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• W2051294804 abstract "Tankyrase is an ankyrin repeat-containing poly(ADP-ribose) polymerase originally isolated as a binding partner for the telomeric protein TRF1, but recently identified as a mitogen-activated protein kinase substrate implicated in regulation of Golgi vesicle trafficking. In this study, a novel human tankyrase, designated tankyrase 2, was isolated in a yeast two-hybrid screen as a binding partner for the Src homology 2 domain-containing adaptor protein Grb14. Tankyrase 2 is a 130-kDa protein, which lacks the N-terminal histidine/proline/serine-rich region of tankyrase, but contains a corresponding ankyrin repeat region, sterile α motif module, and poly(ADP-ribose) polymerase homology domain. TheTANKYRASE 2 gene localizes to chromosome 10q23.2 and is widely expressed, with mRNA transcripts particularly abundant in skeletal muscle and placenta. Upon subcellular fractionation, both Grb14 and tankyrase 2 associate with the low density microsome fraction, and association of these proteins in vivo can be detected by co-immunoprecipitation analysis. Deletion analyses implicate the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10–19 of tankyrase 2 in mediating this interaction. This study supports a role for the tankyrases in cytoplasmic signal transduction pathways and suggests that vesicle trafficking may be involved in the subcellular localization or signaling function of Grb14.AF329696 Tankyrase is an ankyrin repeat-containing poly(ADP-ribose) polymerase originally isolated as a binding partner for the telomeric protein TRF1, but recently identified as a mitogen-activated protein kinase substrate implicated in regulation of Golgi vesicle trafficking. In this study, a novel human tankyrase, designated tankyrase 2, was isolated in a yeast two-hybrid screen as a binding partner for the Src homology 2 domain-containing adaptor protein Grb14. Tankyrase 2 is a 130-kDa protein, which lacks the N-terminal histidine/proline/serine-rich region of tankyrase, but contains a corresponding ankyrin repeat region, sterile α motif module, and poly(ADP-ribose) polymerase homology domain. TheTANKYRASE 2 gene localizes to chromosome 10q23.2 and is widely expressed, with mRNA transcripts particularly abundant in skeletal muscle and placenta. Upon subcellular fractionation, both Grb14 and tankyrase 2 associate with the low density microsome fraction, and association of these proteins in vivo can be detected by co-immunoprecipitation analysis. Deletion analyses implicate the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10–19 of tankyrase 2 in mediating this interaction. This study supports a role for the tankyrases in cytoplasmic signal transduction pathways and suggests that vesicle trafficking may be involved in the subcellular localization or signaling function of Grb14. AF329696 Src homology antibody activation domain binding domain base pair(s) between pleckstrin homology and Src homology 2 cycloheximide 4′, 6-diamidine-2-phenylindole fluorescencein situ hybridization formiminotransferase cyclodeaminase Grb-Mig growth factor receptor-bound glutathione S-transferase high density microsome histidine/proline/serine-rich insulin-like growth factor 1 (receptor) insulin receptor insulin-responsive aminopeptidase kilobase(s) low density microsome mitogen-activated protein polyacrylamide gel electrophoresis poly(ADP-ribose) polymerase phosphate-buffered saline PSD-95/Dlg/ZO1 pleckstrin homology sterile α motif telomere repeat binding factor It is now evident that protein-protein interactions play a critical role in signal transduction, not only mediating recruitment of signaling proteins to receptors and assembly of multiprotein signaling complexes, but also directing the correct subcellular compartmentalization of such complexes and hence providing signal fidelity (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1904) Google Scholar). A variety of protein modules have been identified that mediate these interactions including SH21 domains, which bind specific phosphotyrosine-containing peptide sequences; SH3 domains, which target specific proline-rich motifs with a PXXP core; and PDZ domains, which interact with the C-terminal consensus (S/T/Y)X(V/I) (2Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar, 3Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-76Crossref PubMed Scopus (1224) Google Scholar). Another module, the PH domain, also mediates intermolecular interactions, but here the targets are predominantly specific polyphosphoinositides and inositol polyphosphates (4Rebecchi M.J. Scarlata S. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 503-528Crossref PubMed Scopus (250) Google Scholar). These modules may be found in signaling proteins that possess a catalytic activity (e.g. c-Src and phospholipase C-γ); the adaptor class (e.g. Grb2), which provide a molecular link to separate effector molecules; and proteins that provide an anchoring or scaffolding function, e.g.PSD-95 (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1904) Google Scholar). As well as initiating signaling events, protein-protein interactions are also important in regulating the internalization of cell surface receptors and their subsequent sorting to lysosomal or recycling compartments (5Owen D.J. Luzio J.P. Curr. Opin. Cell Biol. 2000; 12: 467-474Crossref PubMed Scopus (47) Google Scholar, 6Lemmon S.K. Traub L.M. Curr. Opin. Cell Biol. 2000; 12: 457-466Crossref PubMed Scopus (179) Google Scholar). The Grb7 family is a group of related SH2 domain-containing adaptors, comprising Grb7, -10, and -14 (7Daly R.J. Cell. Signal. 1998; 10: 613-618Crossref PubMed Scopus (104) Google Scholar). These proteins share significant sequence homology and a conserved molecular architecture, consisting of a N-terminal region containing the motif P(S/A)IPNPFPEL, a central PH domain-containing region (designated the GM domain), which bears homology to the Caenorhabditis elegans protein Mig10 and a C-terminal SH2 domain. The family members differ in their specificity and modes of receptor recruitment. Grb7 binds via its SH2 domain to a variety of receptor tyrosine kinases and tyrosine-phosphorylated proteins, including erbB2, erbB3, and Shc (7Daly R.J. Cell. Signal. 1998; 10: 613-618Crossref PubMed Scopus (104) Google Scholar, 8Stein D. Wu J. Fuqua S.A.W. Roonprapunt C. Yajnik V. D'Eustachio P. Moskow J.J. Buchberg A.M. Osborne C.K. Margolis B. EMBO J. 1994; 13: 1331-1340Crossref PubMed Scopus (220) Google Scholar, 9Janes P.W. Lackmann M. Church W.B. Sanderson G.M. Sutherland R.L. Daly R.J. J. Biol. Chem. 1997; 272: 8490-8497Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Fiddes R.J. Campbell D.H. Janes P.W. Sivertsen S.P. Sasaki H. Wallasch C. Daly R.J. J. Biol. Chem. 1998; 273: 7717-7724Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In the case of Grb10, most attention has focused on its recruitment by the IR and IGF-1R (7Daly R.J. Cell. Signal. 1998; 10: 613-618Crossref PubMed Scopus (104) Google Scholar, 11He W. Rose D.W. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1998; 273: 6860-6867Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Grb14 is also bound by the IR (12Kasus-Jacobi A. Perdereau D. Auzan C. Clauser E. Van Obberghen E. Mauvais-Jarvis F. Girard J. Burnol A. J. Biol. Chem. 1998; 273: 26026-26035Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and has recently been identified as a fibroblast growth factor receptor 1 target (13Reilly J.F. Mickey G. Maher P.A. J. Biol. Chem. 2000; 275: 7771-7778Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). A 50-amino acid region between the PH and SH2 domains (BPS domain) contributes to binding of Grb10 and Grb14 to the IR (11He W. Rose D.W. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1998; 273: 6860-6867Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 12Kasus-Jacobi A. Perdereau D. Auzan C. Clauser E. Van Obberghen E. Mauvais-Jarvis F. Girard J. Burnol A. J. Biol. Chem. 1998; 273: 26026-26035Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The signaling function of the Grb7 family is poorly understood. One role for Grb10 and -14 may be as negative regulators of IR signaling. For example, overexpression of Grb14 reduces insulin-induced DNA and glycogen synthesis (12Kasus-Jacobi A. Perdereau D. Auzan C. Clauser E. Van Obberghen E. Mauvais-Jarvis F. Girard J. Burnol A. J. Biol. Chem. 1998; 273: 26026-26035Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and inhibition of insulin-induced insulin-like receptor substrate-1 phosphorylation occurs upon overexpression of hGrb10β 2The nomenclature used for particular Grb10 isoforms is that proposed by André Nantel following consultation with workers in the field. This system allows for the possibility that the same variant will be identified in different species, and should therefore be given the same isoform designation (indicated by a Greek letter). (Grb-IR) (14Liu F. Roth R.A. Proc. Natl. Acad. Sci U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (154) Google Scholar) or Grb14 (12Kasus-Jacobi A. Perdereau D. Auzan C. Clauser E. Van Obberghen E. Mauvais-Jarvis F. Girard J. Burnol A. J. Biol. Chem. 1998; 273: 26026-26035Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, data supporting a positive role for mGrb10α in insulin-, IGF-1-, and platelet-derived growth factor BB-stimulated mitogenesis have recently been presented (15Wang J. Dai H. Yousaf N. Moussaif M. Deng Y. Boufelliga A. Rama Swamy O. Leone M.E. Riedel H. Mol. Cell. Biol. 1999; 19: 6217-6228Crossref PubMed Scopus (100) Google Scholar). It is also likely that the functional role of the Grb7 family extends beyond signal modulation. For example, inhibition of Grb7 expression suppresses the invasive potential of esophageal cancer cells (16Tanaka S. Mori M. Akiyoshi T. Tanaka Y. Mafune K. Wands J.R. Sugimachi K. J. Clin. Invest. 1998; 102: 821-827Crossref PubMed Scopus (67) Google Scholar), and overexpression of Grb7 and its targeting to focal contacts correlates with increased cell motility (17Han D.C. Shen T.-L. Guan J.-L. J. Biol. Chem. 2000; 275: 28911-28917Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Definition of the molecular interactions mediated by Grb7 family proteins, particularly those involving the N-terminal and GM domains, may provide a valuable insight into their signaling mechanism and how it is regulated. With regard to the N-terminal region, the SH3 domain of c-Abl binds the conserved proline-rich motif of Grb10 in vitro (18Frantz J.D. Giorgetti-Peraldi S. Ottinger E.A. Shoelson S.E. J. Biol. Chem. 1997; 272: 2659-2667Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), but an in vivo binding partner has yet to be identified. In this paper we describe the identification of a novel tankyrase, tankyrase 2, as a binding partner for the Grb14 N terminus. Tankyrase was originally identified by virtue of an interaction with the telomeric protein TRF1, and consists of a N-terminal HPS region, 24 consecutive ankyrin-type repeats, a SAM module, and a C-terminal region with homology to the PARP catalytic domain (19Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (908) Google Scholar). A small fraction of tankyrase co-localizes with TRF1 at telomeres, and tankyrase can ADP-ribosylate TRF1 in vitro, leading to a reduction in binding of TRF1 to telomeric DNA. Consequently, one function of tankyrase may be in regulation of telomere function via ADP-ribosylation. However, the majority of tankyrase is extranuclear, and a recent study identified it as a peripheral membrane protein associated with the Golgi, where it localizes to Glut4 vesicles via the IRAP cytosolic domain and acts as a substrate for insulin and growth factor-induced MAP kinase activity (20Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Interestingly, tankyrase 2 is also predominantly cytoplasmic and associates with the LDM fraction. The association of tankyrase 2 with Grb14 supports the hypothesis that tankyrases may provide a link between signal transduction pathways and vesicle trafficking. A plasmid construct encoding a Gal4 DNA-BD-Grb14 fusion was generated as follows. The plasmid GRB14/pRcCMVF containing full-lengthGRB14 cDNA (21Daly R.J. Sanderson G.M. Janes P.J. Sutherland R.L. J. Biol. Chem. 1996; 271: 12502-12510Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) was digested with HindIII and Klenow-treated to create blunt ends, and then digested withBclI to release three fragments of ∼1.1, 4.2, and 1.7 kb. The 1.7-kb fragment was isolated and cloned into the NdeI (Klenow-treated) and BamHI sites of the yeast expression vector pAS2–1 (CLONTECH, Palo Alto, CA) to generate GRB14/pAS2–1 containing an in-frame fusion of full-length Grb14 with the Gal4 DNA-BD. This construct was introduced by electroporation into the yeast strain CG1945 selecting for tryptophan prototrophy. Following preparation of yeast cell extracts by trichloroacetic acid protein extraction, the expression of the fusion protein was verified by Western blot analysis with antibodies directed against the Flag epitope or the Gal4 DNA-BD. The recipient strain was then grown to mid-log phase and a human liver cDNA library in the vector pACT2 (CLONTECH) introduced using the LiAc procedure (22Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1776) Google Scholar). Transformants were selected for tryptophan, leucine, and histidine prototrophy in the presence of 5 mm3-aminotriazole and then tested for β-galactosidase activity by either a liquid culture-based method (Galacto-Light, Tropix, Bedford, MA) or colony lift filter assay (CLONTECH). Clones scoring positive in the β-galactosidase assays were subjected to CHX curing to remove the bait plasmid by streaking out on synthetic complete-leu media containing 10 μg/ml CHX (pAS2–1 contains theCYH2 gene which restores CHX sensitivity to CG1945 cells). This enabled confirmation of the bait dependence of LacZactivation and subsequent isolation of the pACT2 plasmids encoding interacting proteins by standard methodology (23Philippsen P. Stotz A. Scherf C. Methods Enzymol. 1991; 194: 170-177Google Scholar). Back transformations were then performed in which these pACT2 plasmids were introduced into CG1945 strains containing the bait plasmid (GRB14/pAS2–1) or constructs encoding non-related Gal4 DNA-BD fusions in order to confirm the specificity of the interactions. The DNA sequences of the cDNA inserts were then obtained by cycle sequencing (Promega, Annandale, New South Wales, Australia) using pACT2-specific and/or clone-specific primers. In order to identify the region of Grb14 that interacts with tankyrase 2, a series of Grb14 deletion mutants were generated by cloning polymerase chain reaction fragments synthesized using the appropriate flanking primers into the vector pAS2–1. These fragments spanned the following regions: N terminus (amino acids 1–110), the central region encompassing the Mig10 homology and the BPS domain (amino acids 110–437), and the N-terminal and central regions (amino acids 1–437). These plasmids were individually transformed into the yeast strain Y190. Following transformation of the resulting yeast strains with the TANKYRASE 2 cDNA clone L1 in pACT-2, the strength of the interaction was determined by either liquid- or filter-based β-galactosidase assays. Expression of the constructs was confirmed by Western blotting of yeast extracts with Gal4 DNA-BD- and Gal4 AD-specific antibodies. In order to investigate the interaction of tankyrase 2 with TRF1, a fragment of tankyrase 2 corresponding to the 10-ankyrin repeat region of tankyrase responsible for TRF1 binding (TR1L12) (19Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (908) Google Scholar) was expressed as a Gal4 AD fusion in pGAD10 (CLONTECH). Binding of this to LexA fusions of full-length TRF1 and TRF1 lacking the tankyrase binding site (amino acids 1–67) was then performed as described previously (19Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (908) Google Scholar). Following the isolation of the original TANKYRASE 2 cDNA, further clones were isolated by standard cDNA library screening methodology (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA probes were labeled by random primer extension (Promega) using [α-32P]dCTP (110 TBq/mmol, Amersham Pharmacia Biotech Pty Ltd, Castle Hill, New South Wales, Australia). Following isolation of phage or phagemid DNA (Promega Wizard kits), sequencing of the cDNA inserts was performed by cycle sequencing. The cDNA cloning strategy was as follows, and further cDNA clone details can be provided upon request. The original TANKYRASE 2 cDNA isolated from the two hybrid screen (L1) was used as a probe to screen a λgt10 human placental cDNA library (5′ Stretch Plus,CLONTECH). This isolated two clones, designated P8 and P12. P8 was ∼2.0 kb and provided the C-terminal end of the tankyrase 2 protein sequence. P12 was ∼3.5 kb and extended the cDNA sequence 0.9 kb in the 5′ direction. Screening of the human placental cDNA library and a λgt11 human small intestine cDNA library (5′ Stretch, CLONTECH) with 5′-located probes led to the isolation of two clones, designated P5 and SI4, respectively, which both extended the sequence further 5′ and provided a putative translation initiation codon. Screening of a λZAP II human fetal brain cDNA library (Stratagene, La Jolla, CA) with a 414-bp probe including the extended sequence isolated two further clones, FB3 and FB11, which confirmed this sequence. Sequence alignments were performed using the program ClustalW. The cDNA was first assembled in the vector Bluescript SK+ (Stratagene) containing alterations to the multiple cloning site (MCS). The MCS was changed by insertion of annealed oligonucleotides 5′-GGCCGCGGATCCCGGCTCGAGCGGGAATTCCATGCCATGGCATGCCAAGCTTTCTAGAG-3′ and 5′-TCGACTCTAGAAAGCTTGGCAT- GCCATGGCATGGAATTCCCGCTCGAGCCGGGATCCGC-3′ into the NotI/XhoI sites to provide the modified cloning site NotI, BamHI,XhoI, EcoRI, NcoI, HindIII,XbaI and to destroy the original XhoI site, creating the vector BSK (ΔMCS). The first 495 bp of TANKYRASE 2 were obtained as a BamHI/XhoI fragment from FB11, and inserted into the BamHI/XhoI sites of BSK (ΔMCS) creating BSK(I). The next 840 bp were obtained as aXhoI/EcoRI fragment from SI4 and cloned into theXhoI/EcoRI sites of BSK(I) creating BSK(II). The following 1104 bp were obtained as a EcoRI/NcoI fragment from L1 and inserted into the EcoRI/NcoI site in BSK(II), creating BSK(III). The final 1361 bp were obtained as a NcoI/HindIII fragment from P8, and cloned into the NcoI/HindIII site in BSK(III), creating BSK(IV). The assembled TANKYRASE 2 cDNA was subcloned into the NotI/XbaI sites of pcDNA 3.1(+) (Invitrogen, Groningen, The Netherlands). Coupled transcription/translation reactions were performed according to the manufacturer's instructions (Promega). The originalTANKYRASE 2 cDNA clone (L1) subcloned into pGEX-4T-2 (Amersham Pharmacia Biotech) was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 15 ng/μl to metaphases from two normal males. The FISH method was modified from that described previously (25Callen D.F. Baker E. Eyre H.J. Chernos J.E. Bell J.A. Sutherland G.R. Ann. Genet. 1990; 33: 219-221PubMed Google Scholar) in that chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a cooled charged coupled device camera using the ChromoScan image collection and enhancement system (Applied Imaging Corporation, Newcastle, United Kingdom). FISH signals and the DAPI banding pattern were merged for figure preparation. Human multiple tissue Northern blots (CLONTECH) were hybridized under conditions recommended by the manufacturer. The radiolabeled probe utilized was the TANKYRASE 2 cDNA clone L1 labeled by random primer extension (Promega) using [α-32P]dCTP (Amersham Pharmacia Biotech). The following regions of tankyrase 2 were expressed as GST fusion proteins; amino acids 324–980 (corresponding to clone L1 and construct 1 in Fig. 8), amino acids 324–870 (construct 2), amino acids 324–630 (construct 3), amino acids 631–980 (construct 4, also used to generate Ab-1), amino acids 486–630 (used to generate Ab-5), and amino acids 871–935 (construct 5). Construct 1 was generated by subcloning aSalI-XhoI fragment from pACT2 into theNdeI site of pGEX-4T-2 (Amersham Pharmacia Biotech). DNA fragments encoding the other regions were synthesized by polymerase chain reaction using flanking primers containing restriction enzyme sites for in-frame directional insertion into this vector. Following cloning and transformation of the resulting plasmids intoEscherichia coli DH5α, GST fusion proteins were purified from isopropyl-β-d-thiogalactopyranoside-induced bacterial cultures as described previously (26Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). DU145 human prostate cancer cells, HEK293 cells, and HEK293 cells stably transfected with theGRB14/pRcCMVF expression vector were maintained as described previously (21Daly R.J. Sanderson G.M. Janes P.J. Sutherland R.L. J. Biol. Chem. 1996; 271: 12502-12510Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Where indicated, the cells were starved overnight in medium containing 0.5% fetal calf serum. These techniques were as described previously (27Janes P.W. Daly R.J. deFazio A. Sutherland R.L. Oncogene. 1994; 9: 3601-3608PubMed Google Scholar), except that the lysis and wash buffers used for detection of Grb14-tankyrase 2 co-immunoprecipitation contained 0.1% Triton X-100. DU145 cells were serum-starved overnight in RPMI/0.5% fetal calf serum and then harvested (1 ml/150-mm dish) in subcellular fractionation buffer (250 mm sucrose, 10 mm Tris, pH 7.5, 0.5 mm EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride). The cell suspensions were subjected to three freeze-thaw cycles and then Dounce homogenization until, by microscopic inspection, the majority of the nuclei were released. The samples were then centrifuged at 800 × g for 10 min (to isolate the low speed pellet), 50,000 × g for 20 min (to isolate the HDM), and 160,000 × g for 70 min (to isolate the LDM). The pellets from each centrifugation step were resuspended in subcellular fractionation buffer at 10% of the original volume, and the remaining supernatant was then concentrated to the same volume using a Microcon YM-10 centrifugal filter device (Millipore Corp., Bedford, MA). Equivalent amounts of each fraction (i.e.normalized for cell number) were then analyzed by Western blotting. GST or the appropriate GST fusion protein were purified on glutathione-agarose beads (Sigma, Castle Hill, New South Wales, Australia) (26Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar) and then cross-linked to the beads using dimethylpimelimidate (Sigma) (28Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 524-525Google Scholar) to generate affinity columns. The rabbit antiserum was diluted 1:10 with 10 mm Tris-HCl, pH 7.5, and applied to the GST column. The flow-through was then applied to the GST fusion protein column. Following washing with 10 mm Tris-HCl, pH 7.5, 500 mm NaCl, the bound antibodies were eluted with 100 mm glycine, pH 2.5, and immediately neutralized with 1m Tris-HCl, pH 8.0. The antibodies were finally subjected to buffer exchange with 10 mm Tris-HCl, pH 7.4, 150 mm NaCl using a Centricon 30 microconcentrator (Amicon, Beverly, MA) and stored in aliquots at −70 °C. Commercially available antibodies used were as follows: M2 monoclonal anti-FLAG antibody (Sigma), monoclonal anti-golgi 58 kDa protein FTCD (29Gao Y.-S. Alvarez C. Nelson D.S. Sztul E. J. Biol. Chem. 1998; 273: 33825-33834Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (Sigma), monoclonal anti-GAL4 AD antibody (CLONTECH), monoclonal anti-GAL4 DNA-BD antibody (CLONTECH), goat polyclonal anti-Grb14 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and D8 polyclonal anti-Flag antibody (Santa Cruz Biotechnology). Five μg of GST or GST fusion protein immobilized on glutathione-agarose beads were incubated with 400 μl of lysate (∼5 mg/ml total protein) from serum-starved HEK/Grb14 cells (21Daly R.J. Sanderson G.M. Janes P.J. Sutherland R.L. J. Biol. Chem. 1996; 271: 12502-12510Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) for 2 h at 4 C. The beads were then washed three times with cell lysis buffer and subjected to SDS-PAGE. Following transfer to a polyvinylidene difluoride membrane, bound Grb14 was detected by Western blotting with antibody D8 against the Flag epitope tag. Cells grown on culture slides in RPMI/10% fetal calf serum were rinsed twice in PBS, fixed at room temperature in 3.7% paraformaldehyde in PBS for 20 min, and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. After extensive washing, fixed cells were blocked in 10% normal goat serum or 2% bovine serum albumin in PBS containing 0.05% Tween 20 at room temperature for 45 min and subsequently incubated with antibodies against tankyrase 2 (Ab-5, 1:50) and Golgi 58-kDa protein (1:50) for 1 h at room temperature or overnight at 4 C. After extensive washes in PBS containing 0.05% Tween 20, cells were incubated with Alexa Fluor™ 594-conjugated goat anti-rabbit IgG antibody (1:50, Molecular Probes Inc, Eugene, OR) and Alexa Fluor™ 488-conjugated goat anti-mouse IgG antibody (1:50, Molecular Probes) for 45 min at room temperature. To detect Grb14, cells were stained as above with anti-Grb14 antibody (1:100), followed by Texas Red-conjugated donkey anti-goat antibody (1:50, Jackson Immunoresearch Laboratories Inc, West Grove, PA). Following washing, samples were mounted in Vectashield plus DAPI (Vector Laboratories Inc., Burlingame, CA). Images were acquired on a Leica DMR microscope (Leica Microsystems Pty Ltd, Gladesville, New South Wales, Australia) using the TCS SP software. In order to identify binding partners for the Grb14 adaptor protein, a human liver cDNA library in the Gal4 AD vector pACT2 was screened using a full-length Grb14 bait expressed as a Gal4 DNA-BD fusion. From a screen of ∼1 × 106clones, 31 colonies were initially selected on synthetic complete −Leu−His−Trp medium and were then tested for β-galactosidase activity. Nine clones gave significant activity in the latter assay and were characterized by DNA sequencing. One of these pACT2 clones harbored a novel cDNA of 1971 bp. This clone encoded a polypeptide of 657 amino acids in frame with the Gal4 DNA-BD and exhibited homology to tankyrase (19Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (908) Google Scholar), but the absence of translation start and stop codons revealed that the cDNA clone was incomplete. Screening of cDNA libraries using the original TANKYRASE 2 clone L1 as probe led to the isolation of a series of overlapping cDNA clones, which provided the full-length TANKYRASE 2 cDNA sequence. 3The nucleotide sequence for the humanTANKYRASE 2 cDNA has been deposited in the GenBank™ database under GenBank accession no. AF329696. We note close matches with sequences deposited under the following accession numbers:AF264912, AX029397, AF305081, and AK023746. This revealed an open reading frame for tankyrase 2 spanning 1166 amino acids, encoding a polypeptide with a predicted molecular mass of 130 kDa. The protein sequence for tankyrase 2 aligned with that of tankyrase (19Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (908) Google Scholar) is shown in Fig. 1. The original cDNA clone isolated by the two hybrid screen, clone L1, spans amino acids 324–980 of the full-length sequence. The major difference between the two proteins is the absence of a HPS domain in tankyrase 2. The molecular architecture of tankyrase 2, starting at the ankyrin repeat region, is then similar to tankyrase. Both proteins possess 24 ankyrin-type repeats, aligned in Smithet al. (19Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (908) Google Scholar), with an overall sequence identity of 83% and sequence similarity of 90%. The major differences between the two proteins in this region occur at the C termini of ankyrin repeats 1, 14, and 24 and the N terminus of repeat 24, where there are five or more non-conservative changes, and the C terminus of repeat 23, where there is a non-conservative change and then an insertion of 7 amino acids in tankyrase 2 relative to tankyrase (Fig. 1). The ankyrin repeat region is then followed by a SAM domain, exhibiting 77% sequence identity and 89% similarity. The most closely related region is the C-terminal PARP homology domain, with 93% sequence identity and 96% similarity. Critical residues required for NAD+ binding and catalysis are entirely conserved. The TANKYRASE 2 gene was localized by FISH. Twenty metaphases from a normal male were examined for fluorescent signal. All of these metaphases showed signal on one or both chromatids of chromosome" @default.
• W2051294804 created "2016-06-24" @default.
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• W2051294804 creator A5041532971 @default.
• W2051294804 creator A5045049850 @default.
• W2051294804 creator A5045185056 @default.
• W2051294804 creator A5046858810 @default.
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• W2051294804 date "2001-05-01" @default.
• W2051294804 modified "2023-09-27" @default.
• W2051294804 title "Identification of a Novel Human Tankyrase through Its Interaction with the Adaptor Protein Grb14" @default.
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