JP4059941B2 - Rho target protein p140mDia - Google Patents

Description

【００９８】

【図面の簡単な説明】
【図１】クローン５０とＲｈｏタンパク質とのツー・ハイブリッド・システムでの結合を示した写真である。Ｖａｌ、Ａｓｎ、ＷＴおよびＣｄｃ４２は、それぞれ、ＲｈｏＡ^Val14 、ＲｈｏＡ^Asn19 、野生型ＲｈｏＡおよびＣｄｃ４２Ｈｓを示している。ΔＡｓｎおよびΔＷＴは、Ａｌａ^１８１でトランケートしたＲｈｏ^Asn19 および野生型ＲｈｏＡを示している。ラミン（Lamin ）および他のタンパク質と融合していないＬｅｘ結合ドメインを、ネガティブコントロールとして用いた。Ａｌａ^１８１でトランケートしたＲｈｏＡ^Val14 は、他のタンパク質と融合していないＶＰ１６活性化ドメインとも結合して高ＬａｃＺ活性を示したので、この実験では使わなかった。
【図２】単離されたｐ１４０ｍＤｉａを模式的に示した図である。
ＯＲＦは点のボックスで示した。ヌクレオチド８７番目のＴからＣへの変換および２２２９番目のＣからＴへのヌクレオチドの変換が、それぞれ５０３およびＥ７３ｃＤＮＡ中に見出された。ライブラリーａ、ｂおよびＣは、それぞれマウス脳ライブラリー９３６３０９（ストラタジーン社）、ＭＬ３０００ａ（クロンテック社）およびマウス胚ライブラリーを示している。Ｅ７３ ｃＤＮＡには９アミノ酸の挿入が認められた。
【図３】ｐ１４０ｍＤｉａの推定アミノ酸配列を示した図である。ツー・ハイブリッド・システムで得られたクローンの配列は太い下線で、プロリンリッチ領域の繰り返し構造は点下線で、ＦＨ−２領域は細い下線で、それぞれ示した。矢じり印は９アミノ酸の挿入箇所を示している。
【図４】ｐ１４０ｍＤｉａおよびＦＨ領域を含む他の蛋白質との模式的な構造比較を示す。ｐ１４０ｍＤｉａの配列をDrosophilaの diaphanous 、S.cerevisiaeのＢｎｉ１ｐ、ラットｆｏｒｍｉｎ、およびDrosophilaのcappucino と比較した。比較は推定Ｒｈｏ結合領域（Ｒｈｏ-binding）、Ｎー末端からプロリンリッチ領域（poly-proline）、ＦＨ−２領域、プロリンリッチ領域からＣ末端領域で行った。アミノ酸の同一性は％で示した。フォルミンとカプチーノのＮー末端領域（＊で示した）はホモロジーが認められなかった。示した全ての配列は、中間部分にポリープロリンストレッチを持ち、Ｃ末端側の半分にホモロジーを持っている。
【図５】ノザンブロッティングによるマウスの種々の組織におけるｐ１４０ｍＤｉａの組織分布を示したＲＮＡブロット（電気泳動写真）である。
ｈｅａｒｔ：心臓、ｌｕｎｇ：肺、ｂｒａｉｎ：脳、ｋｉｄｎｅｙ：腎臓、ｔｅｓｔｉｓ：睾丸、ｓｋｅｌｅｔａｌ ｍｕｓｃｌｅ：骨格筋、ｔｈｙｍｕｓ：胸腺、ｓｐｌｅｅｎ：脾臓、ｌｉｖｅｒ：肝臓、ｓｔｏｍａｃｈ：胃、ｓｍａｌｌ ｉｎｔｅｓｔｉｎｅ：小腸、ｃｏｌｏｎ：結腸。
【図６】抗ｐ１４０ｍＤｉａ抗体の特異性を示した電気泳動写真である。
抗血清（ＡＰ５０）はツー・ハイブリッド・システムで得られたクローン５０がコードするペプチドに対するもので、その特異性を、ＩＰＴＧで誘導前（レーン１）および誘導後（レーン２）の組換えタンパク質を発現している大腸菌（Ｅ．ｃｏｌｉ）の全ライゼート、あるいはＳｗｉｓｓ ３Ｔ３細胞（３Ｔ３ ｃｅｌｌｓ）の全ライゼート（レーン３）に対するウエスタンブロッティングにより検討した。レーン４は細胞の全ライゼートをＣＢＢ染色したものを示している。
【図７】ＧＴＰγＳ結合型Ｒｈｏタンパク質によるｐ１４０ｍＤｉａの沈降を示した電気泳動写真である。Ｓｗｉｓｓ ３Ｔ３細胞の全ライゼートを、ＧＴＰγＳ結合型あるいはＧＤＰ結合型のＧＳＴ−Ｒｈｏ、ＧＳＴ−Ｒａｃ、ＧＳＴ−Ｃｄｃ４２あるいはＧＳＴとともにインキュベートした。結合したタンパク質をグルタチオンアガロースビーズで沈降させ、抗ｐ１４０ｍＤｉａ抗体を用いたイムノブロッティングにより解析した。レーン９（cell lysates）では、細胞の全ライゼート中のｐ１４０ｍＤｉａを抗体で検出した。
【図８】ｐ１４０ｍＤｉａとプロフィリンのインビトロでの結合を示した電気泳動写真である。Ｓｗｉｓｓ ３Ｔ３細胞ライゼートを、ＧＴＰγＳ−またはＧＤＰ結合型のＧＳＴ−Ｒｈｏタンパク質の存在下または非存在下（none）で、プロフィリンあるいはＢＳＡをコンジュゲート（immobilized ）したアガロースビーズとインキュベートし、結合タンパク質を沈降させた。沈降物（Pellet）および上清（Supernatant ）は、抗ｐ１４０ｍＤｉａ抗体を用いてイムノブロッティングにより解析した。
【図９】広がった線維芽細胞の膜ラッフルでのＲｈｏＡとｐ１４０ｍＤｉａおよびプロフィリンの共局在を示した写真（生物の形態の写真）である。（Ａ−Ｄ）ＨＴ１０８０ヒト線維肉腫細胞。（ＥとＦ）Ｓｗｉｓｓ ３Ｔ３マウス線維芽細胞。細胞を培養し、固定し、抗ｐ１４０ｍＤｉａ抗体（ＡとＥ）、抗ＲｈｏＡポリクローナル抗体（Ｃ）あるいはマウス抗プロフィリンモノクローナル抗体（ＢとＤ）又はローダミン−ファロイジン（Ｆ）いずれかで同時に細胞を染色し、標準的な蛍光顕微鏡写真を撮影した。
【図１０】ＰＭＡで刺激した広がったｓＭＤＣＫ細胞の膜ラッフルにおけるｍｙｃ標識化ＲｈｏＡ、ｐ１４０ｍＤｉａおよびプロフィリンの共局在化を示した写真（生物の形態の写真）である。休止期（Resting ）（Ａ−Ｃ）またはphorbol myristate acetate （Ｄ−Ｈ）で刺激した（PMA stimulation ）ｓＭＤＣＫ細胞を固定し、抗ｐ１４０ｍＤｉａ抗体（Ｂ、ＥおよびＨ）またはモノクローナル抗ｍｙｃ抗体（Ｃ、Ｆ）で染色した。Ｈには、過剰量の組換えペプチドで予め吸収させた（preabsorbed ）抗ｐ１４０抗血清を用いた染色が示されている。Ａ、Ｄ、Ｇには、位相差顕微鏡（phase contrast）を用いてそれぞれの細胞を撮影した顕微鏡写真が示されている。写真上段左から、Ａ、Ｂ、Ｃであり、写真中段左からＤ、Ｅ、Ｆであり、写真下段左からＧ、Ｈである。
【図１１】分裂期細胞の分裂溝におけるｐ１４０ｍＤｉａの濃縮を示した写真（生物の形態の写真）である。ＡおよびＢ：Ｓｗｉｓｓ ３Ｔ３細胞、ＣおよびＤ：ＨｅＬａ細胞。抗ｐ１４０ｍＤｉａ抗体（anti-p140 ）で染色（staining）された対数増殖期の細胞（ＡおよびＣ）およびＤＡＰＩ（Ｂ）または２Ｈ１１モノクローナル抗プロフィリン抗体（Ｄ）（anti-profilin ）で同時に染色したものを標準的な蛍光顕微鏡で撮影した。
【図１２】フィブロネクチンでコートされたビーズの回りにＲｈｏ依存的な様式で出現したＲｈｏＡおよびｐ１４０ｍＤｉａのクラスターを示した写真である。４８時間培養したＳｗｉｓｓ ３Ｔ３細胞を用いた。細胞を播き、フィブロネクチン（ＦＮ−coated）（ａ、ｂ）またはポリ−Ｌ−リジン（ＰＬＬ−coated）（ｃおよびｄ）でコートしたラテックス・ビーズとともに１５分間インキュベートした。細胞は固定後、抗ｐ１４０ｍＤｉａ抗体（anti-p140 ）（ａ、ｃ）または抗Ｒｈｏ抗体（anti-Rho）（ｂ、ｄ）で染色（staining）した。
【図１３】ＣＯＳ−７細胞におけるｐ１４０ｍＤｉａの一過性の発現を示した写真である。ＣＯＳ細胞はｐ１４０ｍＤｉａの発現ベクター単独（ＡおよびＢ）またはＣ３菌体外酵素の発現ベクター単独（ＣおよびＤ）でトランスフェクトした。Ｃ３菌体外酵素のトランスフェクションは、ＦＬＡＧエピトープを有したＣ３菌体外酵素発現様ベクターで実施した。細胞を固定し、抗ｐ１４０ｍＤｉａ抗体（anti-p140 ）単独（ＡおよびＣ）あるいは同時にアクチンをローダミン−ファロイジン（phalloidin）染色（staining）した（Ｂ、Ｄ）。[0001]
BACKGROUND OF THE INVENTION
Field of Invention
The present invention relates to a target protein of Rho protein, and more particularly to a protein involved in reconstitution of actin cytoskeleton.
[0002]
Background art
In the living body, a group of low molecular weight GTP-binding proteins (G proteins) having a molecular weight of 20,000 to 30,000 having no subunit structure exists. Currently, more than 50 members have already been found in the superfamily of low molecular weight G proteins from yeast to mammals. Low molecular weight G proteins can be roughly divided into four families, Ras, Rho, Rab, and others, based on amino acid sequence similarity. It has been clarified that this low molecular weight G protein controls various cell functions. For example, Ras protein indicates cell proliferation and differentiation, and Rho protein indicates cell shape change, cell adhesion, cell motility. Etc. are thought to be controlling each.
[0003]
Among them, Rho protein shows GDP / GTP binding ability and endogenous GTPase activity, and is related to cytoskeletal response to extracellular signals such as lysophosphatidic acid (LPA) and certain growth factors. Yes. When a stimulus is given to the inactive GDP-bound Rho protein, the GTP-bound Rho protein (hereinafter referred to as “active Rho protein”) is activated by the action of a GDP / GTP converting protein such as Smg GDS, Dbl or Ost. ”). And it is thought that stress fiber and adhesion spots are formed by this active Rho protein acting on the target protein, and cell adhesion and cell movement are induced (Experimental Medicine vol.12, No.8, 97-102 (1994), Takai, Y. et al. Trends Biochem. Sci., 20, 227-231 (1995)). On the other hand, the active Rho protein is converted to GDP-bound Rho protein by Rho protein endogenous GTPase. This protein that enhances the activity of endogenous GTPase is called GTPase activating protein (GAP) (Lamarche, N. & Hall, A. et al., TIG, 10, 436-440 (1994)).
[0004]
The amino acid sequences of Rho family proteins such as RhoA protein, RhoB protein, RhoC protein, Rac1 protein, Rac2 protein, Cdc42 protein are more than 50% similar to each other. This Rho family protein is involved in reactions that cause the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors. (AJ Ridley & A. Hall, Cell, 70, 389-399 (1992), AJ Ridley & A. Hall, EMBO J., 1353, 2600-2610 (1994)). In addition, Rho protein, which is a subfamily, is associated with cell morphological changes (HFParterson et al., J. Cell Biol., 111, 1001-1007 (1990)) and cell adhesion (Morii, N. et al., J. Biol. Chem. 267, 20921-20926 (1992), T. Tominaga et al., J. Cell Biol., 120, 1529-1537 (1993), Nusrat, A. et al., Proc, Natl. Acad. Sci USA, 92, 10629-10633 (1995), Landanna, C. et al., Science 271, 981-983 (1996)), cell motility (K. Takaishi et al., Oncogene, 9,273-279 (1994)) , Cytokinesis (K. Kishi et al., J. Cell Biol., 120, 1187-1195 (1993), I. Mabuchi et al., Zygote, 1,325-331 (1993)) It is thought to be related to the physiological function associated with the reorganization of In addition, Rho protein can be used for smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992), M. Noda et al., FEBS Lett., 367, 246-250 ( 1995), M. Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996) *), phosphatidylinositol 3-kinase (PI3-kinase) (J. Zhang et al. , J. Biol. Chem., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI 4,5-kinase) (LD Chong et al., Cell, 79, 507-513 ( 1994)) and c-fos expression (CS Hill et al., Cell, 81, 1159-1170 (1995)) has been suggested to be involved.
[0005]
Recently, it has been found that when a Rho protein partially substituted with an amino acid sequence is introduced into a cell, Ras-dependent tumor formation is suppressed, and the Rho protein is transformed into cells by Ras, that is, It has been shown to play an important role in tumorigenesis (GCPrendergast et al., Oncogene, 10, 2289-2296 (1995), Khosravi-Far, R., et al., Mol. Cell. Biol., 15, 6443-6453 (1995), R. Qiu et al., Proc. Natl. Acad. Sci. USA, 92, 11781-11785 (1995), and Lebowitz, P. et al., Mol. Cell Biol., 15, 6613-6622 (1995)). It has also been clarified that cells are transformed when the GDP / GTP converting protein of Rho protein is mutated (Collard, J., Int. J. Oncol., 8, 131-138 (1996)), Hart, M. et al., J. Biol Chem., 269, 62-65 (1994), Horii, Y. et al., EMBO J., 13,4776-4786 (1994)). Furthermore, it has been clarified that Rho protein is involved in cancer cell invasion, that is, cancer metastasis (Yoshioka, K. et al., FEBS Lett., 372, 25-28 (1995)). Cancer cell invasion is closely related to changes in the cell adhesion ability of cancer cells, but Rho protein has been shown to be involved in cell adhesion (see Morii, N. et al. (1992). ), Tominaga, T. et al. (1993), Nusrat, A. et al. (1995), Landanna, C. et al. (1996)).
[0006]
In addition, the involvement of phosphoinositide kinase in Rho signaling has been reported. Rho (Chong, LD et al., Cell, 79, 507-513 (1994)) and other members of the Rho family low molecular weight G proteins Rac (Hartwig, JH et al., Cell, 82, 643-653 ( 1995)) has been demonstrated to stimulate the synthesis of phosphatidylinositol bisphosphate (PIP2) in different cell lines. Since PIP2 binding is thought to regulate the function of many actin-related proteins (Janmey, PA, Ann. Rev. Physiol., 56, 169-191 (1994)), it is focal by its local biosynthesis. Actin reconstitution is thought to be promoted. One protein regulated by PIP2 is profilin, which forms a complex with actin monomers and dissociates actin upon binding to PIP2. Profilin also promotes actin filament conjugation in the presence of thymosin β4 (Pantaloni, D., & Crlier, M-F, Cell, 75, 1007-1014 (1993)). Therefore, accumulation of profilin at the adhesion site is considered to be important in actin reconstitution (Theriot, J.A. & Mitchison, T.J., Cell, 75, 835-838 (1993)).
[0007]
The actin cytoskeleton plays an important role in cell motility, morphology, phagocytosis, and cytokinesis. It is reconfigured spatially and dynamically, providing power for the transformation and cell surface movement of most eukaryotic cells. Actin rearrangement is rapidly triggered by extracellular stimuli, and a series of actin-binding proteins are thought to act in concert on actin filament polymerization, cross-linking and immobilization. The low molecular weight G protein Rho is associated with a number of actin-dependent cellular processes such as platelet aggregation (Morii, N. et al., J. Biol. Chem., 267, 20921-20926 (1992)), lymphocyte adhesion (Tominaga, T. et al., J. Cell. Biol., 120, 1529-1537 (1993)), enhanced cell motility (Takaishi, K. et al., Oncogene, 11, 39-48 (1995)), and Contractile ring formation and cytokinesis (Kishi, K. et al., J. Cell Biol., 120, 1187-1195 (1993), and Mabuchi, I. et al., Zygote, 1, 325-331 (1993) ) Is required. When the Rho protein is microinjected in cultured fibroblasts, it rapidly induces the formation of actin stress fibers and focal adhesion. Conversely, inactivation of Rho by the C3 extracellular enzyme (ADP-ribosyltransferase) of botulinum inhibits this process (Ridley, AJ & Hall, A., Cell, 70, 389-399 ( 1992)). Treatment of C3 extracellular enzyme also inhibits focal adhesion kinase (FAK) and paxillin lysophosphatidic acid (LPA)-, endothelin- or GTPγS-induced tyrosine phosphorylation (Kumagai). , N. et al., J. Biol. Chem., 268, 24535-24538 (1993), Rankin, S. et al., FEBS Lett., 354, 315-319 (1994), Ridley, AJ & Hall, A., Cell, 70, 389-399 (1992), and Seckl, MJ et al., J. Biol. Chem. 270, 6984-6990 (1995)). These results indicate that Rho protein regulates signal transduction pathways that link extracellular stimuli and actin cytoskeleton reorganization.
[0008]
The Rho protein has a large number of target molecules, and is thought to regulate the above-described numerous signal transduction pathways. Most recently, several candidate proteins have been reported in mammals. These proteins include protein kinase N (PKN) (Watanabe, G. et al., Science 271, 645-648 (1996); Amano, M. et al., Sceince 271, 648-650 (1996)), lophyrin. (Watanabe, G. et al., Science 271, 645-648 (1996)), Citron (Madaule, P. et al., FEBS Lett. 377, 243-248 (1995)), ROKα (Leung, T. et al., J. Biol. Chem. 270, 29051-29054 (1995)), Rho binding kinase (Matsui, T. et al., EMBO J. 15, 1885-1893 (1996)), rotekin (Reid, T. et al., J. Biol. Chem., 271, 9816-9822 (1996)), myosin light chain phosphatase (Kimura, K. et al., Science, 273, 245-248 (1996)). Both of these proteins bind to GTP-bound RhoA protein (however, citron alone also binds to GTP-bound Rac1 protein).
[0009]
However, to the best of the inventors' knowledge, no report has been made regarding Rho proteins and Rho target proteins that bind to profilin that regulates the reorganization of the actin cytoskeleton.
[0010]
SUMMARY OF THE INVENTION
The present inventor has now identified a target protein (p140mDia) of the Rho protein using a yeast two-hybrid system. It is a Drosophila diaphanous mammalian homolog required for cytokinesis, containing a stretch of repetitive poly-proline and a formin homology (FH2) domain. p140mDia selectively binds through its amino-terminal region of GTP-linked Rho and binds to profilin, an actin-binding protein. p140mDia, profilin and Rho protein are localized in the ruffle of the spread fibroblast membrane, and in the cleavage furrow of the dividing cell, and the original subbead by latex beads coated with fibronectin. Recruited to the plasma membrane. These results suggested that one of the mechanisms of induction of actin reconstitution by Rho protein is the recruitment of profilactin complex at specific sites of cells via p140mDia. The present invention is based on such knowledge.
[0011]
Accordingly, an object of the present invention is to provide a Rho target protein having an active Rho protein binding ability and binding to profilin.
[0012]
Further, according to the present invention, a base sequence encoding the protein, a vector containing the base sequence, a host cell transformed with the vector, a method for producing the protein, an antibody against the protein, and an active Rho protein The purpose is to provide a screening method for inhibiting the binding to the target protein.
[0013]
The protein according to the present invention has an active Rho protein binding ability and a profilin binding ability.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Definition
In the present invention, “amino acid” is used in the meaning including both optical isomers, that is, L-form and D-form. Therefore, in the present invention, the “peptide” means not only a peptide composed only of L-form amino acids but also a peptide containing a part or all of D-form amino acids.
[0015]
In the present invention, the “amino acid” means not only 20 kinds of α-amino acids constituting natural proteins, but also other α-amino acids, and β-, γ-, δ-amino acids and non-natural amino acids. It shall be used with the meaning including etc. Therefore, the amino acids that are substituted in the peptide or inserted into the peptide as described below are not limited to the 20 α-amino acids constituting the natural protein, and other α-amino acids are not included. In addition, β-, γ-, δ-amino acids and non-natural amino acids may be used. Examples of such β-, γ-, or δ-amino acids include β-alanine, γ-aminobutyric acid, ornithine, and amino acids other than those constituting natural proteins or non-natural amino acids include 3, 4 -Dihydroxyphenylalanine, phenylglycine, cyclohexylglycine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, dipecotic acid, and the like.
[0016]
Further, in the present specification, the term “protein according to the present invention” is used in the meaning including its derivatives.
[0017]
Furthermore, in this specification, the “base sequence” means both a DNA sequence and an RNA sequence.
[0018]
protein
The protein according to the present invention is a protein having an active Rho protein binding ability and a profilin binding ability or a derivative thereof. Here, examples of the Rho protein include a RhoA protein, a RhoB protein, a RhoC protein, and a RhoG protein.
[0019]
In the present invention, the “protein having an active Rho protein binding ability” refers to a protein that is evaluated by a person skilled in the art to be recognized as binding to the active Rho protein. For example, the same conditions as in Examples 1 and 5 It means a protein that is evaluated as binding to the active Rho protein in the experiment.
[0020]
The protein according to the present invention has a poly-proline region and an FH2 region in addition to the Rho protein binding region (Example 2). In addition, the protein according to the present invention is characterized by being strongly expressed in mouse lung, testis, thymus, liver, and stomach.
[0021]
Proteins having active Rho binding ability and profilin binding ability also include p140mDia isoform and homologue (see Example 3).
[0022]
In the present invention, the “protein having the ability to bind profilin” refers to a protein that is evaluated by a person skilled in the art to be recognized as binding to profilin. For example, when the experiment is performed under the same conditions as in Example 6, It shall mean a protein that is evaluated as binding to phosphorus.
[0023]
In the present specification, the Rho protein is intended to include a Rho protein modified so that the binding between the Rho protein and the protein according to the present invention is not substantially impaired. As such a modified Rho protein, a RhoA mutant (RhoA) in which the 14th amino acid is replaced with valine.^Val14).
[0024]
The origin of the protein according to the present invention is not particularly limited, and it may be derived from mammals including mice or may be derived from other sources.
[0025]
When derived from a mouse, the molecular weight of the protein according to the present invention is about 160 kD as measured by SDS-PAGE.
[0026]
The protein according to the present invention can be obtained, for example, by expressing the cDNA sequence of SEQ ID NO: 2 in a host (Examples 4 and 9). Acquisition of the sequence of SEQ ID NO: 2 and expression in the host will be described later.
[0027]
As used herein, “protein derivative” means the amino group at the amino terminus (N terminus) of a protein or part or all of the amino group of the side chain of each amino acid, and / or the carboxyl terminus (C terminus) of a peptide. Or a part or all of the carboxyl group of the side chain of each amino acid, and / or a functional group other than the amino group and carboxyl group of the side chain of each amino acid of the peptide (eg, hydrogen group, thiol group, amide group) Etc.) are partly or wholly modified with other suitable substituents. Modification with other appropriate substituents is performed for the purpose of, for example, protecting functional groups present in the peptide, improving safety and tissue migration, or enhancing activity.
[0028]
Specifically, protein derivatives include:
(1) The amino group at the amino terminus (N-terminus) of the protein or the partial or all of the amino groups in the side chain of each amino acid are substituted or unsubstituted alkyl groups (straight chain, branched chain, or cyclic). (For example, methyl group, ethyl group, propyl group, isopropyl group, isobutyl group, butyl group, t-butyl group, cyclopropyl group, cyclohexyl group, benzyl group), substituted or unsubstituted acyl group (for example, , Formyl group, acetyl group, caproyl group, cyclohexylcarbonyl group, benzoyl group, phthaloyl group, tosyl group, nicotinoyl group, piperidinecarbonyl group), urethane type protective group (for example, p-nitrobenzyloxycarbonyl group, p-methoxybenzyl) Oxycarbonyl group, p-biphenylisopropyloxycarbonyl group, t-but Aryloxycarbonyl group) or urea-type substituents (e.g., those substituted methylaminocarbonyl group, a phenylcarbonyl group, the cyclohexylamino group) and the like, as well as
(2) A carboxyl group at the carboxyl terminus (C terminus) of the protein or a part or all of the carboxyl group of the side chain of each amino acid is modified with an ester type (for example, the hydrogen atom is methyl, ethyl, Isopropyl, cyclohexyl, phenyl, benzyl, t-butyl, those substituted by 4-picolyl), those having an amide type modification (eg, unsubstituted amide, C1-C6 alkylamide (eg, methylamide, ethylamide, Forming isopropylamide), and
(3) A part or all of functional groups (for example, a hydrogen group, a thiol group, an amino group, etc.) other than the amino group and carboxyl group of the side chain of each amino acid of protein are the same as the above-described amino group or Examples thereof include those modified with a trityl group and the like.
[0029]
Examples of the protein according to the present invention include a protein consisting of the amino acid sequence of SEQ ID NO: 1 (referred to herein as “p140mDia”) and derivatives thereof. The amino acid sequence of SEQ ID NO: 1 was determined from a DNA sequence (cDNA sequence) obtained from a cDNA library derived from mouse brain and embryo. This cDNA library is commercially available or Kakizuka, A. et al. (1993), cDNA Libraty Construction (edited by Stein, C. and Holland, P.) Essential Developmental Biology: A Practical Approach, pp223-232, IRL Press , Oxford can be used. The cDNA sequence can be obtained by using an oligonucleotide corresponding to the thick underline (clone 50) in FIG. 3 as a probe.
[0030]
An example of the protein according to the present invention is the amino acid sequence of SEQ ID NO: 1, wherein one or more amino acid sequences are added and / or inserted into the amino acid sequence of SEQ ID NO: 1, and / or the amino acid of SEQ ID NO: 1 Examples include proteins in which one or more amino acids in the sequence are substituted and / or deleted, having active Rho protein binding ability and profilin binding ability. That is, the addition, insertion, substitution, and deletion referred to here are the active Rho protein binding ability and profilin binding ability of the protein consisting of the amino acid sequence of SEQ ID NO: 1. Things that do not damage.
[0031]
According to another aspect of the present invention, there is provided a protein having an amino acid sequence of 63 to 260 of SEQ ID NO: 1 (Rho protein binding region) and an amino acid sequence of 571 to 737 (profylline binding region).
[0032]
The protein according to the present invention has an active Rho protein binding ability and a profilin binding ability. In addition, Rho protein is closely involved in the expression of cell functions such as tumor formation and metastasis, cell morphology, cell motility, cell adhesion, cytokinesis, etc. (Takai, Y., et al., GC Prendergast. et al., Khosravi-Far, R., et al., R. Qiu et al., Lebowitz, P., et al., and Yoshioka, K. et al.). Therefore, the protein according to the present invention is considered useful for elucidating the mechanism of tumor formation and metastasis.
[0033]
Moreover, according to the postscript Example, it was shown that the protein by this invention is concerned in cell adhesion. Therefore, the protein according to the present invention is used to infiltrate and metastasize cancer cells, aggregate and activate leukocytes (T lymphocytes, B lymphocytes, neutrophils, eosinophils, basophils, macrophages, etc.) and platelets. It is useful for elucidating functions such as aggregation and activation.
[0034]
Furthermore, according to the examples described later, it was shown that the protein according to the present invention is involved in cell division (particularly cell proliferation). Therefore, the protein according to the present invention is useful for elucidating functions such as cancer growth.
[0035]
Base sequence
According to the present invention, a base sequence encoding the protein according to the present invention is provided. A typical sequence of this base sequence has a part or all of the DNA sequence of SEQ ID NO: 2.
[0036]
The DNA sequence of SEQ ID NO: 2 was obtained from a mouse brain-derived cDNA library as described above. The sequence of Nos. 94 to 3861 in SEQ ID NO: 2 corresponds to the open reading frame of p140 mDia.
[0037]
If the amino acid sequence of the protein according to the present invention is given, the base sequence encoding it can be easily determined, and various base sequences encoding the amino acid sequence described in SEQ ID NO: 1 can be selected. Accordingly, the base sequence encoding the protein according to the present invention includes a DNA sequence encoding the same amino acid and a codon in a degenerate relationship in addition to part or all of the DNA sequence set forth in SEQ ID NO: 2. As well as RNA sequences corresponding to these sequences.
[0038]
The base sequence according to the present invention may be naturally derived or fully synthesized. Further, it may be synthesized by using a part of a natural product. The nucleotide sequence is obtained by a method commonly used in the field of genetic engineering from a chromosomal library or a cDNA library, for example, a method of screening using an appropriate DNA probe prepared based on partial amino acid sequence information, etc. be able to. The nucleotide sequence according to the present invention can be obtained by screening a mouse-derived cDNA library. A commercially available cDNA library can be used. In addition, screening can be performed by using an oligonucleotide corresponding to the thick underline (clone 50) in FIG. 3 as a probe.
[0039]
When the base sequence is derived from nature, its origin is not particularly limited, and it may be derived from mammals including mice or may be derived from other sources.
[0040]
Examples of the base sequence encoding the protein according to the present invention include the base sequence of SEQ ID NO: 2 and a part of the DNA sequence of SEQ ID NO: 2 (for example, 94-3861 of SEQ ID NO: 2 (corresponding to an open reading frame), 280-800 873 (corresponding to Rho protein binding region) and 1804 to 2304 (corresponding to profilin binding region).
[0041]
Vectors and host cells
According to the present invention, there is provided a vector comprising the above-described base sequence according to the present invention in a state capable of replicating in a host cell and expressing a protein encoded by the base sequence. Furthermore, according to the present invention, a host cell transformed with this vector is provided. This host-vector system is not particularly limited, and a fusion protein expression system with other proteins can be used. Examples of the fusion protein expression system include those using MBP (maltose binding protein), GST (glutathione S transferase), HA (hemaglutinin), polyhistidine, myc, Fas and the like.
[0042]
Examples of vectors include plasmid vectors (for example, expression vectors in prokaryotic cells, yeast, insect cell animal cells, etc.), virus vectors (for example, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus vectors). , HIV vectors, baculovirus vectors), liposome vectors (for example, cationic liposome vectors) and the like.
[0043]
In order to express a desired protein by actually introducing the vector according to the present invention into a host cell, in addition to the base sequence according to the present invention described above, a sequence for controlling the expression and a host cell are selected. Or a genetic marker. Further, this vector may contain the base sequence according to the present invention in a repeated form (for example, in tandem). These may be present in a vector according to a conventional method, and a host cell transformation method using this vector may be any one commonly used in this field.
[0044]
As the procedure and method for constructing the vector according to the present invention, those conventionally used in the field of genetic engineering can be used.
[0045]
Examples of host cells include Escherichia coli, yeast, insect cells, and animal cells (eg, COS cells, lymphocytes, fibroblasts, CHO cells, blood cells, tumor cells, etc.).
[0046]
The transformed host cell is cultured in an appropriate medium, and the above-described protein according to the present invention can be obtained from the culture. Therefore, according to another aspect of the present invention, a method for producing a protein according to the present invention is provided. The culture of the transformed host cell and its conditions may be essentially similar to that for the cell used. In addition, recovery and purification of the protein according to the present invention from the culture medium can also be performed according to a conventional method.
[0047]
Screening method
According to the present invention,
(1) A substance to be screened is present in a screening system comprising an active Rho protein and the protein according to the present invention, and
(2) Measuring the degree of inhibition of binding between the active Rho protein and the protein according to the present invention.
A method for screening a substance that inhibits the binding between an active Rho protein and a protein according to the present invention is provided.
[0048]
According to the invention,
(1) A substance to be screened is present in a screening system containing profilin and the protein according to the present invention, and
(2) Measuring the degree of inhibition of binding between profilin and the protein according to the present invention.
A method for screening a substance that inhibits the binding of profilin and the protein according to the present invention is provided.
[0049]
Here, as a method of “measuring the degree of inhibition of binding”, a method of measuring using an immunoblot with an antibody against a protein according to the present invention, a two hybrid system (M. Kawabata Experimental Medicine 13 , 2111-2120 (1995), ABVojetk et al. Cell 74, 205-214 (1993)), and measurement using an overlay assay (Ishizaki, T. et al., EMBO J., 15, 1885- 1893 (1996)), a method using a protein translated in vitro (Shibata, H. et al., FEBS Lett. 385, 221-224 (1996)), Amano, M., et al., Science 271 , 648-650 (1996), and the like, for example, the degree of inhibition of binding is measured according to the methods described in Examples 1, 5, and 6. be able to.
[0050]
In the present specification, “measuring the degree of inhibition of binding” is used to mean the measurement of the presence or absence of binding. The term “screening” is used to mean including an assay.
[0051]
The screening system may be either a cell system or a cell-free system. Examples of the cell system include yeast cells, COS cells, E. coli, insect cells, nematode cells, lymphocytes, fibroblasts, CHO cells, blood Lineage cells, and tumor cells.
[0052]
The screening target is not particularly limited, and examples thereof include peptides, peptide analogs, microbial culture solutions, and organic compounds.
[0053]
antibody
The antibody according to the present invention can be produced by a method commonly used in the art. For example, the peptide indicated by the bold line in FIG. 3 is injected into an animal body (eg, rabbit, goat, rat, mouse, sheep) together with any carrier (eg, bovine serum albumin), and after a certain period of time, the animal's It can be obtained by purifying serum.
[0054]
The peptide indicated by the bold line in FIG. 3 is a part of p140mDia. Thus, this polyclonal antibody response (ie, immune response) is an indicator of the presence of p140mDia. Therefore, according to another aspect of the present invention, there are provided a protein recognized by the antibody and a protein according to the present invention recognized by the antibody.
[0055]
According to yet another aspect of the invention,
(1) the substance to be detected is present in a detection system comprising an antibody according to the invention, and
(2) There is provided a method for detecting a substance recognized by an antibody according to the present invention, comprising measuring the degree of reaction between the substance to be detected and the antibody according to the present invention.
[0056]
Methods for measuring the degree of reaction with the antibody according to the present invention include ELISA, radioimmunoassay, western blotting, immunoprecipitation, and fluorescent antibody (eg, monoclonal antibody experiment manual, Kodansha, (1987) The degree of reaction can be measured according to the methods described in Examples 4 to 6, for example. In the present invention, “measuring the degree of reaction” also includes measuring the presence or absence of a reaction.
[0057]
The detection system in the detection method according to the present invention may contain, for example, latex particles in addition to the polyclonal antibody according to the present invention.
Furthermore, according to the present invention, there is provided a detection kit for a substance that specifically reacts with the antibody, comprising the antibody according to the present invention. Here, the “detection kit” includes a detection reagent for a disease or the like in which the protein according to the present invention is involved, a diagnostic reagent or a diagnostic kit.
[0058]
Specific examples of the detection method according to the present invention include:
(1) The substance to be detected is present in a detection system containing latex particles carrying the antibody according to the present invention, and
(2) Measuring the degree of agglutination of the latex particles.
And a method for detecting a substance that specifically reacts with the antibody.
The degree of the agglutination reaction of the latex particles can be measured by an optical measurement method such as a turbidimetric method or a specific wax method.
[0059]
The detection kit according to the present invention may contain latex particles as in the above detection system. In this case, the latex particles may carry antibodies on the surface.
Further, the detection kit according to the present invention further containing latex particles can be used in a manner as shown in the specific examples of the detection method described above.
Examples of the substance to be detected include body fluids derived from animals including humans (for example, serum, blood, etc.), urine, feces, tissue sections, cells (for example, tumor cells, etc.) and the like.
[0060]
Examples of the substance that reacts with the polyclonal antibody according to the present invention include the protein according to the present invention (sequence of SEQ ID NO: 1).
[0061]
【Example】
Example 1  Screening of RhoA binding peptide fragments using yeast two-hybrid system
N fused to LexA DNA binding protein¹⁹-RhoAΔC (C-terminal Ala¹⁸¹RhoA truncated at^Asn19) As a bite, a new Rho binding protein was screened by the yeast two-hybrid system. The yeast two-hybrid system is based on the method described previously (Vojtek, A. et al., Cell 74, 205-214 (1993); Modaule, P. et al., FEBS Lett., 377, 243-248 ( 1995); Watanabe, G. et al., Science 271, 645-648 (1996); Reid, T. et al., J. Biol. Chem., 271, 13556-13560 (1996)) .
[0062]
Plasmids pBTM116 and pVP16 (Vojtek, A. et al., Cell 74, 205-214 (1993)) used in the two-hybrid system were obtained from Stan Hollenberg, Rolf Sternglanz, Stan Fields and Paul Bartel. pBTM-RhoA (pBTM116 containing cDNA encoding RhoA) was prepared according to previously described methods (Watanabe, G. et al., Science, 271, 645-648 (1996)). Val on RhoA¹⁴Or Asn¹⁹To generate the mutation, the BamHI-EcoRV fragment of pGEX-RhoA encoding the N-terminus of RhoA was inserted into pBluescript (Stratagene) and the method of Kunkel (Kunkel, T., Proc. Natl. Acad. Sci. USA, 82, 488-492 (1985)). The corresponding wild-type fragment of pGEX-RhoA (Morii, N. et al., J. Biol. Chem., 268, 27160-27163 (1993)) was then replaced with each mutagenized fragment, and pGEX-V¹⁴RhoA and pGEX-N¹⁹RhoA was prepared.
[0063]
pGEX-V¹⁴RhoA and pGEX-N¹⁹A BamHI-EcoRI fragment encoding the full-length coding region of mutant RhoA was excised from RhoA, inserted into pBTM116, and pBTM-V¹⁴RhoA and pBTM-N¹⁹RhoA was obtained. Ala of these mutants¹⁸¹PBTM116 (pBTM-V inserted with a C-terminal deletion mutant in¹⁴RhoAΔC and pBTM-N¹⁹RhoAΔC) was prepared according to previously described methods (Reid, T. et al., J. Biol. Chem., (1996)).
[0064]
pBTM-N¹⁹The yeast L40 strain (Mata trp1 leu2 his3 LYS :: lexA-HIS3 URA3 :: lexA-lacZ) (Vojtek, A. et al., Cell 74, 205-214 (1993)) carrying RhoAΔC was obtained from mouse embryo cDNA. PVP16 fused with the library (Kakizuka, A. et al. (1993), cDNA Libraty Construction (edited by Stein, C. and Holland, P.) Essential Developmental Biology: A Practical Approach, pp223-232, IRL Press, Oxford) (Vojtek, A. et al., Cell 74, 205-214 (1993)). Initial transformation efficiency is 2.2 × 10⁷Which means that it has divided 7 times during 6 hours of culture before plating on HIS (-) plates. 1.5 × 10⁸Of these transformants, 978 clones were isolated as His + and lacZ positive, and 220 clones were isolated to eliminate the byte plasmid. Interaction with other proteins was evaluated by crossing with yeast strain AMR70 with various test bytes. Of 220 clones, 55 clones are N¹⁹-RhoAΔC showed positive lacZ activity and lamin used as a negative control showed negative. 55 clones isolated by this procedure have cDNA inserts of the same size and some of them were sequenced and found to have the same nucleotide sequence.
[0065]
These clones were bred to AMR70 with LexA fused to various RhoA mutants. All of them are RhoA^Val14And strongly, RhoA^Asn19And weakly bound to wild-type RhoA almost negligibly, but they are N¹⁹-Strongly bound to RhoAΔC or wild type RhoAΔC. Similar results were confirmed by the following experiment.
[0066]
In order to test the specificity of the interaction in the two-hybrid system, the pVP16 plasmid recovered from yeast clone 50 (pVP-cl.50) together with the pBTM116 plasmid carrying cDNAs of various proteins into the L40 strain. Cotransformed and the interaction was tested by lacZ assay. PBTM-RhoAΔ and pBTM-lamin (Watanabe, G. et al., Science 271, 645-648 (1996)), pBTM-Rac and pBTM-Cdc42H (Reid, T. et al., J. Biol. Chem., 271, 13556-13560 (1996)) has been described previously.
[0067]
As a result, the specificity of binding was confirmed by cotransforming the L40 strain with plasmids recovered from a representative clone (clone 50) and various LexA-mutant RhoA fusion constructs (FIG. 1). ). That is, when the L40 strain was cotransformed with the plasmid recovered from clone 50) and the LexA-Rac fusion construct or LexA-Cdc42H fusion construct, the peptide encoded by this clone was specific for both Rac and Cdc42H. It turns out that it doesn't bind to. These results indicate that the peptide encoded by this clone specifically binds to RhoA.
[0068]
Example 2  Cloning of full length cDNA of p140mDia
In order to obtain the full-length coding sequence, the present inventors used a 0.6 Kbp cDNA insert (from pVP-cl.50) obtained from clone 50 as a probe.³²Using P-labeled 0.6 kbp cDNA inserts, two mouse brain libraries (936309 library (Stratagene) made in λZAP II) and ML3000a library (Clontech) made in λgt-10 )), And then using the two cDNA fragments obtained from this screen as probes, the mouse embryo library (Kakizuka, A. et al. (1993), cDNA Libraty Construction (Stein, C. and Holland, P. Edit) Essential Developmental Biology: A Practical Approach, pp223-232, IRL Press, Oxford).
[0069]
By this approach, six overlapping clones were isolated (Figure 2). One positive clone (clone 502) and two positive clones (clone 503 and 504) were obtained from the former and latter libraries, respectively. Clones E51, E52, and E73 were isolated from a mouse embryo library using the 5 'portion of 504 and the 3' portion of 503 as probes.
[0070]
The nucleotide sequences of these clones were determined by the dideoxy chain termination method, and from these clones containing a 3,765 bp open reading frame encoding a 1,255 amino acid protein with a calculated molecular weight of 139,336 Da, The complete cDNA sequence was determined (Figure 3). The initial cDNA obtained from clone 50 encodes a 198 amino acid sequence (amino acids 63-260) containing an N-terminal Rho-binding domain. Between amino acids 571-737 in the middle of the molecule, there is a proline-rich region characterized by a 14-repeat structure of IPPPPPLPG (G / S / A / V), that is, a homologous sequence. Comparison of the deduced amino acid sequence with other sequences on the database identified several homologous proteins, all of which share a proline-rich region (Figure 4). These proteins belong to a family of formin-like molecules that have two homologous regions, a proline-rich FH-1 region and an FH-2 region. The FH-2 region was also found between amino acids 945-1010 in the putative sequence. The protein with the highest homology to this sequence is the Drosophila diaphanous (a protein characterized by being required for cytokinesis), which is N- to the identified protein. It showed about 30% identity in the terminal proline-rich region and about 39% identity in the C-terminal region of that region. It also showed about 32% and 57% homology to the identified proteins in each putative Rho binding domain and FH-2 region. Based on these results, we conclude that the protein we have identified is a mammalian homologue of the Drosophila diaphanos protein, and this protein is identified as p140mDia (mammalian diaphanos (mamarianDia(hereinafter referred to as “p140mDia”).
[0071]
p140mDia also showed significant homology to the entire region of Bni1p involved in budding of yeast (Saccharomyces cerevisiae) cells. On the other hand, p140mDia showed homology in the C-terminal half region to formin and Drosophila capuccino. The Rho binding domain of p140mDia was mapped to the N-terminal part (described above), but no sequence similar to the Rho binding domain was found in formin or capsino. This suggests that these proteins (formin and capsino, which do not contain a Rho binding domain) perform the same function as proteins identified in the cell, but only p140mDia, Diaphanos and Bnilp function in a Rho-dependent manner. It was suggested that it is effective.
[0072]
Example 3  Search for tissues expressing p140mDia
Northern blot analysis was performed to search for tissues expressing p140mDia. Standard techniques (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring) using oligo-dT latex beads (Pharmacia Biotech) from several tissues of mature mice. According to Harbor Laboratory Press, Cold Spring Harbor)⁺RNA was prepared. 6 μg of each poly (A)⁺RNA was separated on a 1.0% agarose gel containing 2.1% formaldehyde and transferred to a Biodyne A filter (Pall BioSuport). Then filter³²P-labeled cl. Hybridization with 50 0.6 Kbp cDNA fragments. Finally, the filters were washed with 0.4 × SSC and 0.1% SDS at 65 ° C. and subjected to autoradiography.
[0073]
As a result, a major 6.3 kb transcript was ubiquitously detected in all tissues examined, but particularly strong expression was detected in lung, testis, thymus, liver and stomach. In addition, another transcript of 5 kb was found in testis and lung (FIG. 5).
[0074]
Example 4  Preparation of anti-p140Dia antibody
In order to investigate the localization of p140mDia in cells and the binding to other proteins, the peptide encoded by cDNA of clone 50 (including amino acid sequences 63 to 260 of SEQ ID NO: 1) was used as a His-labeled protein. By expressing and preparing an antibody against this peptide, a specific antibody against p140mDia was obtained. Specifically, the experiment was performed according to the following method.
[0075]
pVP-cl. Using the BamHI and EcoRI sites adjacent to the 50 cDNA inserts, cl. 50 cDNAs were ligated into pQE11 (QIAGEN) and pGEX-3X (Pharmacia) vectors. His6 labeled cl. 50 was expressed in E. coli strain JM109 and purified with Ni-NTA resin (QIAGEN) according to the manufacturer's protocol. The purified protein was mixed with Freund's adjuvant and injected into rabbits. According to the method described in Reid, T. et al., J. Biol. Chem., 271, 13556-13560 (1992), GST-cl. The antibody was affinity purified using 50 fusion proteins. Specifically, GST-cl. Inclusion bodies containing 50 were isolated from E. coli, solubilized in Laemmli buffer, separated by SDS-PAGE and transferred to a nitrocellulose membrane. GST-cl. 50 bands were cut as a strip, and the antibody absorbed in this strip was treated with 100 mM glycine-HCl buffer (pH 2.3), 100 mM monoethanolamine buffer (pH 11.5), and 10% 1,4-dioxane at 4 ° C. Was eluted successively with 100 mM glycine-HCl (pH 2.5). The eluate was immediately neutralized with 0.25 volumes of 250 mM sodium phosphate buffer (pH 8.8 for the first and third eluates and pH 7.0 for the second eluate). These eluates were combined and used as affinity purified antibody AP50. For the control immunofluorescence test, aliquots of AP50 were serially separated into 5 pieces of GST-cl. AP50 solution depleted of antibody was prepared by incubating with 50 blot membrane.
[0076]
The specificity of the resulting polyclonal antibody AP50 was confirmed by its reactivity with the expected molecular weight of 50 kDa protein and its degradation product found in E. coli lysate after IPTG induction (Fig. 6, lanes 1 and 2). The AP50 antibody detected a single protein of approximately 160 kDa in Swiss 3T3 cell lysate, which clearly shows endogenous p140 mDia (lanes 3 and 4).
[0077]
Example 5  Binding of Rho protein and p140mDia in vitro detection
Using AP50 antibody, the binding of wild-type Rho family low molecular weight G protein and native p140mDia was examined in vitro. Specifically, the experiment was performed according to the method described below.
[0078]
The preparation of GDP or GTP-conjugated GST-low molecular weight G protein (GST-Rho, Rac and Cdc42H) was prepared by the method described previously (Reid, T. et al., J. Biol. Chem., 271, 13556- 13560 (1992)). 1x10⁷Cell confluent Swiss 3T3 cells are collected and 3.2 ml of buffer A [10 mM Mes (pH 6.5), 150 mM NaCl, 2 mM MgCl₂, 0.5 mM EDTA, 0.5% Triton X-100, 5 mM DTT, 1 mM PMSF, 5 μg / ml leupeptin] was disrupted by sonication (5 sec, 4 times). The sonicated homogenate was centrifuged at 10,000 g for 20 minutes and the supernatant was saved. Loading each nucleotide by incubating 10 μM GST-low molecular weight G protein with 1 mM nucleotides according to previously described methods (Ishizaki, T. et al., EMBO J., 15, 1885-1893 (1996)). Carried out. 1/10 of the supernatant was then incubated with each GST-low molecular weight G protein loaded with 400 pmol of GTPγS or GDP. After 30 minutes incubation at 30 ° C., 5 μl glutathione-sepharose was added to the solution and the mixture was incubated at 4 ° C. for 1 hour. The immune complex recovered by centrifugation was washed twice with 1 ml of buffer A and boiled in Lemmli sample buffer. The solubilized extract was subjected to SDS-PAGE, and the separated protein was transferred to a PVDF membrane. According to a procedure described in the past (Kumagai, N. et al., J. Biol. Chem., 268, 4535-24538 (1993)), anti-cl. Immunoblotting was performed using 50 antiserum AP50.
[0079]
The result was as shown in FIG. p140mDia was precipitated only by GTPγS-bound Rho, but not by its GDP-bound Rho, or GTPγS-bound Rac or Cdc42. These results support the results in the two-hybrid system and indicate that p140mDia selectively binds to active Rho.
[0080]
Example 6  Detection of binding of profilin to p140mDia in vitrop140mDia has a repeating structure of stretches of poly-proline, and profilin has been shown to bind to poly-L-proline sequences. Therefore, the present inventor examined whether profilin binds to p140mDia and whether this binding depends on Rho. Specifically, the experiment was performed according to the method described below.
[0081]
First, human platelet profilin was purified by poly-L-proline affinity chromatography according to a method described in the past (Janmey, P.A., Ann. Rev. Physiol., 56, 169-191 (1994)). Specifically, 250 mg of poly-L-proline (PLP, molecular weight 12,000, Sigma) was bound to CNBr activated Sepharose 4B (Pharmacia). Washed human platelets were prepared from the buffy coat fraction of 100 units of blood according to the method described previously (Ishizaki, T. et al., EMBO J., 15, 1885-1893 (1996)). Platelets were disrupted by sonication in 200 ml extraction buffer [20 mM Tris pH 7.4, 150 mM KCl, 0.2 mM ATP, 1 mM DTT, 1 mM PMSF] +50 mM benzamidine and 1 mg / ml aprotinin. After centrifugation at 100,000 000 g for a time, the supernatant was applied to a PLP-Sepharose column. This was washed with 4M urea and then eluted with 7M urea to obtain a homogeneous preparation of profilin. Next, 0.96 mg of profilin was complexed with 1 ml of NHS activated Sepharose (Pharmacia) according to the manufacturer's protocol to obtain immobilized profilin beads. As a control, bovine serum albumin was similarly conjugated to NHS activated sepharose.
[0082]
Confluent Swiss 3T3 cells obtained from 12 6 cm culture dishes were solubilized in 2.4 ml of buffer C, the lysate was centrifuged at 10,000 × g for 10 minutes, and the supernatant was collected. A 1/10 volume aliquot of the resulting supernatant was then incubated in the presence or absence of GST-low molecular weight G protein or free GTPγS or GDP. Also 20 μl of immobilized profilin was added. After incubating the mixture at 25 ° C. for 30 minutes, the beads were centrifuged at 1,000 × g for 2 minutes. The supernatant was saved and the beads were washed once with buffer C (100 μl) containing 300 mM NaCl instead of 100 mM NaCl. 50 μl of Remli sample buffer was added to the beads and 1/10 volume of the supernatant was mixed with 1/5 volume of 5 × Remli buffer. Samples were boiled, subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed as described above, and detection was performed with an ECL system (Amersham).
[0083]
The result was as shown in FIG. P140mDia in Swiss 3T3 cell lysate precipitated almost quantitatively with the addition of profilin-agarose but not with BSA-agarose. On the other hand, this binding was not affected by the external addition of RhoA or GTPγS.
[0084]
Example 7  Co-localization of RhoA, p140mDia and profilin in the wavy structure of spreading and migrating cell membranes and the dividing groove of dividing cells
In HT1080 cultured human fibroblastoma cells, Swiss 3T3 cells, sMDCK2 cells stably expressing myc-labeled RhoA (K. Takaishi, K. et al., Oncogene, 11, 39-48 (1995)) or HeLa cells The distribution of p140mDia, profilin and endogenous Rho and their potential for co-localization in vivo were examined by fluorescence microscopy using antibodies specific for each protein.
[0085]
Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). HT1080 cells are 5 x 10^FourCells were seeded at a density of 35 mm dish, cultured overnight and subjected to analysis. For analysis of sMDCK2 cells, 1 × 10 in DMEM containing 10% FCS on cover glass^FourCells were seeded at a density of cells / 35 mm culture dish and incubated for 16 hours. The medium was then changed to DMEM without FCS and incubated for 24 hours. Serum starved cells at 1 x 10^-7Stimulation was carried out at 37 ° C. for 15 minutes in the presence or absence of phorbol myristate acetate (PMA) and fixation.
[0086]
For indirect immunofluorescence, cells were fixed in phosphate buffered saline (PBS) containing 3.7% paraformaldehyde for 20 minutes at room temperature, then PBS containing 0.2% Triton X-100 For 10 minutes to make it permeable. After washing several times with PBS, the cells were incubated for 30 minutes or more at room temperature in buffer B [20 mM Tris pH 7.4, 50 mM NaCl] containing 5% BSA. For staining of p140mDia, cells were incubated with a 1:10 dilution of AP50 in blocking solution for 1 hour at room temperature and washed 3 times with buffer B containing 0.1% Triton X-100. The cells were then stained with Cy2-labeled goat anti-rabbit IgG antibody (Amersham Life Science). For staining of actin, rhodamine phalloidin (Molecular Probe) was added at a 1: 200 dilution with the second antibody. For staining of Myc-epitope, 9E10 monoclonal anti-Myc antibody was added for primary incubation with AP50 at a concentration of 10 μg / ml. Rhodamine anti-mouse IgG antibody was then used for detection at a 1:50 dilution.
[0087]
The results were as described below. Most of the fluorescence obtained with the anti-p140mDia antibody was localized in the thicker part of HT1080 cells, ie, the cytoplasmic region around the nucleus. However, significant fluorescence is also present in surrounding areas known to be highly motile, such as leading lamella and membrane ruffles of cells that spread and move. Observed (FIG. 9A). There was a significant lack of staining for p140mDia between the cytoplasmic region around the nucleus and the leading edge. Binding of p140mDia to focal adhesion and stress fiber was not observed. A distribution pattern similar to the above was also observed in cultured Swiss 3T3 cells (FIG. 9E).
[0088]
Double immunofluorescence microscopy using a monoclonal anti-profilin antibody (2H11) detected profilin accumulation in the cytoplasmic region around the nucleus of HT1080 cells and in expanding lamella an veils. Overlapped with the distribution of p140 mDia (FIG. 9B). This profilin distribution was consistent with previous results in rat fibroblasts (Buβ, F. et al., Cell Mot. Cytoskeleton, 22, 51-61 (1992)). Interestingly, double immunofluorescence observation using a profilin antibody and a polyclonal anti-RhoA antibody revealed that a portion of endogenous RhoA also accumulates with profilin in the membrane veil of motile cells. (FIGS. 9C and D). This suggested that p140mDia, profilin, and RhoA co-localize with the highly motile structure of the cell. In addition, the distribution of p140mDia was examined in Swiss 3T3 cells and compared with the distribution of F-actin. In this cell, p140mDia was also localized in the spread lamella with a well-developed actin skeleton (FIGS. 9E and F).
[0089]
Co-localization of p140mDia, profilin and RhoA also sMDCK cells that stably express myc-labeled RhoA and expand the membrane in response to phorbol esters (Takaishi, K. et al., Oncogene, 11 , 39-48 (1995)). As shown in FIG. 10, both Myc-RhoA and p140mDia were rather homogeneous in the cytoplasm in the thicker part of resting cells. After 2 minutes of stimulation with PMA, a portion of Myc-RhoA migrated around the wavy structure of the membrane co-localized with p140mDia. It became clear that part of profilin also moved to the corrugated structure of the membrane and colocalized with RhoA. Induction of membrane expansion by phorbol esters in these cells is known to occur in a Rho-dependent manner (Takaishi, K. et al., Oncogene, 11, 39-48 (1995)). The results suggest that p140mDia and profilin recruitment are also Rho dependent.
[0090]
In interphase cells, the majority of p140mDia is present in the cytoplasm and some is translocated to the plasma membrane at the spreading edge. On the other hand, in mitotic cells, the intracellular localization of p140mDia was not observed until they undergo cytokinesis. At this time, a part of p140mDia was concentrated in the plasma membrane of the split groove part and appeared as a ring-like structure (FIGS. 11A and C). This enrichment disappeared at the end of cytokinesis and staining moved to the area surrounding the plasma membrane. Staining was not observed in the intercellular cross-linked portion connecting daughter cells. Profilin showed a staining pattern almost overlapping with p140mDia during these processes (FIG. 11D).
[0091]
Example 8  RhoA and p140mDia are clustered around fibronectin (FN) coated beads
In recent studies, both Rho activation and ligation of the integrin of the extracellular matrix protein are required for cell spreading and adherence to the substrate (Hotchin, NA & Hall, A., J. Cell Biol., 131, 1857-1865 (1995)), and ligation of integrins with beads coated with either fibronectin or anti-integrin antibodies has been shown to recruit Rho to the plasma membrane directly under the beads. (Burbelo, P. et al., J. Biol. Chem. 270, 30919-30926 (1995)). Therefore, p140mDia also investigated whether Rho was recruited to the plasma membrane by these beads and whether this recruitment was dependent on Rho activation.
[0092]
Polystyrene latex beads (11.9 μm mean diameter, Sigma) were coated as described in 50 μg / ml human fibronectin (Collabrative Research) or 100 μg / ml poly-L-lysine (Sigma) ( Grinnel & Geiger, Exp. Cell Res., 162, 449-461 (1986)). Trypsinized Swiss 3T3 cells were plated on poly-lysine coated glass slips and allowed to bind to the slips in DMEM containing 10% FCS for 2 hours at 37 ° C. Each different type of bead was then placed on the cell. Cells were fixed after 15 minutes incubation at 37 ° C. p140mDia staining was performed as described above. For immunostaining of endogenous RhoA, rabbit anti-RhoA antibody (Santa Cruz) was used at a 1:40 dilution. The antibody was visualized using a Cy2 labeled goat anti-rabbit IgG antibody as described above.
[0093]
The results were as described below. First, it was confirmed that endogenous Rho was accumulated not directly under beads coated with poly-lysine (FIG. 12d) but directly under beads coated with fibronectin (FN) (FIG. 12b). Under this condition, p140mDia was also recruited directly under the FN-coated beads, and ring-like accumulation was observed around the beads (FIG. 12a). In addition to the above results (accumulation of both Rho and p140mDia), when the cells were pretreated with C3 exoenzyme, these accumulations were inhibited, so these recruitments are Rho activation dependent. It became clear (data omitted).
[0094]
Example 9  Induction of actin polymerization by overexpression of p140mDia
In order to examine the function of p140mDia, according to the method described previously (Ishizaki, T. et al., EMBO J., 15, 1885-1893 (1996)), p140mDia cDNA (# 1-1255 of SEQ ID NO: 1) was used. And / or other protein cDNAs were transiently expressed in COS7 cells, then the cells were fixed and stained with anti-p140mDia antibody.
[0095]
When the cells at 40 hours after transfection were stained with an anti-p140 mDia antibody, the cells were stained well, and the expression of p140 mDia was confirmed. In non-transfected cells, staining was observed around the nucleus. Compared to this, the transfected cells were clearly and evenly stained, indicating that this molecule was localized throughout the plasma membrane when p140mDia was overexpressed. (FIG. 13A). When stained with phalloidin, it was observed that stress fibers disappeared and actin staining was enhanced in accordance with the plasma membrane of the cells (FIG. 13B). It was suggested to induce. When p140mDia was expressed together with the C3 extracellular enzyme, more remarkable results were obtained. That is, when the C3 extracellular enzyme alone was transfected, almost all actin fibers disappeared from the cells and the cells became spherical (FIGS. 13C and D). When p140mDia was transfected with C3 exoenzyme, the cells maintained their morphology and staining of fibrous actin similar to that observed in cells transfected with p140mDia alone was observed (data not shown).
[0096]
Furthermore, p140mDia is changed to Rho.^Val14Alternatively, it was co-expressed with wild type Rho. Rho^Val14Alternatively, when wild-type Rho was expressed alone, stress fiber and contraction induction were observed. However, p140mDia^Val14Alternatively, when co-expressed with wild-type Rho, the above-described p140mDia trait (disappearance of stress fiber and actin staining consistent with the plasma membrane of the cell) traits induced by Rho alone (stress fiber and contraction) Induction of stress fibers and contraction were hardly observed (data not shown). These results suggest that when p140mDia is overexpressed, it automatically shifts to the plasma membrane, that is, shifts independently of actin polymerization by Rho protein.
[0097]
[Sequence Listing]

[0098]

[Brief description of the drawings]
FIG. 1 is a photograph showing binding of clone 50 and Rho protein in a two-hybrid system. Val, Asn, WT and Cdc42 are each RhoA^Val14, RhoA^Asn19, Wild-type RhoA and Cdc42Hs are shown. ΔAsn and ΔWT are Ala¹⁸¹Rho truncated at^Asn19And wild-type RhoA. Lamin and Lex binding domain not fused with other proteins were used as negative controls. Ala¹⁸¹RhoA truncated at^Val14Was not used in this experiment because it also showed high LacZ activity in association with VP16 activation domains that were not fused to other proteins.
FIG. 2 is a diagram schematically showing isolated p140mDia.
The ORF is indicated by a box of dots. A nucleotide 87th T to C conversion and a 2229th C to T nucleotide conversion were found in the 503 and E73 cDNAs, respectively. Libraries a, b and C represent mouse brain library 936309 (Stratagene), ML3000a (Clontech) and mouse embryo library, respectively. An insertion of 9 amino acids was found in the E73 cDNA.
FIG. 3 shows the deduced amino acid sequence of p140mDia. The clone sequence obtained by the two-hybrid system is indicated by a bold underline, the repeat structure of the proline-rich region is indicated by a dotted underline, and the FH-2 region is indicated by a thin underline. The arrowhead mark indicates the insertion position of 9 amino acids.
FIG. 4 shows a schematic structural comparison with other proteins containing p140mDia and FH regions. The sequence of p140mDia was compared to Drosophila diaphanous, S. cerevisiae Bni1p, rat formin, and Drosophila cappucino. The comparison was performed in the putative Rho binding region (Rho-binding), from the N-terminal to the proline rich region (poly-proline), the FH-2 region, and from the proline rich region to the C terminal region. Amino acid identity is expressed in%. No homology was observed in the N-terminal region of formin and cappuccino (indicated by *). All the sequences shown have a polyproline stretch in the middle and homology in the C-terminal half.
FIG. 5 is an RNA blot (electrophoresis photograph) showing the tissue distribution of p140mDia in various tissues of mice by Northern blotting.
heart: lung: lung, brain: brain, kidney: kidney, testis: testicle, skeletal muscle: skeletal muscle, thymus: thymus, liver: liver, stomach: stomach, small intestine: colon .
FIG. 6 is an electrophoresis photograph showing the specificity of an anti-p140mDia antibody.
Antiserum (AP50) is directed to the peptide encoded by clone 50 obtained by the two-hybrid system, and its specificity was determined by comparing the recombinant protein before induction (lane 1) and after induction (lane 2) with IPTG. Western blotting was performed on all lysates of E. coli expressed or all lysates of Swiss 3T3 cells (3T3 cells) (lane 3). Lane 4 shows the whole cell lysate stained with CBB.
FIG. 7 is an electrophoresis photograph showing precipitation of p140mDia by GTPγS-binding Rho protein. All lysates of Swiss 3T3 cells were incubated with GTPγS- or GDP-bound GST-Rho, GST-Rac, GST-Cdc42 or GST. The bound protein was precipitated with glutathione agarose beads and analyzed by immunoblotting using an anti-p140 mDia antibody. In lane 9 (cell lysates), p140mDia in all lysates of cells was detected with an antibody.
FIG. 8 is an electrophoretogram showing the binding of p140mDia and profilin in vitro. Swiss 3T3 cell lysates are incubated with agarose beads conjugated with profilin or BSA in the presence or absence of GTPγS- or GDP-bound GST-Rho protein (none) to precipitate the bound protein I let you. The precipitate (Pellet) and supernatant (Supernatant) were analyzed by immunoblotting using anti-p140 mDia antibody.
FIG. 9 is a photograph showing the co-localization of RhoA, p140mDia and profilin in a membrane raffle of expanded fibroblasts (photo of the morphology of the organism). (AD) HT1080 human fibrosarcoma cells. (E and F) Swiss 3T3 mouse fibroblasts. Cells are cultured, fixed, and stained simultaneously with either anti-p140mDia antibody (A and E), anti-RhoA polyclonal antibody (C), mouse anti-profilin monoclonal antibody (B and D) or rhodamine-phalloidin (F) Standard fluorescence micrographs were taken.
FIG. 10 is a photograph showing the colocalization of myc-labeled RhoA, p140mDia and profilin in membrane raffles of expanded sMDCK cells stimulated with PMA (photo of the morphology of the organism). Resting (AC) or phorbol myristate acetate (DH) stimulated (PMA stimulation) sMDCK cells were fixed and anti-p140 mDia antibody (B, E and H) or monoclonal anti-myc antibody (C, Stained with F). H shows staining with anti-p140 antiserum preabsorbed with an excess of recombinant peptide. A, D, and G show photomicrographs of each cell taken using a phase contrast microscope. From the upper left of the photograph, they are A, B and C, from the left of the middle of the photograph are D, E and F, and from the lower left of the photograph are G and H.
FIG. 11 is a photograph showing the concentration of p140mDia in the dividing groove of mitotic cells (photo of the morphology of the organism). A and B: Swiss 3T3 cells, C and D: HeLa cells. Logarithmically growing cells (A and C) stained with anti-p140mDia antibody (anti-p140) and simultaneously stained with DAPI (B) or 2H11 monoclonal anti-profilin antibody (D) (anti-profilin) Were taken with a standard fluorescence microscope.
FIG. 12 is a photograph showing clusters of RhoA and p140mDia that appeared in a Rho-dependent manner around fibronectin-coated beads. Swiss 3T3 cells cultured for 48 hours were used. Cells were seeded and incubated for 15 minutes with latex beads coated with fibronectin (FN-coated) (a, b) or poly-L-lysine (PLL-coated) (c and d). The cells were fixed and stained with anti-p140mDia antibody (anti-p140) (a, c) or anti-Rho antibody (anti-Rho) (b, d).
FIG. 13 is a photograph showing transient expression of p140mDia in COS-7 cells. COS cells were transfected with the p140mDia expression vector alone (A and B) or the C3 extracellular enzyme expression vector alone (C and D). C3 exoenzyme transfection was performed with a C3 exoenzyme expression-like vector having a FLAG epitope. Cells were fixed and anti-p140mDia antibody (anti-p140) alone (A and C) or simultaneously actin was stained with rhodamine-phalloidin (B, D).

Claims (17)

The following protein (a) or (b):
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 ; or
(B) One or several amino acid sequences are added and / or inserted into the amino acid sequence of SEQ ID NO: 1 and / or one or several amino acids of the amino acid sequence of SEQ ID NO: 1 are substituted and / or deleted A protein having an amino acid sequence and having an active Rho protein binding ability and a profilin binding ability. The protein according to claim 1 , which has the amino acid sequence of 63 to 260 of SEQ ID NO: 1 and the amino acid sequence of 571 to 737 of SEQ ID NO: 1. A nucleic acid molecule encoding the protein according to claim 1 or 2 . The nucleic acid molecule according to claim 3 , which encodes a protein consisting of a part or all of the DNA sequence of SEQ ID NO: 2 and having active Rho protein binding ability and profilin binding ability. The nucleic acid molecule according to claim 4 , wherein a part of the base sequence is the DNA sequence of Nos. 94 to 3861, 280 to 873, and 1804 to 2304 of SEQ ID NO: 2. A vector comprising the nucleic acid molecule according to any one of claims 3-5 . The vector according to claim 6 , wherein the vector is selected from the group consisting of a plasmid vector, a viral vector, and a liposome vector. A host cell transformed with the vector according to claim 6 or 7 (however, in the case of a human cell, it is limited to a cell isolated from a human). The host cell according to claim 8 , wherein the host cell is selected from the group consisting of E. coli, yeast, insect cells, COS cells, lymphocytes, fibroblasts, CHO cells, blood cells, and tumor cells. A method for producing the protein according to claim 1 or 2 , comprising culturing the host cell according to claim 8 or 9 and isolating the protein according to claim 1 or 2 from the culture. . (1) subject to substance screening, the activated Rho protein, is present in the screening system comprising a protein according to claim 1 or 2, and (2) activated Rho protein, according to claim 1 or 2 screening method of a substance that inhibits the binding of a protein described comprises measuring the degree of inhibition of binding of the protein according, the activated Rho protein to claim 1 or 2. (1) subject to substance screening, and profilin, be present in the screening system comprising a protein according to claim 1 or 2, and (2) profilin and the protein of claim 1 or 2 comprising measuring the degree of inhibition of binding of, and profilin, screening methods of substances to inhibit the binding of a protein according to claim 1 or 2. The screening method according to claim 11 or 12 , wherein the screening system is a cell system or a cell-free system.   An antibody against the amino acid sequence of 63 to 260 in SEQ ID NO: 1. The antibody according to claim 14 , which is a polyclonal antibody. (1) a substance to be detected is present in a detection system containing the antibody according to claim 14 or 15 , and (2) a reaction between the substance to be detected and the antibody according to claim 14 or 15 A method for detecting a substance that reacts with the antibody, which comprises measuring the degree of. A detection kit for a substance recognized by the antibody, comprising the antibody according to claim 14 or 15 .

Images