WO2002077623A1 - Probe for visualizing phosphorylation/dephosphorylation of protein and method of detecting and quantifying phosphorylation/dephosphorylation of protein - Google Patents
Probe for visualizing phosphorylation/dephosphorylation of protein and method of detecting and quantifying phosphorylation/dephosphorylation of protein Download PDFInfo
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- WO2002077623A1 WO2002077623A1 PCT/JP2001/002360 JP0102360W WO02077623A1 WO 2002077623 A1 WO2002077623 A1 WO 2002077623A1 JP 0102360 W JP0102360 W JP 0102360W WO 02077623 A1 WO02077623 A1 WO 02077623A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Definitions
- the invention of this application relates to a visualization probe for detecting and quantifying protein phosphorylation dephosphorylation. More specifically, the invention of this application relates to a method of linking a substrate domain having a site to be phosphorylated between two different chromophores and a phosphorylation recognition domain via a linker sequence to arrange protein phosphorylation.
- the present invention relates to a dephosphorylation visualization probe and a method for detecting and quantifying protein phosphorylation / dephosphorylation using the probe.
- Phosphorylation of proteins by intracellular kinases is one of the most important reactions in intracellular signaling, and is known to play an important role in cell survival, proliferation, differentiation and other processes. , 1 1 3-1 27 (2000)).
- the kinase protein catalyzes the transfer of ⁇ -phosphate in ATP and the phosphorylation of the hydroxyl group of serine, threonine, and tyrosine on the substrate protein. These changes trigger the enzymatic activation of substrate proteins and their interaction with the target proteins. Therefore, screening for substances that enhance or suppress intracellular signaling by phosphorylation and dephosphorylation of proteins will not only enable diagnosis of diseases, but will also be important findings in the development of new drugs, etc. Is expected to be obtained.
- the invention of this application has been made in view of the circumstances described above, and solves the problems of the prior art, and achieves the phosphorylation and dephosphorylation of proteins in living cells, animals, plants, and the like.
- the task is to provide a versatile detection and measurement method for intracellular protein phosphorylation and dephosphorylation that can be monitored nondestructively, and that enables spatial and temporal analysis. Disclosure of the invention
- a substrate domain having a site to be phosphorylated between a donor chromophore that causes fluorescence resonance energy transfer and an axepin chromophore is provided.
- the present invention provides a protein phosphorylation / dephosphorylation visualization probe, which is linked to a tandem fusion unit in which a phosphorylation recognition domain is bound via a linker sequence.
- the invention of the present application relates to a fluorescent protein in which a donor chromophore and an axepin chromophore that cause fluorescence resonance energy transfer are fluorescent proteins having different fluorescent wavelengths, and third, a mutant of a green fluorescent protein. Fourth, it was confirmed that the mutants of the green fluorescent protein were the cyan fluorescent protein and the yellow fluorescent protein, and that the mutants of the green fluorescent protein were the cyan fluorescent protein and the yellow fluorescent protein.
- the invention of the present application provides a probe for visualizing the protein phosphorylation-dephosphorylation in which the phosphorylation site of the substrate domain has any one of tyrosine, serine, and threonine amino acids.
- the invention of this application is based on a protein phosphorylation in which the phosphorylation recognition domain is an endogenous domain of any of SH2 domain, phosphotyrosine binding domain and WW domain.
- the present invention provides a protein phosphorylation ⁇ dephosphorylation visualization probe which is a single-chain antibody prepared using a substrate domain in which a phosphorylation recognition domain is phosphorylated as an immunogen.
- the invention of the present application also provides a probe for visualizing any one of the above-mentioned protein phosphorylation and dephosphorylation having an intracellular localization sequence at the terminal end.
- the invention of the present application is characterized in that a protein phosphorylation / dephosphorylation visualization probe according to any one of the first to eighth inventions coexists with a candidate substance, and a change in fluorescence wavelength is measured. Also provides a method of screening for inhibitors.
- the invention of the present application is the protein phosphorylation / dephosphorylation visualization probe according to any one of the first to eighth aspects, wherein the protein phosphorylation / dephosphorylation in which the substrate domain is phosphorylated is provided.
- a screening method for a protein dephosphorylation-enhancing / suppressing substance which comprises coexisting an oxidation visualization probe and a candidate substance and measuring a change in fluorescence wavelength.
- the invention of the present application is, in the first aspect, characterized in that in any one of the above-described screening methods, a protein phosphorylation / desorption oxidation visualization probe is introduced into a cell and coexists with a candidate substance. provide. Furthermore, in the invention of this application, in a second aspect, the protein phosphorylation / dephosphorylation visualization probe according to any one of the first to eighth aspects is introduced into a cell, and the change in fluorescence wavelength is measured. The present invention provides a method for quantifying protein phosphorylated substances. Third, the protein phosphorylation according to any one of the first to eighth aspects of the invention.
- a phosphorylated substrate domain is introduced into a cell, and the change in fluorescence wavelength is detected. Also provided is a method for quantifying a protein dephosphorylated substance, which is characterized by measuring.
- FIG. 1 is a schematic diagram showing the structure and principle of the protein phosphorylation / dephosphorylation visualization probe of the present invention.
- 1 to 8 indicate the following parts.
- 1b Protein phosphorylation / dephosphorylation visualization probe (after phosphorylation)
- 11 tandem fusion unit
- 2b Substrate domain (after phosphorylation)
- 21a Phosphorylation site (before phosphorylation)
- 21b Phosphorylation site (after phosphorylation)
- 3 Phosphorylation recognition site
- FIG. 2 is a schematic diagram showing a specific configuration of each phosphorylated / dephosphorylated visualization probe constructed in the example of the present invention.
- FIG. 3 shows an example of an immunoplot using a phosphotyrosine antibody when the phosphorylation / dephosphorylation visualization probes shown in FIGS. 2 (a) and 2 (b) were introduced into cells in an example of the present invention.
- FIG. 9 is a diagram replacing a confocal laser fluorescence micrograph showing the analysis result.
- FIG. 4A shows the probe for visualizing protein phosphorylation and dephosphorylation shown in Figure 2 (b) introduced into cells, and photographed using a CFP emission filter (480 nm ⁇ 15 nm).
- FIG. 3 is a diagram showing a fluorescence microscope image obtained. . —
- FIG. 4B shows the protein phosphorylation of Fig. 2 (b).
- CFP 480 ⁇ 15 nm
- YFP YFP
- excitation light 450 ⁇ 10 ⁇ m when the cells into which the cells were introduced were stimulated with insulin.
- FIG. 4 is a diagram showing a pseudo-color image showing a temporal change of a fluorescence intensity ratio (hereinafter referred to as CFP / YFP).
- FIG. 4C shows the excitation light of 450 ⁇ 1 O nm in the cytoplasm and nucleus when cells transfected with the protein phosphorylation / dephosphorylation visualization probe in Fig. 2 (b) were stimulated with insulin.
- FIG. 4 is a diagram showing the change over time of CFP / YFP due to aging.
- FIG. 4D shows cells ( ⁇ ) transfected with the protein phosphorylation / dephosphorylation visualization probe of Fig. 2 (b) treated with tyrphostin, an inhibitor of the insulin receptor, and protein phosphorylation / deactivation of Fig. 2 (c).
- FIG. 4 is a diagram showing the time-dependent change of CFP / YFP by excitation light of 450 ⁇ 10 nm in each cytoplasm when cells (mouth) into which phosphorylation visualization probe was introduced were stimulated with insulin.
- FIG. 4 is a view showing a pseudo-color image showing a temporal change of CFP ZY FP by excitation light of FIG.
- Fig. 5B shows the excitation light at 440 ⁇ 10 nm in the nucleus and cytoplasm when the cells into which the protein phosphorylation / dephosphorylation visualization probe of Fig. 2 (d) was introduced were stimulated with various concentrations of insulin.
- FIG. 4 is a diagram showing a change over time of CF PZY FP due to the above.
- FIG. 6A shows the excitation of 440 ⁇ 10 nm in the cytoplasm when the cells transfected with the protein phosphorylation / dephosphorylation visualization probes shown in Figures 2 (b) and 2 (e) were stimulated with insulin. It is a figure which compares the change with time of CFP / YFP by light.
- Fig. 6B shows the simultaneous localization of (e) and insulin receptor when cells transfected with the protein phosphorylation / dephosphorylation visualization prop of Fig. 2 (e) were stimulated with insulin.
- FIG. 3 is a view showing one micrograph of a confocal scanning laser for microscopy. BEST MODE FOR CARRYING OUT THE INVENTION
- phosphorylation of proteins by intracellular kinases is one of the most important steps in intracellular signaling, and is deeply involved in processes such as cell survival, proliferation, and differentiation. Therefore, protein phosphorylation and dephosphorylation are phenomena observed as a cause or symptom in many diseases. That is, if the phosphorylation of a specific protein can be detected or quantified, early diagnosis of various diseases becomes possible. In addition, screening for factors and substances that enhance or inhibit protein phosphorylation or dephosphorylation can greatly contribute to the discovery of substances involved in these diseases and new therapeutic agents.
- the protein phosphorylation 'dephosphorylation visualization probe of the invention of this application is a probe for visualizing, detecting, and quantifying protein phosphorylation by a phosphorylating substance.
- Figure 1 shows a schematic diagram showing the structure and principle of such a protein phosphorylation / dephosphorylation visualization probe.
- the protein phosphorylation / dephosphorylation visualization probe (1a) of the present invention comprises a substrate domain (2a) having a phosphorylation site (21a) and a phosphorylation recognition domain (3).
- a tandem fusion unit (11) connected via a sequence (4), wherein the tandem fusion unit (11) undergoes a fluorescence resonance energy transfer with a donor chromophore (5) and an acceptor. It is characterized by being linked between chromophores (5 ').
- the protein phosphorylation / dephosphorylation visualization probe (1a) of the present invention for example, when the phosphorylation site (21a) of the substrate domain (2a) is phosphorylated by the phosphorylation substance (6), The phosphorylated recognition domain (3) recognizes this and interacts specifically with the phosphorylated substrate domain (21b).
- the donor chromophore (5) and the Axep Yuichi chromophore (5 ') approach each other.
- a dephosphorylation visualization probe (lb) coexists with the dephosphorylated substance (7),
- the phosphorylated site (21b) is dephosphorylated (21a)
- the interaction between the phosphorylation recognition domain (3) and the substrate domain (2b) disappears, and the donor chromophore (5) And Aksep one chromophore (5 ') leaves again.
- the Donna chromophore (5) is excited, and energy transfer to the receptor chromophore does not occur. Therefore, from the change in FRE efficiency, dephosphorylation of the substrate domain (2b) can be detected by fluorescence analysis.
- the tandem fusion unit (11) consists of the substrate domain (2a), the phosphorylation recognition domain (3), and the linker sequence (4) that binds them.
- the substrate domain (2a) may be any as long as it has a site (21a) where phosphorylation occurs, and the sequence, structure, and the like are not particularly limited.
- the site that can be a phosphorylation site (21a) basically has only to have a —0H group. Natural amino acids such as tyrosine (Tyr), serine (Ser), and torenin (Thr) And peptides into which 0 H groups have been introduced by chemical modification.
- the phosphorylation recognition domain recognizes phosphorylation of the substrate domain (2a) and can specifically interact with the phosphorylated substrate domain (2b). It may be any one that satisfies this condition, for example, recognizes a specific phosphorylated substrate such as SH2 domain, phosphotyrosine binding domain, WW domain, etc. Endogenous domains are known.
- the phosphorylated target substrate domain (2b) is used as an immunogen in a single chain.
- An antibody can be prepared and used as a phosphorylation recognition domain (3).
- the linker sequence (4) has moderate flexibility and does not have a site to be phosphorylated.
- the sequence and chain length are not particularly limited.
- the site (2) where the linker sequence (4) is phosphorylated In the case of having 1a), the possibility of phosphorylation by the phosphorylating substance (6) is high, and accurate detection and quantification of protein phosphorylation becomes impossible, which is not preferable.
- the phosphorylation (2b) of the substrate domain (2a) allows the phosphorylation recognition site (3) to interact with the substrate domain (2b), and the donor chromophore (5) )
- the chromophore (5 ') are preferably polypeptides or oligopeptides having a chain length that allows access to the chromophore (5').
- the tandem fusion unit (11) having the above-described configuration is used to produce a donor chromophore (5) that undergoes fluorescence resonance energy transfer and an axceptor coloring. It is linked between the groups (5 '), and when the substrate domain (2a) is phosphorylated, it shows a change in the FRE by the mechanism described above.
- Various fluorescent substances, particularly fluorescent proteins, are considered as the donor chromophore (5) and the receptor chromophore (5 ').
- Both may be substances that exhibit fluorescence at different wavelengths upon irradiation with external light, and preferred examples thereof include a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) which are mutants of green fluorescent protein (GFP). Among them, CFP and YFP are preferred. These fluorescent proteins may be further mutated according to the intended use and used as donor and / or receptor chromophore.
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- GFP green fluorescent protein
- CFP and YFP are preferred.
- These fluorescent proteins may be further mutated according to the intended use and used as donor and / or receptor chromophore.
- the domain adjacent to the donor chromophore is the substrate domain (2a) or the phosphorylation recognition domain (3) is particularly limited. Not done. Since the appropriate linking order differs depending on the structure and steric hindrance of these domains and the linker sequence (4), the appropriate order depends on the combination of the substrate domain (2a) and phosphorylation recognition domain (3). Can choose preferable.
- the protein phosphorylation / dephosphorylation visualization probe of the invention of the present application may further have various localization sequences at its ends.
- a localization sequence can recognize a specific cell or a specific region in a cell, or a specific tissue, and localize a protein phosphorylation / dephosphorylation visualization probe.
- a localization sequence can be obtained by linking a nuclear export signal sequence and a cell membrane binding sequence such as a pleckstrin homology (P H) domain.
- the protein phosphorylation 'dephosphorylation visualization probe of the invention of this application is as described above, but the production method is not particularly limited. Although it may be constructed by total synthesis, a preferred method is to link each domain by a general genetic engineering technique such as polymerase chain reaction (PCR). At this time, various restriction sites may be introduced.
- PCR polymerase chain reaction
- the invention of this application also provides a method for screening for a substance that enhances or suppresses protein phosphorylation using the above-described protein phosphorylation / dephosphorylation visualization probe. That is, when the protein phosphorylation dephosphorylation visualization probe of the invention of this application and the candidate substance coexist, the substrate domain (2a) of the protein phosphorylation / dephosphorylation visualization probe (1a) becomes If phosphorylated, protein phosphorylation can be detected from the change in FRET by the mechanism described above, and a substance that phosphorylates the substrate domain (2a) can be screened.
- Such a candidate substance may directly act as a protein kinase and phosphorylate a protein, or may act at an early stage of intracellular signaling, that is, a substance that acts as a protein kinase activator. Is also good.
- the candidate substance enhances or suppresses dephosphorylation, and when screening for a substance that enhances or suppresses dephosphorylation, the protein phosphorylation-dephosphorylation visualization probe (1a) substrate must be used in advance. It is sufficient to measure the FRET change that occurs when the domain (2a) is phosphorylated (2b), and the phosphorylation / dephosphorylation visualization probe after phosphorylation (1b) coexists with the candidate substance.
- the protein phosphorylation / dephosphorylation visualization probe ( ⁇ a) is used in a solution in which pH, salt concentration, etc. are adjusted. May coexist with the candidate substance, or may be introduced into cells by a general genetic engineering technique and brought into contact with the candidate substance. At this time, the candidate substance may exist outside the cell, may be taken up into the cell, or may be introduced into the cell in advance by a genetic engineering technique. In addition, the candidate substance may be an existing enzyme in a cell, a receptor, or the like.
- protein phosphorylation / dephosphorylation visualization probe of the invention of the present application enables the quantification of protein phosphorylated substances.
- protein phosphorylation can be quantified by introducing the protein phosphorylation / dephosphorylation visualization probe into cells and measuring the change in fluorescence wavelength.
- substance A which is known to phosphorylate a certain protein a
- the FRET change in the protein phosphorylation / dephosphorylation visualization probe when it is brought into contact with phosphorylation substance A at various concentrations in advance Measure and determine the time at which all FRET values reach saturation.
- a phosphorylation '' dephosphorylation visualization probe that has protein a as a substrate domain in cells that is phosphorylated by substance A Prepared and transfected into cells By measuring T, the amount of substance A in these cells can be determined. Similar quantification is possible for dephosphorylated substances.
- an expression vector incorporating a protein phosphorylation / dephosphorylation visualization probe can be introduced into cells by a known method such as an electroporation method, a calcium phosphate method, a liposomal method, and a DEAE dextran method. .
- protein phosphorylation ⁇ Dephosphorylation visualization probe is introduced into cells and co-existed with a phosphorylation (or dephosphorylation) substance, so that protein phosphorylation (or dephosphorylation) can be performed without destroying cells. Oxidation) can be detected and quantified.
- the protein phosphorylation 'dephosphorylation visualization probe of the invention of this application can not only image kinase signaling in a single living cell with high spatial and temporal resolution, but also regulate phosphorylation or dephosphorylation substances.
- the protein phosphorylation / dephosphorylation visualization probe of the invention of the present application is characterized in that a polynucleotide for expressing the protein is introduced into a cell, and the non-human animal totipotent cell is ontogenized to generate the animal or animal.
- a protein phosphorylation / dephosphorylation visualization probe and a phosphorylation (or dephosphorylation) substance in all cells of the progeny animal can be used together with a protein phosphorylation / dephosphorylation visualization probe and a phosphorylation (or dephosphorylation) substance in all cells of the progeny animal.
- Such a so-called transgenic non-human animal can be prepared according to a known preparation method (for example, Proc. Natl. Acad. Sci. USA 77, 7348-, (1980)).
- transgenic—non-human animals provide a visualization of protein phosphorylation / dephosphorylation to all somatic cells. Because it has lobes, it can measure the concentration of phosphorylated (or dephosphorylated) substances in cells and tissues, and can contain phosphorylated (or dephosphorylated) substances, It is also possible to screen candidate substances such as phosphorylation (phosphorylation) enhancers and phosphorylation (or dephosphorylation) inhibitors, which are effective in cells and tissues.
- the non-receptor thymosine kinase / serine / threonine kinase functions through the entire signaling cascade.
- tyrosine kinase receptors such as the insulin receptor and the hormone receptor, function at the onset of many signaling cascades.
- a protein phosphorylation / dephosphorylation visualization probe using an insulin signaling protein was examined in order to detect and quantify protein phosphorylation by an insulin receptor that is also a protein kinase.
- each sample and reagent were as follows.
- Tyrphost in 25 used was manufactured by Sigma Chemical Co. (St. Louis, 0).
- the anti-phosphotyrosine antibody (PY20) and the anti-j8 subunit of the human insulin receptor antibody used were those manufactured by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
- the anti-GFP antibody used was from Clontech (Palo Alto, CA).
- Cy5 labeled anti-Egret IgG antibody was obtained from Jacson ImmunoResearch Lab., Inc. (Pennsylania, PA).
- FIG. 2 is a schematic diagram showing a specific configuration of the prepared probe for visualizing protein phosphorylation / dephosphorylation.
- the visualization probe is a series of two green fluorescent protein mutants in which a substrate domain having a phosphorylated site and a phosphorylation recognition domain are linked via a linker sequence.
- the fusion unit was connected.
- GFP green fluorescent protein
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- a tyrosine phosphorylation domain (Y940: SEQ ID NO: ⁇ ) derived from insulin receptor substrate 1 (IRS-1) was used as a substrate domain.
- insulin receptor Yuichi phosphorylates tyrosine residue 9441 in an insulin-dependent manner. Mol. Cel I Biol. 13, 7418-7428 (1993)).
- the oligonucleotide shown in SEQ ID NO: 2 was used as the linker sequence (Ln).
- the restriction sites shown in FIG. 2 were introduced into CFP, YFP, the substrate domain, and the cDNA of the phosphorylation recognition domain by standard polymerase chain reaction (PCR). All cloning enzymes used were from Takara Biomedical (Tokyo, Japan). The sequence of the PCR fragment was determined with an ABI 310 gene analyzer.
- the cDNAs encoding the protein phosphorylation / dephosphorylation visualization probes were obtained from HindIII and pcDMA3.1 (+) (Invitrogen Co., Carlsbad, CA). Sub-cloned at Xba / site.
- IR cells were cultured in a 6-ml plate, and transfected with a plasmid containing each cDNA of probe (a) and probe (b).
- the CH 0- HIR cells overexpressing human Bok insulin receptions evening one were cultured in Ham 's F-12 medium supplemented with 1 0% ⁇ shea calf serum in 5% C 0 2 in 3 7 ° C.
- Cells were transfected with UpofectAMINE 2000 reagent, and after 12-24 hours, cells were plated on glass bottom dishes, cover slips or plastic culture dishes.
- CHO-IR cells expressing the probe (a) and the probe (b) were stimulated with 100 nM of insulin at 25 ° C for 20 minutes.
- cells were dissolved in ice-cold lysis buffer (Tris-HCI 50 mMs pH 7.4, NaCl 100m, EDTA 1m, 0.1% Triton X-100.
- FIG. 3 shows the results of the immunoblotting.
- FIG. 3 shows that probe (b) was successfully phosphorylated by the insulin receptor, but probe (a) was very unlikely to be phosphorylated. Therefore, in this test, the tandem fusion unit joined in the order shown in Fig. 2 (b) is more effective as a protein phosphorylation probe than the tandem fusion unit joined in the order shown in Fig. 2 (a). It was suggested that it was effective.
- CHO-IR cells were transfected using cDNA encoding probe (b) inserted into a mammalian expression vector.
- the exposure time at 440 ⁇ 10 nm excitation was 100 ms.
- Fluorescence microscopy images were obtained using a 40 ⁇ oil immersion lens (Carl Zeiss, Jena, Germany) through 480 ⁇ 15 nm and 535 ⁇ 12.5 nm filters.
- FIG. 4A shows a fluorescence microscope image of the probe (b) -expressing cells taken using a CFP emission filter (480 ⁇ 15 nm).
- probe (b) was uniformly distributed in both cytoplasmic fraction and nucleus.
- CHO-IR cells expressing the probe (b) were stimulated with 100 nM of insulin in the same manner as in Example 1.
- Figure 4 (a) shows the change over time in the pseudocolor image of the fluorescence intensity ratio of 480 ⁇ 15 nm for CFP to 535 ⁇ 12.5 nm for YFP when excited at 440 ⁇ 10 nm.
- Fig. 4 shows the change over time in the fluorescence intensity ratio between the cytoplasm and the nucleus. Insulin administration caused a rapid and clear decrease in the fluorescence intensity ratio of the cytoplasm with respect to the cells expressing the probe (b), but no change in the fluorescence intensity ratio in the nucleus (Fig. 4 C).
- the non-phosphorylated probe (b) has a loose conformation due to the presence of the linker sequence, but the tyrosine phosphorylated probe (b) It is believed that the conformation is more tightly packed, making it difficult to translocate through the nuclear pore and staying in the cytoplasmic fraction.
- Example 2 in order to prevent the protein phosphorylation probe from transferring to the nucleus where FRET was not changed by insulin stimulation, a protein phosphorylation visualization probe (d) having an extranuclear translocation signal sequence was prepared.
- the nuclear translocation signal sequence a protein derived from a human immunodeficiency virus, a nuclear translocation signal sequence (nes: SEQ ID NO: 3) derived from Rev (EMBO J. 16, 5573-5581) is used as a protein phosphate. Bound to the end of the probe. .
- Plasmid formation and transfection were performed in the same manner as in Example 1. No fluorescence was observed from the nuclei of the cells expressing the probe (d), confirming that the probe for visualizing the protein phosphorylation of the probe (d) was removed from the nucleus (FIG. 5A).
- FIG. 5B shows the responsiveness of the probe (d) to various concentrations of insulin in CHO-IR cells.
- the accumulation rate of the probe (d) phosphorylated by the insulin receptor increased with increasing insulin concentration. At 0.1 ⁇ insulin, no accumulation of phosphorylated probe (d) was observed.
- the relationship between the fluorescence intensity ratio of the probe (d) and the insulin concentration was determined by immunoradiography of tyrosine phosphorylation of the native IRS-1 protein in the cells (E-J. J. 16, 5573-5581 ( 1997)).
- the probe (d) is suitable as a visualization probe for multicellular analysis using a fluorescent multiwell plate reader which cannot distinguish between cytoplasmic and nuclear probe proteins.
- probe (d) directly stimulates the kinase activity of insulin receptor from a huge variety of candidate chemicals. 1_-783, l ⁇ MNatureZn, 183-186 ( 1985); Science 284, 974-977 (1999)) is expected to be able to screen high-throughput antidiabetic small molecules.
- IRS-1 an endogenous substrate protein of insulin receptor Yuichi, has a plextrin homology (i ⁇ H) domain and a phosphotyrosine binding (PTB) domain at its N-terminus, Diabetoiogia 40, S2 -S17 (1997)).
- the PH and PTB domains bind to the plasma membrane phosphoinositide and the insulin receptor-stimulated tyrosine-phosphorylated insulin-receptor domain, respectively (/ V ⁇ . Natl. Acad. Sci./ ⁇ 96, 8378-8383) .
- insulin stimulation increases the concentration of IRS-1 around the insulin receptor near the plasma membrane, which is the basis for efficient and selective phosphorylation of IRS-1 by the insulin receptor. (J. Biol. Chem. 270, 11715-11718 (1995)).
- Fig. 2 (e) was constructed by fusing the PH-PTB domain derived from the IRS- "1 protein to the probe (b).
- CHO-IR cells expressing probe (e) were stimulated with 100 nM of insulin at 25 ° C for 7 minutes.
- Cells were fixed with 2% paraformaldehyde and permeabilized with phosphate buffered saline containing 0.2% Triton X-100 for 10 minutes.
- Egret Anti-Subunit (1: 100 dilution)
- the cells were washed with a phosphate buffered saline solution containing 0.2% fish skin gelatin, and the cells were washed with Cy5. Incubation was carried out for 30 minutes together with a labeled anti-Peacock IgG antibody (1: 500 dilution).
- a cover glass was placed on the slide, and the cells were observed using a confocal scanning laser microscope (LSM510, Carl Zeiss).
- Figure 6A shows the cytoplasmic fluorescence intensity of probe (e) and probe (b) in CH 0 r IR cells stimulated with insulin 1 1) 0 ⁇ . 7 shows a comparison of changes in power ratio. The rate of change in the cytoplasmic fluorescence intensity ratio for probe (e) was faster than when probe (b) was used, but there was no significant difference in the fluorescence intensity ratio between the two when saturation was reached.
- the PH-PTB domain contributes to targeting of the probe (e) to the membrane insulin receptor by insulin.
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01915740A EP1304561A4 (en) | 2001-03-23 | 2001-03-23 | PROBE FOR PROBING THE PHOSPHORYLATION / DEPHOSPHORYLATION OF PROTEIN AND METHOD FOR DETECTING AND QUANTIFYING THE PHOSPHORYLATION / DEPHOSPHORYLATION OF PROTEIN |
US10/296,313 US7425430B2 (en) | 2001-03-23 | 2001-03-23 | Probe for visualizing phosphorylation/dephosphorylation of protein and method of detecting and quantifying phosphorylation/dephosphorylation of protein |
PCT/JP2001/002360 WO2002077623A1 (en) | 2001-03-23 | 2001-03-23 | Probe for visualizing phosphorylation/dephosphorylation of protein and method of detecting and quantifying phosphorylation/dephosphorylation of protein |
JP2002575624A JP3888974B2 (ja) | 2001-03-23 | 2001-03-23 | 蛋白質リン酸化・脱リン酸化可視化プローブとそれを用いた蛋白質リン酸化・脱リン酸化の検出および定量方法 |
CA2410460A CA2410460C (en) | 2001-03-23 | 2001-03-23 | Probes for imaging protein phosphorylation and dephosphorylation and method for detecting and determining protein phosphorylation and dephosphorylation using the same |
US11/149,169 US20050221412A1 (en) | 2001-03-23 | 2005-06-10 | Probes for imaging protein phosphorylation and dephosphorylation and method for detecting and determining protein phosphorylation and dephosphorylation using the same |
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PCT/JP2001/002360 WO2002077623A1 (en) | 2001-03-23 | 2001-03-23 | Probe for visualizing phosphorylation/dephosphorylation of protein and method of detecting and quantifying phosphorylation/dephosphorylation of protein |
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US11/149,169 Division US20050221412A1 (en) | 2001-03-23 | 2005-06-10 | Probes for imaging protein phosphorylation and dephosphorylation and method for detecting and determining protein phosphorylation and dephosphorylation using the same |
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WO2002077623A1 true WO2002077623A1 (en) | 2002-10-03 |
WO2002077623A9 WO2002077623A9 (fr) | 2006-01-05 |
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Country Status (5)
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US (2) | US7425430B2 (ja) |
EP (1) | EP1304561A4 (ja) |
JP (1) | JP3888974B2 (ja) |
CA (1) | CA2410460C (ja) |
WO (1) | WO2002077623A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005019447A1 (ja) * | 2003-08-26 | 2005-03-03 | Japan Science And Technology Agency | 単色蛍光プローブ |
WO2007102507A1 (ja) * | 2006-03-06 | 2007-09-13 | The University Of Tokyo | タンパク質リン酸化インディケーター |
JP2009278942A (ja) * | 2008-05-23 | 2009-12-03 | Hokkaido Univ | Bcr−ablチロシンキナーゼ活性測定用試薬 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1264897A3 (en) * | 2001-06-06 | 2003-11-12 | Europäisches Laboratorium Für Molekularbiologie (Embl) | Synthetic sensor peptide for kinase or phosphatase assays |
FI20070171A0 (fi) * | 2007-02-28 | 2007-02-28 | Hidex Oy | Biotesti ja biotestivälineet |
DE102010010052A1 (de) * | 2010-03-03 | 2011-09-08 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Signalgebende Bindemoleküle, Vorrichtungen und Verfahren zu deren Verwendung |
CN101831054B (zh) * | 2010-05-04 | 2011-11-16 | 中国科学院化学研究所 | 一种鉴别真菌和细菌的方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH1156398A (ja) * | 1997-08-11 | 1999-03-02 | Hamamatsu Photonics Kk | 二本鎖核酸分解酵素活性の測定方法 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU6702398A (en) * | 1997-03-14 | 1998-09-29 | Regents Of The University Of California, The | Fluorescent protein sensors for detection of analytes |
AU738194B2 (en) * | 1997-12-05 | 2001-09-13 | Pharmacia & Upjohn Company | Fluorescence-based high throughput screening assays for protein kinases and phosphatases |
WO2000008444A1 (en) * | 1998-08-08 | 2000-02-17 | Imperial Cancer Research Technology Limited | Fluorescent assay for biological systems |
US6441140B1 (en) * | 1998-09-04 | 2002-08-27 | Cell Signaling Technology, Inc. | Production of motif-specific and context-independent antibodies using peptide libraries as antigens |
GB9901072D0 (en) * | 1999-01-19 | 1999-03-10 | Imp Cancer Res Tech | Methods for detecting changes to a macromolecular component of a cell |
GB9904407D0 (en) * | 1999-02-25 | 1999-04-21 | Fluorescience Ltd | Compositions and methods for monitoring the modification of engineered binding partners |
-
2001
- 2001-03-23 JP JP2002575624A patent/JP3888974B2/ja not_active Expired - Fee Related
- 2001-03-23 US US10/296,313 patent/US7425430B2/en not_active Expired - Fee Related
- 2001-03-23 CA CA2410460A patent/CA2410460C/en not_active Expired - Fee Related
- 2001-03-23 EP EP01915740A patent/EP1304561A4/en not_active Withdrawn
- 2001-03-23 WO PCT/JP2001/002360 patent/WO2002077623A1/ja active Application Filing
-
2005
- 2005-06-10 US US11/149,169 patent/US20050221412A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH1156398A (ja) * | 1997-08-11 | 1999-03-02 | Hamamatsu Photonics Kk | 二本鎖核酸分解酵素活性の測定方法 |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005019447A1 (ja) * | 2003-08-26 | 2005-03-03 | Japan Science And Technology Agency | 単色蛍光プローブ |
JPWO2005019447A1 (ja) * | 2003-08-26 | 2006-10-19 | 独立行政法人科学技術振興機構 | 単色蛍光プローブ |
WO2007102507A1 (ja) * | 2006-03-06 | 2007-09-13 | The University Of Tokyo | タンパク質リン酸化インディケーター |
JP2009278942A (ja) * | 2008-05-23 | 2009-12-03 | Hokkaido Univ | Bcr−ablチロシンキナーゼ活性測定用試薬 |
Also Published As
Publication number | Publication date |
---|---|
WO2002077623A9 (fr) | 2006-01-05 |
US20050049396A1 (en) | 2005-03-03 |
CA2410460C (en) | 2010-06-29 |
US20050221412A1 (en) | 2005-10-06 |
JP3888974B2 (ja) | 2007-03-07 |
EP1304561A4 (en) | 2006-01-11 |
EP1304561A1 (en) | 2003-04-23 |
CA2410460A1 (en) | 2002-10-03 |
JPWO2002077623A1 (ja) | 2004-07-15 |
US7425430B2 (en) | 2008-09-16 |
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