JP2008182937A - Mutant of phosphorylation site of protein phosphatase PP2AB'α phosphorylated by GAK and method of using the same - Google Patents

Abstract

Based on the knowledge obtained from the analysis results of GAK and PP2A B′α, a means for elucidating a signal transduction mechanism related to these, and a medicine having a new mechanism of action based on an action function revealed by the means And the development method.
PP2A B′α variant in which threonine phosphorylated by GAK is substituted or deleted by another amino acid, or a fragment thereof containing the substituted or deleted portion; PP2A and phosphorylation In a phosphatase reaction system containing a substance, the substance having reduced degree of dephosphorylation by coexistence with the PP2A B′α mutant or a fragment thereof is identified as a dephosphorylation target of PP2A. A method for screening an oxidation target; a method for screening a substance capable of regulating the transcriptional activity of p53, including evaluating whether a test substance can regulate the expression or activity of a constituent factor of PP2A.
[Selection figure] None

Description

  The present invention relates to a PP2A B′α mutant and a fragment thereof, a method for screening a PP2A dephosphorylation target, a method for screening a substance capable of regulating the transcriptional activity of p53, a regulator of the transcriptional activity of p53, and the like.

  Phosphorylation of the target protein by protein kinases and subsequent signaling cascades controls various aspects of cellular events. Once the target is phosphorylated, the reverse reaction of dephosphorylation catalyzed by various protein phosphatases should proceed. Protein phosphatases, like their cognate kinases, are thought to provide both spatial and temporal regulation for various subcellular events. Nevertheless, in contrast to a well-established understanding of the molecular and subcellular mechanisms of protein phosphorylation, details of the regulatory mechanism of dephosphorylation remain unclear. One of many different types of phosphatases, protein phosphatase 2A (PP2A) represents a large and ubiquitously expressed group of Ser / Thr phosphatases that regulate multiple signaling pathways. This suggests that many possible targets of PP2A play an important role in the growth of eukaryotic cells (Non-Patent Documents 1 and 2). PP2A consists of three subunits: an invariant catalyst (C) and a structure (A) subunit, and a variable regulatory (B) subunit. Three unrelated families of PP2A regulatory subunits (B, B ′ and B ″) have been identified; five different mammalian genes encode members of the B ′ subunit family and many isoforms are Generated from these genes. Among these, the B′α subunit contains three alternative splicing mutants with different subcellular localization (nuclei: B′α1 and B′α2) as we have previously shown (Non-patent Document 3). Cytoplasm: B′α3).

  Cyclin G1, one of the two cyclin G paralogs in mammals, has been shown to play a major role in recruiting PP2A to target proteins (Non-Patent Documents 4 and 5). Cyclin G1 was first identified as a novel member of the cyclin family that possesses sequence homology with the tyrosine phosphorylation site of epidermal growth factor receptor (EGFR) in its C-terminal region (Non-patent Document 6). Cyclin G1 is one of the target genes of p53 tumor suppressor protein (Non-patent Document 7), and is induced in a p53-dependent manner in response to DNA damage. Cyclin G1 was found to be a regulatory component of an active PP2A holoenzyme containing a B ′ subunit that activates Mdm2 via dephosphorylation at T216 causing stabilization of the p53 tumor suppressor protein. (Non-Patent Document 5). Cyclin G1 null mice are normally born and grow healthy (Non-Patent Document 8), but cyclin G1 directly interacts with Mdm2 to promote ARF / Mdm2 complex formation after DNA damage, Accumulation of p53 after injury is enhanced in cyclin G1-/-cells (Non-patent Document 9). These findings suggest that cyclin G is a key regulator of the p53-Mdm2 network (Non-patent Document 10).

  We previously described a cyclin G1 binding protein, cyclin G-associated kinase (GAK), which has motifs such as Ser / Thr kinase domain, tensin / auxilin-like domain, Tyr phosphorylation site and leucine zipper. (Patent document 1, non-patent document 11). As suggested by the high homology other than the kinase domain with the neuron-specific protein auxilin, ubiquitous GAK, like auxilin, is essential for the disassembly of clathrin-coated vesicles. It regulates membrane transport mediated by clathrin as a cofactor (Non-patent Document 12). Suppression of GAK expression (knockdown) by small hairpin RNA not only induces clathrin exchange on clathrin coat pits but also binding of clathrin and adapters to the plasma membrane and trans-Golgi network It was also shown to mediate (Non-Patent Documents 13 and 14). By microscopic observation, after transient transfer of dynamin, GAK transiently migrates to a punctate local location made by clathrin, and this transition is a PTEN-like region to which phospholipids contained in GAK bind. It has been shown to depend on (Non-Patent Document 15). Furthermore, GAK phosphorylates not only the clathrin adapters AP-1 and AP-2, but also the mu2 subunit of the AP2 adapter complex. This suggests a major role of GAK in the assembly of clathrin-coated vesicles (Non-Patent Document 16).

The fact that GAK has several conserved amino acid sequences suggests an unknown role for GAK other than membrane trafficking in the cytoskeletal network through interaction with other regulatory proteins. For example, suppression of GAK expression by small hairpin RNAs causes a dramatic increase in EGFR expression and its tyrosine kinase activity, which significantly alters various events that occur downstream of EGFR signaling (this is in receptor transport). (Non-patent document 17). Constitutive overexpression of nucleophosmin anaplastic lymphoma kinase (NPM-ALK) is a key carcinogenic event in anaplastic large cell lymphoma, and by mass spectrometry (MS / MS), NPM-ALK reciprocal GAK was identified as one of the acting proteins (Non-patent Document 18). Interestingly, GAK has been identified as an androgen receptor (AR) interacting protein that enhances AR activity in a ligand-dependent manner, and GAK expression is significantly enhanced with prostate cancer progression independent of the presence of androgen (Non-Patent Document 19). This report suggests a role for GAK not only in the cytoplasm but also in the nucleus. The recent finding that clathrin heavy chain (CHC) binds to p53 and contributes to p53-mediated transcription in the nucleus (Non-patent Document 20) also suggests the function of GAK in the nucleus. However, no direct evidence has been reported so far.
Japanese Patent Laid-Open No. 10-286086 Janssens V. and Goris, J., Biochem J 353 (2001), pp. 417-439. Janssens V et al., Curr Opin Genet Dev. 2005 Feb; 15 (1): 34-41. Ito et al., EMBO J. 2000 Feb 15; 19 (4): 562-71. Okamoto K et al., Mol Cell Biol. 1996 Nov; 16 (11): 6593-602. Okamoto K et al., Mol Cell. 2002 Apr; 9 (4): 761-71. Tamura et al., Oncogene. 1993 Aug; 8 (8): 2113-8. Okamoto K et al., EMBO J. 1994 Oct 17; 13 (20): 4816-22. Kimura SH et al., Oncogene. 2001 May 31; 20 (25): 3290-300. Kimura SH et al., Genes Cells. 2002 Aug; 7 (8): 869-80. Chen, Dev Cell. 2002 May; 2 (5): 518-9. Kanaoka Y et al., FEBS Lett. 1997 Jan 27; 402 (1): 73-80. Greener T et al., J Biol Chem. 2000 Jan 14; 275 (2): 1365-70. Lee DW et al., J Cell Sci. 2005 Sep 15; 118 (Pt 18): 4311-21. Zhang CX et al., Traffic. 2005 Dec; 6 (12): 1103-13. Lee DW et al., J Cell Sci. 2006 Sep 1; 119 (Pt 17): 3502-12. Korolchuk and Banting, Traffic. 2002 Jun; 3 (6): 428-39. Zhang L et al, Proc Natl Acad Sci US A. 2004 Jul 13; 101 (28): 10296-301. Epub 2004 Crockett DK et al., Oncogene. 2004 Apr 8; 23 (15): 2617-29. Ray MR et al., Int J Cancer. 2006 Mar 1; 118 (5): 1108-19. Enari M et al., Genes Dev. 2006 May 1; 20 (9): 1087-99.

  Analysis of gene function and / or signal transduction mechanism leads to development of drugs having new action mechanisms for various diseases. Based on the knowledge obtained from the analysis results of GAK and PP2A B′α, the present invention has a means for elucidating a signal transduction mechanism related to these, and a new mechanism of action based on an action function clarified by the means. The purpose is to provide medicine and its development.

As a result of intensive studies, the present inventors have found that GAK directly interacts with PP2A B′α, specifically phosphorylates Thr-104, and regulates its phosphatase activity in the nucleus. It was. We also find that assays using PP2A B′α variants or fragments thereof in which threonine phosphorylated by GAK is replaced with other amino acids are useful for finding new dephosphorylation targets of PP2A. As a result, this assay has succeeded in actually identifying a novel dephosphorylation target (eg, p53) of PP2A, thereby completing the present invention.
That is, the present invention is as follows:
[1] A PP2A B′α mutant in which threonine phosphorylated by GAK is substituted or deleted with another amino acid, or a fragment thereof containing the substituted or deleted portion.
[2] The mutant or fragment thereof of [1] above, wherein PP2A B′α is PP2A B′α1.
[3] The mutant of [2] above or a fragment thereof, wherein PP2A B′α1 is derived from a mouse and the threonine is threonine at position 104.
[4] The variant of the above [3] or a fragment thereof, wherein PP2A B′α1 consists of the amino acid sequence represented by SEQ ID NO: 2 or 4.
[5] The mutant or fragment thereof according to any one of [1] to [4] above, wherein the other amino acid is alanine.
[6] A polynucleotide encoding the PP2A B′α mutant or a fragment thereof of any one of [1] to [5] above.
[7] An expression vector comprising the polynucleotide of [6] above.
[8] A transformant introduced with the expression vector of [7] above.
[9] In a phosphatase reaction system containing PP2A and a phosphorylated substance, a substance in which the degree of dephosphorylation is reduced by the coexistence of the PP2A B′α mutant or fragment thereof of any one of [1] to [5] above A method for screening a PP2A dephosphorylation target, comprising identifying a PP2A dephosphorylation target.
[10] PP2A dephosphorylation comprising the PP2A B′α mutant or fragment thereof of any one of [1] to [5], the expression vector of [7], or the transformant of [8] Reagent or kit for target screening.
[11] A screening method for a substance capable of regulating the transcriptional activity of p53, comprising evaluating whether a test substance can regulate the expression or activity of a constituent factor of PP2A.
[12] Evaluation of whether or not a test substance can regulate the activity of a constituent factor of PP2A is based on whether the test substance is a kinase activity of GAK using PP2A B′α as a substrate or PP2A B′α using p53 as a substrate The screening method according to [11] above, which is carried out by evaluating whether phosphatase activity can be regulated.
[13] The screening method of [11] or [12] above, wherein the test substance is a screening method for a substance that can promote the transcriptional activity of p53.
[14] The screening method according to any one of [11] to [13] above, wherein the substance capable of regulating the transcriptional activity of p53 is a substance capable of preventing or treating a disease caused by abnormal cell proliferation.
[15] The screening method of [13] above, wherein the substance capable of promoting the transcription activity of p53 is a drug against cancer.
[16] A regulator of p53 transcriptional activity, comprising a substance capable of regulating the expression or activity of a constituent factor of PP2A.
[17] Detection of PP2A B′α mutation in PP2A B′α, comprising measuring an amino acid mutation of threonine phosphorylated by GAK or a mutation of a nucleotide portion encoding threonine phosphorylated by GAK Method.

The mutants and fragments of the present invention are useful for screening PP2A B′α dephosphorylation targets and inhibiting PP2A B′α dephosphorylation targets (eg, p53).
The regulator and the regulation method of the present invention prevent diseases caused by abnormal cell proliferation such as diseases accompanied by enhanced cell proliferation (eg, epithelial cancer, non-epithelial cancer, cancer in hematopoietic tissue (eg, leukemia)). Or it is useful for treatment.
The screening method of the present invention is useful because it enables the development of drugs and the like for diseases caused by abnormal cell proliferation. The screening method of the present invention is also useful because it enables identification of the dephosphorylation target of PP2A B′α, and hence the development of a drug having a new mechanism of action by using the identified dephosphorylation target. It is.
The detection method of the present invention is useful in assays using the mutants of the present invention or for detecting polymorphisms.

  The present invention provides PP2A B'α variants or fragments thereof.

  The mutant of the present invention may be a PP2A B'α mutant in which the threonine phosphorylated by cyclin G-associated kinase (GAK) is substituted with another amino acid or is deleted. PP2A B'α and GAK can be proteins from any mammalian species. Examples of such mammalian species include primates such as humans and monkeys, rodents such as mice, rats, hamsters, and guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs. . Examples of PP2A B′α include PP2A B′α1, PP2A B′α2, and PP2A B′α3, which are subtypes produced by alternative splicing.

  In mouse PP2A B′α, threonine phosphorylated by GAK in any of α1, α2, and α3 may be threonine at position 104. Since the size of human PP2A B'α is slightly larger than that of the mouse, in human PP2A B'α, the threonine phosphorylated by GAK may be threonine at position 114. For reference, the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of human PP2A B′α1 and the nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of mouse PP2A B′α1 are shown. .

  In the mutant of the present invention, when threonine phosphorylated by GAK is substituted with another amino acid, the other amino acid is not particularly limited as long as it is a natural α-amino acid other than threonine. (Eg, alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, valine), acidic amino acids (eg, aspartic acid, glutamic acid), basic amino acids (eg, arginine) , Histidine and lysine), and alanine is preferable from the viewpoint of dominant negative activity. In addition, it is well known that changing the threonine at the phosphorylation site of a given protein to glutamic acid results in a state equivalent to the activity of the protein being constantly activated. It is also preferable to substitute threonine phosphorylated by GAK with glutamic acid.

  In the mutant of the present invention, when threonine phosphorylated by GAK is deleted, the deletion mutant of the present invention may be missing only threonine, but threonine and its adjacent amino acids are 1 to several. (For example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, most preferably 1 or 2) may be missing. The adjacent amino acids can be N and / or C adjacent amino acids.

  In the mutant of the present invention, amino acid residues other than threonine to be phosphorylated may be the same as the natural protein (eg, SEQ ID NO: 2 or 4) or may be modified.

  For example, in the mutant of the present invention, when an amino acid residue is modified in addition to phosphorylated threonine, the mutant of the present invention has 90% or more of the amino acid sequence represented by SEQ ID NO: 2 or 4 ( Preferably consisting of amino acid sequences having amino acid sequence identity of about 95%, more preferably about 97%, even more preferably about 98%, most preferably about 99% or more (for calculation of amino acid sequence identity) Threonine substitutions or deletions (including deletions if adjacent amino acids are also deleted) are also considered). Amino acid sequence identity can be determined by a method known per se. Unless otherwise specified, amino acid sequence identity (%) is calculated by executing a command of the maximum matching method, for example, using DNASIS (Hitachi Software Engineering), which is sequence analysis software. The parameters at that time are default settings (initial settings). In addition, the amino acid sequence identity (%) can be determined using a program commonly used in the field (for example, BLAST, FASTA, etc.) in the initial setting without depending on the above. In another aspect, identity (%) is determined by any algorithm known in the art, such as Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4: It can be determined using the algorithm of 11-17). The Needleman et al. Algorithm is incorporated into the GAP program of the GCG software package, and the percent identity is, for example, BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, 14, 12, 10, 8, 6 or 4 and length weight: can be determined by using any of 1, 2, 3, 4, 5 or 6. The Myers and Miller algorithms are also incorporated into the ALIGN program that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, for example, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used. For calculation of amino acid sequence identity, a method showing the lowest value among the above methods may be adopted.

  When the amino acid residue is modified in addition to threonine to be phosphorylated, the variant of the present invention has one or more amino acid residues (substitution or deletion) in the amino acid sequence represented by SEQ ID NO: 2 or 4. Amino acid residues other than threonine) may be composed of an amino acid sequence having one or more modifications selected from substitution, addition, deletion and insertion. The number of amino acids to be modified is not particularly limited as long as it is 1 or more. For example, 1 to about 50, preferably 1 to about 40, more preferably 1 to about 30, and even more preferably 1 to about 20 is used. One, most preferably 1 to about 10, 1 to about 5, or 1 or 2.

  A fragment of a PP2A B'α variant can be a partial polypeptide of a PP2A B'α variant, including a substituted or deleted portion of the PP2A B'α variant. The size of the fragment is not particularly limited as long as it is a size capable of exhibiting a predetermined function (eg, immunogenicity or antigenicity, dominant negative effect), but is 8 or more, preferably about 12 or more, more preferably about 30 or more, Even more preferably, it may consist of about 50 or more, most preferably about 100 or more, about 200 or more, about 300 or more, about 400 or more, or about 500 or more amino acid residues.

  The variant or fragment of the present invention may be fused to a polypeptide consisting of a heterologous amino acid sequence. Such polypeptides include polypeptides that facilitate purification or solubilization, and fluorescent polypeptides. Polypeptides that facilitate purification or solubilization include, for example, histidine tag, maltose binding protein (MBP), glutathione-S-transferase (GST), calmodulin binding peptide (CBP), FLAG tag, Fc region of IgG molecule, Examples include Myc tag and HA tag. Examples of the fluorescent polypeptide include GFP, DsRed, Kikume Green-Red, Kaede, Keima-Red, Kusabira-Orange, Midoriishi-Cyan, and Dronpa-Green. When the mutant or fragment of the present invention is fused with a polypeptide comprising a heterologous amino acid sequence as described above, the fused heterologous amino acid sequence is not included in the calculation of amino acid sequence identity described above. Similarly, amino acid residues contained in a fused heterologous amino acid sequence shall not be included in one or more modifications of one or more amino acid residues as described above.

  The mutant of the present invention or a fragment thereof can be produced by a method known per se. For example, the mutant of the present invention or a fragment thereof may be recovered from 1) the following transformant expressing the mutant of the present invention or a fragment thereof or the culture supernatant thereof. 2) Rabbit reticulocyte lysate, wheat It may be synthesized in a cell-free system using embryo lysate, E. coli lysate or the like, or 3) synthesized organically (eg, solid phase synthesis). The variant of the present invention or a fragment thereof is a method that utilizes solubility such as salting-out or solvent precipitation; mainly molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method utilizing difference; Method utilizing difference in charge such as ion exchange chromatography; Method utilizing specific affinity such as affinity chromatography and use of antibody; Difference in hydrophobicity such as reversed phase high performance liquid chromatography A method using a difference in isoelectric point such as isoelectric focusing method; a method combining them, and the like.

  Variants and fragments of the invention may have altered phosphatase activity compared to PP2A B'α. For example, variants and fragments of the present invention cannot have phosphatase activity because they are not phosphorylated by GAK. Therefore, the mutants and fragments of the present invention can reduce the dephosphorylation of the target protein of PP2A B'α due to the dominant negative effect. Examples of such target proteins include Chk2, p53, ATM, and Mdm2. Reduction of dephosphorylation of the target protein allows for stabilization of the target protein (eg, Chk2), regulation of the interaction of the target protein (eg, p53) with other proteins (eg, large T antigen), etc. . The mutants and fragments of the present invention are also useful for screening the dephosphorylation target of PP2A B'α described later and for regulating the transcriptional activity of p53.

  The present invention also provides a polynucleotide encoding the mutant or fragment of the present invention, an expression vector containing the polynucleotide, and a transformant into which such an expression vector has been introduced.

  The polynucleotide of the present invention may be DNA or RNA. The polynucleotide of the present invention is not particularly limited as long as it encodes the mutant or fragment of the present invention. For example, among the nucleotide sequences represented by SEQ ID NO: 1 or 3, threonine phosphorylated by GAK is used. It may be a partial polynucleotide of the PP2A B′α variant gene, wherein the encoding nucleotide residue is substituted or deleted so as to encode another amino acid, or contains the substituted or deleted portion. .

  The expression vector of the present invention may comprise a polynucleotide of the present invention and a promoter operably linked to the polynucleotide. “Promoter is operably linked to a polynucleotide” refers to a polynucleotide encoding the gene such that the promoter allows expression of the polypeptide encoded by the polynucleotide under its control. Means that they are connected.

  The backbone of the expression vector of the present invention is not particularly limited as long as it can produce a target substance in a predetermined cell, and examples thereof include a plasmid vector and a virus vector. When the expression vector is used as a medicine, suitable vectors for administration to mammals include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus, etc. A viral vector is mentioned.

  When prokaryotic cells are used as host cells, expression vectors that can use prokaryotic cells as host cells can be used. Such an expression vector can contain elements such as a promoter-operator region, a start codon, a polynucleotide encoding the SNI polypeptide of the present invention or a partial peptide thereof, a stop codon, a terminator region, and an origin of replication. The promoter-operator region for expressing the polypeptide of the present invention in bacteria contains a promoter, an operator and a Shine-Dalgarno (SD) sequence. As these elements, those known per se can be used.

  When eukaryotic cells are used as host cells, expression vectors that can use eukaryotic cells as host cells can be used. In this case, the promoter used is not particularly limited as long as it can function in eukaryotes such as mammals. Examples of such promoters include SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter, and the like, as well as β-actin gene promoter, PGK gene promoter, transferrin Examples include mammalian component protein gene promoters such as gene promoters.

  The expression vector of the present invention may further contain sites for transcription initiation and transcription termination, a ribosome binding site that may be required for translation in the transcription region, a replication origin, a selectable marker gene, and the like. The selectable marker gene is not particularly limited as long as a transformant can be selected when a specific cell or bacterium is used as a host. For example, ampicillin, tetracycline, kanamycin, spectinomycin, erythromycin, chloram Examples include resistance genes to antibiotics such as phenicol and carbenicillin.

The transformant of the present invention may be a cell into which the expression vector of the present invention has been introduced. The host cell used for the production of the transformant is not particularly limited as long as it is compatible with the expression vector and can express the target polypeptide. For example, natural cells commonly used in the technical field of the present invention are used. Another example is an artificially established cell line. Specifically, examples of such host cells include prokaryotic cells such as Escherichia coli, Bacillus (eg, Bacillus subtilis), actinomycetes, yeast, insect cells, avian cells, and mammalian cells (eg, the above-mentioned). Eukaryotic cells such as cells derived from mammals). The transformant of the present invention can be prepared by a method known per se ^{(e.g., Molecular Cloning, 2 nd edition,} Sambrook et al., See Cold Spring Harbor Lab. Press (1989 )).

  The transformant can be cultured in a nutrient medium such as a liquid medium by a method known per se. The medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Here, examples of the carbon source include glucose, dextrin, soluble starch, and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include inorganic or organic substances such as bean paste and potato extract. Examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Moreover, you may add a yeast extract, vitamins, etc. to a culture medium. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the mutant of the present invention or a fragment thereof is produced in large quantities. The culture temperature is, for example, 30 to 37 ° C.

  The present invention also provides antisense nucleic acids, RNAi-inducible nucleic acids, antibodies, and expression vectors thereof specific for the variants or fragments of the present invention. Such antisense and RNAi inducible nucleic acids comprise a nucleotide sequence encoding a substitution or deletion site for threonine phosphorylated by GAK in a polynucleotide encoding a variant or fragment of the invention, thereby It can specifically inhibit the activity of a variant or fragment of the invention. Such an antibody specifically binds / inhibits the mutant of the present invention or a fragment thereof by specifically recognizing the substitution or deletion site of threonine phosphorylated by GAK. These substances can be useful, for example, as controls in screening methods for PP2A B'α dephosphorylation targets as described below.

  The present invention also provides a screening method for PP2A dephosphorylation target. Such a screening method identifies, as a PP2A dephosphorylation target, in a phosphatase reaction system containing PP2A and a phosphorylated substance, a substance having a reduced degree of dephosphorylation by coexisting the mutant of the present invention or a fragment thereof. Can include.

  PP2A is a serine / threonine phosphatase consisting of three subunits (A, B, C). Of these, the A subunit has catalytic activity, the C subunit has a scaffold function, and the B subunit has a regulatory function. In the B subunit, three subtypes (B, B ′, B ″) are encoded by different genes, and each of them produces a B subunit having various amino acids by multiple variable splicing. The C subunit and the A subunit work in common, and the intracellular localization and substrate specificity of PP2A are determined by exchanging many types of B subunits. Cyclin G is thought to specifically bind to the B 'subunit and serve to accurately carry PP2A to the target. Dephosphorylating enzyme activity is specifically inhibited by okadaic acid, a toxic component that accumulates in marine bivalves. The phosphatase reaction system used in the present invention contains such three subunits of PP2A as constituent factors.

  The phosphatase reaction system containing PP2A and a phosphorylated substance can be a cell system or a reconstitution system (non-cell system). The phosphorylated substance is not particularly limited as long as it is a phosphorylated substance that can be dephosphorylated by PP2A, and examples thereof include phosphorylated proteins. The phosphorylated substance used in this screening method may be prepared by a known organic synthesis method or kinase reaction, or may be extracted from cells or tissues. The phosphatase reaction system containing PP2A and a phosphorylated substance may further contain cofactors necessary for the phosphatase reaction by PP2A. Such cofactors include GAK and cyclin G. The phosphatase reaction with PP2A can be performed by a method known per se (eg, Okamoto et al., Mol. Cell, 2002 Apr; 9 (4) 761-71). Moreover, the phosphatase reaction by PP2A can be easily performed by using a commercially available kit such as PP2A assay kit (manufactured by Molecular Devices) using IMAP technology.

For example, when the phosphatase reaction system is a cell system, expression vectors for PP2A-expressing cells (eg, primary cultured cells, cell lines) or all or part of PP2A components (eg, B′α) have been introduced. The screening method of the present invention can be performed using a transformant (host cell may be the same as described above). Since PP2A expression cells and host cells used for the production of transformants contain various phosphorylated proteins and the like, PP2A expression cells or expression vectors for all or part of PP2A components The transformant introduced with can be used as a phosphatase reaction system containing PP2A and a phosphorylated substance. The coexistence of the mutant of the present invention or a fragment thereof in such a cell line is, for example, the expression vector of the present invention for a transformant into which an expression vector of PP2A-expressing cells or all or part of the constituent elements of PP2A has been introduced. Can be achieved by the introduction of A substance having a reduced degree of dephosphorylation in a cell line into which the expression vector of the present invention has been introduced can be identified as a dephosphorylation target for PP2A compared to a cell line into which the expression vector of the present invention has not been introduced. Detection of substances with a reduced degree of dephosphorylation can be achieved by detecting antibodies specific to phosphorylated amino acids (eg, phosphothreonine, phosphoserine, phosphotyrosine) or non-phosphorylated amino acids (eg, threonine, serine, tyrosine), phosphorylated or non-phosphorylated. It can be done by the use of specific antibodies against phosphorylated proteins, by measurement of phosphatase activity indicators such as radioactivity (eg ³² P) or fluorescence intensity, or by mass spectrometry. For example, when mass spectrometry is used, it is included in the used cell by identifying a series of peaks with reduced molecular weight corresponding to one or more phosphate groups in the presence of the mutant of the present invention or a fragment thereof. A series of targets can be comprehensively identified.

  In addition, when the phosphatase reaction system is a reconstitution system, the degree of dephosphorylation is reduced in the reconstitution system containing the mutant or fragment of the present invention compared to the reconstitution system not containing the mutant or fragment of the present invention. The identified material can be identified as a dephosphorylation target for PP2A. Detection of a substance having a reduced degree of dephosphorylation can be performed by the same method as described above.

  The present invention also provides a reagent or kit for screening a PP2A dephosphorylation target. The reagent or kit of the present invention may contain the mutant of the present invention or a fragment thereof, the expression vector of the present invention, or the transformant of the present invention. The reagent or kit of the present invention is also directed against antibodies specific to phosphorylated amino acids (eg, phosphothreonine, phosphoserine, phosphotyrosine) or non-phosphorylated amino acids (eg, threonine, serine, tyrosine), phosphorylated or nonphosphorylated proteins. Means capable of measuring the degree of phosphorylation, such as specific antibodies, may be included. The reagent or kit of the present invention may also contain an antisense nucleic acid, RNAi-inducible nucleic acid, antibody, and expression vectors thereof specific for the mutant or fragment of the present invention.

  The present invention provides a regulator of p53 transcriptional activity. The agent of the present invention may contain a substance capable of regulating the expression or activity of a constituent factor of PP2A. The constituent factors of PP2A include those described above.

  In one embodiment, the agent of the present invention may be an inhibitor of p53 transcriptional activity. In this case, the agent of the present invention may contain a substance that can promote the expression or activity of a constituent factor of PP2A.

  The expression of the constituent factor of PP2A means that a translation product (ie, protein) is produced from a gene encoding the constituent factor of PP2A and is localized in its functional site in a functional state. Therefore, the substance that promotes the expression of PP2A component may act at any stage, such as transcription, post-transcriptional regulation, translation, post-translational modification, localization and protein folding of PP2A component. Good. As used herein, the promotion of PP2A component expression includes supplementation of PP2A component (protein) itself.

  Examples of the substance that promotes the expression or activity of the constituent factor of PP2A include a constituent factor of PP2A and an expression vector thereof. The component of PP2A can be a natural protein or a recombinant protein. The constituent factor of PP2A can be prepared by a method known per se as described above for the PP2A B ′ mutant.

  In another embodiment, the agent of the present invention may be a promoter of p53 transcriptional activity. In this case, the agent of the present invention may contain a substance capable of suppressing the expression or activity of the constituent factor of PP2A. The substance that suppresses the expression of the constituent factor of PP2A may act at any stage such as transcription, post-transcriptional regulation, translation, post-translational modification, localization and protein folding of the constituent factor of PP2A.

  Examples of substances that suppress the expression of PP2A constituents include, for example, antisense nucleic acids (eg, DNA, RNA, or modified nucleotides, or chimeric molecules thereof), ribozymes, RNAi-inducible nucleic acids (cells) for PP2A constituents Polynucleotides that can induce the RNAi effect by being introduced into them, preferably RNA (eg, siRNA), aptamers, and expression vectors containing the nucleic acids encoding them. An expression vector similar to that described above can be used.

  Examples of the substance that suppresses the activity of the constituent factor of PP2A include an antibody against the constituent factor of PP2A, a dominant negative mutant (eg, the PP2A B ′ mutant of the present invention or a fragment thereof), or an expression vector thereof, or a small molecule. Compounds.

  Substances that inhibit the activity of the constituents of PP2A are also antisense nucleic acids, ribozymes, RNAi-inducible nucleic acids (eg, siRNA), dominant negative mutants (eg, kinase dead mutants), antibodies, aptamers, and An expression vector containing a nucleic acid encoding these may be mentioned.

  The agent of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that modulates the expression or activity of the constituent factor of PP2A. Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Sodium lauryl sulfate lubricant, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder, Fragrance, Sodium benzoate Preservatives such as lithium, sodium hydrogen sulfite, methyl paraben, propyl paraben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Preparations suitable for oral administration include solutions in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, capsules, sachets or tablets containing an effective amount of the substance as solids or granules. A suspension in which an effective amount of a substance is suspended in a suitable dispersion medium, an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified, or a powder, a granule or the like.

  Formulations suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local infusion, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  The dosage of the agent of the present invention includes the activity and type of the active ingredient, the mode of administration (eg, oral and parenteral), the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age Usually, it is about 0.001 mg to about 5.0 g as an active ingredient amount per day for an adult, although it may not be unconditionally different depending on etc. The agent of the present invention can be used in the same manner as the aforementioned medicament of the present invention.

  The agent of the present invention is useful, for example, as a medicine. When the agent of the present invention is used as a medicine, it can be used, for example, for prevention or treatment of diseases caused by abnormal cell proliferation. Diseases caused by abnormal cell proliferation that can be prevented or treated by regulating the transcriptional activity of p53 include diseases associated with increased cell proliferation (eg, epithelial cancer, non-epithelial cancer, cancer in hematopoietic tissues (eg, leukemia) )).

  The present invention also provides a method for regulating the transcriptional activity of p53, using a substance that regulates the expression or activity of a constituent factor of PP2A. The modulating method of the present invention can be performed in vivo or in vitro. When performed in vitro, the transcriptional activity of p53 can be regulated, for example, by treating cells expressing p53 with a substance that modulates the expression or activity of the PP2A component. Regulation of the transcriptional activity of p53 can be confirmed by an index of the transcriptional activity of p53 (eg, expression of a target gene of p53).

  The present invention provides a screening method for a substance capable of regulating the transcriptional activity of p53. The screening method of the present invention may comprise evaluating whether a test substance can modulate the expression or activity of a constituent factor of PP2A.

  The test substance to be subjected to the screening method may be any compound, for example, nucleic acid (eg, nucleoside, oligonucleotide, polynucleotide), carbohydrate (eg, monosaccharide, disaccharide, oligosaccharide, polysaccharide), Made using lipids (eg, saturated or unsaturated linear, branched and / or cyclic fatty acids), amino acids, proteins (eg, oligopeptides, polypeptides), small organic compounds, combinatorial chemistry techniques Compound libraries, random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components (eg, components derived from microorganisms, animals and plants, marine organisms, etc.) can be mentioned.

  The screening method of the present invention can be carried out in any form as long as it can be evaluated whether or not a test substance can regulate the expression or activity of a constituent factor of PP2A. For example, in the screening method of the present invention, the expression or activity of a constituent factor of PP2A can be evaluated using cells or a reconstitution system. In addition, when a cell is used in the screening method of the present invention, the cell may be a cell derived from the mammal described above.

More specifically, a screening method for evaluating the expression of a constituent factor of PP2A using cells may include, for example, the following steps (a) to (d):
(A) contacting the test substance with a cell capable of analyzing the expression of a constituent factor of PP2A;
(B) a step of measuring the expression level of a constituent factor of PP2A in a cell contacted with a test substance;
(C) comparing the expression level with the expression level of a constituent factor of PP2A in a control cell not contacted with a test substance;
(D) A step of selecting a test substance that regulates the expression level of the constituent factor of PP2A based on the comparison result of (c).

  A cell capable of analyzing the expression of a constituent factor of PP2A refers to a cell capable of directly or indirectly evaluating the expression level of a product (eg, transcription product, translation product) of a constituent factor of PP2A. A cell capable of directly evaluating the expression level of the PP2A component product may be a PP2A component expression cell, while a cell capable of indirectly evaluating the PP2A component expression level is , A cell that allows a reporter assay for the transcriptional regulatory region of the PP2A component. The cell capable of analyzing the expression of the constituent factor of PP2A can be the animal-derived cell described above.

  The expression cell of the constituent factor of PP2A is not particularly limited as long as it can potentially express the constituent factor of PP2A. Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used.

  A cell that enables a reporter assay for the transcriptional regulatory region of a PP2A component is a cell that includes a transcriptional regulatory region of a PP2A component and a reporter gene operably linked to the region. The transcriptional regulatory region of PP2A, a reporter gene, can be inserted into an expression vector. The transcriptional regulatory region of the PP2A component is not particularly limited as long as it can control the expression of the PP2A component. For example, the region from the transcription start point of the PP2A component to about 2 kbp upstream, or the region. And a region having one or more bases deleted, substituted, or added, and having the ability to control transcription of a constituent factor of PP2A. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, a GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

  The transcriptional regulatory region of the PP2A component, and the cell into which the reporter gene operably linked to the region is introduced, as long as the transcriptional regulatory activity of the PP2A component can be evaluated, that is, the expression level of the reporter gene is quantified. As long as the analysis is possible, there is no particular limitation. However, since it expresses a physiological transcriptional regulatory factor for PP2A component and is considered to be more suitable for evaluation of expression regulation of PP2A component, expression of PP2A component is introduced as the introduced cell. Cells are preferred.

  The medium in which the test substance is contacted with cells capable of analyzing the expression of the constituent factors of PP2A is appropriately selected according to the type of cells used, and includes, for example, about 5 to 20% fetal bovine serum. Minimum essential medium (MEM), Dulbecco's modified minimum essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the type of cells used, etc. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours.

In addition, a screening method for evaluating the activity of a PP2A component using a cell capable of analyzing the activity of the PP2A component can include, for example, the following steps (a) to (d):
(A) contacting the test substance with a cell capable of analyzing the activity of a constituent factor of PP2A;
(B) measuring the activity of a constituent factor of PP2A in a cell contacted with a test substance;
(C) comparing the activity with the activity of a constituent factor of PP2A in a control cell not contacted with a test substance;
(D) A step of selecting a test substance that regulates the activity of a constituent factor of PP2A based on the comparison result of (c) above.

  A cell capable of analyzing the activity of a constituent factor of PP2A refers to a cell capable of directly or indirectly evaluating the activity of a constituent factor of PP2A (eg, phosphatase activity). Such cells may be cells that express proteins such as PP2A and its substrate proteins (eg, Chk2, p53), GAK. For example, such a cell can be a cell transformed with an expression vector of one or more proteins selected from a constituent factor of PP2A (eg, PP2A B′α) and its substrate protein, and a protein such as GAK. A cell that can directly evaluate the activity of the constituent factor of PP2A can be a cell expressing PP2A and its substrate protein, while a cell that can indirectly evaluate the activity of the constituent factor of PP2A is PP2A B It can be a cell expressing 'α and GAK. This is because, by evaluating whether or not a test substance can regulate the kinase activity of GAK using PP2A B′α as a substrate, a substance that can indirectly regulate the activity of PP2A B′α and thus the activity of PP2A This is because screening is possible.

Further, the screening method for evaluating the activity of the constituent factor of PP2A in the reconstitution system may include, for example, the following steps (a) to (c):
(A) a step of measuring PP2A phosphatase activity or GAK kinase activity in the presence of a test substance;
(B) comparing the activity with the phosphatase activity of PP2A or the kinase activity of GAK in the absence of the test substance;
(C) A step of selecting a test substance that modulates the phosphatase activity of PP2A or the kinase activity of GAK based on the comparison result of (b) above.

The phosphatase activity can be measured by a method known per se as described above. The kinase activity can be measured by an in vitro kinase assay known per se (for example, see Yabuta, N. et al., Genes Cells, 8 (5), 413-421, 2003). In the above assay, necessary cofactors (eg, ATP or magnesium chloride optionally labeled with ³² P) and the like are appropriately supplemented when measuring the kinase activity.

  The present invention also provides a method for measuring a mutation in PP2A B'α. The detection method of the present invention may comprise detecting a mutation in a nucleotide moiety encoding threonine that is phosphorylated by GAK in PP2A B′α. Detection of a mutation in the nucleotide moiety encoding threonine that is phosphorylated by GAK in PP2A B′α can be performed by a method known per se. For example, such methods include DNA sequencing and methods utilizing primers and nucleic acid probes. PP2A B′α may be derived from the transformant of the present invention or contained in a sample derived from a mammal such as a human. The detection method of the present invention may be useful for detection of the mutant in an assay using the mutant of the present invention. The detection method of the present invention also enables detection of such polymorphism when a polymorphism corresponding to the mutant of the present invention exists in nature, for example, a disease associated with PP2A (eg, cell proliferation). This is useful because it makes it possible to evaluate the risk of developing an abnormality-related disease.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Materials and methods Cell culture Human cells were purchased from the American Type Culture Collection. HEF is a gift from Dr. M. Yutsudo. HeLa cells were provided by the Japanese Cancer Research Resources Bank (JCRB). Cells were maintained at 35 ° C. in 5% CO ₂ in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% calf serum (Hyclone Laboratory, UT), penicillin (100 U / ml), streptomycin (100 μg / ml). .

Antibody Preparation Anti-GAK monoclonal antibodies were generated by immunizing mice with rat GST-GAK fusion protein according to standard methods. Monoclonal antibodies that recognize GST were excluded during the screening procedure. Polyclonal antibodies against rat GAK or human cyclin G1 were raised by standard methods after immunizing New Zealand white rabbits with rat GST-GAK or human GST-cyclin G1. The antibody recognizing the GST moiety was removed by affinity chromatography using a GST binding resin. Anti-PP2A B′α polyclonal antibodies that do not distinguish B′α1-α3 subtypes were generated as previously described (Ito et al., 20000). Other antibodies were purchased from the following companies: Myc (Oncogene Science, Unionale, NY), Actin and GFP (MBL, Nagoya, Japan), p53 (DO-1) (Santa Crutz Biotechnology, Santa Cruz, CA) p53P-S9, p53P-S15, p53P-S20, p53P-S46, p53P-S392, Chk2P-T68 (Cell Signaling Technology Inc., Danvers, MA), CHC, p21 (BD bioscience, SanJ, CA, SanJ) Sigma-Aldrich Co., Milwaukee, WI), GAPDH (Fitzgerald Industries International) Inc., Concord, MA), SV40 T antigen (Ab-1) (Calbiochem, La Jolla, CA) and ATM (Abcam, Cambridge, UK).

Fluorescent immunostaining Cells (during synchronization or log phase) were cultured on coverslips soaked in culture dishes (6 cm diameter). They were fixed in succession for 10 minutes at 20 ° C. with 3.7% formamide, 0.5% Triton X-100 and 0.05% Tween 20 in PBS. After removal of the medium, the cells on the coverslip were brought to room temperature with TBST buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20) supplemented with 2% bovine serum albumin (BSA; Sigma). Rinse 3 times (2 ml for 5 minutes each). The microscope coverslip was lifted and layered cell side down against 100 μl TBST spotted on parafilm containing the relevant antibody. After 1 hour incubation at room temperature, the coverslips were rinsed with TBST / BSA as described above in the culture dish with the cell side up. Immunostaining was visualized using FITC- or Texas Red-crosslinked anti-rabbit Ig (donkey-derived; Amersham) or Texas Red-crosslinked anti-mouse Ig (sheep-derived; Amersham) as the second probe. These secondary antibodies were incubated with cells attached to the coverslips for 1 hour and rinsed three times as described above. Pictures were taken and images were recorded with an inverted laser scanning microscope (Zeiss LSM410 or LSM510).

Cell Irradiation and Transfection Cells were subjected to 10 Gy dose gamma irradiation using a ¹³⁷ Cs source at a rate of 1.142 Gy / min in a Gammacell 40 Exactor Research Irradiator (MDS Nordion, Ontario, Canada). UV irradiation was performed by irradiation with a germinal lamp at a rate of 0.22 J / m ² / s on cells with culture medium and cells with the plate cover removed.

Immunoprecipitation and Western blot analysis Immunoprecipitation was performed essentially as described (Kanaoka et al., 1997). Briefly, cells were harvested and lysis buffer (50 mM Tris-HCl [pH 7] supplemented with protease inhibitors (2 μg / ml aprotinin, 2 μg / ml leupeptin, 1 μg / ml pepstatin A, 50 μg / ml PMSF, 1 mM EGTA). 5], 250 mM NaCl, 0.1% Nonidet P-40). After clarification of the extract by centrifugation at 10,000 xg for 5 minutes, an aliquot of the supernatant was immunoprecipitated by the use of protein A-Sepharose only. Subsequently, the clarified lysate was immunoprecipitated with the relevant antibody. An equal volume of fresh or immunoprecipitated cell extract was then absorbed into protein A-Sepharose, pelleted and subjected to 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). For Western blotting, total cellular proteins and immunoprecipitates degraded on the gel were transferred to nitrocellulose filters and probed with related antibodies. Immunoreactive protein bands were visualized using Renaisense ^™ chemiluminescent reagent (Dupont NEN, Boston, Mass.).

Preparation of GFP fusion construct To create an in-frame junction of a GFP (green fluorescent protein) fusion vector, a synthetic linker is fused to the cDNA insert in-frame via the AscI-NotI site. Was inserted into the pEGFP-C1 vector (Clontech, Palo Alto, USA). In order to obtain a cDNA insert of human GAK carrying an in-frame AscI-NotI site, a set of oligonucleotides around the start codon (+ AscI site) and the stop codon (+ NotI site) was synthesized and PCR using the relevant cDNA as a substrate Used as a primer for. The identity of each gene is confirmed by DNA sequencing of four independent clones, plasmid DNA containing no mismatched DNA sequence is selected, cut with AscI and NotI, and the resulting cDNA insert is incorporated into a GFP vector. It is. A similar strategy was applied to make other plasmid constructs. The GFP-GAK plasmid was transfected into HeLa cells using TransIT ^™ polyamine transfection reagent (Pan Vera Corporation, Madison) according to the manufacturer's protocol, and the expressed GFP fusion protein was analyzed using a Zeiss LSM510 confocal light microscope. And visualized.

Pull-down assay GST-PP2A protein It was expressed in E. coli and purified on glutathione sepharose 4B. GLAG-GAK is expressed by using the TNT expression system, dissolved in lysis buffer (Promega, Madison, WI), mixed on GST-PP2A protein and glutathione sepharose 4B, and after thorough washing Western blot analysis was performed. FLAG-GAK expressed in the TNT solution was used as a positive control, and GST alone was used as a negative control.

Example 1: PP2A B'α1 preferentially associates with GAK in the nucleus Since GAK associates with B'α1 in vivo (data not shown), we then selected which of the B'α subunits We examined whether subtypes would preferentially meet with GAK. For this purpose, we transformed 293T cells with plasmids capable of expressing N′-terminal Myc-tagged B′α1, B′α2 or B′α3 with FLAG-tagged GAK. In this experiment and the following experiment, GAK was derived from rat (when kinase activity was measured) or human (other than measurement of kinase activity), and PP2A B′α was derived from mouse. We then collected cell extracts, immunoprecipitated with anti-Myc antibody, and performed Western blot analysis probed with anti-FLAG antibody. We have found that GAK associates most strongly with B′α1, then weakly with B′α2, and very slightly with B′α3 (FIG. 1A). Reverse immunoprecipitation / Western blot analysis confirmed the most preferential association of B′α1 and GAK compared to B′α2 and B′α3 (FIG. 1B).
We then examined the subcellular co-localization of PP2A B'α with GAK by immunostaining and about 25% of the signal co-localized in the nucleus, although the cytoplasmic signal showed a slight merge signal. Localization was found (FIG. 2C). Since B′α1 and B′α2 but not B′α3 can localize in the nucleus, and GAK associates more preferentially with B′α1 than B′α2, this result indicates that GAK in the nucleus Indicates that B′α1 functions.
We then determined which part of GAK associates with B′α1. For this purpose, we divided GAK into three parts and performed similar immunoprecipitation / Western analysis and found that PP2A B′α1 associates with GAK in both the kinase and non-kinase domains (FIG. 1C). ). The association of the GAK fragment with PP2A B′α1 is summarized in the right panel of FIG. 2A.

Example 2: GAK phosphorylates PP2A at Thy104 We then examined which of these association partners of GAK (eg, p53, CHC or PP2A B'α) is phosphorylated by GAK . We have found that only B′α is a target for phosphorylation of GAK (FIG. 3A). In order to accurately determine the phosphorylation site, we divided the B′α cDNA into four fragments, Fragmented B′α protein was expressed in E. coli to yield an affinity purified GST fusion protein to be used as a substrate (FIG. 3B). We found that only fragment α1-1 shows a strong band (FIG. 3C, right panel, lane 2) similar to full length B′α3 (FIG. 3C, right panel, lane 8), which is a positive control. This indicates that there is a single phosphorylation site in the α1-1 fragment. Of the serine and threonine residues contained in this region, we selected four candidates as GAK targets (FIG. 3D, shown as bold in the upper panel). Because they possess a conserved sequence with the GAK target residue we recently determined to be directly phosphorylated by GAK in vitro (our unpublished results). In fact, we have mutated proteins in which the indicated serine or threonine is replaced one by one with alanine. Four plasmid constructs that can be expressed in E. coli are prepared (FIG. 3D, shown as 1-, 2-, 3-, or 4-mutations in the lower panel), these proteins are affinity purified, and affinity purified GST-GAK is obtained. Using them for in vitro kinase assays, we found that only one construct (α1-1-4 mutation) showed the absence of ³² P incorporation (FIG. 3E). This indicates that threonine 104 (T104) is a true phosphorylation target of GAK. We confirmed this by preparing α1-T104A or α3-T104A mutant proteins that were not phosphorylated even in the presence of GST-GAK as opposed to WT protein (FIG. 3F).
In order to gain insight into the physiological importance of this phosphorylation, we prepared rabbit polyclonal antibodies against phospho-T104B′α. The affinity purified antibody recognized phospho T104B′α (FIG. 3G). To confirm the quality of this antibody, we performed an in vitro kinase assay using α1-1-3 mutant or α1-1-4 mutant protein either in the presence or absence of GAK. In fact, we were able to confirm that only the α1-1-3 mutant protein incorporated ³² P only when GAK was present in the reaction mixture (FIG. 3H). This result indicates that this antibody recognized phosphoThy104B′α.

Example 3: GAK enhances phosphatase activity of PP2A B 'by phosphorylation of T104 in vivo. Next, we developed a FLAG-tagged PP2A mutant (F-T104A) in which T104 was replaced with alanine and Myc of GAK. We investigated whether phospho-phosphorylation of PP2A B 'at T104 caused any biological effect in vivo by transfection of a tagged kinase dead mutant (M-kd). As a control, only the vector (MV), GAK (M-K), and M-kd were separately transfected into 293T cells. First, we have the amount of Chk2 (its phosphorylated tyrosine 68 is the dephosphorylation target of PP2A and causes Chk2 degradation) (Dozier C et al., Biol Cell. 2004, 96 (7): 509-17) I investigated. In fact, we found that cell extracts with exogenously expressed M-kd and FT104A show an increase in the amount of Chk2 protein (FIG. 4A). This suggests that Chk2 is stabilized due to reduced dephosphorylation due to the dominant negative effect of the exogenously expressed PP2A mutant (F-T104A). The marked increase in phosphorylated form of Chk2-T68, as revealed by related anti-phospho antibodies, strongly supports this interpretation (arrowhead in FIG. 3A). Overexpression of FT104A alone did not show such an effect (data not shown). This is probably because endogenous GAK activates endogenous PP2A; co-expression of a kinased mutant of GAK (M-kd) was necessary to observe this effect.
The above results suggest that this simple T104A assay is useful for finding new dephosphorylated targets of PP2A. Indeed, when we tested p53 commercial anti-phospho antibodies, we found that S15 and S20 were candidates for PP2A dephosphorylation targets, and that the enhancement was more pronounced than the increased amount of p53 protein itself I found. On the other hand, the band intensities of S9, S46 and S392 did not change significantly. This suggests that they are not targets. Interestingly, CHC also appears to be stabilized; in this case, overexpression of M-kd alone had already enhanced the stability of CHC. Considering that GAK did not directly phosphorylate CHC (FIG. 3A), this result is not a dephosphorylation by PP2A but a phosphorylation of GAK targets such as mu2 (Korolchuk and Banting, Traffic. 2002 Jun 3 (6): 428-39) suggests that this phenomenon may be important.

Example 4: PP2A B'α plays a role in the association of p53 to SV40 large T antigen. P53 of 293T cells is a direct direct of large T antigen (LTTag), the major early gene product encoded by SV40. Association is known to inactivate in the DNA binding domain of p53 (Lilysstrom et al., Genes Dev. 2006 Sep 1; 20 (17): 2373-82). We speculated that the association between p53 and LTag may be affected. This is because the dephosphorylation of S53 and S20 of p53 seems to be prevented by ectopic co-expression of M-kd and FT104A (FIG. 4B). Therefore, we investigated whether ectopic expression of M-kd and FT104A alters p53 stability during DNA damage induced by gamma irradiation of 293T cells. As expected, ectopic co-expression of MV, MK and M-kd did not cause any change in the amount of p53 (FIG. 5A). However, we found p53 stabilization; the amount of p53 increased in the sample 3-12 hours after irradiation and then returned to the initial amount 20 hours after irradiation (FIG. 5A, lower panel). ). This result suggests that the interaction between p53 and LTag was affected by the expression of M-kd and FT104A.
Indeed, when we immunoprecipitated cell extracts with anti-p53 antibody and probed Western blots with anti-LTag antibody, we found that their association was lost by co-expression of M-kd and FT104A. (FIG. 5B). This result suggests that dephosphorylation of p53 by PP2A (possibly on S15 or S20) may be important for the association between p53 and PP2A, as summarized schematically in FIG. 5C. Note that the expression of M-kd alone reduced their association to some extent; this indicates that there is an unknown phosphorylated target of GAK that also affects the interaction between p53 and LTag. To suggest.

Example 5: Overexpression of T104A activates p53 Recent reports suggest that CHC binds to p53 and regulates p53-mediated transcription (Enari M et al., Genes Dev. 2006 May 1; 20 (9): 1087-99). To examine whether GAK is involved in this regulation, we performed a T104A assay using RT-PCR in 293T cells and analyzed the transcription of p53 target genes such as p21 and Puma. As shown in FIG. 6A, we found that p21 and Puma mRNA levels were enhanced by ectopic expression of M-kd and FT104A; this result indicates that p53 function as a transcription factor is active. Indicates that This result was consistent with the disappearance of the p53 / LTTag interaction that could result in p53 activation (FIG. 6B). This result suggests that the kinase activity of GAK is involved in the regulation of p21 transcription.

In summary, the present inventors have shown the following.
1. GAK associates with PP2A (Figure 1)
2. GAK associates with nuclear proteins (cyclin G1, p53, CHC and PP2A B ′) (FIG. 2).
3. GAK phosphorylates PP2A B ′ with threonine 104 (T104) (FIG. 3).
4). Ectopic expression of the T104 variant of PP2A B ′ affects the phosphorylation of factors in the p53 pathway (FIG. 4).
5. Transfection of PP2A B ′ with T104A mutant 293T cells leads to dissociation of p53 from the SV40 large T antigen resulting in reactivation of p53-mediated transcription (FIG. 5).
6). Transcription of the p53 target gene is induced by ectopic expression of the T104A variant of PP2A B ′ (FIG. 6).

GAK preferentially coimmunized with PP2A B'α1 and PP2A B'α3, Myc-tagged GAK, FLAG-tagged PP2A B'α1 and FLAG-tagged PP2A B'α3 over PP2A B'α2 in 293T cells It is a figure which shows settling. Immunoprecipitation (IP) using anti-Myc antibody and anti-FLAG antibody was subjected to Western blot analysis. Abbreviations are used as follows. 3FLAG-GAK (F-K), 6Myc-GAK (M-K), 6Myc-PP2A B′α1 (M-α1), 6Myc-PP2A B′2 (M-α2), 6Myc-PP2A B′α3 (M -Α3), 3FLAG-PP2A B′α1 (F-α1), 3FLAG-PP2A B′α2 (F-α2) and 3FLAG-PP2A B′α3 (F-α3). GST-PP2A B′α1-1, GST-PP2A B′α1-2, GST-PP2A B′α1-3, GST-PP2A B′α1-4, GST-PP2A B′α2-4 and GST-PP2A B ′ α3-4. FIG. 3 shows that PP2A B′α1 and PP2A B′α3 preferentially co-immunoprecipitate with GAK over PP2A B′α2. FLAG-tagged GAK and Myc-tagged PP2A B 'isoforms were transfected into 293T cells. Immunoprecipitation with anti-Myc and anti-FLAG antibodies for Western blotting. FIG. 2 shows that PP2A B′α1 associates in vivo with both the kinase and non-kinase domains of GAK. FLAG-tagged GAK and Myc-tagged PP2A B'α3 were transfected into 293T cells. Immunoprecipitation using anti-Myc antibody was subjected to Western blot analysis probed with anti-Myc or anti-FLAG antibody. FIG. 4 shows a schematic diagram of fragmented GAK (left panel) and an outline of their association with PP2A B ′ (right panel). − Means no association, +/− means weak association, and + means association. FIG. 4 shows a schematic diagram of the subtypes of PP2A B ′ (left panel) and an outline of their association with GAK (right panel). Two or three + indicates a stronger association. It is a figure which shows the immuno-staining (left three panels) of TIG1 cell by anti- GAK (9-13) and anti-PP2A B 'antibody. A bar graph with standard deviation (n = 10) shows the percentage of yellow signal in the merged image (rightmost panel). FIG. 4 shows that GAK phosphorylates GST-PP2A B′α1, but does not phosphorylate CHC (GST-CHC1 to GST-CHC5), GST-p53 N-terminal and GST-p53 C-terminal. The CHC1-CHC5 fragment, PP2A B′α1, p53 N-terminal and p53 C-terminal fragments were used as substrates for the kinase assay. Affinity purified TIGA1 (Yabuta N, et al., Nucleic Acids Res. 2006; 34 (17): 4878-92) was used as a negative control. It is a schematic diagram of the PP2A fragment used in this study. GST-α1-1 (1-121a.a.), GST-α1-2 (122-210a.a.), GST-α1-3 (211-279a.a.), GST-α1-4 (280-) 515a.a.), GST-α2-4 (280-476a.a.) And GST-α3-4 (280-443a.a.). FIG. 3 shows that GAK phosphorylates PP2A B′α1-1 fragment. GST fusion α1-1, GST fusion α1-2, GST fusion α1-3, GST fusion α1-4, GST fusion α2-4, GST fusion α3-4 and GST fusion α3 were used as substrates in this assay. GST-NPM was used as a negative control. It is a schematic diagram of PP2A B'α1 mutant fragment used in this study. There are 11 serine / threonines in the α1-1 fragment. Four types of fragments were prepared (serine or threonine was substituted with alanine); GST-α1-1 4-mutant, GST-α1-1 3-mutant, GST-α1-1 2-mutant, GST- α1-1 1-mutant. The five amino acid sequences in FIG. 3D (SEQ ID NOs: 5-9). FIG. 4 shows that GAK phosphorylates T104 of PP2A B ′ in vitro. The GST-fused B'α1-1 fragment carrying the alanine mutation at the residue shown in FIG. 3D was used as a substrate with the wild-type α1-1 fragment and the intact wild-type B′α3 protein in the kinase assay. Only the α1-1-4 fragment was not phosphorylated. FIG. 4 shows that GAK phosphorylates T104 of PP2A B ′ in vitro. GST fusion intact PP2A B'α1 and B'α3 (with or without T104A mutation each) were used as substrates for kinase assays. B′α1 and B′α3 are indicated by arrows or arrowheads, respectively. FIG. 3 shows that anti-phospho T104 antibody recognizes the phospho T104 peptide of PP2A B ′. Dot blot analysis was performed using phosphopeptides and non-phosphopeptides. Pre-immune serum and anti-phospho T104 antibody were used for this analysis. FIG. 4 shows that GAK phosphorylates T2 of PP2A B′α1. GST-α1-1 4-variant and GST-α1-1 3-variant were used as substrates in this assay. Anti-phospho T104 antibody and anti-GST antibody were used for Western blotting. FIG. 6 shows a Western blot for Chk2 and phospho T68. Chk2 is known to be a dephosphorylated target protein of PP2A. 9Myc vector (MV), Myc-tagged GAK (M-K), Myc-tagged GAK kinase dead (M-Kd) or M-kd + PP2A B'α1 FLAG-tagged T104A variant (M-kd + F-T104A) Were transfected separately into 293T cells. The whole cell lysate was separated by SDS-PAGE (the same applies to FIG. 4B). It is a figure which shows the Western blot analysis about a clathrin heavy chain (CHC), p53, p21, p53 phospho-Ser9, p53 phospho-Ser15, p53 phospho-Ser20, and p53 phospho-Ser392. An asterisk indicates a non-specific band. It is a figure which shows the Western blot analysis by the anti- T antigen with respect to the immunoprecipitate of an anti- p53 antibody, or an anti- p53 antibody. Immunoprecipitates for preimmune serum (IgG) were also analyzed as controls. TE (total extract) was also separated by SDS-PAGE as a control. 9Myc vector (MV), Myc-tagged GAK (M-K), Myc-tagged GAK kinase dead (M-Kd) or M-kd + PP2A B'α1 FLAG-tagged T104A variant (M-kd + F-T104A) Were transfected separately into 293T cells. An asterisk indicates a non-specific band. It is a figure which shows the Western blot analysis about p53 in 293T cell irradiated with the gamma ray of 10 gray (Gy). Cells were irradiated and lysed after each time point (3, 6, 12 and 20 hours). Myc-tagged GAK (M-K), Myc-tagged GAK kinase dead (M-kd), and FLAG-tagged PP2A B'α1T104A mutant were transfected into 293T cells. Whole cell lysates were separated by SDS-PAGE. It is a schematic diagram explaining the mode which T104A expression induces dissociation from SV40 large T antigen of p53. It is a figure which shows RT-PCR about p21 and a PUMA gene. 9Myc vector (MV), Myc-tagged GAK (M-K), Myc-tagged GAK kinase dead (M-Kd) or M-kd + PP2A B'α1 FLAG-tagged T104A variant (M-kd + F-T104A) Were transfected separately into 293T cells. Total RNA extracted from 293T cells was subjected to RT-PCR using appropriate primer sets for p21 and PUMA cDNA. An asterisk indicates a non-specific band. It is a figure which shows the action hypothesis of this research.

Claims (17)

  A PP2A B'α variant in which the threonine phosphorylated by GAK is substituted or deleted by another amino acid, or a fragment thereof containing the substituted or deleted portion.   The variant or fragment thereof according to claim 1, wherein PP2A B'α is PP2A B'α1.   The variant or fragment thereof according to claim 2, wherein PP2A B'α1 is derived from a mouse and the threonine is threonine at position 104.   The variant or fragment thereof according to claim 3, wherein PP2A B'α1 consists of the amino acid sequence represented by SEQ ID NO: 2 or 4.   The mutant or fragment thereof according to any one of claims 1 to 4, wherein the other amino acid is alanine.   A polynucleotide encoding the PP2A B'α variant or fragment thereof according to any one of claims 1 to 5.   An expression vector comprising the polynucleotide according to claim 6.   A transformant into which the expression vector according to claim 7 has been introduced.   In a phosphatase reaction system containing PP2A and a phosphorylated substance, a substance having a reduced degree of dephosphorylation by the presence of the PP2A B′α mutant or fragment thereof according to any one of claims 1 to 5 is used. A screening method for a dephosphorylation target of PP2A, comprising identifying as a dephosphorylation target.   Screening of PP2A dephosphorylation target comprising the PP2A B′α mutant or fragment thereof according to any one of claims 1 to 5, the expression vector according to claim 7, or the transformant according to claim 8. Reagent or kit.   A screening method for a substance capable of regulating the transcriptional activity of p53, comprising evaluating whether a test substance can regulate the expression or activity of a constituent factor of PP2A.   Whether or not the test substance can modulate the activity of the constituent factor of PP2A is determined based on the kinase activity of GAK using PP2A B′α as a substrate or the phosphatase activity of PP2A B′α using p53 as a substrate. The screening method according to claim 11, wherein the screening method is carried out by evaluating whether it can be adjusted.   The screening method according to claim 11 or 12, wherein the test substance is a screening method for a substance capable of promoting the transcriptional activity of p53.   The screening method according to any one of claims 11 to 13, wherein the substance capable of regulating the transcriptional activity of p53 is a substance capable of preventing or treating a disease caused by abnormal cell proliferation.   The screening method according to claim 13, wherein the substance capable of promoting the transcriptional activity of p53 is a drug against cancer.   A regulator of p53 transcriptional activity comprising a substance capable of regulating the expression or activity of a constituent factor of PP2A.   A method for measuring a mutation in PP2A B'α, comprising detecting a mutation in a nucleotide moiety encoding threonine phosphorylated by GAK in PP2A B'α.