Patent application title: PIM-3 Kinase as a Target for Type 2 Diabetes Mellitus
Inventors:
Marc Korn (Offenbach, DE)
Guenter Mueller (Sulzbach, DE)
Rudolf Schneider (Niedernhausen, DE)
Georg Tschank (Essenheim, DE)
Assignees:
SANOFI-AVENTIS DEUTSCHLAND GMBH
IPC8 Class: AC12Q168FI
USPC Class:
506 9
Class name: Combinatorial chemistry technology: method, library, apparatus method of screening a library by measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Publication date: 2014-03-27
Patent application number: 20140087969
Abstract:
The invention relates to isolated nucleic acid molecules and to host
cells comprising such nucleic acid molecules. Moreover, the invention
relates to a polypeptide having PIM-3 activity and having a definite
amino acid sequence, as well as to the use of PIM-3 as a screening agent
for identifying anti-type 2 diabetes mellitus drugs and for preparing a
medicament for the treatment of insulin resistance or type 2 diabetes
mellitus.Claims:
1-5. (canceled)
6. A method of identifying a subject with type 2 diabetes mellitus or insulin resistance, comprising the steps of: providing a sample from a subject; contacting, in vitro, a portion of the sample with an antibody comprising specific binding affinity for a polypeptide comprising the amino acid sequence of SEQ ID NO:2; calculating an amount of the polypeptide based on said step of contacting; and identifying the subject as having type 2 diabetes mellitus or insulin resistance if there is a difference between the amount of polypeptide in the subject sample and a control sample.
7. The method of claim 6, wherein the subject has a normal blood glucose level.
8. The method of claim 6, wherein the subject has normal glycohemoglobin (HbA1c) levels.
9. The method of claim 6, wherein the subject is healthy.
10. The method of claim 6, wherein the subject is undiagnosed with type 2 diabetes mellitus or insulin resistance.
11. A method of identifying a subject with type 2 diabetes mellitus or insulin resistance, comprising the steps of: providing a sample from a subject; contacting, in vitro, a portion of the sample with an oligonucleotide probe comprising specific binding affinity for a nucleic acid comprising SEQ ID NO:1 or a nucleic acid transcribed from SEQ ID NO:1; calculating an amount of the nucleic acid based on said step of contacting; and identifying the subject as having type 2 diabetes mellitus or insulin resistance if there is a difference between the amount of nucleic acid in the subject sample and a control sample.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to a novel use of PIM-3 kinase, to novel PIM-3 kinase subtypes and the use thereof.
BACKGROUND OF THE INVENTION
[0002] The rat PIM-3 (originally termed KID-1, KID "kinase induced by depolarisation"), frog PIM-1, and human and murine PIM-1 are all known to have serine/threonine protein kinase activity in in vitro phosphorylation assays. The high polypeptide sequence similarity between human, murine and rat PIM-3, frog PIM-1, and human and murine PIM-1, demonstrates that human and murine PIM-3 are a serine/threonine protein kinase.
[0003] Rat PIM-3 is described by Feldman, J. D. et al. (J. Biol. Chem. (1998) 273, 16535-16543). Rat PIM-3 is induced in specific regions of the hippocampus and cortex in response to kainic acid and electroconvulsive shock suggesting that PIM-3 is involved in neuronal function, synaptic plasticity, learning, and memory as well as kainic acid seizures and some nervous system-related diseases such as seizures and epilepsy.
[0004] U.S. Pat. No. 6,143,540, Konietzko, U. et al. (EMBO (1999) 18, 3359-3369) and Eichmann, A. (Oncogene (2000) 19, 1215-1224) also refer to PIM-3 kinase.
SUMMARY OF THE INVENTION
[0005] The present invention provides novel PIM-3 encoding sequences and novel uses of PIM-3.
[0006] The present invention provides a novel human and murine PIM-3 sequence. SEQ ID NO. 1 depicts the DNA sequence and SEQ ID NO. 2 the predicted amino acid sequence of human PIM-3. The open reading frame of SEQ ID NO:1 extends from nucleotide 17 to nucleotide 995 (SEQ ID NO. 3).
[0007] SEQ ID NO. 5 depicts the DNA sequence and SEQ ID NO. 6 the predicted amino acid sequence of murine PIM-3. The open reading frame of SEQ ID NO. 5 extends from nucleotide 199 to nucleotide 1177 (SEQ ID NO. 7).
[0008] The present invention demonstrates that expression of the rat PIM-3 gene is decreased in adipocytes in two independent models of insulin resistance. Treatment with an insulin sensitizer causes an increase in PIM-3 gene expression in murine 3T3-L1 cells. Because human and murine PIM-3 is the species ortholog of rat PIM-3, human and murine PIM-3 is involved in some or all of the processes and diseases in which rat PIM-3 is involved. PIM-3, in particular human and murine PIM-3 is involved in development of insulin resistance. In addition PIM-3, in particular human and murine PIM-3 is involved in development of type 2 diabetes mellitus. In addition, the human and murine PIM-3 paralogs, the PIM-1 proteins, are proto-oncogenes. Consequently, PIM-3, in particular human and murine PIM-3 are involved in cell growth regulation, cancer, and related pathways and diseases.
[0009] The present invention relates to the use of PIM-3 encoding nucleic acid molecules, PIM-3 proteins and protein homologs in a) screening assays for identifying compounds that modulate insulin resistance or type 2 diabetes mellitus; b) detection assays for detecting insulin resistance or type 2 diabetes mellitus (e.g. chromosomal mapping, tissue typing, forensic biology); c) predictive medicine (prediction of insulin resistance or type 2 diabetes mellitus by e.g. diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics).
[0010] The present invention relates in particular to an isolated nucleic acid molecule comprising nucleotide sequence SEQ ID. NO. 1. The present invention further relates to an isolated nucleic acid molecule comprising nucleotide sequence SEQ ID NO. 3.
[0011] The present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from
[0012] a) SEQ ID NO. 5,
[0013] b) SEQ ID NO. 7,
[0014] c) a DNA sequence which hybridize to SEQ ID NO. 5 or SEQ ID NO. 7, and
[0015] d) DNA sequences which are degenerated as a result of the genetic code to sequences defined in a), b) and c) and which encode for a polypeptide of the PIM-3 type.
[0016] The present invention furthermore relates to vectors and host cells comprising the respective DNA sequences or parts thereof.
[0017] The present invention relates to polypeptides having PIM-3 activity, said polypeptides being selected from
[0018] a) a polypeptide having amino acid sequence SEQ ID NO. 6,
[0019] b) a polypeptide that in respect to a) is deficient in one or more amino acids,
[0020] c) a polypeptide in which in respect to a) one or more amino acids are replaced with different amino acids,
[0021] d) a polypeptide in which in respect to a) one or more amino acids are added to the sequence,
[0022] e) a fusion polypeptide comprising an amino acid sequence comprising all or a part of the amino acids of sequence SEQ ID NO. 6. Preferably, not more than 10 amino acids are replaced according to c).
[0023] The present invention relates to the use of PIM-3 as a screening agent for identifying anti-diabetes mellitus drugs, e.g. the use of PIM-3 encoding DNA or a polypeptide having PIM-3 activity for such purpose.
[0024] The present invention further relates to the use of PIM-3 for preparing a medicament for the treatment of insulin resistance or type 2 diabetes mellitus and the use of PIM-3 for predicting insulin resistance or type 2 diabetes mellitus.
DETAILED DESCRIPTION OF THE INVENTION
[0025] PIM-3 protein, which interacts with other cellular proteins, can thus be used as a target for developing therapeutic molecules for modulating PIM-3 protein in cells expressing PIM-3 protein or cells involved in the PIM-3 pathway, e.g., adipocytes. Nucleic acid molecules of the invention can be used to express PIM-3 protein (e.g. via a recombinant expression vector in a host cell in gene therapy applications), to detect PIM-3 mRNA (e.g. in a biological sample) or a genetic lesion in a PIM-3 gene, and to modulate PIM-3 activity.
[0026] PIM-3 proteins can be used to screen drugs or compounds which modulate the PIM-3 activity or expression as well as to treat disorders characterized by insufficient or excessive production of PIM-3 protein or production of PIM-3 protein forms which have decreased or aberrant activity compared to PIM-3 wild type protein.
[0027] The invention provides methods (also referred to herein as a "screening assay") for identifying modulators, i.e. candidate or test compounds or agents (e.g. peptides, peptidomimetics, small molecules or other drugs) which bind to PIM-3 proteins or have a stimulatory or inhibitory effect on, for example, PIM-3 expression or PIM-3 activity.
[0028] In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a PIM-3 protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection.
[0029] In an embodiment, an assay of the present invention is a cell-free assay comprising contacting a PIM-3 protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the PIM-3 protein or biologically active portion thereof. Binding of the test compound to the PIM-3 protein can be determined either directly or indirectly.
[0030] In an other embodiment, the assay includes contacting the PIM-3 protein or biologically active portion thereof with a known compound which binds PIM-3 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a PIM-3 protein, wherein determining the ability of the test compound to interact with a PIM-3 protein comprises determining the ability of the test compound to preferentially bind to PIM-3 or biologically active portion thereof as compared to the known compound.
[0031] In another embodiment, an assay is a cell-free assay comprising contacting PIM-3 protein or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the PIM-3 protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of PIM-3 can be accomplished, for example, by determining the ability of the PIM-3 protein to bind to a PIM-3 target molecule, this means determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of PIM-3 can be accomplished by determining the ability of the PIM-3 protein to further modulate an PIM-3 target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined.
[0032] In another embodiment, the cell-free assay comprises contacting the PIM-3 protein or biologically active portion thereof with a known compound which binds PIM-3 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the PIM-3 protein, wherein determining the ability of the test compound to interact with the PIM-3 protein comprises determining the ability of the PIM-3 protein to preferentially bind to or modulate the activity of a PIM-3 target molecule.
[0033] Phosphoaminoacid analysis of the phosphorylated substrate can also be performed in order to determine which residues on the PIM-3 substrate are phosphorylated. Briefly, the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis. The products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to ninhydrin-stained phosphoaminoacid standards.
[0034] In another embodiment of the invention, the cell free assay determines the ability of the PIM-3 protein to phosphorylate a PIM-3 target molecule by, for example, an in vitro kinase assay. Briefly, a PIM-3 target molecule, e.g., an immunoprecipitated PIM-3 target molecule from a cell line expressing such a molecule, can be incubated with the PIM-3 protein and radioactive ATP.
[0035] In another embodiment, an assay is a cell-based assay in which a cell which expresses a soluble form of PIM-3 protein, or a biologically active portion thereof, is contacted with a test compound and the ability of the test compound to bind to a PIM-3 protein determined. The cell, for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the PIM-3 protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the PIM-3 protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex, e.g. with 125 I, 35 S, 14 C, or 3 H, or enzymatically with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase.
[0036] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a soluble form of PIM-3 protein, or a biologically active portion thereof, with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the PIM-3 protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of PIM-3 or a biologically active portion thereof can be accomplished, for example, by determining the ability of the PIM-3 protein to bind to or interact with an PIM-3 target molecule.
[0037] As used herein, a "target molecule" is a molecule with which an PIM-3 protein binds or interacts in nature, for example, a substrate molecule phosphorylated by PIM-3 protein in the interior of a cell which expresses a PIM-3 protein, the intracellular domains of transmembrane receptors, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A PIM-3 target molecule can be a non-PIM-3 molecule or a PIM-3 protein or polypeptide of the present invention. In one embodiment, a PIM-3 target molecule is a component of a signal transduction pathway, which mediates transduction of a signal.
[0038] In an embodiment, determining the ability of the PIM-3 protein to bind to or interact with a PIM-3 target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a PIM-3-responsive regulatory element operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.
[0039] In various formats of the assay methods of the present invention, it may be desirable to immobilize either PIM-3 or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to PIM-3, or interaction of PIM-3 with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/PIM-3 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads or glutathione derivatized microtitre plates, plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or PIM-3 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly. Alternatively, the complexes can be dissociated from the matrix, and the level of PIM-3 binding or activity determined using standard techniques.
[0040] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either PIM-3 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated PIM-3 or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art. Alternatively, antibodies reactive with PIM-3 or target molecules but which do not interfere with binding of the PIM-3 protein to its target molecule can be derivatized to the wells of the plate, and unbound target or PIM-3 trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the PIM-3 or target molecule, as well as enzyme-linked assays, which rely on detecting an enzymatic activity associated with the PIM-3 or target molecule.
[0041] In another embodiment, modulators of PIM-3 expression are identified in a method in which a cell is contacted with a candidate compound and the expression of PIM-3 mRNA or protein in the cell is determined. The level of expression of PIM-3 mRNA or protein in the presence of the candidate compound is compared to the level of expression of PIM-3 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of PIM-3 expression based on this comparison. For example, when expression of PIM-3 mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of PIM-3 mRNA or protein expression. Alternatively, when expression of PIM-3 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of PIM-3 mRNA or protein expression. The level of PIM-3 mRNA or protein expression in the cells can be determined by methods described herein for detecting PIM-3 mRNA or protein.
[0042] In another aspect of the invention, the PIM-3 proteins or polypeptides thereof can be used as "bait proteins" in a two-hybrid assay or three hybrid assay, to identify other proteins, which bind to or interact with PIM-3 ("PIM-3-binding proteins" or "PIM-3-bp") and modulate PIM-3 activity. Such PIM-3-binding proteins are also likely to be involved in the propagation of signals by the PIM-3 proteins as, for example, upstream or downstream elements of the PIM-3 pathway. The invention also provides for the use of proteins that interact with PIM-3, e.g., two-hybrid interactors with PIM-3, as baits in two-hybrid screens and the identification of PIM-3 interacting protein interacting proteins. PIM-3 interacting protein interacting proteins are likely to be involved in the PIM-3 signal transduction pathway.
[0043] The present invention also provides the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining PIM-3 protein and/or nucleic acid expression as well as PIM-3 activity, in the context of a biological sample (e.g., blood, serum, cells, tissue form the individual, preferably human) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant PIM-3 expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with PIM-3 protein, nucleic acid expression or activity. For example, mutations in a PIM-3 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with PIM-3 protein, nucleic acid expression or activity.
[0044] Another aspect of the invention provides methods for determining PIM-3 protein, nucleic acid expression or PIM-3 activity in an individuals biological sample to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)
[0045] Another aspect of the invention provides monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of PIM-3 in clinical trials.
[0046] An exemplary method for detecting the presence or absence of PIM-3 in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting PIM-3 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes PIM-3 protein such that the presence of PIM-3 is detected in the biological sample. An agent for detecting PIM-3 mRNA or genomic DNA can be a labeled nucleic acid probe capable of hybridizing to PIM-3 mRNA or genomic DNA.
[0047] An agent for detecting PIM-3 protein can be an antibody capable of binding to PIM-3 protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
[0048] The term "biological sample" is intended to include tissues, cells and biological fluids isolated from an individual, as well as tissues, cells and fluids present within an individual. That is, the detection method of the invention can be used to detect PIM-3 mRNA, protein, or genomic DNA in a biological sample e.g. in vitro as well as or in vivo. In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A biological sample is e.g. a biopsy from adipose tissue isolated by conventional means from a subject.
[0049] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting PIM-3 protein, mRNA, or genomic DNA, such that the presence of PIM-3 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of PIM-3 protein, mRNA or genomic DNA in the control sample with the presence of PIM-3 protein, mRNA or genomic DNA in the test sample.
[0050] The invention also encompasses kits for detecting the presence of PIM-3 in a biological sample (a test sample). Such kits can be used to determine if an individual is suffering from or is at increased risk of developing a disorder associated with insulin resistance or type 2 diabetes. For example, the kit can comprise a labeled compound or agent capable of detecting PIM-3 protein or mRNA in a biological sample and means for determining the amount of PIM-3 in the sample (e.g., an anti-PIM-3 antibody or an oligonucleotide probe which binds to DNA encoding PIM-3, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO. 5 or SEQ ID NO. 7).
[0051] The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant PIM-3 expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing insulin resistance or type 2 diabetes. Thus, the present invention provides a method in which PIM-3 protein or nucleic acid (e.g., mRNA, genomic DNA) is detected in a test sample from an individual, wherein the presence of PIM-3 protein or nucleic acid is diagnostic for the individual for having or at risk of developing a disease or disorder associated with aberrant PIM-3 expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
[0052] Furthermore, the prognostic assays can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat insulin resistance or type 2 diabetes. For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease PIM-3 activity).
[0053] Agents, or modulators which have a stimulatory or inhibitory effect on PIM-3 activity (e.g., PIM-3 gene expression) as identified by a screening assay can be used for preparing a pharmaceutical which is useful for treating (prophylactically or therapeutically) disorders (e.g., disorders involving cells or tissues in which PIM-3 is expressed, such as adipocytes) associated with aberrant PIM-3 activity. In conjunction with such treatment, the pharmacogenomics (i.e. the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of PIM-3 protein, expression of PIM-3 nucleic acid, or mutation content of PIM-3 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
[0054] Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of PIM-3 (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials.
[0055] An anti type 2 diabetes agent that modulates PIM-3 protein activity can be an agent, such as a small molecule, e.g., a small molecule that modulates the protein kinase activity of PIM-3, a nucleic acid or a protein, a naturally-occurring cognate ligand of a PIM-3 protein, a peptide, or a PIM-3 peptidomimetic. In one embodiment, the agent stimulates one or more of the biological activities of PIM-3 protein. Examples of such stimulatory agents include small molecules that stimulate one or more activities of PIM-3, e.g., the PIM-3 protein kinase activity, active PIM-3 protein and a nucleic acid molecule encoding PIM-3 that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of PIM-3 protein. Examples of such inhibitory agents include a small molecule that inhibits one or more PIM-3 activities e.g., PIM-3 protein kinase activity, antisense PIM-3 nucleic acid molecules and anti-PIM-3 antibodies.
[0056] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.
EXAMPLES
Example 1
Determination of the Nucleotide Sequence of Human and Murine PIM-3
[0057] Rat PIM-3 nucleotide sequence (AF057026, NM--022602, SEQUENCE ID NO:9) was used to query a proprietary database using the BLASTN program with the BLOSUM62 matrix. This proprietary database is based on a proprietary cDNA library, which is constructed in standard cloning vectors. The most closely related cDNA clones identified by this BLASTN were sequenced. The cDNA sequences were assembled into a contig. The human PIM-3 sequence was determined from the consensus sequence of this contig, further analysis of this contig revealed a cDNA sequence 1977 bp in length. That human PIM-3 cDNA contains a 978 base pair open reading frame predicted to encode a novel 326 amino acid protein. Rat PIM-3 nucleotide sequence was also used to query the public UNIGENE Mouse database using the BLASTN program with the BLOSUM62 matrix. Some closely related EST sequences were identified by this BLASTN. Sequence information of this ESTs was used to screen a mouse embryo cDNA library constructed using the Gene Trapper II Technology (Life Technologies, Karlsruhe, Germany). A cDNA clone was sequenced, and the cDNA sequences were assembled into a contig. The murine PIM-3 sequence was determined from the consensus sequence of this contig, further analysis of this contig and exclusion of an intron sequence revealed a cDNA sequence 2236 bp in length. That murine PIM-3 cDNA contains a 978 base pair open reading frame predicted to encode a novel 326 amino acid protein.
Example 2
Characterization of the Human and the Murine PIM-3 Protein
[0058] In this example, the predicted amino acid sequence of human PIM-3 and the murine PIM-3 protein was compared to amino acid sequences of known motifs and/or domains present in proteins and to the polypeptide sequences of known proteins. Polypeptide domains and/or motifs present in human PIM-3 and murine PIM-3 were identified as were proteins with significant amino acid similarities to human PIM-3 and murine PIM-3. In addition, the molecular weight of the human PIM-3 and the murine PIM-3 protein was predicted.
[0059] The human and the murine nucleotide sequences (SEQ ID NO.1; SEQ ID NO.5), identified as described above, encode a 326 amino acid protein (SEQ ID NO.2 and SEQ ID NO.6). Human and murine PIM-3 has a predicted MW of about 35.9 kDa, respectively, not including post-translational modifications. To check for evidences for a putative kinase activity and for possible posttranslational modification sites, the human and the murine polypeptide sequences of SEQ ID NO.2 and SEQ ID NO.6, respectively, were analyzed using the PROSITE database of protein patterns, as well as using IMPALA and PFAM.
[0060] Searching the PROSITE database revealed the presence of one cAMP and cGMP dependent protein kinase phosphorylation site from amino acids 260-263 of SEQ ID NO.2 and SEQ ID NO.6; three Casein kinase II phosphorylation sites from amino acids 202-205, 211-214 and 321-324 of SEQ ID NO.2 and four Casein kinase II phosphorylation sites from amino acids 202-205, 211-214 299-302 and 321-324 of SEQ ID NO.6; ten N-myristoylation sites from amino acids 43-48, 49-54, 52-57, 57-62, 63-68, 80-85, 98-103, 101-106, 295-300 and 316-321 of SEQ ID NO.2 and SEQ ID NO.6; three Protein kinase C phosphorylation sites from amino acids 137-139, 275-277 and 279-281 of SEQ ID NO.2 and SEQ ID NO.6; one tyrosine kinase phosphorylation site from amino acids 33-40 of SEQ ID NO.2 and SEQ ID NO.6; one protein kinase signature and profile (ATP binding site) from amino acids 46-69 of SEQ ID NO.2 and SEQ ID NO.6; one serine/threonine protein kinase active site signature from amino acids 166-178 of SEQ ID NO.2 and SEQ ID NO.6. The search using IMPALA revealed the presence of one eukaryotic protein kinase domain from amino acid 40-293 (SEQ ID NO.4 and SEQ ID NO.8, respectively), of SEQ ID NO.2 and SEQ ID NO.6; with a score of 186 bits and Expect value of 8e-49 and with a score of 184 bits and Expect value of 4e-48, respectively. The search using PFAM revealed also the presence of one eukaryotic protein kinase domain from amino acid 40-293, of SEQ ID NO.2 and SEQ ID NO.6; with a score of 262,5 and E-value of 5.7e-75 and with a score of 261,1 bits and E-value of 1.5e-74, respectively.
[0061] The human, murine and rat PIM-3 polypeptide sequences (SEQ ID NO.2, SEQ ID NO.6 and SEQ ID NO.10) were aligned in a pair wise Clustal W alignment analysis using blosum as the protein weight matrix. Thereby, human PIM-3 was found to be 95% identical to murine and rat PIM-3 (AF057026, NM--022602, SEQ ID NO.10) with a score of 2011, murine PIM-3 was found to be 99% identical to rat PIM-3 (AF057026, NM--022602, SEQ ID NO.10) with a score of 2074.
[0062] The human and the murine PIM-3 polypeptide sequences of SEQ ID NO.2 and of SEQ ID NO.6 was also used to query the Swissprot database of protein sequences using the BLASTP program with the BLOSUM62 matrix. The four most closely related proteins to human and murine PIM-3 identified by this BLASTP analysis are listed: Human and murine PIM-3 was found to be 76% identical to Xenopus laevis (frog) PIM-1 (Q91822; SEQ ID NO.11) with a score of 518, 72% identical to rat PIM-1 (P26794; SEQ ID NO.12) with a score of 442, 71% identical to human PIM-1 (P11309; SEQ ID NO.13) with a score of 441 and 71% identical to murine PIM-1 (P06803; SEQ ID NO.14) with a score of 436 and 438, respectively.
Example 3
Gene Expression of PIM-3 in an In Vivo Model of Insulin Resistance and Type 2 Diabetes Mellitus
[0063] Zucker diabetic fatty (ZDF) rats are well known animal model carrying a homozygous defect in the leptin receptor fa gene. This rat strain develops age dependent an insulin resistant/hyperinsulinaemic state which than progresses to overt type 2 diabetes mellitus/hyperglycaemic state. To identify genes the expression of which is either induced or repressed and so may contribute or mark the development of insulin resistance or type 2 diabetes mellitus, gene expression profiles of ZDF rats and their lean heterozygous control littermates were collected using oligonucleotide array based profiling technique.
Material and Methods
[0064] Zucker Diabetic Fatty (ZDF/Gmi®-fa/fa) male rats as well as their lean male fa+/fa- counterparts, used as healthy controls, aged 6, 8 and 13 weeks were obtained from Genetic Models Inc. (Indianapolis, Ind., US).
[0065] Before the animals were included into the study they were kept under standard animal house conditions for one week. For collection of epididymal fat tissue and blood samples the animals were killed by cervical dislocation. 6 ZDF rats and 6 lean fa+/fa- littermates per age group were used for gene expression analysis as described below.
Tissue Collection and RNA Isolation
[0066] Following cervical dislocation, epididymal fat pads were surgically removed, portioned and quickly transferred in suitable tubes containing sufficient volumes of RNA later (Ambion, Tex., US). Samples from each animal were stored individually. Long term storage of the samples was performed at minus 80°C.
[0067] Total cellular RNA was extracted from adipose tissue using Rneasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturers recommendations for RNA isolation from fat tissue. RNA was eluted twice in 50.mul RNAse free water, RNA concentration was determined spectroscopically (A260).
[0068] For further purification, RNA solution was filled up to 100 μl total volume with RNase free water. RNA clean up was performed according to the manufacturers instructions using Qiagen Rneasy Mini kit. RNA was eluted twice in 50 μl RNase free water, RNA concentration was determined spectroscopically (A260).
[0069] For concentration of the RNA eluate, NH4 acetate and ethanol were added, and RNA was precipitated overnight in an ethanol-dry ice bath. RNA was collected by centrifugation at maximum speed at 4°C. Pelleted RNA was washed twice with 80% ethanol, air dried and dissolved in a small volume of RNAse free water. Rnase concentration was determined spectroscopically (A260).
Gene Expression Profiling
[0070] The general use of oligonucleotides arrays for gene expression monitoring has been described in U.S. Pat. No. 6,177,248. In our practical application, the used microarrays contain desoxynucleotide sequences that represent approximately 8000 known genes or EST clusters. Each gene or EST sequence is represented by up to 20 pairs of oligonucleotides, each pair consisting of one oligo that matches to a segment of the transcript, and a control oligo that contains a centrally located 1 bp mismatch. For rat, 3 arrays (RG U34A, RG U34B and RG U34C) representing approximately 24000 gene and EST sequences in total, derived from a database of known genes or EST sequences are provided by Affymetrix, Santa Clara, Calif., US.
cRNA Preparation for Hybridization
[0071] RNA was obtained from epididymal fat as described above. An oligo dT primer containing a T7 promotor site was added to total cellular RNA (10μg)+-. After annealing of the primer, the RNA was subsequently reverse transcribed using Superscript choice reverse transcriptase, following the manufacturers instructions. After extraction with phenol:chloroform:isoamylalcohol, using phase lock gel tubes (Eppendorf, Hamburg, Germany), and ethanol precipitation, the cDNA was collected by centrifugation and washed twice with 80% ethanol. The pellet was dissolved in Rnase free water and transcribed into biotinylated cRNA using the Enzo High Yield labeling transcription kit (Enzo Diagnostics, Farmingdale N.Y., US) or MEGAscript T7 high yield transcription kit (Ambion, Austin, Tex., US) according the manufacturers instructions. For the latter application, biotin labeled UTP and CTP (Sigma, Munich, Germany) plus unlabelled ATP and GTP was used in a molar ratio of 1:3 (labeled vs unlabeled). cRNA was then precipitated and washed as described above. Finally, the precipitated, air dried cRNA was dissolved in a small volume of RNAse free water. RNA concentration was determined spectroscopically (A.sub260), and size distribution was checked by agarose gelelectrophoresis. Subsequently, cRNA was hydrolized to an average size of 50 nucleotides in length by incubating for 25 minutes in 40 mM Tris-acetate pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate at 94°C. 20 μg of the fragmented cRNA was used to set up of a hybridisation cocktail according to the instructions of Affymetrix. Prior hybridization, RNA samples were heated in the hybridization cocktails to 99°C. for 10 min, placed on ice for 5 min, and allowed to equilibrate to room temperature before being placed in the hybridization flow cell. The hybridization cocktail was then hybridized to a microarray at 45° C. and 60 rpm in a hybridization oven overnight. After hybridization, the hybridization cocktail was removed and stored at minus 80° C.) for further use. The arrays were washed and stained in a Affymetrix fluidics station using the phycoerythrin-steptavidin-antibody amplification protocol EukWS2 according to the manufacturers instructions. Data were collected using a scanning confocal microscope made for Affymetrix by Hewlett Packard (Commercially available through Affymetrix, Santa Clara, Calif., US.). The scanner uses an argon ion laser as the excitation source, with the emission detected by a photomultiplier tube through either a 530 nm band pass filter (fluorescein) or a 560 nm longpass filter (phycoerythrin).
Quantitative Analysis of Hybridization Patterns and Intensities
[0072] Following a quantitative scan of an array, a grid is aligned to the image using the known dimensions of the array and the corner control regions as markers. The image is reduced to a simple text file containing position and intensity information using software developed at Affymetrix (available with the confocal scanner). This information is merged with another text file that contains information relating physical position on the array to probe sequence and the identity of the RNA (and the specific part of the RNA) for which the oligonucleotide probe is designed. The quantitative analysis of the hybridization results involves a simple form of pattern recognition based on the assumption that, in the presence of a specific RNA, the PM (PM means "perfect match" in U.S. Pat. No. 6,177,248) probes will hybridize more strongly on average than their MM (MM means "mismatch" in U.S. Pat. No. 6,177,248) partners. The number of instances in which the PM hybridization signal is larger than the MM signal is computed along with average of the logarithm of the PM/MM ratios for each probe set. These values are used to make a decision (using a predefined decision matrix) concerning the presence or absence of an RNA. To determine the quantitative RNA abundance, the average of the differences (PM minus MM) for each probe family is calculated, The advantage of the difference method is that signals from random cross-hybridization contribute equally, on average, to the PM and MM probes, while specific hybridization contributes more to the PM probes. By averaging the pairwise differences, the real signals add constructively while the contributions form cross-hybridization tend to cancel. When assessing the differences between two different RNA samples, the hybridization signals from side-by-side experiments on identically synthesized arrays are compared directly. The magnitude of the changes in the average of the difference (PM-MM) values is interpreted by comparison with the results of spiking experiments as well as the signals observed for the internal standard bacterial and phage RNAs spiked into each sample at a known amount. Data analysis programs developed at Affymetrix perform these operations automatically.
TABLE-US-00001 TABLE 1 ZDF rats, 7 weeks old ZDF # 1 ZDF # 2 ZDF # 3 ZDF # 4 ZDF # 5 vs control vs control vs control vs control vs control Comparison #1 #2 #3 #4 #5 Fold change -4.3 -4.8 -3.1 -5.3 -4.6
TABLE-US-00002 TABLE 2 ZDF rats, 9 weeks old ZDF 7 vs ZDF 8 vs ZDF 9 vs ZDF 10 vs ZDF 11 vs control control control control control Comparison #7 #8 #9 #10 #11 Fold change -5.4 -3.3 -2.9 -1.7 -1.3
TABLE-US-00003 TABLE 3 ZDF rats, 14 weeks old ZDF 13 vs ZDF 14 vs ZDF 15 vs ZDF 16 vs ZDF 17 vs control control control control control Comparison #13 #14 #15 #16 #17 Fold change 1 -1.7 -3.0 -2.4 -1.7
Example 4
Gene Expression of PIM-3 in an In Vitro Model of Insulin Resistance
[0073] Adipocytes were isolated from epididymal fat pads of 160-180 g male Sprague Dawley rats and incubated as described (Muller, G. and Wied, S., Diabetes (1993) 42: 1852-1867). Briefly, adipocytes were isolated from the pooled epididymal fat of 20 male rats. The isolated adipocytes were split into two pools. One pool was made insulin resistant by incubating the cells for 5 hours in medium containing 25 mM D-glucose plus 10 nM insulin.
[0074] The second pool was incubated for 5 h in medium containing 5 mM D-glucose, which kept the cells in the insulin sensitive state. At the end of the incubation insulin sensitivity and insulin resistance where checked by measuring insulin dependent glucose uptake with an aliquot of the two pools as described (Muller, G. and Wied, S. Diabetes (1993) 42: 1852-1867). The majority of the adipocytes was used for isolating RNA as described in example 3.
[0075] Subsequently RNA was used for gene expression monitoring using Affymetrix technology as described in example 3. Fold changes of gene expression for PIM-3 were analyzed using the Affimetrix qualifier AF086624_S_AT. The results of these analysis are summarized in table 4:
TABLE-US-00004 TABLE 4 In vitro insulin resistant adipocytes, 5 h incubated Resistant Resistant Resistant Resistant Resistant 1 vs 2 vs 3 vs 4 vs 5 vs Comparison control 1 control 2 control 3 control 4 control 5 Fold change -2.4 -2.7 -4.0 -2.1 -2.4
Experiments 5
Gene Expression of PIM-3 in 3T3-L1 Adipocytes Treated with Antidiabetic Drugs (PPARγ Agonists)
Material and Methods
[0076] 3T3-L1 preadipocytes were grown at 37° C. in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 1 g/l glucose and 10% fetal calf serum (FCS). For differentiation into mature adipocytes, confluent preadipocytes were cultured for four days in DMEM supplemented with 4.5 g/l glucose, 10% FCS, 50 μg/ml ascorbic acid, 1 μM biotin, 17 μM pantothenic acid (=basal medium), 500 μM 3-isobutylmethylxanthine, 0.25 μM dexamethasone and 1 μg/ml human recombinant insulin. During the four day treatment the medium was changed once. Finally, 3T3-L1 cells were treated for three days with basal medium containing 1 μg/ml insulin whereupon approximately 90% of the cells were converted into adipocytes.
[0077] Differentiated 3T3-L1 cells were maintained in basal medium for one additional day and subsequently kept in serum-free basal medium for four hours. Then, PPARγ agonists diluted in basal medium were added to the adipocytes as described below (concentrations and conditions see table 5):
TABLE-US-00005 TABLE 5 duration of probe no. compound* concentration treatment 1 rosiglitazone 1 μM 6 hours 2 rosiglitazone 5 μM 6 hours 3 troglitazone 1 μM 6 hours 4 troglitazone 5 μM 6 hours 5 DMSO control 0.02% (v/v) 6 hours 6 rosiglitazone 1 μM 24 hours 7 rosiglitazone 5 μM 24 hours 8 troglitazone 1 μM 24 hours 9 troglitazone 5 μM 24 hours 10 DMSO control 0.02% (v/v) 24 hours 11 rosiglitazone 1 μM 48 hours 12 rosiglitazone 5 μM 48 hours 13 troglitazone 1 μM 48 hours 14 troglitazone 5 μM 48 hours 15 DMSO control 0.02% (v/v) 48 hours *5 mM and 25 mM stock solutions of rosiglitazone and troglitazone dissolved in DMSO were made and diluted 5000fold to the final concentrations (1 μM/5 μM) in the culture medium. For the controls, DMSO without compound was equally diluted 5000fold to a final concentration of 0.02% (v/v).
[0078] RNA was isolated using the TRIzol reagent (Life Technologies, Karlsruhe, Germany) and treated with DNase I by applying the DNA-free kit (Ambion, Austin, Tex., US). Further purification of the RNA was achieved using the RNeasy mini kit (Qiagen, Hilden, Germany) and quality/quantity control was done with the 2100 Bioanalyzer (Agilent, Boblingen, Germany). 10 μg of total RNA was converted into biotinylated cRNA according to the GeneChip expression analysis technical manual (Affymetrix, Santa Clara, Calif., US). Briefly, first and second strand synthesis was performed by applying the SuperScript double stranded cDNA synthesis kit (Life Technologies, Karlsruhe, Germany) and biotin labeled cRNA was produced with the BioArray RNA transcript labeling kit (Enzo Diagnostics, Framingdale, N.Y., US). 10 μg of cRNA was fragmented by heat, added to the GeneChip eukaryotic hybridization control solution (Affymetrix) and hybridized to a GeneChip MG-U74Av2 array (Affymetrix) by rotating for 16 hours at 45° C. Washing, staining and scanning of the array was carried out with standard procedures using the hardware provided by Affymetrix. Raw data was analyzed by applying the microarray suite version 4.0.1 software (Affymetrix, see above).
[0079] The entire experiment was repeated twice to provide three biological replicates.
Data Analysis
[0080] Data analysis including the estimation of fold changes was performed as described above. Fold change values for PIM-3 were obtained by comparing the compound treated samples against the untreated controls for each time point. Therefore, Affymetrix qualifier 96841_AT was used.
[0081] The results are summarized in table 6 a)-c).
TABLE-US-00006 TABLE 6 a) 6 hours 24 hours 48 hours Rosi Rosi Tro Tro Rosi Rosi Tro Tro Rosi Rosi Tro Tro 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM comparison 1/5 2/5 3/5 4/5 6/10 7/10 8/10 9/10 11/15 12/15 13/15 14/15 Fold change 2.1 2.2 1.7 2.1 2.9 2.9 1.8 2.7 2.6 2.6 1.8 2.4
TABLE-US-00007 TABLE b) 6 hours 24 hours 48 hours Rosi Rosi Tro Tro Rosi Rosi Tro Tro Rosi Rosi Tro Tro 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM comparison 1/5 2/5 3/5 4/5 6/10 7/10 8/10 9/10 11/15 12/15 13/15 14/15 Fold change 1.5 1.3 1.7 1.7 2.2 1.9 1.6 1.9 2.0 1.9 1.6 1.9
TABLE-US-00008 TABLE c) 6 hours 24 hours 48 hours Rosi Rosi Tro Tro Rosi Rosi Tro Tro Rosi Rosi Tro Tro 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM 1 μM 5 μM comparison 1/5 2/5 3/5 4/5 6/10 7/10 8/10 9/10 11/15 12/15 13/15 14/15 Fold change 2.0 2.2 1.8 2.3 1.9 1.9 1.6 2.0 2.1 2.0 1.6 2.3 "Rosi" stands for "rosiglitazone", "Tro" stands for "troglitazone".
Example 6
Gene Expression of Pim-3 in ZDF Rats Treated with Anti-Diabetic Drugs (PPARγ Agonists)
[0082] Zucker Diabetic Fatty (ZDF/Gmi®-fa/fa) male rats as well as their lean fa+/fa- counterparts, aged 5 weeks (control 1-5, ZDF 1-10) were obtained from Genetic Models Inc. (Indianapolis, Ind.). The rats were given free access to food and water. The ZDF animals were split into 2 groups: group 1 (animals ZDF 1-5) was not treated with any agent, whereas the second group was treated for 14 weeks with the antidiabetic drug Rosiglitazone (3 mg/kg/day, animals 6-10, Rosi 1-5.). For the collection of epididymal fat pads and also blood samples, in each case 5 animals were sacrificed by cervical dislocation. To monitor the success of the antidiabetic treatment, HbA1c as marker of long-term blood glucose levels were measured by standard methods after finishing of the experiment. The results of these measurements are summarized in table 7:
TABLE-US-00009 TABLE 7 animal C1 C2 C3 C4 C5 Z1 Z2 Z3 Z4 Z5 R1 R2 R3 R4 R5 HbAlc (%) 4.20 4.28 4.21 4.16 4.09 5.12 8.01 7.59 8.71 9.51 4.30 4.27 4.14 4.14 4.13 % HbAlc in different rats: control (x) rats (c), ZDF rats untreated (Z), ZDF rats Rosiglitazone treated (R)
[0083] Collection of epididymal adipose tissue, RNA isolation and Affymetrix experiments were performed as described in experiment 1. Data analysis included the comparison of expression data of samples derived from the lean control animals versus the untreated ZDF rats as well as the comparison of the expression data derived from Rosiglitazone treated ZDF animals versus non-treated ZDF animals. Data analysis including the estimation of fold changes and weighing of the statistical significance of those fold changes was performed using a proprietary software developed in Aventis pharmaceuticals. Fold changes of gene expression for Pim-3 were analyzed using the Affymetrix qualifier AF086624_S_AT. The results of those analysis are summarized in the following tables 8a und 8b:
TABLE-US-00010 TABLE 8a ZDF rats, 20 weeks old ZDF 1 vs ZDF 2 vs ZDF 3 vs ZDF 4 vs ZDF 5 vs control control control control control comparison #1 #2 #3 #4 #5 Fold change -1.5 -1.8 -1.6 1 -1.7
TABLE-US-00011 TABLE 8b ZDF rats Rosiglitazone treated, comparison to untreated ZDF rats, 20 weeks old Rosi 1 Rosi 2 Rosi 3 Rosi 4 Rosi 5 comparison vs ZDF 1 vs ZDF 2 vs ZDF 3 vs ZDF 4 vs ZDF 5 Fold change 1.6 1.3 1.6 1.5 1.4
Sequence CWU
1
1
1411973DNAHomo sapiens 1gagggccgtc gcccgcgatg ctgctctcca agttcggctc
cctggcgcac ctctgcgggc 60ccggcggcgt ggaccacctc ccggtgaaga tcctgcagcc
agccaaggcg gacaaggaga 120gcttcgagaa ggcgtaccag gtgggcgccg tgctgggtag
cggcggcttc ggcacggtct 180acgcgggtag ccgcatcgcc gacgggctcc cggtggctgt
gaagcacgtg gtgaaggagc 240gggtgaccga gtggggcagc ctgggcggcg cgaccgtgcc
cctggaggtg gtgctgctgc 300gcaaggtggg cgcggcgggc ggcgcgcgcg gcgtcatccg
cctgctggac tggttcgagc 360ggcccgacgg cttcctgctg gtgctggagc ggcccgagcc
ggcgcaggac ctcttcgact 420ttatcacgga gcgcggcgcc ctggacgagc cgctggcgcg
ccgcttcttc gcgcaggtgc 480tggccgccgt gcgccactgc cacagctgcg gggtcgtgca
ccgcgacatt aaggacgaaa 540atctgcttgt ggacctgcgc tccggagagc tcaagctcat
cgacttcggt tcgggtgcgc 600tgctcaagga cacggtctac accgacttcg acggcacccg
agtgtacagc cccccggagt 660ggatccgcta ccaccgctac cacgggcgct cggccaccgt
gtggtcgctg ggcgtgcttc 720tctacgatat ggtgtgtggg gacatcccct tcgagcagga
cgaggagatc ctccgaggcc 780gcctgctctt ccggaggagg gtctctccag agtgccagca
gctgatccgg tggtgcctgt 840ccctgcggcc ctcagagcgg ccgtcgctgg atcagattgc
ggcccatccc tggatgctgg 900gggctgacgg gggcgccccg gagagctgtg acctgcggct
gtgcaccctc gaccctgatg 960acgtggccag caccacgtcc agcagcgaga gcttgtgagg
agctgcacct gactgggagc 1020taggggacca cctgccttgg ccagacctgg gacgccccca
gaccctgact ttttcctgcg 1080tgggccgtct cctcctgcgg aagcagtgac ctctgacccc
tggtgacctt cgctttgagt 1140gccttttgaa cgctggtccc gcgggacttg gttttctcaa
gctctgtctg tccaaagacg 1200ctccggtcga ggtcccgcct gccctgggtg gatacttgaa
ccccagacgc ccctctgtgc 1260tgctgtgtcc ggaggcggcc ttcccatctg cctgcccacc
cggagctctt tccgccggcg 1320cagggtccca agcccacctc ccgccctcag tcctgcggtg
tgcgtctggg cacgtcctgc 1380acacacaatg caagtcctgg cctccgcgcc cgcccgccca
cgcgagccgt acccgccgcc 1440aactctgtta tttatggtgt gaccccctgg aggtgccctc
ggcccaccgg ggctatttat 1500tgtttaattt atttgttgag gttatttcct ctgagcagtc
tgcctctccc aagccccagg 1560ggacagtggg gaggcagggg agggggtggc tgtggtccag
ggaccccagg ccctgattcc 1620tgtgcctggc gtctgtcctg gccccgcctg tcagaagatg
aacatgtata gtggctaact 1680taaggggagt gggtgaccct gacacttcca ggcactgtgc
ccagggtttg ggttttaaat 1740tattgacttt gtacagtctg cttgtgggct ctgaaagctg
gggtggggcc agagcctgag 1800cgtttaattt attcagtacc tgtgtttgtg tgaatgcggt
gtgtgcaggc atcgcagatg 1860ggggttcttt cagttcaaaa gtgagatgtc tggagatcat
atttttttat acaggtattt 1920caattaaaat gtttttgtac atagtggaaa aaaaaaaaaa
aagggcggcc gcc 19732326PRTHomo sapiens 2Met Leu Leu Ser Lys Phe
Gly Ser Leu Ala His Leu Cys Gly Pro Gly 1 5
10 15 Gly Val Asp His Leu Pro Val Lys Ile Leu Gln
Pro Ala Lys Ala Asp 20 25
30 Lys Glu Ser Phe Glu Lys Ala Tyr Gln Val Gly Ala Val Leu Gly
Ser 35 40 45 Gly
Gly Phe Gly Thr Val Tyr Ala Gly Ser Arg Ile Ala Asp Gly Leu 50
55 60 Pro Val Ala Val Lys His
Val Val Lys Glu Arg Val Thr Glu Trp Gly 65 70
75 80 Ser Leu Gly Gly Ala Thr Val Pro Leu Glu Val
Val Leu Leu Arg Lys 85 90
95 Val Gly Ala Ala Gly Gly Ala Arg Gly Val Ile Arg Leu Leu Asp Trp
100 105 110 Phe Glu
Arg Pro Asp Gly Phe Leu Leu Val Leu Glu Arg Pro Glu Pro 115
120 125 Ala Gln Asp Leu Phe Asp Phe
Ile Thr Glu Arg Gly Ala Leu Asp Glu 130 135
140 Pro Leu Ala Arg Arg Phe Phe Ala Gln Val Leu Ala
Ala Val Arg His 145 150 155
160 Cys His Ser Cys Gly Val Val His Arg Asp Ile Lys Asp Glu Asn Leu
165 170 175 Leu Val Asp
Leu Arg Ser Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser 180
185 190 Gly Ala Leu Leu Lys Asp Thr Val
Tyr Thr Asp Phe Asp Gly Thr Arg 195 200
205 Val Tyr Ser Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr
His Gly Arg 210 215 220
Ser Ala Thr Val Trp Ser Leu Gly Val Leu Leu Tyr Asp Met Val Cys 225
230 235 240 Gly Asp Ile Pro
Phe Glu Gln Asp Glu Glu Ile Leu Arg Gly Arg Leu 245
250 255 Leu Phe Arg Arg Arg Val Ser Pro Glu
Cys Gln Gln Leu Ile Arg Trp 260 265
270 Cys Leu Ser Leu Arg Pro Ser Glu Arg Pro Ser Leu Asp Gln
Ile Ala 275 280 285
Ala His Pro Trp Met Leu Gly Ala Asp Gly Gly Ala Pro Glu Ser Cys 290
295 300 Asp Leu Arg Leu Cys
Thr Leu Asp Pro Asp Asp Val Ala Ser Thr Thr 305 310
315 320 Ser Ser Ser Glu Ser Leu
325 3978DNAHomo sapiens 3atgctgctct ccaagttcgg ctccctggcg cacctctgcg
ggcccggcgg cgtggaccac 60ctcccggtga agatcctgca gccagccaag gcggacaagg
agagcttcga gaaggcgtac 120caggtgggcg ccgtgctggg tagcggcggc ttcggcacgg
tctacgcggg tagccgcatc 180gccgacgggc tcccggtggc tgtgaagcac gtggtgaagg
agcgggtgac cgagtggggc 240agcctgggcg gcgcgaccgt gcccctggag gtggtgctgc
tgcgcaaggt gggcgcggcg 300ggcggcgcgc gcggcgtcat ccgcctgctg gactggttcg
agcggcccga cggcttcctg 360ctggtgctgg agcggcccga gccggcgcag gacctcttcg
actttatcac ggagcgcggc 420gccctggacg agccgctggc gcgccgcttc ttcgcgcagg
tgctggccgc cgtgcgccac 480tgccacagct gcggggtcgt gcaccgcgac attaaggacg
aaaatctgct tgtggacctg 540cgctccggag agctcaagct catcgacttc ggttcgggtg
cgctgctcaa ggacacggtc 600tacaccgact tcgacggcac ccgagtgtac agccccccgg
agtggatccg ctaccaccgc 660taccacgggc gctcggccac cgtgtggtcg ctgggcgtgc
ttctctacga tatggtgtgt 720ggggacatcc ccttcgagca ggacgaggag atcctccgag
gccgcctgct cttccggagg 780agggtctctc cagagtgcca gcagctgatc cggtggtgcc
tgtccctgcg gccctcagag 840cggccgtcgc tggatcagat tgcggcccat ccctggatgc
tgggggctga cgggggcgcc 900ccggagagct gtgacctgcg gctgtgcacc ctcgaccctg
atgacgtggc cagcaccacg 960tccagcagcg agagcttg
9784254PRTArtificialRecombinant Plasmid 4Tyr Gln
Val Gly Ala Val Leu Gly Ser Gly Gly Phe Gly Thr Val Tyr 1 5
10 15 Ala Gly Ser Arg Ile Ala Asp
Gly Leu Pro Val Ala Val Lys His Val 20 25
30 Val Lys Glu Arg Val Thr Glu Trp Gly Ser Leu Gly
Gly Ala Thr Val 35 40 45
Pro Leu Glu Val Val Leu Leu Arg Lys Val Gly Ala Ala Gly Gly Ala
50 55 60 Arg Gly Val
Ile Arg Leu Leu Asp Trp Phe Glu Arg Pro Asp Gly Phe 65
70 75 80 Leu Leu Val Leu Glu Arg Pro
Glu Pro Ala Gln Asp Leu Phe Asp Phe 85
90 95 Ile Thr Glu Arg Gly Ala Leu Asp Glu Pro Leu
Ala Arg Arg Phe Phe 100 105
110 Ala Gln Val Leu Ala Ala Val Arg His Cys His Ser Cys Gly Val
Val 115 120 125 His
Arg Asp Ile Lys Asp Glu Asn Leu Leu Val Asp Leu Arg Ser Gly 130
135 140 Glu Leu Lys Leu Ile Asp
Phe Gly Ser Gly Ala Leu Leu Lys Asp Thr 145 150
155 160 Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr
Ser Pro Pro Glu Trp 165 170
175 Ile Arg Tyr His Arg Tyr His Gly Arg Ser Ala Thr Val Trp Ser Leu
180 185 190 Gly Val
Leu Leu Tyr Asp Met Val Cys Gly Asp Ile Pro Phe Glu Gln 195
200 205 Asp Glu Glu Ile Leu Arg Gly
Arg Leu Leu Phe Arg Arg Arg Val Ser 210 215
220 Pro Glu Cys Gln Gln Leu Ile Arg Trp Cys Leu Ser
Leu Arg Pro Ser 225 230 235
240 Glu Arg Pro Ser Leu Asp Gln Ile Ala Ala His Pro Trp Met
245 250 52236DNAMus musculus
5tgacactata gaaggtacgc ctgcaggtac cggtccggaa ttcccgggtc gacccacgcg
60tccgcgccgc gcggctgccc cgctgagcgc tcggcctcgg ggccgtggga tccgccgcgc
120tgtctgcggt caggaagacc gccctcccgc gtccttgccg gacgggtcag aggcggcacc
180gcacgcgagg ccacccgcga tgctgctgtc caagttcggc tccctggcgc acctctgcgg
240gcctggcggc gtggaccacc tcccagtgaa gatcctacag ccagccaagg ctgacaagga
300gagcttcgag aaggtgtacc aggtgggcgc cgtgctgggc agcggcggct tcggcacggt
360ctacgcgggc agccgcatcg ccgacggact cccggtggct gtgaagcacg tggtgaagga
420gcgggtgacc gagtggggca gtctcggcgg agtggccgtg cccctggagg tggtgctgct
480gcgcaaggtg ggcgcggcgg gcggcgcgcg cggcgtcatc cgcttgctgg actggttcga
540gcggcccgac ggcttcttgt tggtgctgga gcgacccgag ccggcacagg acctcttcga
600cttcatcact gaacgaggcg ccctggacga gccgctggcg cgtcgcttct tcgcgcaggt
660gcttgccgct gtgcggcact gccacaattg tggggtcgtg caccgcgaca tcaaggacga
720gaacctgctg gtggacctgc gctcgggaga gctgaagctc atcgacttcg gctcgggcgc
780ggtgctcaag gacacggtct acactgactt tgatggcacc cgtgtgtaca gccccccaga
840gtggatccga tatcaccgat atcacgggcg gtctgccact gtgtggtctc tgggtgtact
900gctctacgac atggtgtgtg gggacattcc ctttgagcag gatgaggaga tcttgcgcgg
960caggctcttt ttccggagga gggtctcccc agagtgccag cagcttattg agtggtgtct
1020ctccctgagg ccctcagaga ggccctccct ggaccaaatt gctgcccacc cctggatgct
1080ggggacagag gggagcgttc cagagaactg tgaccttcgg ctttgtgccc tggatactga
1140cgacggagcc agtaccactt ccagcagtga gagcttgtga ggaggagaag gggcctgggc
1200tcggcctagc cagcgctctc ccagaattga acactttctg cctgggatgt ctgctgcaaa
1260agcagtgacc tctgacccct ggtgaccttt gctctcggca ccgggcctgt ttcctttgct
1320ttgagtgcct ttttgaacgc tgctccacag ggcctgggtt ttcttgagct cttctgtcca
1380aagatggctg cgggctaagc aaggtcctgc cctgggtgga tacttgaacc agagatcccg
1440accctgctgc tccatctcag gaggcagcct tcctgaccaa gtgtgtttga catggagcgc
1500cctgtggtgc ccacctccaa ccctccagtc tcctggtgtt catctgggca tgtctgcaca
1560agcaatgcaa cgctgggcca ctgctgcccg tctgcctccc cggcacggca cggctccgca
1620cgcaacctaa gcgtgccacc acggtctctt atttatggtg tgatcaccct ggagggcgcc
1680cccgccctgc tggggctatt tattgtttaa tttatttgct gaggttcctc caagcaacca
1740ccttctccag gcccctgggg tgttgaaagt caaatgtggc tgttgagtcc acagaccccc
1800atcctaattc ctgcacctgg aggagttccc caacccccgt gtttgcggga ggaagcattt
1860gtacagtggc taatttaagg ggagtgggag accctgtcac cctgagcact ctgcgctggg
1920gaggggttta aattattgac cttgtacagt ctgcttgctg gctctgaaag ctggggttgg
1980gggacagagt ctcaagccct taatttattt tagcagctgt gtttctgtga ccctggtgtg
2040actaagcatc aggggtgggg ttgtataagt tcaaaagtgt gaaatgtctg aagatcatat
2100tttttataca ggtatttcaa ttaaatgttt tggtatataa aaaaaaaaaa aaaaaaaaaa
2160aagggcggcc gctctagagg atccctcgag gggcccaagc ttacgcgtgc atgcgacgtc
2220atagctctct ccctat
22366326PRTMus musculus 6Met Leu Leu Ser Lys Phe Gly Ser Leu Ala His Leu
Cys Gly Pro Gly 1 5 10
15 Gly Val Asp His Leu Pro Val Lys Ile Leu Gln Pro Ala Lys Ala Asp
20 25 30 Lys Glu Ser
Phe Glu Lys Val Tyr Gln Val Gly Ala Val Leu Gly Ser 35
40 45 Gly Gly Phe Gly Thr Val Tyr Ala
Gly Ser Arg Ile Ala Asp Gly Leu 50 55
60 Pro Val Ala Val Lys His Val Val Lys Glu Arg Val Thr
Glu Trp Gly 65 70 75
80 Ser Leu Gly Gly Val Ala Val Pro Leu Glu Val Val Leu Leu Arg Lys
85 90 95 Val Gly Ala Ala
Gly Gly Ala Arg Gly Val Ile Arg Leu Leu Asp Trp 100
105 110 Phe Glu Arg Pro Asp Gly Phe Leu Leu
Val Leu Glu Arg Pro Glu Pro 115 120
125 Ala Gln Asp Leu Phe Asp Phe Ile Thr Glu Arg Gly Ala Leu
Asp Glu 130 135 140
Pro Leu Ala Arg Arg Phe Phe Ala Gln Val Leu Ala Ala Val Arg His 145
150 155 160 Cys His Asn Cys Gly
Val Val His Arg Asp Ile Lys Asp Glu Asn Leu 165
170 175 Leu Val Asp Leu Arg Ser Gly Glu Leu Lys
Leu Ile Asp Phe Gly Ser 180 185
190 Gly Ala Val Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr
Arg 195 200 205 Val
Tyr Ser Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr His Gly Arg 210
215 220 Ser Ala Thr Val Trp Ser
Leu Gly Val Leu Leu Tyr Asp Met Val Cys 225 230
235 240 Gly Asp Ile Pro Phe Glu Gln Asp Glu Glu Ile
Leu Arg Gly Arg Leu 245 250
255 Phe Phe Arg Arg Arg Val Ser Pro Glu Cys Gln Gln Leu Ile Glu Trp
260 265 270 Cys Leu
Ser Leu Arg Pro Ser Glu Arg Pro Ser Leu Asp Gln Ile Ala 275
280 285 Ala His Pro Trp Met Leu Gly
Thr Glu Gly Ser Val Pro Glu Asn Cys 290 295
300 Asp Leu Arg Leu Cys Ala Leu Asp Thr Asp Asp Gly
Ala Ser Thr Thr 305 310 315
320 Ser Ser Ser Glu Ser Leu 325 7978DNAMus musculus
7atgctgctgt ccaagttcgg ctccctggcg cacctctgcg ggcctggcgg cgtggaccac
60ctcccagtga agatcctaca gccagccaag gctgacaagg agagcttcga gaaggtgtac
120caggtgggcg ccgtgctggg cagcggcggc ttcggcacgg tctacgcggg cagccgcatc
180gccgacggac tcccggtggc tgtgaagcac gtggtgaagg agcgggtgac cgagtggggc
240agtctcggcg gagtggccgt gcccctggag gtggtgctgc tgcgcaaggt gggcgcggcg
300ggcggcgcgc gcggcgtcat ccgcttgctg gactggttcg agcggcccga cggcttcttg
360ttggtgctgg agcgacccga gccggcacag gacctcttcg acttcatcac tgaacgaggc
420gccctggacg agccgctggc gcgtcgcttc ttcgcgcagg tgcttgccgc tgtgcggcac
480tgccacaatt gtggggtcgt gcaccgcgac atcaaggacg agaacctgct ggtggacctg
540cgctcgggag agctgaagct catcgacttc ggctcgggcg cggtgctcaa ggacacggtc
600tacactgact ttgatggcac ccgtgtgtac agccccccag agtggatccg atatcaccga
660tatcacgggc ggtctgccac tgtgtggtct ctgggtgtac tgctctacga catggtgtgt
720ggggacattc cctttgagca ggatgaggag atcttgcgcg gcaggctctt tttccggagg
780agggtctccc cagagtgcca gcagcttatt gagtggtgtc tctccctgag gccctcagag
840aggccctccc tggaccaaat tgctgcccac ccctggatgc tggggacaga ggggagcgtt
900ccagagaact gtgaccttcg gctttgtgcc ctggatactg acgacggagc cagtaccact
960tccagcagtg agagcttg
9788254PRTArtificialEukaryotic Protein Kinase Domain 8Tyr Gln Val Gly Ala
Val Leu Gly Ser Gly Gly Phe Gly Thr Val Tyr 1 5
10 15 Ala Gly Ser Arg Ile Ala Asp Gly Leu Pro
Val Ala Val Lys His Val 20 25
30 Val Lys Glu Arg Val Thr Glu Trp Gly Ser Leu Gly Gly Val Ala
Val 35 40 45 Pro
Leu Glu Val Val Leu Leu Arg Lys Val Gly Ala Ala Gly Gly Ala 50
55 60 Arg Gly Val Ile Arg Leu
Leu Asp Trp Phe Glu Arg Pro Asp Gly Phe 65 70
75 80 Leu Leu Val Leu Glu Arg Pro Glu Pro Ala Gln
Asp Leu Phe Asp Phe 85 90
95 Ile Thr Glu Arg Gly Ala Leu Asp Glu Pro Leu Ala Arg Arg Phe Phe
100 105 110 Ala Gln
Val Leu Ala Ala Val Arg His Cys His Asn Cys Gly Val Val 115
120 125 His Arg Asp Ile Lys Asp Glu
Asn Leu Leu Val Asp Leu Arg Ser Gly 130 135
140 Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Val
Leu Lys Asp Thr 145 150 155
160 Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro Pro Glu Trp
165 170 175 Ile Arg Tyr
His Arg Tyr His Gly Arg Ser Ala Thr Val Trp Ser Leu 180
185 190 Gly Val Leu Leu Tyr Asp Met Val
Cys Gly Asp Ile Pro Phe Glu Gln 195 200
205 Asp Glu Glu Ile Leu Arg Gly Arg Leu Phe Phe Arg Arg
Arg Val Ser 210 215 220
Pro Glu Cys Gln Gln Leu Ile Glu Trp Cys Leu Ser Leu Arg Pro Ser 225
230 235 240 Glu Arg Pro Ser
Leu Asp Gln Ile Ala Ala His Pro Trp Met 245
250 92133DNARattus norvegicus 9cgctcggcca gctgccgtct
acgggcttcc gcgcggccac cgggcaactg cgccgcgcgg 60ctgccccact gagcgctcgg
cctcggggcc gtgggatccg ccgcgctgtc tgcggtcagg 120aagaccgccc tcccgcgtcc
gtgccggacg ggtcagaggc ggcgccgcac gcgaggccac 180ccgcgatgct gctgtccaag
ttcggctccc tggcgcacct ctgcgggcct ggcggcgtgg 240accacctccc agtgaagatc
ctacagccag ccaaggcgga caaggagagc ttcgagaagg 300tgtaccaggt gggcgccgtg
ctcggcagcg gcggcttcgg cacggtctac gcgggcagcc 360gcatcgccga cggactcccg
gtggctgtga agcacgtggt gaaggagcgg gtgaccgagt 420ggggcagtct cggcggaatg
gccgtgcccc tggaggtggt gctgctgcgc aaggtgggcg 480cggcgggcgg cgcgcgcggc
gtcatccgcc tgctggactg gttcgagcgg cccgacggct 540tcctgctggt gctggagcga
cccgagccgg cacaggacct cttcgacttc atcactgaac 600gcggcgccct ggacgagcct
ctggctcgtc gcttcttcgc gcaggtgctc gccgctgtgc 660ggcactgcca caattgtggg
gtcgtgcacc gcgacatcaa ggacgagaac ctgctggtgg 720acttgcgctc gggcgagctg
aagctcatcg acttcggctc gggcgcggtg ctcaaggaca 780cggtctacac tgactttgat
ggcacccgtg tgtacagccc cccagagtgg atccggtatc 840atcgatatca cgggcggtct
gccactgtgt ggtctctggg tgtactgctc tacgacatgg 900tgtgtgggga cattcccttt
gagcaggatg aggagatctt gcgcggcagg ctctttttcc 960ggaggagggt ctccccagag
tgccagcagc ttattgagtg gtgtctctcc ctgcggccct 1020cagagaggcc ctcgctggac
caaattgctg cccatccctg gatgctgggg acagagggca 1080gcgttccaga gaactgtgac
cttcggctct gtgccctgga tactgatgac ggagccagta 1140ccacttccag cagtgagagc
ttgtgaggag gaggaggggc ctggactcca cactgggggc 1200ctgggctcag cctagccagc
cctctcccag aatgaacatt ttctgcctgg gatgtctcct 1260gcaaaagcag tgacctctga
cccctggtga cctttgctct cggcaccggg cctgtttcct 1320ttgctttgag tgcctttttg
aacgctgctc cacagggcct gggttttctt gagctcttct 1380gtccaaagat ggctgcgggc
taagcaaggt cccgcctgcc ctgggtggat acttgaaccc 1440gagaccctac cctgctgctc
catcttgcgg cagccttcct gaccaagtgt gtttgacatg 1500gagcgccctg tggtgcccac
ctccaaccct ccagtctcct ggtcttcgtc tgggcatgtc 1560tgcacaagca atgcaacgct
gggccactgc tgcccgcctg cctccctggc accgcacgca 1620acgagcgtgc cacggtctct
tatttatggt gtgatcaccc tggagggcgc ccctgccctg 1680ctggggctat ttattgttta
atttatttgc tgaggttact tcctccaagc aaccaccttc 1740tccaggcccc tggggtgttc
aggaaagcca agggtggccg ttcagtccac agacggcatc 1800ctggttcctg cacctgcagt
aggtccctaa ccccatgttt gtgggaggag gaatttgtac 1860agtggctaat ttaaggggag
tgggagaccc tgtcaccctg ggcactctgc gctggggagg 1920gggtttaaat tattgacctt
gtacagtctg cttgctggct ctgaaagctg gggtggggga 1980cagagtctca agcccttaat
ttattttagc aactgtgttc tgtgaccctg gtgtgagtag 2040gcatcagggg tggggttgta
taagttcaaa agtgtgaaat gtctggagat catatttttt 2100atacaggtat ttcaattaaa
tgttttggta tat 213310326PRTRattus
norvegicus 10Met Leu Leu Ser Lys Phe Gly Ser Leu Ala His Leu Cys Gly Pro
Gly 1 5 10 15 Gly
Val Asp His Leu Pro Val Lys Ile Leu Gln Pro Ala Lys Ala Asp
20 25 30 Lys Glu Ser Phe Glu
Lys Val Tyr Gln Val Gly Ala Val Leu Gly Ser 35
40 45 Gly Gly Phe Gly Thr Val Tyr Ala Gly
Ser Arg Ile Ala Asp Gly Leu 50 55
60 Pro Val Ala Val Lys His Val Val Lys Glu Arg Val Thr
Glu Trp Gly 65 70 75
80 Ser Leu Gly Gly Met Ala Val Pro Leu Glu Val Val Leu Leu Arg Lys
85 90 95 Val Gly Ala Ala
Gly Gly Ala Arg Gly Val Ile Arg Leu Leu Asp Trp 100
105 110 Phe Glu Arg Pro Asp Gly Phe Leu Leu
Val Leu Glu Arg Pro Glu Pro 115 120
125 Ala Gln Asp Leu Phe Asp Phe Ile Thr Glu Arg Gly Ala Leu
Asp Glu 130 135 140
Pro Leu Ala Arg Arg Phe Phe Ala Gln Val Leu Ala Ala Val Arg His 145
150 155 160 Cys His Asn Cys Gly
Val Val His Arg Asp Ile Lys Asp Glu Asn Leu 165
170 175 Leu Val Asp Leu Arg Ser Gly Glu Leu Lys
Leu Ile Asp Phe Gly Ser 180 185
190 Gly Ala Val Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr
Arg 195 200 205 Val
Tyr Ser Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr His Gly Arg 210
215 220 Ser Ala Thr Val Trp Ser
Leu Gly Val Leu Leu Tyr Asp Met Val Cys 225 230
235 240 Gly Asp Ile Pro Phe Glu Gln Asp Glu Glu Ile
Leu Arg Gly Arg Leu 245 250
255 Phe Phe Arg Arg Arg Val Ser Pro Glu Cys Gln Gln Leu Ile Glu Trp
260 265 270 Cys Leu
Ser Leu Arg Pro Ser Glu Arg Pro Ser Leu Asp Gln Ile Ala 275
280 285 Ala His Pro Trp Met Leu Gly
Thr Glu Gly Ser Val Pro Glu Asn Cys 290 295
300 Asp Leu Arg Leu Cys Ala Leu Asp Thr Asp Asp Gly
Ala Ser Thr Thr 305 310 315
320 Ser Ser Ser Glu Ser Leu 325 11323PRTXenopus
laevis 11Met Leu Leu Ser Lys Phe Gly Ser Leu Ala His Ile Cys Asn Pro Ser
1 5 10 15 Asn Met
Glu His Leu Pro Val Lys Ile Leu Gln Pro Val Lys Val Asp 20
25 30 Lys Glu Pro Phe Glu Lys Val
Tyr Gln Val Gly Ser Val Val Ala Ser 35 40
45 Gly Gly Phe Gly Thr Val Tyr Ser Asp Ser Arg Ile
Ala Asp Gly Gln 50 55 60
Pro Val Ala Val Lys His Val Ala Lys Glu Arg Val Thr Glu Trp Gly 65
70 75 80 Thr Leu Asn
Gly Val Met Val Pro Leu Glu Ile Val Leu Leu Lys Lys 85
90 95 Val Pro Thr Ala Phe Arg Gly Val
Ile Asn Leu Leu Asp Trp Tyr Glu 100 105
110 Arg Pro Asp Ala Phe Leu Ile Val Met Glu Arg Pro Glu
Pro Val Lys 115 120 125
Asp Leu Phe Asp Tyr Ile Thr Glu Lys Gly Pro Leu Asp Glu Asp Thr 130
135 140 Ala Arg Gly Phe
Phe Arg Gln Val Leu Glu Ala Val Arg His Cys Tyr 145 150
155 160 Asn Cys Gly Val Val His Arg Asp Ile
Lys Asp Glu Asn Leu Leu Val 165 170
175 Asp Thr Arg Asn Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser
Gly Ala 180 185 190
Leu Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr
195 200 205 Ser Pro Pro Glu
Trp Val Arg Tyr His Arg Tyr His Gly Arg Ser Ala 210
215 220 Thr Val Trp Ser Leu Gly Val Leu
Leu Tyr Asp Met Val Tyr Gly Asp 225 230
235 240 Ile Pro Phe Glu Gln Asp Glu Glu Ile Val Arg Val
Arg Leu Cys Phe 245 250
255 Arg Arg Arg Ile Ser Thr Glu Cys Gln Gln Leu Ile Lys Trp Cys Leu
260 265 270 Ser Leu Arg
Pro Ser Asp Arg Pro Thr Leu Glu Gln Ile Phe Asp His 275
280 285 Pro Trp Met Cys Lys Cys Asp Leu
Val Lys Ser Glu Asp Cys Asp Leu 290 295
300 Arg Leu Arg Thr Ile Asp Asn Asp Ser Ser Ser Thr Ser
Ser Ser Asn 305 310 315
320 Glu Ser Leu 12313PRTRattus norvegicus 12Met Leu Leu Ser Lys Ile Asn
Ser Leu Ala His Leu Arg Ala Ala Pro 1 5
10 15 Cys Asn Asp Leu His Ala Asn Lys Leu Ala Pro
Gly Lys Glu Lys Glu 20 25
30 Pro Leu Glu Ser Gln Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly
Gly 35 40 45 Phe
Gly Ser Val Tyr Ser Gly Ile Arg Val Ala Asp Asn Leu Pro Val 50
55 60 Ala Ile Lys His Val Glu
Lys Asp Arg Ile Ser Asp Trp Gly Glu Leu 65 70
75 80 Pro Asn Gly Thr Arg Val Pro Met Glu Val Val
Leu Leu Lys Lys Val 85 90
95 Ser Ser Gly Phe Ser Gly Val Ile Arg Leu Leu Asp Trp Phe Glu Arg
100 105 110 Pro Asp
Ser Phe Val Leu Ile Leu Glu Arg Pro Glu Pro Val Gln Asp 115
120 125 Leu Phe Asp Phe Ile Thr Glu
Arg Gly Ala Leu Gln Glu Glu Leu Ala 130 135
140 Arg Ser Phe Phe Trp Gln Val Leu Glu Ala Val Arg
His Cys His Asn 145 150 155
160 Cys Gly Val Leu His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp
165 170 175 Leu Asn Arg
Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu 180
185 190 Leu Lys Asp Thr Val Tyr Thr Asp
Phe Asp Gly Thr Arg Val Tyr Ser 195 200
205 Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr His Gly Arg
Ser Ala Ala 210 215 220
Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile 225
230 235 240 Pro Phe Glu His
Asp Glu Glu Ile Val Lys Gly Gln Val Tyr Phe Arg 245
250 255 Gln Arg Val Ser Ser Glu Cys Gln His
Leu Ile Arg Trp Cys Leu Ser 260 265
270 Leu Arg Pro Ser Asp Arg Pro Ser Phe Glu Glu Ile Gln Asn
His Pro 275 280 285
Trp Met Gln Asp Val Leu Leu Pro Gln Ala Thr Ala Glu Ile His Leu 290
295 300 His Ser Leu Ser Pro
Ser Pro Ser Lys 305 310 13313PRTHomo sapiens
13Met Leu Leu Ser Lys Ile Asn Ser Leu Ala His Leu Arg Ala Ala Pro 1
5 10 15 Cys Asn Asp Leu
His Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu 20
25 30 Pro Leu Glu Ser Gln Tyr Gln Val Gly
Pro Leu Leu Gly Ser Gly Gly 35 40
45 Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Ser Asp Asn Leu
Pro Val 50 55 60
Ala Ile Lys His Val Glu Lys Asp Arg Ile Ser Asp Trp Gly Glu Leu 65
70 75 80 Pro Asn Gly Thr Arg
Val Pro Met Glu Val Val Leu Leu Lys Lys Val 85
90 95 Ser Ser Gly Phe Ser Gly Val Ile Arg Leu
Leu Asp Trp Phe Glu Arg 100 105
110 Pro Asp Ser Phe Val Leu Ile Leu Glu Arg Pro Glu Pro Val Gln
Asp 115 120 125 Leu
Phe Asp Phe Ile Thr Glu Arg Gly Ala Leu Gln Glu Glu Leu Ala 130
135 140 Arg Ser Phe Phe Trp Gln
Val Leu Glu Ala Val Arg His Cys His Asn 145 150
155 160 Cys Gly Val Leu His Arg Asp Ile Lys Asp Glu
Asn Ile Leu Ile Asp 165 170
175 Leu Asn Arg Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu
180 185 190 Leu Lys
Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser 195
200 205 Pro Pro Glu Trp Ile Arg Tyr
His Arg Tyr His Gly Arg Ser Ala Ala 210 215
220 Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp Met Val
Cys Gly Asp Ile 225 230 235
240 Pro Phe Glu His Asp Glu Glu Ile Ile Arg Gly Gln Val Phe Phe Arg
245 250 255 Gln Arg Val
Ser Ser Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala 260
265 270 Leu Arg Pro Ser Asp Arg Pro Thr
Phe Glu Glu Ile Gln Asn His Pro 275 280
285 Trp Met Gln Asp Val Leu Leu Pro Gln Glu Thr Ala Glu
Ile His Leu 290 295 300
His Ser Leu Ser Pro Gly Pro Ser Lys 305 310
14313PRTMus musculus 14Met Leu Leu Ser Lys Ile Asn Ser Leu Ala His Leu
Arg Ala Arg Pro 1 5 10
15 Cys Asn Asp Leu His Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu
20 25 30 Pro Leu Glu
Ser Gln Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly 35
40 45 Phe Gly Ser Val Tyr Ser Gly Ile
Arg Val Ala Asp Asn Leu Pro Val 50 55
60 Ala Ile Lys His Val Glu Lys Asp Arg Ile Ser Asp Trp
Gly Glu Leu 65 70 75
80 Pro Asn Gly Thr Arg Val Pro Met Glu Val Val Leu Leu Lys Lys Val
85 90 95 Ser Ser Asp Phe
Ser Gly Val Ile Arg Leu Leu Asp Trp Phe Glu Arg 100
105 110 Pro Asp Ser Phe Val Leu Ile Leu Glu
Arg Pro Glu Pro Val Gln Asp 115 120
125 Leu Phe Asp Phe Ile Thr Glu Arg Gly Ala Leu Gln Glu Asp
Leu Ala 130 135 140
Arg Gly Phe Phe Trp Gln Val Leu Glu Ala Val Arg His Cys His Asn 145
150 155 160 Cys Gly Val Leu His
Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp 165
170 175 Leu Ser Arg Gly Glu Ile Lys Leu Ile Asp
Phe Gly Ser Gly Ala Leu 180 185
190 Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr
Ser 195 200 205 Pro
Pro Glu Trp Ile Arg Tyr His Arg Tyr His Gly Arg Ser Ala Ala 210
215 220 Val Trp Ser Leu Gly Ile
Leu Leu Tyr Asp Met Val Cys Gly Asp Ile 225 230
235 240 Pro Phe Glu His Asp Glu Glu Ile Ile Lys Gly
Gln Val Phe Phe Arg 245 250
255 Gln Thr Val Ser Ser Glu Cys Gln His Leu Ile Lys Trp Cys Leu Ser
260 265 270 Leu Arg
Pro Ser Asp Arg Pro Ser Phe Glu Glu Ile Arg Asn His Pro 275
280 285 Trp Met Gln Gly Asp Leu Leu
Pro Gln Ala Ala Ser Glu Ile His Leu 290 295
300 His Ser Leu Ser Pro Gly Ser Ser Lys 305
310
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