-4884Crossref PubMed Scopus (81) Google Scholar, 9Stone S.R. Hofsteenge J. Hemmings B.A. Biochemistry. 1987; 26: 7215-7220Crossref PubMed Scopus (121) Google Scholar, 10Da Cruz e Silva O.B. Cohen P.T.W. FEBS Lett. 1987; 226: 176-178Crossref PubMed Scopus (48) Google Scholar, 11Arino J. Woon C.W. Brautigan D.L. Miller Jr., T.B. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4252-4256Crossref PubMed Scopus (117) Google Scholar), and three isoforms of PP2B with >80% identity (12Da Cruz e Silva E.F. Hughes V. McDonald P. Stark M.J.R. Cohen P.T.W. Biochim. Biophys. Acta. 1991; 1089: 269-272Crossref PubMed Scopus (28) Google Scholar, 13Kincaid R.L. Nightingale M.S. Martin B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8983-8987Crossref PubMed Scopus (100) Google Scholar, 14Ito A. Hashimoto T. Hirai M. Takeda T. Shuntoh H. Kuno T. Tanaka C. Biochem. Biophys. Res. Commun. 1989; 163: 1492-1497Crossref PubMed Scopus (100) Google Scholar, 15Kincaid R.L. Giri P.R. Higuchi S. Tamura J. Dixon S.C. Marietta C.A. Amorese D.A. Martin B.M. J. Biol. Chem. 1990; 265: 11312-11319Abstract Full Text PDF PubMed Google Scholar, 16Guerini D. Krinks M.H. Sikela J.M. Hahn W.E. Klee C.B. DNA (N. Y.). 1989; 8: 675-682Crossref PubMed Scopus (52) Google Scholar, 17Kuno T. Takeda T. Hirai M. Ito A. Mukai H. Tanaka C. Biochem. Biophys. Res. Commun. 1989; 165: 1352-1358Crossref PubMed Scopus (86) Google Scholar, 18Muramatsu T. Giri P.G. Higuchi S Kincaid R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 529-533Crossref PubMed Scopus (91) Google Scholar) have been identified. Molecular studies have also identified three additional PPases designated as PP4 (PPX; Refs. 19Brewis N.D. Street A.J. Prescott A.R. Cohen P.T.W. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (199) Google Scholar and20Brewis N.D. Cohen P.T.W. Biochim. Biophys. Acta. 1992; 1171: 231-233Crossref PubMed Scopus (38) Google Scholar), PP5 (21Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar, 22Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 12: 4278-4290Crossref Scopus (252) Google Scholar, 23Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar), and PP6 (PPV; Refs. 21Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar and 24Bastians H. Ponstingl H. J. Cell Sci. 1996; 109: 2865-2874Crossref PubMed Google Scholar). PP4 and PP6 are structurally related to PP2A, sharing 65 and 57% identity at the level of their primary amino acid sequence, respectively (19Brewis N.D. Street A.J. Prescott A.R. Cohen P.T.W. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (199) Google Scholar, 20Brewis N.D. Cohen P.T.W. Biochim. Biophys. Acta. 1992; 1171: 231-233Crossref PubMed Scopus (38) Google Scholar, 24Bastians H. Ponstingl H. J. Cell Sci. 1996; 109: 2865-2874Crossref PubMed Google Scholar). PP5 contains a catalytic domain common to the PP1/PP2A/PP2B/PP4/PP6 family of enzymes and an extended N-terminal domain containing four 34-amino acid tetratricopeptide repeat motifs (21Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar, 22Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 12: 4278-4290Crossref Scopus (252) Google Scholar, 23Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar). The known PPases can also be classified based on their sensitivities to several natural toxins (25Cohen P. Methods Enzymol. 1991; 201: 389-397Crossref PubMed Scopus (204) Google Scholar, 26Honkanen R.E. Codispoti B.A. Tse K. Boynton A.L. Toxicon. 1994; 32: 339-350Crossref PubMed Scopus (190) Google Scholar). PP1, PP2A, PP4, and PP5 are all sensitive to okadaic acid and microcystin-LR (19Brewis N.D. Street A.J. Prescott A.R. Cohen P.T.W. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (199) Google Scholar, 22Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 12: 4278-4290Crossref Scopus (252) Google Scholar, 26Honkanen R.E. Codispoti B.A. Tse K. Boynton A.L. Toxicon. 1994; 32: 339-350Crossref PubMed Scopus (190) Google Scholar, 27Bialojan C. Takai A. Biochem. J. 1988; 250: 283-290Crossref Scopus (1509) Google Scholar, 28Honkanen R.E. Zwiller J. Moore R.E. Daily S. Khatra B.S. Dukelow M. Boynton A.L. J. Biol. Chem. 1990; 265: 19401-19404Abstract Full Text PDF PubMed Google Scholar). In contrast, PP2B is relatively resistant to the known inhibitors (okadaic acid, microcystin, nodularin, tautomycin, cantharidin, and calyculin A), and PP2C is insensitive to these compounds (4Walter G. Mumby M. Biochim. Biophys. Acta. 1993; 1155: 207-226PubMed Google Scholar, 25Cohen P. Methods Enzymol. 1991; 201: 389-397Crossref PubMed Scopus (204) Google Scholar, 26Honkanen R.E. Codispoti B.A. Tse K. Boynton A.L. Toxicon. 1994; 32: 339-350Crossref PubMed Scopus (190) Google Scholar, 27Bialojan C. Takai A. Biochem. J. 1988; 250: 283-290Crossref Scopus (1509) Google Scholar, 28Honkanen R.E. Zwiller J. Moore R.E. Daily S. Khatra B.S. Dukelow M. Boynton A.L. J. Biol. Chem. 1990; 265: 19401-19404Abstract Full Text PDF PubMed Google Scholar, 29Cohen P. Holmes C.F.B. Tsukitani Y. Trends Biochem. Sci. 1990; 15: 98-102Abstract Full Text PDF PubMed Scopus (1259) Google Scholar, 30Honkanen R.E. FEBS Lett. 1993; 330: 283-286Crossref PubMed Scopus (252) Google Scholar). In addition, both PP2B and PP2C have an absolute requirement for divalent cations, which are not required by PP1, PP2A, PP4, or PP5. PP2C activity has an absolute requirement for Mg2+, and PP2B is activated by the association of a calcium-bound calmodulin complex (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar,2Shenolikar S. Nairn A.C. Adv. Second Messenger Phosphoprotein Res. 1991; 23: 1-119PubMed Google Scholar, 4Walter G. Mumby M. Biochim. Biophys. Acta. 1993; 1155: 207-226PubMed Google Scholar). PP6 has not yet been characterized biochemically. Several studies suggest that the expression of certain PPases, particularly isoforms of PP1, are altered in human tumor cells (31Saadat M. Mizuno Y. Takizawa N. Kakinoki Y. Rikuchi R. Cancer Lett. 1995; 94: 165-170Crossref PubMed Scopus (14) Google Scholar, 32Sogawa K. Yamada T. Oka S. Kawasaki K. Mori S. Tanaka H. Norimatsu H. Cai Y. Kuwabara H. Shima H. Nagao M. Matsumoto K. Cancer Lett. 1995; 89: 1-6Crossref PubMed Scopus (11) Google Scholar, 33Saadat M. Kitamura K. Mizuno Y. Saeki H. Kudo T. Kikuchi K. Cancer Detect. Prev. 1994; 18: 115-122PubMed Google Scholar). Thus, the aim of this study was to characterize the heterogeneity of related PPases in Y-79 human retinoblastoma cells for comparison with those contained in normal human retina. In the course of these studies, we identified a novel human PPase that is directly activated by calcium and dependent on Mg2+ for activity. First strand human retinal cDNA was generously provided by Dr. S. Pittler and Dr. M. Ardell. Degenerate oligonucleotide primers matching to highly conserved regions of known PPases (RGNHE, DILWSDP, GDY/FVDR) were synthesized and employed in combination with an oligo(dT)15 primer to amplify human first-strand cDNA produced from human retina, Y-79 cells, and bovine brain by PCR. PCR reactions were allowed to proceed for 35 cycles (1 min at 95 °C, 45 s each at 40 and 45 °C; 1 min at 72 °C) in a solution containing 50 mm KCl, 2 mm MgCl2, 0.2 mm dNTP, 10 mm Tris-HCl, pH 8.3, and 100 ng of each primer. The PCR products produced were then analyzed by agarose gel electrophoresis, and ∼125 bands were isolated and cloned into pT7/T3α-18 or pBluescript for sequence analysis. The PCR products were sequenced, and the sequences obtained were searched for homologous regions contained in known PPases. The expressed sequence tag data base was also searched for homologous sequences. One sequence identifying a human cDNA clone isolated from fetal brain (GenBankTM accession numberH18854) was homologous to the Drosophila retinal degeneration C gene product (rdgC). The human brain clone (ID51064) was obtained from Research Genetics, Inc. (Huntsville AL), and sequence analysis of this clone confirmed the similarity withrdgC (37.4%). A ∼600-bp HindIII digest fragment from clone 51064 was radiolabeled (DECAprime IITM, Ambion) and used as a probe to screen ∼4 × 106clones from a human retina cDNA library constructed in λgt10. Four positive clones were isolated and completely sequenced. The full-length PP7 open reading frame was amplified with PCR employing a nested primer pair, (RH108, 5′-AACTCTGCAGTTAGCCAAGGTTGGTGACATCAGG-3′, and RH112, 5′-AGCCGGATCCATGGGATGCAGCAGTTCTTC-3′), which contained restriction sites (BamHI and PstI) added to the 5′ ends, respectively. The PCR product was then digested withBamHI/PstI and ligated into a baculovirus transfer vector, pFastBacTMHTb (Life Technologies, Inc.). The construct adds six consecutive His+ residues to the N-terminal region of PP7 to aid in the purification of the recombinant enzyme. After sequencing the insert, to confirm the fidelity of the construct, the recombinant plasmid was used to transformEscherichia coli (DH10Bac) containing a helper plasmid and bacmid. Recombinant bacmids were identified by the color phenotypes of the host E. coli colonies growing on Blue-gal plates (i.e. white color). Further confirmation was obtained by PCR analysis with PP7-specific primers. Approximately 5 μg of bacmid DNA was then purified and used to transfect CellFECTIN® (Life Technologies)-treated SF21 cells. Recombinant baculovirus (∼9 × 105) was harvested 72 h later, and the initial stock was reamplified and used to infect three 125-mm flasks of SF21 cells at a multiplicity of infection of 1. Cells were harvested 48 h after the infection, collected by centrifugation at 1000 rpm, sonicated in a binding buffer (5 mm imidazole, 0.5 m NaCl, 20 mm Tris-HCl, pH 7.9, 2 mm phenylmethylsulfonyl fluoride), and subjected to ultracentrifugation at 400,000 ×g for 20 min. The supernatant was then passed over a Ni+-charged chelating His-tag column (Novagene). The column was washed with 10 volumes of binding buffer and then with 6 volumes of washing buffer (60Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google Scholar mm imidazole, 0.5 m NaCl, 20 mm Tris-HCl, pH 7.9). The recombinant PP7 was then eluted with the addition of 3 column volumes of elution buffer (1m imidazole, 0.5 m NaCl, 20 mmTris-HCl pH 7.9). Fractions containing PPase activity were pooled and concentrated and subjected to fast protein liquid chromatography gel filtration on a Superose 12 high resolution 10/30 column equilibrated with binding buffer. The active fractions were identified by absorbance at 280 nm and confirmed by assaying for PPase activity. Active fractions were then pooled and subjected to a second round of affinity purification employing a Ni+-charged chelating His-tag column as described above. The purity of the active fractions obtained were then analyzed by SDS-polyacrylamide gel electrophoresis. Total RNA from Y-79 cell cultures or human retina was prepared using TRI reagent according to the methods of the manufacturer (Life Technologies, Inc.). Poly(A)+ RNA was obtained by affinity chromatography with an oligo(dT) spin column (Poly(A) spinTM mRNA isolation kit; New England Biolabs). Poly(A)+ RNA was separated on formaldehyde-agarose (1.2%) gels and transferred to a nylon membrane (Duralon-UV, Stratagene). The RNA was fixed to the membrane by ultraviolet irradiation (UV cross-linker; Stratagene). Northern blots containing 2 μg of Poly(A)+ RNA from eight different human tissues was also purchased from CLONTECH((Palo Alto, CA; MTN Blots). The full-length coding region of human PP7 was amplified by PCR and random-radiolabeled with [32P]dATP using a DECAprime IITM labeling kit (Ambion). The radiolabeled PCR product was then employed as a probe to detect the expression of PP7 mRNA. A human multiple tissue cDNA (MTCTM) panel containing prenormalized cDNAs from eight different tissues was purchased from CLONTECH). cDNA from human retina and Y-79 cells was produced from the appropriate mRNA (Y-79 mRNA was prepared as described above (Northern blot analysis), and human retina mRNA was purchased fromCLONTECH) by incubating 1 μg of poly(A)+ RNA in a solution (20 μl total volume) containing 50 mm Tris-HCl, pH 8.3, 75 mm KCl, 3 mm MgCl2, 10 mm dithiothreitol, 0.5 mm dNTP mix, 100 ng of random primer, and 200 units of reverse transcriptase (Superscript II, Life Technologies, Inc.) at 42 °C for 50 min. After normalizing the concentration of Y-79 and human retina cDNA to that of the other tissues, 5 μl aliquots from each tissue type was employed as template in PCR reactions containing PP7 or GAPDH-specific primer pairs. The PCR reactions were carried out in a volume of 25 μl that contained 20 mmTris-HCl, pH 8.4, 50 mm KCl, 1.5 mmMgCl2, 0.2 mm dNTP mix, 100 ng each of the primers, and 25 units of Taq DNA polymerase (Promega) on a PTC-100 programmable thermal cycler (MJ Research Inc.). The reaction mixture was denatured at 94 °C for 5 min, and the amplification reaction consisted of 25 (for GAPDH) or 38 (for PP7) cycles of denaturation (94 °C, 30 s), annealing (55 °C, 50 s), and extension (72 °C, 2 min) with a final extension at 72 °C for 10 min. The sequences for PP7 and GAPDH-specific primers pairs were 5′-CAGAGCATGAATGGGAACAGA-3′ and 5′-ACATGGCACGAAATTCTTCC-3′ and 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and 5′-CATGTGGGCCATGAGGTCCACCAC-3′, respectively. The PCR products were then resolved on a 2% agarose gel. The activity of PP7 was assessed against [32P]phosphorylase a, [32P]phosphohistone, and pNPP. Phosphohistone and phosphorylase a were prepared as described by Honkanenet al. (34Honkanen R.E. Dukelow M. Moore R.E. Zwiller J. Khatra B.S. Boynton A.L. Mol. Pharmacol. 1991; 40: 577-583PubMed Google Scholar) and assayed as described by Critz and Honkanen (35Critz S.D. Honkanen R.E. Neuroprotocols. 1995; 6: 78-83Google Scholar). pNPP activity was measured at 30 °C and determined from the change in absorbance (A 410). Reactions were conducted in a final volume of 150 μl as described previously (30Honkanen R.E. FEBS Lett. 1993; 330: 283-286Crossref PubMed Scopus (252) Google Scholar,34Honkanen R.E. Dukelow M. Moore R.E. Zwiller J. Khatra B.S. Boynton A.L. Mol. Pharmacol. 1991; 40: 577-583PubMed Google Scholar). The reaction contained 50 mm Tris-HCl, pH 8.1 (or as indicated), 10 mm MgCl2, 30 mm KCl, 20 μm pNPP, and the indicated concentrations of Ca2+. Reactions rates were determined using a Beckman DU 640 spectrophotometer over a 15-min period using ∼0.5–2.0 μg of PP7. With both pNPP and phosphohistone, the concentrations of enzymes were adjusted to ensure that the dephosphorylation of substrate was <10% total available substrate, and the reaction was linear with respect to enzyme concentration and time. A human leukocyte genomic library constructed in λ EMBL-3 (CLONTECH) was screened using a [α-32P]dATP-labeled cDNA probe complementary to ∼200 bp of the C-terminal region of PP7 (bp 1998–2220). Phage DNA from λ clones that hybridized with the PP7 cDNA probe were then isolated using a Wizard λ DNA purification system (Promega) according to the method of the manufacturer. Sequence analysis of one positive clone revealed that it contains a complete exon encoding a portion of the C-terminal region of PP7. PCR with nested primers contained in this exon (5-GGAAGAATTTCGTGCCATGT-3, sense, nucleotides 1999–2018; and 5-TTAGCCAAGGTTGGTGACATCAGG-3, antisense, nucleotides 2173–2196) amplifies a 199-bp intronless fragment of PP7 from human genomic DNA that is not amplified in rodent DNA. PCR analysis of a panel of human/rodent somatic cell hybrid DNAs (National Institute of General Medical Sciences (NIGMS) human/rodent somatic cell hybrid mapping panel 2, Coriell Cell Repositories, Camden, NJ) was then employed to determine the chromosomal localization of PP7. Each 25-μl PCR reaction contained 50 mm Tris-HCl, pH 8.4, 50 mm NaCl, 1.5 mm MgCl2, 0.1% Triton X-100, 200 μm dNTP, 100 ng of each primer, 25 unit of Taq DNA polymerase (Promega), and 100 ng of template DNA. DNA amplification was performed on a thermal cycler (Perkin-Elmer 2400) with 30 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 1 min), with an initial denaturation (95 °C, 5 min) and a final extension (72 °C, 10 min). The products were analyzed by electrophoresis on 2.5% agarose gels. The same primers and PCR conditions were used to amplify PP7 from a panel of radiation hybrid DNAs that contain different portions of chromosome X (NIGMS regional mapping panel for chromosome X, Coriell Cell Repositories, Camden, NJ). In an effort to characterize the PPase expressed in Y-79 retinoblastoma cells, several degenerate oligonucleotide primers corresponding to highly conserved regions of known PPases were constructed in the sense orientation. These primers were then employed in combination with oligo(dT)15 antisense primers to amplify first-strand cDNA produced from human retina or Y-79 cell RNA employing essentially the same strategy developed by Wadzinski et al. (36Wadzinski B.E. Heasley L.E. Johnson G.L. J. Biol. Chem. 1990; 265: 21504-21508Abstract Full Text PDF PubMed Google Scholar). Electrophoretic analysis of the PCR products from ∼200 reactions yielded numerous bands of different sizes, and ∼125 were purified and ligated into pT7/T3α-18 or pBluescript. Systematic sequencing of these clones resulted in the identification of PP1α, PP1β, PP1γ1, PP1γ2, PP2Aα, PP4, PP5, PP6 and several unidentified sequences (data not shown). To distinguish additional promising clones from the numerous PCR artifacts produced, we performed a BLAST computer search of the expressed sequence tag data base with the sequences of known PPases and those obtained via PCR. One sequence identified in the expressed sequence tag data base was a 875-bp fragment from human fetal brain (GenBankTM accession numberH18854). This clone (ID51064) was obtained from Research Genetics, Inc. (Huntsville, AL), and the sequencing of clone 51064 confirmed the similarity with a DNA fragment produced by PCR. An ∼600-bp probe produced from clone 51064 was then constructed and used to screen a human retina cDNA library. After three rounds of purification, four positive clones were isolated and characterized by sequencing. All of the clones contained the same sequence, and the largest clone contained a 2,658-bp insert. Nucleotide sequence analysis of this clone revealed a 1,959-bp open reading frame flanked by a region containing 234 bp of the 5′-untranslated sequence upstream of a translation start site AGTCATGG that is compatible with the Kozark (37Kozark M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar) consensus sequence. The clone, designated as PP7, also contains a 465-bp 3′-untranslated region ending in a poly(A) tail. The PP7 open reading frame encodes a protein of 653 amino acids with a predicted molecular mass of 75 kDa. A computer-aided search for homology of the deduced amino acid sequence for PP7 with the GenBankTM/EMBL data bank (GCG; Genetics Computer Group, Madison, WI) revealed greatest homology with the gene product encoded by the Drosophila retinal degeneration C (rdgC) gene (38Steel F.R. Washburn T. Rieger R. O'Tousa J.E. Cell. 1992; 69: 669-676Abstract Full Text PDF PubMed Scopus (132) Google Scholar). Direct comparison between PP7 and rdgC using “gap routine” (GCG) indicates that they share 42.1% identity and 54.3% similarity when conservative substitutions are considered (Fig.1). In addition, two putative functional domains were identified through a motif search using “motif routine” (GCG). One is a serine/threonine phosphatase catalytic core domain, which is located between amino acids 166 and 436. This core is shared by PP1-PP6 (39Barton G.J. Cohen P.T.W. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (153) Google Scholar, 40Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (743) Google Scholar), and the overall identity of PP7 is 28–35% with these PPases. The similarity increases to 35–44% when conservative substitutions are included. In addition, PP7 contains all 53 of the amino acids that are absolutely conserved in PP1-PP6 (Fig.2) and all of the 16 amino acids predicted to form the active site of these enzymes based on the crystal structure of PP1 (40Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (743) Google Scholar).Figure 2Homology of PP7 with other serine/threonine PPases. A, a schematic representation of PP7 amino acid (a.a.) similarity with the catalytic subunits of other human serine/threonine PPases. Most PPases contain a common catalytic core domain that is conserved with λ-PPase (53Barik S. Proc. Natl. Acad. Sci. U. S. A. 1993; 22: 10633-10637Crossref Scopus (42) Google Scholar, 59Palczewski K. Farber D.B. Hargrave P.A. Exp. Eye Res. 1991; 53: 101-105Crossref PubMed Scopus (14) Google Scholar). PP1, PP2A, PP4, and PP6 are highly homologous enzymes, differing primarily in their C- and N-terminal regions. PP2B differs in that it contains a Ca2+-calmodulin (CaM) binding domain in its C-terminal region and two small divergent regions (indicated by open boxes) in the catalytic domain near the okadaic acid/microcystin binding domain. PP5 possesses four tetratricopeptide (TPR) domains in the N-terminal region. PP7 differs from all of the other PPase families in that it contains EF-hand motifs in the C-terminal region (indicated by filled squares) and a 43-amino acid insert in the catalytic core domain (indicated by anopen box). B, sequence similarity between the catalytic core domain of PP7 and other human PPases. Residues that are identical in all of the PPases compared are indicated inboldface type. PP2A is human protein phosphatase type 2A (9Stone S.R. Hofsteenge J. Hemmings B.A. Biochemistry. 1987; 26: 7215-7220Crossref PubMed Scopus (121) Google Scholar), PP1 is human protein phosphatase type 1α (5Song Q. Khanna K.K. Lu H. Lavin M.F. Gene. 1993; 129: 291-295Crossref PubMed Scopus (20) Google Scholar), PP2B is human protein phosphatase type 2B (16Guerini D. Krinks M.H. Sikela J.M. Hahn W.E. Klee C.B. DNA (N. Y.). 1989; 8: 675-682Crossref PubMed Scopus (52) Google Scholar), PP4 is human protein phosphatase type 4 (19Brewis N.D. Street A.J. Prescott A.R. Cohen P.T.W. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (199) Google Scholar), PP5 is human protein phosphatase type 5 (25Cohen P. Methods Enzymol. 1991; 201: 389-397Crossref PubMed Scopus (204) Google Scholar), PP6 is human protein phosphatase type 6 (24Bastians H. Ponstingl H. J. Cell Sci. 1996; 109: 2865-2874Crossref PubMed Google Scholar), and PP7 is the human retinal protein phosphatase designated as type 7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The second domain identified in PP7 is comprised of putative Ca2+ binding EF-hand motifs located in the C-terminal region. Two EF-hand motifs (amino acids 579–591 and 619–631) are excellent matches to the consensus sequence (41Kretsinger R.H. CRC Crit. Rev. Biochem. 1980; 8: 119-174Crossref PubMed Scopus (709) Google Scholar), and three other EF-hand-like motifs with weaker identity were recognized in the PP7 sequence (Figs. 1 and 2). These EF-hand and EF-hand-like motifs are conserved between PP7 and rdgC (Fig. 2). PP7 also contains an inserted domain made up of 43 amino acids (305–347) in the PPase core region that is not found in PP1-PP6. The rdgC gene sequence predicts that rdgC contains a 15-amino acid insert in a corresponding region, and 6 of the 15 amino acids in the rdgC insert are conserved in PP7. The N-terminal region of PP7 is structurally divergent from that of PP1-PP6 (Fig. 2). The expression of PP7 poly(A)+ RNA in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, retina, and Y-79 retinoblastoma cells was analyzed by PCR amplification of normalized human cDNA and Northern analysis. PP7 mRNA was only detected in retina and Y-79 cells by PCR analysis, suggesting that PP7 is not ubiquitously expressed like PP1, PP2A, PP4, or PP5. Northern analysis employing 2 μg of poly(A)+ RNA did not identify PP7 mRNA in any of the tissues tested, identifying a single ∼2.8-kilobase transcript only in mRNA produced from Y-79 cells (Fig. 3 B). Whe" @default.
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• W2059778621 title "Molecular Cloning, Expression, and Characterization of a Novel Human Serine/Threonine Protein Phosphatase, PP7, That Is Homologous toDrosophila Retinal Degeneration C Gene Product (rdgC)" @default.
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