JPWO2007060899A1 - Use of actin-binding protein for cell motility-related diseases - Google Patents

Abstract

An object of the present invention is to identify a protein involved in the molecular mechanism of Akt and cell movement, elucidate its function, and use it. Therefore, in the present invention, any one of cell motility, cell migration and angiogenesis is activated using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof. A compound or a salt thereof that is converted or inhibited is screened.

Description

  The present invention relates to the use of an actin-binding protein, and more particularly to the use of Girdin (Akt Phosphorylation Enhancer), a novel substrate and actin-binding protein of serine / threonine kinase Akt, for diseases associated with the protein.

  Control mechanisms of cell movement are closely related to morphogenesis in development, wound healing and angiogenesis, as well as pathological conditions such as cancer cell infiltration, arteriosclerosis, and immune diseases. Many regulatory molecules for cell movement have been identified, including low molecular weight G proteins (Ridley A, al., Science (2003) 302, p1702-1709). Akt (also known as protein kinase B: PKB), a serine / threonine kinase, is known as an important factor for controlling cell survival and proliferation. It has been shown in many species such as fungi (Higuchi M, al., Curr. Biol (2001) 11, p1958-1962, Merlot S, al., J. Cell Sci (2003) 116, p3471-3478). Malignant tumors with marked expression of Akt tend to be highly invasive.

  However, nothing is known about the molecular mechanism by which Akt promotes cell motility. An object of the present invention is to identify a protein involved in the molecular mechanism of Akt and cell movement, elucidate its function, and use it.

  The present inventors have discovered a novel substrate for Akt using the yeast two-hybrid method, and this substrate is an actin-binding protein, which is transferred to the leading edge (leading edge) of a cell that moves when phosphorylated by Akt. On the other hand, the localization of this actin-binding protein was found to be altered in cells when the actin-binding protein mutant was expressed in the cell, resulting in impaired cell movement depending on growth factor stimulation. Was found to be essential for actin cytoskeleton integration and cell motility, completing the present invention. That is, according to the knowledge of the present inventors, the following means are provided.

(1) Any one of cell motility, cell migration and angiogenesis characterized by using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof A screening method for a compound or a salt thereof that activates or inhibits.
(2) The screening method according to (1), wherein the protein is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
(3) The screening method according to (1), wherein the partial peptide is a C-terminal region (CT1 region and / or CT2 region) of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
(4) In the protein (1) or (2), the serine residue at position 1416 of the amino acid sequence represented by SEQ ID NO: 2 or a serine residue corresponding to the serine residue is phosphorylated The screening method described.
(5) Promotion or inhibition of binding activity between serine / threonine kinase Akt and a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1 or a part thereof, as an index (1 The screening method according to any one of (4) to (4).
(6) The screening method according to any one of (1) to (4), wherein promotion or inhibition of phosphorylation of the protein by serine / threonine kinase Akt is used as an index.
(7) The screening method according to any one of (1) to (4), wherein promotion or inhibition of actin filament binding ability is used as an index.
(8) The screening method according to any one of (1) to (4), wherein promotion or inhibition of actin stress fiber forming ability is used as an index.
(9) The screening method according to any one of (1) to (4), wherein promotion or inhibition of actin meshwork formation ability in lapolimedia is used as an index.
(10) The screening method according to any one of (1) to (4), wherein cell membrane binding promotion or inhibition is used as an index.
(11) The screening method according to any one of (1) to (4), wherein the promotion or inhibition of phosphoinositide binding is used as an index.
(12) Any of cell motility, cell migration and angiogenesis, characterized by using a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. A method for screening a compound or a salt thereof that activates or inhibits.
(13) The screening method according to any one of (1) to (12), which is a screening method for a prophylactic / therapeutic agent for a disease associated with any of cell motility, cell migration, and angiogenesis.
(14) The screening method according to any one of (1) to (13), wherein the disease is any one selected from cancer, arteriosclerotic disease, and central and peripheral neuropathy.
(15) A compound containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof that expresses any one of the following reactivities, or a salt thereof: A prophylactic / therapeutic agent for a disease associated with any of cell motility, cell migration and angiogenesis.
a) Inhibitory activity of serine / threonine kinase Akt binding to the protein b) Inhibitory activity of phosphorylation of the protein by serine / threonine kinase Akt c) Inhibitory activity of binding to actin filaments d) Inhibitory activity of actin stress fiber formation e) Inhibitory activity of actin meshwork formation in lapolimedia f) Inhibitory activity of cell membrane binding g) Inhibitory activity of binding to phosphoinositide (16) Same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2 A prophylactic / therapeutic agent for a disease associated with cell motility, cell migration or angiogenesis, which is a compound that suppresses the expression level of a protein containing the same amino acid sequence.
(17) The compound is a polynucleotide having a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2, or a part thereof. (16) The preventive / therapeutic agent described in 1.
(18) The prophylactic / therapeutic agent according to (17), which is RNA that expresses RNA interference.
(19) The prophylactic / therapeutic agent according to (16), which is a DNA encoding the RNA according to (18).
(20) The prophylactic / therapeutic agent according to (16), which is a recombinant vector containing the DNA according to (19).
(21) The prophylactic / therapeutic agent according to any one of (16) to (20), wherein the disease is any one selected from cancer, arteriosclerotic disease, and central and peripheral neuropathy.
(22) A cell in which the expression level of a gene encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 is suppressed.
(23) An animal in which the expression level of a gene encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 is suppressed.
(24) The animal according to (23), wherein the expression level of the gene is suppressed by knockdown.
(25) The animal according to (23) or (24), which is used for research of a disease associated with any of cell motility, cell migration and angiogenesis, and for screening for a prophylactic / therapeutic agent.
(26) Cell motility, cell migration, and blood vessel formation using the reaction of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof with serine / threonine kinase Akt A method for qualitative analysis of a compound that activates or inhibits any of the above.
(27) Cell motility, cell migration, and blood vessel formation using the reaction of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof with serine / threonine kinase Akt A method for quantifying a compound that activates or inhibits any of the above.
(28) An antibody specific for a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof.
(29) A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, wherein the serine residue at position 1416 of the amino acid sequence represented by SEQ ID NO: 2 or the serine residue An antibody specific for a protein in which a serine residue corresponding to a group is phosphorylated or a partial peptide containing the phosphorylated moiety.
(30) A reagent for studying activation or inhibition of cell motility, cell migration and angiogenesis, comprising the antibody according to (28) or (29).
(31) A diagnostic agent for a disease associated with any of cell motility, cell migration and angiogenesis, comprising the antibody according to (28) or (29).
(32) Cell motility utilizing any of the following reactivities for a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 in the disease site cell or a partial peptide thereof, A method for diagnosing a disease associated with either cell migration or angiogenesis.
a) Binding activity to serine / threonine kinase Akt and the protein b) Phosphorylation activity of the protein by serine / threonine kinase Akt c) Binding activity to actin filament d) Actin stress fiber formation activity e) Actin mesh in Lapolymedia Work formation activity f) Cell membrane binding activity g) Binding activity to phosphoinositide

FIG. 1a shows various bait constructs (expression vectors incorporating various genes) used in the yeast two-hybrid assay. Various cDNA fragments of human Akt1 were constructed in the pAS2 vector so as to be fused to the DNA binding domain of the GAL4 transcription factor. PH, pleckstrin homology domain; KD, kinase active domain; RD, regulatory domain. FIG. 1b shows the mode of binding of human Akt1 to clones F-10 and F-12 in the yeast two-hybrid assay. pAS, negative control; pVA3-pTD1, positive control, His, histidine. FIG. 1c shows the primary structure of Girdin and the structure of Girdin predicted by the COILS algorithm. FIG. 1d shows universal expression of Girdin messenger RNA (mRNA) in various human tissues. Clontech multi-tissue northern blot membranes were hybridized with probes in the 3 'region of Girdin cDNA. FIG. 1e shows the results of detection of endogenous Girdin expression in human brain and testis using anti-Girdin polyclonal antibody by Western blotting under reducing conditions. Shown not detected in (control) rabbit IgG. FIG. 1f shows the detection of large protein complexes formed by endogenous Girdin. Western blotting was performed using an anti-Girdin antibody under conditions in which COS7 cells excluding nuclear components were crosslinked with 100 μM BS3 and under non-crosslinked conditions. After cross-linking (arrow), a considerably larger band was detected compared to the case where cross-linking was not performed (arrow head). FIG. 1g shows that Girdin's N-terminal domain (NT) forms multimers. When VOS tag (NT-V5) and myc-tagged NT (NT-myc) were transfected into COS7 cells and immunoprecipitated with anti-myc antibody, NT-V5 co-precipitated with NT-myc. The multiple bands seen in the lower panel may indicate degradation products of NT-myc. The estimated amino acid sequence of Girdin is shown. The amino acid residues at positions a and d in 135 heptad repeat (abcdefg) 135, which are thought to form an α-helical coiled coil structure, are shown in red and green, respectively. The putative binding site to phosphoinositide is shown underlined. Boxes indicate putative Akt phosphorylation sites. Girdin forms oligomers in cells and forms large protein complexes with actin filaments, A is gel filtered with COS7 cell lysate Superose 6PC3.2 / 20 (Amersham), after fractionation, It is the figure which confirmed each fraction by Western blot analysis with the anti-Girdin antibody (upper stage) and the anti-actin antibody (lower stage), and confirmed those elution characteristics. Actin eluted as two peaks, the first one eluted in the void volume, corresponding to a polymer of actin (F-actin). The main elution peak of actin is at a position corresponding to a low molecular weight of less than 67 kDa, suggesting either partially dissociated or monomeric (G-actin). These data indicate that Girdin forms large complexes with various lengths of F-actin. B transfected COS7 cells with either V5 epitope tagged GFP-Girdin N-terminal fragment (NT) (GFP-NT-V5) (top) or myc epitope tagged NT (NT-myc) (bottom). The cell lysate was then treated with Superose 6PC for gel filtration. After fractionation, each fraction was analyzed by Western blot using anti-V5 antibody and anti-myc antibody. The calculated molecular weight of GFT-NT-V5 or NT-myc monomer was 58 kDa or 28 kDa, respectively, and these data suggested that the NT domain formed a dimer. Method: COS7 cells in the dish were washed with cold PBS and 0.5 ml of 25 mM Tris-HCl, pH 8.0, 250 mM, NaCl 5 mM, 1 mM EDTA, 1 mM DTT. The suspension was sonicated and centrifuged at 100,000 g for 60 minutes at 4 ° C. The supernatant was treated with Superose 6PC3.2 / 20 (Amersham). Elution was performed at a flow rate of 40 μl / min. 50 μl fractions were collected. The protein markers used were thyroglobulin (669K), ferritin (440K) and bovine serum albumin (67K). In FIG. 2a, a indicates that the 1416th serine of the Girdin C-terminal domain (CT) is an Akt phosphorylation site. b, CIR7 cells transfected with Girdin CT-V5 wild type (WT) and SA mutants (mutants in which the 1416th serine was replaced with alanine), immunoprecipitated with anti-V5 antibody, and radiolabeled Incubated with active and inactive recombinant Akt in the presence of ATP. It shows that phosphorylated Girdin CT was confirmed by autoradiography (upper panel) and immunoprecipitation product was confirmed by Western blotting (lower panel). In FIG. 2b, a shows various Girdin fragments to which a V5 tag transfected into COS7 cells was added. b shows the result of incubation with active Akt in the presence of radiolabeled ATP after immunoprecipitation. It shows that the immunoprecipitation product was confirmed by Western blotting (left) and phosphorylated Girdin CT was confirmed by autoradiography (right). FIG. 2c shows Western blotting using anti-phosphorylated Girdin antibody (anti-P-Girdin) after Girdin CT WT and SA were transfected into COS7 cells together with various Akt mutants, and after immunoprecipitation (upper panel). The expression of Girdin CT and various Akts shows the results of monitoring by Western blotting (middle and lower panels). FIG. 2d stimulated COS7 cells with epidermal growth factor (EGF) in the presence of DMSO, PD98059, LY294002 and Wortmannin. After immunoprecipitation with anti-Girdin antibody, Western blotting was performed using anti-phosphorylated Girdin antibody and anti-Girdin antibody. Akt activation was detected using an anti-phosphorylated Akt antibody. FIG. 2e shows the induction of Girdin phosphorylation in COS7 transformants of Akt WT and Akt CT and Akt DN. COS7 cells were transfected with various variants of Akt. Endogenous Girdin was immunoprecipitated using anti-Girdin antibody, and then Western blotting was performed using anti-phosphorylated Girdin antibody and anti-Girdin antibody. In FIG. 2f, a shows the effect of Akt knockdown on endogenous Girdin phosphorylation. COS7 cells were transfected with control or Akt siRNA and cultured for 48 hours. The cells were then stimulated with EGF for 30 minutes and Girdin phosphorylation was detected using the method described in (E). b shows suppression of Akt expression by siRNA in COS7 cells. In FIG. 3a, a and b are double-stained with Vero cells before stimulation (a) or after stimulation with EGF (b) with Alexa488-labeled phalloidin (a reagent that binds to actin fibers), anti-Coractin antibody, and anti-Girdin antibody. The result of having performed is shown. Arrowheads indicate lamellipodia (foliate pseudopods: actin-rich structure formed at the tip of cell movement) at the leading edge (tip of moving cells). FIG. 3b shows the intracellular localization of various Girdin fragments. The following shows the results of expressing various fragments of Girdin fused with GFP (green fluorescent dye gene) in Vero cells, fixing, and staining with each probe. The arrows indicate aggregates of the expressed protein. FIG. 3c shows that the Girdin CT2 fragment binds directly to actin filaments in vitro. Purified GST, GST-CT2 and alpha-actinin were incubated with actin fibers prepared in vitro. Subsequently, F-actin fibers are pelleted by ultracentrifugation, and various proteins precipitated together with F-actin are detected by gel CBB staining (Coomassie staining). FIG. 3d shows the binding mode of GST-CT2 and actin fibers. A certain amount of GST-CT2 was mixed with various amounts of F-actin, followed by ultracentrifugation. Unbound and bound F-actin were quantified and bound GST-CT2 was plotted against the concentration of F-actin. Kd values were calculated by Scatchard analysis. In FIG. 3e, a shows a summary of localization and function of various domains of Girdin. b shows the proposed Girdin structure. FIG. 4 a shows Girdin expression suppression Akt by siRNA in Vero cells. Cell extracts of Vero cells transfected with control siRNA and Girdin siRNA were detected using anti-Girdin antibody, anti-Akt antibody, and anti-actin antibody. FIG. 4b. Vero cells were transfected with control siRNA and Girdin siRNA, fixed 72 hours later, and double-stained with Alexa488-labeled phalloidin and anti-Girdin antibody. The arrowhead indicates lamellipodia at the tip of the protrusion. FIG. 4c shows the number of cells forming a plurality of protrusions after transfection with various siRNAs was quantified by staining with anti-Girdin antibody and Alexa488-labeled phalloidin. More than 100 cells were counted in each group. Results are shown as mean ± standard error. In FIG. 4d, Vero cells were transfected with GFP-actin and various siRNAs and cultured for 48 hours. Cells were plated on glass coverslips coated with fibronectin and wounded with a pointed tip on the cell layer to induce cell movement. Acquisition of cell images started 2 hours after the wound of the cell layer, and was performed every 90 seconds for 5-6 hours. The arrow indicates the direction in which the cell moves toward the direction of the wound. FIG. 4e shows the effect of Girdin knockdown on stress fiber formation and lamyolipia of RET tyrosine kinase SK-N-MC cells. In A, SK-N-MC (RET) cells were collected 72 hours after transfection of siRNA. The cell lysate is analyzed by SDS-PAGE, and the results of Western blot analysis using an anti-Girdin antibody and an anti-actin antibody are shown. B, SK-N-MC (RET) cells transfected with siRNA were stimulated with 50 ng / ml GDNF for 60 minutes, fixed and then stained with Alexa-488 phalloidin. Cells in which Girdin was knocked down exhibited marked morphological changes with a large number of processes compared to control cells. The arrow indicates lamellipodia at the leading edge. Girdin knockdown cells form lamellipodia, but are less polar and less extensible than control cells. FIG. 5a shows the results of analysis by an immunoelectron microscope using an anti-Girdin antibody. FIG. 5b shows an enlarged view of the colloidal immune gold signal of the Girdin molecule indicated by a circle. The arrow indicates the immune gold colloid signal. FIG. 5c shows the fine structure of actin filaments in Vero cells transfected with control or Girdin siRNA. FIG. 6a shows Vero cells fixed before stimulation, after stimulation with EGF, or transfected with Girdin siRNA, and double-stained with Alexa488-labeled phalloidin and anti-phosphorylated Girdin antibody. Arrows indicate lamellipodia in which phosphorylated Girdin is accumulated. FIG. 6b shows the result of double staining of moving Vero cells with anti-coractin antibody and anti-phosphorylated Girdin antibody. In FIG. 6c, a represents the assumed inositol phospholipid binding site located upstream of Girdin's Akt phosphorylation site. b, GFP-CT1 or GFP-CT1 ΔPB (excluding the assumed inositol phospholipid binding site) was transfected into Vero cells. The arrow indicates the GFP signal in the cell membrane. FIG. 6d shows GST-CT purification and protein-lipid binding assay. a shows the result of analyzing purified GST-CT or GST-CT ΔPB by Western blotting using an anti-Girdin antibody. b shows phosphorylation of GST-CT by recombinant Akt in vitro. Phosphorylation of GST-CT in the in vitro kinase assay was detected with an anti-phosphorylated Girdin antibody. FIG. 6e shows a protein-lipid binding assay using GST-CT, GST-CT ΔPB and phosphorylated GST-CT. Binding of GST-CT and GST-CT ΔPB to lipid was detected with an anti-Girdin antibody, and binding of phosphorylated GST-CT to lipid was detected with an anti-phosphorylated Girdin antibody. Various lipids spotted on the membrane are shown in (Ed). FIG. 6f shows that phosphorylated GST-CT binds to F-actin. GST-CT was phosphorylated by Akt in vitro and subsequently used in the actin precipitation assay. A certain amount of GST-CT was phosphorylated, mixed with various amounts of F-actin, and ultracentrifuged. Phosphorylated GST-CT bound to F-actin contained in the precipitate fraction was detected by Western blotting using an anti-phosphorylated Girdin antibody. FIG. 6g shows that active Akt and phosphorylated Girdin colocalize with lamellipodia in EGF-stimulated Vero cells. Vero cells in serum-starved state for 24 hours were treated with either untreated A and 50 ng / ml EGF-treated B for 30 minutes, fixed, anti-phosphorylated Girdin monoclonal antibody (green, cell signaling technology) and anti-phosphorylated The result of staining with Girdin antibody (red) is shown. Images were visualized with a confocal microscope. Arrows indicate the colocalization of the leading edge of phosphorylated Akt and phosphorylated Girdin in lamellipodia. Enlarged area in white box. FIG. 6h shows the effect of amino acid substitutions in the CT1 domain on Girdin function. A indicates that a positive charge at the putative phosphoinositide binding (PB) site is required for CT1 domain localization in the cell membrane. Various CT1 mutants, that is, mutants in which one of the positively charged amino acid residues of lysine and arginine is substituted with alanine (PBala, PBalaF, PBalaR) Ser-1416 is substituted with alanine or aspartic acid (SA, SD) was made as shown in a and the localization in these Vero cells was examined (b). The arrowhead indicates the localization of GFP-CT1 in the cell membrane, and the arrow indicates that the mutant is not localized in the cell membrane. B shows the results of the protein-lipid overlay assay. GST-CT wild type (ST) and GSTCT-PBala in which the positively charged amino acid residue of the PB site is substituted with alanine are expressed in BL-21 CodonPlus cells (Stratagene), purified (a), and protein-lipid overlay assay (B, c) shows the results subjected to. C was co-transfected with GFP (0.5 μg), siRNA (20 pmol) and the indicated siRNA resistance construct (siRNAr) (1 μg), incubated for 48 hours and assayed by Boyden chamber method. An asterisk indicates a statistically significant difference (Student t-test * P <0.05) compared to WT. FIG. 6i shows the modulation of all Girdin membrane interactions by EGF in Latrunculin treated cells. A transfected COS7 cells with GFP-full length Girdin wild type (WT) or SA mutants with Ser-1416 replaced by alanine and incubated on glass dishes for 24 hours. Cells incubated for 60 minutes in the presence or absence of EGF (50 ng / ml) were treated with Latrunculin (1 μM, Calbiochem) for 10 minutes and fixed. Girdin WT or Girdin SA overexpressed in COS7 cells were dispersed throughout the cells excluding the nucleus (upper panel). When treated with Latrunculin that destroys actin (middle stage), Girdin WT and SA were detected in the cell membrane as well as the cytoplasm (yellow arrowhead). When cells stimulated with EGF were treated with Latrunculin (bottom), most of Girdin WT dissociated from the cell membrane and accumulated in the cytoplasm in the form of dots (arrows). On the other hand, Girdin SA maintained the interaction with the cell membrane as before even after stimulation with EGF (open arrowhead). B shows the result of COS7 cells transfected with GFP-full length Girdin PBala mutant and subsequently treated with Latrunculin. Little interaction of the mutant with the cell membrane was observed after treatment with Latrunculin even in the absence of EGF. FIG. 7a shows that Girdin is phosphorylated at the tip (leading edge) during cell movement. a. After scratching a monolayer of Vero cells (cells in a monolayer), cells facing the damaged surface were fixed and stained with an anti-phosphorylated Girdin antibody. Arrowheads indicate Girdin phosphorylation signals. b indicates that phosphorylation of Girdin at the leading edge increases in a time-dependent manner. FIG. 7 b shows the results of culturing Vero cells transfected with the GFP gene, various siRNAs and Girdin CT for 48 hours, and using them in the Boyden chamber assay. The lower chamber was subjected to the conditions where EGF was added and not added. An asterisk indicates that there is a significant difference compared to the control. Boxes indicate suppression of Akt expression by siRNA in Vero cells. The results are shown as mean ± standard error. FIG. 7c shows the generation of Girdin siRNA-resistant mutants and the evaluation results using them. a shows the base substitution for preparation of the target sequence of Girdin siRNA and siRNA resistant mutant. b, COS7 cell lysates transfected with the various siRNAs and constructs shown above were analyzed by Western blotting using anti-V5 antibodies (Virtag was added to the C-terminus of various Girdin constructs). (This method can be detected by this method). Fig. 7d shows the results of Boyden chamber assay after 48 hours of transfection of GFP gene and various siRNAs and constructs transfected into Vero cells. Expression of various Girdin mutants was analyzed by Western blotting prior to cell migration assay to confirm that expression levels were similar (results not shown). The results are shown as mean ± standard error. FIG. 7e shows a proposed model in which cell motility is controlled by Girdin phosphorylation. In quiescent cells, Girdin crosslinks actin fibers and anchors cell actin (cortical actin) to the cell membrane. On the other hand, during cell movement, Girdin changes its localization to the leading edge due to phosphorylation by Akt, and bridges short branched actin fibers in lamellipodia. FIG. 7f shows that it is essential for directional migration in the human fibrosarcoma cell line HT-1080 cells. A shows Girdin depletion in HT1080 cells. Total cell lysates from control cells without SiRNA and HT-1080 cells transfected with siRNA were subjected to Western blot analysis using anti-Girdin, anti-Akt, and anti-actin antibodies. B shows that Girdin depletion impairs directional cell migration of HT-1080 cells. HT-1080 cells transfected with siRNA were subjected to Boyden chamber method. Serum (5%) was added to the lower chamber. * P was <0.05 versus control. C shows that cells with impaired cell migration due to Girdin depletion recover by re-adding siRNA-resistant (siRNAr) forms of Girdin. GFP, the indicated siRNA (20 pmol) and various constructs (2.5 μg) were co-transfected into HT-1080 cells and subjected to Boyden chamber method for 48 hours after transfection. SiRNAr-Girdin wild type (WT) completely restored cell mobility, while SA and ΔPB did not. The results shown are representative of two independent experiments. FIG. 7g shows the effect of expression of the Girdin mutant pair on the directional mobility of Vero cells. A shows the observation result of migration of Vero cells expressing Girdin mutants by damage healing assay. Confluent Vero cells on glass dishes coated with fibronectin (10 μg / ml) were transformed into Girdin siRNA and Girdin siRNA resistant (siRNAr) wild type GFP-Girdin (WT) mutants (SA, ΔPB). And Pbala) are co-transfected (a), 48 hours after transfection, the cell monolayer is scratched to initiate cell migration to the damaged part (b), and expresses GFP-Girdin, Were analyzed by low speed imaging (c). B shows low-speed imaging of migration of Vero cells expressing wild type Girdin (WT) and various mutants (SA, ΔPB and Pbala). Images were acquired every 90 seconds for 7 hours after 2 hours from scratch. The white dashed line indicates the damage line and the arrowhead indicates the nucleus of the moving cell. These results are representative images of two independent experiments. FIG. 8a shows the quantification result of the migration ability by treating the HUVEC cells with Girdin siRNA and Boyden chamber method. FIG. 8b shows an image of stained migrated cells. FIG. 9a shows the observation results of the lumen forming ability. FIG. 9b shows the results of Western blot analysis using an anti-Girdin antibody. FIG. 10a shows the restoration of lumen formation. FIG. 10b shows the results of Western blot analysis using an anti-Girdin antibody. FIG. 11 is a diagram showing the results of the Matrigel plug assay, in which the upper row is a control and the lower row is a result of injecting Matrigel containing a knockdown vector. FIG. 12 is a figure which shows the tissue immunostaining result of Girdin in breast cancer and cervical cancer.

  The present invention relates to Girdin, which is a protein (Girdin: referred to as Girdin by the present inventors) and actin-binding protein that is phosphorylated by serine / threonine kinase Akt (hereinafter simply referred to as Akt). A protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 (Girdin amino acid sequence) or a protein containing substantially the same amino acid sequence or a partial peptide thereof (hereinafter referred to as the protein and the part) Peptides are collectively referred to as this protein). Girdin is normally involved in cell movement, cell movement and blood vessel formation in the process of forming body parts from human fertilized eggs. Specifically, tumor cell movement and migration are related to cancer progression, metastasis, and invasion, nerve cell migration is related to neurogenesis, and migration of vascular constituent cells such as vascular endothelial cells is related to angiogenesis. Related. Therefore, by using this protein, it is possible to search and provide a prophylactic / therapeutic agent effective for a disease associated with any of cell motility, cell migration and angiogenesis. In addition, by using this protein, it becomes possible to diagnose these diseases.

  The present invention also relates to a compound or a salt thereof that promotes or inhibits various activities appearing in cell movement or the like in the relationship between the present protein and Akt. Such a compound or a salt thereof can be used as a compound or a salt thereof that promotes or inhibits any of cell motility, cell migration, and angiogenesis. Such a compound of the present invention can activate or inhibit any of cell motility, cell migration, and angiogenesis, and thus is useful, for example, in the treatment and prevention of diseases associated with these cell kinetics. It is also possible to screen for compounds that are useful as preventive / therapeutic agents for diseases related to cell motility, cell migration, angiogenesis, etc. by supplying test compounds to this protein and detecting the effects on this protein It becomes. Furthermore, since an antibody specific for this protein can detect quantitative measurement or localization of this protein in cells, it can be used for the prevention and treatment of diseases associated with cell motility, cell migration and angiogenesis. Diagnosis is also possible. Hereinafter, embodiments of the present invention will be described in detail.

(This protein) This protein can contain the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. It may consist of these amino acid sequences. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 is about 50% or more, preferably about 60% or more, more preferably about 70% or more when represented by SEQ ID NO: 2, more An amino acid sequence having a homology of preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is mentioned. The homology (identity) (%) can be determined using a homology search program (for example, BLAST, FASTA, etc.) commonly used in this field by default. The homology (%) is determined by any algorithm known in the art, for example, Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4:11). It may be determined using the algorithm of -17).

  This protein is one or more in the amino acid sequence represented by SEQ ID NO: 2 (preferably about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) An amino acid sequence in which the same number of amino acids are added, an amino acid sequence in which the same number of amino acids are inserted, an amino acid sequence in which the same number of amino acids are substituted, or the same number of amino acids is deleted, It may have an amino acid sequence modified in a combination of two or more selected from addition, insertion and substitution, or may consist of such an amino acid sequence. Such amino acid modification sites are not particularly limited as long as the activity of the protein is not lost.

  Moreover, it is preferable that the protein having substantially the same amino acid sequence represented by SEQ ID NO: 2 has the same quality of activity as the protein comprising the amino acid sequence represented by SEQ ID NO: 2. Homogeneous activity means that the degree of activity does not matter. Therefore, the degree of activity may be less than that of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or may be about the same or more. Such activity is, for example, the activity of the protein as an Akt substrate, and in particular, has the activity that the serine at position 1416 in the amino acid sequence of SEQ ID NO: 2 or the serine corresponding thereto is phosphorylated by Akt. It is preferable. In addition, as the activity of the same quality, the above phosphorylation activity can be mentioned as preferable, but it is not limited to the above activity, and any other activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 It may be a combination of two or more. These activities will be described in detail later.

  Moreover, this protein includes the partial peptide as long as it has the same quality of activity as the protein represented by SEQ ID NO: 2. Although the region of the partial peptide is not particularly limited, the amino acid at positions 1376 to 1870 (CT1) of the amino acid sequence of SEQ ID NO: 2 which is the C-terminal region CT region of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 Region and / or CT2 region). The CT1 region is a region of the first half of the amino acid sequence of SEQ ID NO: 2, and includes at least the 1389th to 1407th positions or a sequence corresponding to the amino acid sequence and the 1411th to 1416th positions. Preferably, for example, it can have the amino acid sequence from position 1375 to position 1622. The CT2 region is the latter half of the CT region, and can have, for example, the amino acid sequence at positions 1623 to 1870. The partial peptide may have both of these regions, or only one of them. A preferred CT1 region has an Akt phosphorylation site and interacts with the cell membrane, and a preferred CT2 region can bind to actin filaments.

  Examples of such proteins include orthologs or homologs of Girdin in humans and Girdin (the amino acid sequence can be obtained from the NCBI homepage (http://www.ncbi.nlm.nih.gov/, accession number BAE44387). In addition, as a transcriptional variant of Girdin, a protein having the amino acid sequence described in Accession No. NP_060554 on the NCBI homepage (residue 1843) is also mentioned. It is done.

  This protein also includes a protein or partial peptide in a mode in which the serine residue at position 1416 of the amino acid sequence represented by SEQ ID NO: 2 or a serine residue corresponding to the serine residue is phosphorylated. This phosphorylation is due to Akt, and it is inferred that Girdin is concentrated in the leading edge of the cell due to this phosphorylation and is involved in cell movement. In particular, when distinguishing between a non-phosphorylated mode and a phosphorylated mode, these are expressed as a non-phosphorylated main protein and a phosphorylated main protein, respectively.

  Examples of the present protein include the above-mentioned Girdin, which is a naturally-occurring protein consisting of the amino acid sequence represented by SEQ ID NO: 2, but the protein or peptide included in the present protein is not composed of only natural amino acids. In addition, various modifications may be applied as long as the activity of the protein is not lost. Various modifications to proteins and amino acids are well known. For example, for carboxyl groups, carboxylate (—COO—), amide (—CONH 2), or ester (—COOR) can be used. . Here, as R in the ester, for example, a C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, for example, a C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α -C6-12 aryl groups such as naphthyl, for example, C7-14 aralkyl groups such as phenyl-C1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl, pivalo An yloxymethyl group or the like is used. When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, an amino group such as an N-terminal amino acid residue (eg, methionine residue) is protected with a protecting group (eg, a C1-6 acyl group such as C1-6 alkanoyl such as formyl group or acetyl group). It is also possible that the N-terminal glutamine residue produced by cleavage in vivo is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino group, imidazole group, Indole group, guanidino group, etc.) may be protected with an appropriate protecting group (for example, C1-6 acyl group such as C1-6 alkanoyl group such as formyl group, acetyl group, etc.) It may be a so-called glycoprotein.

  In this non-phosphorylated protein, the serine at position 1416 of the amino acid sequence of SEQ ID NO: 1 or the serine corresponding thereto is modified to the extent that phosphorylation by Akt is possible.

  The protein may be in the form of a salt. For example, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  This protein can be obtained by a conventionally known method. For example, since this protein is ubiquitous in mammalian cells, it can also be obtained from these cells or tissues containing these cells by a known protein purification method. Furthermore, it can also be obtained by obtaining a DNA encoding the present protein, preparing a transformant containing the DNA, and culturing it. Proteins can be purified and isolated by combining chromatography such as ammonium sulfate precipitation, ethanol precipitation, acid extraction, ion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, reverse phase chromatography, gel filtration chromatography, etc. . Further, the protein of the present invention may be synthesized by a known peptide solid-phase synthesis method (Haraaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977), etc.). . The protection of the functional group used for peptide synthesis, the removal of the protecting group and its protecting group, the activation of the functional group involved in the reaction, etc. may be appropriately selected from known groups or known means. The protein can also be obtained by culturing cells that hold foreign DNA that expresses the protein, as described later, or from cells or tissues of transgenic animals that hold the foreign DNA.

  Next, the activity of this protein, that is, the activity of Girdin, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, will be described. Girdin has the following activities, and the present protein has any of these activities. (1) Akt activity as a substrate (Akt of this protein phosphorylates serine at position 1416 in SEQ ID NO: 2 or the corresponding site), (2) Actin-binding protein activity (into actin filaments) And (3) cell membrane binding activity. The Akt substrate activity in (1) is understood as binding to Akt (complex formation) and phosphorylation by Akt, and the actin-binding protein activity in (2) is related to the formation of actin stress fibers by this protein and in lapoly media It is grasped as the formation of actin meshwork, and the cell membrane binding activity of (3) is grasped as binding (interaction) to phosphoinositide in the cell membrane.

  According to the present inventors, these various activities of (1) to (3) are related to cell motility control by Akt, and further to cell migration and angiogenesis, and activation and promotion of these cell kinetics. It has been confirmed that it contributes. The activities (1) and (3) are those of Girdin that is not phosphorylated, and the activities (2) are activities that Girdin has regardless of the presence or absence of phosphorylation. Any of these various activities can be detected in an in vitro or in vivo experimental system, as will be described in detail in the Examples below.

(Nucleotide encoding this protein)
In the present invention, a nucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof has a base sequence encoding each protein or the like. There is no particular limitation. The nucleotide may be DNA, RNA, or DNA / RNA chimera having such a base sequence, and may be single-stranded or double-stranded. In the case of a double strand, a DNA / RNA hybrid may be used in addition to a DNA double strand and an RNA double strand. In the case of a single strand, it may be either a sense strand or an antisense strand. Specifically, such nucleotides can be obtained in the form of genomic DNA, cDNA, mRNA, PCR product, chemically synthesized DNA, or the like.

  Examples of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 include the base sequence represented by SEQ ID NO: 1. This base sequence is a base sequence encoding Girdin. As described above, the nucleotide encoding this protein is not limited to the same base sequence as the base sequence represented by SEQ ID NO: 1, but a base sequence encoding the same amino acid sequence by a codon usage different from this base sequence It may be. The nucleotide sequence of this nucleotide is a nucleotide such as DNA that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, and encodes a protein having the same activity as this protein. May be.

  Examples of DNA that can hybridize under stringent conditions include, for example, about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably, the nucleotide sequence represented by each SEQ ID NO. A DNA containing a base sequence having a homology of about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used. Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions. The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

  The nucleotide encoding Girdin has, for example, a nucleotide having the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 under highly stringent conditions. Any protein may be used as long as it encodes a protein having the same activity as the protein represented by 2.

  Nucleotides encoding this protein can take the form of a nucleic acid construct for transformation. That is, various forms (Naked DNA, various expression vectors, artificial chromosomes, etc.) as nucleic acid constructs for expressing the present protein in host cells such as mammalian cells can be employed. Such a nucleic acid construct can appropriately include various elements such as a promoter, a terminator and the like so that the present protein can be expressed.

(An antibody specific for this protein) An antibody specific for this protein is directed against a protein containing the same or substantially the same amino acid sequence as SEQ ID NO: 2, such as Girdin, or a partial peptide thereof. A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, such as a specific antibody and phosphorylated Girdin, It includes an antibody specific for a serine residue at position 1416 or a protein in which the serine residue corresponding to the serine residue is phosphorylated or a partial peptide containing the phosphorylated moiety.

  The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. An antibody can be obtained from a warm-blooded animal by administering an appropriate antigen substance to the warm-blooded animal and then obtained from the warm-blooded animal. A monoclonal antibody can be obtained from a finally produced monoclonal-producing hybridoma.

  Polyclonal antibodies, for example, create a complex of an immunogenic antigen (protein antigen) and a carrier protein, immunize warm-blooded animals as appropriate, collect polyclonal antibody-containing materials from the immunized animals, It can be produced by performing separation and purification. As the carrier protein, conventionally known proteins can be used, but bovine serum albumin, bovine thyroglobulin, hemocyanin and the like can be used, and a complex may be formed with the immunogen at an appropriate ratio. In general, an active ester reagent containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, or dithiobilidyl group can be used for coupling between the hapten and the carrier. In administering such a complex, complete Freund's adjuvant or incomplete Freund's adjuvant can be administered simultaneously as appropriate. The administration can usually be carried out about once every 2 to 6 weeks, about 3 to 10 times in total. Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of warm-blooded animals immunized by the above method. The polyclonal antibody titer in the antiserum may be measured using an EIA such as ELISA as appropriate, and the separation and purification of the antibody may be performed according to known antibody separation and purification methods.

  Monoclonal antibodies are included in, for example, immunizing warm-blooded animals in the same manner as polyclonal antibodies, selecting individuals with antibody titers, and collecting spleen or lymph nodes several days after the final immunization. The antibody-producing cells are fused with myeloma to prepare a monoclonal antibody-producing hybridoma, the screened monoclonal antibody-producing hybridoma is cultured, and the monoclonal antibody is separated and purified from the culture. In addition, conventionally well-known method can be used for cell fusion operation and selection of a monoclonal antibody.

  The antibody of the present invention can take a form immobilized on a carrier. The form of the carrier is not particularly limited, and may be a bead shape, a flat plate shape, a microtiter plate, or the like. The antibody immobilized on the bead-shaped carrier can be used for immunoprecipitation, affinity chromatography, etc. The antibody immobilized on the plate-like carrier, microtiter plate, etc. can be used for EIA or RIA such as an antibody array or ELISA. It can be used as an assay plate.

(Nucleotide having a base sequence complementary to or substantially complementary to the base sequence of the nucleotide encoding the protein or a part thereof) One nucleotide in this embodiment includes an antigene for DNA encoding the protein such as Girdin. Examples include nucleotides (preferably DNA) or antisense nucleotides (preferably RNA) against mRNA encoding the present protein. Such nucleotides can take various forms similar to the nucleotides encoding the present protein, and the modified form of the length, base, sugar moiety, etc. is not particularly limited as long as the expression of the present protein can be suppressed. Various modifications can be adopted. Here, the term “complementary base sequence” means, for example, about 40% or more, preferably about 60% or more, more preferably, the total base sequence or partial base sequence of DNA or mRNA encoding the protein or partial peptide. Examples include base sequences having a homology of about 80% or more, more preferably about 90% or more. In particular, of the total base sequence of the DNA or mRNA of the present invention, the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, the base sequence near the start codon) is about 40% or more, preferably about 60%. %, More preferably about 80% or more, and still more preferably about 90% or more of the antisense DNA. These antisense DNAs can be produced using a known DNA synthesizer.

  In addition, another nucleotide in this embodiment includes a nucleotide that expresses RNA interference with a gene transcription product encoding the present protein such as Girdin. These nucleotides are constructed so that the expression of these Girdins can be suppressed by targeting at least a part of the transcript of the present protein, in particular, mRNA of DNA encoding Girdin. One embodiment of such a nucleotide is a nucleotide having a double-stranded structure of oligoribonucleotides that hybridize to each other. Specifically, a relatively short double-stranded oligoribonucleotide (siRNA) with or without a protruding 3 ′ end and a single oligoribonucleotide (short) that forms (or has) a hairpin structure. hairpin RNA: shRNA). Note that single-stranded oligonucleotide ribonucleotides that do not form a hairpin structure can also express RNA interference.

The length of the duplex in which the sense sequence and the antisense sequence in the nucleotide of this embodiment are paired is not particularly limited as long as an RNA interference effect is obtained, but is preferably 50 base pairs or less, typically 13 to It is preferable that it is 28 base pairs, More preferably, it is the range of 13-27 base pairs, More preferably, it is 19-21 base pairs. Most preferred is 19 or 20 base pairs. In addition, the sense sequence and antisense sequence including the 3′-side structure that does not form a duplex are typically preferably 15 to 30 nt, more preferably 15 to 29 nt, and still more preferably 21 to 23 nt. Most preferably, it is 21 or 22 nt. Moreover, it is preferable that 3 'terminal which protrudes in siRNA is 2-4 nt, More preferably, it is 2 nt. Furthermore, the loop site | part in shRNA should just be provided with the length of the loop site | part of the grade which does not prevent forming and maintaining the duplex (stem in shRNA) and 3 'terminal structure of such an aspect.

  siRNA and shRNA may be designed based on a target sequence determined by appropriately applying published rules and the like. Furthermore, siRNA design, including methods for determining target sequences, can be found at http: // design. rnai. jp / sidirect / index. php, http: // www. rockfeller. edu / labheads / tuschl / sirna. html and Rational siRNA design for RNA interference (Nature Biotechnology, vol.22,326-330 (2004), Angela Reynolds, Devin Leake, Queta Boese, Stephen Scaringe, William S Marshall & Anastasia Khvorova), Improved and automated prediction of effective siRNA (Biochem Biophys Res Commun. 2004 Jun 18; 319 (1): 264-74, Chalk AM, Wahlesttedt C, Sonnhammer EL.) Can be appropriately applied.

  Nucleotide that expresses RNA interference is not particularly limited to modifications such as base and sugar moiety, and various modifications can be adopted.

(Nucleotides encoding nucleotides that express RNA interference)
According to the present invention, nucleotides (preferably DNA) encoding nucleotides that express RNA interference with gene transcripts encoding the present protein such as Girdin and vectors containing these nucleotides can be included. That is, it is an embodiment of a vector that encodes siRNA or shRNA for a gene transcription product encoding the present protein so that it can be expressed. Nucleotides of such an embodiment are preferable in that continuous RNA interference can be realized and knockdown animals can be produced. In this embodiment, the shRNA expression vector can be constructed such that a continuous single-stranded RNA capable of constructing shRNA by intracellular transcription is transcribed from the antisense sequence and the sense sequence in addition to the loop sequence. The siRNA expression vector may be configured so that RNA having a predetermined sense sequence and antisense sequence is transcribed. In the siRNA expression vector, the sense sequence and the antisense sequence may be expressed by a single vector, or may be expressed by different vectors.

  The expression vector can take the form of a plasmid vector or a viral vector. The type of the vector is not particularly limited and can be selected according to the cell to be introduced. For example, in mammalian cells, for example, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, lentivirus vectors, herpes virus vectors, alphavirus vectors, EB virus vectors, papilloma virus vectors, foamy virus vectors, etc. These viral vectors are mentioned. In addition, the promoter used in such an expression vector may be either a pol II system or a pol III system as long as it can produce a corresponding RNA from each of the above DNAs.

(Cell) As one aspect, the present invention includes a cell in which the expression level of a gene encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 such as Girdin is suppressed. It is out. This cell can be obtained by introducing a knockout vector prepared based on the base sequence encoding this protein, a nucleotide that expresses RNA interference as described above, or a nucleotide (vector) encoding the nucleotide into the cell, or a non-human to be described later. It can be obtained from a mammalian knockout animal or a non-human mammal knockdown animal.

  Moreover, the present invention includes, as another aspect, a cell holding a foreign DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, such as Girdin. Yes. Such cells can be obtained by introducing a nucleic acid construct for expressing the present protein into the cells or using a non-human mammal transgenic animal described later as an acquisition source.

  Furthermore, the present invention provides, as another aspect, a foreign DNA encoding the present mutant protein in which a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 such as Girdin is mutated. Containing cells. Such cells can be obtained by introducing a nucleic acid construct for expressing the mutant protein into the cells or using a non-human mammalian transgenic animal described later as an acquisition source.

  These various cells can be, for example, humans (not including transgenic animals, knockout animals, and knockdown animals) and non-human mammalian cells. Further, it may be a non-human mammalian embryonic stem cell such as a rodent such as a mouse or a rat, or a somatic stem cell or a somatic cell. Furthermore, it may be a target cell for diseases to which the present protein is related, such as nerve cells, vascular endothelial cells, cancer cells and the like. Such various cells of the present invention are useful for screening, evaluation, cell motility and the like of the preventive / therapeutic agents of the present invention.

  Hereinafter, the use of the present protein and nucleotides, antibodies, cells and the like obtained in connection with the present protein will be described.

(Prophylactic / therapeutic agent for diseases associated with this protein) This protein has the activity of any one of (1) to (3) above, and these activities activate cell motility, cell migration and angiogenesis. Therefore, a compound having an activity that inhibits any one of the above-mentioned (1) to (3) or two or more kinds of the protein or a salt thereof is a disease associated with the protein, such as Girdin, specifically a cell. It can be used as a prophylactic / therapeutic agent for diseases associated with any of exercise, cell migration and angiogenesis. In addition, a compound that suppresses the expression level of the protein or a salt thereof can also be used as a prophylactic / therapeutic agent for diseases associated with the protein, such as Girdin, cell motility, cell migration, and angiogenesis.

  Here, the diseases associated with any of cell motility, cell migration, and angiogenesis include all of the diseases associated with such a phenomenon. Examples of the diseases include the following diseases.

(1) Cancer For example, primary, metastatic or recurrent breast cancer, prostate cancer, pancreatic cancer, gastric cancer, lung cancer, colon cancer (colon cancer, rectal cancer, anal cancer), esophageal cancer, duodenal cancer, head and neck cancer ( Tongue cancer, pharyngeal cancer, laryngeal cancer), brain tumor, schwannoma, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer (endometrial cancer, cervical cancer), ovarian cancer, Bladder cancer, skin cancer, hemangioma, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, hemangioma, hemangiofibroma, retinal sarcoma, penile cancer, childhood solid cancer, Kaposi sarcoma, Kaposi sarcoma due to AIDS, maxilla Sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, uterine fibroid, osteoblastoma, osteosarcoma, chondrosarcoma, tumor such as cancerous mesothelioma, leukemia, Hodgkin disease, etc.

(2) Arteriosclerotic diseases For example, acute coronary syndrome (eg, acute myocardial infarction, unstable angina), peripheral arterial occlusion, coronary intervention (percutaneous coronary angioplasty (PTCA), atherectomy (DCA) Restenosis after stent placement, restenosis after coronary artery bypass surgery, other peripheral arterial intervention (angioplasty, atherectomy, stent placement, etc.) and restenosis after bypass surgery, ischemic heart disease ( Eg, myocardial infarction, angina pectoris), myocarditis, intermittent claudication, lacunar infarction, arteriosclerosis (eg, atherosclerosis, etc.), heart failure (acute heart failure, chronic heart failure including congestive), arrhythmia, Atherosclerotic lesion development, thrombosis, hypertension, hypertensive tinnitus, hypotension, etc.

(3) Central and peripheral nerve disorders For example, head injury, spinal cord injury, brain edema, sensory dysfunction, sensory dysfunction, autonomic dysfunction, autonomic dysfunction, whiplash etc.

Specific examples of the index for obtaining a compound or a salt thereof that can be used as the preventive / therapeutic agent of the present invention include activities that inhibit the following activities. The following a) to b) are related to the inhibition of the Akt substrate activity of (1) above, the following c) to e) are related to the inhibition of the actin binding protein activity of (2) above, and f) below ~ G) relates to the inhibition of the cell membrane binding activity of (3) above.
a) Binding to serine / threonine kinase Akt and the protein b) Phosphorylation of the protein by serine / threonine kinase Akt c) Binding to actin filaments d) Formation of actin stress fibers e) Formation of actin meshwork in Lapolymedia f ) Binding to cell membrane g) Binding to phosphoinositide

  As described later, the compound having such inhibitory activity can be obtained based on a screening method for obtaining a compound that inhibits the above a) to g). Examples of such compounds include antibodies specific for the present protein. Moreover, the variant of this protein (for example, Girdin etc.) which has a variation | mutation in the phosphorylation site by Akt, and the variant of this protein (for example, Girdin etc.) which has a variation | mutation in a phosphoinositide binding site may be sufficient.

  As a salt of such a compound, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal) is used, and a physiologically acceptable acid addition salt is particularly preferable. . Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  When the compound obtained from the screening method or a salt thereof is used as the prophylactic / therapeutic agent of the present invention, the formulation form is not particularly limited. Any form such as oral preparations, injection preparations, parenteral preparations such as injections, and external preparations suitable for topical application can be employed. For example, tablets, capsules, elixirs, microcapsules and the like, as needed, or aseptic solutions or suspensions with water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. These various preparations are also an embodiment of the preventive / therapeutic agent of the present invention. For example, it can be manufactured by mixing with physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in unit dosage forms generally required for practicing recognized formulations. it can. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained. In addition, what is necessary is just to select suitably various additives for obtaining the said formulation suitably in the said field | area. The preparation thus obtained can be administered to humans and non-human animals such as rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, and the like. . The dosage of the above compound can be set in consideration of symptoms and individual differences among patients.

  In addition to the screened compound, the prophylactic / therapeutic agent of the present invention may be a compound that suppresses the expression level of the present protein or a salt thereof. The compound that suppresses the expression level of the protein in the cell or a salt thereof is included in “nucleotide having a base sequence complementary to or substantially complementary to the base sequence of the nucleotide encoding the protein or a part thereof”. And various forms of nucleotides. Since these nucleotides suppress the expression of the present protein by antigene, antisense, and RAN interference in the cell, as a result, the above a) to g) can be inhibited, and according to the above a) to g). It suppresses activation of cell motility, cell migration, and angiogenesis, and can thus act as a prophylactic / therapeutic agent for any of these related diseases.

  Moreover, as a compound or its salt which suppresses the expression level of this protein, the DNA construct which can destroy the gene which codes this protein is mentioned. Such a DNA construct can be prepared based on the nucleotide sequence of the nucleotide encoding this protein.

  The prophylactic / therapeutic agent containing such a nucleotide construct can be more easily introduced into cells by being used in an appropriate carrier. For this reason, the preventive / therapeutic agent of the present invention includes those containing such a carrier. Examples of the carrier include vectors suitable for introduction into cells, liposomes, metal particles, positively charged polymers, calcium phosphate, DEAE dextran and the like. The carrier may be a liposome, metal particles, positively charged polymer, calcium phosphate, or DEAE dextran that carries the vector carrying the nucleotide of the present invention. As a vector for introduction into a cell, a non-viral vector or a viral vector can be used. Furthermore, the present preventive / therapeutic agent may contain usual pharmacologically acceptable carriers, excipients, binders, stabilizers, buffers, solubilizers, isotonic agents and the like.

  The administration is not particularly limited as long as it can achieve introduction of the target cell and suppress the expression level of the present protein, and examples thereof include the following methods. That is, ex vivo transplanted to a target site after introducing the gene of the present invention into various cells such as fibroblasts outside the body and selecting a cell that has been successfully introduced (also a cell of the present invention). Examples thereof include a method and an approach in which the gene of the present invention is introduced in vivo by direct injection into a target site directly using a viral vector or liposome. Furthermore, there is a method of preparing a sustained-release preparation and implanting it near the affected area. As a method for introducing a prophylactic / therapeutic agent containing such a nucleotide construct into cells, tissues, etc., a phosphate-calcium coprecipitation method; a nucleic acid direct injection method using a micro glass tube; Method: Gene transfer method using electrostatic liposomes; HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method); receptor-mediated gene transfer method; A method of directly introducing naked-DNA; a method of introducing a positively charged polymer, and the like.

  The prophylactic / therapeutic agent containing such a nucleotide construct can be appropriately set within a range in which the expression level of the present protein is reduced, depending on the purpose of administration, the age, weight, condition, etc. of the individual.

(Prophylaxis / treatment methods for diseases associated with this protein)
The prophylactic / therapeutic agent of the present invention can also be implemented as a prophylactic / therapeutic method for diseases associated with any of cell motility, cell migration, and angiogenesis. That is, administering the prophylactic / therapeutic agent of the present invention in an effective amount for the prophylaxis or treatment of human or non-human warm-blooded animals is a method for the prophylaxis / treatment of the above diseases.

(Screening method) According to the present invention, screening of a compound or a salt thereof that promotes or inhibits various activities of the protein, or increases or decreases the expression level of the protein, using the protein and a nucleotide encoding the protein. A method is provided. The compound obtained by this screening method or a salt thereof can be used as a compound that activates or inhibits any of cell motility, cell migration and angiogenesis. In addition, a compound or a salt thereof that inhibits various activities of the protein or decreases the expression level can be used as a prophylactic / therapeutic agent for a disease associated with the protein, and in particular, any of cell motility, cell migration and angiogenesis. It can also be used as a prophylactic / therapeutic agent for kana related diseases

  In this screening method, the above-described activities (1) to (3) of the present protein are specifically compared with the case where the test compound is supplied to the present protein and cells expressing the present protein, and compared to when the test compound is not supplied. Can obtain a compound or a salt thereof that promotes or inhibits the activity of the protein by using as an index whether to promote or inhibit the above (a) to (g). In addition, by supplying a test compound to cells expressing the protein and detecting whether the expression level of the protein is increased or decreased compared to when the test compound is not supplied, A compound or a salt thereof that increases or decreases the amount can be obtained. In the detection of the action, the test compound supplied to the screening system may be a substance that acts on the protein, a nucleotide that encodes the protein, or a substance that acts on the nucleotide. Since a compound or salt thereof that inhibits the above activity of the protein or reduces the expression level of the protein suppresses cell motility, cell migration and angiogenesis, prevention of diseases associated with any of these cell kinetics. -It can be used as a therapeutic agent.

  Examples of such test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, genes (genomic DNA, cDNA), and the like. May be a novel compound or a known compound.

  In this screening method, in order to detect the expression level of the present protein and the various indicators a) to g), for example, the following methods can be employed. For example, in order to use the binding between Akt and the unphosphorylated protein as an index, phosphorylation of the protein not phosphorylated by Akt is preferably used as an index. For example, Girdin (expressing tagged Girdin is expressed. A cell lysate from COS7 cells to be purified with a tag) and an in vitro kinase assay system comprising an active Akt such as constitutively active and ATP labeled with P32 etc. For example, and the resulting phosphate can be detected by autoradiography. Alternatively, EGF is added to cells expressing Girdin to activate Akt, a test compound is supplied and incubated, and then the cell lysate is immunoprecipitated with anti-Girdin antibody and then immunoprecipitated with phosphorylated Girdin antibody. Western blot analysis may be performed using an anti-phosphorylated Girdin antibody or the like. When the binding of this protein to an actin filament is used as an index, a test compound is supplied to the actin coprecipitation assay system containing Girdin's CT2 domain or Girdin tagged with GST, and after incubation, coprecipitation is performed. After the collected proteins are collected, they may be separated by SDS-PAGE and subjected to Western blot analysis using Coomassie Brilliant Blue (CBB), anti-GST antibody (Cell Signaling Technology) or anti-phosphorylated Girdin antibody. In addition, when actin stress fiber formation is used as an index, for example, a Girdin-expressing cell is added with a stimulus that activates Akt such as EGF and a test compound, and then the Girdin-expressing cell is specifically phosphorylated Girdin. The formation of actin meshwork by phosphorylated Girdin may be observed by immunostaining with an anti-phosphorylated Girdin antibody or the like that recognizes the protein. Furthermore, a test compound is applied to cells expressing Girdin in a confluent state. When the layer cell culture is damaged by scratch (damage healing assay), phosphorylated Girdin at the leading edge may be detected by immunostaining. Phosphorylated Girdin at the leading edge is present in the actin meshwork. Furthermore, Girdin-expressing cells may be detected as cell migration in the Boyden chamber method. In addition, when binding to cell membranes or phosphoinositide is used as an index, a test compound is supplied to a protein-lipid overlay assay system containing Girdin to which a tag is added, and the protein binds to phospholipid. Alternatively, the test compound may be supplied to cells expressing Girdin, and Girdin may be observed using an anti-Girdin antibody.

  Examples of the compound or salt thereof obtained by this screening method include antibodies that specifically bind to the present protein. In addition, a compound that suppresses Akt phosphorylation by changing the three-dimensional structure of the phosphorylation site of this protein to reduce the binding activity to Akt, the stability of the complex with Akt-this protein, etc., and an Akt substrate Examples include Akt inhibitors that reversibly bind to the binding site. Further, a compound or salt thereof that inhibits cross-linking of the protein to actin filaments or promotes release of the cross-linked state, a compound or salt thereof that inhibits formation of actin stress fibers or promotes release of actin stress fibers, A compound or salt thereof that inhibits the formation of actin meshwork or promotes its release, a compound or salt thereof that inhibits cell membrane binding or promotes the release of binding, or inhibits the binding to phosphoinosidide or releases the bond. Examples include a promoting compound or a salt thereof. Furthermore, as already explained, various nucleotide constructs that suppress the expression of the present protein by knockout or knockdown can be mentioned.

(Transgenic animals)
According to the present invention, a non-human mammal having an exogenous DNA encoding this protein is provided. According to this transgenic animal, since the present protein is expressed by exogenous DNA, it is suitable for studying the activity of the present protein, screening and evaluation of preventive / therapeutic agents for diseases related to the present protein, and the like. It has become. Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice and rats. Of these, rodents such as mice and rats are preferred.

  Transgenic animals can be used for unfertilized eggs, fertilized eggs, sperm and embryonic cells including primordial cells thereof, such as calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran. It can be obtained by introducing an exogenous DNA or the like by a method or the like and preparing an embryo using the cells.

  The exogenous DNA used for the production of the present transgenic animal has various regions capable of expressing DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) encoding the protein. The DNA construct can be a DNA construct that is under the control of a promoter and further includes a terminator. Such a DNA construct can be appropriately selected according to the type of DNA introduction method, can be converted to Naked DNA, and can take the form of a conventionally known expression vector.

  The present transgenic animal can have the present foreign DNA heterozygously or can be homozygous. In addition, the present transgenic animal may be one in which such foreign DNA is randomly introduced into the host chromosome, or may be introduced in a knock-in form at a desired site. Since this transgenic animal has a tendency to overexpress this protein more than usual, for example, when the hyperfunction of this protein develops by using this transgenic animal, Sometimes it can be used.

  Further, according to the present invention, a foreign DNA encoding a mutant protein of the present protein, for example, a mutant protein in which serine at position 1416 is substituted with alanine and cannot be used as a substrate for Akt and does not have the same activity as the present protein is obtained. Retained transgenic animals are also provided. The transgenic animal expressing this mutant protein may be heterozygous or homozygous, as is the case with the transgenic animal introduced with the foreign DNA of this protein, but the mutant foreign DNA was randomly introduced. In many cases, the mutant protein is expressed in addition to the protein, and when the mutant foreign DNA is introduced so as to destroy the host chromosome encoding the protein, Only protein will be expressed. Such a mutant protein-expressing transgenic animal can be used for elucidation of the pathological mechanism of functional inactive refractory disease of this protein and for research use of such diseases. It can also be used for screening and evaluation of therapeutic agents for functionally inactive refractories of this protein.

  Such a transgenic animal can also be used as a cell source for tissue culture, a tissue source for tissue observation, a cell source for drug screening, an acquisition source of the present protein or a mutant protein thereof.

(Knockout animal)
According to the present invention, a non-human animal in which the expression of DNA encoding this protein is suppressed is provided. According to these animals, since the expression of the protein is suppressed, various activities related to the protein may be lost, and a model of a disease caused by inactivation of the biological activity of the protein can do.

  In addition, as a non-human mammal, the thing similar to the said transgenic animal can be used. In addition, in order to obtain a knockout animal in which the DNA encoding this protein is destroyed by knockout, a targeting vector is constructed using the genome encoding the protein, the coding region, or the base sequence in the vicinity thereof, and this is used as an embryo. What is necessary is just to use for manufacture. In embryo production, it is preferable to use somatic cells capable of producing ES cells or non-human cloned animals. In addition, a knockdown animal in which the expression of DNA encoding this protein is suppressed by RNA interference can be obtained by producing an embryo into which the nucleic acid construct capable of expressing RNA interference is introduced.

(Analytical methods, diagnostic agents, diagnostic methods, diagnostic kits)
The antibody of the present invention can specifically recognize the present protein or the present phosphorylated protein. By using the antibody of the present invention, the present protein or phosphorylated present protein in a test sample can be quantified by a measuring method such as an enzyme antibody method using an antibody such as EIA such as ELISA in particular. . That is, the antibody of the present invention can be used as a reagent for quantifying the present protein or the phosphorylated present protein. Of course, it can also be used as a qualitative reagent for this protein. Furthermore, the amount of the protein or phosphorylated protein in a test sample such as a cell or tissue is associated with the diagnosis of a disease associated with the protein, eg, a disease associated with cell motility, cell migration and angiogenesis. Therefore, the antibody of the present invention can be used as a diagnostic agent for such diseases.

  Further, by utilizing phosphorylation of this protein by Akt, the concentration of the compound that activates and inhibits various activities of this protein can be qualitatively detected from the degree of activation or inhibition of this phosphorylation. That is, an assay system containing Akt and the present protein and capable of phosphorylating the present protein by Akt is prepared, and a test compound is supplied to the assay system to provide the present protein or reaction product as a substrate. By detecting this phosphorylated protein, it can be qualitatively detected whether this test compound is a compound that activates or inhibits phosphorylation of the protein by Akt. The detection of the present protein or the phosphorylated present protein can be performed by an enzyme antibody method using an antibody specific for the present protein or an antibody specific for the phosphorylated present protein. In addition, by using a similar system and preparing a standard curve or the like, the test compound can be quantified when the test compound is a compound that activates or inhibits phosphorylation of the protein by Akt. Qualitative or quantitative analysis of a test compound by such an assay system can be used in the screening method of the present invention and the diagnosis method of a disease associated with this protein.

  The method for quantifying the protein of the present invention and phosphorylated antibody using the antibody of the present invention is not particularly limited, and an antibody corresponding to the amount of antigen (for example, the amount of protein) in the solution to be measured, Any measurement method can be used as long as the amount of the antigen or antibody-antigen complex is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. May be used. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but sandwich method is particularly preferable in terms of sensitivity and specificity. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. Examples of the radioisotope include [125I] and [131I], and the enzyme preferably has a stable and high specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, apple For example, fluorescamine and fluorescein isothiocyanate are used as fluorescent substances, and luminol, luminol derivatives, luciferin, lucigenin, and the like are used as luminescent substances. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass. In such detection, the antibody molecule itself may be used, or a part thereof may be used.

  The diagnostic kit of the present invention may comprise one or more selected from the Akt phosphorylation assay system element comprising the antibody of the present invention and the present protein such as Akt and Girdin. The antibody and Akt phosphorylation assay system of the present invention described above can be used not only for diagnostic purposes but also for research purposes of cell motility, cell migration and angiogenesis.

  Hereinafter, the present invention will be described by way of example, but the present invention is not limited to the following examples. The experimental methods used in the following examples will be described below, and then individual examples will be described.

1. To identify proteins that interact with the yeast two-hybrid test Akt, the full-length cDNA of Akt1 was inserted into the pAS2 vector (Clontech). The construct thus obtained was used as a material (bait) for screening the above-described human fetal brain MARCHMAKER cDNA library (Murakami et al., 2002). Two positive plasmids containing the inserted cDNA were selected and sequenced. These two plasmids contained a cDNA fragment encoding the C-terminal region of Girdin (residues 1217-1870). The full-length cDNA encoding Girdin was separated from human fetal brain poly A + RNA by 5 ′ rapid amplification (5′-RACE system, Invitrogen) at its ends. Furthermore, a yeast two-hybrid binding test was performed with purified pAS and pAC containing fragments of human Akt1 and Girdin cDNA.

2. Plasmid Wild human Akt1 with constitutively active and dominant inhibitory properties Obtained by courtesy of Mr. Gotoh (University of Tokyo). Then, constructs of pcDNA3.1-, pGEX-, and pEGFP-Girdin fragments were prepared (Murakami et al., 2002). EGFP was fused to the amino terminus of the protein, and V5 and myc tag were fused to the carboxyl terminus of the protein. Girdin mutants were generated using the Quick Change site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. Anti-siRNA Girdin was prepared by introducing two silent mutations into Girdin nucleotides 780-800 (5′-AAGAAGGCTACGGCAGGGAATT-3 ′) (underlined in the mutated gene).

3. A rabbit anti-Girdin polyclonal antibody was generated against the 19 amino acids at the carboxyl terminus of the antibody Girdin and affinity purified with an immunizing peptide. Anti-phosphorylated Girdin polyclonal antibodies are available from Kumamoto Immunochemical Laboratory, Transgenic, Inc. (Kumamoto Prefecture). Obtained by immunizing rabbits with phosphopeptides bound to keyhole limpet hemocyanin corresponding to Girdin amino acid sequence 1408-1420 (CDINRRQKpSLTLT). The antiserum was purified as a fraction bound to a column bound to phosphopeptide. Examples of other antibodies used in the experiment include anti-Akt polyclonal antibody (Cell Signaling Technology), anti-phosphorylated Akt polyclonal antibody (Cell Signaling Technology), and anti-coatctin monoclonal antibody (Upstate).

4). Kinase assay Immunoprecipitates with anti-V5 antibody (Invitrogen) taken from COS7 cells expressing Girdin CT-V5 WT, SA or generated GST-ST, with active or inactive recombinant Akt (500 ng) (Upstate) The cells were cultured in 10 μCi [γ-32P] ATP (3000 Ci / mmol, Amersham) in a kinase buffer (20 mM MOPS, 25 mM β-glycerophosphate, 5 mM EGTA, 15 mM MgCl 2). The mixture was incubated at 30 ° C. for 30 minutes and Laemmli sodium dodecyl sulfate (SDS) sample dilution buffer (20 mM Tris-HCl [pH 6.8], 2 mM EDTA, 2% SDS, 10% sucrose, 20 μg / ml bromophenol blue, 80 mM The reaction was terminated by adding dithiothreitol).

5. Immunofluorescence staining method Vero cells were fixed on a coverslip or glass dish coated with fibronectin (10 μg / ml, Sigma) and collagen I (10 μg / ml, Upstate), and stained with the antibody described above. The fluorescence staining was confirmed with a confocal laser microscope (fluoview FV500, Olympus).

6). Actin coprecipitation assay The F-actin coprecipitation test was performed according to the manufacturer's protocol (Cytoskeleton). Purified GST fusion protein, GST, α-actin (Cytoskeleton) was incubated with 40 μg of purified actin filament for 30 minutes at room temperature. The final concentration of F-actin was 18 μM. The filaments were then pelleted by centrifugation (100,000 × g) (Beckman). The coprecipitated protein was separated by SDS-PAGE and confirmed by Western blot analysis using Coomassie Brilliant Blue (CBB), anti-GST antibody (Cell Signaling Technology) or anti-phosphorylated Girdin antibody. For quantitative assay, a predetermined concentration of GST-Girdin CT2 (1 μM) was mixed with various concentrations of F-actin (0 to 2.5 μM) in a polymerization buffer, and incubated at room temperature for 30 minutes. The protein was centrifuged as described above, and all pellets and supernatants were each subjected to SDS-PAGE. Protein bands were detected and scanned by CBB staining and analyzed with WinROOF (Mitani Corp., Fukui, Japan).

7). RNA interference Girdin and Akt siRNA knockdown was performed using the previously described method (Watanabe et al., 2004). Target sequences that mediated silencing of Girdin expression were 5-AACCAGGTCATGCTCCAAAATT-3 (145-165 nucleotides, GirdinsiRNA A) and 5-AAGAAGGCTTTAGGCAGGAATT-3 (780-800 nucleotides, GirdinsiRNA B) as follows. These 21 nucleotide synthetic duplexes were made by Qiagen. Moreover, siRNA specific for human Akt1 was obtained from Qiagen. Vero cells were transfected with these siRNAs or irrelevant 21 RNA (Qiagen) as a control using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

8). Cryogenic replica electron microscopy of the cell membrane surface (cytoplasmic side) Electron microscopy of the cytoplasmic membrane surface of the cytoplasmic membrane followed previously reported methods (Heuser, 1989, 2000; Usukura, 1993). Vero cells cultured on glass cover slips (diameter 3 mm, standard # 1, Matunami, Osaka, Japan) were transfected with siRNA. After cleaving (unroofing) from the cell membrane on the upper layer side, the cells were immediately washed with a 2.5% glutaraldehyde buffer A solution (buffer A; 70 mM KCl, 5 mM MgCl2, 3 mM EDTA, 30 mM HEPES buffer, pH 7.4 (to KOH). )) Immobilized in. After washing with a mixed solution of buffer and distilled water, the sample was immediately frozen with liquid helium in a quick freezer (Eiko, Tokyo, Japan). Samples were then rotary shadowed with platinum-carbon using a freeze etch apparatus (FR2000, HITACHI, Ibaraki, Japan). For immunolabeling of the Girdin molecule, the unroofed cells were immobilized in 4% paraformaldehyde / 0.5% glutaraldehyde buffer A solution. After washing 3 times with buffer B (100 mM NaCl, 30 mM HEPES, 2 mM caCl 2), the sample is quenched and blocked, and at 37 ° C. with primary or secondary gold-binding antibody (Amersham) in buffer B containing 1% BSA. Labeled for 1 hour. Finally, it was quickly frozen and freeze-etched by the same method as described above.

9. Scratch-induced cell migration and low-speed imaging Directional cell migration in Vero cells was stimulated in a monolayer using an in vitro scratch injury assay (Watanabe et al., 2004). Vero cells were seeded on fibronectin-coated coverslips or 35 mm glass dishes and transfected with siRNA. After 48 hours from transfection, confluent Vero cells were scratched with the tip of a 200 μl disposable pipette tip to make them movable in the direction of damage. Cells were fixed for immunostaining at the indicated times. For low speed observation, Vero cells were transfected with both siRNA and GFP-actin and subjected to a scratch damage assay. Cells on the damaged edge were observed with a confocal laser scanning electron microscope (Fluoview, FV500, Olympus).

10. Three-dimensional cell migration assay To evaluate the mobility of cells transfected with various constructs and siRNA, the Boyden chamber cell migration assay was modified to include HTS FluoroBlok Insert (8.0 μM pores 24-well, BD). It was made possible to measure the movement of GFP-labeled cells passing through the fluorescent dye blocking micropore membrane to be used. That is, both sides of the membrane were coated with 10 μg / ml fibronectin for 12 hours at 37 ° C. and washed with PBS. The chamber was then placed in a 24-well dish filled with DMEM containing 0.1% BSA and with or without human recombinant EGF (20 ng / ml). For migration assays using HT-1080, 10% FBS was added to the lower chamber. In a 24-well plate, cells (1 × 10 5) were transfected with GFP (0.5 μg (vs. individual cells), the indicated construct (2.5 μg) and siRNA (20 pmol) and placed at the top of the chamber. The cell mobility was determined by counting the number of GFP positive cells that had migrated through the membrane with a fluorescence microscope.

11. Protein-lipid overlay assay GST fusion protein was expressed in DH5α or BL21-CodonPlus cells (Stratagene) and purified by conventional methods. Binding of purified GST-CT to phospholipids was confirmed at 4 ° C. using PPIP-Stripe (Echolen Biosciences) according to the manufacturer's protocol. Bound GST-CT was detected using either anti-Girdin antibody or anti-phosphorylated Girdin antibody.

(Example 1: Girdin identification, primary structure and expression mode thereof)
In order to identify a novel substrate of Akt, the yeast two-hybrid method was performed using the full length of human Akt1 as a bait construct, and proteins interacting with each other were searched using a human fetal brain cDNA library (see FIG. 1a). As a result, two clones (F-10, F-12) were detected. FIG. 1b shows the binding mode between human Akt1 and clones F-10 and F-12. These cDNAs were then expressed in yeast and were found to interact only with the Akt1 C-terminal regulatory region. Furthermore, 5′-RACE was performed on the 5′-end of the cDNA to obtain a clone having the entire coding region containing a 3.6 kb cDNA continuous to the 5 ′ end. The resulting full-length cDNA contained a 5610 bp open reading frame and encoded a protein having 1870 amino acid residues. Moreover, as a result of database search, it was found that homologs exist in mice, rats and Drosophila.

  FIG. 1c shows the primary structure of Girdin and the Girdin structure predicted by the COILS algorithm. As shown in FIG. 1c, Girdin was predicted to form a dimer with an N-terminal region (NT) and an α-helical coiled-coil region (Ala253 to Lys-1375). The predicted coiled coil region had 135 typical 7 amino acid repeats in the region (see FIG. 1h).

  In addition, the expression of Girdin mRNA was analyzed by Northern blotting. The result is shown in FIG. As shown in FIG. 1d, Girdin was found to be ubiquitously expressed in various human tissues. Furthermore, Western blotting was performed using a polyclonal antibody against the 19 amino acids at the C-terminal of Girdin. As a result, a 250 kDa protein was confirmed in the brain and testis lysate (see FIG. 1e).

  In order to confirm the oligomeric form of Girdin, the COS7 cell lysate was analyzed by Western blotting using an anti-Girdin antibody before and after crosslinking with 100 μM sulfosuccinimide. As shown in FIG. 1f, the results strongly suggested that Girdin exists as a polymer complex in intact cells after treatment with a crosslinking agent. Furthermore, according to the result of subjecting the COS7 cell lysate to gel filtration, it was found that Girdin formed a large protein complex. Furthermore, in order to examine whether NT region and CT region are involved in oligomer formation, when V5 epitope-tagged NT (NT-V5) and myc epitope-tagged NT region (NT-myc) are expressed in COS7 cells As shown in FIG. 1g, complexes of these two NT regions were observed in the cells. This suggested that the NT region contributed to Girdin oligomer formation. On the other hand, it was found that the CT region did not contribute to oligomer formation (data not shown). According to the gel filtration result of the COS7 cell lysate expressing the NT region, the NT region was predicted to form a dimer (see FIG. 1i).

(Example 2: Phosphorylation of Girdin in vitro and in vivo by Akt)
Whether Akt and Girdin physically interacted was confirmed by in vivo and in vitro assays. However, a stable complex of both proteins could not be detected in vitro or by immunoprecipitation (data not shown). This suggested that both had normal interactions in a very transient manner as observed, for example, in protein kinase and its substrate interactions. Akt phosphorylation site substrate contains Rx-RxxS / T site, and the amino acid sequence (RERQKS) close to serine (underline) at position 1416 in the CT region of Girdin It corresponded to this amino acid sequence. This site was the only consensus area. Since the CT region, which is a presumed phosphorylation region, was also conserved in other types of Girdin homologs, it was decided to investigate whether Akt phosphorylates Girdin.

  According to the in vitro kinase assay shown in FIG. 2a, GirdinCT wild type (WT) is phosphorylated by Akt, but the SA mutant in which Ser-1416 is replaced with Ala is not phosphorylated. Akt directly phosphorylates 1416-Ser. It can be said that it is oxidizing. On the other hand, neither the NT region nor the coil region was phosphorylated by Girdin in vitro (see FIG. 2b).

  Next, an antibody that specifically binds to a peptide containing Ser-1416 that is phosphorylated to confirm that Ser-1416 is a phosphorylation site catalyzed by Akt in vivo is used and this antibody is used. The western blotting analysis shown in FIG. As shown in FIG. 2c, the anti-phosphorylated Ser-1416 antibody recognized Girdin CT (WT) under co-expression of wild type Akt (Akt WT) or constitutively active Akt (Akt CA). However, it was not recognized by dominant negative (dominant inhibition) Akt (Akt DN). Further, the anti-phosphorylated Ser-1416 antibody did not recognize the Girdin CT (SA) mutant even under the co-expression of Akt CA. From these results, it was found that the anti-phosphorylated Ser-1416 antibody specifically recognizes Girdin CT in which Ser-1416 is phosphorylated.

  It was then investigated whether anti-phosphorylated Ser-1416 antibody could recognize phosphorylated endogenous Girdin in response to an external stimulus that physiologically activates endogenous Akt. As shown in FIG. 2d, when EGF was supplied, phosphorylation of a 250 kda protein presumed to be Girdin was induced by immunoprecipitation. The time course of this induction was similar to the time course of Akt activation. Furthermore, Girdin phosphorylation was inhibited when cells were treated with the PI3K inhibitors LY294002 and Wortmannin, but not when treated with the mitogen activated protein kinase kinase 1 (MEK1) inhibitor PD98059. From these results, it was concluded that Girdin phosphorylation is induced in a PI3K-dependent manner. Moreover, as shown in FIG. 2e, the expression of Akt WT and Akt CA induced significant phosphorylation of Girdin, whereas Akt DN did not induce phosphorylation. Only active Akt was phosphorylated in cells. It can be induced. Furthermore, Girdin phosphorylation was not observed in cells into which Akt RNA interference short RNA (Akt siRNA) was introduced. These findings supported that Akt activation is required for Girdin phosphorylation.

(Example 3: Binding to actin fibers via Girdin C-terminal domain) In order to confirm the intracellular localization of Girdin, Vero fibroblasts were immunofluorescently stained with an anti-Girdin antibody. As shown in FIG. 3a, Girdin was localized with actin stress fibers in quiescent cells. When cells were stimulated by EGF (50 ng / ml), they began to become localized, after which a directional elongation of lamellipodia (foliate foot) occurred. In cells stimulated by EGF, Girdin was localized not only to actin stress fibers but also to lamellipodia at the leading edge. The leading edge is shown as the stained area with the Arp2 / 3 binding protein coatactin in FIG.

  Since the localization of Girdin suggests that Girdin is an F-actin binding protein, various fusion GFP proteins encoding fragments, NT (GFP-NT) are used to confirm the actin binding domain of Girdin. N-terminal half (GFP-M1) and C-terminal half (GFP-M2) for the coiled-coil region, N-terminal half (GFP-CT1) and C-terminal half (GFP-CT2) for the CT region, These intracellular localizations were confirmed (see a in FIG. 3b). As shown in FIGS. 3b-d, GFP-NT, GFP-M1 and GFP-M2 were localized in the cytoplasm when expressed in Vero cells. As shown in e of FIG. 3b, GFP-CT1 was localized in both the nucleus and the cell membrane as shown by the co-localization of the carbocyanine membrane probe CM-Dil. On the other hand, as shown in f of FIG. 3b, GFP-CT2 was clearly localized on the stress fiber. These results suggested that Girdin localizes to actin filaments via its CT2 region, but interacts with the cell membrane via the CT1 region. In addition, since accumulation of endogenous Girdin in the nucleus was not observed (see FIG. 3a), it was considered that the nuclear localization of GFP-CT1 was artificial.

  Next, the actin binding ability of the CT2 domain was examined by actin coprecipitation assay. As shown in FIG. 3c and FIG. 3d, purified glutathione S transferase (GST) fusion CT2 (GST-CT2) co-precipitated with F-actin as well as α-actinin, but not with GST alone. This indicated that GST-CT2 was directly bound to F-actin. As the amount of F-actin increased, the amount of GST-CT2 binding to F-actin was saturated, as shown in FIG. As shown in b of FIG. 3d, the dissociation coefficient (Kd) of F-actin was 1.03 μM. This indicates that Girdin has a relatively low affinity for F-actin. The various domains of Girdin, predicted functions and the predicted structure of Girdin are shown in FIG. 3e.

(Example 4: Formation of Girdin Stress Fiber and Importance in Cell Movement) In order to evaluate the function of Girdin, RNA interference was employed to suppress (knock down) Girdin expression in Vero cells. Some Girdin siRNA (21 nucleotides) and control siRNA were introduced into Vero cells. As shown in FIG. 4a, according to Western blot analysis, Girdin expression level was suppressed by 90% or more in cells introduced with Girdin siRNA without affecting the activities of Akt and actin. In order to confirm the specificity of the knockdown effect, Western blot analysis using two types of Girdin siRNAs (A and B) was performed, and other functional assays were performed.

  In order to confirm whether Girdin functions to crosslink actin filaments, the knockdown effect of Girdin in the organization of the actin cytoskeleton was examined. As shown in a of FIG. 4b, according to immunofluorescence staining with an anti-Girdin antibody, Girdin was very small in cells into which Girdin siRNA was introduced. Moreover, as shown in b of FIG. 4b, according to the staining of the F-actin rich structure with phalloidin, the actin stress fiber was broken in the cells into which Girdin siRNA was introduced. This indicates that Girdin is essential for the formation of actin stress fibers.

  When a cell into which Girdin siRNA was introduced was observed at a high magnification, a remarkable decrease in thin and short stress fiber was observed in this cell. Furthermore, they lost their original form and formed a bumpy boundary due to the formation of a number of protrusions (leading edges) (see the lower side of FIG. 4b and FIG. 4c).

  Furthermore, to clarify the role of Girdin in actin dynamics, the behavior of Girdin-inhibited cells in cell migration in an in vitro wound healing assay was examined. As shown in FIG. 4d, cells introduced with Girdin siRNA in contact with the wound cannot form lamellipodia elongated at the leading edge, and exhibit abnormal cell motility such that the protrusions repeatedly expand and contract. It was. These results indicated that Girdin is essential for the organization of actin filaments during cell migration.

  In order to confirm the specificity of the knockdown effect of Girdin, Girdin siRNA was introduced into SK-N-MC neuroblastoma cells that stably express RET receptor tyrosine kinase, which is another cell line. SK-N-MC (RET) cells into which Girdin siRNA was introduced showed disruption of stress fibers and the formation of restrictive lamellipodia in response to RET ligand, a glial cell line-derived nerve growth factor (GDNF).

(Example 5: Analysis of the influence of Girdin localization and its suppression on actin mechanism by electron microscope)
Furthermore, in order to confirm the role of Girdin and actin filaments in vivo and their role in actin organization, ultrastructural analysis was performed on “unroof” Vero cells by deep etch electron microscopy. As shown in FIG. 5a, immunogold colloid label analysis revealed that Girdin molecules were localized with actin filaments. As shown in FIG. 5b, it was found from the high-magnification image that Girdin was localized together with the connecting portion between the actin filaments. Consistent with the results of this immunofluorescence analysis, the actin filament density on the surface layer observed in the electron microscopic image at the cytoplasmic interface in contact with the cell membrane of Vero cells depleted of Girdin was lower than that of the control cells (see FIG. 5c). In the control cells, actin fibers that were organized more strongly predominated. In the cells into which the control siRNA was introduced, the filaments were directed in the same direction without being separated from each other, forming a thick cable that was firmly cross-linked by many molecules. Some filaments were oriented perpendicular to other actin cables, resulting in a woven and dense actin network.

Example 6 Effect of Girdin Phosphorylation on Localization and Interaction with Inositol Phospholipid (Phosphoinositide) To obtain knowledge about the role of Girdin phosphate by Akt, staining with anti-phosphorylated Girdin antibody Was used to examine the localization of phosphorylated Girdin in Vero cells. As shown in the top diagram of FIG. 6a, little Girdin phosphorylation was observed throughout the cells in serum-starved quiescent cells. This result is consistent with the Western blot analysis results shown in FIG. On the other hand, when cells are stimulated with EGF (50 ng / ml), immunostaining shows that Girdin phosphorylated on lamellipodia at the leading edge of migrating cells appears (middle panel in FIG. 6a). In addition, phosphorylated Girdin colocalizes with the coatactin present at the leading edge (see FIG. 6b). In addition, Vero cells stimulated with EGF observed enhanced accumulation and localization of Akt activated with phosphorylated Girdin in lame lipodia at the leading edge (FIG. 6g). In cells introduced with Girdin siRNA, when stimulated with EGF, a number of leading edges are induced. At these leading edges, a phosphorylated Girdin signal exists as expected. There was no (bottom of FIG. 6a). This result supports the specificity of immunostaining.

  Next, in order to elucidate the mechanism of determining the localization difference between phosphorylated Girdin and non-phosphorylated Girdin, the Girdin CT1 domain must be localized in the cell membrane and have a phosphorylation site. Therefore, the experiment was performed on the assumption that the interaction of Girdin with the cell membrane is controlled by Girdin phosphorylation. As shown in FIG. 6c, the present inventors consist of 19 amino acid residues (Arg-1389 to Lys-1407) that are positively charged upstream of the phosphorylation site, and phosphatidylinositol 4,5- A sequence similar to the consensus sequence for binding bisphosphate was found. When the GFP-CT1 fusion protein deficient in this phosphoinositide binding site was introduced into Vero cells, localization of this derived protein in the cell membrane was not observed (see b in FIG. 6c). This result suggests that the CT1 domain is anchored to the cell membrane via binding to phosphoinositide.

  In order to confirm Girdin's ability to bind phosphoinositide, a protein-lipid binding assay was performed using a purified GST fusion protein containing Girdin's phosphoinositide binding site. In this experiment, since the GST-CT1 fusion protein is easily decomposed in the expression and purification operations, the CT domain and GST were fused and used instead of the CT1 domain. As shown in FIG. 6e a, GST-CT selectively binds to PI (4) P and weakly binds to PI (3) P, but binds to other phosphoinositides and phospholipids. I did not. Also, as shown in FIG. 6e b, the ability of the GST-CT fusion protein to bind to PI (4) P and PI (3) P was lost when the phosphoinositide binding site was removed. Further, it was found that a mutant of CT1 domain (a basic amino acid residue having a positive charge substituted with alanine) cannot bind to phosphoinositide and has no cell membrane localization. These results suggested that a positive charge generated by basic amino acid residues in the phosphoinositide binding motif is required for Girdin's interaction with the cell membrane (see FIG. 6h).

  Next, the phosphoinositide binding site is present in the vicinity of the Akt phosphorylation site (see a in FIG. 6c). Akt controls the localization of Girdin by regulating its phosphoinositide binding ability. In order to confirm whether or not the phosphoinositide binding ability of the phosphorylated GST-CT fusion protein was examined. The purified GST-CT fusion protein was naturally phosphorylated in vitro by Akt (see b in FIG. 6d). This phosphorylated GST-CT was then subjected to a protein-lipid binding assay. This protein-lipid bond was detected with an anti-phosphorylated Girdin antibody. As shown in c of FIG. 6e, phosphorylated GST-CT was found not to bind to PI4P or PI3P. According to the actin coprecipitation assay, phosphorylation of GST-CT did not attenuate the affinity for F-actin, and its binding kinetics was similar to that of GST-CT2 (Fig. 6f, see FIG. 3d). These findings suggested that Girdin binding to phosphoinositide was attenuated by phosphorylation but binding to F-actin was not attenuated.

  Furthermore, whether or not the cell membrane interaction of Girdin was regulated by EGF treatment was examined using COS7 cells expressing either a fusion protein of GFP and full-length Girdin (WT) or an SA mutant. As shown in FIG. 6 i, Ser-1416 phosphorylation by Akt was found to be required for Girdin delocalization from the cell membrane.

Example 7 Regulation of Cell Migration by Girdin Phosphorylation In order to confirm whether phosphorylation of Girdin at the leading edge is caused by cell motility, the following experiment was performed. . That is, after damaging the confluent monolayer of Vero cells and immunostaining with anti-phosphorylated Girdin antibody, the level of Girdin phosphorylation in the cells quickly increases after the damage at the damaged end, The maximum was reached after 8 hours and then continued for at least 12 hours (see FIG. 7a). This suggested that Girdin phosphorylation plays an important role in cell motility. Therefore, the role of Girdin in cell motility was investigated by the Boyden chamber method. As shown in FIG. 7b, Girdin knockdown significantly inhibited Vero cells from migrating to downstream chambers in response to fed EGF (50 ng / ml). Further, as shown in FIG. 7b, it was found that the expression of CT domain in Vero cells was significantly attenuated by cell migration. This supports that the interaction between endogenous Girdin and actin filaments is important for integrated cell migration. Consistent with previous studies, depletion of Akt by siRNA also attenuated cell migration (Figure 7b). This suggested that intrinsic Akt activity was required for Vero cells to migrate through the pores of the assay method.

  Next, it was examined whether or not exogenous Girdin was supplied to knockdown cells to restore cell migration injury in the cells. Therefore, a siRNA resistant Girdin (siRNAr Girdin) construct was constructed with silent mutation (see FIG. 7c). As shown in FIG. 7d, siRNAr Girdin (WT) was expressed in knockdown cells to fully restore EGF-responsive cell mobility. On the other hand, siRNAr Girdin (SA) which is a siRNAr Girdin mutant in which Ser-1416 is substituted with Ala, siRNAr Girdin (ΔPB) which is a siRNAr Girdi mutant lacking a phosphoinositide binding site, phosphoi SiRNAr Girdin (PB ala), a siRNAr Girdin mutant in which the nocitide binding site was replaced with alanine, could not compensate for cell migration injury. (See FIG. 7d and FIG. 6h c). These results were confirmed by a cell migration assay using the human fibrosarcoma cell line HT1080 (FIG. 7f).

  Finally, migration of Vero cells expressing the Girdin mutant was directly confirmed using a wound healing assay (see FIG. 7g). Cells expressing Girdin WT migrated easily or quickly to the site of injury, while cells expressing Girdin SA only showed an extended morphology. Furthermore, the nuclei in the latter cells seemed fixed and not mobile. This suggests that these cells cannot be separated from the matrix. Cells expressing GirdinΔPB and cells expressing Girdin PBala were less capable of migration than cells expressing Girdin WT. These results indicate that Girdin mutant expression impairs the proper direction of cell migration. Considering the results in FIGS. 6a-f and 6i together, these data indicate that regulation of the interaction between Girdin and the cell membrane by Akt is essential for cell migration.

(Example 8: Inhibition of migration ability of human umbilical vascular endothelial cell HUVEC by Girdin siRNA) HUVEC cells were treated with control siRNA and Girdin siRNA for 48 hours, and then VEGF (vascular endothelium) by Boyden chamber method (Boyden Chamber method). The migration ability by growth factors) was quantified. As shown in FIG. 8a, it was revealed that the migration ability of HUVEC cells treated with Girdin siRNA was significantly reduced in the presence of VEGF. FIG. 8b shows a diagram in which migrated cells are stained purple. As shown in FIG. 8b, it was clear that the number of migratory cells of HUVEC cells treated with Girdin siRNA was reduced.

(Example 9: Inhibition of lumen formation ability of human umbilical cord vascular endothelial cell HUVEC by Girdin siRNA) After treating HUVEC cells with Girdin siRNA in the same manner as in Example 8, the lumen formation ability was observed. The observation results are shown in FIG. As shown in FIG. 9a, untreated HUVEC cells and HUVEC cells treated with control siRNA show luminal formation under normal culture conditions, whereas luminal formation is markedly inhibited in HUVEC cells treated with Girdin siRNA. It was. FIG. 9b shows Western blotting results in which Girdin expression in each cell of FIG. 9a was detected with an anti-Girdin antibody. As shown in FIG. 9b, Girdin expression was observed in HUVEC cells into which Girdin siRNA was introduced. Was significantly suppressed.

(Example 10: Recovery of lumen formation ability by expression of siRNA-resistant Girdin gene) A siRNA-resistant Girdin gene cDNA was introduced into an adenovirus vector (Ad-rGirdin WT-V5). In addition, a vector into which Girdin cDNA in which serine 1416, which is the Akt phosphorylation site of Girdin, was substituted with alanine was introduced was also prepared (Ad-rGirdin SA-V5). As shown in FIG. 10a, when siRNA and Ad-rGirdin WT-V5 were simultaneously introduced into HUVEC cells, lumen formation was restored (middle of FIG. 10a). When the phosphorylation site by Akt was mutated, tube formation was not recovered (right side of FIG. 10a), and it was revealed that phosphorylation of Girdin by Akt is important for its function. FIG. 10b shows the results of Western blotting in which the expression of Girdin in each cell of FIG. 10a was detected with anti-Girdin antibody and anti-V5 antibody. As a control, the expression of VEGFR2 (vascular endothelial growth factor receptor 2) is shown.

  The above results indicate that Girdin plays an important role in angiogenesis, and the possibility of developing an antitumor drug by developing an inhibitor compound of the Akt-Girdin system aimed at inhibiting angiogenesis in the tumor. There is.

(Example 11: Matrigel plug assay)
A knockdown vector for short hairpin Girdin siRNA was constructed using an adenovirus vector. The target sequence in the Girdin gene was gaaggagaggcaactggat (SEQ ID NO: 3). 100 μl of this adenovirus (1 × 10 ¹⁰ cells / ml) was mixed with 400 μl of Matrigel (BD Matrigel (product name)) containing 100 ng / ml of VEGF, and the entire amount was injected subcutaneously into the abdomen of the mouse. After 1 week, the matrigel injection site was removed and stained with hematoxylin and eosin (HE). Moreover, it operated similarly to the Example except having used the adenovirus holding EGFP instead of the knockdown cassette as control. The results are shown in FIG.

  In FIG. 11, the control result is shown in the upper part, and the result of the present example is shown in the lower part. In the control, angiogenesis was observed, whereas in the Examples, almost no angiogenesis was observed. As a result of suppression of Girdin expression, it was found that angiogenesis was inhibited. In addition, it has been confirmed that the knockdown vector used in this example suppresses Girdin expression.

(Example 12: Expression of Girdin in tumor blood vessels) Human breast and cervical cancer tissues were collected, and the results of immunostaining Girdin by a conventional method are shown in FIG. In Girdin tissue immunostaining, tissue sections paraffin-embedded with xylene were deparaffinized and then dehydrated, and the endogenous peroxidase activity was inactivated with 0.3% hydrogen peroxide. And blocked. Then, add anti-Girdin antibody (diluted 50 times with PBS containing BSA), incubate in a moisturizing box (4 times, overnight), wash with PBS, add secondary antibody and incubate at room temperature (15 min) )did. After washing with PBS, enzyme-labeled streptavidin was added, incubated for 15 minutes at room temperature, washed, and DAB substrate was added. The reaction was terminated by immersion in PBS, followed by nuclear staining with hematoxylene staining, washing and dehydration, and xylene treatment. In FIG. 12, it can be seen that Girdin is strongly stained in vascular endothelial cells (the arrow portion is a strong stained portion based on Girdin). From these results, it was found that Girdin is strongly expressed in tumor blood vessel endothelial cells.

  The present invention can be used as a drug, screening and reagent for prevention / treatment of various diseases.

Sequence number 3: Target sequence of Girdin siRNA [Sequence Listing]

Claims (32)

Activating any of cell motility, cell migration and angiogenesis, characterized by using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof, or A screening method for inhibiting compounds or salts thereof. The screening method according to claim 1, wherein the protein is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The screening method according to claim 1, wherein the partial peptide is a C-terminal region (CT1 region and / or CT2 region) of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The screening method according to claim 1 or 2, wherein the protein is phosphorylated at the serine residue at position 1416 of the amino acid sequence represented by SEQ ID NO: 2 or a serine residue corresponding to the serine residue. . The promotion or inhibition of the binding activity between the serine / threonine kinase Akt and a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or a part thereof, as an index. 5. The screening method according to any one of 4. The screening method according to any one of claims 1 to 4, wherein the promotion or inhibition of phosphorylation of the protein by serine / threonine kinase Akt is used as an index. The screening method according to any one of claims 1 to 4, wherein promotion or inhibition of actin filament binding ability is used as an index. The screening method according to any one of claims 1 to 4, wherein promotion or inhibition of actin stress fiber forming ability is used as an index. The screening method according to any one of claims 1 to 4, wherein the promotion or inhibition of actin meshwork formation ability in Lapolimedia is used as an index. The screening method according to any one of claims 1 to 4, wherein cell membrane binding promotion or inhibition is used as an index. The screening method according to any one of claims 1 to 4, wherein the promotion or inhibition of phosphoinositide binding is used as an index. Activation of one of cell motility, cell migration and angiogenesis, characterized by using a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. Or a screening method for a compound or a salt thereof that inhibits the compound. The screening method according to any one of claims 1 to 12, which is a screening method for a prophylactic / therapeutic agent for a disease associated with any of cell motility, cell migration and angiogenesis. The screening method according to any one of claims 1 to 13, wherein the disease is any one selected from cancer, arteriosclerotic disease, and central and peripheral neuropathy. A cell, which is a compound or a salt thereof that expresses one or more of the following reactivity with respect to a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof A prophylactic / therapeutic agent for diseases associated with any of exercise, cell migration, and angiogenesis.
a) Inhibitory activity of serine / threonine kinase Akt binding to the protein b) Inhibitory activity of phosphorylation of the protein by serine / threonine kinase Akt c) Inhibitory activity of binding to actin filaments d) Inhibitory activity of actin stress fiber formation e) Inhibitory activity of actin meshwork formation in lapolimedia f) Inhibitory activity of cell membrane binding g) Inhibitory activity of binding to phosphoinositide Prevention of diseases associated with cell motility, cell migration or angiogenesis, which is a compound that suppresses the expression level of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2.・ Therapeutic drugs. 17. The compound according to claim 16, wherein the compound is a polynucleotide having a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2, or a part thereof. Preventive and therapeutic drugs. The prophylactic / therapeutic agent according to claim 17, which is RNA that expresses RNA interference. The prophylactic / therapeutic agent according to claim 16, which is a DNA encoding the RNA according to claim 18. The prophylactic / therapeutic agent according to claim 16, which is a recombinant vector containing the DNA according to claim 19. The prophylactic / therapeutic agent according to any one of claims 16 to 20, wherein the disease is any one selected from cancer, arteriosclerotic disease and central and peripheral neuropathy. A cell in which the expression level of a gene encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 is suppressed. An animal in which the expression level of a gene encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 is suppressed. 24. The animal according to claim 23, wherein the suppression of the gene expression level is effected by knockdown. The animal according to claim 23 or 24, which is used for research of a disease associated with any of cell motility, cell migration and angiogenesis, and for screening for a prophylactic / therapeutic agent. Any of cell motility, cell migration, and angiogenesis using the reaction of a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 and serine / threonine kinase Akt For qualitative analysis of a compound that activates or inhibits. Any of cell motility, cell migration, and angiogenesis using the reaction of a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 and serine / threonine kinase Akt For quantifying a compound that activates or inhibits. An antibody specific for a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof. A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 and corresponding to the serine residue at position 1416 of the amino acid sequence represented by SEQ ID NO: 2 or the serine residue An antibody specific for a protein in which a serine residue is phosphorylated or a partial peptide containing the phosphorylated moiety. A reagent for studying activation or inhibition of cell motility, cell migration and angiogenesis, comprising the antibody according to claim 28 or 29. A diagnostic agent for a disease associated with any of cell motility, cell migration, and angiogenesis, comprising the antibody according to claim 28 or 29. Cell motility, cell migration, and protein using any of the following reactivities for a protein containing the same or substantially the same amino acid sequence as SEQ ID NO: 2 in the cell at the disease site or a partial peptide thereof: A method for diagnosing a disease associated with any of angiogenesis.
a) Binding activity to serine / threonine kinase Akt and the protein b) Phosphorylation activity of the protein by serine / threonine kinase Akt c) Binding activity to actin filaments d) Actin stress fiber formation activity e) Actin mesh in Lapolymedia Work formation activity f) Cell membrane binding activity g) Binding activity to phosphoinositide

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