EP0941324A1 - Protein das auf glykogen auspürt - Google Patents

Protein das auf glykogen auspürt

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Publication number
EP0941324A1
EP0941324A1 EP97938246A EP97938246A EP0941324A1 EP 0941324 A1 EP0941324 A1 EP 0941324A1 EP 97938246 A EP97938246 A EP 97938246A EP 97938246 A EP97938246 A EP 97938246A EP 0941324 A1 EP0941324 A1 EP 0941324A1
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ser
leu
val
lys
asp
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French (fr)
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Matthew Jemail Brady
John Andrew Printen
Alan Robert Saltiel
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Warner Lambert Co LLC
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Warner Lambert Co LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • This invention relates to isolated murine and human genomic and cDNA molecules that encode for a protein called the Protein Targeting to Glycogen (PTG) protein.
  • PTG Protein Targeting to Glycogen
  • This invention also relates to the PTG protein and to methods of increasing the amount of glycogen in a cell.
  • PPl has been shown regulate cell cycle progression (Hisamoto N., et al., Mol. Cell. Biol. 1994;14:3158; Zang S., et al., Mol. Cell. Biol., 1995; 15:2037), chromosome segregation (Francisco L., et al., Mol. Cell. Biol., 1994; 14:4731), protein synthesis (Wek R.C., et al., Mol. Cell. Biol., 1992;12:5700), and glycogen metabolism (Cannon J.F., et al., Genetics, 1994;136:485).
  • PPl In mammalian cells, PPl also has multiple physiological roles, such as regulation of glycogen metabolism (Bollen M. and Stalmans W., Crit. Rev. Biochem. Mol. Bio., 1992;27:227), protein synthesis (Cohen P., Ann. Rev. Biochem., 1989;58,453), and muscle contraction (Shenolikar S., Annu. Rev. Cell Biol., 1994;10:55). Many of the metabolic effects of insulin are thought to occur via activation of PPl (Saltiel A.R., Am. J. Physiol, 1996;33:E375).
  • two targeting subunits M and G direct the catalytic subunit of PPl, PPIC, to different subcellular locations.
  • the M subunit directs PPIC to myofibrils, acting to facilitate dephosphorylation of myosin (Dent P., et al., Eur. J. Biochem., 1992;210: 1037), whereas the G subunit localizes PPIC to both the glycogen particle and the membranes of the sarcoplasmic reticulum, where glycogen metabolizing enzymes and SR proteins serve as substrates for PPIC (Stralfors P., et al., Eur. J. Biochem., 1985;149:295; Hubbard M.J.
  • PPIC is also known to interact with proteins in nuclei, an inhibitor protein, NIPP1 (Beullins M., et al, J. Biol. Chem., 1992;267: 16538), and the
  • PPIC has also been shown to interact with the product of the tumor suppressor gene, Rb, implicating PPIC in the control of tumorigenesis and cell cycle progression (Durfee T., et al., Genes Dev., 1993;7:555).
  • Rb tumor suppressor gene
  • phosphorylation of site 1 following insulin stimulation leads to a higher affinity of PPIC for RGl, leading to activation of glycogen metabolizing enzymes by dephosphorylation, while phosphorylation of site 2 by cAMP activated protein kinase A (PKA) causes a reduced affinity and subsequent release of PPIC from the glycogen pellet.
  • PKA protein kinase A
  • G A glycogen binding subunit expressed exclusively in liver, G , was recently cloned and was found to encode a predicted protein product of only 33 kD. G was shown to also differ from RGl with respect to enzymatic activity towards various substrates and in the ability of G to serve as a substrate for PKA (Doherty M.J., et al., FEBS Lett., 1995;375:294).
  • a 3T3-L1 adipocyte cDNA 2-hybrid library was screened for PPIC interacting proteins.
  • PTG a novel glycogen binding subunit of PPIC, called PTG, that may act as a scaffold for the localization of critical enzymes in glycogen metabolism, including phosphorylase b, glycogen synthase, and phosphorylase kinase.
  • PTG is expressed predominantly in insulin-sensitive tissues and was found to mediate the hormonal control of glycogen accumulation in intact cells.
  • Figure 1 shows the sequence comparison of PTG with glycogen localizing subunits of PPIC.
  • Figure 2 shows PPIC binding and glycogen localizing activity of PTG protein.
  • Figure 3 shows the tissue distribution of PTG expression.
  • Figure 4 shows the binding of phosphorylase a, glycogen synthase, and phosphorylase kinase to PTG.
  • Figure 5 shows glycogen synthesis in CHO-IR cells overexpressing PTG.
  • the present invention provides an isolated murine protein comprising the amino acid sequence:
  • the present invention also provides an isolated human protein protein comprising the amino acid sequence; Met He Gin Val Leu Asp Pro Arg Pro Leu Thr Ser Ser Val Met Pro
  • the present invention also provides an isolated murine genomic DNA molecule comprising the sequence:
  • the present invention also provides an isolated human genomic DNA molecule comprising the sequence:
  • the present invention also provides an isolated murine cDNA molecule comprising the sequence:
  • the present invention also provides an isolated human cDNA molecule comprising the sequence:
  • PTG is a PPIC binding protein with homology to known glycogen binding proteins:
  • a 3T3-L1 adipocyte cDNA library fused to the Gal4p transcriptional activation domain was screened for proteins that could interact with a Gal4p-PPlC DNA binding domain fusion.
  • Library plasmids containing interacting proteins were identified by the ability to induce transcription of the integrated GALI-lacZ reporter. Of approximately 3.5 x 10 ⁇ primary tranformants, 27 were positive for ⁇ -galactosidase activity.
  • One class of interacting cDN As typified by clone B 1 - 1 , consistently gave the highest levels of ⁇ -galactosidase activity when plated on X-gal containing media.
  • PTG can direct PPIC localization to glycogen both in vivo and in vitro:
  • the homology of the predicted PTG protein with G j ⁇ , RGl and Gael suggests that PTG might bind simultaneously to both PPl C and glycogen.
  • a FLAG epitope tagged PTG construct (FLAG-PTG) was transiently transfected into CHO cells over-expressing the insulin receptor (CHO-IR), followed by immunoprecipitation with ⁇ FLAG antibodies and subsequent immunoblotting with ⁇ PPIC antibodies.
  • PPIC could be co-immunoprecipitated from cell lysates with the antibodies directed against FLAG-PTG ( Figure 2A), demonstrating direct association of PPIC with PTG in vivo. This association was unaffected by treatment of cells with insulin. Moreover, PTG did not appear to undergo phosphorylation in response to insulin, and was not a substrate in vitro for cAMP-dependent protein kinase, or other protein kinases in lysates from insulin stimulated cells (not shown). To determine the subcellular localization of PTG, similarly transfected CHO-IR cells were fractionated by differential centrifugation followed by SDS-PAGE and immunoblotting with ⁇ FLAG antibodies.
  • RGl is expressed in muscle tissue (diaphragm, skeletal muscle, and heart) (Tang P.M., supra., 1991), whereas GL is expressed exclusively in liver tissue (Doherty M.J., supra., 1995).
  • a rat multi-tissue northern blot (Clontech) was hybridized with a probe prepared from the cDNA insert of clone Bl-1.
  • An mRNA of approximately 2.3 kb was detected in all tissues except testis ( Figure 3A).
  • the PTG mRNA was most abundant in skeletal muscle, liver, and heart.
  • PTG transcript was also detected in RNA prepared from rat adipose tissue (not shown).
  • 3T3-L1 fibroblasts into adipocytes is correlated with a significant increase in insulin sensitivity, including the stimulation of glycogen synthesis.
  • the expression of many genes critical to insulin action is increased during adipocyte differentiation, including the insulin receptor, GLUT4 and others
  • PTG can associate with multiple proteins involved in regulating glycogen metabolism: Glycogen synthesis and breakdown is regulated by the reciprocal actions of protein phosphatases and kinases. To determine whether PTG is involved in the localization of the metabolizing enzymes, and of the kinases and phosphatases involved in their regulation, a series of in vitro binding assays were performed with a bacterially expressed PTG fusion protein. First, we examined the ability of PTG to bind to phosphorylase a, the phosphorylated, active form of the enzyme that directly catalyzes glycogen breakdown. GST-PTG bound to glutathione-Sepharose beads was incubated with [32p]-phosphorylated phosphorylase a.
  • GST-PTG efficiently bound to phosphorylase a, but did not bind an unrelated GST-fusion protein, GST-PTP1B ( Figure 4A).
  • glutathione-Sepharose bound GST-PTG was incubated with purified glycogen synthase. Glycogen synthase activity was specifically associated with GST-PTG, and not with an irrelevent GST-fusion protein, PTP1B ( Figure 4B).
  • Phosphorylase kinase converts phosphorylase from the inactive b form to the active a form. This activation can be reversed via dephosphorylation by PPIC.
  • Phosphorylase kinase can also be directly inactivated by PPl. Because of the central role of phosphorylase kinase in regulating glycogen metabolism, it is also a candidate for association with PTG.
  • GST-PTG-glutathione-Sepharose beads were incubated with 3T3-L1 adipocyte lysates, washed extensively, and assayed for phosphorylase kinase activity with [32p]- ⁇ -ATP and phosphorylase b as substrate.
  • Calcium-stimulated phosphorylase kinase activity was associated with GST-PTG, but not with GST-PTP 1 B
  • PTG overexpression cannot only increase basal glycogen synthesis, but also dramatically elevate maximally insulin-stimulated glycogen accumulation in a poorly responsive cell line to a level comparable to that observed in insulin target cells (Lazar D.F., supra., 1995). However, because the sensitivity of these transfected cells to insulin remains unchanged, and because insulin does not appear to modulate PP 1 C-PTG binding, PTG itself is not likely to be a direct target of insulin signaling.
  • DNA sequences such as PTG cDNA can be subcloned into a variety of plasmid shuttle vectors, allowing rapid amplification and manipulation of recombinant DNA sequences in bacterial and mammalian hosts.
  • Plasmid vectors used for routine manipulation of DNA such as Bluescript SK (GenBank
  • Plasmid vectors that are used to introduce desired DNA fragments into mammalian cells contain, in addition to those components required for bacterial vectors, a promoter sequence and a gene coding for resistance to eukaryotic antibiotics.
  • the promoter region is typically a viral promoter (CMV, Simian Virus 40, SV40) that directs high expression of the cloned gene in mammalian cells, and the antibiotic resistance gene is typically Neomycin phosphotransferase (Neo r ), which confers resistance to the eukaryotic antibiotic neomycin.
  • Viral (retrovirus and adenovirus) vectors typically contain all of the above mentioned components, in addition to viral sequences that allow recombinant DNA to be efficiently packaged into viral particles and infect the mammalian host cells. These types of viral vectors are widely used to introduce recombinant DNA into mammalian tissue culture cells and in gene therapy, where recombinant viral particles are used to infect tissues in vivo.
  • 3T3-L1 and 3T3-F442A cell lines derived from a variety of tissues are used as model systems to examine intracellular processes in the laboratory.
  • the 3T3-L1 and 3T3-F442A cell lines cloned from Swiss mouse embryo fibroblast 3T3 cultures (Green and Kehinde, 1974, Cell, 1 :1 13-116), differentiate into adipocytes and are useful in studying adipogenesis and insulin action (Garcia de Herreros and Birnbaum, 1989, J. Biol. Chem., 264 -.19994- 19999V
  • Other cell lines commonly used include NIH 3T3 fibroblasts (ATCC #CRL-1658), rat muscle cell line L6 (Proc. Natl. Acad. Sci.
  • CHO-K1 Chinese hamster ovary cells
  • ATCC #CRL-9618 Chinese hamster ovary cells
  • Primary cells derived from isolated mammalian tissues can also be cultured in the laboratory, however these cells have a limited lifespan in culture and usually die after 7 to 10 days.
  • Recombinant DNA can be introduced (transfected) into mammalian cells either in culture or in vivo by a number of techniques.
  • Calcium Phosphate- mediated transfection Choen and Okayama 1987, Mol. Cell. Biol.. 7:2745-2752
  • Liposome-mediated transfection Lipofectamine; Gibco-BRL
  • adenoviral -mediated gene transfer has been used to infect terminally differenciated cells and tissue (Becker et al. 1994, Methods Cell Biol.. 43:161-189), since adenoviral infection does not require actively dividing cells, as does retroviral gene transfer.
  • Retroviral-mediated gene transfer has been successfully used to correct adenosine deaminase (ADA) deffiency in humans (Blaese, et al., 1995, Science. 270:475-480; Kohn, et al., 1995. Nature Med.. 1 :1017-1023).
  • ADA adenosine deaminase
  • the present invention is also useful in that new drugs can be identified by screening librarys of chemical compounds for agonists or antagonists (inhibitors) of the PTG protein.
  • CHO cells expressing >1 x 10 ⁇ human insulin receptors were grown in alpha-minimal essential medium containing nucleotides,
  • 3T3-L1 Adipocyte 2-Hybrid Library Construction of 3T3-L1 Adipocyte 2-Hybrid Library. 3T3-L1 Fibroblasts were differentiated to adipocytes, as described previously
  • Stratogene cDNA synthesis kit First strand synthesis utilized an oligo ⁇ T-Xho ⁇ primer, whereas the 5' end was ligated to an EcoRl adapter following second strand synthesis. cDNA Fragments were then ligated unidirectionally into EcoRl/Xhol digested pGAD-GH GAL4 activation domain plasmid (Clontech, Palo Alto, CA). Ligations were electroporated into E. coli D12S to yield >2 x
  • a Gal4p- DNA binding domain (BD) fusion of PPIC was constructed by cloning the entire PPIC open reading frame (a generous gift of A. Nairn), contained within a 1.0 kb EcoRl/BamHl fragment, into the Eco ⁇ /Bam sites of pGBT9 (Clontech), creating BD-PPIC.
  • Strain Y190 was transformed first with BD-PP1C, Trp+ prototrophs selected, and then transformed with 150 ⁇ g of 3T3-L1 adipocyte library DNA.
  • Transformants were selected by plating cells on synthetic medium lacking tryptophan, leucine, and histidine (SD-Trp-Leu-His) and containing 25 mM 3-aminotriazole (ATZ). Yeast transformations were performed by the lithium acetate procedure of Geitz, et al.,
  • GST S-transferase
  • Sub-Cellular Fractionation of PPIC Activity Following a 3-hour serum deprivation in KRBH/0.5% BSA/2.5 mM glucose, Ll adipocyte cells were washed three times with ice cold PBS. Cells were scraped in homogenization buffer (50 mM HEPES, pH 7.2/2 mM EDTA 2 mg/mL glycogen/0.2% 2-ME/+ protease inhibitors). Samples were sonicated and centrifuged at 2500 x g to remove nuclei and unlysed cells. The PNS was removed, and glycogen bound PTG was added. Samples were incubated at 4°C for 60 minutes with gentle mixing.
  • homogenization buffer 50 mM HEPES, pH 7.2/2 mM EDTA 2 mg/mL glycogen/0.2% 2-ME/+ protease inhibitors
  • Lysates were subjected to centrifugation for 15 minutes at 10,000 x g and 1 hour at 100,000 x g to pellet plasma membranes and glycogen pellets, respectively. The final supernatant was called cytosol.
  • the glycogen pellets were resuspended in homogenization buffer by 10 passes through a 23 gauge needle. Protein concentrations and PPl activity were measured in the PNS, plasma membrane, glycogen pellet, and cytosolic fractions, as described previously (Lazar D.F., supra., 1995). Fractionation of pFPTG transfected CHO-IR cells into cytosol and glycogen pellet was performed similarly.
  • CHO cells expressing insulin receptor were transfected with Lipofectamine (Gibco-BRL) according to manufacturer recommendations. Typically, 1 ⁇ g of pFPTG/6 ⁇ L Lipofectamine was used per 60 mm dish to achieve 20% to 30% transfection efficiency, as determined by a CMV-lacZ reporter vector transfected in parallel.
  • CHO-IR cells transfected with pFPTG were sonicated in homogenization buffer and subjected to 14000 x g centrifugation for 10 minutes at 4°C to remove nuclei and cell debris.
  • FLAG-PTG was immunoprecipitated from the supernatant by incubation with 10 ⁇ g of ⁇ FLAG antibody (IBI) for 1 hour at 4°C.
  • Immune complexes were precipitated by incubation with Protein A G-agarose for 1 hour at 4°C and washed four times with homogenization buffer prior to the addition of SDS -sample buffer. Immunoprecipitates and subcellular fractions were separated on SDS- polyacrylamide gels and transferred to nitrocellulose.
  • Immunoblots were performed with either FLAG monoclonal antibody or with PPIC polyclonal antibody (a generous gift from Dr. J. Lawrence).
  • the primary monoclonal and polyclonal antibodies were detected with horseradish peroxidase-conjugated anti- mouse or anti-chicken IgG, respectively, and visualized by the enhanced chemiluminescence detection system (Amersham).
  • RNA was washed at 65°C in 2 x SSC/0.1% SDS for 15 minutes, then washed twice in 0.1 x SSC/0.1% SDS at 65°C for 15 minutes each time.
  • Equal loading of RNA was determined by ethidium bromide staining of rRNA and by probing for ⁇ -actin, as described above.
  • Glycogen Synthase and Glycogen Synthesis Assays Glycogen synthase activity associated with immobilized GST-PTG was determined as described previously (Lazar D.F., supra., 1995).
  • the agarose beads were washed four times with glycogen synthase buffer, brought to a final volume of 300 ⁇ L and 50 ⁇ L assayed for glycogen synthase activity by measuring the incorporation of UDP-[ ⁇ C]glucose into glycogen, both in the presence and absence of 10 mM glucose-6-phosphate (Sigma).
  • the accumulation of glycogen in intact pFPTG transfected CHO-IR cells was determined by an adaptation of the method of Lawrence J.C., et al., J. Biol. Chem., 1977;252:444 as described previously (Lazar D.F., supra., 1995).
  • Phosphorylase kinase 50 ⁇ L of GST-PTG fusion protein beads was added to 750 ⁇ L homogenization buffer containing 0.15 M NaCl, 0.1 % BSA, and 25 ⁇ g of [ 32 P]-labeled phosphorylase a. The tubes were incubated at 37°C for 20 minutes, washed four times with homogenization buffer, and proteins separated by SDS-PAGE, followed by autoradiography (Lawrence J.C., supra., 1977).
  • glycogen synthase buffer 50 mM HEPES, pH 7.8/100 mM NaF/10 mM EDTA
  • 25 ⁇ g (0.1 U) purified glycogen synthase Sigma
  • the agarose beads were washed four times with glycogen synthase buffer, brought to a final volume of 300 ⁇ L and 50 ⁇ L assayed for glycogen synthase activity (Lazar D.F., supra., 1995) by measuring the incorporation of UDP- [ ⁇ Cjglucose into glycogen, both in the presence and absence of 10 mM glucose- 6-phosphate (Sigma).
  • Phosphorylase Kinase Fifty microliters of fusion protein beads were incubated with 10 ⁇ g purified phosphorylase kinase (Gibco) in homogenization buffer plus 0.15 M NaCl and 0.1 % BS A, or with 3T3-L 1 adipocyte cell lysate, incubated 30 minutes at 4°C and washed four times with the same buffer. Ten microliter beads were assayed (Lazar D.F., supra., 1995) in 50 mM HEPES, pH 7.4, 10 mM MgCl, 1 ⁇ M okadaic acid, in the absence (1 mM EGTA) or presence
  • Murine and human genomic PTG sequences were obtained by screening the respective genomic Bacterial Artificial Chromosome (BAC) (Shizuya H, et al., Proc. Natl.
  • BAC Bacterial Artificial Chromosome
  • BAC clone 201D24 were chosen for further characterization following southern analysis with the labeled 1.0 kb cDNA fragment from clone B2-2 to confirm the presence of hybridizing DNA sequences.
  • 255E4 (5 ⁇ g) was digested with EcoRI (1 unit) at 37°C for 1 hour prior to separation of the resulting DNA fragments by electrophoresis through a 0.6% agarose gel. DNA fragments were transferred to nylon membrane (Hybond, Amersham) by capillary diffusion and probed with cDNA fragment encompassing the PTG coding sequence from clone B2-2. The transfer membrane was pre- hybridized for 1 hour in FBY hybridization buffer ( 10% PEG/1.5 x SSPE/7%
  • a 5.0 kb EcoRI fragment was found to hybridize to the Bl-1 PTG probe and was subsequently cloned into the EcoRI site of vector pBluescript II SK" (Stratogene, La Jolla, CA), creating the plasmid pJPD23.
  • Preliminary sequence analysis of subcloned 5.0 kb fragment was performed by using T3 and T7 primers complementary to vector sequences flanking the inserted fragment. Complete sequence information was obtained by synthesizing oligonucleotide primers complementary to both positive and negative strands of the inserted human genomic DNA. Sequencing was performed at The University of Michigan DNA sequencing core facility with an Appligen fluorescent dye terminator kit (Perkin- ⁇ lmer) and an ABI 8700 automated sequencer.
  • BAC clone 201D24 was subjected to southern analysis, as described for the human genomic BAC clone, except Bam ⁇ (1 unit, 37°C, 2 hours) was used to digest 5 ⁇ g of DNA. A 7.0 kb hybridizing fragment was identified and subcloned into the BamHl site of pBluescript II Sk", creating pJPD27. An overlapping 5.0 kb EcoRI fragment 3' to the PTG open reading frame was identified by restriction digest of BAC clone 201D24 (5 ⁇ g) with EcoRI (1 unit, 37°C, 2 hours), followed by southern analysis using a 0.8 kb S.stl-Ba Vll fragment from the extreme 3' of genomic DNNA of pJPD27.
  • PTG Knockout Vector To further characterize the physiological role of PTG in overall glycogen metabolism in vivo, a targeted replacement vector was constructed to delete the PTG coding sequences from a mouse genome.
  • pKO Scrambler V901 vector (Lexicon Genetics, Ine, The Woodlands, TX) forms the backbone of the targeting vector, as this vector has scrambled polylinkers, for insertion of 5' and 3' homologous genomic DNA, flanking a unique restriction site for insertion of a positive selectable marker (neo r for selection of transfected ⁇ S cells on the antibiotic G418).
  • pKO Scrambler V901 also contains a unique restriction site for the insertion of a negative selection element (Thymidine Kinase) for positive-negative selection strategies, which has been reported to increase targeting efficiency to the desired locus 2- to 20-fold (Hasty P. and Bradley A. in Gene Targeting: A Practical Approach, 1993, A.L. Joyner, ⁇ d., IRL Press, Oxford).
  • Neomycin Phosphotransferase gene under the control of the PGK promoter was excised from plasmid pKO selectN ⁇ O V800 (Lexicon Genetics, Ine, The Woodlands, TX) by digestion with the restriction enzyme Ascll (New England Biolabs, Beverly, MA) and subcloned into the unique Ascll site of pKO Scrambler V901 , creating plasmid pKO-neo.
  • a negative selection cassette containing the thymidine kinase gene under the control of the MCI promoter was subcloned into the unique Rsr ⁇ l site of pKO-neo by digestion of plasmid pKO SelectTK V800 (Lexicon Genetics, Ine, The Woodlands, TX) withforll (New England Biolabs, Beverly, MA) followed by separation and isolation of the appropriate restriction fragment (2.0 kb) by electrophoresis through a 1.0% agarose gel, creating plasmid pKO-TK/neo.
  • a 2.0 kb region of DNA 5' to the PTG coding region was amplified by Polymerase Chain Reaction (PCR) using primers 5'-CGAGGATCCTTGTCTTCTCTGCAGATG-3' (SEQ ID NO.: 7) and 5'-GCTGGTACCTGAATGAGCCAAGCAAATCCTC-3' (SEQ ID NO.: 8), which contain BamHl and Kpnl sites, respectively.
  • the amplified DNA product was then cloned into the Bgl ⁇ l-Kpnl of plasmid pKO-TK/neo, creating the plasmid pKO-TK/neo-5'.
  • the 3.5 kb 3' homology region of genomic PTG DNA was cloned into the EcoRI-S ⁇ /I sites of pKO-TK/neo-5' by first digesting pJPD27 (1 ⁇ g) with Smal (1 unit, 22°C, 2 hours), and inserting a Sail oligonucleotide linker (5'-CCGG CGACCGG-3') (S ⁇ Q ID NO.: 9), creating plasmid pJPD27 ⁇ Sma.
  • the 3.5 kb EcoRI-S ⁇ fl fragment from pJPD27 ⁇ Sma was then ligated into the EcoRI -Sa l sites of pKO-TK/Neo-5' to create the targeting vector pKO-PTG.
  • a 0.5 kb 5' DNA probe was generated by PCR amplification from plasmid pJPD27 with the T3 specific primer, complementary to DNA sequences contained within the vector pBluescript II Sk " and a primer specific to the extreme 5' region of mouse genomic DNA sequence (5'-GCAGAGAAGACAAAACCAC-3') (S ⁇ Q ID NO.: 10).
  • the 3' DNA probe was generated by digestion of plasmid p201-3' with Bam l and isolation of the resulting 0.8 kb fragment following electrophoretic separation on a 1.5% agarose gel.
  • FIG. 1 Sequence comparison of PTG with glycogen localizing subunits of PPIC.
  • BESTFIT sequence comparison program was used to align and compare the primary amino acid sequences of PTG, GL, RGl and Gael. The boxed regions represent areas of similarity and the sites of conservation are indicated by shading.
  • PPIC binds PTG in vivo.
  • pCI-neo expressing FLAG-PTG from the CMV promoter was transiently transfected into CHO-IR cells and immunoprecipitated from cell lysates with antibodies directed against the FLAG epitope. Precipitates were analyzed by SDS-PAGE on a 4% to 20% gel, transferred to nitrocellulose and blotted with ⁇ PPIC polyclonal antibodies. Immunoreactive proteins were visualized by
  • ECL Enhanced Chemiluminescence
  • PTG targets PPIC to glycogen in vitro PTG dependent localization of PPl C to the glycogen pellet was determined by incubated 3T3-L1 adipocyte cell lysates with bacterially expressed GST-PTP1B or GST-PTG prior to subcellular fractionation as above. PPIC activity in the glycogen pellet was measured as described previously (Lazar D.F., supra., 1995).
  • A PTG is expressed in insulin responsive tissues.
  • a multi-tissue northern blot (Clonetech) was hybridized overnight at 65°C with a 1.0 kb EcoRI fragment of clone Bl-1, which was labeled with [ ⁇ -32p]dCTP by random priming, and exposed to film for 24 hours.
  • B PTG expression is induced by adipocyte differentiation. 3T3-L1 fibroblast and fully differentiated 3T3-L1 adipocyte total RNA was isolated and electrophoresed (15 ⁇ g) in 1.2% agarose/2.2 M formaldehyde/ 1 x MOPS, followed by transfer to nylon membrane by capillary diffusion. The transfer membrane was hybridized and probed as in (A). Equal loading of RNA was determined by ethidium bromide staining of rRNA and by probing for ⁇ -actin transcript. Molecular size markers (kb) are indicated on the left.
  • glycogen synthase buffer 50 mM HEPES, pH 7.8/100 mM NaF/10 mM EDTA
  • 25 ⁇ L purified glycogen synthase (Sigma), followed by incubation at 4°C for 1 hour with gentle mixing.
  • the agarose beads were washed four times with glycogen synthase buffer and assayed for glycogen synthase activity.
  • C, D Phosphorylase kinase binds to PTG.
  • Fifty microliters of bacterially expressed GST-PTG bound to glutathione-agarose beads was incubated with of 3T3-L1 adipocyte cell lysate (C) or 10 ⁇ g purified phosphorylase kinase (Gibco) (D) in homogenization buffer. Samples were incubated 30 minutes at 4°C and washed four times with the same buffer.
  • Ten microliter beads were assayed for phosphorylase kinase activity using 2 ⁇ g phosphorylase b per sample in the absence (1 mM EGTA) or presence (0.5 mM) of Ca + ⁇ .
  • Complexed proteins were separated on a 10% SDS-polyacrylamide gel and radiolabeled phosphorylase a was visualized by autoradiography.
  • FIG. 1 Glycogen synthesis in CHO-IR cells overexpressing PTG.
  • CHO-IR cells were grown to 40% to 50% confluency in 6-well dishes and transiently transfected with pFLAG-PTG. Forty-eight hours after transfection, cells were serum deprived for 3 hours and glycogen accumulation in intact pFLAG-PTG or lacZ transfected cells, in the presence or absence of 100 nM insulin, was determined. Results are expressed as means of triplicate determinations, of SD, and were repeated in two separate experiments.
  • MOLECULE TYPE DNA (genomic)
  • AAGTCATTCT TTCTTTTAAC AAGCGTCACC TACTGTCACT CTAAGGACAG CATGACATTT 1920 TAAGAATTGC TTCATTT ⁇ TT GTTTCCCAAG TGGATTACTT CTCCTGAGAA GTAAAACCGG 1980
  • MOLECULE TYPE DNA (genomic)
  • TCTCTCCCAG CGACCGCCGC GGGGGCAAGG CCTGGAGCTG TGGTTCGAAT TTGTGCAGGC 2100 AGCGGGTGCT GGCTTTTAGG GTCCGCCGCC TCTCTGCCTA ATGAGCTGCA CCAGGTAGGT 2160
  • AATCGAACCC TTTTATTTCT CAGATGGGGA AACTGAGACC CCCATCACCC TCT ⁇ AGTGTT 4020 TTAAGCAATT AATAGCCTTT ACCGGCCAAG GGTAGAGGTA GACATAGAAG ATCTGATCAC 4080
  • TTAATACTGT TCTCTTTTAC TACATATGAT AGCACCTGCC TGATATCTAG TGCACTGGCT 140
  • ATCCAGGTGA TAATCCCTCT TCTTTTTGCA TTCCAGA ATG ATC CAG GTT TTA GAT 4255
  • TCA TGT CTC AAT ATA AAA CAC AAA GCC AAA TCA CAG AAT GAC TGG AAG 4447 Ser Cys Leu Asn He Lys His Lys Ala Lys Ser Gin Asn Asp Trp Lys 350 355 360 365
  • TTAGAGTCAA CAATCTTTGG CAGTCCGAGG CTGGCTAGTG GGCTCTTCCC AGAGTGGCAG 900

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EP97938246A 1996-08-30 1997-08-22 Protein das auf glykogen auspürt Withdrawn EP0941324A1 (de)

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Application Number Priority Date Filing Date Title
US2510796P 1996-08-30 1996-08-30
US25107P 1996-08-30
US5524397P 1997-08-12 1997-08-12
US55243P 1997-08-12
PCT/US1997/014142 WO1998008948A1 (en) 1996-08-30 1997-08-22 Protein targeting to glycogen

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US5939284A (en) 1996-12-05 1999-08-17 Smithkline Beecham Corporation Protein phosphatase 1 binding protein, R5

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Title
See references of WO9808948A1 *

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AU4062397A (en) 1998-03-19
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