JP2002360265A - Protein having binding activity to p85, nucleic acid encoding the protein, etc. - Google Patents

Abstract

[Problem] To purify BCAP, a novel protein, analyze the molecular structure and function of the protein as a mediator between BCR-associated PTKs and PI3K, and react specifically with the protein To provide antibodies. SOLUTION: In order for a BCR-associated protein tyrosine kinase to control the activity of a downstream effector molecule, tyrosine phosphorylation of an associated protein is required. Here we describe BCAP, a novel B-cell associated protein that associates with PI3K
Report on Tyrosine phosphorylation of BCAP is Syk and Btk
Through which BCAP provides a binding site for the p85 subunit of PI3K. Deletion of the BCAP gene in the DT40B cell line inhibits BCR-mediated biosynthesis of PIP3,4,5-3 phosphate, resulting in a suppressed Akt response. Furthermore, PI3K
The transition to Glycolipid-rich microdomains (GEMs)
Significantly weakened by loss of BCAP. Therefore, these data indicate that BCAP controls the localization of PI3K,
It suggests that it is responsible for mediating signals between the associated kinase and the PI3K pathway.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel protein, BCAP, which binds to p85.

[0002]

BACKGROUND OF THE INVENTION The B cell antigen receptor (BCR) is a trigger of a biochemical cascade to B cell activation that causes B cell proliferation and differentiation. BCR activates Igα and non-receptor protein tyrosine kinases (PTKs) to activate them.
Utilizes the Igβ immunoreceptor tyrosine-based activation motifs (ITAMs). PTKs belonging to three different classes are involved in the regulation of signaling pathways downstream of BCR.
(Src-PTKs, Syk, Btk) must be activated in order. Indeed, deficiency of any of these three PTK families would impair B cell function and development. If so, the specific content is summarized in <References>.)
(DeFranco 1997; Reth and Wienands, 1997; Tamir an
d Cambier, 1998; Kurosaki 1999). Therefore, these
Analysis of the mechanism by which BCR-associated PTKs regulate the generation of second messengers downstream of the signal has been actively performed to date.

In order for B cells to acquire function normally,
There is a need for an efficient and ordered synthesis of many second messengers. These second messengers include IP ₃ (inositol [3,4,5] triphosphate) and PI (3,
4,5) P ₃ (phosphatidylinositol [3, 4, 5] triphosphate) is included. Phosphoinositide 3-kinase (PI
3K) is responsible for the production of PI (3,4,5) P ₃ and is involved in cell growth, survival, metabolism, cytoskeletal rearrangement, and cell membrane exchange (Leevers et al., 1999; Rameh and Cantley, 1
999). Heterodimeric (class Ia) PI3K consists of a catalytic subunit (p110) and regulatory subunits encoded by at least three different genes (p85α, p85β,
p55γ). p85α is the most frequently expressed control subunit isoform.The importance of this α-type subunit in B cell proliferation and differentiation has recently been elucidated by the gene targeting method using mice, and has attracted attention. I have. Indeed, the proliferative response of B cells to BCR stimulation is attenuated by the deletion of the p85α subunit (Fruman et al., 1999; Suzuki et al, 199).
9).

[0004]

Although the p85 subunit plays a key role in BCR signaling, the mechanism by which BCR-associated PTKs regulate the PI3K pathway has not yet been elucidated. This mechanism appears to involve two SH2 domains of the p85 subunit. Both SH2 domains bind with high affinity to phosphorylated tyrosine in the YxxM motif. Thus, SH2 domain-dependent binding to associated proteins is thought to be important for PI3K to translocate to cell membranes and exert enzymatic activity (Shoelson et al.,
1993; Fruman et al., 1998). One such association partner is CD19, a B cell-specific membrane protein with two YxxM motifs in the cell membrane region. In fact, CD19 is tyrosine phosphorylated when stimulated by BCR. Thereby, CD19 is thought to provide a binding region for the p85 subunit (Tuveson et al., 1993; Buh
l and Cambier, 1999). However, B cell development and dysfunction in p85α ^{− / −} mice is more prominent than that in CD19 ^{− / −} mice, so the CD19-mediated activation mechanism alone fully explains the PI3K activation mode. (Engel et al., 1995; Rickert et al., 199
5; Fruman et al., 1999; Suzuki et al., 1999).

[0005] The present invention has been made in view of the above circumstances, and based on the hypothesis that there may be other associated molecules as mediators between BCR-associated PTKs and PI3K,
It is intended to purify a novel protein, BCAP, to analyze the molecular structure and function of the protein, and to provide an antibody that specifically reacts with the protein.

[0006]

Means for Solving the Problems, Effects of the Invention, and Effects of the Invention According to the first invention, one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 17. Consisting of an amino acid sequence, and
It is a protein that has binding activity to p85. “One or more amino acids are deleted, substituted and / or added” means that the number of amino acids can be deleted, substituted and / or added by a method well known to those skilled in the art such as site-directed mutagenesis. It means that amino acids are deleted, substituted and / or added. "P85"
The two subunits that make up the heterodimeric PI3K (ie, the catalytic subunit (p110) and at least 3
Among the control subunits (p85α, p85β, p55γ) encoded by different types of genes, it means the control subunit. The control subunit has
Although there are three different molecules, the molecule to which the protein of the present invention exhibits binding activity is at least p85α.

[0007] The term "p85 binding activity" means that the protein of the present invention is in direct contact with p85 at least in an environment close to the in vivo environment. Examples of in-vitro conditions simulating the in-vivo environment include, but are not limited to, about 150 mM NaCl and about 20 mM Tris [pH 7.5]. "Protein" means a polymer in which the amino acid residues that are monomers are joined together by amide bonds. As used herein, the term "polypeptide" or "protein" includes any amino acid sequence,
And modified amino acid sequences such as glycoproteins. "Polypeptide" also includes naturally-occurring proteins as well as proteins formed by recombinant or chemical synthesis. In addition, the protein of the present invention was prepared by the inventors using BCAP (B cell adapter for pho
sphoinositide 3-kinase (PI3K)).

[0008] A second invention is a nucleic acid encoding the protein according to the first invention. "Encode" means that the protein of the present invention is expressed with p85-binding activity. The term "encode" includes encoding the protein of the present invention as a continuous structural sequence (exon) or encoding the protein of the present invention via an appropriate intervening sequence (intron). The term “nucleic acid” includes ribonucleic acid, deoxyribonucleic acid, or a modified form of any nucleic acid. In addition, the nucleic acid includes single-stranded or double-stranded DNA.

A third invention is a nucleic acid which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 16 under stringent conditions and encodes a protein having p85 binding activity. "Stringent conditions"
For example, a temperature of about 40 ° C. or higher and a salt concentration of about 5 × SS
C (1 × SSC has a composition of 15 mM sodium citrate buffer [pH 7.0] and 150 mM sodium chloride), preferably at about 50 ° C. or higher, about 2 × SSC,
More preferably, it refers to hybridization conditions at about 65 ° C. or higher and about 0.1 × SSC, but is not limited thereto. In addition to these conditions, molecular cloning (2nd edition), Current Protocols in Molecular Biology, DNA Cloning1: Core T
echniques, A Practical Approach, Second Edition, O
Hybridization conditions described in xford University (1995) and the like can be used.

A fourth invention is a nucleic acid comprising an amino acid sequence having at least 70% homology with the amino acid sequence shown in SEQ ID NO: 17 and encoding a protein having p85 binding activity. "Homology" is a numerical value obtained by comparing the difference between two amino acid sequences, for example,
In databases such as DDBJ, GenBank, and EMBL, regarding homology between a predetermined amino acid sequence and an amino acid sequence recorded in an existing amino acid sequence database, for example,
It can be calculated using software such as t2 sepuence research. The homology of the protein encoded by the nucleic acid of the present invention to the amino acid sequence shown in SEQ ID NO: 17 is preferably 70% or more (or the same), more preferably 80% or more, and still more preferably 90% or more, especially homology. Preferably, they are 95% or more homologous.

[0011] A fifth invention is a recombinant expression vector to which the DNA according to the second invention to the fourth invention is operably linked. In the second to fourth inventions, nucleic acids containing DNA are described, but in the fifth invention, a vector to which DNA of those nucleic acids is ligated is described. Note that, in the present invention, the expression vector for obtaining the protein of the present invention is described. Those skilled in the art will very easily think that a ligated recombinant vector is sufficient. `` Expressable '' means that a DNA encoding a predetermined amino acid sequence, under predetermined conditions,
It means that it has the ability to express a protein having that amino acid sequence. When a DNA encoding a predetermined amino acid sequence is operably linked to the DNA,
Under a given condition, a given protein is expressed. Specifically, the DNA is linked to an expression control sequence in a vector. Here, “expression control sequence” means a nucleic acid sequence that regulates the expression of another nucleic acid sequence, and controls and regulates the transcription (preferably, translation) of another nucleic acid sequence. . Expression control sequences include appropriate promoters, enhancers, transcription terminators, initiation codons (ie, ATG) in the gene encoding the protein, splicing signals for introns, and stop codons.

The term "promoter" means a minimum sequence necessary for performing transcription. The promoter also includes a promoter element that controls gene expression in a cell type-specific, tissue-specific, or promoter-dependent manner by an external signal or a regulator. The promoter element is linked to either the 5 'or 3' region of the expressed DNA. In addition, the promoter includes both a constitutive one and an inductive one. For example,
When producing an expression vector in bacteria,
Bacteriophage γ p as an inducible promoter
Promoters such as L, plac, ptrp, ptac (ptrp-lac hybrid promoter) can be used. When an expression vector is produced in a mammalian cell, a promoter derived from a mammalian cell (eg, a metallothionein promoter), a promoter derived from a mammalian virus (eg, a retroviral terminal repeat (LTR), an adenovirus Late promoters, vaccinia virus 7.5K promoter, etc.) can be used.

"Recombinant vector" includes a plasmid or a virus. Expression recombinant vectors generally include an origin of replication, a promoter, and a special gene that permits selection when the expression recombinant vector is introduced into cells. Suitable vectors for the present invention include T7-based expression vectors for bacterial systems, pMSXND expression vectors for mammalian cell systems, retroviral vectors, adenovirus vectors, baculovirus-derived vectors for insect cell systems, Cauliflower mosaic virus (CaM
V), tobacco mosaic virus (TMV) and the like. In addition, those skilled in the art can very easily select other appropriate vectors for carrying out the present invention. Depending on the vector used, many suitable transcription and translation elements can be used, such as constitutive or inducible promoters, transcription enhancer elements, transcription terminators, etc., which are known to those of skill in the art. is there. In yeast, too, many vectors containing constitutive or inducible promoters can be used. For example, there are constitutive yeast promoters such as ADH or LEU2, and inducible promoters such as GAL. In addition, vectors that facilitate the incorporation of foreign DNA sequences into yeast chromosomes can be used.

A sixth invention is a transformant containing the recombinant vector according to the fifth invention. "Transformation"
Introducing new (foreign) DNA to a cell means that the cell undergoes a permanent genetic change. If the cell is a mammalian cell, a permanent genetic change is caused by the presence of DNA in the cell's genome.
Is usually achieved by introducing A “transformant” means that the cell or a cell that is an ancestor of the cell is a cell into which DNA (including a vector) encoding the protein of the present invention has been introduced by recombinant DNA technology. Means. Transforming a host cell with the recombinant DNA can be performed by a method well known to those skilled in the art. When the host is a prokaryotic cell (eg, Escherichia coli), competent cells having the ability to take up DNA can be prepared by a calcium chloride method according to a procedure well known to those skilled in the art. Transformation can also be performed by alternative methods such as electroporation.

The protein of the present invention can be obtained by expressing a nucleic acid encoding the protein in a prokaryote. Examples of prokaryotes include, but are not limited to, microorganisms (eg, bacteria) transformed with a recombinant vector for expressing bacteriophage, plasmid DNA, or cosmid DNA containing the nucleic acid sequence encoding the protein of the present invention. is not. The protein of the present invention can be expressed by culturing Escherichia coli on a large scale in an in vitro test system. Purification of the protein from bacteria can be simplified by including a label in the protein sequence for purification in one step, for example using nickel chelate chromatography. As such a label, for example, a polyhistidine label consisting of a plurality of (preferably 5 to 15) histidine residues can be incorporated into the amino terminal or carboxyl terminal of the protein of the present invention. Such polyhistidine labeling enables efficient protein isolation in one step using nickel chelate chromatography. The protein of the present invention can also be designed to include an appropriate enzyme cleavage site for the purpose of improving recovery.

When the host is eukaryotic, the calcium phosphate method, microinjection, electroporation, liposome, viral vector
DNA can be transfected. Also,
Eukaryotic cells contain a DN encoding the protein of the present invention.
A molecule and a second encoding a selectable trait (eg, the herpes simplex virus thymidine kinase gene).
The DNA molecule can be co-transfected. Alternatively, a eukaryotic viral vector (eg, Simian Virus 40 (SV40) or bovine papilloma virus) is used to transiently infect eukaryotic cells with the viral vector to transform and transform the protein. It can be expressed. Preferably, eukaryotic cells are utilized as host cells, as described herein.

Eukaryotic cells (more preferably, mammalian cells)
In, appropriate post-translational modifications occur after the protein is expressed. In eukaryotic cells that have the proper processing of primary transcripts (eg, glycosylation, phosphorylation) or that have an intracellular mechanism to secrete gene products, host cells whose expression can be confirmed by fluorescent indicators Can be used as Such host cell strains include, for example, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-2
93 and WI38. In order to produce a recombinant protein in a high yield over a long period of time, it is preferable to construct a stable expression system. To do so, rather than using an expression vector containing the viral origin of replication, the host cell is controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) In addition, it is preferable that the DNA is transformed with a DNA encoding the protein of the present invention and a selectable marker. A selectable marker on the recombinant plasmid confers resistance to selection on the cell, stably incorporates the plasmid into their chromosomes, allows for sequential cloning and establishment as a cell line. Those capable of forming cell colonies are preferred. For example, exogenous DNA is introduced into host cells, and the cells are cultured for 1-2 days in a nutrient-rich medium, and then changed to a selection medium and cultured. Such selection systems include, for example, herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase. In each selection system, the selection genes are tk-, hgprt-, and aprt-, respectively. Dhfr, gpt, n
There are eo, hygro, trpB, etc. dhfr is a gene that confers resistance to methotrexate, gpt is a gene that confers resistance to mycophenolic acid, neo is a gene that confers resistance to aminoglycoside G-418, and hygro confers resistance to hygromycin Is a gene. Also, tr
pB is a gene that allows cells to use indole instead of tryptophan, hisD is a gene that allows cells to use histinol instead of histidine, and ODC (ornithine decarboxylase gene) is ornithine. Is a decarboxylase inhibitor
It is a gene that confers resistance to 2- (difluoromethyl) -DL-ornithine (DFMO), and such a selection gene can also be used. Techniques for isolation or purification after expression of the protein of the present invention include conventionally known means, for example, chromatographic separation and immunological separation using an antigen or an antibody (monoclonal antibody, polyclonal antibody). It can be performed by such means.

According to a seventh aspect, a protein encoded by the nucleic acid according to the second to fourth aspects is used,
This is a screening method for screening a substance that inhibits the signaling function of the amino acid. "A substance that inhibits the signal transduction function" refers to a substance that inhibits the binding between BCAP and Syk, a substance that inhibits the binding between BCAP and p85, and
Includes substances that act on BCAP to inhibit signal transduction to p85 (PIK). "Screening methods" include:
By adding certain substances, BCAP and Syk or BCA
A method for confirming whether the binding between P and P85 is inhibited (in that case, (i) BCAP should be labeled in advance with a predetermined labeling molecule (eg, fluorescent substance, radioactivity, etc.) Or (ii) by labeling Syk or p85 with a predetermined labeling molecule, it becomes possible to confirm the inhibition.) Or by adding a certain substance, PI (phosphatidylinositol), A method for confirming whether or not phosphoric acid is inhibited from being added to PIP (phosphatidylinositol monophosphate) or PIP ₂ (phosphatidylinositol monophosphate). Labeling (for example, ³² P, ³³ P, etc.) makes it possible to confirm inhibition.).

An eighth aspect of the present invention is an immune function modulator obtainable by the screening method according to the seventh aspect. “Immune function modulator” means an agent that enhances or reduces immune activity. However, in view of the fact that the protein (BCAP) of the present invention binds to p85 and affects the activity of PI3K, immunomodulators obtainable by the screening method of the present invention often require antibody production. Therefore, it is highly likely that the suppression of the function will reduce the immune function.

A ninth invention is an antibody or an antibody fragment which binds to a protein encoded by the nucleic acid according to the second to fourth inventions or a partial amino acid sequence of the protein. `` Antibody '' is a generic term for those that have the activity of specifically binding to an immunogen,
A, IgD, IgE, IgG, IgM are included. “Antibody fragment” means a fragment comprising an amino acid sequence of a part of a protein constituting an antibody, and at least a part having a function of binding to the protein of the present invention (for example, Fab fragment, F (ab ′ ) Means ₂ fragments).

According to a tenth aspect, in the ninth aspect, the antibody is polyclonal. The term "polyclonal" antibody refers to a group of antibody molecules produced by antibody-producing cells of various different clones. Say. According to an eleventh aspect, in the ninth aspect, the antibody is monoclonal. A “monoclonal” antibody is an antibody produced by a single clone of antibody-producing cells and has a uniform amino acid sequence, and is a group of antibody molecules having a uniform class, subclass, light chain type, and idiotype. Say.

According to a twelfth aspect, in the eleventh aspect,
The monoclonal antibody is produced by the fusion cell 4C8. `` Fused cell 4C8 ''
Is a cell received by the National Institute of Advanced Industrial Science and Technology on April 13, and has accession number FERM P-18.
292 means the cell deposited. A thirteenth invention is an antibody-producing cell, which produces the antibody or antibody fragment according to the tenth invention or the eleventh invention. In the present invention, “antibody-producing cell” means a cell that produces an antibody or an antibody fragment. This cell is a natural cell obtained from an animal given the protein of the present invention (or a partial amino acid sequence thereof) as an immunogen (and
The cells are cultured to form a cell line), and the natural cells are used in a method known to those skilled in the art (for example, a method using a compound such as lysolecithin, glycerol oleate, polyethylene glycol (PEG), or electrical stimulation). , A method using a virus (Sendai virus, measles virus, paramyxovirus), etc.) and other cells (eg, a cancer derived from an animal of the same species as the animal from which the natural cells were obtained). Cell lines). First
A fourth invention is characterized in that the antibody-producing cell according to the thirteenth invention is a fused cell 4C8.

[0023]

Next, an embodiment of the present invention will be described in detail with reference to the drawings. However, the technical scope of the present invention is not limited by the following embodiment. Various modifications can be made without changing the gist. The technical scope of the present invention extends to an equivalent range. <Function of BCAP> FIG. 11 is a schematic diagram showing the relationship between BCR and BCAP. BCAP3, a novel protein of the present invention, is considered to be located between Syk2 and PI3K4 in cells. BCAP3 is PI3K4
Has binding activity to p85 molecule 4A among the two types of molecules (p110 molecule 4B and p85 molecule 4A). When a signal enters BCR1 (that is, when a plurality of BCR1s associate), the signal is transmitted to PI3K4 via Syk2 and BCAP3 in that order. Then, PI3K4 phosphorylates various molecules (PI, PIP, PIP ₂ ).

<About BCAP of chicken and mouse> Test Method <Example 1. Cells and Antibodies> Wild-type and mutant DT40 cells derived therefrom are 10% fetal calf serum (FCS),
The cells were cultured in RPMI1640 medium containing 1% chicken serum, 50 μM 2-mercaptoethanol and 2 mM L-glutamine / penicillin / streptomycin. COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS) and 2 mM L-glutamine penicillin streptomycin. Preparation of mouse spleen B cells was performed as previously described (Hashimoto et a
l., 2000). Anti-BCAP and anti-CD19 antibodies were obtained by immunizing rabbits. The immunogens include chicken BCAP (SEQ ID NO: 2: 410-540 amino acid region), mouse BCAP (SEQ ID NO: 10: 603-797 amino acid region) and mouse CD19 (C
A GST fusion protein containing (terminal 49 amino acids) expressed in bacteria was used. Anti-chicken IgM antibody, M
4. Anti-mouse IgM antibody, anti-phosphorylated antibody (4G10) and anti-chicken Lyn antibody have been described previously (Ishiai et al,
1999; Hashimoto et al., 2000). The following antibodies were purchased (anti-p85 antibody; Upstate Biotechnology, anti-Akt1 and anti-α tubulin antibodies; Santa Cruz Biotechnology, anti-JNK1 antibody; Farmingen).

<Embodiment 2. Purification and peptide sequencing of chicken pp100> DT40 cells (1.5 × 10 ¹⁰ )
At a concentration of 10 ⁸ cells / ml, it was performed M4 (4 μg / ml) stimulated for 3 minutes (in RPMI medium, 37 ° C.). The cells are transformed as previously described (Ta
kata et al., 1994) 50 mM NaF and 0.5 mM pervanada
The cells were dissolved in an NP-40 lysis solution containing te and a proteolysis inhibitor (1% NP-40, 150 mM NaCl, 20 mM Tris [pH 7.5]). After centrifugation at 13,000 × g, the supernatant was pretreated with Sepharose 4B and incubated with a p85-SH2 (N) affinity column. The p85-SH2 (N) affinity column was created by binding 5 mg GST-p85-SH2 (N) protein with 4 ml of GSH-Sepharose with dimethyl pimmelidate. Thereafter, the beads were washed with a 10-fold amount of NP-40 solution, and eluted with 1% SDS containing 50 mM Tris-HCl at 95 ° C. for 5 minutes. Dilute the eluate with lysate and add 2 ml of 4G10-bound Pr
Incubated with otein A Sepharose for 12 hours at 4 ° C. The beads are washed with 50 volumes of NP-40 lysis solution, and 0.1% CH
Elution was performed with TBS containing APS, 50 mM phenyl phosphate, 0.1 mM sodium vanadate and a proteolysis inhibitor.
After concentration and desalting with Centricon-10 (Amicon), the final sample was developed on a 7% SDS-PAGE gel, transferred to a PVDF membrane (Applied Biosystems), and stained with Ponceau S. The pp100 band was cut out and digested with Achromobacter protease I. The digested peptide fragments were developed by reverse phase HPLC (Wako Pure Chemical Industries, Ltd.), and amino acid sequencing was performed with a gas phase sequencer (Shimadzu; model PPSQ-23). As shown in the description of FIG. 1, six types of peptide sequences (SEQ ID NOS: 3 to 8)
I got

<Embodiment 3> cDNA cloning and sequencing> Based on peptide sequence TPGQRQLITLQEQV (SEQ ID NO: 4) and IVPYSIEALDK (SEQ ID NO: 5)
A modified oligonucleotide was synthesized. Using the ³² P-labeled oligonucleotide at the 5 'end as a probe, plasmid DT40 c
DNA library (pJG4-5 vector, GenBank accession number U8
9961). Eight clones were isolated and sequenced. The sequence of the longest insert (clone 2: SEQ ID NO: 1) was registered in GenBank (accession number AF2
93805). Using the obtained BCAP cDNA (clone 2) as a probe, a further DT40 cDNA library was screened, and clone 11 having a selective 5 'sequence was isolated (SEQ ID NO: 11; accession number AF315784). A homology search of the chicken BCAP cDNA sequence was performed using the NCBI database, and a mouse EST clone (accession number AA178577) was found. EST
A λZAP mouse cDNA library was screened using DNA as a probe, and 17 clones were isolated. The longest insert had a translation region encoding 811 amino acids (SEQ ID NO: 9 and SEQ ID NO: 10: accession number AF29380).
6).

<Embodiment 4> Establishment of BCAP-deficient DT40 cells> PC
DT40 genomic DNA containing exons corresponding to chicken BCAP amino acid residues 223-800 was isolated by the R method. Amino acid 3
The targeting vectors pBCAP-bsr and pBCAP-puro were constructed by replacing the 11 kb genomic DNA fragment containing the exon corresponding to 38-721 with the bsr or puro cassette (Sugawara et al., 1997). These cassettes corresponded to 3.5 kb each 5 'and 3' of the BCAP genomic sequence. According to literature, pBCAP-bsr or pBCAP-puro
DT40 cells transfected were screened (Sugawara et al., 1997). Lyn, Syk, and Btk deficiency
DT40 cells were previously reported (Takata et al., 1994; Tak
ata and Kurosaki, 1996). BCAP (Y4F) mutant
It was constructed by replacing the codons encoding tyrosine residues (199, 423, 448 and 463) of the chicken BCAP cDNA clone (clone 2) with codons encoding phenylalanine. Mutant chicken BCAP cDN with wild-type or C-terminal flanking FLAG epitope tag
A for each pcDNA3.1 vector (Invitrogen)
Incorporated into This was transfected into BCAP-deficient DT40 cells and selected in the presence of 2 mg / ml G-418. 5 transformations
The electroporation was performed at 50 V and 25 μF.
Expression of the transformed cDNA was confirmed by Western blotting using an anti-FLAG antibody.

<Embodiment 5> Microscopic analysis by immunofluorescence staining> The constructed FLAG-labeled chicken BCAP cDNA was transfected into COS-7 cells by the calcium phosphate method. Four
After 8 hours, the transformed COS cells were cultured on cover glass for 6 hours. COS cells on a cover glass or DT40 cells in the culture solution were fixed with PBS containing 3.7% paraformaldehyde, and permeated with PBS containing 0.1% Triton X-100. 100
μg / ml RNase treatment for 10 minutes and PBS containing 10% FBS (FBS / PB
After blocking by S), the cells were incubated with anti-chicken BCAP antibody for 1 hour and washed three times with FBS / PBS. The stained cells were mounted with Fluoromount-G (Southern Biotechnology Associates) and FLUOV
Analysis was performed using an IEW confocal microscope (Olympus).

<Embodiment 6> Flow cytometry analysis>
BCR expression and apoptosis were assessed by FACScan as previously described.
(Becton Dickinson)
suda et al., 2000). The X-axis and Y-axis of the histograms (FIGS. 4 (A) and 5 (D)) indicate the fluorescence intensity (log scale) and the relative cell number, respectively.

<Embodiment 7> Northern blot analysis> Mouse tissue RNA was obtained from various tissues of 8-week-old C57 / BL6 mice by RN.
It was prepared using Azol B (Tel-Testinc.). Northern blots of various mouse cell lines are from Drs. Goitsuka and Kitamur
a (Tokyo University of Science). Twenty micrograms of total RNA was developed on a 1% agarose formamide gel, transferred to a Hybond-N ⁺ membrane (Amersham Pharmacia Biotech), and detected with a ³² P-labeled mouse BCAP or β-actin cDNA probe.

Embodiment 8 FIG. Immunoprecipitation and Western blot analysis> For immunoprecipitation, cells were lysed with an NP-40 lysate containing a protease inhibitor and a phosphatase inhibitor according to the literature (Takata et al., 1994),
After pretreatment, incubate with the appropriate antibody, then
Incubated with Protein A-agarose. The cell lysate or immunoprecipitation product is developed on an SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF) membrane, and the indicated antibody and chemiluminescence sensitizing system (ECL; Amersham Pharmacia Biotech) are used. Detected.
For the in vitro kinase assay for Akt, the product immunoprecipitated with the anti-Akt1 antibody was added four times with 0.1 ml
Assay buffer (20 mM HEPES [pH 7.4], 10 mM Mg
Cl ₂ , 10 mM MnCl ₂ , 10 mM Sodium vanadate, 2 mM DT
T, 10 μM PKA inhibitor and 10 μM PKC inhibitor). Half of each immunoprecipitated product was treated with 5 μg of histone (H2
B), used for the kinase reaction in 40 μl of assay buffer containing 5 μM ATP and 10 μCi [γ- ³² P] ATP. After incubation at 30 ° C. for 20 minutes, the reaction was stopped by adding SDS sample buffer and boiling for 5 minutes. Sample is SDS-PAGE
It was developed on a gel (15%) and subjected to autoradiography. The other half of the immunoprecipitated product is
Used for Western blot analysis to determine the amount.
JNK's in vitro kinase assay was performed as previously described (Hashimoto et al., 1998).

<Embodiment 9> Cell fractionation method and preparation of GEM fraction> The cytoplasmic and membrane fractionation of DT40 cells was performed as previously described (Ishiai et al., 1999). Fractionation of DT40 cell extracts by sucrose gradient centrifugation for the preparation of GEM fractions was previously described (Ishiai et al., 1999).

Embodiment 10 FIG. PI (3, 4, 5) P ₃ and I
P ₃ Analysis> DT40 cells (10 ⁷ cells / ml) to 1 mCi / ml ^[32 P]
Calcium buffer (10 mM HEPES [pH 7.4]) containing orthophosphate (H ₃ ³² PO ₄ : NEN Life Science)
Radioisotope labeling was performed by incubation in 135 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 5.6 mM glucose) for 1 hour. After washing once with calcium buffer, cells (10 ⁷ cells / time point)
Was stimulated with M4 (4 μg / ml). Then methanol 750 μ
The reaction was stopped by adding l / 1.2 N HCl (1: 1) and lipids were extracted with 600 μl of chloroform. ³² P
-Label-PI (3,4,5) P ₃ marker reacts with PI (3,4,5) P ₂ (Sigma) and [γ- ³² P] ATP in the presence of anti-p85 antibody immunoprecipitated product It was adjusted by doing. For thin layer chromatography (TLC), samples were spotted on silica gel plates (EM Science) previously treated with potassium oxalate, and chloroform / acetone / methanol / acetic acid / water (40: 15: 13: 12: 7) was used. ).
The radioactivity of the PI (3,4,5) P ₃ spot was quantified using a Fuji FLA2000 bioimaging analyzer and normalized by the total radioactivity in the sample phospholipids.
The values obtained therefrom were normalized to the values at 0 hours after BCR stimulation for three independent experiments, respectively.
The values for the batches were averaged. PI ₃ measurements were performed as previously described (Ishiai et al., 1999).

B. Test results Molecular structure analysis of BCAP BCAP was purified using the property that BCAP protein associates with the SH2 domain of the PI3K p85 subunit following crosslinking of BCR. Both SH2 domains of the p85 subunit have been shown to bind with high affinity to similar phosphorylated tyrosine-containing sequences (Songyang
et al., 1993), we employed a two-step affinity purification method. In this method, using the immobilized N-terminal SH2 domain of p85 and an anti-phosphorylated tyrosine monoclonal antibody,
Pp10 from chicken DT40 B cells activated by BCR
0 was isolated (see experimental method). The microsequencing of pp100 resulted in six intermediate peptide sequence fragments (FIG. 1A), the sequences TPGQRLITLQEQV (SEQ ID NO: 4) and I
VPYSIEALDK (SEQ ID NO: 5) was used for the synthesis of altered oligonucleotides. A DT40 cDNA library was screened using a mixture of these oligonucleotides,
A cDNA clone of kb (SEQ ID NO: 1 clone 2) was isolated. The translated region of this clone encoded 800 amino acids (SEQ ID NO: 2), and the molecular weight was calculated to be 90,152 Da (FIG. 1B). All six peptides (SEQ ID NO: 3 to SEQ ID NO: 8) derived from pp100 were present in the translation region. The expected molecular weight from the results of SDS-polyacrylamide gel electrophoresis was 100,000 Da, which was slightly higher than the value calculated from the sequence, which is considered to be due to the acidic nature of the BCAP polypeptide (calculation Isoelectric point; 4.85).

A mouse homolog corresponding to chicken BCAP was also obtained from the mouse spleen cDNA library. The isolated clone encodes 811 amino acids (SEQ ID NO: 10),
It has 65% homology with chicken BCAP (FIG. 1B). BCAP
When the primary structure of the deduced amino acid sequence is compared on the NCBI protein database, it shows weak homology to the Drosophila Dof protein (FIG. 1C) (Vincent et al., 199).
8). Both BCAP and Dof share a short sequence (amino acids 633-666 of chicken BCAP with amino acids 336-404 of chicken BCAP and amino acids 366-440 of Dof) expected to form a coiled-coil structure. Dof
703-741 amino acids). Chicken BCAP has 31 tyrosine residues that can be phosphorylated. SH
BCAP has four YxxM motifs (PI3Kp85 subunit) as two candidate binding regions (Songyang et al., 1993, 1994).
And one YxNx motif (Grob2) and one YxxV motif (SHP-2), while Dof has one YxxM motif and 5
It has one YxNx motif and one YxxV motif (FIG. 1C). In addition, BCAP includes Src-PTKs, PLC-γ, and
There are three conserved proline-rich regions presumed to bind to the Grb2 N-terminal SH3 region, but Dof lacks this (FIG. 1C) (Sparks et al., 1996).

To clarify the tissue expressing BCAP, Northern blot analysis was performed using various mouse tissues and cell lines (FIG. 2A). The BCAP transcript was strongly expressed in the spleen and weakly expressed in the thymus, liver, and lung. BCAP mRNA is expressed in B cell lines (WEHI279 and L121
0), pre-B cell line (18-81), and macrophage cell line (P388D1 and WEHI3), but T cell line (EL-4 and BW5147), mast cell line (P815), blood cell line (B813), and some non-hematopoietic cell lines (B1
6, Y1, NIH3T3, and ES-E14). From these data, we concluded that BCAP was mainly expressed in B cells and macrophages.

Using a polyclonal antibody against chicken BCAP (410-540 amino acids) and mouse BCAP (603-797 amino acids), BCAP protein expression analysis in B cells was performed. As a result of Western blot analysis, four proteins (BCAP4, BCAP3, BCAP2, and BCAP1) having molecular weights of 100, 98, 72, and 70 kD were detected in both the DT40 cell line and the mouse spleen B cell extract (BCAP4, BCAP3). (Figure 2B). Excision of the chicken BCAP gene
Since the synthesis of all kinds of proteins does not occur, it is considered that these four kinds of proteins are due to the selective transcription and / or post-translational modification of the BCAP gene. To explore this possibility, we will add another DT40
Screen the cDNA library and use a different 5 '
A new 2.5 kb cDNA clone containing the sequence (SEQ ID NO: 1)
1: Clone 11) was obtained. The translated region of clone 11 encoded 632 amino acids (calculated molecular weight; 70,904 Da) lacking the N-terminus (see description for FIG. 1B). When cDNA clone 11 was expressed in COS cells, this clone
When 1 / BCAP2 protein was expressed, while cDNA clone 2 was expressed, BCAP3 / BCAP4 was expressed (FIG. 2B). Furthermore, the presence of an alternative exon present in the 5 ′ untranslated region of clone 11 was confirmed by genomic PCR analysis (FIG. 2).
C). Therefore, BCAP3 / BCAP4 and BCAP1 / BCAP2 are likely to indicate the presence of two different transcripts due to alternative translation initiation or alternative splicing. In contrast to the immunoprecipitation results, BCAP2 / BCAP4 was more predominantly detected in chicken NP-40 cell extract than BCAP1 / BCAP3 (FIG. 2B). This result suggests that the anti-chicken BCAP antibody used in this study has a higher affinity for BCAP1 / BCAP3 than for BCAP2 / BCAP4. The localization of BCAP in DT40B cells was analyzed by immunofluorescence. BCAP was present in the cytoplasm and plasma membrane of DT40B cells, but not in the nucleus (Figure 2).
D). No staining was detected in BCAP-deficient DT40 cells. Localization at the cytoplasm and cell membrane was also observed in COS cells transfected with BCAP cDNA (FIG. 2D). Consistent with the microscopic observation, BCAP was also present in the cytoplasmic fraction and the cell membrane fraction in the cell fraction analysis of DT40 cells (FIG. 2E).

2. Tyrosine phosphorylation of BCAP is Syk and B
Haptic and BCR-activated DT40 B cell-derived BCAP via tk
The results of immunoprecipitation experiments showed that all four types of BCAP were receptor-dependently tyrosine-phosphorylated, peaking at 1 minute after the start of stimulation and returning to the original level at 30 minutes (Fig. 3A; data not shown). The degree of tyrosine phosphorylation of BCAP3 / BCAP4 was at least 5 times that of BCAP1 / BCAP2, based on the ratio of antiphosphorylated tyrosine signal to anti-BCAP signal. Furthermore, after BCR cross-linking, two new BCAP proteins (denoted as BCAP5 and BCAP6) were detected in DT40B cells in addition to these four BCAP proteins,
The time required for these phosphates to reach their peaks is
It was longer than BCAP3 / BCAP4 (10 minutes). These observations suggest that there is structural heterogeneity in the tyrosine phosphorylation state of BCAP after BCR crosslinking. The heterogeneity of the tyrosine phosphorylation state of BCAP after BCR stimulation is different from that of chickens, although the degree of tyrosine phosphorylation of mouse BCAP1 and BCAP2 is higher than that of chickens.
It was also detected in B cells. For mouse spleen B cells,
Tyrosine phosphorylation of BCAP decreased more rapidly than in DT40 cells (FIG. 3A).

To examine the involvement of PTKs in BCAP tyrosine phosphorylation in the BCR cross-linked state, Lyn, Syk, or Bt
BCAP phosphorylation was analyzed using the chicken DT40 cell line deficient in k (Figure 3C) (Takata et al., 1994; Taka
ta and Kurosaki, 1996). In Lyn-deficient cells, BCAP5 / BC
Tyrosine phosphorylation of AP6 was significantly increased, while phosphorylation of BCAP3 and BCAP4 was attenuated. Conversely, Syk deficient DT40
In cells, incomplete phosphorylation of BCAP3 / BCAP4 was observed. In Btk-deficient DT40 cells, tyrosine phosphorylation of BCAP occurred to the same extent as that of the wild-type cells 1 minute after BCR stimulation, but this phosphorylation returned to the original level 10 minutes after the start of BCR stimulation (Fig. 3C). These results are based on Syk and Btk
Suggest that they contribute to the initiation and continuation of BCAP phosphorylation in BCR signaling, respectively. Bt
k is also activated to some extent in Syk-deficient DT40 cells (Kurosaki and Kurosaki, 1997).
The incomplete phosphorylation of BCAP seen in 40 cells may be due to Btk compensating for Syk activity at the initiation of BCAP phosphorylation. Our results also indicate that Lyn plays an inhibitory role in BCR-mediated tyrosine phosphorylation of BCAP, in contrast to the promoting role of Syk and Btk.

3. Association of BCAP with PI3K p85 in B cells BCAP was purified in vitro as a binding target that binds to the N-terminal SH2 domain of the PI3K p85 subunit.
The possibility of the interaction between BCAP and p85 occurring in vivo (in B cells) was examined. PI3K from DT40 B cells activated by dormancy and BCR by immunoprecipitation of BCAP
The presence or absence of a meeting between p85 and BCAP was examined. As shown in Figure 3A,
In both chicken DT40 cells and mouse spleen DT40 cells, p85 is inducibly associated with BCAP by BCR stimulation, and BC85
P85 was not detected by immunoprecipitation from AP-deficient DT40 cells. The degree of association of p85 with BCAP correlated well with the tyrosine phosphorylation status of BCAP (Figure 3A). As previously reported (Tuveson et al., 1993; Buhl and Cambier, 1999), in spleen B cells, p85 also associated with CD19 after BCR stimulation (FIG. 3B).

4. BCAP is involved in PI3K activation by BCR In order to clarify the function of BCAP, we established a BCAP-deficient DT40 strain by the gene targeting method (see the procedure of Example 4). The lack of BCAP protein expression was confirmed by Western blot analysis (FIG. 3A). The expression of cell surface BCR in BCAP-deficient DT40 cell clones was basically equivalent to CBAP-deficient DT40 cells (FIG. 4A). B
Little difference was detected in the degree of CR-induced tyrosine phosphorylation of whole cells between wild-type and BCAP-deficient DT40 cells (Fig. 4B), which indicates that BCR-associated PTKs such as Lyn and Syk are normally free of BCAP. Is activated below.

To investigate the effects of BCAP on PI3K signaling, PI in wild-type and BCAP-deficient DT40 cells
(3, 4, 5) was examined the accumulation of in vivo of P _3. BCR stimulation increased the amount of radioisotope-labeled PI (3,4,5) P ₃ in DT40 cells, but this was inhibited by 100 nM wortmannin treatment prior to BCP stimulation. As shown in Figure 5A, B
CR-mediated accumulation of PI (3,4,5) P ₃ was reduced by 50% in BCAP-deficient cells, resulting in a reduction in Akt activation by half (Fig.
5B). JNK is also shown to be activated in a PI3K-dependent manner in many systems (Klippel et al., 1996; Timo
khina et al., 1998), we also discuss BCAP in JNK activation.
The effect of was examined. In fact, BNK-induced activation of JNK was inhibited by wortmannin treatment, and only incomplete activation of JNK was detected in BCAP-deficient DT40 cells (FIG. 5C). It is well known that Akt inhibits apoptosis (Datta et al., 1999), but as predicted by suppression of Akt activation in BCAP-deficient DT40 cells, Apoptosis through BCR was significantly promoted (FIG. 5D). Furthermore, PI
It has been reported that the accumulation of (3,4,5) P ₃ involves PLC-γ activation through Tec kinases including Btk (Sc
harenberg and Kinet, 1998), was measured IP ₃ synthesis through the BCR in BCAP deficient DT40 cells, found that it is also markedly inhibited (Figure 5E). These results justify a critical role for BCAP in PI3K pathway activation. However, the accumulation of the remaining PI (3,4,5) P ₃ in BCAP deficient DT40 cells, BCAP in B-cell
This suggests the existence of an independent PI3K activation mechanism.

5. Phosphorylation of BCAP YxxM motif is PI3K
To examine the association of the YxxM motif of BCAP (clone 2) with PI3K during PI3K activation, which is essential for activation, we examined a mutant of chicken BCAP (Y4M) in which four YxxM motifs were replaced by the sequence FxxM. It was created. When this protein was expressed in BCAP-deficient DT40B cells and BCR stimulation was applied, still significant BCAP (Y4M) tyrosine phosphorylation was observed.
This suggested that BCAP may have other sites that undergo tyrosine phosphorylation (Fig. 6A). However, BCAP5 / BCAP6 phosphorylation of BCAP (Y4M) is wild-type.
Decreased compared to BCAP. Despite the fact that BCAP (Y4M) is still phosphorylated, this mutant BCAP failed to associate with the PI3K p85 subunit and failed to restore the inhibited Akt activation (FIG. 6A). And 6B).
Similarly, BNK-mediated activation of JNK could be restored by wild-type BCAP, but not by BCAP (Y4M) (Fig. 6).
C). Thus, these data support our conclusion that BCAP supplies a binding region to the p85 SH2 domain after undergoing phosphorylation, thereby contributing to PI3K activation. IP ₃ synthesis completely by the wild-type BCAP, BCAP (Y
4M) partially recovered (data not show
n) This suggests that BCAP is involved in PLC-γ activation by YxxM motif-dependent and -independent mechanisms.

6. Transfer of p85 to GEMs in BCAP-deficient cells is attenuated PI3K translocation to the cell membrane is caused by growth factor receptors
An essential process for PI3K control (Klippel et
al., 1996; Gillham et al., 1999). In recent years, BCR
In downstream signaling, PI3K is redistributed into special subdomains of the cell membrane known as glycolipid-rich microdomains (GEMs) (Xavier et al., 1998), whereby PI3K becomes PI (3,4,5) ) Access to substrates such as P ₂ (phosphatidylinositol [4,5] diphosphate) (Pike
and Miller, 1998). Based on these observations, we believe that BCAP is GCRs of PI3Ks induced by BCR.
I assumed that they would be involved in the transition to. To examine this possibility, for each of wild-type and BCAP-deficient DT40 cells, BCR-mediated relocation of PI3K p85 was examined using cell fractionation by sucrose gradient centrifugation. As a result, previous reports (Cheng et al., 1999; Aman
and Ravichandran, 2000), Lyn was increased in tyrosine-insoluble GEMs (major fraction number 4) and α-tubulin was removed from GEMs (FIG. 7). In pre-stimulated wild-type DT40B cells, a small amount of p85 is localized in GEMs and BC
R stimulation increased this. In contrast, in BCAP-deficient cells, p85 localization to GEMs occurred at low levels but was significantly weakened (FIG. 7). These results indicate that BCAP
It indicates that it is involved in the meeting to GEMs.

The above observation results suggest that BCAP, like LAT in T cells, is structurally localized in GEMs and functions as a protein that plays a role in BCR signal transduction ( Zhang et al., 1998). However, in the case of BCAP, a portion of the protein is reproducibly observed to be localized in GEMs, but most of it is present outside of GEMs before stimulation. The total amount of BCAP protein in GEMs was hardly changed by BCR association. On the other hand, phosphorylated BCAP occurs after BCR stimulation (Fig. 7).

C. Discussion PI3K p85 in B cell development and BCR signaling
Despite the importance of the α subunit, the exact mechanism of signaling between BTK-activated PTKs and PI3K is not yet clear. Here we report that we have isolated and characterized BCAP, a novel PTK substrate that links BCR and PI3K activation. Analysis using a DT40 cell line deficient in Lyn, Syk and Btk revealed that Syk and Btk were BCAP
It has been found that it contributes to tyrosine phosphorylation. Therefore,
Earlier reports that BCR-mediated PI3K activation was attenuated in Syk-deficient B cells indicate that BCAP is phosphorylated by Syk.
The need for k can at least partially explain (Beitz et al., 1999). Bt in contrast to the lack of prominent and global BCAP phosphorylation in Syk-deficient B cells
k-deficient cells showed specific suppression during the sustained process of BCAP phosphorylation (FIG. 3C). Given these data, Syk and Btk may be involved in the initiation and persistence of BCAP phosphorylation, respectively.

Syk in promoting BCAP tyrosine phosphorylation
In contrast to the role of Btk and Btk, Lyn was found to play a totally inhibitory role (Figure 3C). One explanation for this is that Lyn phosphorylates the ITIMs motif of inhibitory receptors such as CD22 and PIR-B to introduce a tyrosine phosphatase with an SH2 domain into the BCR signal complex. (Tamir et al.,
2000). In the case of CD22, Lyn is tyrosine phosphorylated shortly after BCR crosslinking, thereby associating with SHP-1 (Cornall e
t al., 1998; Nishizumi et al., 1998; Smith et al.,
1998). Given the presence of such inhibitory receptors in DT40B cells, further enhancement of BCAP phosphorylation in Lyn-deficient cells can be explained by a deficiency of inhibitory receptor function. This inhibitory function may not necessarily be performed by inhibitory receptors. In fact, cytoplasmic signaling molecules, such as Gab1 / 2, are also known to associate with SHP-2 in a SH2-phosphorylated tyrosine-dependent manner, thereby activating these phosphatases. (Tamir et al., 2000).

When the amino acid sequence of BCAP was estimated, there was one ankyrin repeat-like region, one coiled-coil region and three proline-rich regions (FIG. 1).
C). In Drosophila, Dof has been isolated as a regulator downstream of the signal and has been identified as a fibroblast growth factor receptor (FG).
It has been shown to specifically act on signaling pathways via FR) (Vincent et al., 1998). However, the overall homology between BCAP-Dof is weak, and these proteins show only 54% and 44% homology in the ankyrin repeat-like and coiled-coil regions, respectively. These regions may be important for Dof and BCAP to interact with other proteins and localize within the cell. While Dof has been shown to be involved in the Ras-ERK pathway (Vincrnt et al., 1998), BCAP-deficient DT
BRK-mediated ERK activation occurs normally in 40 cells (data not shown). This difference is due to the G
This may be due to the fact that the binding region for the SH2 domain of rb2 (YxNx) is much more numerous than BCAP (Dof has five, whereas BCAP has only one).

There are 31 potential tyrosine residues that can be phosphorylated in chicken BCAP, four of which are SH3 of PI3K p85.
Exists in the region that binds to the domain. The importance of phosphorylation of these four tyrosines is BCAP (Y4F)
Failed to complement Akt activation and was indirectly able to bind inductively to p85 (Figure 6).
Our finding that BCAP (Y4F) still undergoes tyrosine phosphorylation clearly shows that there are other sites that undergo phosphorylation after BCR stimulation. However, we did not pursue the importance of these areas in this study.
It cannot be ruled out that some other tyrosine phosphorylated proteins could still co-precipitate with BCAP (Y4F) (Fig. 6).
A; data not shown), it is possible that proteins with other SH2 domains associate with these regions and, as a result, have at least partially contributed to the function of BCAP.

GEMs, also called detergent-insoluble lipid rafts (surfactant-insoluble lipid suspensions), have been shown to function as a basis for the formation of multi-component signal complexes (Langlet et al. ,
2000). BCAP, like LAT and Src family PTKs, does not accumulate in GEMs in the plasma membrane. Nevertheless, a small fraction of the BCAP fraction was localized to GEMs prior to BCR stimulation, indicating that BCAP is heterogeneously localized within the cell. Thus, it is possible that some BCAP may be involved in post-translational modification and / or association with other membrane proteins and, therefore, may be localized to GEMs.

Similar to the rearrangement of PI3K p85 to GEMs observed after TCR stimulation (Xavier et al., 1998), p85
Transition to GEMs after BCR stimulation in 40B cells. This transition is inhibited by BCAP deficiency (Figure 7). BCAP (Y4
F) failed to complement the migration of p85 to GEMs (data not
Thus, the phosphorylation-dependent interaction between BCAP and PI3K p85 may be aimed at translocating PI3K to GEMs. PI (3,4,5) P ₂ is abundant in GEMs (Pike and Miller, 1998), resulting in P
I3K will become so accessible to PI (3,4,5) P ₂ is the substrate. In addition, the binding of PI3K to BCAP and vice versa is probably due to a conformational change, as previously reported by other p85 binding proteins.
May increase the enzyme activity (Shoelson et al.,
1993; Buhl et al., 1997). These phenomena are considered to be important for the synthesis of PI (3,4,5) P ₃ via BCAP after BCR crosslinking.

Following BCR crosslinking in BCAP-deficient DT40B cells
The suppression of JNK activation is thought to be due to a decrease in PI3K activation. Rac and Cdc42 are activated by the guanine nucleotide exchange factor Vav with a PH domain, which
It is thought to cause activation (Crespo et al.,
1997). Therefore, the importance of the PI (3,4,5) P ₃ in Vav activation through the PH domain (Han et al., 1998) , which enters the JNK pathway, a sufficient amount to activate the Vav
PI (3,4,5) P ₃ is considered to be necessary. Akt is known to play an apoptosis-suppressing role through phosphorylation of BAD, caspase 9, IκB kinase (IKK) and forkhead transcription factors (Datta et al., 1999). The promotion of apoptosis mediated by Akt is partially explained by a decrease in Akt activation. Furthermore, recent findings indicate that JNK is also involved in the anti-apoptotic process (Leppa and Bohmann, 199
9), The further promotion of apoptosis observed in BCAP-deficient DT40 cells may be due to decreased activation of both Akt and JNK.

The generation of PI (3,4,5) P ₃ and subsequent incomplete activation of Akt still observed in BCAP-deficient DT40B cells is due to the complete activation of PI3K in B cells. Clearly indicates that the involvement of other molecules is required. A likely possibility is that both insulin receptor substrates (IRS) -1 and IRS-2 are involved in insulin metabolism (Withers et al., 19
99) It is the theory that other BCAP isoforms exist and function as excess molecules. Another candidate molecule is YxxM
Gab, Cbl and CD19 having motifs. Indeed, these molecules have been shown to be tyrosine phosphorylated by BCR stimulation, thereby associating with PI3K p85 (reviewed in Gold, 2000). Of these three molecules, Cbl was identified because Cbl-deficient DT40 cells displayed apparently normal BCR-mediated PI3K activation (Yasuda et al., 2000)
Probably not as important in DT40 cells. Therefore, considering that chicken homologs of Gab and CD19 are expressed in DT40B cells, BCAP
To still remains deficient cells PI (3,4,5) P ₃ Synthesis and Akt activation, is believed to be due to CD19 and / or Gab-dependent activation of PI3K. In fact, in mouse spleen B cells, BCR
After stimulation, PI3K associates with both BCAP and CD19 (Figure 3A an
d 3B). Decreased PI (3,4,5) P ₃ synthesis in BCAP-deficient cells is not due to a mere quantitative change in PI3K activation during BCR signaling, but to PI3K in mutant cells.
The activation pathway may be undergoing a qualitative change. For example, if two sequential steps, phosphorylation initiation and persistence, are required for full activation of the PI3K pathway, phosphorylation initiation can occur via molecules such as CD19. BCAP may be responsible for maintaining or amplifying PI3K activation. From this perspective, it PI (3, 4, 5) only a temporary analysis by P ₃ and Akt assay system may not be sufficient to distinguish between these differences.

<About human BCAP> Test method Example 11 Search for human BCAP gene> Next, human BCAP
In order to search for a gene, a homology search was performed by DNASIS software (Hitachi Software Engineering Co., Ltd.) using the mouse BCAP nucleotide sequence (SEQ ID NO: 9) or amino acid sequence (SEQ ID NO: 10).
Genbank, EMBL, DDJB, NCBI were used as databases. As a calculation method of homology search, Bl
ast2 sequence research was used.

Embodiment 12 FIG. Preparation of Monoclonal Antibody Reacting with Human BCAP Protein> Preparation of Immunogen Using mouse murine BCAP cDNA as a template with 1 μl of a homemade mouse cDNA library (prepared from normal animal blood) as a template
It was incorporated into a fusion protein expression vector pGEX-5X-3 (Pharmacia) and introduced into Escherichia coli XL-1 blue. The integrated sequence is almost identical (more than 95%) only in the region of homology (more than 95%) between human cDNA and mouse cDNA (specifically, in the amino acid sequence shown in FIG. The corresponding human region is 540-727), that is, human BCA
It contains almost the same (95% or more) amino acid sequence as the P protein. Escherichia coli culture containing BCAP gene
500 ml was cultured at 37 ° C. until OD600 = 0.6, IPTG was added at a final concentration of 0.3 mM, and then cultured at 25 ° C. overnight. The next day, the bacteria were collected by centrifugation, and GST-fused BCAP
The protein was purified.

Immunization of mice 0.1 ml of the GST-fused BCAP protein solution prepared above and 0.1 ml of complete Freund's adjuvant were mixed well to prepare a suspension, and this suspension was used for two mice (BALB / c).
Was intraperitoneally administered in an amount of 25 μg per animal. Every other week, a well-mixed suspension of 0.1 ml of GST-fused BCAP protein solution and 0.1 ml of incomplete Freund's adjuvant was added to the suspension.
Three times after the last administration, the spleen was removed, and cell fusion was performed as shown below.

Cell fusion and preparation of a fusion cell producing the desired monoclonal antibody
SP-2 / 0 Ag-14) was mixed at a ratio of about 10: 1, and cell fusion was performed using 50% polyethylene glycol 4000 as a fusion promoter. The cells after fusion are suspended in a medium containing 10% FCS-containing hypoxanthine, aminopterin, and thymidine (HAT medium) to a cell concentration of 1x10 ⁶ cells / ml, and placed in a 96-well microtiter plate per well. 0.1 ml
Each was dispensed. The fused cells are in a CO ₂ incubator (5% CO ₂ ,
The cells were cultured at 37 ° C) for 1-2 weeks, and selection was performed using a HAT medium. Using a microplate on which GST-fused BCAP protein was immobilized, the supernatant of the fused cells was detected by ELISA to confirm the presence or absence of the antibody. For positive wells, cloning by limiting dilution was repeated twice,
One clone having reactivity to the GST-fused BCAP protein was selected and the fused cell clone 4C8 (Accession No. F
ERM P-18292). 10% of clone 4C8 obtained
The cells were suspended in 90% FCS containing DMSO and stored in liquid nitrogen. The monoclonal antibody 4C8-AB produced by 4C8 was obtained by growing clone 4C8 in the abdominal cavity of a BALB / c mouse and then purifying the ascites from the ascites using a protein A sepharose gel.

Reaction Specificity of Monoclonal Antibody The specificity of monoclonal antibody 4C8-AB was confirmed by Western blotting. That is, 5 ml each of GST-fused p53 protein, GST protein and Raji cell extract was added to a 10% agarose gel, and electrophoresed. A labeled anti-mouse IgG was reacted, and a substrate reaction was performed to confirm a band. In addition, monoclonal antibody 4C8-
AB and B cells (chicken-derived (DT40), mouse-derived (A2
0) and the reactivity with the contents of human-derived (C1LCL, B104) cell lines) were confirmed by Western blotting.

B. Test results <About human BCAP> Results of homology search, human BC
The nucleotide sequence encoding AP (SEQ ID NO: 16) and the protein encoded by the nucleotide sequence (SEQ ID NO: 17) were detected. In mouse BCAP and human BCAP, the base sequence homology is 83%, and the amino acid sequence homology is 90%.
(Figure 8 shows mouse BCAP protein and human BCAP
(Comparison with protein is shown). <About monoclonal antibody reactive to human BCAP protein> The results of Western blotting were as shown in FIGS. 9 and 10. As shown in FIG.
In the fused BCAP, one band was observed at about 45 kDa,
No band was observed for the protein. In the Raji cell extract, a band was observed at a position of about 97 kDa. As shown in FIG. 10, it was confirmed that the monoclonal antibody 4C8-AB specifically reacted with mouse and human BCAP.
As shown in the two right lanes of FIG. 10 (B), mouse and human B cell lysates contained monoclonal antibody 4
Multiple bands reacting with C8-AB were observed, indicating that BCAP has several isoforms with different molecular weights. In FIG. 10, IP
First, after immunoprecipitation with monoclonal antibody 4C8-AB, this precipitate was run on a gel, and
This indicates that Western blotting was performed using 4C8-AB.

<References> References in the prior art and in the detailed description of the invention are shown below. Aman. MJ, and Ravichandran, KS (2000). A requi
rement for lipid raftsin B cell receptor induced C
a ²⁺ flux. Curr. BioI. 10, 393-396. Beitz, LO, Fruman, DA, Kurosaki, T., Cantley,
LC., And Scharenberg, AM (1999) .Syk is upstream
of phosphoinositide 3-kinase in B cell receptor si
gnaling. J. Biol. Chem. 274. 32662-32666. Buhl, AM, and Cambier, JC (1999). Phosphorylat.
ion of CD19 Y484 and Y51 5, and linked activation
of phosphatidylinositoI 3-kinase, are required for
B cell antigen receptor-mediated activation of Br
uton's tyrosinekinase. J. lmmunol. 162, 4438-444
6. Buhl, AM, Pleiman, CM. Rickert, RC, and Camb
ier, JC (1997) .Qualitative regulation of B cell
antign receptor signaling by CD19: selective requi
rement for PI3-kinase activation, inositol-1,4,5-
triphosphate production and Ca ²⁺ mobilization. J.
Exp. Med. 186, 1897-1910. Cheng, PC, Dykstra, ML, MitchelI, RN, and Pi
erce, SK (1999) .A role for lipid rafts in B cell
antigen receptor signaling and antigentargeting.
J. Exp. Med. 190, 1549-1560.Cornall, RJ., Cyster, JG, Hibbs, ML, Dunn, AR.,
Otipoby, KL, Clark.EA, and Goodnow, CC (199
8) .Polygenic autoimmune traits: Lyn, CD22, and SHP
-1 are limiting elements of a biochemical pathway
regulating BCRsignaling and selection.Immunity 8,
497-508. Crespo, P., Schuebel, KE, Ostrom. AA, Gutkind.
JS, and Bustelo, XR (1997). PhosphotyrosiMed.e
pendent activation of Rac-1 GDP / GTP exchange by t
he Vav proto-oncogene product.Nature 385, 169-17
2. Datta, SR, Brunet. A., and Greenberg. ME (199
9) .Cellular suvival: aplay in three Akts. Genes D
ev. 13, 2905-2927. DeFranco, AL (1997) .The complexity of signaling.
pathways activated by the BCR.Curr.Opin.lmmunol.
9, 296-308.Engel, P., Zhou, LJ., Ord, DC, Sato, S., Koller,
B., and Tedder, TF (1995) .Abnormal B lymphocyte
development, activation, and differentiation in m
ice that lack or overexpress the CD19 signal trans
duction molecule.Immunity 3, 39-50.Frauman, DA, Meyers, RE, and Cantley, LC (19
98). Phosphoinositidekinases. Annu. Rev. Biochem.
67, 481-507. Fruman, DA, Snapper, SB, Yballe. CM, Davidson,
L, Yu, JY, Alt, FW, and Cantley, LC (1999).
Impaired B cell development and proliferation in
absence of phosphoinositide 3-kinase p85α. Scienc
e 283, 393-397.Gillham, H..Golding, MC, Pepperkok.R .. and Gul
lick. WJ (1999) .Intracellular movement of green
fluorescent protein-tagged hosphatidylinositorl 3-
kinase in response to growth factor receptor signa
ling. J. Cell. Biol. 146, 869-880. Gold, MR (2000) .lntemediary signaling effectors
coupling the B-cell receptor to the nucleus. In C
urrent Topics in Microbiology and lmmunology, Volu
me 245, LB Justement and KA Siminovitch, ed.
(Berlin Heidelberg: Springer-verlag), pp. 78-134.Han, J., Luby-Phelps, K., Das, B., Shu, X., Xia.
Y .. Mosteller, RD, Krishna. UM, Falck, JR, Whi
te, MA, and Broek, D. (1998) .Role of substrates
and products of PI3-kinase in regurationg activat
ion of of Rac-related guanosine triphosphates by V
av. Science 279, 558-560. Hashimoto, A., Okada. H., Jiang, A .. Kurosaki, M.,
Greenberg, S., Clark.EA, and Kurosaki, T. (199
8). Involvement of guanosine triphosphatasesand p
hospholipase C-γ2 in extracellular signal-regurat
ed kinase, c-JunNH2-terminal kinase, and p38 mitog
en-activated protein kinase activation by the B ce
ll antigen receptor. J. Exp. Med. 188, 1287-1295. Hashimoto, A., Takeda, K., Inaba. M., Sekimata,
M., Kaisho, T., Ikehara, S..Homma Y., Akira, S., a
nd Kurosaki. T. (2000) .Cutting edge; essential rol
e of phospholipipase C-γ2 in B cell development a
nd function. J. Immunol. 165, 1738-1742. Ishiai, M., Kurosaki, M .. Pappu, R., Okawa, K., Ro
nko.I., Fu, C., Shibata, M., Iwamatsu, A., Chan,
AC, and Kurosaki, T. (1999) .BLNK requiredfor co
upling Syk to PLC γ2 and Rac1-JNK in B cells.Imm
unity 10, 117-125.lshiai, M., Kurosaki, M..Inabe, K., Chan, AC, S
ugamura, K., and Kurosaki, T. (2000) .Involvement
of LAT, Gads, and Grb2 in compartmentation of SLP-
76 to the plasma membrane.J. Exp.Med. 192, 847-8
56. Klippel, A .. Reinhard, C., Kavanaugh, WM, Apell,
G., Escobedo.MA, and Williams, LT (1996).
brane localization of phosphatidylinositol 3-kinas
e is sufficient to activate multiple signal-transd
ucing kinase pathways. Mol. Cell. Biol. 16, 4117-4
127. Kurosaki, T. (1999). Genetic analysis of B cell an
tigen receptor signaling. Annu. Rev. Immunol. 17,
555-592.Kurosaki, T., and Kurosaki, M. (1997) .Transphosph.
orylation of Bruton'styrosine kinase on tyrosine 5
51 is critical for B cell antigen rcceptorfunctio
n. J. Biol. Chem. 272. 15595-15598. Langlet. C., Bermard, AM, Drevot, P., and He, H.
T. (2000) .Membrane rafts and signaling by the mul
tichain immune recognition receptors. Curr. Opin. I
mmunol. 12, 250-255. Leevers, SJ, Vanhaesebroeck, B., and Waterfield,
MD (1999) .Signling through phosphoinositide
3-kinases: the lipids take center stage.Curr.Opi
n. Cell. Biol. 11. 219-225. Leppa. S., and Bohmann, D. (1999) .Diverse functio
ns of JNK signaling and c-Jun in stress response a
nd apoptosis.Oncogene 18, 6158-6162.Lupas, A (199
6) .Prediction and analysis of coiled-coil structu
res. Methods Enzymol. 266, 513-525. Nishizumi. H., Horikawa. K., Minaric-Rascan, I., a
nd Yamamoto, T. (1998) .A double-edged kinase Lyn: a
positive and negative regulator for antigen recep
J. Exp.Med. 187, 1343-1348.Pike, LJ, and Miller, JM (1998) .Cholesterol d.
epletion delocalizes phosphatidylinositol bisphosp
hate and inhibits horomone-stimulated phosphatidyl
inositol turnover. J. BioI. Chem. 273, 22298-2230
4. Rameh, LE, and Cantley, LC (1999). The role of
phosphoinositide 3-kinase lipid products in cell
function. J. BioI. Chem. 274. 8347-8350. Reth, M., and Wienands, J. (1997). Initiation and
processing of signalsfrom B cell antigen receptor.
Annu. Rev. Immunol. 15, 453-479. Richert, RC, Rajewsky, K., and Rose, J. (1995).
airment of T-cell-dependent B-cell responses and B
-1 cell development in CD19-deficinent mice.Nature
376, 352-355. Scharenberg, AM, and Kinet, JP (1998) .Ptdlns-
3,4.5-P3: a regulatorynexus between tyrosine kinas
es and sustained calcium signals.Cell 94, 5-8. Shoelson, SE, Sivaraja, M., Williams.KP, Hu,
P., Schlessinger, J., and Weiss, MA (1993) .Specif
ic phosphopeptide binding regulates a conformation
al change in the PI 3-kinase SH2 domain associated
with enzyme activation. EMBO J. 12, 795-802. Smith, KGC, Tarlinton, DM, Doody. GM, Hibb.
s.ML, and Fearon, DT (1998) .Inhibition of the
B cell by CD22: a requirement for Lyn. J. Exp.Me
d. 187, 807-811. Songyang, Z., Shoelson, SE, Chaudhuri. M., Gish,
G., Pawson, T., Haser, WG, King, F., Roberts,
T., Ratnofsky, S., Lechleider, RJ, et al. (199
3). SH2 domains recognize specific phosphopeptide
sequences.Cell 72,767-778.Songyang, Z., Shoelson, SE, McGlade, J., Olivie
r, P., Pawson, T., Buestelo, XR, Barbacid, M., S
abe, H., Hanafusa. H., Yi, T., et al. (1994) .Speifi
c motifs recognized by the SH2 domains of Csk, 3BP
2, fps / fes, GRB-2, HCP, SHC, Syk, and Vav. Mol. Ge
ll. Biol. 14, 2777-2785. Sparks, AB, Rider, JE, Hoffman, NG, Fowlkes,
DM, Quillam, LA, and Kay.BK (1996) .Distinc
t ligand preferences of Src homology 3 domains fro
m Src.Yes, Abl, Cortactin, p53bp2, PLCγ, Crk, an
Natl. Acad. Sci. USA 93, 1540-1544. Sugawara, H., Kurosaki, M., Takata, M., and Kurosa d Grv2.
ki, T. (1997) .Geneticevidence for involvement fo
type 1, type 2 and type 3 inositol 1,4,5-trisphosp
hate receptors in signal transduction through the
B-cell antigenreceptor. EMBO J. 16, 3078-3088. Suzuki. H., Terauchi, Y., Fujiwara, M., Aizawa,
S., Yazaki, Y..Kadowaki, T., and Koyasu, S. (199
9) .Xid-like immunodeficiency in mice with disrupt
ion of the p85α subunit of phosphoinositide 3-kin
ase. Science 283, 390-392. Takata. M., and Kurosaki, T. (1996) .A role for Br.
uton's tyrosine kinasein B cell antigen receptor-m
ediated activation of phospholipase C-γ 2.J. Exp.
Med. 184. 31-40. Takata. M., Sabe, H., Hata. A., Inazu, T., Homma,
Y., Nukada, T., Yamamura, H., and Kurosaki, T. (19
94) .Tyrosine kinases Lyn and Syk regulate Bcell r
eceptor-coupled Ca ²⁺ mobilization through distinct
pathways. EMBOJ. 13, 1341-1349. Tamir, I., and Cambier, JC (1998). Antigen recep.
tor signaling: integration of protein tyrosine kin
ase functions. Oncogene 17, 1353-1364. Tamir, I., DaI Porto, JM, and Cambier, JC (200
0) .Cytoplasmic protein tyrosine phosphatases SHP-
1 and SHP-2: regulators of B cell signal transducti
on. Curr. Opin. Immunol. 12, 307-315. Thompson, JD, Gibson. TJ, Plewniak, F., Jeanmo
ugin, F., and Higgins.DG (1997) .The CLUSTAL_X w
indows interface: frexible strategies for multiple
sequence alignmmt aided by quality analysis tool
s. Nucleic Acids Res. 25. 4876-4882. Timokhina, I., Kissel, H., Stella. G., and Besmer,
P. (1998) .Kit signaling through PI 3-kinase and
Src kinase pathways: an essential role for rac1 an
d JNK activation in mast cell proliferation.EMBO
J. 17, 6250-6262. Tuvenson, DA, Carter, RH, Soltoff, SP, and F
earon.DT (1993) .CD19 of B cells as a surrogate
kinase insert region to bind phosphatidylinositol
3-kinase. Science 260. 986-989. Vincent, S., Wilson, R .. Coelho. C., Affolter, M.,
and Leptin, M. (1998) .The Drosophila protein Dof
is specifically required for FGF signaling.Mol. C
ell 2. 515-525. Withers, DJ, Burks, DJ, Towery, HH, Altamur
o, SL, Flint, CL, andWhite, MF (1999).
oordinates IGF-1 receptor-mediated beta-celldevelo
pment and peripheral insulin signaling. Nat.Gene
t.23, 32-40.Xaver, R., Brennau, T., Li.Q., McCormack, C., and
Seed. B. (1998). Membrane compartmentation is req
uired for efficient T cell activation. Immunity 8,
723-732. Yasuda, T., Maeda, A., Kurosaki, M., Tezuka, T., H
ironaka, K., Yamamoto, T., and Kurosaki, T. (2000).
Cbl suppresses B cell receptor mediated phospholi
pase C (PLC) -γ2 activation by regulating B cell l
inker protein-PLC-γ2 binding. J. Exp. Med. 191, 6
414-650.Zhang, W., Trible, RP, and Samelson, LE (199
8) .Lat palmitoylation: its essential role in membr
ane microdomain targeting and tyrosine phosphoryla
tion during T cell activation.Immunity 9,239-246.

[0061]

[Sequence List] SEQUENCE LISTING <110> Kurosaki, Tomohiro Medical & Biological Laboratories Co., LTD. <120> BCAP: The Tyrosine Kinase Substrate that Connects B Cell Receptor to Phosphoinositide 3-Kinase Activation <130> B cell adaptor for PI3K < 140> P-01004MBL <141> 2001-06-12 <160> 17 <170> PatentIn Ver. 2.1 <210> 1 <211> 2403 <212> DNA <213> chicken <400> 1 atgacggcct ccggaaccca tgggggatat gatgtgctga ttctgtatgc aagtgacgct 60 gctgagtggt gtcagtacct tcagaacctc ttcctctcta ctcggcacat ccgaaagcat 120 cacattcaga gctaccagct ggagggcgag tctgccatct ctgaccaaga gctggacctt 180 ttcaacagaa gtcggagcat catcattttg ctatctgctg agctggtgca gaacttttat 240 tgccctcctg tcctgcaaag tcttcaggaa gctttatggc ccccacacaa agtagtcaag 300 ctgttctgcg gtgttacaga ttgtgatgac tatttgacct ttttcaagga ctggtatcag 360 tggcaagaac tcacgtatga tgatgagcct gatgcttacc tagaagctgt caagaaggct 420 atttcagaag actcaggctg cgattctgta actgatacag aaacagaaga cgagaaaacc 480 tcagtttact cctgtcaact tgccatgaat gaagagcatg aatcatccaa gagcactggg 540 gagcatctg g tggtgcagcc tgatcacata cgctgtgggg tacagaccac tgtttatatt 600 atcatgaaat gtagactgga tgacaaagtg aaaactgaag ttgaattctc tccagagaac 660 tcatcatctg tgcgtgtgct ggctgagttg gagaatgaat acacaatatc tgtggaggct 720 ccaaatttaa cttctggaac agtgcctctg cagatatatt caggggactt gatggtggga 780 gaaacgagtg tcacctatca tactgacatg gaggaaatca gcagtctgtt ggctaatgca 840 gccaacccag tacagttcat gtgccaggcc tttaaaattg tgccatacag catagaagca 900 ctggacaaac tgttgactga gtcactgaag aagaacattc ctgccagtgg cttacacttg 960 tttggaatca accagctgga agaagatgat atgacaacaa atcagaggga tgaggagttg 1020 ccaacgctgc ttcacttttc tgcaagatat gggctgaaaa acctaactgc tttgctgctg 1080 acatgccctg gagcactgca agcctacagt gtagccaaca aatacggtca ctatccaaac 1140 acgatagcag aaaagcatgg ctttaaggac ctccggcaat tcattgatga atacgtggaa 1200 actgcagata tgctgaagag tcacattaag gaagagctga tgcaaggcga agaggatgag 1260 tctgtttatg agtccatggc tcatctttct actgatctgt taatgaaatg ttcgttaaat 1320 ccaggttctg atgaagagct ttatgaatct atggctggat ttgtccctgg tgccccagaa 1380 gacctttatg tagaaatgcttcagtccaag ccagatactc caatttctgg agatgaaatc 1440 tctctaactg tgaaagactc gatgctccgc aagtttttag aaggaggtag cacggatgca 1500 ccagattctg gggaaggagt ttcccagcag tatggtgaag acctatatta ctcagtggaa 1560 aaagatactt ttccacaaga gatggctagt agacctccag ttcctgttcc aagaccagaa 1620 tccagctctc cgcagccaga caatgagttg tacatctcca aagtttttgc acagaaagct 1680 caaagacctg agaatctcta tgtccctaga ggaaaggtga ggaaagagac gatagttagg 1740 cctgttagag acctgtctca atcaagtata tacgatccat ttgctggaat gaaaacccca 1800 ggtcagcggc agctgatcac attacaagaa caagtgaaaa tgggcatact caacgttgat 1860 gaagctgtac ttcactttaa ggagtggcaa ctgaaccaga agaagagatc agaatcattc 1920 cgtttccagc aggaaaacct gaaacggcta cgagacagca taaccaggag gcaaatggag 1980 aagcaaaaat cagggaaaag tgcagatctg gaaattacag tacccatacg acgttctcat 2040 aatactttgg ggaaaccaga atgtggcata tatgaatatg ccccgaggaa aaacatattc 2100 ccaccaaaaa aggagctaaa gcgaggagac tggaaaacag aaagcacatc cagcacaaca 2160 agcagtgcaa gcaaccgctc cagcactcgg agcatactga gtgtcagcag cgggatggaa 2220 ggagacagtg aggacaatga agtctc agaa gcaagcagaa gccgcagccc tatccccagc 2280 caagcagaga ggctgcctct ccccctgcct gaaaggcctc ccagagtgcc ccctcgaggt 2340 gcaagcaggc ctgtgaactg tgaaggcttt tatcctccac ctgtgc2c <tg> Tg tcc2c tggtcc2c tggcccc2g Tyr Asp Val Leu Ile Leu Tyr 1 5 10 15 Ala Ser Asp Ala Ala Glu Trp Cys Gln Tyr Leu Gln Asn Leu Phe Leu 20 25 30 Ser Thr Arg His Ile Arg Lys His His Ile Gln Ser Tyr Gln Leu Glu 35 40 45 Gly Glu Ser Ala Ile Ser Asp Gln Glu Leu Asp Leu Phe Asn Arg Ser 50 55 60 Arg Ser Ile Ile Ile Leu Leu Ser Ala Glu Leu Val Gln Asn Phe Tyr 65 70 75 80 Cys Pro Pro Val Leu Gln Ser Leu Gln Glu Ala Leu Trp Pro Pro His 85 90 95 Lys Val Val Lys Leu Phe Cys Gly Val Thr Asp Cys Asp Asp Tyr Leu 100 105 110 Thr Phe Phe Lys Asp Trp Tyr Gln Trp Gln Glu Leu Thr Tyr Asp Asp 115 120 125 Glu Pro Asp Ala Tyr Leu Glu Ala Val Lys Lys Ala Ile Ser Glu Asp 130 135 140 Ser Gly Cys Asp Ser Val Thr Asp Thr Glu Thr Glu Asp Glu Lys Thr 145 150 155 160 Ser Va l Tyr Ser Cys Gln Leu Ala Met Asn Glu Glu His Glu Ser Ser 165 170 175 Lys Ser Thr Gly Glu His Leu Val Val Gln Pro Asp His Ile Arg Cys 180 185 190 Gly Val Gln Thr Thr Val Tyr Ile Ile Met Lys Cys Arg Leu Asp Asp 195 200 205 Lys Val Lys Thr Glu Val Glu Phe Ser Pro Glu Asn Ser Ser Ser Val 210 215 220 Arg Val Leu Ala Glu Leu Glu Asn Glu Tyr Thr Ile Ser Val Glu Ala 225 230 235 240 Pro Asn Leu Thr Ser Gly Thr Val Pro Leu Gln Ile Tyr Ser Gly Asp 245 250 255 Leu Met Val Gly Glu Thr Ser Val Thr Tyr His Thr Asp Met Glu Glu 260 265 270 Ile Ser Ser Leu Leu Ala Asn Ala Ala Asn Pro Val Gln Phe Met Cys 275 280 285 Gln Ala Phe Lys Ile Val Pro Tyr Ser Ile Glu Ala Leu Asp Lys Leu 290 295 300 Leu Thr Glu Ser Leu Lys Lys Asn Ile Pro Ala Ser Gly Leu His Leu 305 310 315 320 Phe Gly Ile Asn Gln Leu Glu Glu Asp Asp Met Thr Thr Asn Gln Arg 325 330 335 Asp Glu Glu Leu Pro Thr Leu Leu His Phe Ser Ala Arg Tyr Gly Leu 340 345 350 Lys Asn Leu Thr Ala Leu Leu Leu Thr Cys Pro Gly Ala Leu Gln Ala 355 360 365 Tyr Ser Va l Ala Asn Lys Tyr Gly His Tyr Pro Asn Thr Ile Ala Glu 370 375 380 Lys His Gly Phe Lys Asp Leu Arg Gln Phe Ile Asp Glu Tyr Val Glu 385 390 395 400 Thr Ala Asp Met Leu Lys Ser His Ile Lys Glu Glu Leu Met Gln Gly 405 410 415 Glu Glu Asp Glu Ser Val Tyr Glu Ser Met Ala His Leu Ser Thr Asp 420 425 430 Leu Leu Met Lys Cys Ser Leu Asn Pro Gly Ser Asp Glu Glu Leu Tyr 435 440 445 445 Glu Ser Met Ala Gly Phe Val Pro Gly Ala Pro Glu Asp Leu Tyr Val 450 455 460 Glu Met Leu Gln Ser Lys Pro Asp Thr Pro Ile Ser Gly Asp Glu Ile 465 470 475 480 Ser Leu Thr Val Vals Lys Asp Ser Met Leu Arg Lys Phe Leu Glu Gly Gly 485 490 495 Ser Thr Asp Ala Pro Asp Ser Gly Glu Gly Val Ser Gln Gln Tyr Gly 500 505 510 Glu Asp Leu Tyr Tyr Ser Val Glu Lys Asp Thr Phe Pro Gln Glu Met 515 520 525 Ala Ser Arg Pro Pro Val Pro Val Pro Arg Pro Glu Ser Ser Ser Pro 530 535 540 Gln Pro Asp Asn Glu Leu Tyr Ile Ser Lys Val Phe Ala Gln Lys Ala 545 550 555 560 Gln Arg Pro Glu Asn Leu Tyr Val Pro Arg Gly Lys Val Arg Lys Glu 565 570 570 Thr 575 Ile Va l Arg Pro Val Arg Asp Leu Ser Gln Ser Ser Ile Tyr Asp 580 585 590 Pro Phe Ala Gly Met Lys Thr Pro Gly Gln Arg Gln Leu Ile Thr Leu 595 600 605 Gln Glu Gln Val Lys Met Gly Ile Leu Asn Val Asp Glu Ala Val Leu 610 615 620 His Phe Lys Glu Trp Gln Leu Asn Gln Lys Lys Arg Ser Glu Ser Phe 625 630 635 640 Arg Phe Gln Gln Glu Asn Leu Lys Arg Leu Arg Asp Ser Ile Thr Arg 645 650 655 Arg Gln Met Glu Lys Gln Lys Ser Gly Lys Ser Ala Asp Leu Glu Ile 660 665 670 Thr Val Pro Ile Arg Arg Ser His Asn Thr Leu Gly Lys Pro Glu Cys 675 680 685 Gly Ile Tyr Glu Tyr Ala Pro Arg Lys Asn Ile Phe Pro Pro Lys Lys 690 695 700 Glu Leu Lys Arg Gly Asp Trp Lys Thr Glu Ser Thr Ser Ser Thr Thr 705 710 715 720 Ser Ser Ala Ser Asn Arg Ser Ser Thr Arg Ser Ile Leu Ser Val Ser 725 730 735 Ser Gly Met Glu Gly Asp Ser Glu Asp Asn Glu Val Ser Glu Ala Ser 740 745 750 Arg Ser Arg Ser Pro Ile Pro Ser Gln Ala Glu Arg Leu Pro Leu Pro 755 760 765 Leu Pro Glu Arg Pro Pro Arg Val Pro Pro Arg Gly Ala Ser Arg Pro 770 775 780 Val Asn Cys Gl u Gly Phe Tyr Pro Pro Pro Val Pro Pro Arg Gly Arg 785 790 795 800 <210> 3 <211> 7 <212> PRT <213> Chicken <400> 3 Ala Gln Arg Pro Glu Asn Leu 1 5 <210> 4 <211> 14 <212> PRT <213> Chicken <400> 4 Thr Pro Gly Gln Arg Gln Leu Ile Thr Leu Gln Glu Gln Val 1 5 10 <210> 5 <211> 11 <212> PRT <213> Chicken < 400> 5 Ile Val Pro Tyr Ser Ile Glu Ala Leu Asp Lys 1 5 10 <210> 6 <211> 14 <212> PRT <213> Chicken <400> 6 Phe Leu Glu Gly Gly Ser Thr Asp Ala Pro Asp Ser Gly Glu 1 5 10 <210> 7 <211> 11 <212> PRT <213> Chicken <400> 7 Glu Thr Ile Val Arg Pro Val Arg Asp Leu Ser 1 5 10 <210> 8 <211> 11 <212> PRT <213> Chicken <400> 8 Met Gly Ile Leu Asn Val Asp Glu Ala Val Leu 1 5 10 <210> 9 <211> 2615 <212> DNA <213> mouse <400> 9 ctgcttctaa ccccgcgcca gcgacgtcgc ctaccgcggc tgtgggcacc ggataccgtg60g agccggggat gcgagctgct cccgggacgg catggcagcc tcagggtggg 120 gccggggatg cgacatcctc atcttctaca gcccagatgc ggaggaatgg tgtcagtacc 180 tacaggatct tttcgtgtgt tgccggcagg ttcgtagcca gaagaaccgca ggtcccgga tgcctctttc tctgcccagg acctgtgggt cttcagggat gcgcgatgcg 300 tgctggtgct actgtcggcg gggctggtag gatgcttcgg ccagccagga ctgctgccaa 360 tgctgcagcg tgcctgccat ccgccgcagc gtgtggtgcg gctgttgtgt ggagtgcagc 420 caggcgacga ggacttccag gccttctttc ctgactgggc ccactggcag gaaatgactt 480 gtgatgatga gccagagacc tacctggcgg ctgtgaggaa ggccatttct gaagattctg 540 gctgtgactc agtaaccgac acggagcccg aggacgagag agagcttcct ttctcaaagc 600 agacaaacct gcctccggag atatcgcctg ggaacctgat ggtggtacag ccggaccgca 660 ttcgctgtgg ggcagaaacc actgtgtata tcattgtgag atgcaagctg gacgagaagg 720 tatcaacaga agcagagttc tctcctgagg attctccctc catccgggtg gaaggcacac 780 tggagaatga gtacacggtc tctgtgaaag caccagacct ctcctcgggg aatgtttctc 840 tgaaggtcta ttctggggac ctggtggtct gcgaaaccac cgtcagctat tataccgaca 900 tggaggaaat tgggaactta ctgtcgagtg ctgcaaatcc tgtggagttt atgtgtcagg 960 cctttaaaat cgtgccctac aacacagaga ctctcgataa actgctcacc gagtccctga 1020 agaacaacat ccccgcaagt ggactgcacc tctttgggat caaccaactg gaagaagacg 1080 atatgatgac aaatcaga gg gacgaagagc tgcccacgct gttgcatttt gctgccaagt 1140 atggactgaa gaacctcact gccttgctgc tcacctgtcc aggggctctc caggcctaca 1200 gtgtagccaa caagcacggc cactatccca acaccattgc cgagaaacac ggcttcaggg 1260 acctgcgcca gttcatcgac gagtatgtgg agacggtgga catgctgaag acacacatta 1320 aggaggagtt gatgcaaggg gaggaggcgg acgatgtgta cgagtccatg gcccacctat 1380 ccacagacct gctgatgaag tgctccctca accctggctg tgacgatgag ctctacgagt 1440 ccatggctgc cttcgcccca gcagccacag aagacctcta tgttgaaatg cttcaggcca 1500 gtgcaggtaa cccagtctcg ggagaaagtt tctctcggcc cactaaagac tcgatgatcc 1560 gcaagtttct agaaggtaac agtgtgaaac cggccagttg ggagagagaa caacaccatc 1620 cctatgggga ggaactttat cacattgtgg atgaagatga gaccttctct gtggacctag 1680 ccaacaggcc ccctgtccct gtgcccaggc cagaggccag cgctcctggc cctcccccac 1740 cgcctgacaa tgagccatac atttctaaag tttttgcaga aaaaagtcag gagcggctcg 1800 ggaatttcta cgtttcttct gagagcatca gaaaagagcc acttgtgagg ccatggaggg 1860 acaggccgcc atcgagcatc tacgacccat ttgctggaat gaagaccccg ggccagcggc 1920 agctcatcac cctgcaggag cag gtgaagc tgggcatcgt caacgtggac gaggccgtgc 1980 tgcatttcaa agagtggcaa ctcaatcaga agaagcgatc cgagtccttc cgcttccagc 2040 aggaaaatct taaaagacta agagagagca tcacccgaag acggaaagag aagccaaagt 2100 ctgggaagca cacagacctg gagatcacag tcccaatccg acattcacag cacctgccag 2160 agaaagtgga gtttggcgtg tatgagagcg gccccaggaa gagtgtcttg ccggccagga 2220 cagagctgag acgtggggac tggaaaacgg acagcatgtc cagcacagca agcagcacga 2280 gtaaccggtc gagcacacgg agcctcctca gtgtgagcag tggaatggag ggcgacaacg 2340 aggacaacga aatccccgaa attacccgga gtcgtggccc aggtcctacg caggtggacg 2400 gagctccagt tgtgaccgga acaccagtcg ggacccttga gagaccccct agggtgcctc 2460 ctcgagccgc ttctcagagg cctctaaccc gggagtcctt ccatcctcct ccgccggttc 2520 cacccagagg tcgctgatcc cgcctcctat atctggccaa cttcaggact tttcagacta 2580 gcaattctca gcctgttgat gaagtcttca tattg 2615 <210> 10 <211> 811 <212> PRT <213> mouse <400> 10 Met Ala Ala Ser Gly Trp Gly Arg Gly Cys Asp Ile Leu Ile Phe Tyr 1 5 10 15 Ser Pro Asp Ala Glu Glu Trp Cys Gln Tyr Leu Gln Asp Leu Phe Val 20 25 30 Se r Cys Arg Gln Val Arg Ser Gln Lys Thr Gln Thr Tyr Arg Leu Val 35 40 45 Pro Asp Ala Ser Phe Ser Ala Gln Asp Leu Trp Val Phe Arg Asp Ala 50 55 60 Arg Cys Val Leu Val Leu Leu Ser Ala Gly Leu Val Gly Cys Phe Gly 65 70 75 80 Gln Pro Gly Leu Leu Pro Met Leu Gln Arg Ala Cys His Pro Pro Gln 85 90 95 Arg Val Val Arg Leu Leu Cys Gly Val Gln Pro Gly Asp Glu Asp Phe 100 105 110 Gln Ala Phe Phe Pro Asp Trp Ala His Trp Gln Glu Met Thr Cys Asp 115 120 125 Asp Glu Pro Glu Thr Tyr Leu Ala Ala Val Arg Lys Ala Ile Ser Glu 130 135 140 Asp Ser Gly Cys Asp Ser Val Thr Asp Thr Glu Pro Glu Asp Glu Arg 145 150 155 160 Glu Leu Pro Phe Ser Lys Gln Thr Asn Leu Pro Pro Glu Ile Ser Pro 165 170 175 Gly Asn Leu Met Val Val Gln Pro Asp Arg Ile Arg Cys Gly Ala Glu 180 185 190 Thr Thr Val Tyr Ile Ile Val Arg Cys Lys Leu Asp Glu Lys Val Ser 195 200 205 Thr Glu Ala Glu Phe Ser Pro Glu Asp Ser Pro Ser Ile Arg Val Glu 210 215 220 Gly Thr Leu Glu Asn Glu Tyr Thr Val Ser Val Lys Ala Pro Asp Leu 225 230 235 240 Ser Ser Gly Asn Val Ser Leu Lys Val Tyr Ser Gly Asp Leu Val Val 245 250 255 Cys Glu Thr Thr Val Ser Tyr Tyr Thr Asp Met Glu Glu Ile Gly Asn 260 265 270 Leu Leu Ser Ser Ala Ala Asn Pro Val Glu Phe Met Cys Gln Ala Phe 275 280 285 Lys Ile Val Pro Tyr Asn Thr Glu Thr Leu Asp Lys Leu Leu Thr Glu 290 295 300 Ser Leu Lys Asn Asn Ile Pro Ala Ser Gly Leu His Leu Phe Gly Ile 305 310 315 320 Asn Gln Leu Glu Glu Asp Asp Met Met Thr Asn Gln Arg Asp Glu Glu 325 330 335 Leu Pro Thr Leu Leu His Phe Ala Ala Lys Tyr Gly Leu Lys Asn Leu 340 345 350 Thr Ala Leu Leu Leu Thr Cys Pro Gly Ala Leu Gln Ala Tyr Ser Val 355 360 365 Ala Asn Lys His Gly His Tyr Pro Asn Thr Ile Ala Glu Lys His Gly 370 375 380 Phe Arg Asp Leu Arg Gln Phe Ile Asp Glu Tyr Val Glu Thr Val Asp 385 390 395 400 Met Leu Lys Thr His Ile Lys Glu Glu Leu Met Gln Gly Glu Glu Ala 405 410 415 Asp Asp Val Tyr Glu Ser Met Ala His Leu Ser Thr Asp Leu Leu Met 420 425 430 Lys Cys Ser Leu Asn Pro Gly Cys Asp Asp Glu Leu Tyr Glu Ser Met 435 440 445 Ala Ala Ala Phe Ala Pr o Ala Ala Thr Glu Asp Leu Tyr Val Glu Met Leu 450 455 460 Gln Ala Ser Ala Gly Asn Pro Val Ser Gly Glu Ser Phe Ser Arg Pro 465 470 475 480 480 Thr Lys Asp Ser Met Ile Arg Lys Phe Leu Glu Gly Asn Ser Val Lys 485 490 495 Pro Ala Ser Trp Glu Arg Glu Gln His His Pro Tyr Gly Glu Glu Leu 500 505 510 Tyr His Ile Val Asp Glu Asp Glu Thr Phe Ser Val Asp Leu Ala Asn 515 520 525 Arg Pro Pro Val Pro Val Pro Arg Pro Glu Ala Ser Ala Pro Gly Pro 530 535 540 Pro Pro Pro Pro Asp Asn Glu Pro Tyr Ile Ser Lys Val Phe Ala Glu 545 550 555 560 Lys Ser Gln Glu Arg Leu Gly Asn Phe Tyr Val Ser Ser Glu Ser Ile 565 570 575 Arg Lys Glu Pro Leu Val Arg Pro Trp Arg Asp Arg Pro Pro Ser Ser 580 585 590 Ile Tyr Asp Pro Phe Ala Gly Met Lys Thr Pro Gly Gln Arg Gln Leu 595 600 605 Ile Thr Leu Gln Glu Gln Val Lys Leu Gly Ile Val Asn Val Asp Glu 610 615 620 Ala Val Leu His Phe Lys Glu Trp Gln Leu Asn Gln Lys Lys Arg Ser 625 630 635 640 Glu Ser Phe Arg Phe Gln Gln Glu Glu Asn Leu Lys Arg Leu Arg Glu Ser 645 650 655 Ile Thr Arg Arg Ar g Lys Glu Lys Pro Lys Ser Gly Lys His Thr Asp 660 665 670 Leu Glu Ile Thr Val Pro Ile Arg His Ser Gln His Leu Pro Glu Lys 675 680 685 Val Glu Phe Gly Val Tyr Glu Ser Gly Pro Arg Lys Ser Val Leu Pro 690 695 700 Ala Arg Thr Glu Leu Arg Arg Gly Asp Trp Lys Thr Asp Ser Met Ser 705 710 715 720 Ser Thr Ala Ser Ser Thr Ser Asn Arg Ser Ser Thr Arg Ser Leu Leu 725 730 735 Ser Val Ser Ser Gly Met Glu Gly Asp Asn Glu Asp Asn Glu Ile Pro 740 745 750 Glu Ile Thr Arg Ser Arg Gly Pro Gly Pro Thr Gln Val Asp Gly Ala 755 760 765 Pro Val Val Thr Gly Thr Pro Val Gly Thr Leu Glu Arg Pro Pro Arg 770 775 780 Val Pro Pro Arg Ala Ala Ser Gln Arg Pro Leu Thr Arg Glu Ser Phe 785 790 795 800 His Pro Pro Pro Pro Val Pro Pro Arg Gly Arg 805 810 <210> 11 <211> 2540 <212> DNA <213> chicken <400 > 11 actcttgtat tcaccttctt gcttgaggat caggatccct tcagactact gccttacatt 60 ttgcttatag acacagtagt gaattcagga gttggagcaa gcaggatcca gataattaaa 120 gatactgctg ataggaaata aaagtaggtt tctttcatta actttgaaga gg cgttctacag tgcactcagt gtggcaggaa tcagatgagt ccatttgcag 240 atctttctta tatgagactc aggctgcgat tctgtaactg atacagaaac agaagacgag 300 aaaacctcag tttactcctg tcaacttgcc atgaatgaag agcatgaatc atccaagagc 360 actggggagc atctggtggt gcagcctgat cacatacgct gtggggtaca gaccactgtt 420 tatattatca tgaaatgtag actggatgac aaagtgaaaa ctgaagttga attctctcca 480 gagaactcat catctgtgcg tgtgctggct gagttggaga atgaatacac aatatctgtg 540 gaggctccaa atttaacttc tggaacagtg cctctgcaga tatattcagg ggacttgatg 600 gtgggagaaa cgagtgtcac ctatcatact gacatggagg aaatcagcag tctgttggct 660 aatgcagcca acccagtaca gttcatgtgc caggccttta aaattgtgcc atacagcata 720 gaagcactgg acaaactgtt gactgagtca ctgaagaaga acattcctgc cagtggctta 780 cacttgtttg gaatcaacca gctggaagaa gatgatatga caacaaatca gagggatgag 840 gagttgccaa cgctgcttca cttttctgca agatatgggc tgaaaaacct aactgctttg 900 ctgctgacat gccctggagc actgcaagcc tacagtgtag ccaacaaata cggtcactat 960 ccaaacacga tagcagaaaa gcatggcttt aaggacctcc ggcaattcat tgatgaatac 1020 gtggaaactg cagatatgct gaagagtc ac attaaggaag agctgatgca aggcgaagag 1080 gatgagtctg tttatgagtc catggctcat ctttctactg atctgttaat gaaatgttcg 1140 ttaaatccag gttctgatga agagctttat gaatctatgg ctggatttgt ccctggtgcc 1200 ccagaagacc tttatgtaga aatgcttcag tccaagccag atactccaat ttctggagat 1260 gaaatctctc taactgtgaa agactcgatg ctccgcaagt ttttagaagg aggtagcacg 1320 gatgcaccag attctgggga aggagtttcc cagcagtatg gtgaagacct atattactca 1380 gtggaaaaag atacttttcc acaagagatg gctagtagac ctccagttcc tgttccaaga 1440 ccagaatcca gctctccgca gccagacaat gagttgtaca tctccaaagt ttttgcacag 1500 aaagctcaaa gacctgagaa tctctatgtc cctagaggaa aggtgaggaa agagacgata 1560 gttaggcctg ttagagacct gtctcaatca agtatatacg atccatttgc tggaatgaaa 1620 accccaggtc agcggcagct gatcacatta caagaacaag tgaaaatggg catactcaac 1680 gttgatgaag ctgtacttca ctttaaggag tggcaactga accagaagaa gagatcagaa 1740 tcattccgtt tccagcagga aaacctgaaa cggctacgag acagcataac caggaggcaa 1800 atggagaagc aaaaatcagg gaaaagtgca gatctggaaa ttacagtacc catacgacgt 1860 tctcataata ctttggggaa accagaatgt ggc atatatg aatatgcccc gaggaaaaac 1920 atattcccac caaaaaagga gctaaagcga ggagactgga aaacagaaag cacatccagc 1980 acaacaagca gtgcaagcaa ccgctccagc actcggagca tactgagtgt cagcagcggg 2040 atggaaggag acagtgagga caatgaagtc tcagaagcaa gcagaagccg cagccctatc 2100 cccagccaag cagagaggct gcctctcccc ctgcctgaaa ggcctcccag agtgccccct 2160 cgaggtgcaa gcaggcctgt gaactgtgaa ggcttttatc ctccacctgt gccccccaga 2220 ggtcgctgaa tctcacaatg cctgtggaat atgagatttt tctatatata tgtgtgtgtg 2280 tttgtacaga tatttttggg ttgtcttaaa ttatttataa gtgggagctg aattttttta 2340 tatcttcttc cctaaagctc tggtagctct gcccatggtt ttcactgaga ttttttggtc 2400 tactgtagct tttttgtttg gaagaatttc ggtgctttga gggagaaatc atggttgaaa 2460 aggagaatgc tgatacatct tggcagccct gcaccaacat ggggtttccg cttgatgaag 2520 aaagaagtca tcctcttaga 2540 <210> 12 <211> 632 <212> PRT <213> chicken <400> 12 Met Asn Glu Glu His Glu Ser Ser Lys Ser Thr Gly Glu His Leu Val 1 5 10 15 Val Gln Pro Asp His Ile Arg Cys Gly Val Gln Thr Thr Val Tyr Ile 20 25 30 Ile Met Lys Cys Arg Leu Asp Asp Lys Val Lys Thr Glu Val Glu Phe 35 40 45 Ser Pro Glu Asn Ser Ser Ser Val Arg Val Leu Ala Glu Leu Glu Asn 50 55 60 Glu Tyr Thr Ile Ser Val Glu Ala Pro Asn Leu Thr Ser Gly Thr Val 65 70 75 80 Pro Leu Gln Ile Tyr Ser Gly Asp Leu Met Val Gly Glu Thr Ser Val 85 90 95 Thr Tyr His Thr Asp Met Glu Glu Ile Ser Ser Leu Leu Ala Asn Ala 100 105 110 Ala Asn Pro Val Gln Phe Met Cys Gln Ala Phe Lys Ile Val Pro Tyr 115 120 125 Ser Ile Glu Ala Leu Asp Lys Leu Leu Thr Glu Ser Leu Lys Lys Asn 130 135 140 Ile Pro Ala Ser Gly Leu His Leu Phe Gly Ile Asn Gln Leu Glu Glu 145 150 155 160 Asp Asp Met Thr Thr Asn Gln Arg Asp Glu Glu Leu Pro Thr Leu Leu 165 170 175 His Phe Ser Ala Arg Tyr Gly Leu Lys Asn Leu Thr Ala Leu Leu Leu 180 185 190 Thr Cys Pro Gly Ala Leu Gln Ala Tyr Ser Val Ala Asn Lys Tyr Gly 195 200 205 His Tyr Pro Asn Thr Ile Ala Glu Lys His Gly Phe Lys Asp Leu Arg 210 215 220 Gln Phe Ile Asp Glu Tyr Val Glu Thr Ala Asp Met Leu Lys Ser His 225 230 235 240 Ile Lys Glu Glu Leu Met Gln Gly Glu Glu A sp Glu Ser Val Tyr Glu 245 250 255 Ser Met Ala His Leu Ser Thr Asp Leu Leu Met Lys Cys Ser Leu Asn 260 265 270 Pro Gly Ser Asp Glu Glu Leu Tyr Glu Ser Met Ala Gly Phe Val Pro 275 280 285 Gly Ala Pro Glu Asp Leu Tyr Val Glu Met Leu Gln Ser Lys Pro Asp 290 295 295 300 Thr Pro Ile Ser Gly Asp Glu Ile Ser Leu Thr Val Lys Asp Ser Met 305 310 315 320 Leu Arg Lys Phe Leu Glu Gly Gly Ser Thr Asp Ala Pro Asp Ser Gly 325 330 335 Glu Gly Val Ser Gln Gln Tyr Gly Glu Asp Leu Tyr Tyr Ser Val Glu 340 345 350 Lys Asp Thr Phe Pro Gln Glu Met Ala Ser Arg Pro Pro Val Pro Val 355 360 365 365 Pro Arg Pro Glu Ser Ser Ser Pro Gln Pro Asp Asn Glu Leu Tyr Ile 370 375 380 Ser Lys Val Phe Ala Gln Lys Ala Gln Arg Pro Glu Asn Leu Tyr Val 385 390 395 400 Pro Arg Gly Lys Val Arg Lys Glu Thr Ile Val Arg Pro Val Arg Asp 405 410 415 Leu Ser Gln Ser Ser Ile Tyr Asp Pro Phe Ala Gly Met Lys Thr Pro 420 425 430 Gly Gln Arg Gln Leu Ile Thr Leu Gln Glu Gln Val Lys Met Gly Ile 435 440 445 Leu Asn Val Asp Glu Ala Val Leu His Phe LysGlu Trp Gln Leu Asn 450 455 460 Gln Lys Lys Arg Ser Glu Ser Phe Arg Phe Gln Gln Glu Asn Leu Lys 465 470 475 480 Arg Leu Arg Asp Ser Ile Thr Arg Arg Gln Met Glu Lys Gln Lys Ser 485 490 495 Gly Lys Ser Ala Asp Leu Glu Ile Thr Val Pro Ile Arg Arg Ser His 500 505 510 Asn Thr Leu Gly Lys Pro Glu Cys Gly Ile Tyr Glu Tyr Ala Pro Arg 515 520 525 Lys Asn Ile Phe Pro Pro Lys Lys Glu Leu Lys Arg Gly Asp Trp Lys 530 535 540 540 Thr Glu Ser Thr Ser Ser Thr Thr Ser Ser Ala Ser Asn Arg Ser Ser 545 550 555 560 Thr Arg Ser Ile Leu Ser Val Ser Ser Gly Met Glu Gly Asp Ser Glu 565 570 575 Asp Asn Glu Val Ser Glu Ala Ser Arg Ser Arg Ser Pro Ile Pro Ser 580 585 590 Gln Ala Glu Arg Leu Pro Leu Pro Leu Pro Glu Arg Pro Pro Arg Val 595 600 605 Pro Pro Arg Gly Ala Ser Arg Pro Val Asn Cys Glu Gly Phe Tyr Pro 610 615 620 Pro Pro Val Pro Pro Arg Gly Arg 625 630 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: sense primer "a" <400> 13 ggcaagaact cacgtatgat gatgag 26 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: sense primer "b" <400> 14 ggatcaggat cccttcagac tactg 25 <210> 15 <211> 26 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: antisense primer "c" <400> 15 ggcaagttga caggagtaaa ctgagg 26 <210> 16 <211> 2802 <212> DNA <213> human <400 > 16 ggtggcagat cagggctggt ttcccgagga gtggggcgca cgtggcgcga agcccccgcc 60 ttctcgctgc ccctgagcag ggggccgtgg ggggagctgg ccggcggccg gcatggggtt 120 ctcacttccc ctccccaacg gccgcgggtg caggtgccgc gggccgagtc ccgcaggcgg 180 ggcggactct gtggacacgc cctcgtggcg aggccgggct gccctgaggg aggaagcgcc 240 agagcggcgg ccggtcccgc gcggagcccg gcgcccctcc agcccgagcc aggacgccgc 300 cggccccggt cccggccccg ggcacgcagc gagccaggga tgtgagcggc gccccgcggc 360 atggcagcct caggggtgcc cagaggatgc gacatcctca tcgtctacag cccggatgcc 420 gaggaatggt gccagtacct gcagaccctg ttcctgtcca gtcggcaggt ccgcagccag 480 aagatactga ctcacaggct gggccccgag gcctccttct cggcagagga cctaagcctt 540 ttcctcagca cccgctgtgt cgtggtgctg ctgtccgcgg agctggtgca gcacttccac 600 aagcccgcct tgctgcccct gctgcagaga gctttccatc ctccgcaccg cgtggtcagg 660 ctgctctgcg gcgtgcggga cagcgaggag ttcctagact tctttccaga ttgggcccat 720 tggcaggagc tcacctgtga cgatgagcca gagacctacg tggcagctgt gaaaaaagcc 780 atttccgaag attctggctg tgactcagtc actgacactg agcctgagga cgagaaggtt 840 gtttcctact cgaagcagca gaacctgccg acggtgactt cacctgggaa cctgatggtg 900 gtgcagccgg accgcattcg ctgtggggca gaaaccactg tctatgttat cgtgagatgt 960 aagctggatg acagggtggc gacagaagca gagttttctc ctgaggattc tccctctgta 1020 aggatggaag ccaaggtgga gaatgagtac accatttcag tgaaggctcc caacctttca 1080 tctgggaacg tttctctgaa gatatattct ggagacttag tggtgtgtga aaccgttatc 1140 agctattata ctgacatgga agaaattggg aatttattgt ccaatgccgc gaatcctgtg 1200 gaattcatgt gtcaggcctt taaaattgtg ccctacaaca cagagaccct tgataaactg 1260 ctaaccgaat ccctgaagaa caatatccct gcaagcggac tgcacctctt tggaatcaac 1320 cagctggaag aagaagatat gatgacaaat cagagggatg aagagctgcc caccctgttg 1380 cattttgctg cgaagtatgg actgaagaac ctcactgcct tgttgctcac ctgcccagga 1440 gccctgcagg cgtacagcgt ggccaacaag catggccact accccaacac catcgctgag 1500 aaacacggct tcagggacct gcggcagttc atcgacgagt atgtggaaac ggtggacatg 1560 ctcaagagtc acattaaaga ggaactgatg cacggggagg aggctgatgc tgtgtacgag 1620 tccatggccc acctttccac agacctgctt atgaaatgct cgctcaaccc cggctgtgac 1680 gaggatctct atgagtccat ggctgccttt gtcccagctg ccactgaaga cctctatgtt 1740 gaaatgcttc aggccagtac atctaaccca atccctggag atggtttctc tcgggccact 1800 aaggactcta tgatccgcaa gtttttagaa ggcaacagca tgggaatgac caatctggag 1860 agagatcagt gccatcttgg tcaggaagaa gatgtttatc acacggtgga tgacgatgag 1920 gccatttctg tggacttggc cagcaggccc cctgtcccag tgcccagacc agagaccact 1980 gctcctggtg ctcaccagct gcctgacaac gaaccataca tttttaaagt ttttgcagaa 2040 aaaagtcaag agcggcctgg gaatttctac gtttcctcag agagcatcag gaaagggccg 2100 cccgtcagac catggaggga caggccccag tcaagtatat atgacccttt tgcgggaatg 2160 aaaacgccag gccagcggca gcttatcacc ctccaggagc aggtgaagct gggcattgtc 2220 aacgtggatg aggctg tgct ccacttcaaa gagtggcagc tcaaccagaa gaaacgatcg 2280 gagtcctttc gtttccagca ggaaaatctt aaacggctaa gagacagcat cacccgaaga 2340 cagagagaga agcaaaaatc aggaaagcag acagacttgg agatcacggt cccaattcgg 2400 cactcacagc acctgcctgc aaaagtggag tttggagtct atgagagtgg ccccaggaaa 2460 agtgtcattc cccctaggac ggagctgaga cgaggagact ggaaaacaga cagcacctcc 2520 agcacagcaa gtagcacaag taaccgctcc agcacccgga gcctcctcag tgtgagcagc 2580 gggatggaag gggacaacga ggataatgaa gtccctgagg ttaccagaag tcgcagtcca 2640 ggccccccac aagtggatgg gacacccacc atgtccctcg agagaccccc cagggtgcct 2700 ccgagagctg cctcacagag gcctccgacc agggagacct tccatcctcc tccacctgtt 2760 ccacccagag gacgctgatt ccacctccta aaacctgcct ac 2802 <210> A <la> A <la> G Leu Ile Val Tyr 1 5 10 15 Ser Pro Asp Ala Glu Glu Trp Cys Gln Tyr Leu Gln Thr Leu Phe Leu 20 25 30 Ser Ser Arg Gln Val Arg Ser Gln Lys Ile Leu Thr His Arg Leu Gly 35 40 45 Pro Glu Ala Ser Phe Ser Ala Glu Asp Leu Ser Leu Phe Leu Ser Thr 50 55 60 Arg Cys Val Val Val Leu Leu Ser Ala Glu Leu Val Gln His Phe His 65 70 75 80 Lys Pro Ala Leu Leu Pro Leu Leu Gln Arg Ala Phe His Pro Pro His 85 90 95 Arg Val Val Arg Leu Leu Cys Gly Val Arg Asp Ser Glu Glu Phe Leu 100 105 110 Asp Phe Phe Pro Asp Trp Ala His Trp Gln Glu Leu Thr Cys Asp Asp 115 120 125 Glu Pro Glu Thr Tyr Val Ala Ala Val Lys Lys Ala Ile Ser Glu Asp 130 135 140 Ser Gly Cys Asp Ser Val Thr Asp Thr Glu Pro Glu Asp Glu Lys Val 145 150 155 160 Val Ser Tyr Ser Lys Gln Gln Asn Leu Pro Thr Val Thr Ser Pro Gly 165 170 175 Asn Leu Met Val Val Gln Pro Asp Arg Ile Arg Cys Gly Ala Glu Thr 180 185 190 Thr Val Tyr Val Ile Val Arg Cys Lys Leu Asp Asp Arg Val Ala Thr 195 200 205 Glu Ala Glu Phe Ser Pro Glu Asp Ser Pro Ser Val Arg Met Glu Ala 210 215 220 Lys Val Glu Asn Glu Tyr Thr Ile Ser Val Lys Ala Pro Asn Leu Ser 225 230 235 240 Ser Gly Asn Val Ser Leu Lys Ile Tyr Ser Gly Asp Leu Val Val Cys 245 250 255 Glu Thr Val Ile Ser Tyr Tyr Thr Asp Met Glu Glu Ile Gly Asn Leu 2 60 265 270 Leu Ser Asn Ala Ala Asn Pro Val Glu Phe Met Cys Gln Ala Phe Lys 275 280 285 Ile Val Pro Tyr Asn Thr Glu Thr Leu Asp Lys Leu Leu Thr Glu Ser 290 295 300 Leu Lys Asn Asn Ile Pro Ala Ser Gly Leu His Leu Phe Gly Ile Asn 305 310 315 320 Gln Leu Glu Glu Glu Asp Met Met Thr Asn Gln Arg Asp Glu Glu Leu 325 330 335 Pro Thr Leu Leu His Phe Ala Ala Lys Tyr Gly Leu Lys Asn Leu Thr 340 345 350 Ala Leu Leu Leu Thr Cys Pro Gly Ala Leu Gln Ala Tyr Ser Val Ala 355 360 365 Asn Lys His Gly His Tyr Pro Asn Thr Ile Ala Glu Lys His Gly Phe 370 375 380 380 Arg Asp Leu Arg Gln Phe Ile Asp Glu Tyr Val Glu Thr Val Asp Met 385 390 395 400 Leu Lys Ser His Ile Lys Glu Glu Leu Met His Gly Glu Glu Ala Asp 405 410 415 Ala Val Tyr Glu Ser Met Ala His Leu Ser Thr Asp Leu Leu Met Lys 420 425 430 Cys Ser Leu Asn Pro Gly Cys Asp Glu Asp Leu Tyr Glu Ser Met Ala 435 440 445 Ala Phe Val Pro Ala Ala Thr Glu Asp Leu Tyr Val Glu Met Leu Gln 450 455 460 Ala Ser Thr Ser Asn Pro Ile Pro Gly Asp Gly Phe Ser Arg Ala Thr 465 Four 70 475 480 Lys Asp Ser Met Ile Arg Lys Phe Leu Glu Gly Asn Ser Met Gly Met 485 490 495 Thr Asn Leu Glu Arg Asp Gln Cys His Leu Gly Gln Glu Glu Asp Val 500 505 510 Tyr His Thr Val Asp Asp Asp Glu Ala Ile Ser Val Asp Leu Ala Ser 515 520 525 Arg Pro Pro Val Pro Val Pro Arg Pro Glu Thr Thr Ala Pro Gly Ala 530 535 540 His Gln Leu Pro Asp Asn Glu Pro Tyr Ile Phe Lys Val Phe Ala Glu 545 550 555 560 Lys Ser Gln Glu Arg Pro Gly Asn Phe Tyr Val Ser Ser Glu Ser Ile 565 570 575 Arg Lys Gly Pro Pro Val Arg Pro Trp Arg Asp Arg Pro Gln Ser Ser 580 585 590 Ile Tyr Asp Pro Phe Ala Gly Met Lys Thr Pro Gly Gln Arg Gln Leu 595 600 605 Ile Thr Leu Gln Glu Gln Val Lys Leu Gly Ile Val Asn Val Asp Glu 610 615 620 Ala Val Leu His Phe Lys Glu Trp Gln Leu Asn Gln Lys Lys Arg Ser 625 630 635 640 Glu Ser Phe Arg Phe Gln Gln Glu Asn Leu Lys Arg Leu Arg Asp Ser 645 650 655 Ile Thr Arg Arg Gln Arg Glu Lys Gln Lys Ser Gly Lys Gln Thr Asp 660 665 670 Leu Glu Ile Thr Val Pro Ile Arg His Ser Gln His Leu Pro Ala Lys 675680 685 Val Glu Phe Gly Val Tyr Glu Ser Gly Pro Arg Lys Ser Val Ile Pro 690 695 700 Pro Arg Thr Glu Leu Arg Arg Gly Asp Trp Lys Thr Asp Ser Thr Ser 705 710 715 720 720 Ser Thr Ala Ser Ser Thr Ser Asn Arg Ser Ser Thr Arg Ser Leu Leu 725 730 735 Ser Val Ser Ser Gly Met Glu Gly Asp Asn Glu Asp Asn Glu Val Pro 740 745 750 Glu Val Thr Arg Ser Arg Ser Pro Gly Pro Pro Gln Val Asp Gly Thr 755 760 765 Pro Thr Met Ser Leu Glu Arg Pro Pro Arg Val Pro Pro Arg Ala Ala 770 775 780 Ser Gln Arg Pro Pro Thr Arg Glu Thr Phe His Pro Pro Pro Pro Val 785 790 795 800 Pro Pro Arg Gly Arg 805

[Brief description of the drawings]

FIG. 1. Cloning of a novel protein, BCAP. (A) Ponceau S staining of pp100. The part corresponding to pp100 cut out for microsequencing is indicated by [] on the right side. (B) CLUSTAL X program (Thompson et al., 1997)
Was used to show the sequence of chicken and mouse BCAP. Homologous amino acids are outlined. Chicken sequence clone
2 shown. The arrow indicates the start position of the open reading frame of clone 11. The peptide sequence obtained by microsequencing is as follows (amino acid number): AQRP
ENL (560-566), TPGQRQLITLQEQV (599-612), IVPYSIE
ALDK (293-303), FLEGGSTDAPDSGE (492-505), ETIVRP
VRDLS (576-586) and MGILNVDEAVL (612-624). (C) The motif and structure predicted for chicken BCAP and Drosophila Dof protein are shown in the schematic diagram. Ankyrin repeats (BCAP amino acids 336-404), coiled coils (636-666), proline-rich regions (532-5
38, 773-779, and 792-798), the boxed "AR"
Indicated by "CC" and "P" respectively. Candidate tyrosine phosphorylation region known as SH2 binding motif (()
Indicates the corresponding amino acid number): YxxM corresponding to SH2 of PI3K, YxNx corresponding to SH2 of Gab2, and YxxV corresponding to SH2 of SHP-2. The coiled-coil region was estimated based on the algorithm of A. Lupas (Lupas, 1996).

FIG. 2 BCAP expression (A) Northern blot analysis of BCAP RNA expression in mouse tissues and cell lines (upper photo). As a sample volume control, ethidium bromide stained image of tissue RNA and cell line using β-actin cDNA probe
RNA blots are shown at the bottom of each photograph. (B) Western blot analysis of BCAP protein expression in DT40 cells (left photo) and mouse spleen B cells (right photo). The cell lysate and the immunoprecipitated product of the anti-chicken BCAP antibody or the anti-mouse BCAP antibody were developed on a 7.5% SDS-PAGE gel and detected with the anti-BCAP antibody. In the picture on the left,
BCAP immunoprecipitates from BCAP-deficient DT40 cells and COS cells transfected with chicken BCAP cDNA clone 2 or clone 11 were also examined. Various B
CAP: The location of BCAP 1-4 is indicated. (C) chicken B producing two alternative transcripts
Schematic diagram of CAP gene fragment. Exons are indicated by squares.
The exon encoding the translation initiation methionine of clone 2 is located far upstream from the region shown here. The middle exon containing the primer "b" sequence corresponds to the alternative 5 'sequence found in clone 11;
Eleven translation initiation methionines (methionine 169 in FIG. 1B) are encoded in exons containing primer "c". ,
Of the 800 amino acid polypeptides shown in FIG. 1B, the linkage of the exons encoding clone 2 forms a codon that specifies aspartic acid (amino acid residue 144). The ethidium bromide-stained images show those amplified after reverse transcription from DT40 RNA and DNA fragments amplified from DT40 genomic DNA. For amplification, use the sense primer "a"(5'-GGCAAG
AACTCACGTATGATGATGAG-3 ') or "b"(5'-GGATCAG
GATCCCTTCAGACTACTG-3 ') and the sequence of the antisense primer "c"(5'-GGCAAGTTGACAGGAGTAAACTGAGG-3') were used. (D and E) Subcellular localization of BCAP. Wild-type (Wt) and BCAP-deficient DT40 cells (BCAP ⁻ ) with anti-BCAP antibody (green), nuclei with propidium iodide (red)
Each was stained and imaged with a confocal microscope (upper photograph of [D]). In the lower part of the photograph, COS cells transfected with BCAP cDNA clone 2 (COS / BCAP) and untransfected COS cells (COS) were stained with an anti-BCAP antibody and propidium iodide, respectively. The cytoplasmic and cell membrane fractions of unstimulated (−) or (+) DT40 cells stimulated with 4 μg / ml M4 for 2 minutes were developed by SDS-PAGE,
Western plotting was performed with BCAP antibody or M4 (E).

FIG. 3. Tyrosine phosphorylation of BCAP induced by BCR (A) Tyrosine phosphorylation of BCAP in DT40 cells (left photo) and mouse spleen B cells (right photo) and their association with p85. BCR stimulation (DT40 cells 4 μg / ml M4, mouse spleen B
Cells were stimulated with anti-mouse IgM (Fab) ₂ fragments, respectively), and after indicated times, anti-chicken or anti-mouse
The immunoprecipitation products (5 × 10 ⁶ DT cells / lane and 1 × 10 ⁷ mouse spleen B cells / lane) with BCAP antibody were developed on 7.5% SDS-PAGE gel, and 4G10, anti-BCAP antibody or anti-p85 antibody was developed. Was used for Western blotting. As a negative control, BCAP-deficient DT40 cells were also analyzed. The positions of various BCAP types BCAP1-6 are displayed. (B) Tyrosine phosphorylation of CD19 in mouse spleen B cells and association with p85 (C) BCAP tyrosine phosphorylation in Lyn, Syk or Btk-deficient DT40 cells

FIG. 4. Inactivation of BCAP gene in chicken DT40 cells. Deletion of BCAP expression by targeting of BCAP gene was already shown in FIGS. 2 and 3A. (A) Cell surface expression of BCR in wild-type and BCAP-deficient DT40 cells. Wild-type chicken BC in BCAP-deficient cells
AP (Wt / BCAP ⁻ ) or mutant chicken BCAP (Y4F)
The results of analysis of transformed cells expressing (Y4F / BCAP ⁻ ) are also shown here (also shown in FIG. 6). (B) Tyrosine phosphorylation induced by BCR in wild-type and BCAP-deficient DT40 cells. M4 stimulation (4μg / ml)
Later, at the indicated time, the cell lysate obtained from 2 × 10 ⁵ cells was
It was developed on S-PAGE (8% gel) and analyzed by Western blot using 4G10.

FIG. 5. Abnormality of PI3K signaling induced by BCR in the absence of BCAP (A) PI (3,4,5) in wild-type and BCAP-deficient DT40 cells
P ₃ amount. [ ³² P] orthophosphate-labeled cells were treated with 100 nM wortma
After incubation for 10 minutes in the presence or absence of nnin, the cells were stimulated with M4 (4 μg / ml). The spot of PI (3,4,5) P ₃ marker shown by the arrowheads. A typical example of these results is shown in the upper row of the photograph. In the lower panel, PI (3,4,5) after BCR stimulation
Indicating those standardized P ₃ amount or increase many times the total phospholipid amount. The results were shown as the average value of the experiments performed three times and the standard error of the average value (see Experimental Method). PI (3,4,5) P _{3 in} total phospholipids at time 0 after BCR stimulation
The actual percentage of radioactivity is based on wild-type DT40 cells, BCAP-deficient DT
In 40 cells and wortmannin-treated wild-type cells, they were 0.15% ± 0.03%, 0.13% ± 0.04% and 0.07% ± 0.01%, respectively. (B and C) in wild-type and BCAP-deficient DT40 cells
BCR-induced activation of Akt (B) and JNK (C). 100 n
Cells incubated for 10 minutes in the presence or absence of M wortmannin were stimulated with 4 μg / ml M4 for the indicated time. Akt and JNK are immunoprecipitated, respectively, and histone H2B
And GST-cJun were used in assays for in vitro kinase activity using each as a substrate. The reaction product was developed and exposed on SDS-PAGE (upper photograph). The amounts of Akt and JNK proteins in the immunoprecipitated product were examined by Western blot analysis (lower photograph). (D) Apoptosis induction in wild-type and BCAP-deficient DT40 cells. The cells were cultured for 24 hours in the presence (+) or absence (-) of M4 (10 μg / ml), stained with 50 μg / ml propidium iodide, and analyzed by flow cytometry. The percentage of cells in which DNA has undergone nuclear fragmentation is shown as a percentage. (E) generating IP ₃ which BCR induced in wild-type and BCAP deficient DT40 cells (2 × 10 ⁵ cells / sample).

FIG. 6. Requirement of phosphorylation of YxxM motif for BCAP function (A) Tyrosine phosphorylation of wild-type BCAP or BCAP (Y4M) and their association with p85. DT40 cells were stimulated with M4 (4 μg / ml) and immunoprecipitated using an anti-chicken BCAP antibody. Using 4G10 (upper photo), anti-chicken BCAP antibody (in the photo) or anti-p85 antibody (lower photo), a Western blot of the immunoprecipitated product was performed. The positions of BCAP3, BCAP4, BCAP5 and BCAP6 are indicated. (B and C) Activation of Akt (B) and JNK (C). After M4 stimulation (4 μg / ml), each DT40 cell was replaced with Akt and JNK
Was used for the in vitro kinase assay.

FIG. 7: BCAP is involved in the transfer of PI3K to GEMs through BCR. Unstimulated (−) or 4 μg / ml M4 stimulated (+) lysed wild-type and BCAP-deficient DT40 cells, and sucrose gradient centrifugation. Fractionated by the method. Each fraction (30 μl / lane; numbered from low to high density) was developed by SDS-PAGE and subjected to Western blotting with anti-p85 antibody, anti-chicken BCAP antibody, anti-Lyn antibody and anti-tubulin antibody. went.

FIG. 8 is a diagram comparing homology between mouse BCAP and human BCAP.

FIG. 9 is a diagram of Western blotting showing the reaction specificity of the anti-BCAP monoclonal antibody. GST-BCAP lane 2. GST lane 3. Raji Cell extract

FIG. 10 is a photograph of Western blotting showing the reaction specificity of the anti-BCAP monoclonal antibody. (A) Mouse B cells (A20) and human B cells (C1LCL, B10
4) Photo showing reactivity with (B) Photo showing reactivity with chicken B cells (DT40), mouse B cells (A20), and human B cells (C1LCL, B104)

FIG. 11 is a schematic diagram showing the relationship between BCR and BCAP.

[Explanation of symbols]

 1 ... BCR 2 ... Syk 3 ... BCAP 4 ... PI3K

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 4C085 1/21 G01N 33/15 Z 4H045 5/10 33/50 Z 15 / 02 A61K 39/395 D G01N 33/15 H 33/50 N // A61K 39/395 U 48/00 A61P 37/02 C12P 21/08 48/00 C12N 15/00 ZNAA A61P 37/02 5/00 A C12P 21/08 15/00 C 5/00 B (72) Inventor Mineyo Okada United States California 94122 San Francisco, Ortegastreet 1900B (72) Inventor Akito Maeda 2-13-3, Oeda Kitatsukakecho, Nishikyo-ku, Kyoto ( 72) Inventor Masao Shibata 8306-1052 F-term (reference) 2G045 AA40 DA12 DA13 DA36 FB02 FB03 4B024 AA11 BA43 BA44 BA80 CA04 DA06 EA 04 GA11 HA12 HA15 4B064 AG01 AG27 CA02 CA10 CA19 CA20 CC24 CE12 DA01 DA13 4B065 AA26X AA90Y AA91X AA92X AA92Y AA93Y AB01 AB05 AC14 BA02 CA24 CA25 CA44 CA46 4C084 AA13 AA16 CA17 CA19 NA14 ZB071A0 CC14 A04 ZB07A4 CC AA30 BA10 CA40 DA76 DA86 EA22 EA50 FA72 FA74 GA26

Claims (14)

[Claims] 1. An amino acid sequence represented by SEQ ID NO: 17 wherein one or more amino acids are deleted, substituted, and / or
Or a protein comprising an added amino acid sequence and having p85-binding activity. 2. A nucleic acid encoding the protein according to claim 1. 3. D consisting of the nucleotide sequence shown in SEQ ID NO: 16
Hybridizes with NA under stringent conditions,
And a nucleic acid encoding a protein having p85 binding activity. 4. The amino acid sequence of SEQ ID NO: 17 and 70
% Of an amino acid sequence having a homology of at least
5. A nucleic acid encoding a protein having an activity of binding to 5. 5. A recombinant vector for expression, to which the DNA according to claim 2 is operably linked. 6. A transformant containing the recombinant vector according to claim 5. 7. A screening method for screening a substance that inhibits the signaling function of the amino acid, using a protein encoded by the nucleic acid according to claim 2. 8. An immune function modulator obtainable by the screening method according to claim 7. 9. An antibody or antibody fragment that binds to a protein encoded by the nucleic acid according to claim 2 or a partial amino acid sequence of the protein. 10. The antibody or antibody fragment according to claim 9, wherein said antibody is polyclonal. 11. The antibody or antibody fragment according to claim 9, wherein said antibody is monoclonal. 12. The antibody according to claim 11, wherein the monoclonal antibody is produced by a fused cell 4C8. 13. An antibody-producing cell which produces the antibody or the antibody fragment according to claim 10 or 11. 14. The antibody-producing cell according to claim 13,
An antibody-producing cell, which is a fused cell 4C8.