WO1995034201A1 - pp60PIK: A DOWNSTREAM ELEMENT IN INSULIN SIGNALING - Google Patents
pp60PIK: A DOWNSTREAM ELEMENT IN INSULIN SIGNALING Download PDFInfo
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- WO1995034201A1 WO1995034201A1 PCT/US1995/007312 US9507312W WO9534201A1 WO 1995034201 A1 WO1995034201 A1 WO 1995034201A1 US 9507312 W US9507312 W US 9507312W WO 9534201 A1 WO9534201 A1 WO 9534201A1
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- pik
- peptide
- protein
- insulin
- metabolism
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to insulin metabolism and more specifically to pp60 PIK , a protein which mediates insulin regulation of 3'-phosphatidyl-inositol (PI-3) kinase, and to the gene that encodes pp60 PIK .
- PI-3 3'-phosphatidyl-inositol
- the invention features, a substantially pure nucleic acid, e.g., a DNA, which includes a sequence encoding a peptide having pp60 PI activity.
- the sequence is at least 50%, more preferably at least 60%, yet more preferably at least 70%, and most preferably at least 80, 90, 95, or 99% homologous with the sequence from SEQ ID NO:l.
- the invention features, a substantially pure nucleic acid, e.g., a DNA, which includes a nucleic acid sequence which hybridizes under high or low stringency to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence as the peptide of SEQ ID NO: 1.
- the invention features, a substantially pure nucleic acid which includes a sequence encoding a peptide of 20 or more amino acids in length, the peptide having at least 90% homology with an amino acid sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO:l.
- the invention includes a vector which includes a nucleic acid of the invention, preferably a substantially pure DNA of the invention, which encodes a peptide of the invention.
- the invention also includes: a cell containing a nucleic acid of the invention; a cell which is capable of expressing a peptide of the invention; an essentially homogeneous population of cells, each of which includes a nucleic acid of the invention; and a method for manufacture of a peptide of the invention including culturing a cell which includes a nucleic acid of the invention in a medium to express the peptide.
- the invention features a pp60 PIK peptide of the invention, preferably a purified peptide of the invention, e.g.: a peptide having pp60 PI activity; a peptide encoded by a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO:l; a peptide of essentially of the same sequence as the amino acid sequence described in SEQ ID NO: 1 ; a peptide of at least 20, preferably at least 30, more preferably at least 40, more preferably at least 50, and most
- RECTIFIED SHEET (RULE 91) preferably at least 60, amino acid residues in length; and, a peptide having at least 50%, more preferably at least 60%, yet more preferably at least 70%, yet more preferably at least 80%, and most preferably at least 90 or 99% homology with an amino acid sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO:l.
- the peptide is a recombinant peptide, e.g., a protein expressed from a nucleic acid of the invention.
- the invention features: a therapeutic composition which includes a peptide of the invention and a pharmaceutically acceptable carrier; and, a therapeutic composition which includes a substantially pure nucleic acid of the invention and a pharmaceutically acceptable carrier.
- the invention features a transgenic animal, e.g., a transgenic mammal, e.g., a mouse, having a transgene which includes an pp60 PIK encoding DNA.
- the pp60 PIK gene or DNA includes a mutation, e.g., a mutation that results in misexpression of pp60 PIK .
- the animal includes a second transgene, e.g., a transgene which includes the IRS-1 gene, e.g., an IRS-1 knockout.
- the invention features a method of determining if a subject, e.g., a mammal, e.g., a human, is at risk for a disorder, e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes by evaluating an aspect of pp60 PIK metabolism.
- the method includes: determining an aspect of pp60 PIK metabolism in the subject, an abnormal level of pp60 PI metabolism being predictive of risk for the disorder.
- the aspect of pp60 PIK metabolism determined can include: the amount, distribution or structure of intracellular or extracellular pp60 PIK ; the level of phosphorylation of pp60 PIK ; the level of kinase activity of pp60 PIK ; or the amount, distribution, or structure of pp60 PIK encoding RNA.
- the invention features a method of determining if a subject, e.g., a mammal, e.g., a human, is at risk for a disorder, e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes, by determining the structure of the pp60 PIK gene.
- a subject e.g., a mammal, e.g., a human
- a disorder e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes
- the method includes: determining if the structure of an allele of the pp60 PIK gene in the subject differs from wild type, an other than wild type structure being predictive of risk for the disorder.
- the structure of the pp60 PIK gene can be determined, e.g., by hybridization based methods, e.g., by Southern analysis, or by DNA sequencing. Gross abnormalities, e.g., deletions, inversions, or translocations, or point mutations, can be predictive of risk.
- the method can be used to predict risk in the individual tested or in the individual's offspring. The method can be performed prenatally.
- the invention also provides for a system to evaluate or screen treatments for their usefulness in the treatment a disorder, e.g., an insulin related disease, e.g., an insulin resistant disease, e.g., Type II diabetes.
- a disorder e.g., an insulin related disease, e.g., an insulin resistant disease, e.g., Type II diabetes.
- the invention includes a method of evaluating a treatment for use in treating a disorder. The method includes administering a treatment, e.g., adniinistering compounds to a cultured cell or a test organism e.g., a mammal, and determining the effect of the agent on an aspect of pp60 PIK metabolism. A change in an aspect of pp60 PIK 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 response to blood glucose levels.
- Transgenic cells and animals of the invention also provide for a system to evaluate or screen treatments for their usefulness in the treatment a disorder, e.g., an insulin related disorder, e.g., an insulin-resistant disease, e.g., type II diabetes.
- the invention includes a method of evaluating an effect of a treatment, e.g., a therapeutic agent, on insulin metabolism in a transgenic cell or a transgenic animal having a pp60 PIK transgene.
- the method includes: aclministering a treatment to a transgenic cell or animal having a transgene which includes pp60 PIK , e.g., a cell or animal heterozygous or homozygous for a pp60 PIK knockout; and determining the effect of the therapeutic agent on an aspect of insulin or pp60 PIK metabolism.
- a change in the determined parameter being indicative of the usefulness or therapeutic value of the treatment.
- restoration of an aspect of insulin metabolism impaired by a misexpressed pp60 PIK gene is indicative of the effectiveness of a treatment.
- 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 pp60 PIK ) without mimicking a second effect of insulin.
- the method includes: adniinistering the agent to a cell grown in culture or to a test organism, e.g., a mammal; evaluating the agent's ability to mimic the first, pp60 PIK - mediated effect of insulin; and evaluating the agent's ability to niirnic the second effect of insulin.
- evaluating the agent's ability to mimic the first, pp60 PIK - mediated effect of insulin includes: measuring a change in an aspect of pp60 PIK metabolism, e.g., the level of pp60 PI expression, the kinase activity of pp60 PIK , the cellular or intra-cellular distribution of pp60 PIK , or the level of the pp60 PIK phosphorylation.
- the invention also features a method of treating a subject, e.g., a mammal e.g., a human, suffering from a disease caused by an abnormality of pp60 PIK metabolism, e.g., a disease characterized by lower than desirable levels of pp60 PIK activity.
- the method includes administering to the subject, a therapeutically effective amount of an agent, e.g., pp60 PI , which alters an aspect of insulin metabolism, e.g., the level of PI 3' kinase phosphorylation.
- an agent e.g., pp60 PI
- pp60 PI alters an aspect of insulin metabolism, e.g., the level of PI 3' kinase phosphorylation.
- the invention also features a method of treating a subject, e.g., a mammal, e.g., a human, suffering from a disease caused by unwanted tyrosine kinase activity.
- the method includes administering to the subject a therapeutically effective amount of a therapeutic agent, e.g., pp60 PIK antagonist, e.g., a pp60 PIK peptide which is an antagonist of pp60 PIK mediated phosphorylation of a kinase, e.g., PI 3' kinase, a pp60 PIK antisense molecule, or an anti-pp60 PIK mAb, which modifies the ability of pp60 PIK to alter the phosphorylation ofthe tyrosine kinase, thereby altering the tyrosine kinase activity.
- a therapeutic agent e.g., pp60 PIK antagonist, e.g., a
- the tyrosine kinase activity results from a 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.
- the method includes administering to said mammal a therapeutically effective amount of a therapeutic agent, e.g., a pp60 PIK antagonist, e.g., a pp60 PIK peptide which is an antagonist of pp60 PIK mediated phosphorylation of a kinase, e.g., PI 3' kinase, a pp60 PIK antisense molecule, or an anti- pp60 PIK mAb, which alters an aspect of pp60 PIK metabolism.
- a therapeutic agent e.g., a pp60 PIK antagonist, e.g., a pp60 PIK peptide which is an antagonist of pp60 PIK mediated phosphorylation of a kinase, e.g., PI 3' kinase, a pp60 PIK antisense molecule, or an anti- pp60 PIK mAb, which alters an aspect of
- Methods of the invention can be used to diagnose the presence of diseases characterized by an abnormality in the structure or metabolism of pp60 PIK .
- the invention allows for the analysis of various aspects of insulin metabolism, e.g., for the detection of insulin-stimulated PI 3' kinase phosphorylation.
- the invention also provides useful tools for the testing and development of therapeutic agents used to treat insulin or pp60 PIK related diseases.
- the invention also features a method of evaluating an effect of a therapeutic agent which alters the ability of a tyrosine kinase to phosphorylate a substrate which includes the amino acid sequence YFIN.
- the method includes administering the agent to a cultured cell or test organism, e.g., a mammal, and measuring the level of phosphorylation of a substrate which includes the amino acid sequence YFIN, e.g., a naturally occurring YFIN- containing substrate of a tyrosine kinase or a YFIN-containing synthetic substrate.
- a cultured cell or test organism e.g., a mammal
- the invention also includes a method of treating a mammal e.g., a human, suffering from a disease, disorder, or condition caused or characterized by the phosphorylation of a substrate of a tyrosine kinase, the substrate including the amino acid sequence YFIN.
- 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 fins gene product.
- 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
- an oncogene product e.g
- the method includes administering a therapeutically effective amount of a therapeutic agent, e.g., a peptide which includes the sequence YFIN, e.g., pp60 PIK or a fragment thereof which includes the amino acid sequence YFIN, mimetic or a YFIN-continuing peptide.
- a therapeutic agent e.g., a peptide which includes the sequence YFIN, e.g., pp60 PIK or a fragment thereof which includes the amino acid sequence YFIN, mimetic or a YFIN-continuing peptide.
- the invention includes peptides capable of inhibiting an interaction, preferably a site specific interaction, of an SH2-doma -containing protein, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor or the PDGF receptor, or a protein which is active in the regulation of cell proliferation, e.g., an oncogene product with a second protein containing the sequence YFIN, e.g., a protein capable of altering the state of phosphorylation of a tyrosine residue, e.g., a tyrosine kinase, e.g., pp60 PI .
- an SH2-doma -containing protein e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the
- Peptides of the invention include the sequence R FIN (wherein Rl is tyrosine, phosphotyrosine, or more preferably, an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety).
- Rl is tyrosine, phosphotyrosine, or more preferably, an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety.
- the invention also includes mimetics of these peptides.
- the invention features a method of inhibiting an interaction, preferably a site specific interaction, between a first molecule which includes an SH2 domain, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor or the PDGF receptor, or a protein which is active in the regulation of cell proliferation, e.g., an oncogene product, and a second molecule, e.g., a protein containing the sequence YFIN, a protein capable of altering the state of phosphorylation of a tyrosine residue, e.g., a tyrosine kinase, e.g., pp60 PIK .
- the method includes contacting the first molecule with an inhibitor molecule which includes a peptide of the invention or which includes a mimetic of a peptide of the invention.
- the invention features a method of treating a mammal, e.g., a human, having a condition characterized by unwanted cell proliferation including administering to the mammal an amount of a YFIN-containing peptide of the invention, or a mimetic of a peptide of the invention, sufficient to prevent or inhibit the unwanted cell proliferation.
- the invention also includes methods of increasing the affinity of a YFIN- containing phosphopeptide for its substrate, e.g., a protein containing a SH2-domain.
- the method includes replacing the phosphotyrosine of the YFIN motif with a moiety which is more electronegative than the phosphate moiety of phosphotyrosine such as in RI-OPO3H2 where R 1 can be CHF, CF , CHC1, CC1 2 , or CC1F.
- Methods of the invention allow the treatment of a variety of diseases, e.g., insulin related diseases, insulin resistant diseases, diseases characterized by abnormal cellular proliferation, and diseases caused by the phosphorylation of a substrate by a tyrosine kinase, by intervening in aspects of pp60 PIK metabolism.
- diseases e.g., insulin related diseases, insulin resistant diseases, diseases characterized by abnormal cellular proliferation, and diseases caused by the phosphorylation of a substrate by a tyrosine kinase, by intervening in aspects of pp60 PIK metabolism.
- FIG. 1 is a diagram of the DNA sequence of a pp60 PIK cDNA (SEQ ID NO:l) and the amino acid sequence of pp60 PIK (SEQ ID NO:l and 2).
- Fig. 2 is a depiction of the results of expression/screening of an IRS-1 associated protein (51.1.1) from a mouse adipocyte library using the following probes: IRS-1 protein probe, p85 ⁇ cDNA probe, and p85 ⁇ cDNA probe.
- Fig. 3 is a schematic diagram of the three clones ( ⁇ ZAP 2.1, ⁇ Exlox, ⁇ ZAP 52.1) from which the DNA sequence of pp60 PIK cDNA was derived.
- Fig.4 is a diagram of the amino acid sequences of pp60 PIK , p85 ⁇ and p85 ⁇ , which shows the homologous regions among the three proteins.
- Figs. 5 A, 5B, and 5C are depictions, of the results of experiments performed to determine the distribution of pp60 PIK in a variety of mouse tissues.
- Fig. 5A specifically depicts hybridization analysis;
- Fig. 5B competitive PCR experiments;
- Fig. 5C relative levels in various tissues.
- Figs. 6A and 6B are depictions(Fig. 6A: pi 10 immunoblot; Fig. 6B: p85 immunoblot) of the results of experiments performed to determine the expression of p 110 and pp60 PIK in insect Sf-9 cells.
- Fig. 7 is a depiction of the results of experiments performed to determine the association of pi 10 and pp60 PIK in insect Sf-9 cells.
- Figs. 8A, 8B and 8C are depictions of the results of experiments performed to determine the expression of pp60 PIK in CHO cells.
- Fig. 8 A anti-p85 preciptiation
- Fig. 8B anti-FLAG precipitation
- Fig. 8C anti-IRS-1 precipitation.
- Figs. 9 A and 9B are depictions of the results of experiments performed to show phosphorylation of pp60 PIK by the insulin receptor.
- Fig. 9A In Vitro Phosphorylation
- Fig. 9B Tryptic Digest.
- Figs. 10A and 1 OB are depictions of the results of experiments performed to identify the tyrosine phosphorylation site in pp60 PIK .
- Fig. 10A showing the predicted cycles for tryptic peptides;
- Fig. 10B showing the graphical results of the experiments.
- Fig. 11 is a depiction of the results of experiments performed to determine the effect of insulin on PI 3'-kinase activity on mammalian cells (Chinese hamster ovary cells) overexpressing pp60 PIK .
- the invention features, a substantially pure nucleic acid, e.g., a DNA, which includes (or consists essentially of) a nucleic acid sequence encoding pp60 PIK , or a pp60 PIK peptide.
- the encoded peptide has pp60 PIK activity;
- the nucleic acid sequence is at least 50%, more preferably at least 60%, yet more preferably at least 70%, and most preferably at least 80, 90, 95, or 99%, homologous with DNA from SEQ ID NO:l;
- the encoded amino acid sequence is such that it can be encoded by a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence as the peptide of SEQ ID NO: 1 ;
- the pp60 PIK peptide is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 120 amino acid residues in length;
- the pp60 PIK peptide is at least 50%, more preferably at least 60%, more preferably at least 70%, yet more preferably at least 80%, and most preferably at least 90 or 99% homologous with an
- the encoded peptide has one or more of the following properties: it can bind to IRS-1; it can bind to the PI 3'-kinase 110 Kd catalytic subunit; it does not contain an SH3 domain; it can be phosphorylated by the insulin receptor or other tyrosine kinases; it does not contain a BCR-homology region (such as is found in p85s); it contains one and preferably two, SH2 domains; it contains one tyrosine phosphorylation site.
- the invention features, a substantially pure nucleic acid, e.g., DNA, which includes (or consists essentially of) a nucleic acid sequence which hybridizes under high or low stringency to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence as the peptide of SEQ ID NO: 1.
- the DNA sequence is at least 50%, more preferably at least 60%, yet more preferably at least 70%, and most preferably at least 80, 90, 95, or 99% homologous with DNA from of SEQ ID NO: 1 ;
- the substantially pure DNA encodes a peptide at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 120 amino acid residues in length;
- the substantially pure DNA encodes a peptide at least 50%, more preferably at least 60%, yet more preferably at least 70%, and most preferably at least 90 or 99% homologous with an amino acid sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO:l;
- the substantially pure DNA encodes a peptide having essentially the same amino acid sequence, or a fragment of the amino acid sequence, described in SEQ ID NO: 1.
- the encoded peptide has one or more of the following properties: it can bind to IRS-1; it can bind to the PI 3'-kinase 110 Kd catalytic subunit; it does not contain an SH3 domain; it can be phosphorylated by the insulin receptor or other tyrosine kinases; it does not contain a BCR-homology region (such as is found in p85s); it contains one and preferably two, SH2 domains; it contains one tyrosine phosphorylation site.
- the invention features, a substantially pure nucleic acid, e.g., a DNA, which includes (or consists essentially of) a sequence encoding a peptide of 20 or more amino acids in length, the peptide having at least 90% homology with an amino acid sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO:l.
- the substantially pure nucleic acid encodes: a peptide which is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, amino acid residues in length; a peptide which is at least 50%, more preferably at least 60%, yet more preferably at least 70%, yet more preferably at least 80%, and most preferably at least 90 or 99% homologous with an amino acid sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO:l; and, a peptide with pp60 PIK activity.
- the encoded peptide has one or more of the following properties: it can bind to IRS-1 ; it can bind to the PI 3'-kinase 110 Kd catalytic - - subunit; it does not contain an SH3 domain; it can be phosphorylated by the insulin receptor or other tyrosine kinases; it does not contain a BCR-homology region (such as is found in p85s); it contains one and preferably two, SH2 domains; it contains one tyrosine phosphorylation site.
- the invention includes a vector which includes nucleic acid, e.g., a DNA, of the invention, preferably a substantially pure nucleic acid of the invention, which encodes a peptide of the invention, e.g.: DNA which includes a sequence encoding a peptide having pp60 PIK activity; DNA which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence of SEQ ID NO: 1 ; DNA which encodes a peptide of essentially the sequence described in SEQ ID NO:l; DNA which includes a sequence encoding a peptide of at least 20, preferably at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, amino acid residues in length; and DNA which encodes a peptide at least 50%, more preferably at least 60%, yet more preferably at least 70%, yet more preferably at least 80%, and most preferably at least 90
- the invention also includes: a cell containing a nucleic acid, e.g., a DNA, preferably a substantially pure nucleic acid, of the invention, preferably, a cell which is capable of expressing a peptide of the invention; an essentially homogeneous population of cells, each of which includes a sequence, preferably a substantially pure nucleic acid, of the invention, and a method for manufacture of a peptide of the invention including culturing a cell which includes a nucleic acid, preferably a substantially pure nucleic acid of the invention in a medium to express the peptide.
- a nucleic acid e.g., a DNA, preferably a substantially pure nucleic acid, of the invention, preferably, a cell which is capable of expressing a peptide of the invention
- an essentially homogeneous population of cells each of which includes a sequence, preferably a substantially pure nucleic acid, of the invention, and a method for manufacture of a peptide of
- the invention features a peptide of the invention, preferably a purified peptide of the invention, e.g.: a peptide having pp60 PIK activity; a peptide encoded by a DNA which hybridizes under high or low stringency conditions to a nucleic acid which encodes peptide sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO: 1 ; a peptide of essentially the sequence described in SEQ ID NO: 1 ; a peptide of at least 20, preferably at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, amino acid residues in length; and, a peptide having at least 50%, more preferably 60%, yet more preferably 70%, yet more preferably 80%, and most preferably at least 90 or 99% homology with an amino acid sequence which is the same, or essentially the same, as the amino acid sequence of SEQ ID NO:l.
- a purified peptide of the invention e
- the peptide has one or more of the following properties: it can bind to IRS-1; it can bind to the PI 3'-kinase 110 Kd catalytic subunit; it does not contain an SH3 domain; it can be phosphorylated by the insulin receptor or other - - tyrosine kinases; it does not contain a BCR-homology region (such as is found in p85s); it contains one and preferably two, SH2 domains; it contains one tyrosine phosphorylation site.
- the invention features a peptide of the invention, preferably a purified peptide of the invention, produced by expression of a nucleic acid of the invention, preferably a substantially pure nucleic acid of the invention, e.g.: a peptide produced by the expression of : a purified DNA encoding a peptide having pp60 PIK activity; a peptide expressed from DNA which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide with the same, or essentially the same, amino acid sequence as the peptide of SEQ ID NO: 1 ; a peptide expressed from DNA which encodes a peptide of essentially the sequence described in SEQ ID NO: 1 ; a peptide expressed from a purified DNA which includes a sequence encoding a peptide of at least 20, preferably at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, amino acid residues in length; and a
- the encoded peptide has one or more of the following properties: it can bind to IRS-1 ; it can bind to the PI 3'-kinase 110 Kd catalytic subunit; it does not contain an SH3 domain; it can be phosphorylated by the insulin receptor or other tyrosine kinases; it does not contain a BCR-homology region (such as is found in p85s); it contains one and preferably two, SH2 domains; it contains one tyrosine phosphorylation site.
- the invention features: a therapeutic composition which includes a peptide of the invention and a pharmaceutically acceptable carrier; and, a therapeutic composition which includes a purified DNA of the invention and a pharmaceutically acceptable carrier
- the invention features a transgenic animal, e.g., a transgenic mammal, e.g., a mouse, having a transgene which includes a pp60 PIK encoding DNA.
- the pp60 PIK gene or DNA includes a mutation, e.g., a mutation that results in misexpression of pp60 PIK .
- the transgenic animal includes a second transgene, e.g., a transgene which includes the IRS-1 gene, e.g., an IRS-1 knockout.
- the invention features a method of determining if a subject, e.g., a mammal, e.g., a human, is at risk for a disorder, e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes.
- a subject e.g., a mammal, e.g., a human
- a disorder e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes.
- the method includes evaluating or measuring an aspect of pp60 PIK metabolism in the subject, an abnormal level of pp60 PI metabolism being predictive of the disorder.
- Preferred embodiments include those in which: the evaluation or measurement includes determining the level of pp60 PIK ; the evaluation or measurement includes - 10 - determining the level of phosphorylation of the pp60 PIK ; the evaluation or measurement includes determining the level of kinase activity of pp60 PIK ; and the evaluation or measurement includes determining the amount of pp60 PIK encoding RNA.
- the invention features a method of determining if a subject, e.g., a mammal, e.g., a human, is at risk for a disorder, e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes, by determining the structure of the pp60 PIK gene.
- a subject e.g., a mammal, e.g., a human
- a disorder e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes
- the method includes determining, preferably prenatally, if a subject, e.g., a mammal, e.g., a human, is at risk for a disorder, e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes, in a mammal, e.g., a human, by determining the structure of the gene which expresses pp60 PIK , an abnormal structure being predictive of risk.
- a subject e.g., a mammal, e.g., a human
- a disorder e.g., an insulin-related disease, e.g., an insulin resistant insulin-related disease, e.g., Type II diabetes
- the invention also provides for a system to evaluate or screen treatments of usefulness in the treatment of a disorder, e.g., an insulin-related disorder.
- the invention includes a method of evaluating a treatment for use in treating a disorder. The method includes administering the treatment to a cultured cell or a mammal, and measuring the effect of the treatment on an aspect of pp60 PIK metabolism, e.g., measuring the level of pp60 PIK expression, the cellular or intra-cellular distribution of pp60 PIK , or the level of the pp60 PIK phosphorylation.
- a change in an aspect of pp60 PIK metabolism indicates an effect of the treatment.
- the insulin-related disease is an insulin resistant disease and the change in an aspect of metabolism is a change in the response to blood glucose levels.
- Transgenic cells and animals of the invention also provide for a system to evaluate or screen treatments of usefulness in the treatment a disorder, e.g., an insulin related disorder.
- the invention includes a method of evaluating an effect of a treatment, e.g., a therapeutic agent, on insulin metabolism in a transgenic cell or a transgenic animal having a pp60 PIK transgene.
- the method includes administering a treatment to a transgenic cell or animal, e.g., a transgenic mammal, e.g., a mouse, having a transgene which includes pp60 PIK , and determining the effect of the therapeutic agent on an aspect of insulin metabolism.
- the aspect ofinsulin metabolism followed can include, e.g., measuring responsiveness to blood glucose levels or by the phosphorylation of PI 3' kinase in response to insulin administration.
- the pp60 PIK gene or DNA includes a mutation, e.g., a deletion or other mutation which results in misexpression of pp60 PIK -
- a change in the determined parameter for insulin metabolism being indicative of the usefulness of therapeutic value of the treatment, e.g., restoration of an aspect ofinsulin metabolism impaired by a misexpressed pp60 PIK gene is indicative of the effectiveness of a treatment.
- the invention also includes a method of assaying an effect of a therapeutic agent which mimics a first effect ofinsulin (the first effect mediated by pp60 PIK) without mimicking a second effect ofinsulin.
- the method includes: administering the agent to a cell - - grown in culture or to a test organism, e.g., a mammal; evaluating the agent's ability to mimic the first, pp60 PIK - mediated effect ofinsulin; and evaluating the agent's ability to mimic the second effect ofinsulin.
- evaluating the agent's ability to mimic the first, pp60 PI - mediated effect ofinsulin includes: and measuring a change in an aspect of pp60 PI metabolism, e.g., the level of pp60 PIK expression, the kinase activity of pp60 PIK , the cellular or intra-cellular distribution of pp60 PIK , or the level of the pp60 PIK phosphorylation.
- the invention also features a method of treating a subject, e.g., a mammal e.g., a human, suffering from a disease caused by an abnormality of pp60 PIK metabolism, e.g., a disease characterizing lower than desirable levels of pp60 PIK activity.
- the method includes administering to the subject, a therapeutically effective amount of an agent, e.g., pp60 PIK , which alters an aspect ofinsulin metabolism, e.g., the level of PI 3' kinase phosphorylation.
- an agent e.g., pp60 PIK
- an aspect ofinsulin metabolism e.g., the level of PI 3' kinase phosphorylation.
- the invention also features a method of treating a subject, e.g., a mammal, e.g., a human, suffering from a disease caused by unwanted tyrosine kinase activity.
- the method includes administering to the subject a therapeutically effective amount of a therapeutic agent, e.g., pp60 PIK antagonist, e.g., a pp60 PIK peptide which is an antagonist of pp60 PIK mediated phosphorylation of a kinase, e.g., PI 3' kinase, a pp60 PIK antisense molecule, or an anti- pp60 PI mAb, which modifies the ability of pp60 PIK to alter the phosphorylation of the tyrosine kinase, thereby altering the tyrosine kinase activity.
- a therapeutic agent e.g., pp60 PIK antagonist, e.g., a
- the tyrosine kinase activity results from a 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.
- the method includes administering to said mammal a therapeutically effective amount of a therapeutic agent, e.g., a pp60 PIK antagonist, e.g., a pp60 PIK peptide which is an antagonist of pp60 PIK mediated phosphorylation of a kinase, e.g., PI 3' kinase, a pp60 PIK antisense molecule, or an anti- pp60 PIK mAb, which alters an aspect of pp60 PIK metabolism.
- a therapeutic agent e.g., a pp60 PIK antagonist, e.g., a pp60 PIK peptide which is an antagonist of pp60 PIK mediated phosphorylation of a kinase, e.g., PI 3' kinase, a pp60 PIK antisense molecule, or an anti- pp60 PIK mAb, which alters an aspect of
- the invention also features a method of evaluating an effect of a therapeutic agent which alters the ability of a tyrosine kinase to phosphorylate a substrate which includes the amino acid sequence YFIN.
- the method includes administering the agent to a cultured cell or test organism, e.g., a mammal, and measuring the level of phosphorylation of a substrate which includes the amino acid sequence YFIN, e.g., a naturally occurring YFIN- containing substrate of a tyrosine kinase or a YFIN-containing synthetic substrate.
- the substrate is a peptide which includes the sequence YFIN
- the substrate is pp60 PIK or a YFIN containing any fragment thereof.
- the peptide or fragment is between 4 and 30 amino acids in length; the peptide or fragment is between 4 and 15 amino acids in length; the peptide or fragment is at least 40%, preferably at least 80%, and more preferably at least 95% homologous, with a segment of a naturally occurring protein, e.g., tyrosine phosphatase substrate, e.g., pp60 PIK , which interacts with an SH2 containing protein.
- tyrosine phosphatase substrate e.g., pp60 PIK
- the invention also includes a method of treating a mammal e.g., a human, suffering from a disease, disorder, or condition caused or characterized by the phosphorylation of a substrate of a tyrosine kinase, the substrate including the amino acid sequence YFIN.
- 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 fins gene product.
- 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
- an oncogene product e.g
- the method includes administering a therapeutically effective amount of a therapeutic agent, e.g., a peptide which includes the sequence YFIN, e.g., pp60 PIK or a fragment thereof which includes the amino acid sequence YFIN, or a mimetic of a YFIN-containing peptide.
- a therapeutic agent e.g., a peptide which includes the sequence YFIN, e.g., pp60 PIK or a fragment thereof which includes the amino acid sequence YFIN, or a mimetic of a YFIN-containing peptide.
- the substance blocks phosphorylation of the naturally occurring substrate by competitive or non-competitive inhibition of the naturally occurring substrate.
- the peptide or fragment is between 4 and 30 amino acids in length; the peptide or fragment is between 4 and 15 amino acids in length; the peptide or fragment is at least 40%, preferably at least 80%, and more preferably at least 95% homologous, with a segment of a naturally occurring protein, e.g., tyrosine phosphatase substrate, e.g., pp60 PIK , which interacts with an SH2 containing protein.
- a segment of a naturally occurring protein e.g., tyrosine phosphatase substrate, e.g., pp60 PIK
- the invention includes peptides capable of inhibiting an interaction, preferably a site specific interaction, of an SH2-domain-containing protein, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor or the PDGF receptor, or a protein which is active in the regulation of cell proliferation, e.g., an oncogene product, with a second protein containing the sequence YFIN, e.g., a protein capable of altering the state of phosphorylation of a tyrosine residue, e.g., a tyrosine kinase, e.g., pp60 PIK .
- an SH2-domain-containing protein e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor
- Peptides of the invention include the sequence R FIN (wherein Ri is tyrosine, phosphotyrosine, or more preferably, an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g. phosphonomethylphenylalanine (Pmp), or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, mono- or difluorophosphonomethylphenlalanine (FPmp or F2Pmp, respectively).
- the invention also includes mimetics of these peptides.
- the peptide is between 4 and 30 amino acids in length; the peptide is between 4 and 15 amino acids in length; the peptide is at least 40%, preferably at least 80%, and more preferably at least 95% homologous, with a segment of a naturally occurring protein, e.g., tyrosine phosphatase substrate, e.g., pp60 PIK , which interacts with an SH2 containing protein.
- a segment of a naturally occurring protein e.g., tyrosine phosphatase substrate, e.g., pp60 PIK
- the invention features a method of inhibiting an interaction, preferably a site specific interaction, between a first molecule which includes an SH2 domain, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor or the PDGF receptor, or a protein which is active in the regulation of cell proliferation, e.g., an oncogene product, and a second - - molecule, e.g., a protein containing the sequence YFIN, a protein capable of altering the state of phosphorylation of a tyrosine residue, e.g., a tyrosine kinase, e.g., pp60 PIK .
- a signal transduction protein e.g., a cytoplasmic or a transmembrane signal transduction protein
- a receptor protein e.g., the insulin receptor or the
- the method includes contacting the first molecule with an inhibitor molecule which includes a peptide of the invention or which includes a mimetic of a peptide of the invention.
- Preferred embodiments include those in which: the first molecule is a molecule which transmits a signal, e.g., an extracellular signal, across a membrane and the second molecule is an enzyme which can alter the phosphorylation state of tyrosine, e.g., a tyrosine kinase; the first molecule is an oncogene protein and the second molecule is an enzyme which can alter the phosphorylation state of tyrosine, e.g., a tyrosine kinase, e.g., pp60 PIK ; the first molecule is the insulin receptor and the second molecule is an enzyme which can alter the phosphorylation state of tyrosine, e.g., a tyrosine kinase; the inhibitor molecule inhibits the first
- the invention features a method of treating a mammal, e.g., a human, having a condition characterized by unwanted cell proliferation including administering to the mammal an amount of a Y7IN-containing peptide of the invention, or a mimetic of a peptide of the invention, sufficient to prevent or inhibit the unwanted cell proliferation.
- the peptide prevents the association of an SH2 domain containing oncogene with a second molecule, e.g., a protein containing the sequence YFIN, e.g., a protein capable of altering the state of phosphorylation of a tyrosine residue, e.g., a tyrosine kinase.
- the invention also includes methods of increasing the affinity of a YFIN- containing phosphopeptide for its substrate, e.g., a protein containing a SH2-domain.
- the method includes replacing the phosphotyrosine of the YFIN motif with a moiety which is more electronegative than the phosphate moiety of phosphotyrosine such as in RI-OPO3H2 where R 1 can be CHF, CF2, CHC1, CCI2, or CC1F.
- the invention includes peptides which have been modified to make them more resistant to proteolytic degradation and include e.g., depsipeptide derivatives of the peptides disclosed herein, e.g., peptides which have been modified by the reduction of amide bonds, the inclusion of D-amino acids, or end methylation.
- An interaction between an SH2 domain containing protein and a second molecule e.g., a protein, as used herein, refers to any of: binding characterized by noncovalent or covalent interactions; an interaction which includes the alteration of the phosphorylation state of either the SH2 domain containing or another molecule, e.g., the second molecule; or to an interaction which includes an alteration of a catalytic ability of the SIC domain containing proteins, or another molecule, e.g., the second molecule.
- Analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety refers to an amino acid with a side chain having a moiety of the formula
- R is a moiety which renders the phosphate group more resistant to enzymatic
- R and R are preferably H.
- a preferred example of R is CR ⁇ R ⁇ , wherein R ⁇ is H, or a small electronegative atom, e.g., F or Cl, and R5 is H or a small electronegative atom, e.g., F or Cl.
- Particularly preferred embodiments of the invention include hydrolysis resistant phosphorous moieties that are more electronegative than the phosphate moiety of phosphotyrosine, for example, where R! is -CHF, -CF2, -CC1F, -CHC1, or CC-2.
- Increasing the electronegativity of the phosphate moiety in an analog of phosphotyrosine increases the binding affinity of peptides containing the analog to a substrate such as protein containing an SH2 domain. In some cases, the affinity can exceed that of peptides containing phosphotyrosine.
- SH2 domain containing proteins are involved in cellular signaling, e.g., in the signal transduction mediated by insulin and the insulin receptor and by several classes of oncogenes.
- the invention provides for inhibitors of these cellular signal transduction systems by inhibiting an interaction between the SH2 domain of the signal transduction protein and a YFIN motif present on another protein.
- the invention provides for interference with the transduction of growth signals and thereby allows for control of unwanted cellular proliferation.
- the invention also provides peptide inhibitors of PTPases.
- the peptide inhibitors, or analogs, which may or may not have homology with naturally occurring protein tyrosine phosphatase substrates, include an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g., phosphonomethylphenylalanine or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp.
- a hydrolysis resistant phosphorous moiety e.g., phosphonomethylphenylalanine or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp.
- the phosphotyrosine analog includes a phenyl group substituted with an -O-PO3H2 analog, e.g., -R-PO3H2, where R can be any group which confers greater resistance to hydrolysis than does -O-, e.g, CR ⁇ RS wherein R ⁇ can be H, F or Cl and R ⁇ can be H, F or Cl.
- Preferred phosphotyrosine analogs are phosphonomethylphenylalanine and mono- or difluorophosphonomethylphenylalanine.
- Peptide inhibitors of the invention can have homology with PTPase substrates. The sequence of these inhibitors can be based on the amino acid sequences of kinase autophosphorylation and endogenous substrate phosphorylation sites.
- Phosphonomethylphenylalanyl peptides and mono- and difluorophosphonomethylphenylalanyl peptides constitute classes of compounds that potently inhibit PTPase activity.
- Pmp-peptides appear to act as direct substrate mimics, as binding affinity closely matches that of the corresponding phosphopeptides and inhibition is competitive.
- F2Pmp peptides also appear to act as direct substrate mimics. However, binding affinity of the F2Pmp-peptides can match or exceed that of the corresponding phosphopeptides.
- Inhibitors of the invention allow the inhibition of cellular PTPases and can be used in controlling metabolic processes, e.g., abnormal processes associated with diabetes, and as therapeutic modalities for selected malignancies.
- Signal transduction protein refers to a protein involved in transferring a signal from the cell surface into the cell and includes, e.g., membrane bound receptors, e.g., cell surface receptors, ligands of such receptors, and intracellular proteins which interact with either with a receptor, or with another intracellular protein to transfer a signal.
- SH2 domain refers to a conserved apparently noncatalytic sequence of approximately 100 amino acids found in many signal transduction proteins including Fps, Stc, Abl, GAP, PLC ⁇ , v-Crk, Nek, p85, and Vav. See Koch et al., 1991, Science 252:668. hereby incorporated by reference. The amino acid sequences of the SH2 domain of 27 proteins is given in Koch et al., 1991. The SH2 domain mediates protein- protein interactions between the SH2 containing protein and other proteins by recognition of a specific site on a second protein. The SH2/second protein site interaction usually results in an association of the SH2 contacting protein and the second protein.
- SH2 domain refers to any sequence with at least 70%, preferably at least 80%, and more preferably at least 90% sequence homology with a naturally occurring SH2 domain, and to any analog or fragment of an SH2 domain which exhibits at least 50% of the binding activity of a naturally occurring variant of that domain, when binding is measured as the ability to bind a YFIN containing peptide.
- Abnormal cell proliferation includes both neoplastic and non-neoplastic diseases, and thus includes diseases such as cancer and atherosclerosis.
- 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.
- a mutation means an alteration, either gross or small, in the nucleic acid which encodes pp60 PIK .
- Examples of common mutations are nucleotide deletions and insertions.
- the mutation further can be a mutation of the DNA encoding pp60 PI which results in misexpression of pp60 PIK .
- a therapeutic agent can be any substance or treatment.
- the metabolism of a substance means any aspect of the, expression, function, action, or regulation of the substance.
- the metabolism of a substance - 16 - includes modifications, e.g., covalent or noncovalent modifications of the substance.
- the metabolism of a substance includes modifications, e.g., covalent or noncovalent modification, the substance induces in other substances.
- the metabolism of a substance also includes changes in the distribution of the substance.
- the metabolism of a substance includes changes the substance induces in the distribution of other substances.
- An insulin-related disease is a disease, disorder, or condition in which some aspect ofinsulin 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 ofinsulin 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.
- a purified preparation of pp60 PIK means pp60 PIK that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs.
- the pp60 PIK is also separated from substances, e.g., antibodies or gel matrix, e.g, polyacrylamide, which are used to purify it.
- the purified preparation of pp60 PIK constitutes at least 10% dry weight of the purified preparation.
- the purified preparation contains sufficient pp60 PIK to allow protein sequencing.
- a substantially pure nucleic acid e.g., a substantially pure DNA
- the term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.
- Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional pp60 PIK sequence. Homologous refers to the sequence similarity between two pp60 PIK molecules or between two nucleic acid molecules.
- the molecules are homologous at that position.
- the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
- the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology. - 17 -
- transgene is defined as a piece of DNA which is inserted by artifice into a cell and becomes a part of the genome of an animal which develops in whole or part from that cell. Such a transgene may be partly or entirely heterologous to the transgenic animal. transgene can include a deletion.
- a peptide has pp60 PIK biological activity if it has one or more of the following properties: 1.
- the peptide is capable of binding to IRS-1 ; 2.
- the peptide is capable of binding the PI 3'-kinase; 3.
- the peptide is capable of competitively inhibiting the binding of native pp60 PIK to IRS-1; 4.
- the peptide is capable of inhibiting pp60 PIK binding to PI 3'-kinase.
- Misexpression refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the size, amino acid sequence, post-transitional modification, or biological activity of pp60 PIK ; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
- a transgenic animal e.g., a transgenic mouse
- a transgenic cell is a cell which includes a transgene.
- a pp60 PIK peptide is a peptide which is encoded by a nucleic acid of the invention, preferably it has at least 80% homology with native pp60 PIK and has pp60 PIK activity.
- PI 3 '-kinase The phosphatidylinositol (PI) 3 '-kinase is implicated in receptor-stimulated mitogenesis and differentiation, the oxidative burst in neutrophils, membrane ruffling and insulin-stimulated glucose transport.
- PI 3'-kinase is ordinarily composed of a 110 kDa catalytic subunit in association with an 85 kDa regulatory protein that contains two Src homology-2 (SIC) domains (p85).
- SIC Src homology-2
- the PI 3'-kinase is activated when phosphorylated YXXM motifs in IRS-1 bind to the SH2 domains of p85.
- pp60 PIK contains two SH2 domains with similarity to p85, and forms a stable complex with the catalytic subunit of the PI 3'- - 18 - kinase (pi 10). However, pp60 PIK lacks the SH3 domain and the break chain region (BCR)- homology region of the p85s, and contains a unique and short amino terminus.
- BCR break chain region
- Example 1 Isolation of cDNA encoding pp6QEIK and Characterization of pp6QBIK pp60 PIK was identified in a mouse fat cDNA expression library by screening with recombinant [ 32 P]IRS-1. The cDNA was confirmed in a mouse embryo library by DNA screening. See Figure 2.
- IRS-1 has no extrinsic enzymatic activity. IRS-1 must, therefore, be labeled by incubation with an activated insulin receptor in the presence of [ ⁇ - 32 P] ATP and Mn2 + . IRS-1 was obtained from Sf-9 cells infected with a recombinant baculovirus containing the cDNA of IRS- 1. A 100 ml culture of Sf-9 cells yields approximately 1 mg of phosphotyrosine-free IRS-1 that is purified to > 95% by gel exclusion chromatography on SK300HR (Pharmacia, Piscataway, N.J.).
- [32p]IRS-l is prepared by incubating IRS-1 with human insulin receptor partially purified from Chinese hamster ovary cells (CHO) expressing wild-type insulin receptor cDNA.
- the insulin receptor (5 mg of protein in a wheat germ agglutinin eluate) is activated by autophosphorylation during a twenty minute incubation with 100 nM insulin, 50 mM [ ⁇ -32P_ATP (67,00 cpm/pmol, NEN) and 5 mM MnC-2- IRS-1 (1 mg: 8 pmol) was added to the active kinase mixture and incubated at 4°C overnight.
- the [32p]lRS-l in this reaction was immunoprecipitated completely with anti-phosphotyrosine antibody indicating that each labeled molecule contains phosphotyrosine.
- the [ 32 p]iRS-l was reduced with 0.1M dithiothreitol in 50 mM Tris-HCl buffer (pH 7.4) containing 250 mM NaCl and 6M guanidinium chloride for five hours at 55°C, and then carboxymethylated with iodoacetamide (Pierce, Rockford, IL).
- Expression screening with recombinant [3-2p]IRS-l To identify IRS-1 binding proteins, an oligo(dT) primed subtraction library enriched with cDNA clones from F442a adipocytes was prepared in Uni-Zap XR as is known in the art. Adipose tissue was selected because it is a major site of insulin action in mammals. Recombinant [32p]lRS-l was used to screen F442a ⁇ ZAP expression library (500,000 plaques) prepared from mRNA enriched with transcripts expressed in F442a adipose cells and depleted for transcripts predominant in ⁇ differentiated F442a fibroblasts.
- the plates were incubated for 3.5 hours at 42°C, then overlaid with nitrocellulose filters (Millipore, HATF) that were impregnated - - with 10 mM isopropyl-b-D-thiogalactopyranoside (IPTG, BRL), and incubated for 10 hours at 37°C.
- the filters were removed, briefly washed TNT buffer (lOmM Tris-HCl), pH 8.0, 150 mM NaCl, 0.05% Tween 20) at room temperature, and then blocked in TNT buffer containing 5% Carnation instant dry milk for six hours.
- the filters were incubated overnight at 4°C with [ 3 2p]iRS-l (50 mg/ml), and then washed three times at room temperature with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.01% Tween 20.
- the dry filters were exposed to Kodak XAR-5 film with an intensifying screen at -70°C for twenty-four hours. Thirty primary positive plaques were selected, and fifteen remained positive during subsequent hybridizations with [ 32 P]IRS-1. Fourteen hybridized strongly with a cDNA probe for p85 ⁇ , whereas one (clone-52.1.1) was unique and did not cross-react with cDNA probes to p85 ⁇ or p85 ⁇ under ordinary screening conditions. See Figure 2.
- a full length cDNA probe of clone 52.1.1 was used to screen the F442a ⁇ ZAP library and a ⁇ Exlox mouse embryo (day 13) library, which revealed two independent and strongly hybridizing clones ( ⁇ ZAP 2.1, and ⁇ Exlox 2.2) ( Figure 3). Based on the complete DNA sequence of the three clones, a contiguous cDNA containing 5737 bases was obtained ( Figure 1). One of the clones ( ⁇ ZAP 2.1) contained an apparent deletion in the 5'- untranslated region, which is presumably derived from an alternative splicing event.
- the complete DNA sequence contains a 5'-untranslated region of 1366 bp, an open reading frame of 1383 bp, and a 3 '-untranslated regions of 2987 bp with six putative mRNA destabilization motifs (ATTTA), and five polyadenylation signals (AATAAA).
- ATTTA putative mRNA destabilization motifs
- AATAAA polyadenylation signals
- the primary amino acid sequence encoded by the cDNA is related to the C- terminal half of the regulatory subunits of the phosphatidylinositol (P ⁇ )-kinase ( Figure 4).
- the open reading frame encodes a 461 -amino acid sequence with two SH2 domains, one at its N-terminus (residues 65-163) and one at its C-terminus (residues 358-452).
- a Genebank search revealed a high homology at hte protein level between pp60 PIK and the carboxyl terminal half of p85 ⁇ and p85 ⁇ ( Figure 4).
- the N-terminal SH2 domain of pp60 PIK was 88.9% and 83.8% identical to the corresponding region in p85 ⁇ and p85 ⁇ , respectively.
- the carboxy-terminal SH2 domain of pp60 PIK was 81.1% and 73.7% identical to the corresponding region in p85 ⁇ and p85 ⁇ , respectively.
- a region between the SH2 domains of p85 ⁇ (residues 479-492) which is suggested to mediate the association between p85 and pi 10 is completely conserved in pp60 PIK (residues 210-224).
- SH3 domain or BCR (break chain region)-homology region ordinarily found in p85 ⁇ or p85 ⁇ is replaced in pp60 PIK by a short unique amino terminus. Unlike, p85, pp60 PIK is tyrosine phosphorylated at insulin stimulation ( Figure 4, tyrosine 341).
- the Sf-9 cells were infected with recombinant viruses and the PI kinase activity in pp60 PIK and pi 10 immunoprecipitates was measured.
- the antibody against the N-terminal SH2 domain of p85 ⁇ recognized pp60 PIK because of a similar amino acid sequence between p85 ⁇ and pp60 PI - This antibody was used for immunoprecipitation and immunoblotting of pp60 PIK -
- the successful expression of pp60 PIK and pi 10 by infection of pp60 PIK virus and pi 10 virus was confirmed by Western blotting (Figure 6).
- Example 4 Activation of PT 3'-kinase associated with pp ⁇ QEI -during insulin stimulation
- FLAG-tag sequence was added by using polymerase chain reaction (PCR) to the C-terminal end of pp60 PIK to distinguish pp60 PIK from endogenous p85 ⁇ .
- the cDNA encoding pp60 PIK with FLAG-tag was subcloned into the CAGG expression vector.
- Parental CHO cells and CHO cells overexpressing insulin receptors (CHO/IR) were transfected with this expression vector and hygromycin-resistant DNA, and selected in the medium containing 300 ⁇ g/ml hygromycin B.
- expressed pp60 PIK was recognized by the FLAG-tag antibody with its molecular size of 63kDa.
- Example 5 Phosphorylation of p ⁇ OBI by the insulin receptor pp60 PIK was isolated from CHO cells expressing pp60 PIK by immunoprecipitation with anti-FLAG antibodies. The immuncomplex was collected on protein A-Sepharose and washed in HEPES (50 mM, pH 7.5) containing 0.1% Triton. Partially purified insulin receptor was added to the immuncomplex together with Mn + (5mM) and [ ⁇ - 32 P]ATP. After incubation for thirty minutes, the phosphorylated proteins were separated by SDS-PAGE and transferred to nitrocellulose. A strong phosphoprotein was detected in a sample from the CHO cells expressing pp60 PI , whereas a weak band was obtained from the control cells. ( Figure 9).
- nitrocellulose containing [ 32 P]labeled pp60 PIK was excised and treated with 0.5% (w/v) PVP-40 (Sigma, St. Louis, MO) in 100 ml acetic acid for one hour at 37°C.
- the paper was then washed extensively with water and digest with 10 ⁇ g of TPCK-trypsin (Worthington, Freehold, N. J.) in NaHCO 3 (pH 8.0) containing acetonitrile at 37°C for twenty-four hours. An additional 10 ⁇ g TPCK-trypsin was added, and the digestion was continued for an additional twenty-four hours. This technique consistently eluted 90-95% of the phosphopeptides.
- the trypsinization was terminated by the addition of sample buffer and boiling for three minutes.
- the phosphopeptides were separated by Tricine/SDS/PAGE using a 32-cm acrylamide gel consisting of a 3% stacker, 10% spacing, and 16.5% resolving gel. After electrophoresis, the gels were sealed with plastic wrap and exposed to film at -80°C. A single phosphopeptide was detected which contained exclusively phophotyrosine. This phosphopeptide was eluted from the Tricine/SDS/PAGE gel and dialyzed against water for forty-eight hours. The phosphopeptide was lyophilized and resuspended in 50% acetonitrile.
- peptide sample was dissolved in water and digested with 7 ⁇ g of protease V8 of Asp-N (Boehringer Mannheim, Indianapolis, IN) for eighteen hours at 22°C.
- Peptide fragments were covalently coupled to SequelonTM-AA discs (Millipore, Bedford, MA) with l-ethyl-3-(3-dinthylaminopropyl)carboc imide and washed extensively with water, methanol, and trifluoroacetic acid. Manual Edman degradation of the phosphopeptides was performed as follows.
- Example 6 Insulin stimulates tyrosine phosphorylation of p ⁇ 6 ⁇ P-IK attyrosine 341
- the insulin-stimulated phosphorylation site in pp60 PIK was characterized by phosphoamino acid analysis, tryptic digestion, and manual Edman degradation to isolate the major site within the amino acid sequence.
- An immuncomplex containing pp60 PIK was prepared by incubating extracts from quiescent CHO cells or CHO/pp60 PIK cells with an anti-FLAG antibody. The immuncomplex was collected on protein-A-Sepharose.
- pp60 PIK was phosphorylated by a brief incubation with activated insulin receptors and [ 32 P]ATP (Figure 9, left panel); phosphoamino acid analysis revealed that this was exclusively tyrosine phosphorylation (data not shown). Tryptic digestion of phosphorylated pp60 PIK yields a single phosphorylated band following separation of the peptides by Tricine SDS-PAGE. To determine the location of the phosphorylated tyrosine in the pp60 PIK sequence, the tryptic fragment was characterized by additional digestion with V8 protease or endo-Asp-N. The original tryptic peptide and the products of these additional digests were immobilized on disks and subjected to manual Edman degradation.
- Radioactive phosphate was released from the tryptic peptide after cycle 8, where radioactivity was released after cycles 2 or 4 from the V8 and endo-Asp-N secondary digests, respectively. This pattern of digestion unambigously predicts the phosphorylation of Tyr-341 in the pp60 PIK molecule. See Figure 10.
- Example 7 Effect ofinsulin on PI 3'-kinase activity on Chinese hamster ovary (CEO) cells overexpressing pp ⁇ OEl ⁇
- CHO/IR/pp60 PIK was very similar to that of pp60 PI in CHO/pp60 PIK .
- Various concentrations ofinsulin were added and PI 3'-kinase activity in the immunoprecipitates by FLAG-tag antibody or IRS-1 antibody were measured (Figure 11). As shown in Figure 10,
- tyrosine kinase receptors mediate biological responses by using their tyrosine autophosphorylation sites to engage downstream signaling molecules with Src- - - homology-2 domains (SH2-proteins).
- EGF and PDGF receptors undergo a ligand-induced autophosphorylation at multiple tyrosine residues which associate directly with various SH2 proteins, including phosphatidylinositol 3'-kinase, p 21 ras GAP, phospholipase C ⁇ , GRB-2, c- fyn, c-src, and probably other SH2-proteins.
- these receptors phosphorylate an intermediate adapter molecule, IRS-1, which directly associates with multiple SH2-proteins.
- these receptors phosphorylate intermediate "docking proteins" such as IRS-1 or 4PS, which function as tyrosine phosphorylated docking protein to recruit the SH2-proteins into a signaling complex.
- the interleukin-4 receptor (IL-4r) illustrates an additional complexity since it phosphorylates IRS-1 even though it does not contain an intrinsic tyrosine kinase activity.
- the IL-4r like other receptors in the hematopoietic family such as those for erythropoeitin, growth hormone and interferon, presumably recruit cytoplasmic tyrosine kinases, possibly similar to JAK-1, tyk-2, fyn, into a multimeric signaling complex to phosphorylate IRS-1 or related docking elements.
- a number of other lower molecular weight proteins become tyrosine-phosphorylated in insulin-treated cells.
- a 60 kDa tyrosine-phosphorylated protein has been observed in several laboratories after insulin treatment of primary rat adipocytes and Chinese hamster ovary cells expressing human insulin receptors. More recently, a 60 kDa tyrosine-phosphorylated protein has been observed in p 21 ras GAP or PI 3'-kinase immunoprecipitates.
- pp60 PIK associates with the PI 3'- kinase in a complex that appears to be distinct from IRS-1.
- pp60 PIK :PI 3'-kinase complex was located in both the soluble and membrane fractions, whereas IRS-1 was entirely cytosolic. Since recombinant SH2 domains of p85 precipitate pp60 PIK from insulin- stimulated cells, Levan and Lienhard concluded that the most likely mode of association of pp60 PIK with PI 3'-kinase is through the binding of its tyrosine phosphorylation site to p85.
- pp60 PIK was obtained in high percentage yield from insulin-stimulated rat adipocytes, but the low amount of the protein obtained (about 3 ng form the adipocytes of one rat) precluded sequence analysis.
- This protein associates with p21 ras GAP and appears to be distinct from the better known GAP-associated protein, pp62.
- Ras a monomeric membrane-bound GTP- binding protein, may be a downstream element in the insulin receptor signaling pathway which mediates certain biological functions.
- Insulin stimulates the accumulation of activated rasGTP in cells, and appears to play a role in insulin-stimulated Glut-4 translocation. Moreover, microinjection of anti-ras antibodies inhibits insulin-induced maturation of Xenopus oocytes and overexpression of a dominant inhibitory ras mutant blocks insulin action on both gene expression and differentiation of 3T3-L1 to adipocytes. Thus, 60 kDa - 24 - substrates of the insulin receptor are implicated in two important signaling pathways, the PI 3'-kinase and the p21 ras signaling pathways. The molecular structure of a 60 kDa insulin receptor substrate, pp60 PIK , which partially resembles the better known p85 is described herein.
- the peptides or nucleic acids of the invention may be administered to a mammal, particularly a human, in one of the traditional modes (e.g., orally, paternally, transdermally, or transmucosally), in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, pumps, e.g., osmotic drug delivery pumps, gels and liposomes or by transgenic modes.
- traditional modes e.g., orally, paternally, transdermally, or transmucosally
- a sustained release formulation e.g., a biodegradable biocompatible polymer
- micelles e.g., osmotic drug delivery pumps, gels and liposomes or by transgenic modes.
- pumps e.g., osmotic drug delivery pumps, gels and liposomes or by transgenic modes.
- allelic variations include allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes the peptide of SEQ ID NO:l (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incorporated by reference); and, peptides or proteins specifically bound by antisera to pp60 PIK , especially by antisera to an active site or binding domain of pp60 PIK -.
- chimeric pp60 PIK s that include a pp60 PIK peptide or protein and a second peptide, e.g., a toxin, e.g., a chimeric molecule.
- the invention also includes biologically active fragments or analogs of pp60 PIK .
- a biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of the pp60 PIK shown in SEQ ID NO:l, e.g., one or more of the biological activities described above.
- a useful pp60 PIK fragment or pp60 PIK analog is one which exhibits a biological activity in any biological assay for pp60 PIK activity. Most preferably the fragment or analog possesses 10%, preferably 40%, or at least 90% of the activity of pp60 PIK (SEQ ID NO: 1), in any in vivo or in vitro pp60 PIK assay.
- Analogs can differ from naturally occurring pp60 PIK in amino acid sequence or in ways that do not involve sequence, or both. Analogs of the invention will generally exhibit at least 90%, preferably 95% or even 99%, homology with a segment of 20 amino acid residues, preferably more than 40 amino acid residues, or more preferably the entire sequence of a naturally occurring pp60 PIK sequence.
- Non-sequence modifications include in vivo or in vitro chemical derivatization of pp60 PIK s.
- Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.
- Glycosylation can be modified, e.g., by modifying the glycosylation patterns of a pp60 PIK during its synthesis and processing or in further processing steps, e.g., by exposing the pp60 PIK to glycosylation affecting enzymes derived from cells that normally provide such - -
- phosphorylation can be modified by exposing the pp60 PIK to phosphorylation-altering enzymes, e.g., kinases or phosphatases.
- phosphorylation-altering enzymes e.g., kinases or phosphatases.
- Preferred analogs include pp60 PIK (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the pp60 PIK 's biological activity.
- Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be taken from the table below.
- Glutamine Q D-GIn, Asn, D-Asn, Glu, D-Glu, Asp,D-Asp
- Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln,D-Gln
- Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D- Met, He, D-He,Orn, D-Orn
- Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5- phenylproline
- Tyrosine Y D-Tyr Phe, D-Phe, L-Dopa, His, D-His
- analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids; and cyclic analogs.
- fragment as applied to a pp60 PIK , will ordinarily be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length.
- Fragments of pp60 PIK can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of pp60 PIK can be assessed by methods known to those skilled in the art as described herein. Also included are pp60 PIK 's containing residues that are not required for biological activity of the peptide or that result from alternative mRNA splicing or alternative protein processing events.
- a purified antibody preparation preferably a monoclonal antibody preparation, directed against a protein or peptide of the invention.
- Nucleic acid encoding all or part of the pp60 PIK gene can be used to transform cells.
- the pp60 PIK gene e.g., a misexpressing or mutant form of it e.g., a deletion, or other DNA encoding an pp60 PIK protein or peptide can be used to transform a cell and to produce a cell in which the cell's genomic pp60 PIK gene has been replaced by the transformed gene, producing, e.g., a cell deleted for the pp60 PIK gene.
- This approach can be used with cells capable of being grown in culture, e.g., cultured stem cells, to investigate the function of the gene.
- nucleic acid encoding all or part of the pp60 PIK gene can be used to transform a cell which subsequently gives rise to a transgenic animal, e.g. a transgenic mouse.
- This approach can be used to create, e.g., a transgenic animal in which the pp60 PIK gene is, e.g., inactivated, e.g., by a deletion.
- Homozygous transgenic animals can be made by crosses between the offspring of a founder transgenic animal.
- Cell or tissue cultures can be derived from a transgenic animal.
- pp60 PIK -encoding DNA is introduced into an expression vector, the vector introduced into a cell suitable for expression of the desired protein, and the peptide recovered and purified, by prior art methods.
- Antibodies to the peptides an proteins can be made by immunizing an animal, e.g., a rabbit or mouse, and recovering anti-pp60 PIK antibodies by prior art methods.
- Fragments of pp60 PIK can be made by expressing pp60 PIK DNA which has been manipulated in vitro to encode the desired fragment; e.g., by restriction digestion of the DNA sequence of SEQ ID NO:l.
- Analogs can be made, e.g., by in vitro DNA sequence modifications of the sequence of SEQ ID NO: 1.
- in vitro mutagenesis can be used to convert the DNA sequence of SEQ ID NO:l into a sequence which encodes an analog - - in which one or more amino acid residues has undergone a replacement, e.g., a conservative replacement as described in Table 1.
- Fragments or analogs can be tested by methods known to those skilled in the art for the presence of pp60 PIK activity.
- the invention also provides for the generation of pp ⁇ O ⁇ K.
- mimetics e.g. peptides or non-peptide agents, which are able to modulate, e.g., inhibit, binding of a p ⁇ O ⁇ IK to another protein.
- Various forms of mutagenesis are generally applicable for mapping the determinants of the pp6 ⁇ PIK which participate in protein-protein interactions involved in binding to a second protein.
- homologs of pp60 ⁇ K (b om agonist and antagonist forms) can be generated and screened using, for example, alanine scanning mutagenesis and the like (Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al.
- the critical residues of peptides of the invention which are involved in molecular recognition of e.g., an SH2 domain, can be determined and used to generate peptidomimetics which competitively inhibit binding of pp60 p ⁇ y ⁇ fa other proteins (see, for example, "Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein" European patent applications EP-412,7624 and EP-531,080A).
- peptidomimetic compounds can be generated which mimic those residues of pp ⁇ O ⁇ IK ascertained to be involved in binding to the other protein, and which therefore can be used to inhibit binding of the authentic pp ⁇ O ⁇ K protein to the protein.
- non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R.
- ADDRESSEE LAHIVE & COCKFIELD
- B STREET: 60 STATE STREET, SUITE 510
- CTTCCTCTCC ATTCTCTCTT CTCCGAAGGA CACAAAAGTG GCTTCCGCTG AAAGATTAGG 1020 AGGCGGTGGG AGCTTTTCCC TTTGGAGAGC GATTGTGTAG GAAGGATTTT CGGGAAGCTG 1080
- CCTTGTCCTC ATCCTAGTAG TTTGGCTGGA CTTGTACTGG CCGTTGGAAA CCCCCAAGTA 1260
- Lys Asp Ser Phe lie Ser Leu Gin Asp Ala Glu Trp Tyr Trp Gly Asp 50 55 60
- ATC TAC AGC ACG GCT CGA GGA TAT GGC TTT GCA GAA CCC TAC AAC CTG 2629 lie Tyr Ser Thr Ala Arg Gly Tyr Gly Phe Ala Glu Pro Tyr Asn Leu 400 405 410
- CAGCCAGTCC CCAACAGGAA AGCAGCTTTG GGTTGGCTAG ATACAGTTTT TAAAATAAAA 5239
- Lys Ser Arg Leu Gly Glu lie His Asp Ser Lys Leu Arg Leu Glu Gin 260 265 270
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU27698/95A AU710107B2 (en) | 1994-06-10 | 1995-06-08 | PP60 pik-: a downstream element in insulin signaling |
EP95923012A EP0768821A4 (en) | 1994-06-10 | 1995-06-08 | pp60PIK : A DOWNSTREAM ELEMENT IN INSULIN SIGNALING |
JP8502339A JPH10505227A (en) | 1994-06-10 | 1995-06-08 | pp60 ▲ upper PIK ▼: downstream element in insulin signaling |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/259,264 | 1994-06-10 | ||
US08/259,264 US5650293A (en) | 1994-06-10 | 1994-06-10 | Nucleic acid encoding pp60PIK and the methods of making pp60PIK |
Publications (1)
Publication Number | Publication Date |
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WO1995034201A1 true WO1995034201A1 (en) | 1995-12-21 |
Family
ID=22984248
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1995/007312 WO1995034201A1 (en) | 1994-06-10 | 1995-06-08 | pp60PIK: A DOWNSTREAM ELEMENT IN INSULIN SIGNALING |
Country Status (6)
Country | Link |
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US (1) | US5650293A (en) |
EP (1) | EP0768821A4 (en) |
JP (1) | JPH10505227A (en) |
AU (1) | AU710107B2 (en) |
CA (1) | CA2192486A1 (en) |
WO (1) | WO1995034201A1 (en) |
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US20030044803A1 (en) * | 2000-09-22 | 2003-03-06 | Pedersen Finn Skou | Methods for diagnosis and treatment of diseases associated with altered expression of JAK1 |
US20030077590A1 (en) * | 2000-09-22 | 2003-04-24 | Pedersen Finn Skou | Methods for diagnosis and treatment of diseases associated with altered expression of neurogranin |
US20020115058A1 (en) * | 2000-09-22 | 2002-08-22 | Pedersen Finn Skou | Methods for diagnosis and treatment of diseases associated with altered expression of Pik3r1 |
US20070098728A1 (en) * | 2001-09-24 | 2007-05-03 | Pedersen Finn S | Novel compositions and methods in cancer |
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US5260200A (en) * | 1991-01-18 | 1993-11-09 | Joslin Diabetes Center, Inc. | Isolated DNA encoding an insulin receptor substrate |
AU667803B2 (en) * | 1991-01-18 | 1996-04-18 | New York University | A receptor tyrosine kinase target protein cDNA cloning method and hGRB proteins |
US5336615A (en) * | 1992-01-06 | 1994-08-09 | Yale University | Genetically engineered endothelial cells exhibiting enhanced migration and plasminogen activator activity |
-
1994
- 1994-06-10 US US08/259,264 patent/US5650293A/en not_active Expired - Lifetime
-
1995
- 1995-06-08 AU AU27698/95A patent/AU710107B2/en not_active Ceased
- 1995-06-08 JP JP8502339A patent/JPH10505227A/en active Pending
- 1995-06-08 CA CA002192486A patent/CA2192486A1/en not_active Abandoned
- 1995-06-08 WO PCT/US1995/007312 patent/WO1995034201A1/en not_active Application Discontinuation
- 1995-06-08 EP EP95923012A patent/EP0768821A4/en not_active Withdrawn
Non-Patent Citations (5)
Title |
---|
BIOCHEMICAL JOURNAL, Volume 308, issued 1995, MILARSKI et al., "Detection of a 60 kDa Tyrosine-Phosphorylated Protein in Insulin-stimulated Hepatoma Cells that Associates with the SH2 Domain of Phosphatidylinositol 3-Kinase", pages 579-583. * |
GENE, Volume 93, issued 1990, DENG et al., "A Novel Expression Vector For High-Level Synthesis and Secretion of Foreign Proteins in Escherichia Coli: Overproduction of Bovine Pancreatic Phospholipase A2", pages 229-234. * |
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, No. 8, issued 15 March 1993, LAVAN et al., "The Insulin-Elicited 60-kDa Phosphotyrosine Protein in Rat Adipocytes is Associated with Phosphatidylinositol 3-Kinase", pages 5921-5928. * |
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 269, No. 15, issued 15 April 1994, HOSOMI et al., "Characterization of a 60-Kilodalton Substrate of the Insulin Receptor Kinase", pages 11498-11502. * |
See also references of EP0768821A4 * |
Also Published As
Publication number | Publication date |
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CA2192486A1 (en) | 1995-12-21 |
JPH10505227A (en) | 1998-05-26 |
EP0768821A4 (en) | 1999-10-27 |
US5650293A (en) | 1997-07-22 |
EP0768821A1 (en) | 1997-04-23 |
AU2769895A (en) | 1996-01-05 |
AU710107B2 (en) | 1999-09-16 |
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