JP2001231576A - Tyrosine-phosphorylated protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new tyrosine-phosphorylated protein Cbp functioning as a regulatory factor with Src-PTKs activity bearing an important role in cell differentiation, proliferation, adhesion, configurational change, etc., and to provide a genetic engineering material and antibody each for making use of the above protein. SOLUTION: This tyrosine-phosphorylated protein having a specific amino acid sequence, a polynucleotide encoding the above protein, a recombinant vector holding the above polynucleotide, and an antibody to the above protein, are provided respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a novel tyrosine-phosphorylated protein Cbp (Csk bind) involved in the control of cell functions such as differentiation, proliferation, adhesion and morphological changes of animal cells.
ing protein) and genetic engineering materials for obtaining and using this protein Cbp.

[0002]

2. Description of the Related Art Src family protein tyrosine kinases (Src-PTKs) play an important role in the differentiation, proliferation, adhesion and morphological changes of animal cells. Protein C that specifically phosphorylates tyrosine residues at its C-terminal tail
It is negatively regulated by sk (C-terminal src kinase) (References 1-3).

However, Csk and Src-PTKs
Are localized mainly in the cytoplasm and in the vicinity of the membrane, respectively. Therefore, it is assumed that there is a molecular mechanism involved in the interaction between these enzymes, but such a molecule has not been known at all.

[0004] Src-PTKs activity and C responsible for its regulation
To elucidate the molecular mechanism that mediates sk, for example,
It may lead to elucidation of the mechanism of invasion and metastasis of carcinoma, and is also expected to be applied to the diagnosis of malignancy of carcinoma and the development of therapeutic methods and therapeutic agents.

[0005] The inventors of the present application disclose that Csk can translocate from the cytoplasm to a membrane structure in which Src is active (Reference 9), and that the SH2 and / or SH3 domains of Csk are responsible for the suppression of Src-PTKs. Based on previous findings that are essential (References 10 and 11), inhibition of membrane-bound Src-PTKs by Csk requires Csk and Src
Based on the hypothesis that the presence of a Csk-binding molecule having close proximity to PTKs is necessary, a novel tyrosine-phosphorylated protein Cbp having Csk-binding ability was isolated from neonatal rat brain, and the invention of this application Was completed.

[0006] An object of this application is to provide a novel tyrosine phosphorylated protein Cbp isolated by the present inventors in a form which can be used industrially.

[0007] Another object of the present invention is to provide a material for producing the protein by genetic engineering.

[0008]

This application provides the following inventions (1) to (5) to solve the above-mentioned problems. (1) A tyrosine phosphorylated protein having the amino acid sequence of SEQ ID NO: 2. (2) A polynucleotide encoding the tyrosine phosphorylated protein of the invention (1). (3) The polynucleotide of the invention (2) having the base sequence of SEQ ID NO: 1. (4) A recombinant vector having the polynucleotide of the invention (2) or (3). (4) An antibody against the tyrosine phosphorylated protein of the invention (1).

Hereinafter, embodiments of each of the above-described inventions will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The tyrosine phosphorylated protein Cbp of the invention (1) is a protein encoded by the polynucleotide (genomic gene) of the invention (2) and is shown in SEQ ID NO: 2.
It has the amino acid sequence of the amino acid residue (aa). From the mRNA transcribed from this genomic gene, a bp cDNA (polynucleotide of the invention (3)) whose nucleotide sequence is shown in SEQ ID NO: 1 is synthesized.

The protein Cbp of the invention (1) is isolated from rat tissues by a known method, that is, a purification method described later, and a method for preparing a peptide by chemical synthesis based on the amino acid sequence provided by the invention. Alternatively, it can be obtained by a method of producing the recombinant DNA technology using the polynucleotide provided by the invention (3). For example, when Nectin-3 is obtained by recombinant DNA technology, RNA is prepared by in vitro transcription from a recombinant vector having the polynucleotide of the invention (3) (invention (4)), and this is used as a template for in vitro translation. Can be expressed in vitro. If each polynucleotide is recombined into an appropriate expression vector by a known method, Escherichia coli, Bacillus subtilis, yeast,
The protein C encoded by the polynucleotide in animal cells, etc.
bp can be expressed in large amounts.

When the protein Cbp of the invention (1) is produced by expressing a DNA fragment by in vitro translation, for example, the translation region of the polynucleotide of the invention (3) may be replaced with RNA.
Recombined into a vector having a polymerase promoter,
The protein Cbp of the invention (1) can be produced in vitro by adding it to an in vitro translation system such as a rabbit reticulocyte lysate or a wheat germ extract containing an RNA polymerase corresponding to the promoter.

When the protein Cbp is to be expressed in a microorganism such as Escherichia coli, an expression vector having an origin, a promoter, a ribosome binding site, a DNA cloning site, a terminator, and the like, which can be replicated in the microorganism, is used.
A recombinant expression vector is prepared by inserting and binding the polynucleotide of (3), and after transforming a host cell with this expression vector, the resulting transformant is cultured, whereby the polynucleotide encodes Nectin-3 can be mass-produced in microorganisms. Alternatively, it can be expressed as a fusion protein with another protein. By cleaving the obtained fusion protein with an appropriate protease, only the protein portion encoded by the polynucleotide can be obtained.

When the protein Cbp is expressed in animal cells, the polynucleotide of the invention (3) is recombined into an animal cell expression vector having an animal cell promoter, a splicing region, a poly (A) addition site, and the like. When introduced into animal cells, the protein Nectin-3 of the present invention can be expressed in animal cells.

The Cbp of the invention (1) includes a peptide fragment (5 amino acid residues or more) containing any partial amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2. These peptide fragments can be used, for example, as an antigen for producing an antibody.

The polynucleotide of the invention (2) is a genomic gene encoding the above-mentioned protein Cbp. For example, using the polynucleotide of the invention (3) or a partial sequence thereof as a probe, it is possible to obtain a polynucleotide from an existing genomic library or the like. Can be isolated.

[0017] The polynucleotide of the invention (3) has a nucleotide sequence represented by SEQ ID NO: 1;
A. Since the protein Cbp is expressed in almost all tissues of animals, the invention (3) can be performed by screening a cDNA library of a rat or the like using an oligonucleotide probe synthesized based on the nucleotide sequence of SEQ ID NO: 1. And the same cDNA clone as the above polynucleotide can be easily obtained. Alternatively, the target polynucleotide can also be synthesized by using these oligonucleotides as primers and using the polymerase chain reaction (PCR) method.

Generally, polymorphism due to individual differences is frequently observed in mammalian genes. Therefore, in SEQ ID NO: 1, 1
Alternatively, a polynucleotide in which a plurality of nucleotides are added, deleted and / or substituted by other nucleotides is also included in the present invention.

Similarly, proteins in which one or more amino acid residues have been added, deleted and / or substituted by other amino acid residues due to these changes,
As long as it has the activity of the protein having the amino acid sequence represented by SEQ ID NO: 2, it is included in the present invention.

The polynucleotide of the invention (3) also includes a cDNA fragment (10 bp or more) containing any partial nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 1, or a polynucleotide comprising an antisense strand thereof.

The antibody of the invention (5) can be obtained as a polyclonal antibody or a monoclonal antibody by a known method using the protein Cbp itself or a partial peptide thereof as an antigen.

The results of the experiment for obtaining the protein Cbp and confirming its function are described below. 1. Materials and Methods 1.1 Antibodies and Cells Anti-Csk antibodies are derived from the Csk C-terminal 14 amino acids (aa
437-450) were prepared by immunizing rabbits with conjugated to keyhole limpet hemocyanin. The anti-Csk antibody was purified by affinity on a resin to which the peptide was bound. The anti-Cbp antibody is GST-Cbp (aa of SEQ ID NO: 2).
275-419) GST-C using fusion protein as immunogen
Purified by affinity on bp bound resin. Antibody to Fyn, Antibody to Fak, Paxill
The antibodies against in are from Transduction Laboratories, the anti-Src antibody and PP2 are from Calbiochem Novabiochem, the anti-Src (pY527) is from Biosource QCB, and the anti-phosphotyrosine antibody (anti-pY, 4G10) is Upstate Bi.
Obtained from otechnology. Western blotting and immunoprecipitation were performed as described in reference (22). Cholera toxin (CTX) B subunit conjugated to horseradish peroxidase (HRP) is Sigm
Obtained from a. 293T cells, COS7 cells, NIH
3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. For cell adhesion experiments, cells were detached by trypsin / EDTA treatment (non-adherent cells) and then cultured in collagen-coated dishes as described in reference (10) (adherent cells). 1.2 Immunoprecipitation method, pull-down method, and intracellular fractionation method Tissues and cells were treated with ODG buffer (50 mM TrisHCl, pH
8, 1 mM EDTA, 0.15 M NaCl, 20 mM NaF, 1 mM Na ₃ V
O ₄ , 2% octyl-D-glucoside, 5 mM β-mercaptoethanol, 10 μg / ml aprotinin, 10 μg / ml leupeptin, 1 mM PMSF, and 5% glycerol)
In a ratio of 8: 1 (volume / weight). The homogenate was converted to protein G Sepharose (Amer
(sham pharmacia biotech) for 1 hour and clarified by centrifugation at 20,000 g for 20 minutes. The supernatant was incubated with the given antibody and protein G Sepharose for 1 hour. Immunoprecipitates were washed with ODG buffer and analyzed by Western blotting. For pull-down analysis, the clarified lysate was incubated with the corresponding GST fusion protein bound to glutathione sepharose beads for 1 hour, after which the protein bound to the beads was eluted and analyzed by Western blotting. Small-scale centrifugation as described below (4 ml-35% 40% sucrose in a Beckman SW 41 rotor).
% Sucrose-5% sucrose 1 ml) was used to analyze the binding of the DIM fraction to the protein. After centrifugation at 200,000 g for 12 hours at 4 ° C, fractions of 1.0 ml each were collected from the top of the gradient and analyzed by Western blotting. 1.3 Purification of Cbp, peptide analysis, and cDNA
Cloning of 1-day-old rat-derived whole brain (250 brains in total) in a lysis buffer containing Triton X100 (50 mM TrisHCl, pH 8.0, 10 mM
M MgCl ₂ , 0.15 M NaCl, 20 mM NaF, 1 mM Na ₃ VO ₄ , 1%
Triton X100, 5 mM β-mercaptoethanol, 10 μg
/ Ml aprotinin, 10 μg / ml leupeptin, 1 mM PM
(SF and 5% glycerol) at a ratio of 8: 1 (volume / weight) and stirred at 4 ° C. for 1 hour. The homogenate (15 ml per test tube) was mixed with the same volume of buffer A (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM).
Mixed with 80% sucrose in 0 mM MgCl ₂ , 1 mM Na ₃ VO ₄ ). After transferring the mixture to a centrifuge tube, the 35
25 ml of 5% sucrose and 5 ml of 5% sucrose in buffer A were successively overlaid. After centrifugation at 100,000 g for 3 hours at 40 ° C. in a Beckman 35 rotor, material localized at the interface between 35% and 5% sucrose was collected. The collected material was diluted 3-fold with buffer A and centrifuged at 100,000 for 1 hour at 4 ° C.
The pellet was solubilized in ODG buffer, centrifuged at 100,000 g for 1 hour at 4 ° C., and the clear supernatant was then GST-Csk
The solution was passed through an affinity column bound to SH2. Bound material was eluted using 1% SDS in 25 mM Tris-HCl, pH 6.8. After concentration under reduced pressure, the sample was subjected to SDS-PA
Separated further by GE. An 80-90 kDa Csk-binding protein was excised from an SDS-PAGE gel and digested in a gel with lysyl endopeptidase (Reference 23). One-tenth of the recovered peptide was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-T
OFMS) was used for peptide mass fingerprinting, and the remainder was separated by HPLC. The three peptides were subjected to gas-phase microsequencing, and oligonucleotide primers for PCR (forward primer: SEQ ID NO: 3, reverse primer: SEQ ID NO: 4) were designed using the obtained sequences. The template cDNA was a random hexamer, poly (A) + RN from neonatal rat brain.
A was used together with A. By this PCR, a specific DNA fragment of 435 bp was obtained.
5 'and 3'-R using plification Kit (Clontech)
The full-length Cbp cDNA was isolated by the ACE method. After sequencing the cDNA, the amino acid sequence of the putative ORF was deduced. Possible post-transcriptional modifications of Cbp include:
MS-Match, a software for supporting protein identification by MS, using Cbp peptide mass fingerprints (http: // ww
w.protein.osaka-u.ac.jp/organic) and determined by comparison with that of the primary structure of the cDNA predicted. Protein expression CbpcDNA at 1.4 in vivo and in vitro and cloned in pBluscript II (Stratagene) vector at T _N T binding reticular contact erythrocyte lysate system (Promega), in vitro translation in the presence of ³⁵ S- methionine did. His-tagged Cbp protein is Cb
pTrcHis vector carrying pcDNA (Invitrogen)
Was produced in E. coli DH5α transformed with E. coli. Transient transfections in 293T and COS7 cells were performed using Lipofectamine 2000 (Gi
bco BRL). Cbp (CbpΔN, aa53 to 424 of SEQ ID NO: 2) and GEP tag Cbp (Cb
p △ N) was expressed using pCMV-tag (Stratagene) and pEGFP-N1 vector (Clontech), respectively. Tyr is Phe
The Cbp mutant having a single substitution at was prepared by the PCR method. Csk and c-Src were obtained from pME18Sneo (donated by Dr. Maruyama of the University of Tokyo) and pcDNA3 vector (Invi
trogen). Recombinant Csk is Sf
Baculoviru purified from 9 cells and carrying Csk DNA
It was stably transfected using the s vector. 1.5 In Vitro Kinase Assay DIM fractions were extracted in ODG buffer and clarified lysate was combined with 50 μM ATP in kinase buffer (50 mM).
TrisHCl, were incubated at pH 7.4 and 10 mM MgCl ₂₎ in. After 10 minutes incubation at 30 ° C,
The reaction was stopped by the addition of SDS sample buffer. Protein phosphorylation was analyzed by Western blotting using an anti-pY antibody. The analysis of substrate phosphorylation mediated Src, and the c-Src immunoprecipitated and incubated in the presence of [gamma over ³² p] ATP of 10μM with enolase. Protein phosphorylation levels were determined by BAS2000 Bioimage
Visualization was performed using Analyzer (Fuji). 1.6 Immunofluorescence staining and confocal microscopy COS7 cells transfected with pEGFP-N1 and pEGFP-Cbp were grown for 2 days on sterile coverslips in a conventional manner. pME18Sneo-Csk and pcDNA-cS
Cells transfected with rc were grown overnight prior to antibody staining. Immunofluorescence staining was performed using a rhodamine-conjugated anti-rabbit IgG antibody (Amercham pharmacia biotech) (Cs
k) and Cy3-conjugated anti-mouse IgG antibody (Amercham pha
rmacia biotech) (c-Src). The specimen was observed with a confocal microscope using LSM510 (Carl Zeiss). 2. Results 2.1 Immunoprecipitation, pull-down, and subcellular fractionation Immunoprecipitation of Csk from neonatal rat brain lysates enriched for Csk and Src-PTKs resulted in migration as a diffuse band of approximately 80-90 kDa in molecular size. The tyrosine phosphorylated protein was co-precipitated (FIG. 1a, lane 1). The addition of the recombinant Csk protein (rCsk) was 80-
It enhanced the yield of the 90 kDa protein (Figure la, lane 3) and other known proteins that interact with Csk, such as Fak and Paxillin (Reference 10). However, 80-
Only the 90 kDa protein remained tightly bound to Csk even in the presence of increasing concentrations of NaCl (1 M). Pull-down analysis using a GST-Csk fusion protein containing different domains of Csk revealed that the 80-90 kDa protein and the Csk S
A specific interaction with the H2 domain was revealed (FIG. 1b). GST fusion proteins containing the SH2 domain of Src and Fyn were able to bind to the 80-90 kDa protein, in addition to several other proteins,
Immunoprecipitation analysis demonstrated that neither the Src nor the Fyn intact protein could significantly interact with the 80-90 kDa protein (FIG. 3b). These data indicate that Csk and the 80-90 kDa phosphoprotein (Cb
This suggests a unique interaction with p). 2.2 Purification of Cbp In optimizing the conditions for the purification of Cbp, this protein was concentrated in the Triton X-100 insoluble fraction, and phosphorylation of Cbp was achieved by adding MgCl ₂ to the lysis buffer. Was found to be enhanced. Furthermore, Triton X-1
Further fractionation of the cell lysate according to Example 1 on a discontinuous sucrose density gradient, phosphorylated Cbp resulted in cholera toxin (CTX) binding gangliosides GM1 and Src
-PTKFyn was shown to be mainly localized in the so-called DIM (surfactant insoluble membrane) fractions (References 4, 5) (Figure 1c, fractions 2 and 3). Cbp
Was localized in the DIM fraction and interacted with the SH2 domain of Csk. Therefore, Cbp was purified by the following means. That is, separation of the DIM fraction from neonatal rat brain, solubilization with octyl-D-glucose, followed by affinity chromatography on a resin to which the GST-Csk SH2 fusion protein was bound (FIG. 2a). Cbp was further purified by SDS-PAGE, excised from the gel, and digested in a gel for microsequencing. 2.4 Peptide analysis and cloning of cDNA Based on the amino acid sequence of the peptide from Cbp, PCR
A cDNA of Cbp was prepared by the method, and the primary structure of the open reading frame (ORF) was estimated (FIG. 2).
b). The ORF contains 242 amino acids and the calculated molecular mass is 45,912 daltons. The deduced sequence is expressed in T-line cells, and the DIM compartment (literature
L, a linker protein that is significantly localized in
Very weak but significant homology with AT. In addition, the domain structures of both are highly similar (FIG. 2c). That is, both of these proteins have short extracellular domains (Cb
p: aa1-17, LAT: aa 1-4) followed by a putative transmembrane domain (Cbp: aa 18-38, LAT: aa)
5-27), a putative palmitoylation site (Cbp: aa39)
And 42, LAT: aa 26-29), and has a long cytoplasmic domain. In a partial analysis using a mass spectrometer, at least five tyrosine residues (aa 165, 183, 22
4, 381 and 409), but it is presumed that these tyrosine residues are phosphorylated. Cbp has nine potentially tyrosine phosphorylated sites (FIGS. 2b and c). 4 sites (aa 165, 183, 381 and 409; pYE
The sequence around XI / V / L) contains a Src binding motif (pYEE
L) (Literature 14), but the other sites are different. Western blotting with anti-Cbp antibody showed that protein Cbp was expressed in various tissues of rats, but highly expressed in developing brain, lung, thymus, spleen, and testis (data Is not shown). 2.4 In Vitro and In Vivo Expression Cb In Bitro in Rabbit Reticulocyte Lysate
Translation of the pmRNA or expression in bacteria resulted in an unphosphorylated protein with a molecular size of about 68 kDa (Fig. 3a, lanes 1-2). Estimated molecular size of Cbp (49 kDa) and SD of unphosphorylated Cbp
The difference from mobility on S-PAGE could be explained by the large number of negatively charged amino acids (30 Asp and 41 Glu). The molecular size of the phosphorylated Cbp precipitated from the DIM fraction with anti-Cbp antibody varied between 70 and 90 Kda (FIG. 3a, lanes 3, 5), indicating that the cytoplasmic portion of Cbp It may be explained by the presence of multiple phosphorylation sites. Indeed, treatment of the Cbp immunoprecipitate with alkaline phosphatase results in a single Cbp band of 68 kDa (FIG. 3a, lanes 4, 6). Using an anti-Cbp antibody, D
The specific interaction between Csk and phosphorylated Cbp in IM was confirmed (FIG. 3b). The properties of the protein tyrosine kinase responsible for the phosphorylation of Cbp were
It was examined both in itro and in vivo. In using DIM fractionation
Kinase activity in vitro shows strong Mg-ATP-dependent phosphorylation of Cbp, and Cbp and its kinase
It was suggested that they coexist in the IM fraction (FIG. 3c,
Lanes 1, 2, 7, and 8). Phosphorylation is rCsk
Or PP2, a selective inhibitor of Src-PTKs
Addition of (Reference 15) was dose-dependently suppressed (FIG. 3c, lanes 3-6 and 9-12, respectively), implying the involvement of Csk-sensitive Src-PTKs in Cbp phosphorylation. . Cbp phosphorylation could also be performed by co-incubation with purified Src or Fyn (data not shown). Furthermore, phosphorylation of Cbp in NIH3T3 cells
Inhibited by incubation with P2 (FIG. 3d). Exogenous c-Src in 293T cells
And Cbp resulted in phosphorylation of Cbp and binding to Csk (FIG. 3e). Using an expression system in 293T cells and a Cbp mutant having a single substitution of tyrosine by phenylalanine, the residue important for binding to Csk was identified to be tyrosine 314 (Tyr-314) (FIG. 3f). These results indicate that Src-PTK
suggests that s is at least one of the kinases responsible for phosphorylation of Cbp to create a binding site for Csk, although other types of kinases may be involved. It could not be ruled out.

The in vivo interaction between Csk and Cbp was examined in a transient expression system using COS7 cells that moderately express endogenous Cbp. Csk overexpressed in these cells was dispersed in the cytoplasm by diffusion (FIG. 4a). In contrast, c-Src and Cbp coexisted on the cytoplasmic side of the plasma membrane (FIG. 4b). When Csk was co-expressed with Cbp, most Csk
Became localized together with Cbp on the cytoplasmic side of the plasma membrane (FIG. 4c). C with Phe in place of Tyr-314
The bp mutant (F314) and the mutant lacking the amino-terminal membrane binding region (CbpΔN) were unable to recruit Csk to the plasma membrane (FIGS. 4d and e). The results of the Csk-Cbp interaction on Src-PTKs activity in vivo were determined using the COS expressing exogenous Cbp.
Analyzed in 7 cells (FIG. 5a). Overexpressed C
bp was phosphorylated and bound to endogenous Csk. Cb
The amount of Csk bound to p and Cbp depends on the endogenous c-
It was inversely related to Src activity. In contrast, for F314, tyrosine was phosphorylated but did not bind to Csk or affected the activity of c-Src. CbpΔN does not phosphorylate tyrosine, and CskΔN
And did not affect the activity of c-Src.
Furthermore, overexpression of Cbp in a cell line derived from csk knockout mouse (D5) (Reference 15) is due to c-Src
Had no significant effect on the activity of These data, together with the results of the immunofluorescence analysis (FIG. 4), were combined with Csk
Demonstrates an essential role for Cbp phosphorylation of Tyr-314 and membrane localization for recruitment. 2.5 Cell Adhesion The function of Cbp as a regulator of Src-PTKs activity was examined in a model of cell adhesion to substrates. The activation and inhibition of Src-PTKs over time during cell adhesion has been recognized as a good example of the dynamic regulation process of Src-PTK (10, 18). Csk also uses Sr
It has been shown to be involved in the regulation of cell spread and adhesion through regulation of c activity (Reference 18). Under the experimental conditions employed here, detachment of normal COS7 cells from the substrate induced activation of whole cell c-Src, which then gradually became inactive as the cells spread. Wild type Cbp
Overexpression suppressed adhesion-dependent changes in c-Src activity, but not that of the F314 mutant (FIG. 5).
b). Cbp phosphorylation is down-regulated by detachment of cells from the substrate (FIG. 5c), thus dephosphorylation of Cbp in non-adherent cells is c-Src
May be activated. In fact, c-Src
Phosphorylation level of the important tyrosine (Tyr-527) was significantly reduced in non-adherent cells. Even when Cbp was overexpressed, down-regulation of Cbp phosphorylation due to cell detachment occurred, but sufficient amounts of Cbp were present.
Remained phosphorylated and bound to Csk to maintain phosphorylation of Tyr-527. F3 unable to bind to Csk
17 did not show any inhibitory effect on c-Src.
These results indicate that c-mediated Csk-mediated c-
This suggests that Cbp may be involved in the regulation of Src. 3. Conclusion Summarizing the above data, the activation of Src-PTKs results in the phosphorylation of Cbp,
It is proposed that a negative feedback signaling loop exists in which sk is recruited to the membrane and Src-PTKs are suppressed. This process is considered reversible. In such a situation, SHP-2
Tyrosine phosphatases such as (19) or PTPalpha (20) appear to dephosphorylate Cbp, followed by Csk migration and Src-PTKs reactivation. Recently, it has been shown that Src-PTK is down-regulated by a ubiquinone-dependent mechanism (Reference 21). In order to fully understand the life span of Src-PTKs, Csk / Cb
The functional relationship between p and the ubiquinone system must be clarified. The limited localization of Cbp in the DIM fraction indicates a unique role for this membrane domain in the dynamic regulation of ligand-activated Src-PTKs activity. Finally, the relatively ubiquitous expression of Cbp in various cell types suggests the importance of Cbp in regulating other physiological processes.

[0024]

As described above in detail, according to the invention of this application, a novel tyrosine phosphorylation functioning as a regulator of Src-PTKs activity, which plays an important role in cell differentiation, proliferation, adhesion, morphological change, etc. Protein C
bp, and genetic engineering materials and antibodies for utilizing this protein are provided. This protein Cbp plays an important role in the expression of functions in various cells.
Since it is an important signal molecule in the nervous system and immune system, it is not only a diagnostic and therapeutic agent for carcinoma, but also a therapeutic and diagnostic agent for diseases such as immunodeficiency, neurodevelopmental disorders, and osteoporosis.
Alternatively, it is useful for developing gene therapy technology and the like.

[0025]

[Sequence List] SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Tyrosine kinase <130> NP00008-NT <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 1275 <212> DNA <213> Rat <220> <221> CDS <222> (1) .. (1275) <400> 1 atg gga cct gca gga agc gcc ctg agc agc ggg cag atg cag atg cag 48 Met Gly Pro Ala Gly Ser Ala Leu Ser Ser Gly Gln Met Gln Met Gln 1 5 10 15 atg gtg ctg tgg gga agt ctg gct gcg gtg gcc atg ttc ttc ctc atc 96 Met Val Leu Trp Gly Ser Leu Ala Ala Val Ala Met Phe Phe Leu Ile 20 25 30 acc ttc ctc atc ctt ctg tgc tcc agt tgt gac aga gac aag aag cca 144 Thr Phe Leu Ile Leu Leu Cys Ser Ser Cys Asp Arg Asp Lys Lys Pro 35 40 45 cgg cag cat agt gga gac cat gag agc ctg atg aat gtg cct tcc gac 192 Arg Gln His Ser Gly Asp His Glu Ser Leu Met Asn Val Pro Ser Asp 50 55 60 aag gag atg ttc agc cat tca gcg acc agc ctg acc aca gat gca ctg 240 Lys Glu Met Phe Ser His Ser Ala Thr Ser Leu Thr Thr Asp Ala Leu 65 70 75 80 gcc agc agc gaa cag aat ggg gtg ctc acc aac ggc g ac att ctt tca 288 Ala Ser Ser Glu Gln Asn Gly Val Leu Thr Asn Gly Asp Ile Leu Ser 85 90 95 gaa gac agc aca atg acc tgc atg cag cat tat gag gaa gtc cag acc 336 Glu Asp Ser Thr Met Thr Cys Met Gln His Tyr Glu Glu Val Gln Thr 100 105 110 tca gct tca gat ctg ctg gac tcc cag gac agc acg gga aag gcc aag 384 Ser Ala Ser Asp Leu Leu Asp Ser Gln Asp Ser Thr Gly Lys Ala Lys 115 120 125 tgc cac cag agc cgg gaa ctg ccc aga atc ccg cct gag aac gca gtg 432 Cys His Gln Ser Arg Glu Leu Pro Arg Ile Pro Pro Glu Asn Ala Val 130 135 140 gac gcg atg ctc acc gcc agg gcc gca gat ggg gac tcc ggg ccc ggc 480 Asp Ala Met Leu Thr Ala Arg Ala Ala Asp Gly Asp Ser Gly Pro Gly 145 150 155 160 gtg gag ggg ccc tac gag gtg ctc aag gat agt tcc tcc cag gag aac 528 Val Glu Gly Pro Tyr Glu Val Leu Lys Asp Ser Ser Ser Gln Glu Asn 165 170 175 atg gtg gag gac tgc ttg tat gag aca gtg aag gaa atc aag gag gtg 576 Met Val Glu Asp Cys Leu Tyr Glu Thr Val Lys Glu Ile Lys Glu Val 180 185 190 gca gac aaa agc cag ggc gg aag tcc act tct gcc ctg aag 624 Ala Asp Lys Ser Gln Gly Gly Lys Ser Lys Ser Thr Ser Ala Leu Lys 195 200 205 gag ctt cag ggg gcc cac gcc gag ggc aag gcc gac ttt gct gaa tac 672 Glu Leu Gln Gly Ala His Ala Glu Gly Lys Ala Asp Phe Ala Glu Tyr 210 215 220 gcc tct gtg gac aga aac aaa aag tgc cgc cac agc aca aat gca gag 720 Ala Ser Val Asp Arg Asn Lys Lys Cys Arg His Ser Thr Asn Ala Glu 225 230 235 240 agc att ctg gga acg tcc agt gac ctg gat gag gaa acc cca ccg cct 768 Ser Ile Leu Gly Thr Ser Ser Asp Leu Asp Glu Glu Thr Pro Pro Pro 245 250 255 gtc cca gtt aaa ctt ctg gat gag aat gcc aac ctt cca gag aag gga 816 Val Pro Val Lys Leu Leu Asp Glu Asn Ala Asn Leu Pro Glu Lys Gly 260 265 270 gag cac ggg gca gaa gag caa gct cca gaa gca ccc agt gga cac agc 864 Glu His Gly Ala Glu Glu Gln Ala Pro Glu Ala Pro Ser Gly His Ser 275 280 285 aag aga ttt agt tcc ttg tca tac aag tct cga gaa gaa gac cca act 912 Lys Arg Phe Ser Ser Leu Ser Tyr Lys Ser Arg Glu Glu Asp Pro Thr 290 295 300 ctt aca gaa gag gag atc tcagca atg tat tct tca gtg aat aag cct 960 Leu Thr Glu Glu Glu Ile Ser Ala Met Tyr Ser Ser Val Asn Lys Pro 305 310 315 320 gga caa tcg gca cac aaa cct ggg ccg gaa tct acc tgc cag tgc ccg 1008 Gly Gln Ser Ala His Lys Pro Gly Pro Glu Ser Thr Cys Gln Cys Pro 325 330 335 cag ggt ccc cct cag agg tca tca tct tcc tgt aac gac ctc tat gcc 1056 Gln Gly Pro Pro Gln Arg Ser Ser Ser Ser Cys Asn Asp Leu Tyr Ala 340 345 350 act gtg aaa gac ttc gag aaa act ccc aac agc atc agc atg ctg cca 1104 Thr Val Lys Asp Phe Glu Lys Thr Pro Asn Ser Ile Ser Met Leu Pro 355 360 365 cca gca agg agg ccc ggt gag gaa cca gag cct gac tat gaa gcc ata 1152 Pro Ala Arg Arg Pro Gly Glu Glu Pro Glu Pro Asp Tyr Glu Ala Ile 370 375 380 cag act cta aac aga gag gac gac aag gtc cct ctg ggg acc aat ggc 1200 Gln Thr Leu Asn Arg Glu Asp Asp Lys Val Pro Leu Gly Thr Asn Gly 385 390 395 400 cac ctt gtc ccc aag gag aat gac tat gag agc att ggg gac ttg cag 1248 His Leu Val Pro Lys Glu Asn Asp Tyr Glu Ser Ile Gly Asp Leu Gln 405 410 415 caa gg c agg gat gtc acc agg ctc tag 1275 Gln Gly Arg Asp Val Thr Arg Leu 420 425 <210> 2 <211> 424 <212> PRT <213> Rat <400> 2 Met Gly Pro Ala Gly Ser Ala Leu Ser Ser Gly Gln Met Gln Met Gln 1 5 10 15 Met Val Leu Trp Gly Ser Leu Ala Ala Val Ala Met Phe Phe Leu Ile 20 25 30 Thr Phe Leu Ile Leu Leu Cys Ser Ser Cys Asp Arg Asp Lys Lys Pro 35 40 45 Arg Gln His Ser Gly Asp His Glu Ser Leu Met Asn Val Pro Ser Asp 50 55 60 Lys Glu Met Phe Ser His Ser Ala Thr Ser Leu Thr Thr Asp Ala Leu 65 70 75 80 Ala Ser Ser Glu Gln Asn Gly Val Leu Thr Asn Gly Asp Ile Leu Ser 85 90 95 Glu Asp Ser Thr Met Thr Cys Met Gln His Tyr Glu Glu Val Gln Thr 100 105 110 Ser Ala Ser Asp Leu Leu Asp Ser Gln Asp Ser Thr Gly Lys Ala Lys 115 120 125 Cys His Gln Ser Arg Glu Leu Pro Arg Ile Pro Pro Glu Asn Ala Val 130 135 140 Asp Ala Met Leu Thr Ala Arg Ala Ala Asp Gly Asp Ser Gly Pro Gly 145 150 155 160 Val Glu Gly Pro Tyr Glu Val Leu Lys Asp Ser Ser Ser Gln Glu Asn 165 170 175 Met Val Glu Asp Cys Leu Tyr Glu Thr Val Lys Glu Ile Lys Glu Val 180 185 190 Ala Asp Lys Ser Gln Gly Gly Lys Ser Lys Ser Thr Ser Ala Leu Lys 195 200 205 Glu Leu Gln Gly Ala His Ala Glu Gly Lys Ala Asp Phe Ala Glu Tyr 210 215 220 Ala Ser Val Asp Arg Asn Lys Lys Cys Arg His Ser Thr Asn Ala Glu 225 230 235 240 Ser Ile Leu Gly Thr Ser Ser Asp Leu Asp Glu Glu Thr Pro Pro Pro 245 250 255 Val Pro Val Lys Leu Leu Asp Glu Asn Ala Asn Leu Pro Glu Lys Gly 260 265 270 Glu His Gly Ala Glu Glu Gln Ala Pro Glu Ala Pro Ser Gly His Ser 275 280 285 Lys Arg Phe Ser Ser Leu Ser Tyr Lys Ser Arg Glu Glu Asp Pro Thr 290 295 300 Leu Thr Glu Glu Glu Ile Ser Ala Met Tyr Ser Ser Val Asn Lys Pro 305 310 315 320 Gly Gln Ser Ala His Lys Pro Gly Pro Glu Ser Thr Cys Gln Cys Pro 325 330 335 Gln Gly Pro Pro Gln Arg Ser Ser Ser Ser Cys Asn Asp Leu Tyr Ala 340 345 350 Thr Val Lys Asp Phe Glu Lys Thr Pro Asn Ser Ile Ser Met Leu Pro 355 360 365 Pro Ala Arg Arg Pro Gly Glu Glu Pro Glu Pro Asp Tyr Glu Ala Ile 370 375 380 Gln Thr Leu Asn Arg Glu Asp Asp Lys Val Pro Leu Gly Thr Asn Gly 385 390 395 400 His Leu Val Pro Lys Glu Asn Asp Tyr Glu Ser Ile Gly Asp Leu Gln 405 410 415 Gln Gly Arg Asp Val Thr Arg Leu 420 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthesized oligonucleotide <400> 3 ggngcngarg arcargcncc nga 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthesized oligonucleotide <400> 4 cgnccytgyt gnarrtcncc dat 23

[Brief description of the drawings]

FIG. 1 shows the results of experiments in which the detection and localization of Csk-binding protein (Cbp) were examined. a. Cs in rabbit brain
It is the result of confirming the specific binding between k and a phosphoprotein of 80 to 90 kDa. Neonatal brain lysates are transformed with recombinant Csk (r) in the presence or absence of Csk peptide (pep.).
Incubated with anti-Csk antibody with or without Csk). The immunoprecipitated protein is used as an anti-pY
Analysis was performed by western blotting using the antibody. The amount of Csk in the immunoprecipitate was adjusted by Western blotting using anti-Csk antibody (lower panel). Square brackets indicate the position of Cbp. b. Cbp,
This is the result of confirming the binding of Csk to SH2 domain. Lysate of neonatal rat brain was used for GST-Csk
SH2, GST-Src SH2, GST-Fyn SH
2, and GST-Csk SH3 fusion protein,
Alternatively, GST alone was incubated (lane 1 to lane 1).
6). The binding proteins are indicated above. The lower panel shows the input amount of GST fusion protein. c. It is the result which shows the remarkable localization of Cbp in the DIM fraction of a brain lysate. Neonatal rat brains were homogenized in lysis buffer containing Triton X-100 in the presence of MgCl ₂ and separated on a discontinuous sucrose gradient (12). Aliquots of the fractions were resolved by SDS-PAGE and the anti-p
Analysis was performed by Western blotting using the Y, anti-Csk, anti-Fyn antibody, and the B subunit of cholera toxin (CTX).

FIG. 2 shows the results of purification and the structure of Cbp. a. Cb
It is a purification result of p. Neonatal rat brain was transformed with Triton X-
Homogenize (lysate) in a buffer containing 100,
The DIM fraction was further separated by a discontinuous sucrose density gradient (DIM). The DIM fraction was solubilized using ODG buffer, and Cbp was purified by affinity chromatography on a resin bound to GST-Csk SH2 (CsK-SH2). Samples from the purification stage were analyzed by Western blotting with anti-pY (left panel) and protein staining with silver (right panel). b. It is an amino acid sequence of rat Cbp and human LAT (Reference 11). Boxes for putative transmembrane domains.
Arrowheads indicate putative palmitoylated cysteine residues (Reference 11). Tyrosine residues in Cbp and L
An important tyrosine residue of AT (Reference 11) is framed. The amino acid sequence of Cbp determined by microsequencing is underlined. Alignment was performed using the ClustalW program from the DNA Data Bank of Japan. c. 2 shows the domain structure of rat Cbp and human LAT proteins. Amino acid residues (aa) at positions 39 and 42 and aa at positions 26 and 29 are Cbp and L, respectively.
Corresponds to the putative palmitoylation site of AT;
Y, tyrosine residue; P, tyrosine phosphorylation site; DI
M, surfactant insoluble membrane microdomain. The region used for antibody (anti-Cbp) production is underlined (thick line).

FIG. 3 shows the results of confirming the characteristics of the Cbp protein.
a. Cbp mRNA was synthesized and translated in vitro, Cbp protein was degraded by SDS-PAGE and visualized by autoradiography (lane 1).
The His-tagged Cbp protein expressed by bacteria was replaced with anti-C
Detection was performed by Western blotting using a bp antibody (lane 2). The Cbp protein was immunoprecipitated from the DIM fraction prepared in the presence of MgCl ₂ (lane 3) and treated with alkaline phosphatase (CIP) (lane 4).
Cbp was detected as described above, and phosphorylation of immunoprecipitated Cbp was analyzed by Western blotting using anti-pY antibody (lanes 5 and 6). b. It is the result of confirming the interaction in the DIM fraction between Csk and phosphorylated Cbp. The solubilized DIM fraction prepared as described above was used for non-immune IgG, anti-Cbp, anti-Cs
Immunoprecipitated using k, anti-Src, or anti-Fyn antibody. DIM fractions and immunoprecipitates were purified with anti-pY (upper left),
Analysis was performed by Western blotting using anti-Cbp (upper right), anti-Csk (lower left), anti-Src (lower center), and anti-Fyn (lower right) antibodies. c. Cb in DIM fractionation
This is the result of confirming the phosphorylation of p. MgCl ₂ and Na ₃ VO
Rat brain DIM fractions prepared without ₄ were combined with increasing amounts of rCsk (0,1) with 50 μM ATP and 10 mM MgCl _2.
0, 20, 40, and 80 ng) (left panel, lanes 2
6) or PP2 (0, 1, 2, 5, and 10 μM,
Lanes 8-12) (right panel).
Protein phosphorylation in the DIM fraction was analyzed by Western blotting using an anti-pY antibody. d. Cb
It is the result of confirming the effect of PP2 on the phosphorylation of p in vivo. NIH3T3 cells were treated with increasing amounts of P
Cultured in the presence of P2 (0, 1, 2, 5, and 10 μM). After 10 hours incubation, whole cell lysate (left panel) and immunoprecipitated Cbp (right panel)
Was subjected to Western blotting using an anti-pY antibody. The Cbp protein in the immunoprecipitate was confirmed by Western blotting using an anti-Cbp antibody. e. in
It is the result which confirmed the phosphorylation of Cbp in vivo. 239T cells were transformed into the expression vector pCMV-tag
(Lane 1), pCDNA3-c-Src (lane 2), pCMV-Cbp (lane 3), and pCDN
A3-c-Src was used to transiently transfect with pCMV-Cbp (lane 4). Protein expression was analyzed by western blotting of cell lysates with anti-Src, anti-Csk, and anti-Cbp antibodies (top three rows). Lysates of transfected cells were immunoprecipitated with anti-Csk antibody and immunoprecipitates were analyzed by Western blotting with anti-Cbp, anti-pY, or anti-Csk antibody (lower part).
f. It is the identification result of the Csk binding site in Cbp.
A Cbp mutant with Phe instead of Tyr was co-expressed with c-Src in 239T cells as described above. Tyrosine phosphorylation of Cbp protein and Cbp was detected by Western blotting using anti-Cbp and anti-pY antibodies, respectively. Tyrosine phosphorylated Cbp bound to Csk was analyzed by Western blotting of Csk immunoprecipitates using anti-pY antibody.
The amount of Csk in the Csk immunoprecipitate was adjusted by western blotting using an anti-Csk antibody.

FIG. 4 shows the results of confirming the coexistence of Cbp and Csk in COS7 cells. pME18Sneo-Csk
To pEGFP (a), pEGFP-Cbp (c), p
EGFP-F314, (d) or pEGFO-Cbp
Used in combination with the ΔN (e) expression vector, COS7 cells were transiently co-transfected. Twenty-four hours later, the cells were fixed, the fixed cells were incubated with the anti-Csk antibody, and Csk was revealed by staining with a goat anti-rabbit antibody conjugated with rhodamine. The region where Csk (red) and GFP-Cbp (green) coexist is yellow. In addition, COS7 cells were
It was transiently co-transfected with DNA3-c-Srg and pEGFP-Cbp expression vector (b). c-Src was revealed by staining with goat anti-mouse antibody conjugated with Cy3 after incubation with anti-Src antibody. The region where Src (red) and GFP-Cbp (green) coexist is yellow.

FIG. 5 shows the results of confirming the correlation between Csk-Cbp interaction and c-Src kinase activity. a. Cbp
Is the result of confirming that the overexpression of cS7 inhibits c-Src kinase activity in COS7 cells. pCM
V-Cbp, pCMV-F314, pCMV-CbpΔ
Increasing amounts of N were mixed with pCMVtag vector DNA and transfected into COS7 cells (5 × 10 ⁵ ) such that the total amount of transfected DNA was equal to 4 μg. DNA4 of pCMVtag vector only
Cells transfected with μg were used as controls. pCMV-Cbp was also transfected into D5 cells (csk-/-) as described for COS7 cells. Transfected cells were lysed in ODG buffer, cell lysates were incubated with anti-Cbp or anti-Csk antibody, and immunoprecipitated Cbp protein and its phosphorylation were western blotted using anti-Cbp and anti-PY antibodies, respectively. Was analyzed by Endogenous c-Src was immunoprecipitated from the lysate and kinase activity was assessed in an in vitro assay using enolase as a substrate. The c-Src protein in the immunoprecipitate was detected by Western blotting using an anti-Src antibody. The amounts of phosphorylated enolase and immunoprecipitated c-Src were determined by Bioimage Analyzer. b. It is the result of confirming the influence of Cbp overexpression on c-Src regulation depending on cell adhesion. CO
S7 cells were transfected with pCMVtag, pCMV-Cbp, or pCMV-F314 as described above, harvested with trypsin / EDTA, and then washed with serum-free medium in the presence of trypsin inhibitor. One quarter of the cells were incubated for 1 hour in a non-adherent test tube, then used as non-adherent cells (N1h), the rest was seeded on a collagen-coated dish and grown for the indicated time to adhere cells. (A1h, A2h,
A 3h, and A 4h). Cells were lysed in ODG buffer and Cbp expression was analyzed by Western blotting using anti-Cbp antibody. As described above, c-Sr
The activity of c was calculated, and the relative specific activity of c-Src was calculated as
It was calculated by dividing the amount of phosphorylated enolase by the amount of immunoprecipitated c-Src protein. c. c-Sr
It is the result of confirming the effect of Cbp overexpression on the phosphorylation of C at Tyr-527. Transfected C
OS7 cells were detached by trypsin / EDTA treatment (N, non-adherent cells), seeded on collagen-coated dishes and incubated for 12 hours (A, adherent cells). Cells were lysed and Cbp expression was analyzed by Western blotting. Cbp was recovered using anti-Cbp and anti-Csk antibody, and tyrosine phosphorylation of Cbp was detected using anti-pY antibody. C-Src using anti-Src antibody
Immunoprecipitation, in vitro kinase assay and anti-S
Western blotting using rc and anti-Src (pY527) antibody was performed. In non-adherent cells
The ratio of phosphorylation of Tyr-527 to that in adherent cells, N (527) / A (527), was calculated by densitometry and is shown at the bottom of the graph.

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──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 19/10 A61P 25/28 25/28 37/04 37/04 C12N 15/00 ZNAA (72) Inventor Yasushi Shimonishi 3-9-22 Kamikoshien, Nishinomiya-shi, Osaka F-term (reference) 4B024 AA01 AA11 BA10 CA04 CA07 EA04 GA13 HA03 4B050 CC01 CC03 DD11 FF13E FF14E LL01 LL03 4C085 AA13 CC04 DD33 4H045 AA11 CA45 DA50 FA30 GA30 EA20 GA30

Claims (5)

[Claims] 1. A tyrosine phosphorylated protein having the amino acid sequence of SEQ ID NO: 2. 2. A polynucleotide encoding the tyrosine phosphorylated protein according to claim 1. 3. The protein having the nucleotide sequence of SEQ ID NO: 1.
Polynucleotide. 4. A recombinant vector having the polynucleotide according to claim 2 or 3. 5. An antibody against the tyrosine phosphorylated protein of claim 1.