WO1992013083A1 - Acide nucleique codant le substrat-1 recepteur d'insuline (irs-1), proteine d'irs-1, maladies et therapie associees au metabolisme de l'irs-1 - Google Patents

Acide nucleique codant le substrat-1 recepteur d'insuline (irs-1), proteine d'irs-1, maladies et therapie associees au metabolisme de l'irs-1 Download PDF

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WO1992013083A1
WO1992013083A1 PCT/US1992/000437 US9200437W WO9213083A1 WO 1992013083 A1 WO1992013083 A1 WO 1992013083A1 US 9200437 W US9200437 W US 9200437W WO 9213083 A1 WO9213083 A1 WO 9213083A1
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irs
insulin
phosphorylation
metabolism
ser
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PCT/US1992/000437
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English (en)
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C. Ronald Kahn
Morris F. White
Paul Louis Rothenberg
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Joslin Diabetes Center, Inc.
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Publication of WO1992013083A1 publication Critical patent/WO1992013083A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • IRS-1 NUCLEIC ACID ENCODING INSULIN RECEPTOR SUBSTRATE-l (IRS-1), IRS-1 PROTEIN, DISEASES, THERAPY ASSOCIATED WITH THE METABOLISM OF IRS-1
  • the invention relates to insulin metabolism and
  • Tyrosine phosphorylation of several cellular proteins and enzymes has been observed during the initial cellular response to some receptor tyrosine kinase-linked polypeptide growth factors, e.g., PDGF-induced phosphorylation of phospholipase C (Meisenhelder et al., 1989, Cell 52:1109-1122, hereby incorporated by reference), 3'-phosphatidyl-inositol (PI-3) kinase (Kaplan et al., 1987, Cell 5 :1021-1029, hereby incorporated by reference) , and the raf-1 kinase (Morrison et al.
  • PDGF-induced phosphorylation of phospholipase C e.g., PDGF-induced phosphorylation of phospholipase C (Meisenhelder et al., 1989, Cell 52:1109-1122, hereby incorporated by reference)
  • PI-3 kinase
  • the invention features a purified nucleic acid encoding IRS-1.
  • the purified nucleic acid is from a mammal, e.g., a rat or a human, the purified nucleic acid is present in a vector, and the purified nucleic acid is present in a cell, the purified nucleic acid is under the transcriptional control of a heterologous promoter.
  • the invention also includes a homogeneous population of cells, preferably eukaryotic cells, wherein each of the cells contains cloned nucleic acid encoding IRS-1.
  • the invention features a purified preparation of IRS-l, preferrably produced from nucleic acid encoding IRS-1.
  • the invention also includes a method of producing IRS-1, including the steps of: culturing a cell which contains purified nucleic acid which encodes IRS-1 in medium to form a population of cells which expresses IRS-1 and purifying IRS-1 from the cells or from the culture medium.
  • the invention features a method of purifying a phosphoprotein including in the following order: (a) providing a sample containing the phosphoprotein in the phosphorylated state; (b) contacting the sample with denaturant and a reducing agent under conditions, e.g., heating, or boiling, that inhibit the removal of phosphate groups from the phosphoprotein; (c) decreasing the concentration of the denaturant sufficiently to allow an antiphospho-amino antibody e.g., an anti-phosptyrosive antibody, to bind to the phosphoprotein; and (d) contacting the phosphoprotein with an anti-phosphoamino antibody and purifying the phosphoprotein by virtue of its affinity for the antibody.
  • the antibody is bound to a substrate, the sample is contacted with the bound antibody, and the phosphoprotein is eluted from the bound antibody.
  • the invention features a method of diagnosing a disease, e.g., insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g.. Type II diabetes, in a mammal, e.g., in a human.
  • a disease e.g., insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g.. Type II diabetes
  • the disease is characterized by an abnormality in IRS-1 structure or metabolism.
  • the method includes measuring an aspect of IRS-1 metabolism in the mammal, an abnormal level of IRS-1 metabolism being diagnostic of the disease.
  • the metabolism of a substance means any aspect of the metabolism, expression, function, or action of the substance.
  • the measurement includes measuring the level of IRS-1 in a tissue sample (a tissue sample as used herein means any suitable sample e.g., a sample including classic insulin sensitive tissue, e.g., muscle, fat or liver tissue, or a sample including more easily accessible tissue, e.g., circulating blood cells or fibroblasts) , taken from the mammal; the measurement includes measuring the level of phosphorylation of the IRS-1 in a tissue sample taken from the mammal; the measurement includes measuring the level of kinase activity of IRS-1; and the measurement includes measuring the amount of IRS-1 encoding RNA in a tissue sample taken from the mammal.
  • a tissue sample as used herein means any suitable sample e.g., a sample including classic insulin sensitive tissue, e.g., muscle, fat or liver tissue, or a sample including more easily accessible tissue, e.g., circulating blood cells or fibroblasts
  • An insulin-related disease is a disease, disorder, or condition in which some aspect of insulin expression metabolism, or action is disrupted or, a disease in which insulin action contributes to the disease.
  • An insulin resistant insulin-related disease is any disease, disorder, or condition in which a normal amount of insulin results in a less than normal biological response. Examples of insulin resistant diseases include Type II diabetes, obesity, aging related insulin resistance, and insulin resistance that arises secondary to infections, hormonal disorders, or other causes.
  • the invention also features a method of diagnosing, preferably prenatally, an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes, in a mammal, e.g., a human, including determining the structure of the gene which expresses IRS-1, an abnormal structure being diagnostic of the disease.
  • an insulin-related disease e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes
  • the invention also includes a method of assaying an effect of a therapeutic agent on IRS-1 metabolism, e.g., an agent used to treat an insulin-related disease in a mammal, e.g., a human.
  • a therapeutic agent as used herein, can be any substance or treatment.
  • the method includes administering the agent to a test organism e.g., a cultured cell or a mammal, and measuring the effect of the drug on an aspect of IRS-1 metabolism, e.g. , measuring the level of IRS-1 expression, the cellular or intra-cellular distribution of IRS-1, or the level of the IRS-1 phosphorylation.
  • a change in an aspect of IRS-1 metabolism indicates an effect of the agent.
  • the insulin-related disease is an insulin resistant disease and the change in an aspect of metabolism is a change in the level of IRS- 1 phosphorylation.
  • the invention also includes a method of assaying an effect of a therapeutic agent which mimics a first effect of insulin, the first effect mediated by IRS-1, without mimicking a second effect of insulin.
  • the method includes administering the agent to a test organism, e.g., a cell grown in culture or a mammal, and measuring a change in an aspect of IRS-1 metabolism, e.g., the level of IRS-1 expression, the kinase activity of IRS-1, the cellular or intra-cellular distribution of IRS-1, or the level of the IRS-1 phosphorylation.
  • a change in an aspect of IRS-1 metabolism indicates an effect of the agent.
  • the invention also features a method of assaying an effect of a therapeutic agent which alters the ability of a tyrosine kinase to phosphorylate a substrate which includes the amino acid sequence YMXM (Seq. I.D. No. 1).
  • the method includes administering the drug to a test organism, e.g., a cultured cell or a mammal, and measuring the level of phosphorylation of a substrate, which includes the amino acid sequence YMXM (Seq. I.D. No. 1), e.g., a naturally occurring substrate of the tyrosine kinase or a synthetic substrate.
  • the invention also includes a method of treating a mammal e.g., a human, suffering from a disease, disorder, or condition caused by the phosphorylation of a substrate of a tyrosine kinase, the substrate including the amino acid sequence YMXM (Seq. I.D. No. 1) .
  • the tyrosine kinase may be, e.g., a receptor tyrosine kinase, e.g., insulin receptor, epidermal growth factor (EGF) receptor, platelet derived growth factor, (PDGF) receptor, or insulin-like growth factor (ILG) receptor, or an Oncogene product, e.g., the src, abl, or fms gene product.
  • the method includes administering a therapeutically effective amount of a therapeutic agent, e.g., IRS-1, which includes the amino acid sequence YMXM (Seq. I.D. No. 1) .
  • the substance blocks phosphorylation of the naturally occurring substrate by competitive or non-competitive inhibition of the naturally occurring substrate.
  • the invention also features a method of treating a mammal e.g., a human, suffering from a disease caused by IRS-1, e.g., by an abnormality of IRS-1 metabolism.
  • the method includes administering to the mammal a therapeutically effective amount of a therapeutic agent, e.g., IRS-1, agent which alters an aspect of the metabolism of IRS-1, e.g., the level of IRS-1 phosphorylation.
  • the abnormality includes the inability of the insulin receptor to respond to insulin by phosphorylating IRS-1.
  • the agent increases the phosphorylation of IRS-1, e.g., by increasing the activity of a kinase or decreasing the activity of a phosphatase.
  • the agent decreases the phosphorylation of IRS-1, e.g., by decreasing the activity of a kinase or increasing the activity of a phosphatase.
  • the invention also features a method of treating a mammal, e.g., a human, suffering from a disease caused by a tyrosine kinase.
  • the method includes administering to the mammal a therapeutically effective amount of a therapeutic agent which modifies the ability of IRS-1 to alter the phosphorylation of the tyrosine kinase, thereby altering the activity of the tyrosine kinase.
  • the tyrosine kinase is the product of an oncogene.
  • the invention also features a method of treating a mammal, e.g., a human, suffering from a disease characterized by abnormal cell proliferation.
  • Abnormal cell proliferation includes both neoplastic and non-neoplastic diseases, and thus includes diseases such as cancer and atherosclerosis.
  • the method includes administering to said mammal a therapeutically effective amount of a therapeutic agent, e.g., IRS-1, which alters an aspect of IRS-1 metabolism.
  • a therapeutic agent e.g., IRS-1
  • IRS-1 which alters an aspect of IRS-1 metabolism.
  • the aspect of IRS-l metabolism is IRS-1 phosphorylation.
  • the aspect of IRS-1 metabolism is the level of kinase activity of IRS-1.
  • IRS-1 insulin receptor substrate, e.g., mammalian insulin receptor substrate, e.g., rat or human insulin receptor substrate.
  • Purified nucleic acid means nucleic acid which is separated from other nucleic acid with which it is naturally joined covalently.
  • a vector as used herein, is an autonomously replicating nucleic acid molecule.
  • a heterologous promoter as used herein is a promoter which is not naturally associated with a gene or a purified nucleic acid.
  • a nucleic acid encoding IRS-1 is a nucleic acid, preferrably a DNA molecule, which encodes a protein which, in its natural state, is phosphorylated by the insulin receptor, preferably in an insulin dependent fashion, and which has at least 70%, preferably 80%, and more preferably 90% homology to IRS-1 or which hybridizes to IRS-1 under conditions of high stringency.
  • An anti-phosphoamino antibody is an antibody directed against a phosphorylated amino acid, e.g., an antibody directed against phosphotyrosine or phosphoserine.
  • a denaturant is an agent, e.g., a detergent, e.g., SDS, which disrupts the tertiary structure of a protein and thereby inhibits the enzymatic activity of the protein.
  • a detergent e.g., SDS
  • a reducing agent as used herein, is an agent which disrupts S-S bonds.
  • a phosphoprotein as used herein, is a phosphorylated protein or polypeptide.
  • a purified preparation of IRS-1 means IRS-1 that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs.
  • the IRS-1 is also separated from substances, e.g., antibodies or gel matrix, e.g, polyacrylimide, which are used to purify it.
  • the IRS-1 constitutes at least 10% dry weight of the purified preparation.
  • the preparation contains sufficient IRS-1 to allow protein sequencing.
  • the method of the invention can be used to diagnose the presence of diseases charaterized by an abnormality in the structure or metabolism of IRS-1 or the insulin receptor.
  • the invention allows for the analysis of various aspects of insulin metabolism, e.g. , for the determination of insulin receptor function, e.g., the detection of insulin-stimulated substrate phosphorylation.
  • the invention also provides useful tools for the testing and development of therapeutic agents used to treat insulin or IRS-1 related diseases.
  • Methods of the invention allow for rapid and high yield purification of phosphoproteins.
  • the denaturation step prevents dephosphorylation and thus allows efficient anti-phosphoamino antibody based purification.
  • Methods of the invention also allow the treatment of a variety of diseases, e.g., insulin related diseases, insulin resistant diseases, diseases charaterized by abnormal cellular proliferation, and diseases caused by the phosphorylation of a substrate by a tyrosine kinase, by intervening in aspects of IRS-1 metabolism.
  • diseases e.g., insulin related diseases, insulin resistant diseases, diseases charaterized by abnormal cellular proliferation, and diseases caused by the phosphorylation of a substrate by a tyrosine kinase, by intervening in aspects of IRS-1 metabolism.
  • Fig. 1 is a graph of the effect of insulin on phosphorylation of ppl75 and the 0-subunit of the insulin receptor.
  • Fig. 2 is a diagram of two dimensional phosphopeptide maps of ppl75 and the /3-subunit of the insulin receptor.
  • Fig. 3 is a graph of the effect of vanadate on blood glucose levels in ob/ob mice.
  • Fig. 4 is a graph of the effect of vanadate on blood glucose level in ob/ob mice.
  • Fig. 5 is a graph of the effect of vanadate on blood glucose level in db/db mice.
  • Fig. 6 is a comparison of PTPase activity in the cytosolic liver fraction of ob/+ and ob/ob mice.
  • Fig. 7 is a comparison of the PTPase activity in the particulate and WGA purified fractions of livers from ob/ob and ob/+ mice.
  • Fig. 8 is a comparison of the PTPase activity in cytosolic liver fraction from db/db and control mice.
  • Fig. 9 is a comparison of the PTPase activity in the particulate fraction from the livers of db/db and control mice.
  • Fig. 10 is graph of the effects of vanadate treatment on PTPase activity in particulate fractions from ob/ob mouse livers.
  • Fig. 11 is a map of probes 138 (Seq. I.D. No. 2) and 80" (Seq. I.D. No. 3).
  • Fig. 12 is the sequence of rat IRS-1 (Seq. I.D. No. 4) .
  • Fig. 13 is a diagram of overlapping cDNA inserts from two rat liver cDNA libraries.
  • Fig. 14 is a map of three mRNA molecules.
  • Fig. 15 is a diagram of structural features of rat IRS-1.
  • Fig. 16 is a map of the putative phosphorylation sites in IRS-1.
  • Fig. 17 is a graph of the effect of insulin stimulation of phosphorylation of synthetic peptides.
  • Fig. 18 is a graph of the effect of insulin stimulation on phosphatidyl inositol 3-kinase. Purification and Partial Sequence Analysis of PP185 ⁇ the Manor Cellular Substrate of the Insulin Receptor Tyrosine Kinase
  • pp!85 ppl85 was purified from liver using SDS denaturation/TCA precipitation, coupled with preparative- scale anti-phosphotyrosine antibody immunoaffinity chromatography. Following infusion of insulin total liver extracts of denatured proteins were prepared from SDS-homogenates. After dissolution in base and neutralization, each liver extract was passed through a column of immobilized anti-phosphotyrosine antibody. The column was washed, and the adsorbed phosphotyrosyl proteins were eluted from the affinity matrix with p- nitrophenyl phosphate (pNPP) . (The method is described in detail below) .
  • pNPP p- nitrophenyl phosphate
  • the eluted phosphotyrosyl proteins were analyzed by anti-phosphotyrosine Western blotting and by direct silver-staining, as described below.
  • M 120 kDa protein
  • insulin-receptor 0-subunit The eluted phosphotyrosyl proteins were analyzed by anti-phosphotyrosine Western blotting and by direct silver-staining, as described below.
  • the eluate was made 5 mM in DTT, and then simultaneously dialyzed (against 0.05% SDS, 5 mM DTT, 50 mM Tris, pH 7.2) and concentrated 125-fold _in vacuo at 22 C° in a Micro-ProDiCon apparatus using PA-15 membranes (Bio-Molecular Dynamics Co., Beaverton, Oregon).
  • the concentrated sample was made 10% in sucrose, 50 mM DTT, and heated at 100 C° for 3 minutes.
  • liver extract Since only about half of the phosphotyrosyl protein content of the original liver extract was removed by a single pass over the aPY-Ab column under the conditions just described, the liver extract was therefore recycled through the column, and the adsorption, column washing, hapten elution, dialysis and concentration procedure was repeated, and the final sample combined with the first, and stored at -70 C°.
  • Dry liver protein precipitate was prepared as follows. Male rats (100 to 250 g) were fed ad libitum with Purina Laboratory Rodent Chow, except where indicated. Rats were injected with sodium amobarbital (150 mg/kg body weight, intraperitoneal) and were used in experiments 10 to 15 minutes later as soon as anesthesia was assured by loss of pedal and corneal reflexes. The abdominal cavity was opened, the portal vein or inferior vena cava exposed, and normal saline (0.9% NaCl) with or without hormone (10 ⁇ 6 M insulin) was infused through a 27 g needle connected to a mechanical syringe pump driven at 1 cc/min for 0.5 minutes.
  • solubilization buffer maintained at 100 C° in a water bath with a Polytron PTA 20S generator (Brinkmann Inst. Co., Model PT10/35) operated at maximum speed (Setting 10) .
  • the solubilization buffer was composed of 2% SDS, 100 mM HEPES (pH 7.8 at 22 C°) , 100 mM NaCl, 10 mM EDTA and 50 mM DTT. The homogenate was further heated to boiling with gentle stirring for 2 minutes, and then left to cool to 22 C°.
  • the precipitate was washed once with 25 volumes of 10% TCA at 4 C°, and the TCA was then extracted by three washes, each with 25 volumes of ethanol:diethyl ether (1:1, v/v) at 4 C°.
  • the precipitate was dried m vacuo for 4 to 18 hours.
  • the final yield of dry precipitate was about 0.05 gram per gram of liver (wet wt.).
  • the precipitate was thoroughly pulverized to a fine powder in a porcelain mortar. In this form the extracted proteins can be stored for at least 1 year at -70 C° without apparent degradation or significant loss of phosphotyrosine content.
  • Alternate methods of removing the SDS from the initial tissue extracts were evaluated. These include: precipitation of the insoluble potassium salt of SDS by KC1 addition (Suzuki et al., 1988, Anal. Biochem. 172:259-263. hereby incorporated by reference, Zaman et al., 1979, Anal. Bioche . 100:64-69. hereby incorporated by reference) , dilution of the SDS with an excess of Triton X-100 (Clarke, 1981, Biochem. Biophys. Acta 670:195-202. hereby incorporated by reference), selective precipitation of proteins with cold organic solvents (Hager et al., 1980, 109:76-86.
  • the immunoprecipitated proteins were solubilized in 50 ul of SDS-PAGE sample buffer (Laemmli) with 50 mM DTT at 100°C for 3 minutes.
  • the immunoprecipitated phosphotyrosyl proteins were competitively eluted from the antibody-bead pellet by incubating with lOOmM pNPP, 50 mM Tris, pH 7.4, 0.05% SDS for 1 hour at 22°C.
  • the eluate was desalted by centrifugal passage (Helmerhorst et al., 1980, Analytical Biochemistry 104.:130-135, hereby incorporated by reference) over a micro-column of G-25 Sephadex, pre- equilibrated with Laemmli sample buffer.
  • the anti-phosphotyrosine antibody affinity-matrix was prepared as follows. 38 milligrams of affinity- purified rabbit anti-phosphotyrosine antibody (aPY Ab) was adsorbed to 12 mis (settled gel volume) of Protein A TrisAcryl, by slow mixing at 4 C° overnight in 150 mM NaCl, 50 mM HEPES, pH 7.8. The gel matrix was washed three times with 100 mis of 0.2 M sodium borate, pH 9.0 at 22 C°, and resuspended in 45 mis of 0.2 M sodium borate, pH 9.0 also containing 2 mM pNPP (to bind and protect the antibody combining site) for 2 hours at 22 C°.
  • Dimethylpimelimidate was then added (20 mM final concentration) , and the matrix gently mixed at 22 C° for 30 minutes to covalently link the antibody to Protein A (Simanis et al., 1985, Virology 144:88-100. hereby incorporated by reference) .
  • the antibody-matrix was then washed with excess 0.2 M ethylamine, pH 8.0 at 22 C° and incubated 2 hours further in the same buffer to quench unreacted dimethylpimelimidate.
  • the cross-linked matrix was washed extensively and stored at 4 C° in 10 mM Tris, pH 7.5, 150 mM NaCl, 0.02% NaN 3 .
  • PVDF membranes were incubated in 0.1% Coomassie Blue R-250, 50% methanol, for 5 minutes, and following destaining (in 50% methanol, 10% acetic acid) , the visible protein bands were individually excised. Proteins blotted onto PVDF membranes were placed in 6 x 50 mm tubes previously baked at 1000 F for 16 hours. The tube(s) were placed in a Waters hydrolysis vial, 200 ul of constant boiling HC1 added, and the vial evacuated and flushed with argon. After a final exposure to vacuum, the vial was sealed and heated at 110 C° for 22 hrs. Following hydrolysis, the samples were dried in vacuo.
  • the PVDF membrane was wet with 10 ul MeOH, then extracted twice with 100 ul of 0.1 M HCl/20% MeOH. This extract was taken to dryness, dissolved in 4 mM EDTA, and loaded onto an Applied Biosystems 420A derivatizer/analyzer for amino acid analysis.
  • Phosphotyrosyl protein yields were also estimated by direct silver staining of SDS-PAGE gels (Heukeshoeven et al., 1985, Electrophoresis .6:103-112, hereby incorporated by reference) , or alternatively by colloidal-gold staining of nitrocellulose electroblots (Li et al., 1989, Analytical Biochemistry 182:44-47. hereby incorporated by reference), (Hunter et al., 1987, Analytical Biochemistry 164:430-433 , hereby incorporated by reference) with comparison of band intensities to those of standard reference proteins included in the same gel or blot.
  • Electrophoresis and immunoblotting were performed as follows. Immunoprecipitated proteins were separated on 0.5 mm thick, 1-D SDS-PAGE (5% T acrylamide) using the formulations of Laemmli (Laemmli, 1970, Nature 227:680- 685, hereby incorporated by reference) in a BioRad miniature slab gel apparatus (Mini-Protean) at 175 V (constant) . Standard molecular weight protein markers were: myosin (200 kDa) , 0-galactosidase (116 kDa) , phosphorylase b (97.4 kDa) BSA (66.2 kDa) and ovalbumin (42.7 kDa).
  • Electrotransfer of proteins from the gel to nitrocellulose was performed for 2 hours at 100 V (constant) at 5-15°C in the BioRad miniature transfer apparatus (Mini-Protean) , as described by Towbin (Towbin et al., 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354, hereby incorporated by reference), but with 0.05% SDS added to the transfer buffer to enhance elution of high molecular weight proteins.
  • Preliminary experiments using alternate transfer buffers (Szewczyk et al., 1985, Anal. Biochem. 150:403-407, hereby incorporated by reference) or transfer times varying from 0.5 to 4 hours gave qualitatively identical results.
  • Non-specific protein binding to the nitrocellulose was reduced by pre- incubating the filter overnight at 4°C in blocking buffer (5% BSA, 1% ovalbumin in TNA [10 mM Tris, pH 7.2, 0.9% NaCl, 0.02% NaN 3 ]).
  • the nitrocellulose blot was incubated with anti-phosphotyrosine antibodies diluted in blocking buffer (2 ug/ml) for 2 hours at 22°C and then washed twice for 10 min in TNA, once for 10 minutes in TNA containing 0.05% NP-40, and twice further for 10 minutes each in TNA.
  • the blots were then incubated with 50 uCi of 125 I-Protein A (6-30 uCi/ug) in 10 mis of blocking buffer for 1 hour at 22°C, and then again washed as described above.
  • Bound anti-phosphotyrosine antibodies were detected by autoradiography using pre- flashed (Laskey et al., 1975, Eur. J. Biochem. 56:335- 342, hereby incorporated by reference) Kodak XAR film with Cronex Lightning Plus intensifying screens at -70°C for 12 to 72 hours. Band intensities were quantitated by optical densitometry (Hoefer Instruments Model GS300) of the developed autoradiogram or by direct gamma scintillation spectrometry of bands excised from the nitrocellulose blots.
  • TCA and diethyl ether were from Fisher Scientific. Wheat germ agglutinin-agarose was from Vector Labs. Male Sprague-Dawley rats were from Charles River, Wilmington, MA. Nitrocellulose (BA85, 0.2um) was from Schleicher and Schuell. PVDF membranes were from Millipore. Reagents for SDS-PAGE, including molecular weight standards were from BioRad. Silver-stain reagent kit was from Sigma, and colloidal gold-stain from Janssen. Sequencing grade bovine trypsin was obtained from Boehringer Mannheim.
  • HPLC grade trifluoroacetic acid was obtained from Applied Biosystems, Inc.; HPLC grade acetonitrile and water from Burdick and Jackson; and Vydac HPLC columns from The Nest Group. Automated sequencer and analyzer reagents were provided by the manufacturer. All other reagents were of at least analytical grade purity. Polyclonal anti- phosphotyrosine antibodies were raised in rabbits and affinity-purified on phosphotyramine columns as described by Pang et al., 1985, Arch. Biochem. Biophys. 242:176- 186, hereby incorporated by reference) .
  • Phosphotyrosyl proteins are susceptible to rapid phosphatase-mediated dephosphorylation both in vivo (Lau et al., 1989, Biochem J. 257:23-36, hereby incorporated by reference) and during cell extraction procedures (Kamps et al., 1988 Oncogene 2:305-315, hereby incorporated by reference) .
  • TCA was removed by organic extraction, the protein precipitate redissolved in 0.1N NaOH (conditions where phosphotyrosine is stable, (Cooper et al., 1983, Methods Enzymol. 99.:387-402, hereby incorporated by reference), and following neutralization, the phosphotyrosyl proteins were quantitatively precipitated with anti- phosphotyrosine antibodies.
  • the immunoprecipitated proteins were then resolved by 1-D SDS-PAGE, electr ⁇ blotted to nitrocellulose, and detected with additional anti-phosphotyrosine antibody and 125 I-Protein A.
  • FaO hepatoma cells (Deschatrette et al., 1979, Somatic Cell Gen. 5:697-718, hereby incorporated by reference) were cultured in Falcon 150 mm diameter plasticware dishes in Dulbeccos Modified Essential Medium supplemented with 10% heat-inactivated fetal calf serum (Gibco) and penicillin/streptomycin (Crettaz et al., 1984, Diabetes 3 ⁇ :477-485, hereby incorporated by reference) at 37 C in a 5% C0 2 incubator. For experiments, 90% confluent cultures (10 7 cells) were serum deprived for 16-18 hours prior to hormone stimulation, extraction and analysis.
  • cultures were metabolically labelled with l mCi of 32 P-orthophosphate in P- ⁇ -free medium for 4 hours prior to analysis by methods described elsewhere (White et al., 1987, J. Biol. Chem. 262:9769-9777. hereby incorporated by reference) .
  • Solutions containing sodium orthovanadate were prepared at neutral pH to avoid loss of phosphatase inhibitory activity, as previously described (Kadota et al., 1987, J. Biol. Chem. 2628252- 8256, hereby incorporated by reference) .
  • Residues in lower case letters represent assignments of less than full confidence. Positions where no assignment was possible are indicated by X.
  • Peak 42 yielded a single, 8 residue sequence. Searching of gene and protein sequence registries reveals this sequence is not identical to any previously reported sequence. Peak 43 yielded an interesting sequence of 18 residues, beginning with Glu, followed by 10 consecutive Gin residues. Peak 43 also contained a secondary (less abundant) sequence of 11 residues. Neither the primary nor the secondary sequence of Peak 43 is identical with reported sequences. Novel sequences were also derived from Peaks 72 , 76, 80, 98 , and 138. Although Peak 58 appeared in the tryptic map of the insulin-treated sample, the sequence of this peak matched that of an internal tryptic fragment of carbamyl phosphate synthetase (CPS) (Nyunoya et al., 1985, J. Biol. Chem. 260:9346-9356, hereby incorporated by reference). CPS is an inner mitochondrial matrix protein which is abundant in fasted rat liver (Schimke, 1963, J. Biol. Chem.
  • any individual tryptic peak is not necessarily homogenous and may contain more than a single polypeptide species.
  • the ultraviolet absorbance of each peak was monitored with a diode array detector, allowing simultaneous detection at 210 nm, 277 nm and 292 nm. Absorbance at 210 nm and 277 nm monitors for peptide bonds and aromatic residues, respectively, while detection at 292 nm distinguishes peptides containing tyrosine from those containing tryptophan. Tyrosine containing peptides have a low 292/277 nm absorbance ratio, whereas tryptophanyl peptides have a high 292/277 nm ratio.
  • Enzymatic cleavage of ppl85 was performed as follows. Anti-phosphotyrosine affinity-purified liver proteins, concentrated by vacuum-dialysis into a solution containing 3% SDS, 50 mM Tris, pH 7.2, 50 mM DTT, 10% sucrose, were made 5% in SDS, then preparatively separated by reducing 1-D SDS-PAGE (5.5% T -0.8% C°) in a BioRad miniature slab gel apparatus, using 1.2 mm thick gels, run at 150 Vosts, with electrophoresis buffers as described by Laemmli (Laemmli, 1970, Nature 222680-685, hereby incorporated by reference) .
  • the proteins were electro-transferred to BA 85 nitrocellulose in transfer buffer (10 mM Tris, pH 8.0, 192 mM glycine, 20% methanol, 0.02% SDS) for 2 hours at 4 C°, and then for an additional 15 minutes in transfer buffer lacking SDS.
  • transfer buffer 10 mM Tris, pH 8.0, 192 mM glycine, 20% methanol, 0.02% SDS
  • the nitrocellulose was stained for 2 minutes in 0.1% Ponceau S, 1% acetic acid, and destained for 4 minutes in 1% acetic acid.
  • the lightly stained bands were excised with a scalpel, washed three times with HPLC-grade water, and stored moist at -20 C°.
  • Peptide fragments of the electrophoretically separated proteins were generated by in situ proteolytic digestion of the nitrocellulose-bound proteins with trypsin, as described by Aebersold (Aebersold et al., 1987, Proc. Natl. Acad. Sci. USA 84.:6970-6974, hereby incorporated by reference) , but omitting the NaOH wash to minimize the loss of protein. After digestion the solution was immediately stored at -20 C° until separation of the resultant peptides by narrow-bore reverse phase HPLC.
  • Reverse phase HPLC separation of peptides was performed as follows. Peptides were separated by narrow- bore reverse phase HPLC on a Hewlett-Packard 1090 HPLC equipped with a 1040 diode array detector, using a Vydac 2.1 mm x 150 mm C18 column. The gradient employed was a modification of that described by Stone et al. (Stone et al., 1989 Techniques in Proteins Chemistry, Hugli ed. , pp. 377-391 Academic Press) .
  • buffer A was 0.06% trifluoroacetic acid/H 2 0 and buffer B was 0.055% trifluoracetic acid/acetonitrile
  • a gradient of 5% B at 0 min, 33% B at 63 min, 60% B at 95 min and 80% B at 105 min with a flow rate of 0.15 ml/min was used.
  • Chromatographic data at 210, 277 nm and ultraviolet spectra from 209 to 321 nm of each peak were obtained. While monitoring absorbance at 210 nm, fractions were manually collected by peak into microfuge tubes and immediately stored without drying at -20 C° in preparation for sequence analysis.
  • Amino terminal peptide sequence analysis was performed as follows. Samples for amino terminal sequence analysis were applied directly to a polybrene pre-cycled glass fiber filter and placed in the reaction cartridge of an ABI Model 477A protein sequencer. The samples were subjected to automated Edman degradation using the program NORMAL-1, which was modified using the manufacturer's recommendations for faster cycle time (36 min) by decreasing dry-down times and increasing reaction cartridge temperature to 53 C° during coupling. The resultant phenylthiohydantoin amino acid fractions were subsequently identified using an on-line ABI Model 120A HPLC and Shimadzu CR4A integrator. Computerized protein and gene sequence database searches were performed using the Intelligenetics FASTDB program. Anti- peptide antibody studies
  • Anti-ppl85 antibodies were prepared as follows. Polyclonal antibodies to peptide segments of ppl85 were raised in young adult New Zealand White rabbits. Synthetic peptides were prepared by solid phase peptide synthesis and purified by reverse-phase high pressure liquid chromatography. Peptides were coupled to RSA carrier by the bis-diazobenzidine method (Gordon et al., 1958, J. Exptl. Med. 108.:37-51, hereby incorporated by reference) and by glutaraldehyde (Reichlin, 1980, Methods Enzymol. 7):159-165, hereby incorporated by reference), and a mixture of these conjugates used to immunize three rabbits.
  • Immunoglobulin fractions of the sera were prepared by ammonium sulfate precipitation and DEAE- Sephacel chromatography (Harlow et al., 1988, Antibodies, Lab Manual, pp. 302-305, Cold Spring Harbor Laboratory Press, hereby incorporated by reference) .
  • Antibodies were further affinity-purified on a column prepared by coupling synthetic peptide to Affigel 10 (according to the manufacturers directions) , with antibody elution using 100 mM glycine, pH 2.5 and rapid neutralization.
  • whole rat liver cytosolic extracts were prepared by homogenizing 1 gram of liver in 25 cc of homogenization buffer (0.25 M sucrose, 5mM EDTA, 5 mM EGTA, 10 mM Na 4 P 2 0 7 , 20 mM NaF, 50 mM HEPES, pH 7.5, 1 mM PMSF, 5 ug/ml leupeptin, 5 ug/ml aprotinin, 1 mg/ml bacitracin, 0.1 mg/ml benzamidine) at 0°C, and clarified by centrifugation at 100,000 x g) for 1 hr. Insulin Metabolism and pp!85 Hepatic insulin response
  • insulin infused at 0.2 cc/min.) the effective insulin concentration within the hepatic sinusoids and at the cell surface is at least 45-fold lower than the infused concentration.
  • the insulin sensitivity is in good agreement with the reported binding constant of the hepatic insulin receptor (Kahn et al., 1974, J. Biol. Chem. 240:2249-2257 r hereby incorporated by reference).
  • the observed results are not dependent on the anti- phosphotyrosine antibody immunoprecipitation/ immunoblotting assay, as control experiments demonstrated the linearity of the method over the range of antibody concentrations used. Regulation of insulin-stimulated tyrosine phosphorylation in vivo
  • Body weights at sacrifice were 304 ⁇ 19 g (mean ⁇ S.D.) in control rats, 250 + 7 g in diabetic rats, and 314 + 7 g in insulin-treated diabetic rats.
  • Anesthetized animals received intraportal infusion of saline without (-) or with 10 ⁇ 6 M insulin (+) for 0.5 min and the entire liver excised and processed for anti-PY immunoprecipitation and immunoblotting as described in above.
  • the hepatic response is essentially the same as previously described, without and with acute insulin stimulation.
  • the intensity of insulin receptor / 8-subunit and ppl85 tyrosine phosphorylation also normalized.
  • insulin receptor kinase mediated tyrosine phosphorylation of endogenous cellular proteins is an obligatory component of the metabolic regulatory effects of insulin in vivo, then this process should occur in organs besides the liver.
  • tissue distribution of phosphotyrosyl proteins male Sprague- Dawley rats were anesthetized and saline without or with 10 ⁇ 6 M insulin was infused into the portal vein (for liver) or the inferior vena cava (other tissues) for 1 minute at 1 cc/min. Tissues were excised and phosphotyrosyl proteins analyzed by anti-PY antibody immunoprecipitation and immunoblotting as described above.
  • Liver, kidney, spleen, brain, and hindlimb skeletal muscle, and epididymal fat pads were examined under basal and insulin-stimulated conditions. In both the absence or presence of insulin, all tissues examined contain an Mr 120 kDa phosphotyrosyl protein and as noted above in the liver, the intensity of this band is unaffected by insulin. In skeletal muscle, a major insulin target tissue, insulin clearly stimulates insulin receptor jB-subunit autophosphorylation together with the appearance of a prominent phosphotyrosyl protein at Mr+185 kDa, comparable in intensity to the muscle insulin receptor and to hepatic ppl85. An analogous pattern of receptor and ppl85-like protein phosphorylation is also observed in rat epididymal fat pads.
  • Spleen and brain do not contain detectable tyrosine phosphorylated insulin receptors and no new phosphotyrosyl proteins appeared following insulin infusion.
  • There is an intense insulin-insensitive band in brain at Mr 190 kDa.
  • rat hepatocytes were labeled with 32 P, and proteins phosphorylated in response to insulin were detected by immunoprecipitation with anti-phosphotyrosine and anti- receptor antibodies and analyzed by SDS-PAGE and autoradiography.
  • insulin rapidly stimulated tyrosine phosphorylation of two proteins: the 95 kDa 0-subunit of the insulin receptor and a 175 kDa phosphoprotein (ppl75) . Both proteins were precipitated by anti-phosphotyrosine antibody, whereas only the insulin receptor was recognized using anti-insulin receptor antibody.
  • both ppl75 and the receptor jS-subunit were found to be phosphorylated on tyrosine and serine residues. Based on precipitation by the two antibodies, receptor phosphorylation was biphasic with an initial increase in tyrosine phosphorylation followed by a more gradual increase in serine phosphorylation over the first 30 minute ' s of stimulation. The time course of phosphorylation of ppl75 was rapid and paralleled that of the jS-subunit of the insulin receptor. ppl75 was clearly distinguished from the insulin receptor as it was detected only when boiling SDS was used to extract cellular phosphoproteins, whereas the insulin receptor was extracted with either Triton X-100 or SDS.
  • ppl75 is a major endogenous substrate of the insulin receptor in liver and that this may be a cytoskeletal- associated protein. Both serine and tyrosine sites are involved in insulin stimulated phosphorylation in hepatocytes.
  • ppl75 M r - 175 kDa
  • phosphorylation of the ⁇ -subunit of the insulin receptor could be detected in the basal state, consistent with the presence of serine and threonine phosphate before insulin stimulation, Kasuga et al., 1982, Science 215:185-87. hereby incorporated by reference.
  • Total phosphate incorporation was increased within 1 min after insulin stimulation due to the increase in tyrosine phosphorylation.
  • anti-receptor antibody the level of phosphorylation of the jS-subunit gradually increased throughout the 30 min stimulation.
  • the j8-subunit of the insulin receptor becomes phosphorylated mainly on tyrosine residues, and this is followed by a more gradual increase in phosphorylation on serine and threonine residues.
  • the band representing the receptor immunoprecipitated by anti-insulin receptor antibody (B-9) was eluted from the gel by trypsinization and subjected to phosphoamino acid analysis. Without insulin, the receptor contained primarily phosphoserine and a small amount of phosphothreonine, but no phosphotyrosine.
  • Hepatocytes were isolated and 32P-labeled as follows. Hepatocytes were isolated from male Sprague- Dawley rats weighing 160-200 g fed ad libitum using a modification, Okamoto et al. , 1982, Endocrinol Jpn
  • Labeling of the hepatocytes with [ 32 P]orthophosphate was accomplished by incubating 0.5 ml aliquots of the cell suspension for 90 min with 1 mCi of [ 32 P]orthophosphate at 37°C in a humidified atmosphere composed of 95% air and 5% C0 2 .
  • Phosphorylation and immunoprecipitation of labeled proteins were performed as follows. After stimulation by insulin for 1 min (except as otherwise indicated) , the reaction was stopped using one of two methods. For Triton X-100 extracts, the reaction was stopped by adding 0.5 ml of ice cold stopping solution composed of 50 mM HEPES, pH 7.4, 1% Triton X-100, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 2 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 mg/ml aprotinin.
  • Triton X-100 extracts the reaction was stopped by adding 0.5 ml of ice cold stopping solution composed of 50 mM HEPES, pH 7.4, 1% Triton X-100, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 2 mM sodium
  • the mixture was vigorously vortexed, cooled on dry ice-methanol until minimal ice was detected in the bottom of the tube, vortexed again and kept on ice for 30 min.
  • For SDS extraction 0.5 ml of boiling stopping buffer was added to give the same concentrations as described above except that 1% SDS replaced the 1% Triton, and PMSF and aprotinin were omitted. After boiling for 15 min, the sample was cooled in an ice bath for 60 min. After both extraction procedures, the samples were centrifuged at 200,00 x g for 45 min at 0-4°C, and the supernatant was used for immunoprecipitation.
  • Immunoprecipitation with anti-insulin receptor antibody B-9 and anti-phosphotyrosine antibody were performed at dilutions of 1:200 and 1:100, respectively, Kasuga et al., 1985, Methods of Enzymol 109:609-621, hereby incorporated by reference.
  • Immunoprecipitated proteins were solubilized in Laemmli buffer with 100 mM dithiothreitol (DTT) and were separated in 7.5% polyacrylamide gels by electrophoresis. The gels were stained with Coomassie blue in 50% trichloroacetic acid, destained in 7% acetic acid, dried and autoradiographed with Kodak X-Omat film. Molecular weights of proteins were calculated by using standard proteins (BioRad) . The incorporation of P into individual bands was quantitated by scanning densitometry of the film.
  • Phosphoamino acid analysis was performed as follows. Tryptic phosphopeptides were obtained from the protein bands in polyacrylamide gel fragments as previously described, White et al., 1985, J Biol Chem 260:9470-78. hereby incorporated by reference. The positions of the phosphorylated proteins were determined by autoradiography, the gel fragments containing the proteins was excised from the gel, washed for 12 h at 37°C in 20% methanol, dried at 80°C for 2 h and digested with 2 ml of 50 mM NH 2 HC0 3 containing 100 ⁇ g of trypsin, pH 8.0.
  • Sprague-Dawley rats were purchased from Charles River.
  • Collagenase Type IV was obtained from Cooper Biomedical; [ 32 P]orthophosphate and Triton X-100 from New England Nuclear; phosphoamino acids from Sigma; porcine insulin from Elanco; reagents for SDS-PAGE from Bio-Rad; Pansorbin from Calbiochem; and cellulose thin-layer plates from Analtech; RPMI 1640 tissue culture medium from GIBCO.
  • Polyclonal anti-phosphotyrosine antibody was prepared in rabbits as previously described and affinity purified on phosphotyramine Sepharose, Pang et al., 1985, J Biol Chem 260:7131-36. hereby incorporated by reference.
  • Anti-insulin receptor antibody was from the serum of patient B-9, Kasuga et al., 1981, J Biol Chem 256:5305-8, hereby incorporated by reference. ' Phosphorylated proteins in SDS extracts of hepatocytes Although autophosphorylation of the ⁇ - subunit insulin receptor was easily detected in Triton X- 100 extracts of intact rat hepatocytes using anti- phosphotyrosine antibodies, no other insulin-stimulated phosphotyrosine-containing proteins were detected. To further pursue potential substrates, SDS extracts of the hepatocytes were prepared as described above.
  • pp!75 Phosphoamino acid analysis was performed on ppl75 before and after insulin stimulation.
  • isolated rat hepatocytes were labeled with [ P]orthophosphate and treated with insulin (1 ⁇ g/ml) for various time intervals.
  • the cells were extracted with 1% SDS, immunoprecipitated with anti-phosphotyrosine antibody, reduced with DTT, and analyzed by SDS-PAGE with a 7.5% resolving gel.
  • the major phosphoamino acid in ppl75 was phosphoserine with a small amount of phosphotyrosine. After insulin stimulation, both phosphoamino acids increased.
  • Fig. 1 isolated rat hepatocytes were labeled with [ 32 P]orthophosphate, and the cells treated with the indicated concentrations of insulin for 1 min. The cells were extracted with 1% SDS, immunoprecipitated with anti-phosphotyrosine antibody, reduced with DTT, and analyzed by SDS-PAGE with a 7.5% resolving gel. Autoradiogram of the SDS gels were subjected to densitometry scanning to quantitate the amount of 32 P incorporated in the proteins. Data are presented as the percent increase above basal.
  • ppl75 could be the precursor of the insulin receptor. This seemed unlikely, however, since ppl75 was not detected in Triton X-100 extracts of cells or precipitated by anti-receptor antibody, and it contained trace amounts of phosphotyrosine in the basal state (both characteristics different from those of insulin receptor) .
  • ppl75 and the 95 kDa j ⁇ -subunit of the insulin receptor were subjected to two dimensional phosphopeptide mapping, Fig. 2. In Fig.
  • hepatocytes were stimulated by insulin (1 ⁇ g/ml) , and the tyrosine phosphorylated proteins in hepatocytes were immunoprecipitated and separated with SDS-PAGE.
  • the gel fragments containing the proteins were incubated with trypsin, and the eluted phosphopeptides were separated on cellulose thin-layer plates by electrophoresis (ph 1.9) and ascending chromatography (pH 3.5) as described above.
  • Fig 2 shows schematic diagrams of the autoradiograms. The phosphoamino acid in individual peptides was also determined. These are indicated as Y for phosphotyrosine and S for phosphoserine. When both amino acids were present the size of the letters represents the relative amount of each phosphoamino acid in the spot.
  • ppl75 is distinct from the insulin receptor /3-subunit and not likely to represent the precursor of the insulin receptor.
  • HPLC separation of the tryptic phosphopeptides was performed as follows. The phosphopeptides were eluted from the gel fragments as described above with 95% efficiency and were analyzed in two ways. Two- dimensional peptide mapping was performed as described by Ellis et al., 1981, Nature (Lond) 292:506-511, hereby incorporated by reference. Peptide mapping was also performed with high performance liquid chromatography system (Waters) equipped with a wide-pore C ⁇ 8 column (Bio ⁇ Rad, RP-0318) as previously described.
  • Vanadate has been shown to be a potent inhibitor of phosphotyrosyl protein phosphatase (PTPase) activity in vitro at concentrations which do not inhibit phosphoserine and phosphothreonine phosphatase activity, Swarup et al., 1982, J. Biol. Chem., 257:7298-7301. hereby incorporated by reference; Swarup et al., 1982, Biochem. Biophys. Res. Commun. , 107:1104-1109. hereby incorporated by reference.
  • PTPase phosphotyrosyl protein phosphatase
  • vanadate may also directly stimulate 3-subunit tyrosine autophosphorylation and in vitro phosphotransferase activity of purified insulin receptors, Tamura et al., 1984, J. Biol. Chem., 259:6650-6658. hereby incorporated by reference; but this action has not been observed in all studies, Machicao et al., 1983, FEBS Lett., 163:76-80, hereby incorporated by reference. Vanadate increases glucose transport in trypsin-treated adipocytes and in cells where the insulin receptor concentration is reduced 60% by receptor down- regulation. Green, 1986, Biochem. J. , 218:663-669, hereby incorporated by reference; suggesting a post-receptor mechanism of vanadate action.
  • mice Two well studied rodent models of Type II diabetes are ob/ob and db/db mice. These homozygous mice are characterized by obesity, hyperglycemia, hyperinsulinemia and a blunted response to insulin at the receptor and post-receptor levels, Mordes et al., 1985, Animal models of diabetes mellitus, In Joslin*s Diabetes Mellitus, Marble et al., editors. Lea and Febiger, Philadelphia, 110-137, hereby incorporated by reference; Seidman et al., 1970, Diabetologia, (5:313-316, hereby incorporated by reference; Belefiore et al. , 1987, Int. J. Obesity, 11:631-646, hereby incorporated by reference; Coleman et al., 1967, Diabetologia, 1:238-248, hereby incorporated by reference; Stengard et al., 1987, Biomed.
  • PTPase hepatic phosphotyrosyl protein phosphatase
  • NIDDM non-insulin dependent diabetes mellitus
  • the data are represented as the mean ⁇ S.E.M. * - p ⁇ 0.001 for ob/ob vs ob/+ and db/db vs db/+.
  • the vanadate treatment also lowered the blood glucose level of lean ob/+ mice from 170 ⁇ 4 mg/dl to 114 ⁇ 1 mg/dl (p ⁇ 0.001).
  • ob/ob and the ob/+ groups there was no difference in body weight in the vanadate-treated mice as compared to their appropriate saline-treated controls (Table II) .
  • the vanadate treatment was for 47 days, controls received saline only in their drinking water. 15 The data are represented as the mean ⁇ S.E.M. Sample size is 5 per group.
  • Circulating levels of vanadate were 5.2 ⁇ 0.9 ⁇ M and 2.7 ⁇ 0.5 ⁇ M in the ob/+ mice after 3 weeks of treatment. These levels are similar to vanadate levels previously reported by us and others, Meyerovitch et al., 1987, supra ; Gil et al., 1988, supra . Untreated mice have no detectable ( ⁇ 7 nM) serum vanadate. Stoop et al., 1982, Clin. Chem., 21:79-82; hereby incorporated by reference. The effect of vanadate was reversible and 20 days after withdrawal of the vanadate, the blood glucose returned to the initial hyperglycemic levels. Fig. 4. Fig. 4 shows that the effect of vanadate is reversible.
  • the vanadate drinking water was changed to control after 20 days.
  • the ob/ob mice are shown in the upper panel and the ob/+ mice in the lower panel. Each point is the mean of 5 animals.
  • the S.E.M. was 2-3 mg/dl.
  • Vanadate treatment increased plasma insulin levels from 254 ⁇ 29 to 338 ⁇ 49 ⁇ U/ml in the ob/ob, and from 66 ⁇ 10 to 92 ⁇ 20 in the ob/+ group (p ⁇ 0.001). Insulin binding to WGA purified insulin receptors was reduced in the untreated ob/ob mice, (8.7 ⁇ 1.8 %/10 ⁇ g protein compared to 17.3 ⁇ 3.8 %/10 ⁇ g protein, in untreated controls, p ⁇ 0.05) (Table II).
  • Fig. 5 shows the effect of oral administration of vanadate on blood glucose level in db/db mice.
  • Db/db mice (solid line) and their matched controls db/+ (dashed line) were treated with vanadate as described in Figs. 3 and 4.
  • the S.E.M. was 2-3 mg/dl.
  • the vanadate treatment was for 60 days, controls received saline only in their drinking water. 15 The data are represented as the mean ⁇ S.E.M.
  • the vanadate treatment also lowered the blood glucose level of the db/+ mice from 126 i 2 to 81 + 1 mg/dl. There was no difference in body weight gain treatment with vanadate or with saline for either the db/db or the db/+ groups (Table IV) . The effect of vanadate was reversible, and 25 days after withdrawal of the vanadate the blood glucose returned to the initial hyperglycemic levels. Fig. 5.
  • Liver fraction derived from ob/ob and ob+ mice were fractionated using isotonic sucrose differential centrifugation. The particulate fraction was than further fractionated on WGA columns.
  • the 1142-1153 peptide was phosphorylated at 4°C in the presence of 100 nM insulin using WGA purified insulin receptor as described in the methods.
  • the P labeled peptide was separated from the 32 P ATP by chromatography on AG 1-X2 acetate column and by SEP-PAK cartridge and lyophilized. Aliquots from each fraction were assayed for PTPase activity towards the phosphorylated peptides (0.14 ⁇ M) in the presence of 2 mM EDTA and ImM DTT for 5 min at 30°C.
  • PTPase activity associated with the particulate fractions was similarly decreased in the ob/ob mice versus the ob/+ controls (1.43 ⁇ 0.2 U/mg and 2.1 ⁇ 0.3 U/mg, p ⁇ 0.02), Fig. 7.
  • the glycoprotein fraction of the membrane had 5- to 8-fold higher specific activity than the particulate fraction, however, specific activities of the PTPase in the WGA purified particulate fractions were not different between ob/ob and ob/+ mice (16.1 ⁇ 1.3 U/mg versus 16.8 ⁇ 1 U/mg, Fig. 7).
  • Fig. 8 shows activity in cytosolic fractions
  • vanadate did not significantly alter particulate PTPase activity in the 17 week old ob/ob mice compared to the age-matched untreated ob/ob mice, Fig. 10.
  • Fig. 10 shows the effects of vanadate treatment on PTPase activity in particulate fractions from ob/ob mice liver.
  • Particulate PTPase activity from the ob/ob mice livers was assayed as described in Figs. 6 and 7.
  • the results represent mean ⁇ S.E.M. of five mice in each group assayed in duplicate.
  • * p ⁇ 0.02.
  • the specific activity of the PTPase activity in the particulate fraction of the ob/ob mice was 55% of ob/+ (2.93 ⁇ 0.4 U/mg versus 5.3 ⁇ 0.9 U/mg, p ⁇ 0.02), Fig. 10. Vanadate treatment did result in a decrease in PTPase activity in the ob/+ mice (3,9 ⁇ 0.5 U/mg in the treated versus 5.3 ⁇ 0.5 in the control animals); however, the change was not statistically significant, Fig. 10. No significant differences in cytosolic PTPase activity were observed in this age groups (data not shown) .
  • mice were treated for 9 weeks with vanadate or control solutions.
  • Phosphotyrosyl proteins from the liver were isolated as described in above. Briefly, mice were anesthetized, the abdominal wall was incised to expose the viscera. Normal saline or 10 ⁇ 6 M insulin was infused for 20 seconds, after when the liver excised and homogenized in 1% SDS, 100 mM HEPES pH 7.5 50 nM DTT at 100°C for 5 min. The denatured proteins were precipitate with TCA, and immunoprecipitated with polyclonal antiphosphotyrosine antibodies.
  • the immunoprecipitated phosphotyrosyl proteins were resolved on 6% SDS-polyacrylamide gels, transferred to nitrocellulose and detected with antiphosphotyrosine antibodies and [I 125]-Protei •n A and subject to autoradiography. The experiment was preformed twice with similar results.
  • the phosphorylation level of the putative endogenous substrate of the insulin receptor, pp 185 was markedly decreased in the ob/ob compared to the ob/+. Vanadate treatment significantly augmented pp 185 phosphorylation in control mice, however, vanadate did not increase the phosphorylation of the pp 185 in the ob/ob mice. There were no new phosphotyrosyl proteins detected in the vanadate treated mice compared with control treated mice.
  • Streptozotocin, HEPES, phenylmethylsulfonyl fluoride (PMSF) , N ⁇ -P-tosyl-L-lysine chlormethyl ketone (TLCK) , N"- tosyl-1-phenylalanine chlormethyl ketone (TPCK) , aprotinin, N-acetyl-D-glucosamine were from Sigma Chemical (St. Louis, MO) .
  • Silicotungstic acid was from J.T. Baker Chemical Co. (Phillipsburg, NJ) .
  • Dithiothreitol (DTT) Coomassie blue Triton X-100 and AG 1-X2 acetate were purchased from Bio-Rad Laboratories, (Richmond, CA) .
  • mice Female obese-hyperglycemic mice (C57B1/6J ob/ob) and the obese diabetic mice (C57Bl/KsJ db/db) and their lean matched controls (ob/+) and (db/+) were purchased from Jackson Laboratory (Bar Harbor, ME) and used at 6-8 weeks of age. Mice were fed ad libitum a standard laboratory chow. The fed mice were anesthetized by ether and then bled through the orbital venous plexus. Venous blood and liver sample were taken between 9-11 a.m. As noted in the figure legends and text, some mice were treated with vanadate (0.25 mg/ml) included in the drinking water.
  • 80 mM NaCl was also included to reduce vanadate toxicity, as previously described, Heyliger et al. , 1985, supra; Meyerovitch et al., 1987, supra.
  • control mice were treated with 80 mM NaCl alone.
  • 32 P-labelled peptide 1142-1153 was prepared as follows. Wheat germ agglutinin purified insulin receptor, Kasuga et al. , 1984, Methods Enzymol., 109:609- 621, hereby incorporated by reference; was incubated with 100 nM insulin for 30 minutes at 4°C after which 100 ⁇ M [ ⁇ - 32 P] ATP (specific activity 14.2 Ci/mol) , 5 mM Mn 2+ and 2 mM 1142-1153 peptide were incubated with the receptor overnight at 4°C. 32 P-peptide was separated from 32 P-ATP by chromatography on AG1-X2 acetate column (29) and on a C-18 SEP-PAK cartridge (Waters Associates, Milford, MA) and lyophilized.
  • Tissue extractions were performed as follows. Mice were sacrificed by cervical dislocation, and livers were rapidly removed. All tissue extractions were performed at 4°C. Livers were homogenized using a Potter-Elvejhem type homogenizer rotating at 1300 rpm for 20 seconds in three columns of buffer A (20 mM Tris-HCL, pH 7.5, 50 mM 2-mercaptoethanol, 250 mM sucrose, 2 mM
  • EDTA 10 mM EDTA, aprotinin 10 ⁇ g/ml, leupeptin 25 ⁇ g/ml, O.lmM TLCK, 0.1 mM TPCK, 0.5 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride and 5 ⁇ g/ml each of pepstatin A, antipain and chymostatin) .
  • Homogenates were centrifuged at 10,000 x g for 20 minutes, and the supernatant was further centrifuged at 100,000 x g for 45 minutes. The final supernatant was designated the cytosolic fraction.
  • the pellet resulting from the 100,000 x g spin was solubilized using a Potter-Elvejhem type homogenizer rotating at 1300 rpm for 5 minutes in buffer A containing 1% (W/V) CHAPSO and was then centrifuged 45 minutes at 100,000 x g to remove insoluble material.
  • the solubilized material in the supernatant was designated the particulate fraction. In some experiments, this fraction was further fractionated by chromatography on WGA-agarose columns.
  • the column was washed with 50 bed volumes of 10 mM HEPES buffer, pH 7.6, 0.1% (v/v) Triton 100, 5 mM EDTA, 0.5 mM benzamidine and 0.2 mM PMSF and 2 mercaptoethanol 0.1% (v/v) (buffer B) and eluted with two bed volumes with 0.3 M N-acetyl glucosamine in buffer B.
  • the WGA purified fractions were designated the glucoprotein-enriched fractions. All preparations were stored at -70°C prior to use. PTPase assays were performed as follows.
  • the dephosphorylation reaction was carried out at 30°C in a final volume of 50 ⁇ l of 50 mM HEPES, pH 7.0, 2 mM EDTA and 1 mM DTT, and was terminated by the addition of 30 ⁇ l 10% trichloroacetic acid and 20 ⁇ l 1% (w/v) bovine serum albumin (BSA). Following incubation at 4°C for 10 minutes and centrifugation to remove precipitated proteins, 32 P A released from 32 P-peptide was measured using organic extraction of P ⁇ , Shacter, 1984, Anal. Biochem, 138:416-420. hereby incorporated by reference.
  • PTPase One unit of PTPase was defined as the amount of enzyme hydrolyzing 1 pMol of phosphate per minute.
  • 125 I-insulin binding to solubilized insulin receptor was assayed as follows. An insulin receptor preparation was prepared as previously described, Kasuga et al., 1984, supra; and diluted in 50 mM HEPES, pH 7.6, 0.1% Triton X-100. Aliquots (5-9 ⁇ g protein) of wheat
  • Hepatic phosphotyrosine containing proteins were identified in vivo as follows. A recently developed procedure was employed to measure in vivo phosphorylation involving the analytical isolation of phosphotyrosine containing proteins which appear in response to hormonal stimulation of intact tissues. Mice were anesthetized with sodium amobarbital (200 mg/kg body weight, intraperitoneal) . The abdominal wall was incised to expose the viscera. Normal saline with or without 10 _6 M insulin (Humulin R, Eli Lilly) was infused into the portal vein for 20 seconds at a rate of 0.2 ml/minutes.
  • the entire liver was than excised and homogenized in 1% sodium dodecyl sulfate, 50 mM dithiothreitol, 100 mM HEPES, ' 2mM EDTA, pH 7.5 at 100°C for 5 minutes.
  • the denatured proteins were precipitated with TCA (10% w/v) .
  • the TAC was removed using 3 washings with 1:1 (v/v) ether: ethanol.
  • the proteins were resuspended in 50 mM TRIS buffer, pH 7.5, and immunoprecipitated with rabbit polyclonal anti-phosphotyrosine antibodies.
  • Immunoprecipitated phosphotyrosyl proteins were resolved on 6% SDS polyacrylamide gels, transferred to nitrocellulose and detected with anti-phosphotyrosine anti •bodi.es and [125I]-Protei•n A.
  • the nitrocellulose membranes were then subjected to autoradiography. Electron microscopy was as follows. After cervical dislocation, the right lobe of the liver was rapidly excised, minced in cold 2.5% glutaraldehyde, 0.1 M phosphate buffer, pH 7.4 and fixed in fresh fixative at 4°C overnight. Tissue was rinsed in the same buffer, fixed with osmium, dehydrated in graded alcohols and embedded in Araldite. Ultrathin sections were picked up on copper grids and stained with uranyl acetate and lead citrate. Areas adjacent to portal triads were viewed using a Phillips 301 electron microscope.
  • Analytic methods were as follows. Blood glucose levels were determined using ACCU-CHEC II (Boehringer Mannheim Diagnostics Division, Indianapolis, IN) . Plasma immunoreactive insulin concentration was determined by radioimmunoassay using the polyethylene glycol method, Desbuquois et al., 1971, supra. Serum concentrations of vanadate were determined by flameless atomic absorption spectroscopy. Stoop et al. , 1982, supra . The lower limit of detection was 7 nM concentration. Protein concentrations were determined by the method of Bradford, Bradford, 1976, Anal. Biochem., 22:248-254, hereby incorporated by reference; using IgG immunoglobulin as a standard.
  • Phosphotyrosine-containing proteins were partially purified from extracts of basal or insulin-stimulated rat liver by affinity chromatography on immobilized anti ⁇ phosphotyrosine antibodies, as described above.
  • the eluted proteins were separated by 1-dimensional SDS-PAGE and transferred to nitrocellulose, from which the ppl85 band was excised and digested with trypsin. Tryptic peptides eluted from the nitrocellulose were separated by reverse-phase HPLC, as described above.
  • Several peptide fractions were subjected to amino acid sequence analysis revealing two classes of peptide sequences.
  • Class I peptides were found only in the insulin-stimulated extracts, whereas Class II peptides were found in both the basal and insulin-stimulated extracts. Based on a search of the translated Genebank, the Class I peptide sequences were unique and attributed to the insulin receptor substrates in the ppl85 band, whereas Class II peptide sequences were identical to rat liver carbamyl phosphate synthase which apparently binds nonspecifically to the affinity matrix. It was assumed provisionally that the Class I peptides were derived from a single 175 kDa phosphotyrosine-containing substrate in the ppl85 band which termed IRS-1; however, the actual relationship between each peptide was unknown.
  • Double-stranded [ P]phosphate-labeled probes were prepared by synthesizing partially overlapping complementary oligonucleotides corresponding to each sequence, and then filling the 3 *-overhanging ends with Klenow using high specific activity [ ⁇ - P]dCTP and [ ⁇ - 32 P]dGTP. The resulting radioactive double-stranded probes (2 mCi/pmol) were used simultaneously to detect cognate coding sequences in rat liver cDNA libraries. Isolation of the cDNA for IRS-1
  • Fig. 12 The complete cDNA sequence of rat liver IRS-1 is shown in Fig. 12. It was constructed from overlapping cDNA fragments obtained from two rat liver cDNA libraries (Lib-1 , Stratagene #936507 and Lib-2 , Stratagene #936512, see below). Approximately 1.5xl0 6 clones from LiJ-1 were screened with the oligonucleotide probes (Fig. ll) . Two positive recombinant phages were identified and labeled C18 and C19 (Fig. 13) . (Fig. 13 is a diagram of overlapping cDNA inserts obtained from the rat liver cDNA libraries. The first two inserts obtained from Lib-1 , C- 18 and C-19, were identified with probe-138 (see Fig.
  • the remaining fragments were identified by two additional screenings of Lib-1 and Lib-2 using specific cDNA probes prepared with the 3200 bp EcoRI insert of C- 18, or the 1300 bp EcoRI fragment from P2-2.
  • the overlapping fragments define a contiguous piece of cDNA indicated in the black box.
  • the cDNA is 5365 bp long and contains an open reading frame which extends from nucleotide 589 to 4293. The start and end of translation is indicated, and the relative locations of the tryptic peptides listed in Table I are shown. EcoRI sites used during the analysis are shown; only the EcoRI site at the end of the C-18 insert and in the overlapping region of the other fragments is actually found in the cDNA.
  • C18 contained a single EcoRI insert of 3200 bp which hybridized with probe-138, but not with probe-80.
  • the structure of C19 is shown in Fig. 13. A fragment from the C19 insert hybridized during Southern analysis with probe-138, whereas no fragment from of C19 hybridized with probe-80.
  • probe-138 identified 2 cDNA molecules, whereas probe-80 identified none.
  • Nucleotide sequence analysis revealed that more than half of the C19 insert overlapped C18 (Fig. 13) .
  • the sequence of the 5'-end of the C19 insert corresponded to rat albumin up to the EcoRI polylinker; beyond this site, the sequence corresponded exactly to the C18 insert as indicated (Fig. 13) .
  • the C18 insert contained an open reading frame beginning with an ATG codon which matched Kozak's criteria for a translation initiation site Kozak, 1985, J. Mol. Biol. 183:1, hereby incorporated by reference; moreover, three in frame stop codons exist on the 5'-side of the ATG (Fig. 12).
  • a third plating and screening of Lib-1 identified two additional clones with unique inserts, P9 and P2-2 (Fig. 13) .
  • the insert of P9 overlapped with that of C18, confirming the putative initiation codon.
  • this clone extended the sequence in the 5'-untranslated region.
  • the P2-2 insert contained two internal EcoRI sites, which yielded three fragments, see Fig. 13.
  • the open reading frame encoded by the unique overlapping inserts of C18, C19 and P2-2 encodes a 131 kDa protein (Fig. 13) .
  • the complete deduced amino acid sequence is shown in Fig. 12.
  • an additional rat liver cDNA library, Lib-2 was screened.
  • Nine additional overlapping clones were obtained from over 2 million plaques which confirmed the original cDNA sequence (Fig. 13) .
  • these clones extended the cDNA sequence in the 3'-direction and revealed 13 inframe stop codons preceding a polyadenylation signal (AATAAA) ; sequencing was not extended to locate the poly-A tail.
  • AATAAA polyadenylation signal
  • cDNA cloning was performed as follows. Two bacteriophage cDNA libraries, Stratagene #936507, or #936512, prepared with oligo dT and random primed cDNA synthesized from rat liver mRNA and inserted into the ⁇ - Zap-II vector using EcoRI linkers were screened with optimal oligonucleotide probes.
  • plaques were plated at a density of 30,000 plaques per 150 mm plate, transferred to nylon filters (New England Nuclear) , and screened with an equimolar mixture of probe-80 and probe-138 (2-6xl0 8 cpm/pg) .
  • Hybridizations were performed in solutions containing 30% formamide, 10% dextran sulfate, 5x NaCl/citrate (lx NaCl/citrate is 0.15 M NaCl/0.015 M trisodium citrate), 2x Denhardt's solution, and 1% SDS at 42°C.
  • the filters were washed with 0.2% SDS, 0.5x NaCl/citrate at 42°C and exposed to Kodak XAR-5 film with a Quanta 111 intensifying screen at -60°C.
  • the Bluescript KS " plasmid containing the cDNA inserts that remained positive after plaque purification were liberated from the A-Zap-II vector by in vivo excision as described in the manufacturer's instructions (Stratagene, Inc.).
  • the inserts were sequenced on both strands as described previously (Williams and Birnbaum, 1989) , and aligned into contiguous sequence. The sequence was confirmed by sequencing the coding strand of independent cDNA inserts which contained all the translated sequences.
  • the sequences were aligned and analyzed using the EUGENE and SAM (Molecular Biology Computing Research Resource, Dana Farber Cancer Institute and Harvard School of Public Health) .
  • [ 32 P]Phosphate, [ 7 - 32 P]ATP (3000 Ci/mmol) , [a- 32 P]dCTP (3000 Ci/mmol), [ ⁇ - 32 P]UTP (800 Ci/mmol), and [ 125 I]protein A were from New England Nuclear (Boston, MA) . Restriction enzymes and other DNA modifying enzymes were purchased from either New England Biolabs (Beverly, MA) or United States Biochemicals (USB, Cleveland, OH) . Other common materials were commercial products of the highest grade available.
  • RNA analysis was performed as follows. Total RNA was isolated by guanidinium isothiocyanate-cesium chloride centrifugation. For Northern blot analysis, RNA was denatured with 6% formaldehyde, size-fractionated by 1% aga-rose gel electrophoresis, and transferred to a Nytran membrane (Schleicher & Schuell) (Sambrook et al., 1989, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, hereby incorporated by reference) .
  • RNA integrity and relative amounts of RNA were assessed by visualization of the ribosomal RNA by UY shadowing. Hybridizations were performed as above except that the formamide concentration was 50%, and the blots were washed in 0.2% SOS, O.lx NaCl/citrate at 51°C.
  • the probes used were either the full-length brain glucose transporter cDNA. (Birnbaum et al., 1986) or the 2.5 kb insert from pSMll-I.
  • the Nytran membranes were exposed to Kodak XAR-5 lilm overnight at -80°C with a Quanta 111 intensifying screen.
  • the 131 kDa open reading frame of IRS-1 migrates as a 165 kDa protein during SDS-PAGE
  • the molecular size of IRS-1, 131 kDA was somewhat smaller than expected for a protein that migrates near 180 kDa during SDS-PAGE.
  • a full length cDNA encoding IRS-1 was constructed from several fragments, as described below, and inserted the contiguous cDNA into pBSII producing pBSII/IRS-1.
  • the mRNA was transcribed in vitro using the t3 promoter, and the mRNA was translated in vitro in a reticulocyte lysate.
  • mRNA species were produced to test the validity of the 131 kDa open reading frame, Fig. 14.
  • a full length mRNA molecule (5365 bp) was synthesized from the pBSII/IRS-1 plasmid that was linearized beyond the 3' end with EcoRV.
  • a shorter mRNA (4810 bp) was transcribed from a modified plasmid linearized with EcoRV that lacked a 555 bp Spel fragment from the 5'-untranslated region.
  • the shortest mRNA molecule which contained the open reading frame (3754 bp) was transcribed from the plasmid lacking the 555 bp Spel fragment but linearized slightly beyond the first stop codon with Aatll.
  • IRS-1 Anti-peptide antibody, ⁇ Pep ⁇ O, immunoprecipitated the major [ 35 S]methionine-labeled translation products, confirming that these protein were IRS-1.
  • Structural features of IRS-1 The amino acid sequence of IRS-1 shows no significant identity to known sequences in the translated Genebank. Serine is its most abundant residue (14.66%), followed by glycine (10.45%), proline (9.55%), and then alanine (7.69%). IRS-1 is largely hydrophilic containing 41.78% polar residues, and nearly twice as many based (14.41%) as acidic (8.83%) residues. It contains a few hydrophobic stretches, but none long enough to qualify as potential transmembrane spanning regions, Fig. 15A. (In Fig.
  • IRS-1 contains 5 consensus sequences for asparagine-linked glycosylation (Asn-Xxx-Ser/Thr) , although there is no evidence that IRS-1 is glycosylated.
  • Lys 156 exists in the motif, Ala-X-Lys 156 -X-Val, which is frequently found in protein kinases.
  • IRS-1 contains many potential phosphorylation sites, Fig. 15C.
  • 33 putative Ser/Thr phosphorylation sites are distributed throughout the protein.
  • at least 10 potential tyrosine phosphorylation sites can be identified, Fig. 15C.
  • the KK-Y 46 -1 peptide (open circles) was a very poor substrate for the insulin receptor compared to the control IR1150 peptide.
  • the YMXM-containing peptides, KK-Y 608 -1 (solid rectangles) and KK-Y 658 -l were at least as good as the IR1150 peptide.
  • the YMXM peptides displayed V jna ⁇ values twice as high as the IR1150 peptide, but they had slightly higher K B values.
  • In vitro kinase assays were performed as follows.
  • the synthetic peptide substrate (IR 1150 ) composed of amino acids 1143-1152 of the human insulin receptor (numbered as per Ullrich et al., 1984, Nature 309:418, hereby incorporated by reference, was purchased from Dr. David Coy, Tulane University, New La, LA.
  • Lectin- purified insulin receptors normalized to insulin binding activity, were incubated for various periods of time in a final volume of 50 ⁇ l containing 50 mM HEPES pH 7.5, 0.1 % Triton X-100, 5 mM MnCl 2 , and the absence or presence of 100 nM insulin.
  • Substrate phosphorylation was initiated by the addition of varying concentrations of peptide for 5 min at 22°C. Reactions were terminated by the sequential addition of 20 ⁇ l 1% bovine serum albumin (BSA) and 50 ⁇ l 10% trichloroacetic acid (TCA) . Precipitated protein was removed by centrifugation, and the supernatant, containing the phosphorylated peptide, was applied to a 1 x 1 inch piece of phosphocellulose paper (Whatman) . The papers were washed with 4 changes (1 liter each) of 75 mM phosphoric acid, and the retained radioactivity was measured by Cerenkov counting.
  • BSA bovine serum albumin
  • TCA trichloroacetic acid
  • CHO cells expressing the wild-type human insulin receptor were labeled with [ 32 P]orthophosphate for 2 h, and then stimulated with 100 nM insulin for 1 min. This time interval was chosen because in previous experiments, the ppl85 band showed maximal phosphorylation after 20 s of insulin stimulation.
  • the antiphosphotyrosine antibody ( ⁇ PY) immunoprecipitated several proteins which were also detected after insulin stimulation. However, insulin stimulated the tyrosine phosphorylation of 2 additional protein bands, the ⁇ - subunit of the insulin receptor (95 kDa) and ppl85.
  • the ppl85 band from CHO cells was previously shown to migrate at 175 kDa during SDS-PAGE, Bjorge et al., 1990, Proc. Natl. Acad. Sci. (USA) 82, 3816-3820, hereby incorporated by reference.
  • Phosphoproteins from an equal portion of control and insulin-stimulated cell extract were immunoprecipitated with an antibody against Pep-80 ( ⁇ Pep ⁇ O) . In the absence of insulin, the major phosphoprotein migrated at 165 kDa. Other minor proteins were also detected at 120 kDa, 100 kDa, and 55 kDa.
  • IRS-1 165 kDa protein
  • IRS-1 was the major phosphoprotein present in the ⁇ Pep ⁇ O immunoprecipitate.
  • the phosphorylation of IRS-1 protein increased about 2-fold and it migrated at a slightly higher molecular weight (170 kDa) , consistent with insulin-stimulated phosphorylation occurring within 1 min.
  • IRS-1 clearly migrated below the center of the ppl85 band immunoprecipitated with the ⁇ PY.
  • ppl85 is a phosphotyrosine-containing protein
  • IRS-1 immunoprecipitated with the ⁇ Pep ⁇ O contained predominantly Ser(P) with a small amount of Thr(P) , but no Tyr(P) was detectable; however, insulin stimulated the appearance of Tyr(P) in IRS-1.
  • the corresponding analysis of the ppl85 band immunoprecipitated with ⁇ PY revealed Ser(P) , Thr(P) and a small amount of Tyr(P) in the basal state, and a larger increase in Try(P) after insulin stimulation.
  • the difference in the amount of Tyr(P) in the IRS-1 and the ppl85 band after insulin stimulation is unknown.
  • Insulin stimulated the tyrosine phosphorylation of the 3-subunit of each receptor as shown by immunoprecipitation with the ⁇ PY.
  • the ppl85 band was strongly phosphorylated only by the wild-type insulin receptor, whereas the IR F960 and IR ⁇ 960 showed no phosphorylation of the ppl85 band.
  • immunoprecipitation of an equal amount of these cell extracts with ⁇ Pep80 showed insulin-stimulated phosphorylation of the IRS-1 in the CHO/HIRc cells; insulin had no effect on the phosphorylation of IRS-1 in the CHO/I pggQ and CH0/IR ⁇ 96t) .
  • IRS-1 is a substrate of the insulin receptor and is a component of the ppl85 band.
  • CHO cells were grown in 10 cm dishes in F12 medium containing 10% fetal bovine serum (Gibco) .
  • Subconfluent CHO cells (10 6 ) were transfected by calcium phosphate precipitation with 1 ⁇ g pSVEneo alone or together with 10 ⁇ g of pCVSVHIRc, pCVSVHIRc/F960, or pCVSVHIRc/ ⁇ 960 as previously described (10-11) .
  • 800 ⁇ g/ml of geneticin (GIBCO) was added to the medium to select for neomycin-resistant cells.
  • Surviving cells were cultured in the presence of geneticin to amplify the cell line.
  • CHO cells that expressed high levels of surface insulin receptors were selected by fluorescence-activated cell sorting (13) , and clonal cell lines were obtained by plating at limiting dilution.
  • Insulin receptor mutants were constructed, using oligonucleotide-directed mutagenesis, in which Tyr 960 was substituted with phenylalanine (IR j , 960 ) or 12 amino acids (A954-D965) were deleted from the juxtamembrane region (IR ⁇ 960 ) .
  • CHO/neo cells expressing only pSVEneo, contained about 3,000 hamster insulin receptors. Following transfection and selection by fluorescence- activated cell sorting, clonal lines of CHO/IR cells and mutant CHO/IR p960 , and CHO/IR ⁇ 960 cells were obtained which expressed approximately 80,000 receptors/cell.
  • Confluent monolayers of transfected CHO cells in 10 or 15 cm dishes (Nunc) at 37°C were labeled for 2 h with 0.5 mCi/ml with [ *"32 P]phosphate (New England Nuclear) as previously described (Backer et al., 1990).
  • the cells were incubated for additional periods of time in the presence of 100 nM insulin, rapidly frozen with liquid nitrogen, and solubilized in 100 mM Tris, pH 8.2, containing 2 mM sodium vanadate, 3.4 mg/ml PMSF, 100 ⁇ g/ml aprotinin, 1 ⁇ g/ml leupeptin, and 1% Triton-X-100.
  • Tyr(P)-containing proteins were immunoprecipitated with antiphosphotyrosine antibody ( ⁇ PY) ; precipitated proteins were reduced with dithiothreitol and analyzed by SDS- PAGE. Immunoprecipitated proteins were identified by autoradiography and the radioactivity in the insulin receptor subunits was quantified by liquid scintillation counting.
  • Immunopreci itates of IRS-1 contain phosphatidyl inositol 3-kinase activity
  • the phosphatidyl inositol 3- kinase (Ptdlns 3-kinase) is activated by several growth factor receptor tyrosine kinases and is thought to be involved in the regulation of DNA synthesis, Kaplan et al., 1987, Cell 50.:1021, hereby incorporated by reference.
  • Several reports suggest that it is a 85 kDa protein which undergoes tyrosine phosphorylation or associates tightly with phosphotyrosine-containing proteins Kaplan et al., 1987, supra.
  • Ptdlns 3-kinase or its association with Tyr(P)-containing proteins is required for its activation.
  • CHO/IR cells were stimulated with insulin (100 nM) for 10 min and extracts were prepared.
  • the Ptdlns 3-kinase activity was assay in immuncomplexes prepared with ⁇ PY (Tyr(P)), two anti-insulin receptor antibodies (B2 and K-14) and ⁇ Pep ⁇ O.
  • Phosphatidyl inositol 3-kinase activity was assayed as follows. In vitro phosphorylation of phosphatidylinositol was measured as previously described (Ruderman et al., 1990, supra. Subconfluent CHO cells grown in 100 mm dishes were made quiescent by an overnight incubation in F-12 medium containing 0.5% BSA.
  • the cells were then incubated in the absence or presence of insulin (100 nM) for 10 min, and washed once with ice cold PBS and twice with 20 mM Tris (pH 7.5) containing 137 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , and 100 ⁇ M Na 3 V0 4 (Buffer A) .
  • the cells were solubilized in 1 ml Buffer A containing 1% NP-40 (Sigma) and 10% glycerol, and insoluble material was removed by centrifugation at 13,000 x g for 10 min.
  • Tyrosyl phosphoproteins were immunoprecipitated from the supernatant with ⁇ PY and protein A-sepharose (Pharmacia) .
  • anti- insulin receptor or control antibodies were used as described in the text.
  • the immunoprecipitates were washed successively in PBS containing 1% NP-40 and 100 ⁇ M Na 3 V0 4 (3 times), and 10 mM Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA and 100 ⁇ M Na 3 V0 4 (2 times) .
  • the pellets were resuspended in 50 ⁇ l of 10 mM Tris (pH 7.5) containing 100 mM NaCl and 1 mM EDTA.
  • IRS-1 Insulin stimulates tyrosine phosphorylation of IRS-1 in CHO cells expressing wild-type human insulin receptor. This leads to a slight retardation of its migration during SDS-PAGE, which is a typical finding following protein phosphorylation. Moreover, most of the immunoprecipitated IRS-1 migrates more slowly after 1 min of insulin stimulation, suggesting the cellular IRS-1 reacts quickly and completely with the insulin receptor. Thus IRS-1 is expected to be a physiologically relevant substrate of the insulin receptor. Phosphoamino acid analysis reveals that both IRS-l and the ppl85 band contain Ser(P) and a small amount of Thr(P) before and after insulin stimulation. In addition, the pplS5 band contains a small amount of Tyr(P) before insulin stimulation.
  • the amount of Tyr(P) in IRS-1 is detectable only after insulin stimulation, and is relatively low compared to the Tyr(P) in the ppl85 band following insulin stimulation. This disparity is consistent with the presence of additional Tyr(P)-containing proteins in the ppl85 band, or the inability of ⁇ Pep80 to recognize the highly tyrosine phosphorylated form of the IRS-1. Expression of the IRS-1 cDNA in CHO cells and the preparation of other antibodies will be necessary to resolve this question.
  • insulin receptor Several structural feature of the insulin receptor are required for tyrosine phosphorylation of the ppl85 band in CHO cells including a functional ATP binding site, an intact regulatory region which contains the major autophosphorylation sites, and a normal juxtamembrane region. Point mutations or a deletion of a portion of the juxtamembrane region inhibits insulin signal transmission and phosphorylation of the ppl85 - ⁇ i - band, without altering autophosphorylation of the insulin receptor. Two insulin receptor molecules containing juxtamembrane mutations, IR p960 and IR ⁇ 960/ did not phosphorylate IRS-1 during insulin stimulation of transfected CHO cells.
  • IRS-1 shows a similar dependance on an intact juxtamembrane region of the insulin receptor for insulin-stimulated phosphorylation.
  • IRS-1 The deduced amino acid sequence of IRS-1 is unique, which hampers attempts to deduce its function by comparison with homologous proteins.
  • IRS-1 is a - ⁇ 2 - hydrophilic protein with no stretch of hydrophobic residues long enough to provide a transmembrane spanning region. It contains an abundance of glycine and proline residues which leads frequently to the motif Gly-Pro-Y, which leads to a weak alignment between IRS-1 and collagen; however, the proteins are clearly distinct.
  • IRS-1 Several structural motifs are found in IRS-1 which may provides clues regarding its role in insulin action including, ⁇ nucleotide binding motifs, a kinase-like ATP-bihding site, tyrosine phosphorylation sites with the motif YMXM, multiple serine and threonine phosphorylation sites, and a run of 10 glutamine residues.
  • IRS-1 Insulin action is thought to be mediated through a cascade of protein phosphorylation and dephosphorylation. Whether IRS-1 plays a direct role in this mechanism as a protein kinase is unknown. Our previous attempts to label the components of the ppl ⁇ 5 band with ATP affinity reagents have been unsuccessful. However, one of the eight nucleotide binding motifs in IRS-1 has the clear appearance of a ATP binding site.
  • the nucleotide binding component has the structure Gly 137 -Val-Gly-Glu-Ala-Gly and 14 (Seq. I.D. No. 27) amino acids away is the essential catalytic lys 15g .
  • the lysine reside is in an Ala-Xxx-Lys-Ile (Seq. I.D. No. 2 ⁇ ) motif, which is conserved among many protein kinases, Hanks et al., 1990, Science 241:42, hereby incorporated by reference. None of the other nucleotide binding motifs show this additional homology. Hanks et al. recently described 11 conserved subdomains commonly found in protein kinase catalytic domains. The ATP binding site is located in region I, and all protein kinases evaluated contain a glutamic acid residue in region II, 13 to 42 residues down-stream from the catalytic lysine residue of region I.
  • IRS-1 contains Clu 194 3 ⁇ residues away from Lys 156 .
  • Region III of kinase catalytic - 63 - domains contains a conserved leucine or isoleucine 10 to 17 residues away from the conserved glutamic acid of region II; IRS-1 has Ile 206 , 11 residues beyond Glu 194 .
  • IRS-1 lacks the Asp-Phe-Gly motif of region VII, and the Ala-Pro-Glu motif of region VIII, which are absolutely predictive of a protein kinase.
  • the Ala-Pro- Glu motif is essential for catalytic activity in pp60 c ⁇ src and is located about 20 residues down-stream from the autophosphorylation sites in protein kinases.
  • the absence of these motifs from IRS-1 rules out the possibility that the IRS-1 is a typical protein kinase.
  • Tyrosine phosphorylation links IRS-1 to this insulin receptor. At least 10 potential tyrosine phosphorylation sites were detected by eye in the deduced sequence, as any Tyr residue with an Asp or Glu residue nearby was considered a possibility. Interestingly six of these Tyr residues resided in a YMXM motif, which is also found in the polyoma middle T antigen (MTag) , Carmichael et al., I960, J. Biol. Chem, 255:230, hereby incorporated by reference, and receptor tyrosine kinases for PDGF, Yarden et al., 19 ⁇ 6, Nature 323.226-232.
  • MTag polyoma middle T antigen
  • IRS-1 synthetic peptides containing the YMXM motif are good substrates for the insulin receptor as their K m values are nearly the same as insulin receptor peptide4. Although we have no information about in vivo sites, all of the YMXM motifs look like good possibilities.
  • the motif Glu-Tyr-Tyr-Glu is not phosphorylated well by the insulin receptor, suggesting that the presence of a Tyr in the YMXM motif with adjacent negatively charged amino acids may define the preferred substrate of the insulin receptor tyrosine kinase.
  • tyrosine phosphorylation sites in MTag and various growth factor receptors have been shown to bind specifically to the Src homology-2 (SH2) domain in certain signal transduction proteins, including phosphoinositide-specific phospholipase C (PLC 1) , GTPase activating protein (GAP) , phosphatidyl inositol 3-kinase (Ptdlns 3'-kinase) and p74 , Anderson et al., 1990, Nature, 250,979-962. hereby incorporated by reference.
  • PLC 1 phosphoinositide-specific phospholipase C
  • GAP GTPase activating protein
  • Ptdlns 3'-kinase phosphatidyl inositol 3-kinase
  • p74 Anderson et al., 1990, Nature, 250,979-962. hereby incorporated by reference.
  • the SH2 domain was first identified in nonreceptor protein tyrosine kinases like Src and Fps, by its apparent ability to interact with the kinase domain and phosphorylated substratesy.
  • Several motifs are highly conserved within the SH2 domain which typically begins with the sequence W-(Y/F)-(H/F)-G-K.
  • Bacterially expressed SH2 domains from PLC 1 or GAP immobilized on Sepharose precipitate the PDGF and EGF receptor, suggesting that ligand-stimulated tyrosine phosphorylation may regulate the interaction between the receptor and cellular protein.
  • tyrosine phosphorylation enable certain proteins to bind to cellular protein containing SH2 domains and potentially altering their activity.
  • the insulin receptor contains Tyr(P), it has not been shown to bind PLC 1, GAP, and p74 r . Although insulin activates Ptdlns 3•-kinase which is found in ⁇ PY immunoprecipitates, the IR weakly binds Ptdlns 3'-kinase ⁇ . This suggests that all Tyr(P) residues are not in the proper configuration to interact with SH2 domains; moreover, the insulin receptor does not contain any tyrosine phosphorylation sites in the YMXM motif, which appears to be strongly recognized by SH2 domains.
  • IRS-1 which contains 6 tyrosine phosphorylation sites in YMXM motifs may serve as a link between the insulin receptor and cellular proteins involved in the regulation of growth and metabolism. This conclusion is supported by the strong immunoprecipitations of Ptdlns 3'-kinase from insulin stimulation cells with our relatively weak IRS-1 antibodies. Thus, IRS-1 may serve as a cytoplasmic ligand that links the insulin receptor to SH2 domain- containing enzymes involved in cellular regulation. ' The regulation of IRS-1 phosphorylation is potentially very complex. IRS-1 contains many potential serine and threonine phosphorylation sites, and it is serine phosphorylated in the basal state.
  • IRS-1 contains 8 putative nucleotide binding sites which could provide additional regulation through other mechanisms. Thus IRS-1 provides a common intermediate through which multiple protein kinases and other signal transduction systems may communicate.
  • IRS-1 contains a run of 10 gluta ine residues beginning at Gln 871 .
  • Glutamine-rich regions have been found in amino acid sequences of several eukaryotic regulatory proteins including the androgen receptor Lubahn et. al., 19 ⁇ 8, Mol. Endocrin. 2:1265, hereby incorporated by reference, mineralocorticoid receptor Arriza et. al., 1987, Science 237:268. hereby incorporated by reference, glucocorticoid receptor Hollenberg et. al., 1991, Nature 318:635. hereby incorporated by reference, human c-myc oncogene Rabbitts et. al., 1983, Nature 306:760.
  • the transcription factor SP1 Courey et. al. , 1988, Cell 5J>:8 ⁇ 7, hereby incorporated by reference, Drosophila zeste gene which binds and activates the Ubx promoter Pirrotta et. al., 1987, EMBO J. .6:791, hereby incorporated by reference, and products of the homoeobox containing genes such as Antp and Cut.
  • the sequence similarity is limited at most to the run of glutamines and a few adjacent amino acids. However, the role and specificity of the glutamine residues is unclear Courey et. al., 198 ⁇ , Cell 55_: ⁇ 7, hereby incorporated by reference. IRS-1 lacks other characteristics suggestive of a DNA binding protein.
  • IRS-1 may provide a molecular link between the insulin receptor and cellular enzymes involved in regulation of cellular growth and metabolism.
  • the amino acid deduced sequence of IRS-1 does not contain any obvious enzymatic function; however, its low abundance, general distribution, and the presence of several common motifs in the sequence suggest that it may play an important role in insulin action.
  • IRS-1 acts a molecular link between the insulin receptor and cellular proteins which contain the SH2 domain.
  • Other tyrosine kinases may also phosphorylated IRS-1, and other signaling systems such as serine/threonine kinases and nucleotides may regulate the signal flux.
  • Human IRS-1 Human IRS-1
  • Human IRS-1 encoding DNA can be obtained by the methods of the invention or, preferably, by homology to DNA encoding rat IRS-1, by methods known to those skilled in the art, e.g., by probing a human genomic or cDNA library, preferrably a liver cDNA library, with nucleic acid encoding rat IRS-1.
  • Human IRS-1 can be obtained by the protein purification methods described herein, or more preferably, by expression from recombinant human DNA - ⁇ 7 - encoding IRS-1, by methods known to those skilled in the art.
  • Dosages of typosine kinase inhibiting substances and other therapeutic substance will vary, depending on factors such as, the disease being treated, the half life of the substance, potency, route of administration, and the condition of the patent.
  • the molecules and methods of the invention can be used to diagnose insulin related diseases, e.g., diseases characterized by insulin resistance, e.g.. Type II diabetes. These diagnostic tests can be based on the use of antibodies to IRS-1 to determine the levels of IRS-1 in a tissue sample taken from a patient. Alternatively they can measure some other significant aspect of IRS-1 metabolism expression or action, e.g. , the extent of IRS- 1 phosphorylation, or the cellular or intracellular distribution of IRS-1. Cloned DNA homologous to DNA that encodes IRS-1 can also be used to measure levels of IRS- 1 expression, e.g., by measuring levels of IRS-1 encoding mRNA.
  • IRS-1 IRS-1 RNA
  • IRS- 1 phosphorylation or other significant parameters of IRS-1 metabolism expression, or action, can be determined by methods known to those skilled in the art.
  • Insulin related-disease states e.g., insulin- resistant diseases, e.g., Type II diabetes
  • insulin receptor substrate gene can be diagnosed by using DNA homologous to the IRS-1 gene to discover the structural defect.
  • Structural defects in a gene can be discovered by methods known to those skilled in the art, e.g., by restriction fragment length polymorphism or DNA sequence analysis. What is claimed is:
  • GGAATTCCCT GGTATTTGGG CGGCTGGTGG CGGCGGGGAC TGTTGGAGGG TGGGAGGAGG 60
  • GAG GCC GGG GGC CCG GCG CGC CTG GAG TAT TAT GAG AAC
  • GAG AAG AAG 744 Glu Ala Gly Gly Pro Ala Arg Leu Glu Tyr Tyr Glu Asn Glu Lys Lys 40 45 50
  • GGT TCC TTC AGG GTG CGT GCC TCC AGC GAT GGC GAA GGC ACC ATG TCC 1464 Gly Ser Phe Arg Val Arg Ala Ser Ser Asp Gly Glu Gly Thr Met Ser 280 285 290
  • GCT GCC AAC ACA GTG TCT TTT GGA GCA GGG GCT GCA GGA GGG GGC AGC 3720 Ala Ala Asn Thr Val Ser Phe Gly Ala Gly Ala Ala Gly Gly Gly Ser 1030 1035 1040

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Abstract

L'invention se rapporte à un acide nucléique purifié constitué essentiellement d'acide nucléique codant le substrat-1 récepteur d'insuline (IRS-1); à une préparation polypeptidique purifiée d'IRS; au diagnostic des maladies associées à l'insuline qui consistent à mesurer un aspect du métabolisme de l'IRS-1; au diagnostic d'une maladie associée à l'insuline observée chez un patient, qui consiste à déterminer la structure du gène IRS-1; à l'analyse de l'effet d'un agent thérapeutique qui modifie la capacité d'une tyrosine-kinase à phosphoryler un tel substrat; à un procédé d'application d'un traitement chez un mammifère souffrant d'une maladie causée par le métabolisme de l'IRS-1; et à l'application d'un traitement chez un mammifère souffrant d'une maladie causée par la phosphorylation d'un substrat d'une tyrosine-kinase.
PCT/US1992/000437 1991-01-18 1992-01-17 Acide nucleique codant le substrat-1 recepteur d'insuline (irs-1), proteine d'irs-1, maladies et therapie associees au metabolisme de l'irs-1 WO1992013083A1 (fr)

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US64398291A 1991-01-18 1991-01-18
US643,982 1991-01-18

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WO1992013083A1 true WO1992013083A1 (fr) 1992-08-06

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EP (1) EP0572508A4 (fr)
WO (1) WO1992013083A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029345A1 (fr) * 1993-06-10 1994-12-22 Novo Nordisk A/S Adn mutant codant le substrat 1 du recepteur d'insuline
WO1998057987A2 (fr) * 1997-06-19 1998-12-23 Incyte Pharmaceuticals, Inc. Substrat de la tyrosine kinase du recepteur de l'insuline
US5861266A (en) * 1994-02-28 1999-01-19 New York University Treatment of diabetes mellitus and insulin receptor signal transduction
DE19739360A1 (de) * 1997-09-09 1999-04-01 Deutsches Krebsforsch Verfahren zur Diagnose von frühen Krebsvorstufen der Leber und Niere
AU710856B2 (en) * 1994-10-03 1999-09-30 Joslin Diabetes Center, Inc. The irs family of genes
WO2002103014A2 (fr) * 2001-06-14 2002-12-27 Gene Signal Oligonucleotides antisens capables d'inhiber la formation des tubes capillaires
US6525185B1 (en) 1998-05-07 2003-02-25 Affymetrix, Inc. Polymorphisms associated with hypertension
US9682144B2 (en) 2011-06-30 2017-06-20 Gene Signal International, Sa Composition comprising inhibitors of IRS-1 and of VEGF
US10052343B1 (en) 2017-02-03 2018-08-21 Gene Signal International Sa Sterile formulation comprising a stable phosphorothioate oligonucleotide
CN111662369A (zh) * 2020-07-10 2020-09-15 华中农业大学 一种中黑盲蝽insulin receptor substrate 1基因及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2264481B (en) * 1992-02-26 1996-06-19 Samsonite Corp Luggage case

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Archives of Biochemistry and Biophysics, Volume 242, No. 1, issued 01 October 1985, D.T. PANG et al., "Purification of the Catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies", pages 176-186, see entire document. *
Biochemical and Biophysical Research Communications, Volume 180, No. 2, issued 31 October 1991, H.J. GOREN et al., "The 180000 molecular weight plasma membrane insulin receptor substrate is a protein tyrosine phosphatase and is elevated in diabetic plasma membranes", pages 463-469, see entire document. *
Biochemistry, Volume 12, No. 24, issued 20 November 1973, B.A. CUNNINGHAM et al., "The complete amino acid sequence of beta2-microglobulin", pages 4811-4822, see entire document. *
Nature, Volume 318, issued 14 November 1985, M.F. WHITE et al., "Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells", pages 183-188, see entire document. *
Nature, Volume 352, issued 04 July 1991, X.J. SUN et al., "Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein", pages 73-77, see entire document. *
Proceedings of the National Academy of Sciences, Volume 78, No. 11, issued 01 November 1981, S.V. SUGGS et al., "Use of synthetic oligonucleotides as hybridization probes", pages 6613-6617, see entire document. *
See also references of EP0572508A4 *
The Journal of Biological Chemistry, Volume 266, No. 12, issued 05 May 1991, P.L. ROTHENBERG et al., "Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase", pages 8302-8311, see entire document. *
The New England Journal of Medicine, Volume 325, No. 13, issued 26 September 1991, F.H. EPSTEIN et al., "Insulin resistance - mechanisms, syndromes, and implications", pages 938-948, see entire document. *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029345A1 (fr) * 1993-06-10 1994-12-22 Novo Nordisk A/S Adn mutant codant le substrat 1 du recepteur d'insuline
US5827730A (en) * 1993-06-10 1998-10-27 Novo Nordisk A/S Mutant DNA encoding insulin receptor substrate 1
CN1070917C (zh) * 1993-06-10 2001-09-12 诺沃挪第克公司 编码胰岛素受体底物1的突变dna
US5861266A (en) * 1994-02-28 1999-01-19 New York University Treatment of diabetes mellitus and insulin receptor signal transduction
AU710856B2 (en) * 1994-10-03 1999-09-30 Joslin Diabetes Center, Inc. The irs family of genes
WO1998057987A2 (fr) * 1997-06-19 1998-12-23 Incyte Pharmaceuticals, Inc. Substrat de la tyrosine kinase du recepteur de l'insuline
WO1998057987A3 (fr) * 1997-06-19 1999-03-18 Incyte Pharma Inc Substrat de la tyrosine kinase du recepteur de l'insuline
DE19739360A1 (de) * 1997-09-09 1999-04-01 Deutsches Krebsforsch Verfahren zur Diagnose von frühen Krebsvorstufen der Leber und Niere
DE19739360C2 (de) * 1997-09-09 1999-12-09 Deutsches Krebsforsch Verfahren zur Diagnose von frühen Krebsvorstufen der Leber und Niere
US6525185B1 (en) 1998-05-07 2003-02-25 Affymetrix, Inc. Polymorphisms associated with hypertension
WO2002103014A2 (fr) * 2001-06-14 2002-12-27 Gene Signal Oligonucleotides antisens capables d'inhiber la formation des tubes capillaires
WO2002103014A3 (fr) * 2001-06-14 2004-02-26 Salman Al-Mahmood Oligonucleotides antisens capables d'inhiber la formation des tubes capillaires
US7417033B2 (en) 2001-06-14 2008-08-26 Gene Signal International Antisense oligonucleotides capable of inhibiting the formation of capillary tubes by endothelial cells
EP2166095A2 (fr) * 2001-06-14 2010-03-24 Gene Signal International Sa Oligonucléotides antisens capables d'inhiber la formation des tubes capillaires par les cellules endothéliales
US7855184B2 (en) 2001-06-14 2010-12-21 Gene Signal International Sa Antisense oligonucleotides capable of inhibiting the formation of capillary tubes by endothelial cells and methods of treating ophthalmic and dermatological diseases
EP2166095A3 (fr) * 2001-06-14 2012-01-04 Gene Signal International Sa Oligonucléotides antisens capables d'inhiber la formation des tubes capillaires par les cellules endothéliales
US8828959B2 (en) 2001-06-14 2014-09-09 Gene Signal International Sa Antisense oligonucleotides capable of inhibiting the formation of capillary tubes by endothelial cells
US9682144B2 (en) 2011-06-30 2017-06-20 Gene Signal International, Sa Composition comprising inhibitors of IRS-1 and of VEGF
US10052343B1 (en) 2017-02-03 2018-08-21 Gene Signal International Sa Sterile formulation comprising a stable phosphorothioate oligonucleotide
CN111662369A (zh) * 2020-07-10 2020-09-15 华中农业大学 一种中黑盲蝽insulin receptor substrate 1基因及其应用

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Publication number Publication date
EP0572508A1 (fr) 1993-12-08
EP0572508A4 (fr) 1995-03-29

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