WO2002014373A1 - Protein monitoring the activity of low-molecular weight gtp-binding protein - Google Patents

Abstract

A protein monitoring the activity of a low-molecular weight GTP-binding protein which makes it possible to measure the activation of a non-invasive low-molecular weight GTP-binding protein; a gene encoding the above protein; an expression vector containing this gene; transformed cells and a transgenic animal expressing the above-described protein and carrying the above-described expression vector which is useful in measuring the activation of a non-invasive low-molecular weight GTP-binding protein; a method of measuring the activation of a low-molecular weight GTP-binding protein with the use of the above-described protein; and a method of screening a substance controlling the activity of a low-molecular weight GTP-binding protein.

Description

 Description Activity monitoring protein for low molecular weight GTP-binding protein

 The present invention relates to a protein for monitoring the activity of a low molecular weight GTP-binding protein, a gene encoding the protein, an expression vector containing the gene, transformed cells and a transgenic animal carrying the expression vector, The present invention relates to a method for measuring the activation of a low-molecular-weight GTP-binding protein using a protein, and a method for screening a substance for regulating the activity of a low-molecular-weight GTP-binding protein. Background art

Numerous types of intracellular signaling molecules are known, and low-molecular-weight GTP-binding proteins (hereinafter sometimes referred to as GTP-binding proteins) are numerous and serve as important molecular switches. Therefore, it has been analyzed in great detail. The low molecular weight GTP-binding proteins are composed of the Ras family, the Rh0 family, the Rab family, the Ran family, etc. (Reference 1). These low-molecular-weight GTP-binding proteins are important molecular switches that control diverse intracellular signal transduction such as cell proliferation, cytoskeleton, intracellular transport, and nuclear transport. Low molecular weight GTP-binding proteins cycle between an inactivated form that binds to GDP and an activated form that binds to GTP (Figure 1). The GTP-binding form binds to a target protein specific to each GTP-binding protein and activates the target protein. The protein that catalyzes the reaction of converting GDP-linked to GTP-linked is a guanine nucleotide exchange factor, and the protein that catalyzes the reaction of converting GTP-linked to GDP-linked is GTPase-activating enzyme (GTPase activator). ). The GTP hydrolysis promoter promotes the hydrolysis of bound GTP, And works to generate GDP.

 Recently, as many small GTP-binding proteins and their activators and inactivators have been isolated, what are the functional differences between these small GTP-binding proteins in cells and in individuals? Is attracting attention. To clarify the functional differences, it is necessary to monitor the activation status of low-molecular-weight GTP-binding proteins in cells and individuals.

 In order to examine the degree of activation of the low molecular weight GTP binding protein in the cell, it is necessary to know the ratio of the GTP binding type to the GDP binding type of the low molecular weight GTP binding protein in the cell. At present, the following two methods are often used to determine the ratio of GTP-bound to GDP-bound low-molecular-weight GTP-binding proteins in cells.

(1) How to use the labeling with radioisotopes ³² P i: Cells ³² P i a low molecular weight GTP-binding protein then labeled and purified, the separated GTP and GDP bound by thin layer chromatography And quantify (Reference 2).

 (2) Pull-down method: A target protein that binds to a low-molecular-weight GTP-bound protein is bound to a solid layer, and mixed with a solubilized cell extract. Since the GTP-bound form binds to the target protein with high affinity, only the GTP-bound form can be selectively recovered. After this is separated on an SDS-PAGE gel, it is quantified by immunoblotting (Reference 3). However, all methods require the cells to be solubilized once, and there has been no method to directly examine the activation of low-molecular-weight GTP-bound proteins in living cells.

In recent years, it has been revealed that not only many cellular organelles exist in cells besides the cell membrane and cytoplasm, but also different biochemical phenomena occur in different places in the cytoplasm. It has also been shown that, at the individual level, low-molecular-weight GTP-binding proteins are very important for higher nerve function and organ formation. Therefore, non-invasive knowledge of the activation state of a low-molecular-weight GTP-binding protein in cells or individuals is essential not only for understanding biological phenomena but also for drug development. is there. However, since conventional biochemical methods solubilize cells, where in the cells the small GTP-binding protein is activated, and in which cells the small GTP-binding protein is active It was not possible to know if it had been converted.

 On the other hand, as a technique for visualizing proteins in living cells, a method using GFP (green fluorescent protein) is known (Reference 4). GFP is a group of proteins isolated from luminescent jellyfish and other proteins that emit mainly green fluorescence. Currently, it is widely used to investigate the localization of proteins in cells. GFPs include CFP (cyan-emitting mutant of GFP) and YFP (yellow-emitting mutant of GFP), and improved proteins such as EGFP (enhanced green fluorescent protein) and ECFP (enhanced green fluorescent protein) CFP), EYFP (enhanced YFP), EBFP (enhanced blue-emitting mutant of GFP) and the like (in the present specification, these are collectively referred to as GFP-related proteins). Each of these is excited by light of a different wavelength and emits fluorescence of a different wavelength.

 Further, there is a technology using FRET (fluorescent resonance energy transfer) as a technology applying GFP (Reference 5). F R E T refers to the following phenomena. It is assumed that fluorescent substances A and B are excited by laex and Ibex, respectively, and emit light of iae ra and Ibem, respectively. At this time, when A and B are very close to each other; when em is sufficiently close to ex, when the mixture of A and B is irradiated with light of λ & β エ ネ ル ギ ー, the energy of the substance Α is absorbed by I; Is observed. This is called FRET. Using this method, the distance between two molecules can also be estimated. At this time, the fluorescent substance A is called donor and the fluorescent substance B is called receptor.

Furthermore, as an application of this technology, it is possible to detect protein structural changes by labeling two fluorescent substances within one protein. Two sets of GFP-related proteins, EBFP and EGFP, ECFP and EYFP, It is known to make donor and receptor combinations for optimal FRET. For example, it is known that the concentration of calcium can be measured by applying this FRET technology to a fusion protein of two proteins, EBFP and EGFP, and the calcium-binding protein calmodulin (Reference 6). However, the measurement method using a single-molecule monitor applying the GFP protein and FRET technology has not been successful at present except for the aforementioned calcium measurement and the measurement of the activity of cyclic AMP-dependent kinase. Disclosure of the invention

 The present invention relates to a protein for monitoring the activity of a low-molecular-weight GTP-binding protein, which enables non-invasive measurement of the activation of a low-molecular-weight GTP-binding protein; a gene encoding the protein; an expression vector containing the gene; Transformed cells and transgenic animals carrying the expression vector that express the protein and are useful for measuring the activation of non-invasive low molecular weight GTP-binding proteins; and the use of the protein. A method for measuring the activation of a low-molecular-weight GTP-binding protein, more specifically, a method for measuring the amount ratio of GTP-binding to GDP-binding low-molecular-weight GTP-binding protein that can be used in living cells; and low-molecular-weight GTP-binding It is an object of the present invention to provide a method for screening a protein activity modulator.

 That is, the gist of the present invention is:

 [1] All or part of the low-molecular-weight GTP-binding protein, all or part of the target protein of the low-molecular-weight GTP-binding protein, all or part of the GFP protein, and GFP donor protein All or a part of the activity monitor of a low molecular weight GTP binding protein consisting of a fusion protein directly or indirectly linked in a state capable of exerting the function of each protein,

(2) a gene encoding the activity monitor protein of the low-molecular-weight GTP-binding protein according to (1), (3) an expression vector containing the gene according to (2),

 (4) a transformed cell holding the expression vector according to (3),

[5] a transgenic animal carrying the expression vector according to the above [3]

(6) a method for measuring activation of a low-molecular-weight GTP-binding protein, comprising a step of monitoring FRET in the activity monitor protein of the low-molecular-weight GTP-binding protein according to (1),

 (7) a method for measuring activation of a low-molecular-weight GTP-binding protein comprising a step of detecting FRET in the cell according to (4) or the transgenic animal according to (5), and

 (8) (a) contacting the test substance with the cell according to (4), and (b) detecting a change in activity of the low-molecular-weight GTP-binding protein,

And a method of screening for an activity modulator of a low-molecular-weight GTP-binding protein. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows a mechanism for controlling the activity of a low molecular weight GTP-binding protein. In this figure, Ras is taken as an example of a low molecular weight GTP-binding protein, and the activity control mechanism of the low molecular weight GTP-binding protein is schematically shown. The low molecular weight GTP-binding protein is inactive when bound to GDP, and when guanine nucleotide exchange factor (GEF) acts on it, GDP is replaced by GTP and becomes activated. The activated GTP-binding protein undergoes a conformational change, binds to its specific target protein, and becomes able to activate it. Activated low molecular weight GTP-bound protein is hydrolyzed to GDP in the presence of GTP hydrolyzing enzyme (GAP), releasing inorganic phosphate (Pi) and returning to its inactive form .

Fig. 2 shows a method for measuring the activation of GTP-bound protein with low molecular weight using FRET. The principle of is shown. In this figure, Ras is taken as an example of a low molecular weight GTP-binding protein, and Raf is taken as an example of a target protein. CFP (cyan-emitting mutant of GFP) exemplified as a GFP donor protein is excited by light at 433 nm and emits light having a maximum at 475 nm. On the other hand, YFP (yellow-emitting mutant of GFP) exemplified as a GFP receptor protein is excited by light at 505 nm and emits light having a maximum at 530 nm. In the present invention, these can also be used as GFP receptor protein and / or GFP donor protein. As shown in the lower diagram in Fig. 2, before the activation of Ras, the monitor—in the protein, the YFP at the amino terminal and the CFP at the carboxyl terminal are separated, so that There is little energy transfer to the country. However, when some kind of stimulus is received [for example, the addition of epidermal growth factor (EGF)], Ras becomes activated and binds to the Ras-binding domain (RBD) of the target protein Raf. Comes to the vicinity, and as a result, the energy transfer from CFP to YFP and the accompanying 530 nm fluorescence from YFP are observed. Therefore, the activation of Ras can be measured by measuring the FRET efficiency before and after stimulation (ie, before and after the activation of Ras).

 FIG. 3 shows the structure of plasmid pRafras1722. The expression vector used was PCAGGS, which has already been reported. In the figure, EYFP—Ras_RafRBD (Ras binding region) —a cDNA coding for the fusion protein in the order of ECFP was bound downstream of the CAG site α 乇.

 FIG. 4 shows the nucleotide sequence and predicted amino acid sequence of the translation region of plasmid pRafras1722.

 FIG. 5 shows the nucleotide sequence of the translation region of plasmid pRafras1722 and the predicted amino acid sequence (continued).

Figure 6 shows the nucleotide sequence and prediction of the translation region of plasmid p Rafras1722. Indicates the measured amino acid sequence (continued).

 FIG. 7 shows the fluorescence profile of the expressed protein Rafras 1722. HEK 293 cells were transfected with 1 ^ & fras 1 722 and guanine nucleotide exchange factor Sos expression vector (pCAGGS-mSos) or GTP hydrolytic enzyme Gap lm expression vector (pEF-Bos-Gap lm). After transfection by the calcium phosphate method, the cells were solubilized after culturing at 48 hours, and the supernatant was obtained after centrifugation. The fluorescence intensity of the supernatant at an excitation wavelength of 433 nm and a wavelength of 450 nm to 550 nm was measured with a fluorescence spectrophotometer. Sos in the right box of the graph shown in Fig. 7 indicates the fluorescence profile of Rafras 1722 when both pRafras 1722 and pCAGGS-mSos were transfected, and Gap lm indicates that pRafras 1722 and pEF- This shows the fluorescence profile of Rafras 1722 when Bos-Gap 1 m was transfected together.

Figure 8 shows the ratio of GTP to GDP on the GTP-binding protein of the expressed protein Rafras 1722 [GTP / (GDP + GTP) (%)] versus the excitation wavelength of 433 nm at 433 nm and 530 nm. Shows the fluorescence intensity ratio (wavelength 530/475). In HEK 293 T cells, pRafras 1722 and various amounts of guanine nucleotide exchange factor Sos expression vector (pCAGGS-mSos) or GTP hydrolysis enzyme Gap lm expression vector (pEF-Bos-Gap lm) ) Was transfected. ³² P i and labeled after 48 hours of culture, the Ra fras 1 72 2 after immunoprecipitation with anti-GFP antibody, separating the G TP and GDP bound to Ra fras 1722 by thin layer chromatography and quantified. On the other hand, the fluorescence profile of the cell lysate treated in the same manner was measured, and the fluorescence intensity ratio between the wavelength of 475 nm at the excitation wavelength of 433 nm and the wavelength of 530 nm was measured. It can be seen that the fluorescence intensity ratio is enhanced depending on the amount of GTP on Rafras1722.

Figure 9 shows that a cell line expressing the expressed protein Rafras 1722 was obtained. Indicates that 1 ^ & fras 172 2 was transfected into N 1 ^ 13 cho3 cells to establish the cell line 3T3-Rafras. The cells were solubilized and analyzed for Rafras722 expression by immunoblotting using an anti-GFP antibody. The molecular weight is shown on the left side of the imnotlotting shown in FIG.

 FIG. 10 shows an analysis of Ras activation using 3T3-Rafras cells.

3 T 3 -Ra fr a s cells were stimulated with EGF (1 ja g / m \),

The fluorescence profile (wavelength 450 ηπ! ~ 550 nm) excited at a wavelength of 3 nm was measured.

 FIG. 11 shows the structure of plasmid pR ai — c hu 311. The structure of the backbone vector is the same as in Fig. 3.

 FIG. 12 shows the nucleotide sequence and predicted amino acid sequence in the translation region of plasmid pRa i — chu311.

 FIG. 13 shows the nucleotide sequence and predicted amino acid sequence (continued) in the translation region of plasmid pR ai—c hu31 1.

 FIG. 14 shows the nucleotide sequence and the predicted amino acid sequence (continued) in the translation region of plasmid pRa i—chu311.

 FIG. 15 shows the fluorescence profile of the expressed protein R ai -c hu 311. HEK293 T cells have pRa i-chu31 1 and guanine nucleotide exchange factor C3G expression vector (pCAGGS-C3G; described in Reference 9) or GTP water-degrading enzyme rap 1 GAP II The expression vector (p CAGGS-rapl GAP II; described in Reference 9) was transfected with the calcium phosphate method, and after culturing for 48 hours, the cells were solubilized and centrifuged to obtain a supernatant. Excitation wavelength for the supernatant

At 433 nm, the fluorescence intensity at a wavelength of 450-550 nm was measured with a fluorescence spectrophotometer. C 3 G in the right box of the graph shown in FIG. 15 is the fluorescence profile of R ai-c hu 31 1 when both pR ai — c hu 31 1 and pCAGGS — C 3 G were transfected Rap 1 GAP II is p This shows the fluorescence profile of Rai-chu311 when both Rai-chu311 and pCAGGS-raplGAPII were transfected.

 FIG. 16 shows the structure of plasmid pRa i-chu 158. The structure of the backbone vector is the same as in Fig. 3.

 FIG. 17 shows the nucleotide sequence and predicted amino acid sequence in the translation region of plasmid pRa i-chu 158.

 FIG. 18 shows the nucleotide sequence and predicted amino acid sequence (continued) of the translation region of plasmid pRa i-chu158.

 FIG. 19 shows the nucleotide sequence and predicted amino acid sequence (continued) of the translation region of plasmid pRa i-chu158.

 FIG. 20 shows the fluorescence profile of the expressed protein Rai-chu158. HEK 293 T cells contain pRa i-chu 158 and a guanine nucleotide exchange factor Cal DAG-GEF III expression vector (pCAGGS-CalDAG-GEF III; described in Reference 10) or GTP lmmolysis promoting enzyme Gap lm expression vector (PEF-Bos-Gap lm) was transfected by the calcium phosphate method. After culturing for 48 hours, the cells were solubilized and centrifuged to obtain a supernatant. The fluorescence intensity of the supernatant at an excitation wavelength of 433 nm and a wavelength of 450 nm to 550 nm was measured with a fluorescence spectrophotometer. Gap 1 m in the right box of the graph shown in FIG. 20 indicates the fluorescence profile of Ra i-chu 158 when both pRa i-chul 58 and pEF-Bos-Gap lm were transfected. DAG-GEF III shows the fluorescence profile of Rai-chu 158 when both pRa i-chul 58 and pCAGGS-Cal DAG-GEF III were transfected.

FIG. 21 shows the nucleotide sequence and predicted amino acid sequence in the translation region of the plasmid pRa i—chu119. FIG. 22 shows the nucleotide sequence and predicted amino acid sequence (continued) of the translation region of plasmid pRa i—chu119.

 FIG. 23 shows the nucleotide sequence and predicted amino acid sequence (continued) of the translation region of plasmid pRa i-chu119.

 FIG. 24 shows the fluorescence profile of the expressed protein Rai-chu119. HEK 293 cells were transfected with 1¾ & i-cu hu 119 or pRafras 1722 and guanine nucleotide exchange factor S os expression vector (pCAGGS-mSo s) by the calcium phosphate method, and cultured for 24 hours. After transferring at 33 and 40 ° C and culturing for an additional 24 hours, the cells were solubilized and centrifuged to obtain a supernatant. The fluorescence intensity of the supernatant at an excitation wavelength of 433 nm and a wavelength of 450 nm to 550 nm was measured with a fluorescence spectrophotometer. The control in the right box of the graph shown in Fig. 21 is the fluorescence profile of Rai-chu119 when both pRafras 1722 and pCAGGS-mSos were transfected. This shows that this is the fluorescence profile of Rai-chu119 when pRai-chu119 and pCAGGS-mSos are transfected together. Ra i-chu 1 19 had increased reactivity to the guanine nucleotide exchange factor compared to the wild type (Rafras 17 22). FIG. 25 shows the results of the change over time in the fluorescence intensity of ECFP and EYFP in cells by the addition of epidermal growth factor (EGF). Using the fluorescence microscope system described in Example 1, excitation light having a wavelength of 430 nm was irradiated to obtain images at fluorescence wavelengths of 475 nm and 530 nm over time, and ECFP and EY FP were obtained from the images. The fluorescence intensity was determined.

FIG. 26 is a graph showing changes in the fluorescence intensity ratio between ECFP and EYFP in the expressed protein Rafrasl722 by various guanine nucleotide exchange factors and GTP hydrolysis promoter. HEK293T cells were transfected with pRafrasl722 and a guanine nucleotide exchange factor expression vector or a GTP hydrolysis promoter expression vector by the calcium phosphate method. After transfection and culturing for 24 hours or more, the cells were solubilized and centrifuged to obtain a supernatant. The fluorescence intensity of the supernatant was measured at an excitation wavelength of 433 nm, a wavelength of 475 nm, and a fluorescence intensity of 530 nm using a fluorescence spectrophotometer. The ratio of the latter to the former (fluorescence intensity ratio) is shown in the graph.

 FIG. 27 is a graph showing changes in the fluorescence intensity ratio between ECFP and EYFP in the expressed protein Rai-chu404 by various guanine nucleotide exchange factors and GTP hydrolysis enzymes. HEK293T cells were transfected with pRai-chu404 and a guanine nucleotide exchange factor expression vector or GTP hydrolysis promoter vector by the calcium phosphate method.After culturing for 24 hours or more, the cells were solubilized and centrifuged. To obtain a supernatant. The fluorescence intensity of the supernatant was measured at an excitation wavelength of 433 nm and at wavelengths of 475 nm and 530 nm using a fluorescence spectrophotometer. The ratio of the latter to the former (fluorescence intensity ratio) is shown in the graph.

FIG. 28 is a photograph showing the time-dependent change in the fluorescence intensity ratio of EYFP to ECFP and the intracellular distribution in C0S1 cells transfected with pRai-chulOIX or pRai-chu404X due to the addition of EGF. The C0S1 cells were transfected with pRai-chulOIX or pRai-chu404X. After culturing for 24 hours or more, the medium was replaced with a medium free of phenol red and serum. Next, an inverted fluorescence microscope with a xenon light source (Carl Zeiss, Axiovert 100) is equipped with a rotating fluorescence excitation filter and a rotating fluorescence emission filter device (LUDL electronic), and a high-sensitivity cooled CCD camera (P hotometrix). The image was acquired using a system that can be controlled and analyzed by Metamorph image analysis software manufactured by Nippon Koupa Co., Ltd. The cells were irradiated with excitation light at 430 nm, and an image at the fluorescence wavelength of the ECFP donor at 475 nm was taken with a CCD camera, followed by an image at the fluorescence wavelength of the 530 thigh EYFP receptor. This was performed at 30 second intervals. After data acquisition, the EYFP / ECFP fluorescence intensity ratio was divided into eight levels for each pixel on the digital image, and colors from blue to red were assigned. On the other hand, video images can be created by assigning ECFP fluorescence intensity as brightness. . In this figure, the images at the times shown in these figures are shown. When EGF is added to Rai-chulOIX-expressing cells, a process is observed in which the fluorescence intensity ratio, which reflects the FRET efficiency, gradually increases from the cell edge toward the center. On the other hand, in the cells expressing Rai-chu404X, the activity increases from the center toward the periphery. As described above, the activity monitor protein of the present invention makes it possible to simultaneously obtain both temporal information on the activation of Ras family G protein and spatial information in a cell.

FIG. 29 is a photograph showing the time-dependent change in the fluorescence intensity ratio of EYFP to ECFP and the intracellular distribution in subconfluent C0S1 cells transfected with pRai-chulOIX by addition of EGF. The experiment was performed by the same method as described in the explanation of FIG. 28, except that C0S1 cells in a subconfluent state were used. When EGF was added to Rai-chulOIX-expressing cells, the process of increasing the fluorescence intensity ratio, which reflects the FRET efficiency, from the margin that did not adhere to other cells was observed. On the other hand, it can be seen that this increase in FRET efficiency is suppressed at the site where it adheres to the adjacent cells. FIG. 30 is a photograph showing a time-dependent change in the fluorescence intensity ratio of EYFP to ECFP and intracellular distribution in PC12 cells transfected with pRai-chulOIX or pRai-chu404X due to the addition of nerve growth factor. Transfect pRai-chulOIX or pRai-chu404X into PC12 cells, and after culturing for 24 hours or more, replace the medium with a medium free of phenol red and serum, and add nerve growth factor. The observation was performed in the same manner as described in the description of FIG. In this figure, only the image at the time shown in the figure among the continuous images is shown. When nerve growth factor was added to Rai-chulOIX-expressing cells, a process was observed in which the fluorescence intensity ratio, which reflects the FRET efficiency, gradually increased from the periphery of the cells toward the center. After 180 minutes, when the development of neurites is apparent, the increase in FRET efficiency is mainly limited to these neurites. On the other hand, in cells expressing Rai-chu404X, the activity increases from the central part to the peripheral part, and the FRET effect is increased in differentiated neurites. It can be seen that the rate is suppressed. This indicates that Ras is activated from the cell periphery and Rapl is activated from the center of the cell during the induction of differentiation, and that Ras maintains high activity in neurites after differentiation is completed. Is shown. This demonstrates that Ras family G proteins are in different activation states at different sites in the cell, and the activity monitor proteins of the present invention have a temporal and spatial relationship with the Ras family — G protein activation. This indicates that it is the optimal molecular probe for obtaining relevant information.

 FIG. 31 shows the structure of plasmid pRai-chulOllX. The structure of the backbone vector is the same as in Fig. 3.

 FIG. 32 shows the structure of plasmid pRai-chul054X. The structure of the backbone vector is the same as in Fig. 3.

 FIG. 33 shows the structure of plasmid pRai-chul214X. The structure of the backbone vector is the same as in Fig. 3.

 FIG. 34 shows the fluorescence profiles of the expressed proteins Rai-chulOllx (wild-type), Rai-chul012X (activated), and Rai-chul013X (non-activated). The <293> cells were transfected with pRai-chulOllx, pRai-chul012X, or pRai-chul013X by the calcium phosphate method, and after 48 hours, the cells were lysed and centrifuged to obtain a supernatant. At a wavelength of 433 nm, the fluorescence intensity at a wavelength of 450 nm to 550 nm was measured with a fluorescence spectrophotometer.

 FIG. 35 shows the fluorescence profiles of the expressed proteins Rai-chul054x (wild type) and Rai-chul052X (activated type). HEK293T cells were transfected with pRai-chul054x or pRai-chu 1052X by the calcium phosphate method. 48 hours later, the cells were solubilized and centrifuged to obtain a supernatant. The fluorescence intensity of the supernatant at an excitation wavelength of 433 nm and a wavelength of 450 mn to 550 nm was measured with a fluorescence spectrophotometer.

FIG. 36 shows the fluorescence profiles of the expressed proteins Rai-chul214X (wild type) and Rai-chul220X (activated). PRai-chul214X or pRai in HEK293T cells -chul220X was transfected by the calcium phosphate method, and after 48 hours, the cells were solubilized and centrifuged to obtain a supernatant. The fluorescence intensity of the supernatant at an excitation wavelength of 433 nm and a wavelength of 45 Onm to 550 nra was measured with a fluorescence spectrophotometer.

 FIG. 37 is a photograph showing the time-dependent change in the fluorescence intensity ratio of EYFP to ECFP and the intracellular distribution in C0S1 cells transfected with pRai-chulOllX by addition of EGF. An experiment was performed in the same manner as described in the description of FIG. When EGF is added to Rai-chulOllX-expressing cells, the fluorescence intensity ratio that reflects the FRET efficiency transiently increases throughout the cell within one minute, and then the fluorescence intensity ratio at the site where cell membrane ruffling occurs. And a decrease in the fluorescence intensity ratio at the center. This is a result that shows a different time course and spatial distribution from the activation of Ras examined using Rai-chulOIX and the activation of Rapl examined using Rai-chu404X. This indicates that the proteins are optimal molecular probes for obtaining temporal and spatial information on the activation of Rho family GTP-binding proteins. BEST MODE FOR CARRYING OUT THE INVENTION

The activity monitoring protein of the low molecular weight GTP-binding protein of the present invention (hereinafter referred to as "monitor protein") utilizes the property that the GTP-binding low-molecular-weight GTP-binding protein specifically binds only to its target protein. It is a very useful protein for measuring non-invasive activation of small GTP-binding proteins. The monitor protein of the present invention is a fusion protein comprising a low molecular weight GTP-binding protein, a target protein of the low molecular weight GTP-binding protein, a GFP receptor protein, and a GFP donor protein. That is, the respective proteins are directly or indirectly linked in such a state that the original conformations are individually formed and the functions of the respective proteins can be fully exhibited. Therefore, the amino acid sequence of such a fusion protein The rows have a structure in which the amino acid sequence portions of the proteins are directly or indirectly linked. Each protein constituting the monitor protein of the present invention may be a part of the protein, as long as the function of the protein can be fully exhibited.

 In the present specification, when referring to each protein contained in a monitor protein, for example, in the case of a target protein, for example, the target protein is distinguished from the target protein itself, and the target protein portion is simply referred to. Expressed as the target protein.

 In the monitor protein of the present invention, activation of a low-molecular-weight GTP-binding protein by binding to GTP (including activation of a low-molecular-weight GTP-binding protein by conversion of a GDP-binding protein to a GTP-binding protein by a guanine nucleotide exchange factor) As a result, the low-molecular-weight GTP-binding protein and its target protein bind in the monitor protein, resulting in a change in the FRET efficiency from the GFP donor protein to the GFP receptor protein. FIG. 2 schematically shows an example of the monitor protein of the present invention, and shows the principle of a method for measuring the activation of a low molecular weight GTP-binding protein using FRET using the monitor protein. In the present specification, the FRET efficiency refers to the fluorescence intensity at the fluorescence wavelength of the GFP donor protein and the fluorescence of the GFP receptor protein when the monitor protein of the present invention is irradiated with excitation light for the GFP donor protein. The ratio with the fluorescence intensity at the wavelength (fluorescence intensity ratio). Details will be described later.

To achieve FRET, i) the emission spectrum of the GFP donor overlaps the absorption spectrum of the GFP receptor, ii) the distance between the donor and the receptor, iii) the donor's luminescence moment and the receptor The three factors of the orientation of the extinction moment must be taken into account. Also, when GFP is fused with another protein, the fusion of the GFP with another protein causes stress, resulting in GFP misfolding. As a result, the efficiency of chromophore formation is reduced, and non-fluorescent GFP is used. Possible Sex must also be considered. As described above, there are strict conditions for using the GFP donor and the GFP receptor to produce good expression of FRET between the two, and certain rules have been found for the arrangement between the two. Nonetheless, realizing FRET is generally difficult. That is, realization of FRET is not something that can be easily achieved based on the common general technical knowledge, but requires trial and error that is more than expected and complicated experiments. The monitor protein of the present invention is obtained by appropriately combining the above proteins so that the desired effect of the present invention can be obtained, and the GTP-bound low-molecular-weight GTP-binding protein specifically binds only to its target protein. By utilizing the properties of GTP when bound, FRET is realized between the GFP donor protein and the GFP receptor protein, which can be changed according to the binding of GTP to low molecular weight GTP-binding protein. Its technical value is very large.

The order of binding of the constituent proteins in the monitor protein of the present invention is appropriately selected in consideration of the increase in the difference in FRET efficiency before and after activation of the low molecular weight GTP-binding protein (hereinafter simply referred to as the difference in FRET efficiency). Can be done. The greater the difference in FRET efficiency before and after activation of the low molecular weight GTP-binding protein, the more accurately the activation state of the protein can be grasped, thus improving the measurement accuracy of the activation of the low molecular weight GTP-binding protein. It is preferable because it can be performed. In a preferred embodiment, the low-molecular-weight GTP-binding protein and the low-molecular-weight GTP-binding protein in the monitor protein are bound to the target protein of the low-molecular-weight GTP-binding protein at the target protein binding site of the low-molecular-weight GTP-binding protein present on the amino-terminal side. An embodiment in which the carboxy terminal is directly or indirectly bound to the amino terminal of the target protein present on the carboxyl terminal side (1), wherein the carboxyl terminal of the target protein present on the amino terminal side is present on the carboxyl terminal side (2) that is directly or indirectly bound to the amino terminus of the target protein binding site of the low molecular weight GTP-binding protein. In particular, when the low-molecular-weight GTP-binding protein belongs to the family of Ras family, the embodiment (1) is the one belonging to the family of Rho family. In this case, embodiment (2) is preferred. The GFP acceptor protein and GFP donor protein have their amino or carboxyl termini directly or indirectly to the amino or carboxyl terminus of a product in which a low molecular weight GTP-binding protein and a target protein are linked (linked product). And connected. Among them, a monitor protein in which the carboxyl terminal of the GFP receptor protein is directly or indirectly connected to the carboxyl terminal of the GFP receptor protein at the amino terminal of the ligated product is preferable. Therefore, as the monitor protein of the present invention, in the monitor protein, a GFP receptor protein, a low molecular weight GTP binding protein, a target protein of the low molecular weight GTP binding protein, and a GFP dona protein from the amino terminal side. Or directly or indirectly from the amino terminus side to become the GFP receptor protein, the target protein of the low molecular weight GTP binding protein, the low molecular weight GTP binding protein, and the GFP donor protein, respectively. Those connected are particularly preferred. The “indirect linking” refers to a mode in which the linking between proteins is performed, for example, via a peptide or the like as a spacer described later.

 The low-molecular-weight GTP-binding protein that is a component of the monitor protein of the present invention is not particularly limited as long as it is known as the protein, but it belongs to the Ras superfamily from the viewpoint of usefulness. Are preferable, and among them, those belonging to the Ras family or the Rho family are more preferable. More specifically, the group consisting of H—Ras, K—Ras, N—Ras, R—Ras, Rap1A, RapB, Rap2A, and Rap2B, or RhoA, R One selected from the group consisting of hoB, RhoC, Rac1, Rac2, and Cdc42 is preferred.

On the other hand, the target protein of the low-molecular-weight GTP-binding protein is not particularly limited as long as each low-molecular-weight GTP-binding protein specifically binds to the GTP-binding protein as exemplified above. . Preferred from the viewpoint of usefulness Is Ra f or Ra 1 GDS, or Pak or mDia.

 Further, as a combination of the low-molecular-weight GTP-binding protein and the target protein, from the viewpoint of utility and specificity, a combination in which the low-molecular-weight GTP-binding protein is H-Ras and the target protein is Raf, or A combination in which the low-molecular-weight GTP-binding protein is Rap1A and the target protein is Ra1GDS, a combination in which the low-molecular-weight GTP-binding protein is Rac1 and the target protein is Pak, and a low-molecular-weight GTP-binding Particularly preferred is a combination in which the protein is Cdc42 and the target protein is Pak, or a combination in which the low molecular weight GTP-binding protein is RhoA and the target protein is mDia.

 As the GFP receptor protein, any of the GFP-related proteins exemplified above can be used, but from a functional viewpoint, EGFP or EYFP is preferable. Similarly, any one of the GFP-related proteins exemplified above can be used as the GFP donor protein, but from the functional viewpoint, it is preferably ECFP or EBFP.

Particularly preferred combinations of the above-mentioned components of the monitor protein of the present invention include, from the viewpoints of efficacy, specificity and sensitivity, the low molecular weight GTP-binding protein is H-Ras, and the target protein is R af, the GFP donor protein is ECFP, the GFP receptor protein is EYFP, or the low molecular weight GTP-binding protein is Rap1A, and the target protein is Ra1 GDS, GFP donor protein is ECFP, GFP receptor protein is EYFP, or low molecular weight GTP binding protein is Rac 1, target protein is Pak, GFP donor protein is ECFP, GFP If the receptor protein is EYFP, or the low molecular weight GTP-binding protein is Cdc42, the target protein is Pak, and the GFP donor protein is ECFP, GFP receptor-protein Is EYFP, or the low-molecular-weight GTP-binding protein is RhA, the target protein is mDia, the GFP donor protein is ECFP, and the GFP receptor protein is EYFP.

 In addition, the order of binding of the low-molecular-weight GTP-binding protein, the target protein, the GFP donor protein, and the GFP receptor protein is preferably determined in the monitor protein of the present invention from the viewpoint of increasing the difference in FRET efficiency. EYFP—H-Ras—Ra f—ECFP or EYFP—R ap 1 A—Ra l GDS—ECFP, or EYFP—Pak—R ac 1—ECFP, EYFP-Pak-Cdc 42— ECFP or EYF P—mDia-RhoA—ECFP. In these, those obtained by exchanging EYFP and ECFP for each other can also be suitably used.

The low-molecular-weight GTP-binding protein may be a part of the protein as long as it can bind to the target protein, and need not necessarily be the entire (full-length) protein. Here, a part of the low-molecular-weight GTP-binding protein is, for example, a method in which the protein molecule is produced in Escherichia coli according to a known method, and bound to GTP in a test tube, whereby the binding to the target protein can be detected. Refers to the protein part. The detection can be carried out, for example, by immunoprecipitation with an antibody against the target protein and examining by immunoblotting whether a part of GTP-bound protein is coprecipitated. For example, in the case of H—Ras and Raρ1A, a protein portion consisting of an amino acid sequence portion preferably corresponding to positions 1-180, more preferably positions 1-172 is represented by R-Ras. If present, preferably a protein portion comprising an amino acid sequence portion corresponding to positions 1 to 204, more preferably 28 to 204; if Rac 1, a protein portion preferably comprising an amino acid sequence portion corresponding to positions 1 to 177 If Cdc42, the amino acid sequence portion consisting of the amino acid sequence portion preferably corresponding to positions 1 to 176 if Cdc42, and the amino acid sequence preferably corresponding to positions 1 to 176 if Rh0A List the protein part consisting of Can be.

 On the other hand, truncating the amino or carboxyl terminus of the amino acid sequence, rather than the entire low molecular weight GTP binding protein, often results in an increase in the difference in FRET efficiency. Therefore, as a part of the protein, at least one, more preferably 1 to 28, and still more preferably 17 to 28 amino acid sequences are preferably present in the amino terminal region and / or the carboxyl terminal region of the amino acid sequence. And those having an amino acid deficiency. The amino acid deletion site in such a region is not particularly limited. For example, in the case of H-Ras, the difference in FRET efficiency was greater when the C-terminus was truncated to position 172 than when it was truncated to position 180. That is, the amino acid sequence preferably has at least one, more preferably 9 to 20, and more preferably 17 amino acids in the carboxyl-terminal region of the amino acid sequence. Also, in the case of R-Ras, the difference in FRET efficiency was greater when the 28 amino acids were deleted from the amino terminal than when the amino acid was not deleted. That is, the amino acid sequence preferably has a deletion of at least 1, more preferably 1 to 28, and even more preferably 28 amino acids in the amino terminal region of the amino acid sequence.

 The amino-terminal region or lipoxyl-terminal region refers to a region, preferably up to 30 amino acids in number, from the amino terminal or carboxyl terminal in the amino acid sequence of the low molecular weight GTP-binding protein.

In addition, the target protein may be a part of the corresponding low molecular weight GTP-binding protein as long as it can bind to the corresponding low molecular weight GTP-binding protein, and does not necessarily need to be the whole (full length). Here, the part of the target protein refers to a protein part in which the binding to the corresponding low molecular weight GTP-binding protein can be detected in the same manner as in the low molecular weight GTP-binding protein. For example, if it is Ra f (GenBank / EBL terminology number: X03484), it is preferably the Ras binding region (RBD), more preferably, the position 51 to 204, more preferably the position 51 to 204. 1 3 1st place If the protein portion comprising the amino acid sequence portion is Ra1 GDS (GenBank / EMBL accession number: U14417), an amino acid sequence corresponding to preferably positions 202 to 309, more preferably positions 211 to 297 Pak1

(GenBank / EMBL completion number: Sat 02576), preferably, a protein region consisting of an amino acid sequence portion corresponding to the Rac1 binding region, more specifically, the amino acid sequence portion corresponding to positions 68 to 150, is mDia1 (GenBank / EMBL accession number: E17361), preferably a protein portion comprising an amino acid sequence portion corresponding to the Rho binding region, more specifically, positions 68 to 240, and more preferably positions 68 to 180. be able to. On the other hand, the GFP donor protein and / or the GFP receptor protein may be a part of the protein as long as the function of pairing with FRET is maintained, and it is not necessarily required to be all (full length). Frequently, shortening the carboxyl termini of their amino acid sequences results in increased differences in FRET efficiency. For example, as a part of the GFP receptor protein and / or the GFP donor protein, those having preferably at least one, more preferably 1 to 11 deletions in the carboxyl terminal region of their amino acid sequences. Can be mentioned. The amino acid deletion site in such a region is not particularly limited. For example, in the case of EYFP, it is preferably at least one, more preferably 1-11, and still more preferably in the carboxyl terminal region of the amino acid sequence.

Those having a deletion of one amino acid are preferred. In the case of ECFP, it is preferable that the amino acid sequence has a deletion of at least one, more preferably 1 to 11, and even more preferably 11 amino acids in the carboxyl terminal region of the amino acid sequence. Here, the carboxyl terminal region refers to the amino acid sequence of the GFP-related protein used in the present invention, from its lipoxyl end to the number of amino acids, preferably from 1 to 20, more preferably 11 The area up to. Whether the FRET pair function is maintained or not is determined by, for example, a pair of protein molecules that are assumed to form a FRET pair according to a known method. Can be produced in Escherichia coli, and the cell extract containing the pair of proteins can be evaluated by observing the fluorescence intensity at the expected excitation wavelength of each of the proteins.

 Further, the GFP receptor protein and / or the GFP donor protein may have a mutation. Such a mutation can be introduced into any site in the amino acid sequence of the GFP receptor protein and / or the GFP donor protein, as long as the function of pairing with FRET is maintained. For example, mutations include substitution of a plurality of amino acids. Specific examples of such amino acid substitutions include, for example, Phe64Leu, Va168Leu, Ser72Ala, Ile67Thr and the like. Is mentioned. It is preferable to introduce such a variation since effects such as an increase in chromophore formation efficiency and an increase in FRET efficiency can be obtained. Mutation can be introduced by a method using a known restriction enzyme or a method using PCR (polymerase chain reaction).

In addition, low-molecular-weight GTP-binding proteins and / or their target proteins into which mutations have been introduced can also be suitably used in the present invention. For example, by introducing a point mutation, a mutant having improved sensitivity to guanine nucleotide exchange factor or GTPase activator can be obtained. Such a mutation can be introduced into any site in the amino acid sequence of the low molecular weight GTP-binding protein and / or its target protein as long as the function of binding to each other is maintained. For example, examples of the mutation include amino acid substitution, insertion, and deletion. Specifically, for example, an embodiment in which I 1 e 36 is changed to Leu in the amino acid sequence of H—Ras (I 1 e 36 L eu). Such mutations in H-Ras make the H-Ras most sensitive to the GTPase activator, among many mutations. As a result, the dynamic range of the monitor protein can be changed. H-Ras having such a mutation can be suitably used in the monitor protein of the present invention. . The mutation can be introduced by a method using a known restriction enzyme or a method using PCR.

 In the monitor protein of the present invention, the spatial arrangement of the constituent proteins is a factor related to the expression of their functions. By changing such an arrangement, the difference in FRET efficiency can be greatly increased. For example, it is possible to adjust the difference in FRET efficiency by inserting a peptide sequence serving as a spacer between the constituent proteins in the monitor protein. Such a spacer is preferably inserted between the low-molecular-weight GTP-binding protein and the target protein from the viewpoint of increasing the difference in FRET efficiency. Preferable peptide sequences include peptides consisting of preferably 1 to 30, more preferably 1 to 10 consecutive arbitrary amino acids. When such a peptide is inserted between the low-molecular-weight GTP-binding protein and the target protein, it is expected that the difference in FRET efficiency will increase, and that the efficiency of folding of the GFP-related protein itself will increase. In addition, from the viewpoint that each constituent protein can take an appropriate conformation in the monitor protein of the present invention, it is preferable to use an amino acid having a property that it is difficult to form a secondary structure with a small molecule mainly composed of glycine. It is preferable to use such a peptide as a sensor.

 It is also a preferred embodiment to fuse another protein or peptide to the amino terminal and / or carboxyl terminal of the amino acid sequence of the monitor protein of the present invention. In particular, by adding an intracellular localization signal, such as a known ER (local endoplasmic reticulum) (ER) translocation signal and a cell membrane localization signal, to the monitor protein, the GTP-binding protein is localized in the cell. Activation can be directly measured, which is preferable. In addition, as will be described later, it is also possible to directly measure the amount ratio (GTPZGDP ratio) (molar ratio) of GTP-bound to GDP-bound GTP-bound proteins in a cell locally.

In the monitor protein of the present invention, GTP binds to the low molecular weight GTP-bound protein. When the protein is activated, the binding of the low-molecular-weight GTP-binding protein to the target protein is induced in the monitor protein, resulting in a change in the overall conformation, and the GFP receptor protein and the GFP donor protein are changed. The distance and the direction change. Then, irradiation with light of a specific wavelength causes the increase in FRET efficiency to be detected between such a protein and the donor protein (Fig. 2). Such changes in the FRET efficiency are affected by the arrangement of the GFP receptor protein and the GFP donor protein after the conformational change of the monitor protein. For example, the shorter the distance between the GFP donor protein and the GFP receptor protein, the higher the FRET efficiency, and the longer the distance, the lower the FRET efficiency. The width of the change in the FRET efficiency, that is, the increase or decrease in the difference in the FRET efficiency can be appropriately adjusted as desired by inserting a spacer peptide or the like, for example, depending on the properties of each constituent protein used.

 The above-described evaluation as to whether the monitor protein of the present invention can exhibit the desired effect of the present invention can be performed, for example, according to the method described in Example 1 described below. .

 The present invention also provides a gene encoding the monitor protein of the present invention. Such a gene is obtained by obtaining the genetic information of each of the constituent proteins of the protein from GenBank or the like, and using a known PCR method or a method using a restriction enzyme and a ligase according to a conventional method. Can be made.

 The accession numbers in GenBankZEMB L of each protein suitably used as a constituent protein of the monitor protein of the present invention are shown below. The accession number is shown in parentheses after each protein name.

 (1) Low molecular weight GTP-binding protein

HR as (V00574), KR as (L00045-L00049), NR as (L00040-L00043), RR as (M14948, M14949), Ra 1 A (X12533), R ap 1 B (X08004), Rap 2A (X12534) , Rap 2B (X52987), R ho A (L250 80), Rh o B (X06820), R ho C (X06821), R ac 1 (M29870), R ac 2 (NM002872), R ac 3 (NM005052), C dc 42 (M57298)

 (2) Target protein

 Raf (X03484), Ra1 GDS (U14417), Pak1 (NM002576), mDia1 (E17361)

 (3) GFP donor protein and GFP receptor protein

 EGFP (U76561), EYFP (AVU73901 1), ECFP (AB041904) EBFP (GFP has the following three mutations: Ph e 64

Leu, Tyr66His, and Tyr145Phe) are described in Ref.

 The present invention further provides an expression vector containing the gene. According to a known method, such a vector may be a known prokaryotic cell expression vector such as pGEX-2T (Amersham-Pharmacia Biotech), a eukaryotic cell expression vector such as pCAGGS (for example, pGEX-2T). Reference 7) or by insertion into a viral vector, for example, p Shutt 1 e (manufactured by CLONTECH). The expression vector is preferably an expression plasmid.

The present invention further provides transformed cells and transgenic animals that carry the expression vector. Such cells can be obtained by introducing the expression vector into target cells. A known transfection method or a virus infection method can be used as a method for introduction into cells, and there is no particular limitation. For example, a calcium phosphate method, a lipofection method, or an electoral poration method can be used. Eukaryotic cells or prokaryotic cells can be used as the cells, and there is no particular limitation. For example, eukaryotic cells include human embryonic kidney-derived HEK293 T cells, monkey kidney-derived COS cells, human umbilical cord-derived HUVEC cells, yeast, etc.Prokaryotic cells include cultured cells such as Escherichia coli, and other various cells. it can. Meanwhile, before Transgenic animals can be obtained by directly introducing the expression vector into an individual such as a mouse by a known method, for example, by microinjecting plasmid DNA into the nucleus of a fertilized mouse egg. it can.

 The present invention further provides a method for measuring the activation of a low molecular weight GTP binding protein using the monitor protein of the present invention. According to such a method, activation of the low molecular weight GTP-binding protein can be measured by detecting FRET in the monitor protein of the present invention. Alternatively, FRET can be detected in the above-described transformed cell or transgenic animal of the present invention, and the activation of a low-molecular-weight GTP-binding protein in the cell or animal can be directly measured. In such a case, the GTP-GDP ratio (or GTPZ (GDP + GTP-GTP) is measured separately by measuring the GTP-bound low-molecular-weight GTP-binding protein and the GDP-bound low-molecular-weight GTP-binding protein produced by the release of inorganic phosphate from GTP. ) Ratio) (all are molar ratios), and if the corresponding FRET efficiency is measured and a calibration curve is prepared in advance, the GTP ratio is calculated based on the FRET efficiency of the cell or animal. be able to.

 For example, the following method is specifically exemplified.

 (1) Measurement method using a spectrophotometer

The transformed cells of the present invention that can express the monitor protein are cultured under conditions that allow expression of the protein. Next, the cells are solubilized. The method for solubilizing the cells is not particularly limited, but a solubilization method using a solution containing the detergent TritonXlOO is preferable. The solubilized solution is irradiated with excitation light (eg, at a wavelength of 433 nm) for GFP donor protein, and the fluorescence profile is measured, for example, at a wavelength in the range of 45 Onm to 550 nm using a known fluorescence spectrophotometer. Based on the obtained fluorescence profile data, for example, the ratio of the fluorescence intensity of the GFP donor protein at a wavelength of 475 nm to the fluorescence intensity of the GFP receptor protein at a wavelength of 530 nm [(Fluorescence at a wavelength of 530 nm) Intensity) / (wavelength 47 Fluorescence intensity at 5 nm)] is calculated, and this is defined as the FRET efficiency from GFP donor protein to GFP receptor protein. FRET efficiency increases after binding (that is, after activation of the low-molecular-weight GTP-binding protein) compared to before GTP-binding to the low-molecular-weight GTP-binding protein. The activation of is measured. The activation and inactivation of the low-molecular-weight GTP-binding protein can be performed, for example, by using the guanine nucleotide exchange factor Sos expression vector (pCAGGS-mSos; described in Reference 9) as the monitor protein of the present invention. By transfecting cells capable of expressing

It can be performed by stimulating the cells with epidermal growth factor (EGF). For the latter, for example, the GTP hydrolyzing enzyme Gap lm expression vector (pEF-Bos-Gap lm; described in Reference 9) is used. This can be done by transfecting the cells. on the other hand, ? 1¾ £ ¾Efficiency is 0? Since the change is caused by a change in the distance and direction between the donor protein and the GFP receptor protein, a change in the structure of the monitor protein can be detected by a change in the FRET efficiency. (2) Measurement method using a microscope

 The transformed cells or transgenic animals of the present invention that express the monitor protein are observed with a fluorescence microscope, and changes in FRET efficiency that occur before and after activation of the low-molecular-weight GTP-binding protein are directly detected. The activation and inactivation of the low-molecular-weight GTP-binding protein can be performed in the same manner as in the above (1) measuring method using a spectrophotometer.

 There is no particular limitation on the fluorescence microscope to be used, but a high-sensitivity cooled CCD equipped with a rotating fluorescence excitation filter and a rotation fluorescence emission filter in a known inverted fluorescence microscope (Carl Zeiss, Axiovert 100) having a xenon light source Those with a camera are preferred. Further, it is desirable that the filter and the camera image be controlled and analyzed by using Metamorph image analysis software manufactured by Nippon Ichi-Par.

Irradiating said cells or animals with excitation light of a GFP donor protein; An image at the fluorescence wavelength of one protein is taken with a CCD camera, and then an image at the fluorescence wavelength of the GFP receptor is taken. The FRET efficiency at each measurement point can be calculated by measuring the ratio of the fluorescence intensities of both images. In addition, for example, the guanine nucleotide exchange factor Sos expression vector can be introduced into cells or animals capable of expressing the protein in various amounts to introduce various activation states (ie, activation) of the low molecular weight GTP-binding protein. States of varying degrees). Next, the cells or animals in each state are observed with a fluorescence microscope, and the FRET efficiency is determined in the same manner as described above. In addition, the cells in each state (including cells derived from the site for which FRET efficiency was obtained from the animal) were solubilized, and GTP-bound low-molecular-weight GTP-binding protein and GDP were separately bound. GTPZGDP ratio is calculated by measuring low molecular weight GTP-bound protein. Specifically, the GTPZGDP ratio is determined by measuring the amount of GTP bound to a low molecular weight GTP-binding protein and the amount of GDP bound by a known method (Reference 2). Then, the obtained GTP DP ratio is related to the FRET efficiency determined in advance. That is, the FRET efficiency and the GTPZGDP ratio at the measurement time point in each state are measured, and a calibration curve is created based on these. If a calibration curve is separately prepared in this way, the FRET efficiency in cells or animals expressing the monitor protein can be measured directly using a fluorescence microscope, and the GTP / GTP / It is possible to calculate the GDP ratio. Therefore, the activation state of the low-molecular-weight GTP-binding protein in a cell or an individual can be easily ascertained noninvasively, and the GTPZGDP ratio in such a state can be specifically obtained. The method using such a calibration curve can be similarly used in the method (1).

According to the present invention, there is provided a protein monitor for the activity of a low-molecular-weight GTP-binding protein that enables non-invasive measurement of the activation of the low-molecular-weight GTP-binding protein, a gene thereof, and the like. In addition, it expresses such a monitor protein and retains the expression vector useful for measuring the activation of a non-invasive low molecular weight GTP-binding protein. Transformed cells and transgenic animals are provided, as well as methods for measuring the activation of low molecular weight GTP binding proteins using the protein. Therefore, it is possible to non-invasively determine the activation state of the low-molecular-weight GTP binding protein in a cell or an individual, and not only to understand life phenomena, but also to develop drugs (for example, pain, autoimmune diseases, (A therapeutic or prophylactic agent for allergic diseases, etc.).

 Further, as another embodiment of the present invention, there is provided a method for screening a substance regulating the activity of a low molecular weight GTP binding protein. That is,

 (a) contacting a test substance with a transformed cell that expresses the activity monitor protein of the low-molecular-weight GTP-binding protein and that has an expression vector containing the gene of the protein; and

 (b) detecting a change in activity of the low molecular weight GTP binding protein,

It is intended to provide a method for screening a low-molecular-weight GTP-binding protein activity modulator, comprising:

 According to the screening method of the present invention, a substance that changes the activity of a low-molecular-weight GTP-binding protein is constructed by constructing a cell that expresses the activity monitor protein of the low-molecular-weight GTP-binding protein of the present invention and using a bioassay system. Alternatively, it is possible to efficiently screen for a salt thereof (ie, a modulator of the activity of a low molecular weight GTP-binding protein). The test substance in the method is not particularly limited, but examples include peptides, proteins, non-peptidic substances, synthetic substances, fermentation products, and the like.

The screening method of the present invention can be carried out in the presence of (i) a low molecular weight GTP-binding protein activator, or (ii) in the absence of the activator. The low-molecular-weight GTP-binding protein activator is a substance that activates a low-molecular-weight GTP-binding protein, and includes, for example, cell growth factors such as epidermal growth factor, and cytodynamics such as interleukin. Can also this It is not limited to them. In the case of (i), the activity regulating substance of the low-molecular-weight GTP-binding protein is a substance that enhances or reduces the activity of the low-molecular-weight GTP-binding protein, and in the case of (ii), the activity of the low-molecular-weight GTP-binding protein is It can be screened as a substance to enhance.

 Specifically, the screening method of the present invention is characterized in that, in the step (a), in the presence or absence of the activating substance, the cell expressing the activity monitor protein of the low-molecular-weight GTP-binding protein of the present invention is treated with the cell. The sample is brought into contact with the test substance (aspect 1). The method of contacting is not particularly limited, and can be performed, for example, by culturing the cells in the presence of a test substance. In parallel, as a control, under the same conditions, the activity monitor of the low-molecular-weight GTP-binding protein of the present invention is performed (case 2), where the cells expressing the protein are not brought into contact with the test substance. Next, in step (b), the activity of the low-molecular-weight GTP-binding protein in each case is measured, and the change in the activity of the low-molecular-weight GTP-binding protein in embodiment 1 compared to embodiment 2 is detected, whereby the low-molecular-weight GTP-binding protein is detected. Screen for protein activity modulators. The activity can be measured by measuring the FRET efficiency in each case.

 That is, in the case of the above (i), the substance that further enhances the activity of the low-molecular-weight GTP-binding protein is an activity-regulating substance that can enhance the activity of the low-molecular-weight GTP-binding protein. A reducing agent is an activity modulator that can decrease the activity of a low molecular weight GTP binding protein. In the case of the above (ii), the substance that enhances the activity of the low-molecular-weight GTP-binding protein is an activity modulator that can enhance the activity of the low-molecular-weight GTP-binding protein.

By the above method, a substance that regulates the activity of a low-molecular-weight GTP binding protein can be obtained simply and quickly. The following is a list of references described in this specification. Such references are incorporated by reference in their entirety. In this specification, the reference number of each reference referred to as [reference (number)] is shown.

 1. Bos, J.L. 1997. "Ras-like GTPases." Biochim. Biophys. Acta 1333: M19-M31.

 2. Satoh, T. and Y. Kaziro. 1995. "Measurement of Ras-bound guanine nucleotid e in stimulated hematopoietic cells." J Method. Enzymol. 255 : 149-155.

. 3. Franke, B., JWN Akkerman , and JL Bos 1997. "C a ²⁺ mediated activation of your Keru rapid R ap 1 in human platelets (Rapid Ca2 + -. Mediated activation of Rapl in human platelets) JE BO J. 15: 252-259.

 4. Tsien, R.Y. and A. Miyawaki. 1998. Seeing the machinery of living cells. Science 280: 1954-1955.

 5. Pollok, B.A. and R. Heim. 1999. "Using GFP in FRET-based applications." J Trends Cell Biol. 9: 57-60.

6. Miyawaki, A., J. Llopis, R. Heim, JM McCaffery, JA Adams, M. Ikura, and RY Tsien. 1997. "Ca ²⁺ fluorescence index based on green fluorescein protein and carmodulin. Yuichi (Fluorescent indicators for

Ca2 + based on green fluorescent proteins and calmodulin) Nature 388: 88

2-887.

 7. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. "Efficient selection for high-expression transfectants with a novel eukaryotic vector. ) '' Gene 108: 193-20 0.

 8. DeClue, J. E., J. C. Stone, R. A. Blanchard, A. G. Papageorge, P. M

3 i artin, K. Zhang, and DR Lowy. “A temperature-sensitive ras effector domain mutant for cell transformation: Interaction between GTPase-activating protein and NF-1 (A ras effector domain mutant which is temperature sensitive f or cellular transformation: interactions with GTPase-activating protein and NF-1.) J Mol. Cell Biol. 11: 3132-3138, 1991.

 9. Ohba, Y., N. Mochizuki, S. Yamashita, AM Chan, JW Schrader, S. Hattori, K. Nagashima, and M. Matsuda. "R_Ras, TC-21 / RR as 2.MR as / Regulatory proteins of R-Ras, TC-21 / -Ras2, and M-as / -as3.) Biol. Chem. 275: 20020-200

26, 2000.

 10. Yamashi ta, S., N. Mochizuki, Y. Ohba,. Tobiurae, Y. Okada, H. Sawa, K. Nagashima, and M. Matsuda. "Gal DAG—Ras, RR as, GEF III, Activation of Rap 1 (GalDAG-GEFIII activation of Ras, R-Ras, and Rapl.) "J. Biol. Chem. 275: 25488-25493, 2000.

 11. T. Gotoh, S. Hattori, S. Nakamura, H. Kitayama, M. Noda, Y. Takai, K. Kaibuchi, H. Matsui, 0. Hatase, H. Takahashi, T. Kurata, and M. Matsuta da. “Identification of Rapl as a target for Crk SH3 domain-binding guanine nucleotide-release ng factor, C3G) J Mol. Cell. Biol. 15: 6746-6753, 1995.

Hereinafter, the present invention will be described with reference to examples, but the scope of the present invention is not limited to only these examples. In the following, human H-Ras is Ras, human c-Raf1 is Raf, human Rap1A is Rap1A, human RalgDs is RalGDS, Human R-Ras is called R-Ras. Also, human Ra cl Is Racl, human Cdc42 is Cdc42, human RhoA is RhoA, human Pakl is Pakl, and human mDia1 is mDia1. Example 1 Measurement of Ras activation by Rafrasl 722

 (1) Creation of chimeric genes encoding Ras and Raf

 (i) Amplification of Ras gene

 Using the cDNA of Ras (Genbank / EMBL approval number: V00574) as type III, the sense primer 1 h R as X h (5′-CTCGAGATGACGGAATATAAGCTGGTGGTG-3 ′) (SEQ ID NO: 1) and the antisense primer 1 Ra s PCR (Polymerase chain reaction) using 1 72Ra f (5'-AGTGTTGCTTGTC TTAGAAGGGGTACCACCTCCGGAGCCGTTCAGCTTCCGCAGCTTGTG-3 ') (SEQ ID NO: 2) and a thermostable DNA replication enzyme PfX (Gibco-BRL Bethesda, USA) The cDNA portion corresponding to the amino acid sequence from position 1 to position 172 of Ras was amplified by the method.

 The sense primer hRas Xh has the nucleotide sequence of the cleavage site of the restriction enzyme XhoI shown underlined at the 5 'end and the nucleotide sequence of the cDNA portion corresponding to the amino acid sequence at positions 1 to 8 of Ras. Consists of On the other hand, the antisense primer Ras172Raf is complementary to the cDNA complementary to the amino-terminal region (from position 61 to position 67) of the amino acid sequence of the Ras binding region of Raf from the 5 'end. It consists of the base sequence, spacer sequence (underlined), and the base sequence of the complementary strand of c • DNA corresponding to the amino acid sequence from position 166 to position 172 of Ras.

 (ii) Ra f gene amplification

Using the cDNA of Raf (Genbank / EMBL case number: X03484) as type III, the sense primer Ra f RBD—F 1 (5′-GGTACCCCTTCTAAGACAAGCAACACT-3 ′) (SEQ ID NO: 3) and antisense Using the sense primer Ra f RBDn 2 (5′-GCGGCCGCCCA GGAAATCTACTTGAAGTTC-3 ′) (SEQ ID NO: 4) and the Pfx, it corresponds to the amino acid sequence of positions 51 to 1.3 of Ra f by PCR method. Amplify the cDNA portion did.

 The sense primer Ra f RBD—Fl is the nucleotide sequence of the cleavage site of the restriction enzyme KρηI underlined at the 5 'end and the nucleotide sequence of the cDNA portion corresponding to the amino acid sequence from position 51 to position 57 of Raf. Consists of On the other hand, the antisense primer Ra f RBD n2 has the base sequence of the cleavage site of the restriction enzyme Not I and the carboxyl terminal region of the amino acid sequence of the Ras binding region of Ra f (position 125 From position 131 to position 131).

 (iii) Amplification of chimeric genes encoding Ras and Raf

 Using a mixture of the genes amplified in the above (i) and (ii) as a 铸 type, Ras was determined by PCR using the sense primer hR as Xh, the antisense primer Raf RBDn2 and the Pfx, and PCR. A cDNA consisting of a chimeric gene encoding Raf was amplified. Next, the obtained DNA fragment was ligated to pCR-b1unitII-TOPO (Invitrogen), and Escherichia coli was transformed with the obtained plasmid construct. After culturing such Escherichia coli, a plasmid was purified by a known alkaline SDS method.

 (2) Construction of vector pFre t 2 expressing EYFP and ECFP

 (i) Construction of pCAGGS-P7

 p7 (5'-CGCCAGGGTTTTCCCAGTCACGAC-3 ') (SEQ ID NO: 5) and primer P8 (5'-AGCGGATAACAATTTCACACAGGAAAC) were added to pB l ue scrip t-SKI I (+) (Stratagene)' s Martinburg roning site. -3 ′) (SEQ ID NO: 6) and amplified by PCR in the same manner as described above to obtain a DNA fragment.

 On the other hand, pCAGGS (Reference 7), a mammalian cell expression vector, was cut with EcoRI and blunt-ended with K1enow enzyme. Next, the DNA fragment and the pCAGGS after the treatment were ligated with T4 DNA ligase. The resulting vector is called pC AGGS-P7.

(ii) Amplification of EYFP gene In this example, six amino acid substitutions (Leu65Phe; Thr 66Gy; Val 69Leu; Gln70Lys; Gln70Lys; Ser73Ala) to a known EGFP (Genbank / EBL accession number: U76561) by a known method using PCR. Thr204Tyr) was used as EYFP. Using this EYFP cDNA as type III, the sense primers GFP—N 2 (5′-GGATCCGGCATGGTGAGCAAGGGCGAGGAG-3 ′) (SEQ ID NO: 7) and antisense primer GFP—N3 (5′-GGATCCGGTACCTCGAGCTTGTACAGCTCGTCCATG-3 ′) — (SEQ ID NO: : 8) and the above-mentioned Pfx, cDNA corresponding to the full-length amino acid sequence of EYFP was amplified by PCR.

 Sense Primer GFP—N2 is the base sequence of the cleavage site of the restriction enzyme BamHI, underlined at the 5 'end, a 3-base spacer, and the amino acid sequence at positions 1 to 7 of EYFP. And the base sequence of the cDNA portion corresponding to On the other hand, the antisense primer GFP-N3 has the nucleotide sequence of each of the cleavage sites of the restriction enzymes BamHI, KnI and XhoI shown underlined at the 5 'end and the amino acid sequence of ECFP described below. And the base sequence of the complementary strand of the cDNA portion corresponding to the carboxyl-terminal region (from 233 to 239) of the

 (iii) Amplification of EC FP gene

 In the present example, EGFP (Genbank / EMBL R / C / No .: U76561) into which four amino acid substitutions (Tyr67Trp; Asnl47Ile; Metl54Thr; Vall64Ala) were introduced by a known method using a PCR method was used. The cDNA of ECF P was used as type II, and the sense primer XFPNott2 (5'-GCGGCCGCATG GTGAGCAAGGGCGAGGAGC-3 ') (SEQ ID NO: 9) and antisense primer XFP-Bg1 (5'- Using AGATCTACAGCTCGTCCATGCCGAGAG-3 ′) (SEQ ID NO: 10) and the aforementioned PfX, cDNA corresponding to the full-length amino acid sequence of ECFP was amplified by PCR.

The sense primer XF PNot 2 corresponds to the base sequence of the cleavage site of the restriction enzyme Not I shown underlined at the 5 'end and the amino acid sequence from position 1 to position 8 of ECFP. It consists of the base sequence of the DNA part. On the other hand, the antisense primer XFP—Bg1 binds to the base sequence of the cleavage site of the restriction enzyme Bg1II and the carboxyl terminal region (from 231 to 237) of the amino acid sequence of ECFP, which are underlined at the 5 'end. And the base sequence of the complementary strand of the corresponding cDNA portion.

 (iv) Construction of p F r e t 2

 The pCAGGS-P7 obtained in the above (i) was cleaved with the restriction enzyme XhoI, and treated with the Klenow enzyme in the presence of dTTP and dCTP. In addition, the EYFP DNA fragment obtained in (ii) above was digested with BamHI and then treated with the K1enow enzyme in the presence of dGTP and dATP. The resulting two gene fragments were ligated with T4 DNA ligase to obtain a plasmid. The plasmid was cleaved with NotI and Bg1II, and then the DNA fragment of ECFP obtained in the above (iii) which had been cleaved with NotI and Bg1II, and a T4 DNA ligase. Ligation was carried out. The obtained plasmid was named pFret2.

 (3) Construction of pRafras 1722, an expression plasmid for the Ras activity monitor protein gene

 The pFret 2 obtained in the above (2) — (iv) was cut with XhoI and NotI, and then in (1)-(iii), which was cut in advance with XhoI and NotI. The obtained chimeric gene was ligated using T4 DNA ligase. The resulting plasmid is called pRafrasl722. The structure of pRafrasl 722, the nucleotide sequence of its translation region (SEQ ID NO: 11) and the predicted amino acid sequence (SEQ ID NO: 12) are shown in FIGS. 3 and 4 to 6, respectively.

 Illustrate such a base sequence and the predicted amino acid sequence:

nt 1-717 EYFP of the jellyfish (Aequorea)

NT 718-723 linker

nt 724-1239 R a s

nt 1240-125 linker nt 1258-1500: Raf

nt 1501-1509: the linker

nt 1510-2220: Owan jellyfish ECFP

 (4) Expression of Ras activity monitor protein (Rafrasl722) in mammalian cells and analysis by spectrophotometry

 HEK 293 T cells derived from human fetal kidney were cultured in DMEM medium (manufactured by Nippon Pharmaceutical Co., Ltd.) containing 10% fetal serum. In the HEK 293 T cells, the pRafrasl 722 obtained in the above (3) and the guanine nucleotide exchange factor Sos expression vector (pCAGGS-mSos) or the GTP water degradation promoting enzyme Gap lm expression vector (pEF — Bo s — Gap lm) was transfected by the calcium phosphate method. The HEK293 T cells after the transfection were cultured in a DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum to express the Ras activity monitor protein. After culturing for 48 hours, the cells were washed with phosphate buffered saline and lysed with a lysis solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCL 5 mM MgCh, 0.1% Triton X-100). The obtained cell lysate was centrifuged at 10,000 xg, and the supernatant was recovered. The supernatant was placed in a 1 ml cuvette of a fluorescence spectrophotometer (FP-750, manufactured by JASCO Corporation), and the fluorescence intensity from 450 nm to 550 nm was measured at an excitation wavelength of 433 nm. The obtained fluorescence profile is shown in FIG.

After transfected HEK 293 T cells were labeled with ³² Pi inorganic phosphate, a lysate of the cells was obtained, and the expressed Ras-active monitor protein was immunized with an anti-GFP antibody. Separation of sedimented and bound GTP and GDP by thin-layer chromatography, FRET efficiency obtained from the fluorescence profile data obtained for the Ras activity monitor protein (excitation at 433 nm wavelength). Then, the value obtained by dividing (the fluorescence intensity at a wavelength of 530 nm) by (the fluorescence intensity at a wavelength of 475 nm) can be associated with the actual degree of GTP binding (Fig. 8). In FIG. 8, the FRET efficiency is expressed as “fluorescence intensity ratio (wavelength 5 30/475) ”and the degree of GTP binding was expressed as rGTP / ⁷ (GDP + GTP) (%) J.

 (5) Expression of Ras activity monitor protein in mammalian cells and analysis by Timelabs fluorescence microscopy

 Monkey kidney-derived COS 7 cells were cultured in a FUNOLED Red-free MEM medium (manufactured by Nippon Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum. PRafras1722 obtained in the above (3) was transfected into the COS 7 cells by the calcium phosphate method. The transfected COS 7 cells were cultured in a phenol-free MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum to express the Ras activity monitor protein. Forty-eight hours after transfection, the cultured cells were subjected to observation with a Timelabs fluorescence microscope.

 Such a microscope is equipped with a rotary fluorescence excitation filter device and a rotary fluorescence emission filter device (manufactured by LUDL electronic) and an inverted xenon light source equipped with a high-sensitivity cooled CCD camera (Photometrix, Micromax450). Type fluorescence microscope (Carl Zeiss, Axiovert 100). When observing, a system that controls the microscope and analyzes the observation results using Metamorph image analysis software manufactured by Nippon Roper Co., Ltd. Using. The fluorescence excitation filter, fluorescence emission filter, and dichroic mirror were purchased from Omega.

 The cultured cells were irradiated with excitation light of 433 nm, an image of the fluorescence wavelength of the ECFP donor of 475 nm was taken with a CCD camera, and then an image of the fluorescence wavelength of the EYFP receptor of 530 nm was taken. The FRET efficiency at each measurement point was calculated by calculating the ratio of the fluorescence intensities of the two based on the image data. Example 2 Acquisition of Cultured Cell Line for Easy Measurement of Ras Activity

Mouse fibroblast NI H3T3 cells were cultured in DMEM medium (manufactured by Nissui Pharmaceutical) containing 10% fetal calf serum. The NIH 3 T3 cells obtained in Example 1 pRfrasl722 and vector pSV2neo (Genbank / EMBL: U02434) containing the G4 18 resistance gene were co-transfected using FuGene 6 (Nippon Roche). The cells were cultured in the above-mentioned medium, and after culturing for 48 hours, re-wound at a dilution ratio of 1:10, and G4 18 (Gibco — manufactured by BRL) was added to the medium to a concentration of 0.5 mgZml. . The medium was changed once every three days. After 2 weeks of culture, well separated colonies were cloned and named 3T3_Rafras cells. The 3T3-Rafras cells are cultured in a DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum and 0.5 mg / m1 of G418, and the Ras activity monitor protein is analyzed. Was expressed. Next, the expression of such a protein was analyzed by the usual immunoblotting method using an anti-Ras antibody (Transduction Lab). As a result, expression of a protein of about 8.0 kDa was observed (Fig. 9).

 Further, the cells were stimulated with epidermal growth factor (EGF) (Sigma), and the FRET efficiency was determined by the method described in (4) of Example 1 and compared before and after the EGF stimulation. FIG. 10 shows the fluorescence profiles before and after the addition of EGF. Example 3 Measurement of Rap A activation by Ra i _c hu 3 11

 (1) Creation of chimeric genes encoding Rap 1 A and Ra 1 GDS

 (i) Rap A gene amplification

 Using the cDNA of R ap 1 A (Genbank / EMBL end accession number: X12533) as type III, sense primer hRapl Xh (5′-GGCTCGAGATGCGTGAGTACAAGCTAGTGG-3 ′) (SEQ ID NO: 13) and antisense primer Ra p 1 72Ra 1 GDS (5′-GCGGATGATACAGCAGTCGCCACCTCCGGATCCGCCGGTACCTCCACCACCGGTTCCACCTCCGGAGCCAT TGATCTTTGACTTTGCAGAAG-3 ′) (SEQ ID NO: 14) The corresponding cDNA portion was amplified.

The sense primer hRap 1 Xh is the restriction enzyme Xh 0 It consists of the nucleotide sequence of the cleavage site of I and the nucleotide sequence of the cDNA portion corresponding to the amino acid sequence at positions 1 to 8 of Rap1A. On the other hand, the antisense primer Rap172Ra1GDS is composed of the amino terminal region (21) of the amino acid sequence of the Rap1A binding region of Ra1GDS (Genbank / EMBL accession number: U14417) from the 5 'end. The nucleotide sequence of the complementary strand of the cDNA portion corresponding to positions 1 to 217), the spacer sequence (underlined), and the cDN corresponding to the amino acid sequence of positions 166 to 172 of Rap 1A It consists of the base sequence of the complementary strand of the A portion.

 (ii) Ra 1 GDS gene amplification

 Using the Ra 1 GDS cDNA (Genbank / EMBL endion number: U14417) as type III, the sense primer Ra l GDS—F (5′-GGCGACTGCTGTATCATCCGC-3 ′) (SEQ ID NO: 15) and the antisense primer Ra 1 Ra1 GDS cDNA was amplified by PCR using GDSR (5'-CGCGGCCGCCCCG CTTCTTGAGGACAAAGTC-3 ') (SEQ ID NO: 16) and the Pfx.

 The sense primer Ra1GDS-F has the nucleotide sequence of the cDNA portion corresponding to the amino terminal region (from position 211 to position 217) of the amino acid sequence of the Rap1A binding region of the cDNA of Ra1GDS. On the other hand, the antisense primer Ra 1 GDSR is composed of the base sequence of the cleavage site of the restriction enzyme N 0 ΐI underlined at the 5 ′ end and the carboxyl terminal region of the amino acid sequence of the Rap 1 A binding region of Ra 1 GDS ( 291 from position 1 to position 297), and the complementary nucleotide sequence of the nucleotide sequence of the cDNA portion.

 (iii) Amplification of chimeric genes encoding Rap 1 A and Ra 1 GDS

Using the mixture of the genes amplified in the above (i) and (ii) as type I, Rap 1A was synthesized by PCR using sense primer hRap1Xh and antisense primer Ra1GDSR and PfX. A cDNA consisting of a chimeric gene encoding Ra1GDS was amplified. Next, the obtained DNA fragment was ligated to pCR-b1untII-TOPO, and the resulting plasmid construct was used to colonize the large intestine. The bacteria were transformed. After culturing such Escherichia coli, a plasmid was purified by a known alkaline SDS method.

 (2) Construction of pR ai-chu 311 which is an expression plasmid for Rap 1 A activity monitor protein gene

 In (ii) of (2) of Example 1, antisense primer GFP-d11R (5'-GGATCCGGTACCTCGAGGGCGGCGGTCACGAACTCCAGCAG-3 ') (SEQ ID NO: 17) was used instead of antisense primer GFP-N3. A similar operation was performed to prepare a vector containing cDNA encoding ECFP and EYFP which lacks 11 amino acids at the carboxyl terminus of the amino acid sequence. This vector was cut with XhoI and NotI. Next, the vector and the chimeric gene obtained in the above (1), which had been cut with XhoI and NotI, were ligated with T4 DNA ligase. The resulting plasmid was named pR ai -c hu 311. The structure of the obtained plasmid, the nucleotide sequence of its translated region (SEQ ID NO: 18) and the predicted amino acid sequence (SEQ ID NO: 19) are shown in FIGS. 11 and 12 to 14. Are shown below.

 Illustrating such a base sequence and the predicted amino acid sequence:

nt 1-684: EYFP of the Jellyfish

nt 685-690: the linker

nt 691-1206: Rap 1 A

nt 1207-1257: linker

nt 1258-1515: Ra 1 GDS

nt 1516-1521: The linker

nt 1522-2235: Owan jellyfish ECFP

 (3) Expression of Rap 1 A activity monitor protein (Ra i-chu 3 11) in mammalian cells and analysis by spectrophotometry

The analysis was performed by the method described in (4) of Example 1. Obtained fluorescent brophy This is shown in Figure 15. Example 4 Measurement of R—Ras activation by Ra i-chu 158

 (1) Construction of pRa i -chu 1 58

 (i) R—Ras gene amplification

 R—Ras cDNA (Genbank / EMBL accession number: M14948, 14949) was used as type III, and the sense primer RRas 28F (5′-CCCCTCGAGACACACAAGCTGGTGGTC-3 ′) (SEQ ID NO: 20) and antisense primer RRa Using s204R (5'-G CCGGTACCGCCACTGGGAGGGCTCGGTGGGAG-3 ') (SEQ ID NO: 21) and Pfx, a cDNA portion corresponding to the amino acid sequence from position 28 to position 204 of R-Ras by PCR method Was amplified.

 The sense primer RRas28F is derived from the nucleotide sequence of the cleavage site of the restriction enzyme XhoI shown underlined at the 5 'end and the nucleotide sequence of the cDNA corresponding to the amino acid sequence from position 28 to position 33 of R-Ras. Become. On the other hand, the antisense primer RRas 204 R. corresponds to the spacer sequence containing the KpnI cleavage site (underlined) from the 5 'end, and the amino acid sequence from position 198 to position 204 of R-Ras. It consists of the base sequence of the complementary strand of the cDNA portion.

 (ii) Preparation of restriction enzyme fragment

 The PCR product obtained in the above (i) was cut with XhoI and KpnI.

 (iii) Construction of pRa i-chu 158, an expression plasmid for the R—Ras activity monitor protein gene

PR afras 1 722 obtained in Example 1 was completely digested with XhoI and then partially digested with Kpn. I to obtain a DNA fragment from which the Ras portion had been removed. The DNA fragment and the DNA fragment obtained in (ii) were ligated with T4 DNA ligase. The resulting plasmid was named pRa i-chu 158. The structure of the plasmid, the nucleotide sequence in its translation region (SEQ ID NO: 22) and the predicted amino acid The acid sequence (SEQ ID NO: 23) is shown in FIG. 16 and FIGS. 17 to 19, respectively. Illustrating such a base sequence and the predicted amino acid sequence:

nt 1-717: EYFP of the Jellyfish

NT 718 – 723: Linker

nt 724-1251: R-R a s

nt 1252-1257: the linker

nt 1258-1500: R a f

nt 1501 one 1509: linker one

nt 1510-2220: Owan jellyfish ECFP

 (2) Expression of R-Ras activity monitor protein (Ra i- chu158) in mammalian cells and analysis by spectrophotometry

 The analysis was performed by the method described in (4) of Example 1. Fig. 20 shows the obtained fluorescent profiles. Example 5 Construction of Gene Encoding Moni Yuichi Protein Having Temperature-Sensitive Mutation in Ras Target Protein Binding Domain

 (1) Construction of pRa i -chu 1 1 9

 (i) Amplification of mutated Ras gene

 Using the Ras cDNA used in Example 1 as type III, sense primer hRasXh (used in Example 1) and antisense primer RasI36LR (5'-G GAATCCTCTAGAGTGGGGTCG-3 ') Using (SEQ ID NO: 24) and the above Pfx, a cDNA portion corresponding to the amino acid sequence from position 1 to position 39 of Ras was amplified by PCR.

The antisense primer RaSI36LR has the sequence of the cDNA portion corresponding to the amino acid sequence from position 35 to position 42 of Ras, and the underlined portion indicates that the I 1 e It has a mutation. This mutation makes Ras activity temperature sensitive. Is known (Ref. 8).

 Similarly, using the Ras cDNA as type II, the sense primer RasI36LF (5'-CGACCCCACTCTAGAGGATTCC-3 ') (SEQ ID NO: 25) and the antisense primer -Ras172Raf (see Example 1) And the above-mentioned P fx, and amplify the cDNA portion corresponding to positions 32 to 172 of the amino acid sequence of Ras by PCR using PCR

 The obtained two DNA fragments were mixed, and PCR was carried out using the sense primer hRasXh and the antisense primer Ras172Raf in the same manner as described above, corresponding to positions 1 to 172 of the amino acid sequence of Ras. And a DNA containing a point mutation of I1e36 to Leu was amplified.

 (ii) Preparation of restriction enzyme fragment

 The PCR product was cut with XhoI and KpnI.

 (iii) pRafrasl722 obtained in Example 1 was completely digested with XhoI and then partially digested with KpnI to obtain a DNA fragment from which Ras was removed. The DNA fragment and the DNA fragment obtained in (ii) were ligated with T4 DNA ligase. The resulting plasmid was named pRai-chu119. The nucleotide sequence (SEQ ID NO: 26) and the predicted amino acid sequence (SEQ ID NO: 27) in the translation region of the plasmid are shown in FIGS. 21 to 23, respectively.

 (2) Expression of monitor protein (Ra i- c hu 119) in mammalian cells and analysis by spectrophotometry

HEK293 T cells derived from human fetal kidney were cultured in a DMEM medium (manufactured by Mizu Seiyaku) containing 10% fetal serum. The HEK 293 T cells were transfected with the pRafras 1722 or pRa i-chu 119 prepared in Example 1 and a guanine nucleotide exchange factor Sos expression vector (pCAGGS_mSos) by a phosphoric acid lupus method. did. After culturing for 24 hours in the same medium, the cells were transferred to an incubator at 33 ° C and 40 ° C, and further cultured for 24 hours. Remove the cells After washing with a phosphate buffered saline, the cells were dissolved in a lysis solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCL 5 mM MgCh, 0.1¾ Triton X-100). The cell lysate obtained is

The supernatant was collected by centrifugation at 100,000 xg.

 The supernatant was placed in a 1 ml cuvette of a fluorescence spectrophotometer (FP-750, manufactured by JASCO Corporation), and the fluorescence intensity from 450 nm to 550 nm was measured at an excitation wavelength of 433 nm. Was. The resulting fluorescence profile is shown in FIG. Example 6 Generation of transgenic mouse expressing Rafras1722 and measurement of Ras activation in cultured cardiomyocytes of this mouse

 (1) pRafras 1722 obtained in Example 1 was digested with restriction enzymes SpE I and BamHI, subjected to agarose gel electrophoresis, and a promoter, an intron, a coding sequence, a polysequence of about 4.5 kb was obtained. A DNA fragment of the region containing the A addition signal was obtained. After removing the DNA from the gel by electroelution, use a Qiagen20 chip

(Qiagen). This DNA was injected into the pronucleus of a mouse fertilized egg (DB Fl, Japan SLC) according to a standard method, and transplanted into the oviduct of a pseudopregnant ICR mouse (Japan SLC). After weaning of the obtained mouse, the tail was cut 1 cm, and kept overnight at 37 ° C. in a DNA extract containing P-tinase K (ABI). From here, phenol and phenol After removing the protein with form, an equal volume of isopropanol was added to recover the precipitated DNA. The recovered DNA was put in water and dissolved at 37 ° C.

(2) Using this mouse DNA as type II, the sense primer Ra f RBDx (5'-C TCGAGCCTTCTAAGACAAGCAACACT-3 ') (SEQ ID NO: 28) and the antisense primer -XFPNs eq (5'-CGTCGCCGTCCAGCTCGACCAG-3') (SEQ ID NO: 29) and the DNA was amplified by PCR. With this primer, DNA corresponding to the connection region between the Raf gene and the ECFP gene in the Rafras1722 gene can be amplified. Ra fras 1 It was determined that 722 DNA had been integrated. This band was confirmed in 7 out of 35 pups.

 (3) Next, the F1 mouse was bred to a C57 / Black mouse (Japan SLC). The ventricle was taken from a newborn F2 mouse (0 day old) and minced with ophthalmic scissors. To this, PBS containing 0.05% trypsin and 0.5 mM EDTA was added, the cells were treated at 37 ° C for 10 minutes, and the isolated cardiomyocytes were collected. This operation was repeated six times, and cardiomyocytes were collected. To this was added a DMEM medium containing 10% fetal calf serum, and cardiomyocytes were precipitated by low-speed centrifugation, and the supernatant was discarded. The recovered cardiomyocytes were cultured in DMEM containing 10% fetal calf serum.

 (4) The obtained cardiomyocytes were transferred to a glass bottom culture dish (ø35 mm), allowed to adhere to the bottom, and cultured in a serum-free medium (Nippon Pharmaceutical) for 6 hours. Next, 100 ng / ml of EGF was added to the cultured myocardial cells, and the cells were observed with the fluorescence microscope system described in (5) of Example 1. FIG. 25 shows the results of the change over time in the fluorescence intensity of ECFP and EYFP in the cells upon addition of EGF. It was confirmed that the activation of Ras can be measured in an EGF-dependent manner even in primary cultured cells derived from transgenic mice. Example 7 Specificity of Raf rasl722 for guanine nucleotide exchange factor and GTP hydrolysis enzyme

In the experiment of (4) in Example 1, the specificity of Rafrasl722 was examined using many types of guanine nucleotide exchange factor and GTP hydrolysis promoter. As GTP water hydrolysis promoting enzyme and guanine nucleotide exchange factor, expression vectors of GAPlm, R-RasGAP, raplGAPII, mSosl, RasGRF, CalDAG-GEFI, C3G, PDZ-GEF1 and KIAA0351 described in (Reference 9) were used. As shown in Fig. 26, GAPlm, a GTP water-degrading enzyme for Ras, shows a decrease in FRET efficiency.However, FRET efficiency for some RTPases, R-RasGAP and raplGAPII, for R-Ras and Rapl can be reduced Drop Absent. In addition, guanine nucleotide exchange factors for Ras such as mSosl, RasGRF, and CalDAG-GEFI I increase FRET efficiency, but guanine nucleotide exchange using other Ras family G proteins such as CalDAG-GEFI, C3G, PDZ-GEF1, and KIAA0351 as substrates. FRET efficiency did not increase for some factors. This indicates that Rafrasl722 is specifically regulated in its FRBT efficiency by the same guanine nucleotide exchange factor and GTP hydrolysis enzyme as Ras. Example 8 Preparation of Rai-chu404, a Rap1 monitor, and its specificity for guanine nucleotide exchange factor and GTP hydrolysis enzyme

 (1) Construction of chimeric gene Rai-chu404 encoding RaplA and Raf

 A known amino acid substitution (Thr66Gly; Val69Leu; Ser73Ala; Metl54Thr; Vall64Ala; Serl76Gly; Thr204Tyr) in which known EGFP (Genbank / EBL accession number: U76561) has been replaced with seven amino acids by a known method using PCR. Was replaced with the method described in Example 3 by digestion with EYFP of pRaichu311 using restriction enzymes EcoRI and Xhol and ligation. Next, the Kpnl / NotI fragment containing the Raf region of Rafrasl722 was replaced with a Kpnl / Notl fragment containing the RalGDS region of the plasmid derived from pRai-chu311. This vector was named pRaichu404. The base sequence of the translation region is shown as SEQ ID NO: 30 and the amino acid sequence predicted from the base sequence is shown as SEQ ID NO: 31.

(2) The effect of guanine nucleotide exchange factor on the FRET efficiency of Rai-chu404 was examined in the same manner as in Example 7. PDZ-GEFK C3G, CalDAG-GEFI, and CalDAG-GEFI II were used as guanine nucleotide exchange factors for Rapl. As controls, a guanine nucleotide exchange factor for Ras, CalDAG-GEFII, and a guanine nucleotide exchange factor for mSosl, RasGRF and Ral, KIAA0351, were used. These guanine nucleotide exchange factors are described in (Reference 9). As shown in Fig. 27, the results of the experiment show that only guanine nucleotide exchange factor for Rapl It was found that chu404 increased the FRET efficiency. Example 9 Preparation of Activity Monitor Proteins Rai-chul01X and Rai-chu404X Having CAAX Box of Ki-Ras Protein

 In Example 1, ECFP was amplified using a primer (SEQ ID NO: 32) containing a recognition site of a restriction enzyme Xbal instead of the antisense primer XFP-Bg1, and the method described in (Reference 11) was used. Then, the CAAX box of Ki-Ras protein was fused to the carboxyl terminus of ECFP. This was replaced by the method using Rafrasl722 and Rai-chu404 ECFP, restriction enzymes and T4 ligase. The resulting vectors were named pRai-chulOIX and pRai-chu404X, respectively. The nucleotide sequences of the respective translation regions are shown as SEQ ID NO: 33 and SEQ ID NO: 35, and the amino acid sequences predicted from the nucleotide sequences are shown as SEQ ID NO: 34 and SEQ ID NO: 36. Example 10 Visualization of Ras Activation in C0S1 Cells Expressing Rai-chulOIX and Rai-chu404X

Rewind the C0S1 cells into a glass bottom culture dish. Rai-chulOIX and Rai-chu404X obtained in Example 9 were transfected into COS1 cells according to a standard method. Twenty-four hours later, observation was performed with the fluorescence microscope system described in Example 1, (5). FIG. 28 shows the results of the change over time in the fluorescence intensity ratio of ECFP and EYFP (EYFP / ECFP) in the cells due to the addition of EGF. In this figure, a high fluorescence ratio is shown in red and a low fluorescence ratio is shown in blue, and the diagram is presented using the IMD mode in which the fluorescence intensity of ECFP is expressed as lightness. That is, in the original figure, the red area indicates the site where Ras or Rapl activation is high. In Fig. 28, Ras was activated from the periphery of cells by stimulation with cell growth factor, and Rapl was activated from the perinuclear area. When the same experiment is performed with the cells slightly confluent, as shown in Fig. 29, the activation of Ras does not occur in the area where the cells are in contact, and the margin is not in the area where the cells are not in contact. It was clear from the section that Ras was activated. Thus, the use of the active protein protein of the present invention allows intracellular Ras-amily G-tannos. Temporal and spatial information on the activity of the protein can be obtained. Example 11 1 Visualization of Ras activation in PC12 cells expressing Rai-chulOIX and Rai-chu404X

 Rai-chulOlX and Rai-chu404X obtained in Example 9 were transfected into PC12 cells growing in a culture dish at the bottom of the glass according to a standard method, and the fluorescence microscope described in (5) of Example 1 was used. Observed on the system. FIG. 30 shows the results of the time-dependent changes in the fluorescence intensity ratio of ECFP and EYFP (EYFP / ECFP) in the cells due to the addition of nerve growth factor. In the original figure, the part with high fluorescence ratio is shown in red and the part with low fluorescence ratio is shown in blue, and the figure is presented using IMD mode, which shows the fluorescence intensity of ECFP as brightness. That is, in the original figure, the red area indicates the site where Ras or Rapl activation is high. In neural-like differentiation of PC12 cells, Ras is activated from the periphery in the cell body during the induction phase of differentiation, whereas Ras activity required for cell survival is obtained at the completion of differentiation. Was found to be maintained only in neurites. In other words, it became clear that activation of Ra s occurred at different sites in the cell depending on the stage of cell differentiation. In sharp contrast to Ras, Rapl was activated by the addition of nerve growth factor from the perinuclear region, and its activity in differentiated neurites was found to be low. This indicates that the activity of Ras family G proteins is regulated differently in different parts of the cell. Example 1 2 Preparation of Rai-chulOlIX, which is an activity monitor of Ra c 1 activity protein (1) Preparation of chimeric gene encoding Ra c 1 and Pak 1

Using the cDNA of Racl (Genbank / EMBL accession number: M29870) and the cDNA of Pak1 (Genbank / EMBL accession number: NM002576) as type III, According to the method of 1 and Example 9, Plasmid KpRai-chulOllX was obtained. The structure of p ai-chulO 11X (FIG. 31), the nucleotide sequence of its translated region (SEQ ID NO: 37) and the predicted amino acid sequence (SEQ ID NO: 38) are shown.

 Illustrating such a base sequence and the predicted amino acid sequence:

nt 1-684: Owan jellyfish EYFP

nt 685-690 linker

nt 691-939 Pak 1

NT 940-969 Linker

nt 970-1497: R a c 1

nt 1498-1506 linker

nt 1507-2217 owan jellyfish ECFP

nt 2218-2229 linker

nt 2230-2289 Carboxyl-terminal region of K-Ras (CAAX box)

 (2) Creation of chimeric gene mutant

 By a known PCR method, the substitution of G at position 1004 in the nucleotide sequence of pRai-chul011X (SEQ ID NO: 37) with T, and the substitution of the predicted amino acid Gly with Val is referred to as pRai-chul012X. Named. Similarly, one in which C at position 109 was substituted with A and the predicted amino acid Thr was substituted with Asn was named pRai-chul013X. In the protein Rai-chul012X, the GTP hydrolytic activity of Rac 1 is expected to decrease and become a constitutively activated form. On the other hand, in Rai-chul013X, the GTP binding ability of Rac1 is expected to decrease and become inactive.

 (3) Expression of Ra c 1 activity monitor protein (Rai-chulOllX) in mammalian cells and analysis by spectrophotometer

Analysis was performed using cells expressing Rai-chul011X. Rai-chul012X or Rai-chul013X by the method described in (4) of Example 1. Fig. 34 shows the obtained fluorescence profile. Wild-type Rai-ch compared to Rai-chul012X, which is expected to be activated With ulOllX and Rai-chul013X, which is expected to be the non-activated form, the efficiency of FRET is low. Example 13 Preparation of Rai-chul054X, a Protein That Monitors the Activity of 3 Cdc42 (1) Preparation of Chimeric Gene Encoding Cdc42 and Pak1

 Using the cDNA of Cdc42 (Genbank / EMBL accession number: M57298) and the cDNA of Pak1 (Genbank / EMBL accession number: NM002576) as type I, by PCR, according to the method of Example 1, The plasmid pRai-chul054X was obtained. The structure of pRai-chul054X (FIG. 32), the nucleotide sequence of its translated region (SEQ ID NO: 39) and the predicted amino acid sequence (SEQ ID NO: 40) are shown.

 Illustrating such a base sequence and the predicted amino acid sequence:

nt 1-684: Owan jellyfish EYFP

nt 685-690 linker

nt 691-939 Pak 1

nt 940-969 linker

nt 970-1494 C d c 42

nt 1495-1503 linker

nt 1504-2214 owan jellyfish ECFP

nt 2215-2226 linker

nt 2227-2286 K—Ras carboxyl terminal region (CAAX box)

 (2) Creation of chimeric gene mutant

 A known PCR method was used to substitute pRai-chul052X with the substitution of G at position 1001 in the nucleotide sequence of pRai-chul054X (SEQ ID NO: 39) and substitution of the predicted amino acid Gly with Val. Named. In the protein Rai-chul052X, GTP hydrolytic activity of Rac1 is expected to decrease and become a constitutively activated form.

(3) Cdc42 activity monitor protein in cultured mammalian cells (Rai-chul054X) Expression and analysis by spectrophotometer

 The analysis was performed by the method described in (4) of Example 1. Analysis was performed using cells expressing Rai-chul054X or Rai-chul052X. The obtained fluorescence profile is shown in FIG. It can be seen that the wild type Rai-chul05 4X has a lower FRET efficiency than the activated Rai-chul052X. Example 14 Preparation of Rai-chul214X, RhoA Activity Monitor Protein

(1) Creation of chimeric gene encoding 1 ^ 110 eight and 1110 i a 1

 Using the cDNA of Rh0A (Genbank / EMBL accession number: L25080) and the cDNA of mDia1 (Genbank / EMBL accession number: E17361) as type 鎵, the plasmid was prepared by the PCR method according to the method of Example 1. De pRai-chul214X was obtained. The structure of pRai-chul214X (FIG. 33), the nucleotide sequence of its translated region (SEQ ID NO: 41) and the predicted amino acid sequence (SEQ ID NO: 42) are shown.

 Illustrating such a base sequence and the predicted amino acid sequence:

nt 1-684: Owan jellyfish EYFP

nt 685-696: Linker

nt 697-1092: mD i a 1

nt 1093-1110 linker

nt 1111-1677 RhoA

nt 1678-1686 linker

nt 1687-2397 Owan jellyfish ECFP

NT 2398-1 2409 Linker

nt 2410-1 2469 K-Ras carboxyl terminal region (CAAX box)

 (2) Creation of chimeric gene mutant

By a known PCR method, the A at position 1298 in the nucleotide sequence of pRai-chul214X (SEQ ID NO: 41) was substituted with T, and the G at position 1299 was substituted with C, and the predicted amino acid was substituted. The product obtained by substituting Gin-noic acid for Leu was named pRai-chul220X. In the protein Rai-ch U1220X, the GTP hydrolytic activity of RhoA is expected to decrease and become a constantly activated form.

 (3) Expression of Rh 0 A activity monitor protein (Rai-chul214X) in mammalian cells and analysis by spectrophotometer

 The analysis was performed by the method described in (4) of Example 1. Analysis was performed using cells expressing Rai-chul214X or Rai-chul220X. The resulting fluorescence profile is shown in FIG. It can be seen that the wild-type Rai-chul214X has a lower FRET efficiency than the activated Rai-chul220X. Example 15 Rai-chulOllX and! Visualization of Rac1 activation in C0S1 cells expressing ai-chul054X

 Rewind the C0S1 cells into a glass bottom culture dish. PRai-chulO 11X obtained in Example 12 was transfected into C0S1 cells according to a standard method. Twenty-four hours later, observation was performed with the fluorescence microscope system described in Example 1, (5). FIG. 37 shows the results of the change over time in the fluorescence intensity ratio of ECFP and EYFP (EYFP / ECFP) in the cells due to the addition of EGF. In Fig. 37, Rac 1 is rapidly activated in the whole cell within 1 minute by EGF stimulation, and the activation converges to a part where the cell membrane is moving, called ruffling at the cell margin. I was able to observe how it was being done. As described above, by using the activity monitor protein of the present invention, it is possible to obtain a temporal and spatial standard method for the activity of the Rh0 family GTP-binding protein in cells. This is also different from the activation of Ras and Rap1, which is a result that confirms the specificity of these active proteins. Sequence listing free text

SEQ ID NO: 1 is the nucleotide sequence of the restriction enzyme Xh0I cleavage site and that of human H-Ras. This is the base sequence of the primer designed based on the base sequence.

 SEQ ID NO: 2 is a nucleotide sequence of a primer designed based on the nucleotide sequence of human c-Raf 1 and the nucleotide sequence of human H-Ras.

 SEQ ID NO: 3 is a nucleotide sequence of a primer designed based on a nucleotide sequence of a cleavage site of restriction enzyme KpnI and a nucleotide sequence of human c-Raf1.

 SEQ ID NO: 4 is a nucleotide sequence of a primer designed based on the nucleotide sequence of the cleavage site of restriction enzyme N0tI and the nucleotide sequence of human c-Raf1.

 SEQ ID NO: 5 is a nucleotide sequence of a primer designed based on the nucleotide sequence on the 5 ′ side of the Martinburg roning site of pB1uescript—SKIII (+).

 SEQ ID NO: 6 is a nucleotide sequence of a primer designed based on the nucleotide sequence on the 3 ′ side of the Martinburg roning site of pB1uescript—SKIII (+).

 SEQ ID NO: 7 is a nucleotide sequence of a primer designed based on the nucleotide sequence of the cleavage site of restriction enzyme BamHI and the nucleotide sequence of EYFP.

 SEQ ID NO: 8 is a nucleotide sequence of a primer designed based on the nucleotide sequence of each cleavage site of restriction enzymes BamHI, KpnI, and XhoI and the nucleotide sequence of ECFP.

 SEQ ID NO: 9 is a nucleotide sequence of a primer designed based on the nucleotide sequence of the cleavage site of restriction enzyme Not I and the nucleotide sequence of ECFP.

 SEQ ID NO: 10 is a nucleotide sequence of a primer designed based on a nucleotide sequence of a cleavage site of restriction enzyme BglII and a nucleotide sequence of ECFP.

 SEQ ID NO: 11 is a nucleotide sequence of a plasmid designed based on each nucleotide sequence of human H-Ras, human c-Rayl, EYFP and ECFP.

SEQ ID NO: 12 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 11. SEQ ID NO: 13 is a nucleotide sequence of a primer designed based on the nucleotide sequence of the cleavage site of restriction enzyme Xh0I and the nucleotide sequence of human Rap1A.

 SEQ ID NO: 14 is a nucleotide sequence of a primer designed based on the nucleotide sequence of human Ra 1 GDS and the nucleotide sequence of human Rap 1A.

 SEQ ID NO: 15 is a nucleotide sequence of one primer designed based on the nucleotide sequence of human Ra1 GDS.

 SEQ ID NO: 16 is the nucleotide sequence of a primer designed based on the nucleotide sequence of the cleavage site of restriction enzyme N0tI and the nucleotide sequence of human Ra1GDS.

 SEQ ID NO: 17 is a nucleotide sequence of a primer designed based on the nucleotide sequence of each cleavage site of restriction enzymes BamHI, KpnI and XhoI and the nucleotide sequence of ECFP.

 SEQ ID NO: 18 is a nucleotide sequence of a plasmid designed based on each nucleotide sequence of human Rap1A, human Ra1GDS, EYFP and ECFP.

 SEQ ID NO: 19 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 18.

 SEQ ID NO: 20 is a nucleotide sequence of a primer designed based on the nucleotide sequence of the cleavage site of restriction enzyme Xh0I and the nucleotide sequence of human R-Ras.

 SEQ ID NO: 21 is a nucleotide sequence of a primer designed based on the nucleotide sequence of the cleavage site of restriction enzyme KpnI and the nucleotide sequence of human R_Ras.

 SEQ ID NO: 22 is a nucleotide sequence of a plasmid designed based on each nucleotide sequence of human R-Ras, human c-Rafl, EYFP and ECFP.

 SEQ ID NO: 23 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 22.

 SEQ ID NO: 24 is a nucleotide sequence of a primer designed based on the nucleotide sequence of human H-Ras.

SEQ ID NO: 25 is a primer designed based on the nucleotide sequence of human H-Ras Is the base sequence.

 SEQ ID NO: 26 is a nucleotide sequence of a plasmid designed based on each nucleotide sequence of human H-Ras, human c-Rafl, EYFP and ECFP.

 SEQ ID NO: 27 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 26.

 SEQ ID NO: 28 is a nucleotide sequence of a primer designed based on a nucleotide sequence of a human H—Ras binding region of human c-Rafl.

 SEQ ID NO: 29 is a nucleotide sequence of a primer designed based on the nucleotide sequence of ECFP.

 SEQ ID NO: 30 is a plasmid base sequence designed based on each base sequence of human Rap1A, human c-Raf1, EYFP and ECFP.

 SEQ ID NO: 31 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 30.

 SEQ ID NO: 32 is a nucleotide sequence of a primer designed based on the nucleotide sequence of ECFP.

 SEQ ID NO: 33 is given as a nucleotide sequence of a plasmid designed based on each nucleotide sequence of human H_Ras, human c_Rafl, EYFP.ECFP and human K-Ras.

 SEQ ID NO: 34 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 33.

 SEQ ID NO: 35 is a nucleotide sequence of a plasmid designed based on each nucleotide sequence of human RaplA, human c-Rafl, EYFP.ECFP and human K-Ras.

 SEQ ID NO: 36 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 35.

SEQ ID NO: 37 is a fragment of human Ra cl, human Pak l, EYFP. This is a plasmid base sequence designed based on each base sequence of human K-Ras. SEQ ID NO: 38 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 37.

 SEQ ID NO: 39 is a nucleotide sequence of a plasmid designed based on each nucleotide sequence of human Cdc42, human Pak1, EYFP, ECFP, and human K-Ras.

 SEQ ID NO: 40 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 39.

 SEQ ID NO: 41 is a base sequence of a plasmid designed based on each base sequence of human RhoA, human mDia1, EYFP, ECFP, and human K-Ras

0

 SEQ ID NO: 42 is an amino acid sequence predicted from the base sequence of the plasmid of SEQ ID NO: 41. Industrial applicability

 According to the present invention, an activity monitor protein of a low-molecular-weight GTP-binding protein capable of measuring the activation of a non-invasive low-molecular-weight GTP-binding protein, a non-invasive low-molecular-weight GTP-binding protein expressing the protein Cells and transgenic animals useful for measuring protein activation, methods for measuring the activation of low-molecular-weight GTP-binding proteins using the proteins, and more specifically, low-molecular-weight GTP-binding proteins that can be used in living cells The present invention provides a method for measuring the amount ratio of GTP-bound to GDP-bound, and a method for screening a low-molecular-weight GTP-binding protein activity modulator.

Claims

The scope of the claims 1. All or a part of the low-molecular-weight GTP-binding protein, all or a part of the target protein of the low-molecular-weight GTP-binding protein, all or a part of the GFP receptor protein, and all or a part of the GFP donor protein An activity monitor for a low-molecular-weight GTP-binding protein consisting of a fusion protein directly or indirectly linked in a state capable of exerting the function of the protein. 2. The low molecular weight GTP-bound protein activity monitor protein according to claim 1, further comprising an intracellular localization signal. 3. The activity monitor protein for a low molecular weight GTP-binding protein according to claim 1 or 2, further comprising a peptide serving as a spacer between the low molecular weight GTP-binding protein and the target protein. 4. The activity monitor for a low-molecular-weight GTP-binding protein according to any one of claims 1 to 3, wherein the low-molecular-weight GTP-binding protein belongs to the Ras superfamily. 5. The activity monitor protein for a low-molecular-weight GTP-binding protein according to any one of claims 1 to 4, wherein the low-molecular-weight GTP-binding protein belongs to the Ras family. 6. The low molecular weight GTP-binding protein is selected from the group consisting of H-Ras, K-Ras, N-Ras, R-Ras, Rap1A, Rap1B, Rap2A, and Rap2B The low-molecular-weight GTP-bound protein according to any one of claims 1 to 5, Activity monitor of parkaceous matter. 7. The activity monitor protein for a low-molecular-weight GTP-binding protein according to any one of claims 1 to 4, wherein the low-molecular-weight GTP-binding protein belongs to Rho family. 8. The low molecular weight GTP according to any one of claims 1 to 4 and 7, wherein the low molecular weight GTP-binding protein is one selected from the group consisting of RhoA, RhoB, RhoC, Rac1, Rac2, and Cdc42. Monitor activity of combined proteins 9. The activity monitor protein for a low-molecular-weight GTP-binding protein according to any one of claims 1 to 8, wherein the target protein is one selected from the group consisting of Raf, Ra1 GDS, Pak, and mDia. . 10. The activity monitor protein of the low-molecular-weight GTP binding protein according to any one of claims 1 to 9, wherein the GFP receptor protein is EGFP or EYFP. 11. The activity monitor protein for a low molecular weight GTP-binding protein according to any one of claims 1 to 10, wherein the GFP donor protein is ECFP or EBFP. 12. The activity monitor protein for a low-molecular-weight GTP-binding protein according to any one of claims 1 to 11, wherein the low-molecular-weight GTP-binding protein and / or the target protein has a mutation. 13. The activity monitor for a low-molecular-weight GTP-binding protein according to any one of claims 1 to 12, wherein the amino acid sequence of the low-molecular-weight GTP-binding protein has a deletion of at least one amino acid in the amino terminal region and / or the carboxyl-terminal region. Evening protein. 14. The low molecular weight GTP according to any one of claims 1 to 13, wherein at least one amino acid is deleted in the carboxyl terminal region of the amino acid sequence of the GFP receptor protein and / or the GFP donor protein. Active monitor protein of bound protein. 1 5. The activity monitor protein for a low-molecular-weight GTP-binding protein according to any one of claims 1 to 6, and 9 to 14, wherein the low-molecular-weight GTP-binding protein is H-Ras and the target protein is Raf. 1 6. The activity monitor protein for a low molecular weight GTP binding protein according to any one of claims 1 to 6 and 9 to 14, wherein the low molecular weight GTP binding protein is Rap1A and the target protein is Ra1 GDS. 1 7. The activity monitor protein for a low molecular weight GTP binding protein according to any one of claims 1 to 4 and 7 to 14, wherein the low molecular weight GTP binding protein is Rac 1 and the target protein is Pak. 18. The low-molecular-weight GTP-binding protein activity monitor protein according to any one of claims 1 to 4 and 7 to 14, wherein the low-molecular-weight GTP-binding protein is Cdc42 and the target protein is Pak. 1 9. The activity monitor protein for a low molecular weight GTP binding protein according to any one of claims 1 to 4 and 7 to 14, wherein the low molecular weight GTP-binding protein is RhoA and the target protein is mDia. 20. The low molecular weight GTP-binding protein is H—Ras, the target protein is Raf, the GFP donor protein is ECFP, and the GFP receptor protein is EYFP. The activity monitor protein of the low molecular weight GTP binding protein according to the above. 21. The activity monitor protein for a low molecular weight GTP-binding protein according to claim 20, which is directly or indirectly linked in the order of EYFP, H-Ras, Raf. 22. The low molecular weight GTP-binding protein is Rap 1A, the target protein is Ral GDS, the GFP donor protein is ECFP, and the GFP receptor protein is EYFP, wherein the GFP receptor is EYFP. 16. A protein for monitoring the activity of a low-molecular-weight GTP-binding protein according to any one of the above items 16. 23. The activity monitor protein for a low molecular weight GTP-binding protein according to claim 22, which is directly or indirectly linked in the order of EYFP, Rap1A.RaI GDS, and ECFP from the amino terminal side. 24. The low molecular weight GTP-binding protein is Rac1, the target protein is Pak, the GFP donor protein is ECFP, and the GFP receptor protein is EYFP. And 17. An activity monitor for a low molecular weight GTP-binding protein according to any one of 17. 25. The activity monitor protein for a low molecular weight GTP-binding protein according to claim 24, which is directly or indirectly linked in the order of EYFP, Pak, Rac1, and ECFP from the amino side. 26. The low molecular weight GTP-binding protein is Cdc42, the target protein is Pak, the GFP donor protein is ECFP, and the GFP receptor protein is EYFP. An activity monitor protein for the low-molecular-weight GTP-binding protein according to any one of the above. 27. The activity monitor protein for a low-molecular-weight GTP-binding protein according to claim 26, which is directly or indirectly linked in the order of EYFP, Pak, Cdc42, and ECFP from the amino terminal side. 28. The low molecular weight GTP binding protein is RhoA, the target protein is mDia, the GFP donor protein is ECFP, and the GFP receptor protein is EYFP, any one of claims 1 to 4, 7 to 14 and 19. A protein monitor for the activity of a low-molecular-weight GTP-binding protein according to the above. 29. The activity monitor protein of a low molecular weight GTP binding protein according to claim 28, which is directly or indirectly linked in the order of EYFP, mDia, RhoA, and ECFP from the amino terminal side. 30. A gene encoding an activity monitor protein of the low molecular weight GTP-binding protein according to any one of claims 1 to 29. 31. An expression vector comprising the gene according to claim 30.  32. The expression vector according to claim 31, which is an expression plasmid. 33. A transformed cell carrying the expression vector according to claim 31 or 32. 34. A transgenic animal carrying the expression vector according to claim 31 or 32. 35. A method for measuring activation of a low-molecular-weight GTP-binding protein, comprising a step of detecting FRET in a monitor protein of the activity of the low-molecular-weight GTP-binding protein according to any one of claims 1 to 29. 36. A method for measuring activation of a low-molecular-weight GTP-binding protein, comprising a step of detecting FRET in the cell according to claim 33 or the transgenic animal according to claim 34. 37. The method further comprises the step of measuring the GTP-bound GTP-bound protein and the GTP-bound GTP-bound protein resulting from the release of inorganic phosphate from GTP to calculate the GTPZGDP ratio (molar ratio). A method for measuring activation of the low molecular weight GTP-binding protein according to claim 36. 38. (a) contacting the cell according to claim 33 with a test substance; and (b) detecting a change in the activity of the low-molecular-weight GTP-binding protein. A method for screening an activity modulator.