JPH10201480A - Rho target protein rho kinase derived from human and its gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a protein, capable of regulating a signal transmission route such as a morphological change in a cell, formation or metastasis of tumor or constriction of vascular smooth muscles, derived from a human and useful for diagnosing malignant tumor, etc., by providing the protein with an active type Rho protein-binding ability, etc. SOLUTION: This protein has characteristics of (i) having an active type Rho protein-binding ability, (ii) having protein kinase activities, (iii) capable of accelerating the protein kinase activities by binding to the active type Rho protein and (iv) having a gene located at a human chromosome 2p24. The protein (derivative) contains (v) an amino acid sequence represented by the formula or (vi) the amino acid sequence represented by the formula in which one or more amino acid sequences are added and/or inserted and/or one or more amino acid sequences are substituted and/or deleted, has the active type Rho protein- binding ability, contains an amino acid sequence having protein kinase activities and is obtained by culturing a host cell containing a nucleic acid capable of coding the protein (derivative).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a human-derived Rho target protein having protein kinase activity and its gene, and more particularly to a diagnostic agent.

2. Description of the Related Art In vivo, a molecular weight of 2 to 3 having no subunit structure
There are a large group of low molecular weight GTP binding proteins (G proteins). At present, more than 50 members have been found in the superfamily of low molecular weight G proteins, from yeast to mammals. The low-molecular-weight G protein has Ras, R
ho, Rab, and other four families. It has been revealed that this low-molecular-weight G protein controls various cell functions. For example, Ras protein regulates cell growth and differentiation by Rho.
Proteins are thought to control morphological changes of cells, cell adhesion, cell motility, and the like, respectively.

[0003] Among them, Rho protein is GDP / G
It exhibits TP binding capacity and endogenous GTPase activity and has been implicated in the cytoskeletal response to extracellular signals such as lysophosphatidic acid (LPA) and certain growth factors. GDP-bound Rho that is inactive
When a certain stimulus is given to a protein, Smg GD
By the action of a GDP / GTP converting protein such as S, Dbl or Ost, it is converted into an active GTP-binding Rho protein (hereinafter referred to as “active Rho protein”). It is considered that the activated Rho protein acts on the target protein to form stress fibers and adhesion spots, thereby inducing cell adhesion and cell movement (Experimental Medicine vol.12, No.8, 97-10
2 (1994), Takai, Y. et al. Trends Biochem. Sci., 2
0, 227-231 (1995)). On the other hand, the activated Rho protein is converted to a GDP-bound Rho protein by the Rho protein endogenous GTPase. This endogenous GTPa
GTPase activating protein (GAP) (Lamarche, N. & Hall, A. eta)
l., TIG, 10, 436-440 (1994)).

[0004] The C-terminus of the natural RhoA protein is C-terminal.
There is a ys-AA-Leu (A is an aliphatic amino acid) structure, a geranylgeranyl group transfer enzyme binds to a Cys residue, and the carboxyl group of the Cys residue is methylated. This post-translational modification by lipids is thought to be necessary not only for binding of the Rho protein to the cell membrane and interaction with the activity control protein, but also for expression of its function (Imazum
i, K. et al., Experimental Medicine 13,646-656 (1995)).

[0005] RhoA protein, RhoB protein, RhoC protein, Rac1 protein, Rac
The amino acid sequences of Rho family proteins, such as the two proteins, Cdc42 protein, are 50
% Or more similarity. This Rho family of proteins responds to extracellular signals such as lysophosphatidylic acid (LPA) and growth factors in response to stress fibers and focal adhesions.
n) It is thought to be involved in the reaction leading to the formation of (AJ Ridley & A. Hall, Cell, 70, 389-399)
(1992), AJ Ridley & A. Hall, EMBO J., 13, 2600-
2610 (1994)). In addition, the subfamily Rho protein is used for cell morphological changes (HF Parterson et a.
l., J. Cell Biol., 111, 1001-1007 (1990)), cell adhesion (Morii, N. et al., J. Biol. Chem. 267, 20921-20926).
(1992), T. Tominaga et al., J. Cell Biol., 120, 15
29-1537 (1993), Nusrat, A. et al., Proc, Natl. Aca
d. Sci. USA, 92, 10629-10633 (1995), Landanna, C. e.
t al., Science 271, 981-983 (1996)), cell motility (K.
Takaishi et al., Oncogene, 9, 273-279 (1994)), cytokinesis (K. Kishi et al., J. Cell Bi
ol., 120, 1187-1195 (1993), I. Mabuchi et al., Zygot
e, 1, 325-331 (1993)). In addition, the Rho protein has been shown to suppress smooth muscle contraction (K. Hirata et a
l., 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 (19
96)), phosphatidylinositol 3-kinase (PI3-kinase) (J. Zhang et al., J. Biol. Che.
m., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI 4,5-
Kinase) (LD Chong et al., Cell, 79, 507-513 (199
4)) and c-fos expression (CS Hill et al., Cell, 81,
1159-1170 (1995)).

Recently, it has been found that the introduction of Rho protein having a partially substituted amino acid sequence into cells suppresses Ras-dependent tumor formation.
Proteins have been shown to play an important role in Ras cell transformation, ie, tumorigenesis (GC Prendergast et al., Oncogene, 1).
0,2289-2296 (1995), Khosravi-Far, R., et al., Mol. Cel
l. Biol., 15,6443-6453 (1995), R. Qiu et al., Proc. Nat.
l. Acad. Sci. USA, 92, 11781-11785 (1995), and Lebowi
tz, P. et al., Mol. Cell. Biol., 15, 6613-6622 (1995)).

Further, it has been revealed that the Rho protein enhances not only cell proliferation, cell motility and cell aggregation but also smooth muscle contraction. According to recent studies, R
The ho protein is known to be involved in smooth muscle contraction (K. Hirata et al., J. Biol. Chem. 267, 8719-872).
2 (1992) and Noda, M. et al., FEBS Lett., 367,
246-250 (1995)). Therefore, it is considered that the activated Rho protein-binding protein is also likely to be involved in smooth muscle contraction.

Myosin light chain phosphorylation is associated with smooth muscle contraction (Ka
mm, KE & Stull, JT, Annu. Rev. Pharmacol. Tox
icol. 25, 593-603 (1985), Hartshorne, DJ, & Jo
hnson, DR, (1987) in Physiology of the Gastroin
testinal Tract, (Johnson, LR, ed) pp. 423-482, R
aven Press, New York, and Sellers, JR & Adels
tein, RSin The Enzyme (Boyer, P., and Erevs, E.
G., eds) Vol. 18, pp. 381-418, Academic Press, San
Diego, CA (1987)), Actin-myosin interaction for stress fiber formation occurring in non-muscle cells (Huttenlocher, A. et al., Curr. Opi. Cell Biol.
7, 697-706 (1995)). It also has effects on cytokinesis and cell motility (Huttenlocher, A. et al., Curr. Opi. Cell B.
iol. 7, 697-706 (1995)).

[0009] Myosin light chain kinase is a component of the myosin light chain S
er-19 is primarily phosphorylated (Kam
m, KE & Stull, JT, Annu. Rev. Pharmacol. Tox
icol. 25, 593-603 (1985), Hartshorne, DJ & Joh
nson, DR, (1987) in Physiology of the Gastroint
estinal Tract, (Johnson, LR, ed) pp. 423-482, R
aven Press, New York, Sellers, JR & Adelstein,
RSin The Enzyme (Boyer, P., and Erevs, EG, e
ds) Vol. 18, pp. 381-418, Academic Press, San Dieg
o, CA (1987), and Ikebe, M. & Hartshorne, DJ
J. Biol. Chem. 260, 10027-10031 (1985)). In addition to specific kinases such as myosin light chain kinase, none of the protein kinases obtained so far phosphorylate this site (Tan, JL et al., Annu. Rev. Bioche
m. 61, 721-759 (1992)).

When smooth muscle is stimulated with an agonist such as a vasoconstrictor, Ca ²⁺ moves into the cytoplasm. Ca ²⁺
Activates calmodulin-dependent myosin light chain kinase. Myosin light chain phosphorylation induces myosin-actin interaction, which activates myosin ATPase (Kamm, KE & Stull, JT, Ann)
u. Rev. Pharmacol. Toxicol. 25, 593-603 (1985), H.
artshorne, DJ, & Johnson, DR, (1987) in Phys
iology of the Gastrointestinal Tract, (Johnson, L.
R., ed) pp. 423-482, Raven Press, New York, and Sellers, JR & Adelstein, RSin The Enzyme (B
oyer, P., and Erevs, EG, eds) Vol. 18, pp. 381-4
18, Academic Press, San Diego, CA (1987)), which then induces smooth muscle contraction (Kamm, KE &
Stull, JT, Annu. Rev. Pharmacol. Toxicol. 25, 5
93-603 (1985), Hartshorne, DJ, & Johnson, D.
R., (1987) in Physiology of the Gastrointestinal Tr
act, (Johnson, LR, ed) pp. 423-482, Raven Pres
s, New York, and Sellers, JR & Adelstein, R.
S.in The Enzyme (Boyer, P. & Erevs, EG, eds) Vo
l. 18, pp. 381-418, Academic Press, San Diego, CA
(1987)). However, cytosolic Ca ²⁺ levels are not always proportional to contraction levels, and other mechanisms that regulate Ca ²⁺ sensitivity to smooth muscle contraction have been proposed (Bradley, AB & Morgan, KG, J. Phys
iol. 385, 437-448 (1987)). Since GTPγS (a non-hydrolysable GTP analog) reduces the Ca ²⁺ concentration required for contraction of permeable (skinned) smooth muscle, GTP-binding proteins were presumed to modulate Ca ²⁺ sensitivity (Kitaza
wa, T. et al., Proc. Natl. Acad. Sci. USA 88,
9307-9310 (1991), Moreland, S. et al., Am. J. Ph.
ysiol. 263, 540-544 (1992)). Rho protein is G
It has been shown to be involved in the Ca ²⁺ sensitivity of smooth muscle enhanced by TP (Hirata, K. et al., J. Biol. Che.
m. 267, 8719-8722 (1992)). Recently, in smooth muscle cells with enhanced permeability, submaximal Ca ²⁺
PγS has been shown to promote phosphorylation of myosin light chain, which promotes the activation of Rho proteins and the enzymatic activity of myosin light chain phosphatase, which is responsible for dephosphorylation of myosin light chain. This was suggested to be due to suppression (Noda, M. etal., FEBS Lett. 367,
246-250 (1995)). However, it remains to be elucidated by what mechanism Rho protein inhibits myosin light chain phosphatase and whether the increase in phosphorylation of myosin light chain by Rho protein is solely due to suppression of myosin light chain phosphatase activity. . Therefore, it has not been elucidated by what mechanism the Rho protein regulates Ca ²⁺ sensitivity of smooth muscle and consequently enhances smooth muscle contraction.

[0011] As described above, the Rho protein regulates many signal transduction pathways such as cell morphology change, cell adhesion, cell motility, cytokinesis, tumor formation and metastasis, and vascular smooth muscle contraction. I understand. From this, it is considered that the Rho protein has many target molecules and regulates the above-mentioned many signal transduction pathways.

Very recently, several candidate proteins have been reported in mammals. These proteins are known as protein kinase N (PKN) (Watanabe, G. et al.,
Science 271, 645-648 (1996); Amano, M. et al., Sc.
ience 271, 648-650 (1996)), porphyrin (Watanab)
e, G. et al., Science 271, 645-648 (1996)), citron (Madaule, P. et al., FEBS Lett. 377, 243-248).
(1995)), bovine Rho kinase (Matsui, T. et al., EMB
O J. 15,2208-2216 (1996)), ROKα (Leung, T. et a
l., J. Biol. Chem. 270, 29051-29054 (1995)), p1
60 ^ROCK (Ishizaki, T., et al., EMBO J., 15, 1885-
1893 (1996)), Lothekin (Reid, T. et al., J. Biol. Che)
m., 271, 13556-13560 (1996)) and the myosin binding subunit (Amano, M. et al., Science 271,648-650 (199).
6)). All of these proteins bind to GTP-bound RhoA protein (however, only citron also binds to GTP-bound Rac1 protein).

Of these, PKN has a catalytic domain having high homology to the protein kinase catalytic domain of protein kinase C, and exhibits serine / threonine protein kinase activity (Mukai, H. & Ono, Y. , Bioche
m. Biopys. Res. Commun. 199, 897-904 (1994); Muka
i, H. et al., Biochem. Biopys. Res. Commun. 204, 3
48-356 (1994)). On the other hand, ROKα (see Leung, T. e.
t al. (1995)) and p160 ^ROCK (Ishizaki, T., et.
al., EMBO J., 15, 1885-1893 (1996)) also has a serine / threonine protein kinase catalytic domain-like amino acid sequence (Leung, T. et al (1995) supra).

On the other hand, in addition to the above-mentioned mammalian proteins,
Recently, in yeast (Saccharomyces cerevisiae), protein kinase C1 (PKC1) has been identified as a target protein of Rho1 protein corresponding to mammalian RhoA (Nonaka, H. et al., EMBO J. 14, 5931-5938).
(1995)). More recently, yeast (Saccharomyces cerevi)
1,3-β-glucan synthase has been identified as a target protein of the Rho1p protein of D. siae) (Drgo
nova, J. et al., Science 272, 277-279 (1996) and
Qadota, H. et al., Science 272, 279-281 (1996)).

Furthermore, very recently, a novel protein BEM4 has been identified as a target protein for Rho1p in yeast (Mack, D. et al., Mol. Cell. Biol., 16, 4387-).
4395 (1996), Hirano, H. et al., Mol. Cell. Biol.,
16, 4396-4403 (1996)).

[0016] However, the mechanism of cell signal transmission involving the active Rho protein, particularly the mechanism relating to tumor formation and smooth muscle contraction, has not been elucidated yet.

Intermediate filaments (IFs) are major constituent molecules of the cytoskeleton and the nuclear membrane of eukaryotic cells, and their structure is dramatically reorganized (reorganized) by the cell signaling system and the cell cycle. (Steinert, PM & Roop,
DR, Annu. Rev. Biophem. 57: 593-625 (1988); Eri
ksson, J. et al., Curr. Opin. Cell Biol. 4: 99-104.
(1992); Fucks, E. & Weber, K., Annu. Rev. Biophe
m. 63: 345-382 (1994)). This reconstitution of IFs is thought to be controlled by site-specific phosphorylation of serine and threonine residues of IF, and some kinases are known to exhibit IF kinase activity in vivo ( Inag
aki, M. et al., BioEssays 18: 481-487 (1996)). Sites that recognize phosphorylated serine and threonine residues and their surrounding sequences and phosphorylation-specific antibodies visualize site-specific phosphorylated IF and IF kinase activity in situ by immunocytochemistry (Nishizaw
a, K. et al., J. Biol. Chem. 266, 3074-3079 (199
1)).

Recently, I expressed in the cytoplasm of astroglia
Using antibodies that specifically recognize the four different phosphorylation sites of GFAP, F, two different types of GFAP
Mitotic phosphorylation of AP has been reported (Matsuoka,
Y. et al., EMBO J. 11, 1895-2902 (1992); Sekiyama,
M. et al., J. Cell Biol. 132: 635-641 (1996)). One is that GFAP is found throughout the cytoplasm during the transition to G2-M phase.
Specific phosphorylation of Ser-8. Another is GF, which is found in the mitotic sulcus during the metaphase transition of mitosis.
Phosphorylation of Thr-7, Ser-13, and Ser-34 of AP. Phosphorylation of GFAP, which is specifically observed in the cleavage furrow, has been observed not only in astroglial cells but also in other cultured cells transfected with GFAP cDNA (Sekiyam
a, M. et al., J. Cell Biol. 132: 635-641 (1996)).
The kinase involved in phosphorylation was named CF kinase (Sekiyama, M. et al., J. Cell Biol. 132: 635-64.
1 (1996)). However, the CF kinase molecule has not been identified, and the regulatory mechanism and its function were still unknown.

Recently, it was shown that Rho plays an important role in cytokinesis by inducing the formation and maintenance of a contractile ring, a cytoskeleton composed of actin (Kishi, K. et al. , J. Cell Biol. 120: 118
7-1195 (1993); Mabuchi, I. et al., Zygote 1: 325-33
1 (1993)). Furthermore, it has been reported that Rho translocates from the cytoplasm to the fissure during the cytokinesis phase (Takaishi, K. e).
t al., Oncogene 11: 39-48 (1994)). But Rho
The control mechanism of cell division by proteins has not been reported as far as the present inventors know.

[0020]

SUMMARY OF THE INVENTION The present inventors have now isolated a protein having the ability to bind to active Rho protein and having protein kinase activity from bovine brain gray matter. The molecular weight of this protein was about 1 as measured by SDS-PAGE.
It was 64 kDa. The present inventors have also suggested that this protein (Rho kinase) binds to the effector region of the activated Rho protein, that Rho kinase exhibits kinase activity, and that the kinase activity is GTPγS.Rh
o protein, and that Rho kinase has a coiled-coil region in the middle. That is, Rho kinase is a serine / threonine kinase targeted by Rho protein,
It was found to be a mediator of the Rho protein-dependent signaling pathway. In addition, the present inventors
It has been found that ho kinase phosphorylates the myosin binding subunit of myosin light chain phosphatase and myosin and contracts vascular smooth muscle.

Further, the present inventors have succeeded in cloning cDNA of human Rho kinase.

The present inventors have further found that microinjection of dominant active-Rho kinase into cultured cells induces the formation of stress fibers and focal adhesions, resulting in dominant negative-R
LPA by microinjection of ho kinase
Or the formation of stress fibers and focal adhesions induced by the Rho protein is inhibited,
And by staurosporine, Rho in vitro
Not only was kinase activity inhibited, but Rho kinase was also found to inhibit cell-induced formation of stress fibers and focal adhesions.

The present inventors have further found that Rho kinase phosphorylates Ser-19 of MLC, that Rho kinase is involved in the formation of stress fibers and focal adhesion, and that Rho kinase is regulated by Rho protein. Involved in SRE activation, which has been demonstrated (Example 19), Rho-kinase binds to Ser- of GFAP.
13.Specific phosphorylation of Ser-34, the kinase activity of Rho kinase to GFAP is dramatically enhanced by GTP-bound RhoA, and the phosphorylation of GFAP by Rho kinase causes GFAP filament formation in vitro. Almost completely inhibited (Example 20), the result of FISH analysis showed that human R
the ho kinase gene is located on p24 of chromosome 2,
Template D with chromosome 2 by panel analysis using PCR
Only from NA, about 132 bp of human Rho kinase specific D
It was found that the NA fragment was amplified (Example 21).
The present invention is based on the above findings.

That is, an object of the present invention is to provide a Rho target protein having protein kinase activity (hereinafter, referred to as “Rho kinase”).

The present invention also relates to a partial protein of the protein, a base sequence encoding the protein including the partial protein, a vector comprising the base sequence, a host cell transformed with the vector, a protein such as Production method, tumor formation or metastasis inhibitor containing the protein, etc., smooth muscle contraction inhibitor, platelet aggregation inhibitor, inflammatory disease or autoimmune disease therapeutic agent, inhibition of binding of active Rho protein to the protein, etc. It is an object of the present invention to provide a screening method for a substance that inhibits protein kinase activity such as the protein, and a screening method for a substance that inhibits stress fiber or focal adhesion.

Another object of the present invention is to provide a probe containing the partial fragment of the base sequence, and a diagnostic agent containing the partial fragment of the base sequence or the probe.

[0027] The protein according to the present invention is a human-derived protein having the following characteristics: (1) It has an active Rho protein binding ability; (2)
(3) Activated Rho having protein kinase activity
Binding to a protein enhances protein kinase activity, and (4) the gene is located on human chromosome 2p24.

[0028]

DETAILED DESCRIPTION OF THE INVENTION Definitions In the present invention, the term "amino acid" is used to include both optical isomers, that is, L-form and D-form. Therefore, in the present invention, the term “peptide” means not only a peptide composed of only L-form amino acids but also a peptide containing some or all of D-form amino acids.

In the present invention, the term "amino acid" means not only the 20 kinds of α-amino acids constituting natural proteins, but also other α-amino acids, β-,
γ- and δ-amino acids, unnatural amino acids and the like are used. Thus, amino acids that are substituted in a peptide or inserted into a peptide as described below include the 20
It is not limited only to species α-amino acids, but may be other α-amino acids and β-, γ-, δ-amino acids and unnatural amino acids. Such β
-, Γ- or δ-amino acids, β-alanine,
γ-aminobutyric acid or ornithine; and amino acids other than those constituting natural proteins or unnatural amino acids include 3,4-dihydroxyphenylalanine, phenylglycine, cyclohexylglycine, 1,2,3,4-tetraethyl Hydroisoquinoline-3
-Carboxylic acid or nipecotic acid.

Further, the term “protein according to the present invention” used herein is meant to include derivatives thereof.

Furthermore, in the present specification, "base sequence"
Means both DNA and RNA sequences.

In the present specification, when a specific mutation is expressed, the original amino acid is first, the position number is second,
And the substituted amino acid is shown third. For example, "Ly
"s121Gly" indicates that Lys (K: lysine), which is the 121st amino acid residue, is substituted with Gly (G: glycine).

Protein In the present invention, the term "protein having the ability to bind to active Rho protein" refers to a protein that has been evaluated by those skilled in the art as having binding to active Rho protein. , 11 or 13 means that the protein is evaluated to have been found to bind to the active Rho protein when the experiment is carried out under the same conditions as those of the above. Here, Rh protein includes Rh
oA, RhoB, RhoC, or RhoG proteins.

In the present specification, the Rho protein is R
It also includes a Rho protein that has been modified so that the binding between the ho protein and the protein according to the present invention is not substantially impaired. Such modified Rho proteins include Rho in which the 14th amino acid is substituted with valine.
A mutant (RhoA ^Val14 ).

In the present invention, the term "protein having protein kinase activity" refers to a protein which has been evaluated by those skilled in the art as having protein kinase activity. For example, Examples 2, 5, 6-9, 13, 19 , Or a protein that is evaluated as having protein kinase activity when tested under the same conditions as in Example 20.

The protein according to the present invention is an active form of Rho
It has the property that its protein kinase activity is enhanced by binding to a protein. Here, the term “protein kinase activity” is used to include serine / threonine protein kinase activity.

The protein according to the present invention can be obtained, for example, according to the method described in Example 1. Alternatively, it can be obtained by expressing the DNA sequence of SEQ ID NO: 2 or 5 in a host.

As used herein, the term “protein derivative” refers to a part or all of the amino group at the amino terminus (N-terminus) of the protein or the amino group at the side chain of each amino acid, and / or the carboxyl terminus of the protein ( C
A terminal or carboxyl group or 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 protein (for example, hydrogen group, thiol group, Amide group) is partially or entirely modified by another suitable substituent. Modification with an appropriate other substituent is performed for the purpose of, for example, protecting a functional group present in a protein, improving protein safety and tissue transportability, or enhancing protein activity.

As the protein derivative, specifically, (1) a part or all of the hydrogen atoms of the amino group at the amino terminal (N-terminal) of the protein or the amino group in the side chain of each amino acid are substituted or non-substituted; Substituted alkyl groups (which may be linear, branched or cyclic) (e.g., methyl, ethyl, propyl, isopropyl, isobutyl, butyl, t-butyl, cyclopropyl,
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 protection Group (for example, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-biphenylisopropyloxycarbonyl group, t-butoxycarbonyl group) or a urea-type substituent (for example, methylaminocarbonyl group, phenylcarbonyl group) , Cyclohexylaminocarbonyl group), and (2) the carboxyl group at the carboxyl terminal (C-terminal) of the protein or part or all of the carboxyl group in the side chain of each amino acid is esterified. And are those (e.g., the hydrogen atom is methyl, ethyl, isopropyl, cyclohexyl,
Phenyl, benzyl, t-butyl, 4-picolyl substituted), amide-modified (for example, unsubstituted amide, C1-C6 alkylamide (for example, methylamide, ethylamide, isopropylamide)) And (3) some or all of the functional groups other than the amino group and carboxyl group (eg, hydrogen group, thiol group, amino group, etc.) in the side chain of each amino acid of the protein, Examples include those modified with the same substituent as the amino group or a trityl group.

According to the present invention there is provided a protein comprising the following amino acid sequence: (1) the amino acid sequence of SEQ ID NO: 4, or (2) one or more amino acid sequences are added and / or inserted, and / or Or the amino acid sequence of SEQ ID NO: 4 in which one or more amino acids have been substituted and / or deleted, wherein the active Rho
An amino acid sequence having protein binding ability and having protein kinase activity. Additions, insertions, substitutions,
The term "deletion" and "deletion" refer to those which do not impair the ability of the protein consisting of SEQ ID NO: 4 to bind to active Rho protein and protein kinase activity.

An example of such a deletion is a region obtained by removing the amino acid sequence at positions 90 to 359 (protein kinase region) and the amino acid sequence at positions 943 to 1068 (Rho protein binding region) from the amino acid sequence of SEQ ID NO: 4. Or a partial deletion thereof. Specifically, 1-89, 360
942 and / or 1069 to 1388 or a partial sequence thereof.

According to another aspect of the invention, 9 of SEQ ID NO: 4
A protein having the amino acid sequence of 0 to 359 (protein kinase region) and the amino acid sequence of 943 to 1068 (Rho protein binding region) is provided.
Rho kinase is mainly expressed in the cerebrum and cerebellum (see Example 3, (4)). Further R
Ho kinase cross-immunizes with the antibody described below (see Example 3 (4) and Example 10).

Here, “mainly expressed in the cerebrum and cerebellum” means that those skilled in the art will evaluate that the expression in the cerebrum and cerebellum is more frequently observed than in other sites. This means that expression was evaluated to be more frequently observed in the cerebrum and cerebellum as compared with other sites when the experiment was performed under the same conditions as (4) of Example 3.

According to the present invention, there is provided a human-derived protein or a derivative thereof having an active Rho protein binding ability and having no protein kinase activity.

[0045] Examples of the above-mentioned protein have the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid sequences are added and / or inserted, and / or one or more amino acids of the amino acid sequence are substituted and / or deleted. Proteins having an amino acid sequence having an active Rho protein binding ability and having no protein kinase activity. That is, the additions, insertions, substitutions, and deletions herein refer to those that do not impair the ability of the protein consisting of the amino acid sequence of SEQ ID NO: 4 to bind to the active Rho protein and impair the protein kinase activity.

An example of such a deletion is shown in FIG.
-359 amino acid sequence (protein kinase region)
Alternatively, it is a deletion of a region including a part thereof having protein kinase activity.

In addition, there may be mentioned a deletion of a region or a part of the amino acid sequence of SEQ ID NO: 4 excluding the amino acid sequence at positions 943 to 1068 (Rho protein binding region) (see Example 11). Specifically, the amino acid sequences of Nos. 1 to 89 and their partial sequences, the amino acid sequences of Nos. 360 to 942 and their partial sequences, and 1069 to
It is the amino acid sequence at position 1388 and its partial sequence.
An example of such a substitution is Lys121Gly.

The amino acid sequence of SEQ ID NO: 4 (provided that
A protein having an active Rho protein binding ability and having no protein kinase activity in addition to the above-described addition, insertion, substitution and / or deletion. May have additions, insertions, substitutions and / or deletions that do not impair the Rho protein binding ability of the protein.

An example of such a deletion is the amino acid sequence of Nos. 943 to 1068 (Rh
o protein binding region) (see Example 11) or a portion thereof is deleted. Specifically, 1 to 89
No. amino acid sequence and its partial sequence, 360-942
Amino acid sequence and its partial sequence, and 1069
1A to 1388 and their partial sequences.

According to another aspect of the present invention, 4 of SEQ ID NO: 4
21st to 1137th, 438th to 1124th, 799 to 11th
A protein having an amino acid sequence at positions 37, 943 to 1068, or 941 to 1075 or a derivative thereof is provided. These proteins have an active Rho protein binding ability and do not have protein kinase activity.

A protein having an active Rho protein binding ability and having no protein kinase activity also inhibits activation of Rho kinase kinase activity by active Rho protein. For example, as described in Example 13, 941-941 of SEQ ID NO: 1, which is an example of a protein having an active Rho protein binding ability and having no protein kinase activity in vitro.
The GST fusion protein containing the amino acid sequence of No. 1075 inhibits activation of the kinase activity of Rho kinase by activated Rho protein. Thus, the activated R
A protein that has an ability to bind to a ho protein and has no protein kinase activity inhibits activation of Rho kinase by an activated Rho protein (ie, activated Rho protein).
to block signal transduction from the ho protein to Rho kinase.

According to another aspect of the present invention, it comprises the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid sequences have been added and / or inserted and / or one or more amino acids have been substituted and / or deleted. A protein, wherein the amino acid sequence is an active Rho protein and / or R
proteins that inhibit the function of ho kinase (ie,
A dominant negative Rho kinase is provided.

A protein or a derivative thereof having an active Rho protein binding ability and not having a protein kinase activity functions dominantly with respect to Rho kinase which is endogenously present in a cell and exerts its action. Negative. Therefore, a protein having an active Rho protein binding ability and no protein kinase activity or a derivative thereof is one form of the “dominant negative Rho kinase”.

Further examples of dominant negative Rho kinase include SEQ ID NO: 4, 941-1075 or 94
3125 amino acid sequence, 1125 of SEQ ID NO: 4
The amino acid sequence of 〜1388 or the Lys of position 121
Is a protein consisting of the amino acid sequence of positions 6 to 553 of SEQ ID NO: 4 substituted by Glu.

Examples of the “function of activated Rho protein and / or Rho kinase” include, for example, (change of cell morphology such as formation of stress fiber and formation of focal adhesion, induction of cell adhesion, induction of aggregation of platelets and leukocytes, smoothness Induction of muscle contraction, induction of cytokinesis, induction of gene transcription activation, induction of tumor formation and invasion / metastasis of cancer cells, etc.).

The dominant negative Rho kinase is
When it is present in a cell, it inhibits the function of an active Rho protein or Rho kinase that is endogenous in the cell. For example, as shown in Examples 15 to 18,
A GS comprising the amino acid sequence of positions 941 to 1075 of SEQ ID NO: 1, which is an example of a dominant negative Rho kinase
T fusion protein (Rho kinase (RB)), GS comprising the amino acid sequence of positions 1125 to 1388 of SEQ ID NO: 1
T fusion protein (Rho kinase (PH)), or 6-55 of SEQ ID NO: 1 with the substitution Lys121Gly
GST fusion protein containing the amino acid sequence of No. 3 (Rh
o-kinase (CAT-KD) was microinjected into Swiss3T3 cells,
Stress fiber formation and focal adhesion formation of 3T3 cells were inhibited. Induction of stress fiber formation and focal adhesion formation of Swiss3T3 cells
It is one of the representative biological functions of the activated Rho protein (Examples 15 and 16, Ridley, A. & Hal
l, A., Cell 70, 389-399 (1992) and Ridley, A. &
Hall, A., EMBO J., 13, 2600-2610 (1994)). As described above, the dominant negative Rho kinase is caused to be present in the cell, whereby the action of the active Rho protein endogenously present in the cell, for example, activation of the endogenous Rho kinase by the active Rho protein and stress It can be used to inhibit fiber formation and focal adhesion formation.

According to the present invention, there is also provided a human-derived protein having protein kinase activity and not having the ability to bind to active Rho protein, or a derivative thereof. Here, “protein kinase activity” includes serine / threonine protein kinase activity.

An example of the above protein is a protein having the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid sequences have been added and / or inserted, and / or one or more amino acids have been substituted and / or deleted. The amino acid sequence has protein kinase activity,
And those having no active Rho protein binding ability. That is, the additions, insertions, substitutions, and deletions herein refer to those that do not impair the protein kinase activity of the protein consisting of the amino acid sequence of SEQ ID NO: 4 and impair the ability to bind to the active Rho protein.

An example of such a deletion is shown in SEQ ID NO: 4 at 94
This is a deletion of the amino acid sequence at positions 3 to 1068 or a region containing a part thereof having an active Rho protein binding ability. In addition, a protein having the protein kinase activity consisting of the amino acid sequence of SEQ ID NO: 4 (having addition, insertion, substitution, and / or deletion) and having no active Rho protein binding ability or a derivative thereof is In addition to the above additions, insertions, substitutions, and / or deletions, the protein may have additions, insertions, substitutions, and / or deletions that do not impair the protein kinase activity of the protein.

Examples of such a deletion include deletion of the amino acid sequence of SEQ ID NO: 4 from which the amino acid sequence at positions 90 to 359 (protein kinase region) has been deleted or a part thereof. Specifically, deletions of the amino acid sequences 1 to 89 and their partial sequences, and the amino acid sequences 360 to 1388 and their partial sequences are deleted.

According to another aspect of the invention, 9 of SEQ ID NO: 4
A protein having an amino acid sequence of 0 to 359 or 6 to 553 (corresponding to the amino acid sequence of 6 to 553 in SEQ ID NO: 1) or a derivative thereof is provided. This protein has protein kinase activity and has the active form R
It has no ho protein binding ability.

According to another aspect of the present invention, the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid sequences have been added and / or inserted, and / or one or more amino acids have been substituted and / or deleted, A protein having kinase activity of the amino acid sequence constitutively activated (ie, dominant active (do
minant active) (Rho kinase) is provided.

Here, “the kinase activity is constitutively activated” means that the kinase activity is always activated irrespective of the presence or absence of other regulatory factors (eg, Rho protein). Examples of deletions include those described above.

The protein or its derivative having the protein kinase activity according to the present invention and not having the ability to bind to the active form of Rho protein, when present in a cell, is resistant to Rho kinase endogenously present in the cell. Dominate and its action is active. Therefore, a protein having protein kinase activity and not having the ability to bind to active Rho protein or a derivative thereof is an embodiment of “dominant active Rho kinase”.

The dominant active Rho kinase is
It shows strong activity even when no active Rho protein is present. For example, as described in Examples 7, 13, 14, 19, and 20, by using a GST fusion protein comprising the amino acid sequence from 6 to 553 of SEQ ID NO: 1, which is an example of dominant active Rho kinase, The degree of phosphorylation of myosin light chain can be measured even in the absence of type Rho protein. The kinase activity of dominant active Rho kinase is
The kinase activity of native Rho kinase in the absence of active Rho protein (a protein having an active Rho protein binding ability and a protein kinase activity) is much stronger than that of purified Rho kinase in the presence of active Rho protein.
stronger than the kinase activity of ho kinase (Examples 7, 1
3 and 14).

When dominant active Rho kinase is present in a cell, its kinase activity is always activated. For example, Embodiment 1
As described in 2, by treating the isolated smooth muscle (skinned smooth muscle) having increased permeability with a GST fusion protein containing the amino acid sequence of Nos. 6 to 553 of SEQ ID NO: 1, strong smooth muscle contraction can be achieved. Can be measured. For example, as described in Examples 15 to 18, a GST fusion protein containing the amino acid sequence of Nos. 6 to 553 of SEQ ID NO: 1 is microinjected into fibroblasts, so that stress fibers in fibroblasts can be reduced. A pronounced appearance of focal adhesion can be observed. In order to observe these effects of dominant active Rho kinase, it is not necessary to administer skinned smooth muscle or fibroblasts together with activated Rho protein. Because, as mentioned above, dominant active Rho
This is because the kinase is in an activated state (constitutively activated) even in the absence of the active Rho protein.

As described above, the dominant active Rh
O-kinase exhibits sufficiently strong kinase activity and biological action (eg, smooth muscle contraction action, stress fiber and focal adhesion formation-inducing action) even in the absence of activated Rho protein. Therefore, these are R
Useful for measuring or observing the activity or action of ho kinase.

The protein according to the present invention is an active form of Rho
It has protein binding ability and protein kinase activity, or has been modified to lose any of these. In addition, Rho protein is closely related to cell formation such as tumor formation, metastasis, aggregation of platelets and leukocytes, cell morphology, cell motility, cell adhesion, formation of stress fibers and focal adhesion, and cytokinesis. (Takai, Y., et al., GCPren, supra.
dergast.et al., Khosravi-Far, R., et al, R. Qiu e
t al., Lebowitz, P., et al., and Yoshioka, K. et.
al.). Therefore, the protein according to the present invention is useful for elucidating the mechanism of tumor formation and metastasis and aggregation of platelets and leukocytes.

The Rho protein is known to be involved in smooth muscle contraction (see K. Hirata et al., Supra).
And M. Noda et al.). Therefore, the protein according to the present invention is useful for elucidating the mechanisms of various circulatory diseases such as hypertension, vasospasm (cardiovascular and cerebral vasospasm), angina pectoris, myocardial infarction, and arteriosclerosis obliterans. Is also useful.

Nucleotide Sequence According to the present invention, a nucleotide sequence encoding the protein of the present invention is provided. A typical sequence of this nucleotide sequence is
It has part or all of the DNA sequence of SEQ ID NO: 5.

The DNA sequence of SEQ ID NO: 5 was obtained from a cDNA library derived from human brain. This D
The NA sequence contains the open reading frame of human Rho kinase, where the open reading frame is 1
Starting from ATG # 3 to T # 4165-4167
End with AA.

Given the amino acid sequence of the protein according to the present invention, the nucleotide sequence encoding it is easily determined, and various nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO: 4 can be selected. Accordingly, the nucleotide sequence encoding the protein according to the present invention refers to a DNA sequence encoding the same amino acid in addition to a part or all of the DNA sequence shown in SEQ ID NO: 5, and a degenerate codon in the DNA sequence. As well as RNA sequences corresponding thereto.

The base sequence according to the present invention may be of natural origin or totally synthesized. In addition, a product synthesized using a part of a product derived from a natural product may be used. The nucleotide sequence is a chromosome library or c
It can be obtained from a DNA library by a method commonly used in the field of genetic engineering, for example, a method of screening using an appropriate DNA probe prepared based on partial amino acid sequence information. The base sequence according to the present invention can be obtained, for example, by using an oligonucleotide corresponding to the peptide indicated by the double line in FIG. 9 as a probe in screening (see Example 3).

Examples of the nucleotide sequence encoding the protein according to the present invention include the DNA sequence of Nos. 1 to 4167 of SEQ ID NO: 5 (corresponding to the open reading frame), the 1261 to 4111 of SEQ ID NO: 5, 1312 to 3372 239
No. 5-3411, 2821-225 or 282
DNA sequence of Nos. 7 to 3204 (corresponding to active Rho protein binding region), DNA sequence of Nos. 268 to 1077 (corresponding to kinase region), DN of 3373 to 4164
A sequence (corresponding to PH region).

Vector and Transformed Host Cell According to the present invention, the above-described nucleotide sequence of the present invention can be expressed in a state that the vector can be replicated in a host cell and the protein encoded by the nucleotide sequence can be expressed. A vector comprising the state is provided. Further, according to the present invention, there is provided a host cell transformed with the vector. The host-vector system is not particularly limited, and a fusion protein expression system with another protein can be used. Examples of the fusion protein expression system include those using MBP (maltose binding protein), GST (glutathione S transferase), HA (hemagglutinin), polyhistidine, myc, Fas and the like.

Examples of the vector include a plasmid vector (eg, an expression vector in prokaryotic cells, yeast, insect cells, animal cells, etc.), a viral vector (eg, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, Sendai virus vector, HIV vector, baculovirus vector), liposome vector (eg, cationic liposome vector) and the like.

The vector according to the present invention requires, in addition to the above-mentioned nucleotide sequence according to the present invention, a sequence for controlling its expression and a host cell in order to actually introduce the vector into a host cell and express the desired protein. It may contain a genetic marker or the like for selection. In addition, this vector is obtained by repeating the base sequence according to the present invention (for example, in tandem).
May be included. These may be introduced into a vector according to a conventional method, and a method of transforming a host cell with this vector may be one commonly used in this field.

The procedure and method for constructing a vector according to the present invention may be those commonly used in the field of genetic engineering.

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.). The transformed host cell is cultured in an appropriate medium, and the protein according to the present invention can be obtained from the culture. Thus, according to another aspect of the present invention, there is provided a method for producing a protein according to the present invention. The culture of the transformed host cells and their conditions may be essentially the same as for the cells used. The recovery and purification of the protein according to the present invention from the culture solution can also be performed according to a conventional method.

The cells to be transformed are, for example, cancer cells (eg, leukemia cells, gastrointestinal cancer cells, lung cancer cells, watermelon cancer cells, ovarian cancer cells, uterine cancer cells, melanoma cells, brain tumor cells) in cancer patients. Cell), the vector according to the present invention is introduced into a cancer cell in a living body, including a human, by a suitable method to express the protein according to the present invention. And the like.

For example, the protein of the present invention (a protein having the activity of binding to the active Rho protein and having no protein kinase activity) is expressed in vivo, including humans, so that the active Rho protein becomes active. Binds (inhibits the binding of Rho kinase to active Rho protein), resulting in the blocking of signal transduction from active Rho protein to Rho kinase,
It can suppress tumor formation or metastasis involving the ho protein. Regarding the vector for gene therapy, reference can be made to Experimental Medicine (Extra Issue), Vol. 12, No. 15, “The Forefront of Gene Therapy” (1994) supervised by Fumima Takaku.

Uses / Pharmaceutical Compositions Dominant negative Rho kinase is an active Rho kinase
Inhibits the action of proteins and Rho kinase.

The active Rho protein promotes tumor formation and metastasis. First, the active Rho protein has weak but oncogenic activity (Perona,
R. et al., Oncogene 8, 1285-1292 (1992)). Second, it is known that the GDP / GTP exchange protein that activates the Rho protein functions as a proto-oncogene. GDP activates Rho protein
/ GTP exchange proteins include Dbl, Vav, Os
t, Lbc, etc. are known (Collard, J., Int. J. On
col., 8, 131-138 (1996)). All of these proteins, when deleted at their N-terminus, exhibit oncogenic effects in the NIH3T3 transformation assay (Ron, D. et al., EMBO J., 7, 2465-2473 (198
8), Eva, A. et al., Nature 316, 273-275 (1985), Kat
zav, S. et al., EMBO J., 8, 2283-2290 (1989), Hori
i, Y. et al., EMBO J., 4776-4786 (1994), Toksoz,
D. etal., Oncogene 9, 621-628 (1994)). Therefore,
Rh activated by these proto-oncogenes
The o protein is thought to promote tumor formation. Third, Rho has recently been downstream of activated Ras protein, an oncogene product involved in about 30% of human tumors.
The signaling pathway is located, and it has been revealed that at least a part of the oncogenic action of the active Ras protein is mediated by the active Rho protein (Prendergast, G. et al.
et al., Oncogene 10, 2289-2296 (1995), Khosravi-Fa
r, R. et al., Mol. Cell. Biol., 15, 6443-6453 (199
5), Qiu, R. et al., 92, 11781-11785 (1995)). Finally, active Rho proteins have been shown to promote cell cycle (Yamamoto, M. et al., Onc.
ogene 10, 1935-1945 (1993), Olson, M. et al., Scie
nce 269, 1270-1272 (1995)). From the above, activated Rh
The o protein promotes tumor formation.

In addition to tumor formation, the active Rho protein promotes tumor metastasis. During metastasis, cancer cells infiltrate the host (patient) barrier such as vascular endothelial cells and mesothelial cell layers. For example, Imamura, F. et al.,
Biophys. Biochem. Res. Commun., 193, 497-503 (199
According to 3), when ascites carcinoma cells (MM1 cells) having high metastatic potential are cultured on the confluent mesothelial cell layer, the MM1 cells enter below the mesothelial cell layer from the gap between the mesothelial cells. And form infiltration foci. This phenomenon is a good indication of cancer cell invasion during metastasis. MM1 cell infiltration is markedly enhanced in the presence of serum or LPA. This serum or LPA-enhancing effect is inhibited by a Rho protein inhibitor (extracellular enzyme C3 of Clostridium botulinum), and thus is known to be mediated by an active Rho protein. This means that MM1 cells into which the gene of the active Rho protein (Rho ^Val14 ) has been introduced infiltrate the mesothelial cell layer in the absence of serum or LPA (Yoshioka, K. et al., FEBS Lett., 372, 25
-28 (1995)). As described above, the activated Rho protein promotes invasion and metastasis of cancer cells.

As described above, active Rho protein has been shown to promote tumor formation and tumor metastasis. It has been found that activated Rho protein promotes transcriptional activation of cellular genes and promotes cytokinesis. That is, the activated Rho protein is (1) serum response factor (SR
F) to promote the transcriptional activation of c-fos serum response factor (SRE) (Hill et al. (1995)), and (2) to promote cytokinesis by activated Rho protein (Kishi, K. et al. (1993) J. Cell Biol. 120: 1
187-1195; Mabuchi, I. et al. (1993) Zygote 1: 325
-331) is known. However, active Rh
The mechanism by which the o-protein activates SRE transcription, promotes cytokinesis, and enhances tumor metastasis has not been known at all.

In human tumors, Rho kinase activity and expression are enhanced, gene transcriptional activation and cytokinesis are promoted, and cell adhesion is enhanced. As a result, cells form tumors and metastasis is increased. It is thought to acquire ability. The rationale is described in detail below.

(1) Activation of SRE Transcription by Rho Kinase and Its Inhibition by Dominant Negative Rho Kinase As described below (Example 19), the inventors of the present invention used activated Rho kinase to activate protooncogene (proto-oncogene). It was found that the activation of c-fos SRE transcription was promoted. Furthermore, it has been found that dominant negative Rho kinase inhibits the activation of SRE transcription by activated Rho kinase.

These results indicate that physiologically, activation of gene transcription by activated Rho protein in cells is mediated by endogenous Rho kinase activated by activated Rho. Furthermore, dominant negative Rho kinase inhibits activation of gene transcription by activated Rho protein,
It is thought to inhibit the formation of tumors with enhanced E transcriptional activation.

(2) Evidence that Rho kinase is the same protein as mitotic cleavage kinase Rho protein is involved in the regulation of mitotic cleavage kinase (CF kinase) and also in the efficient distribution of IFs to daughter cells. In order to investigate the possibility that
We examined whether the kinase phosphorylates the same site as phosphorylation of GFAP by CF kinase. As a result, GFAP
A very suitable substrate for Rho kinase,
Phosphorylation of GFAP by Rho kinase in vitro
It was shown that P inhibits filament formation. In addition, Rho kinase converts G kinase by CF kinase in vitro.
It was revealed that Thr-7, Ser-13, and Ser-34, which are phosphorylation sites of FAP, are specifically phosphorylated.

The above results indicate that Rho kinase is the same protein as CF kinase, and therefore activated R
It has been shown that ho kinase promotes cytokinesis downstream of the active Rho protein. Therefore, Rho
It is thought that the kinase promotes not only activation of gene transcription (described above) but also cytokinesis, and causes cells to become tumors.

(3) Formation of Focal Adhesion by Activated Rho Kinase In order for tumor cells detached from the primary focus into the blood stream to survive in metastatic organs, adhesion of tumor cells to vascular endothelial cells and tissue at the metastasis destination Requires adhesion to cells. For this reason, tumor cells exhibiting high metastatic potential must have high cell adhesion.

The inventors of the present invention have made the following examples
It has been found that microinjection of active Rho kinase into cells or expression of active Rho kinase in cells can induce formation of focal adhesion (Examples 16 to 19). This indicates that the activated Rho kinase enhances cell adhesion ability, that is, enhances metastatic ability. Also,
The present inventors have found that dominant negative Rho kinase inhibits focal adhesion induced by activated Rho protein or activated Rho kinase. This is demonstrated by dominant negative Rho kinase
It shows that the metastasis of the tumor can be suppressed.

As described above, activated Rho kinase promotes not only tumor formation by activation of gene transcription and enhancement of cytokinesis, but also metastasis of tumor by enhancement of cell adhesion ability. And dominant negative Rho kinase inhibits tumor formation and metastasis.

(4) Relationship between Rho kinase locus and tumor formation and metastasis Various chromosomal abnormalities are observed in human tumors (Heim,
S. & Mitelman, F. (1992) Recent Adv. Histopathol.
15: 37-66). Then, the inventors of the present invention described later (Example 2).
As in 1), the position of the human Rho kinase gene on the chromosome was determined, and the association with the chromosomal abnormality in the tumor was examined. FISH analysis using a human Rho kinase cDNA fragment as a probe revealed that the human Rho kinase gene was present at the 2p24 site on the short arm of the second chromosome. In addition, this result
Confirmed by law.

The 2p24 site contains the oncogene MYCN (also called N-myc) (Garson, JA
et al. (1987) Nucleic. Acid Res. 15: 4761-477
0). The MYCN gene is used in human neuroblastoma.
ma), retinoblastoma, lung cell carcinoma and rhabdomyosarcoma may be amplified about 25 to 700-fold (Kohl, N. et al. (1983) Cell 35: 359-367; Kohl ,
N. et al. (1984) Science 226: 1335-1337; Lee, W.-
H. et al. (1984) Nature 309: 73-77; Schwab, M. Tren
ds Genet. 1: 271-275 and Naotoshi Kanda / Appi Kaneko (199
5) History of Medicine 173: 227-229). In neuroblastoma cell lines, metaphase paired punctate microchromosomes (DMs: d
ouble minute). DMs are often identifiable even with a microscope, and such DMs are usually
It is more than 1,000 kb, and some of them are more than 10,000 kb. DMs are circular and often enter unspecified sites on the chromosome, and HSRs (homogeneously staining region)
s). The amplification of the MYCN gene occurs in DMs and HSRs. In the DMs and HSRs, not only the MYCN gene but also the gene existing around the MYCN gene (2p24) is amplified. Therefore, it is considered that the Rho kinase gene also present in 2p24 is also amplified.

When the Rho kinase gene is amplified, the amount and activity of Rho kinase in the cell increases significantly.
As described above, the present inventors have found that Rho kinase activates cell gene transcription and promotes cytokinesis. In addition, cell adhesion ability is enhanced (focal adhesion is enhanced) by Rho kinase. Therefore, in cells in which the Rho kinase gene has been significantly amplified, it is considered that cell division is accelerated and metastatic potential is increased. Indeed, it is known that in neuroblastoma, in cases where DMs and HSRs appear (that is, MYCN and Rho kinase genes are amplified), the prognosis is extremely poor and the metastatic ability of the tumor is high. Since MYCN is known as one of the transcription factors, MYCN is thought to act synergistically with the transcriptional activation of Rho kinase to promote cell proliferation (ie, tumor formation).

From the above, the Rho kinase gene is associated with malignant tumors, especially tumor neuroblastoma, retinoblastoma, lung cell carcinoma, and rhabdomyosarcoma. Therefore, the nucleotide sequences and probes according to the present invention described below are useful for diagnosis of the above-mentioned diseases.

The present inventors have determined in the following examples that
Activated R by dominant negative Rho kinase
It has been found that the activation of Rho kinase by the ho protein can be inhibited, and the formation of stress fibers and focal adhesion induced in cells by LPA can be inhibited. Since the function of the active Rho protein can be inhibited, the dominant negative Rho kinase can be used as an inhibitor of the above-mentioned tumor formation or metastasis promoted by the active Rho protein (hereinafter, referred to as “an inhibitor of tumor formation or the like”). In addition, dominant negative Rho kinase can be generally used as an inhibitor of metastasis of a tumor with enhanced cell adhesion, because it inhibits the enhancement of cell adhesion.

Here, as tumor formation and metastasis, R
ho involved tumor formation, other low molecular weight G protein (eg Ras, Rac, Cdc42, Ral, etc.) involved tumor formation, low molecular weight G protein GDP /
Tumor formation involving GTP exchange proteins (eg, Dbl, Ost, etc.), lysophosphatidic acid (LP
A) Involvement of tumor formation, receptor-type tyrosine kinase (eg, PDGF receptor, EGF receptor, etc.), transcription control protein (myc, p53, etc.) or formation of tumors involving various human tumor viruses. Can be

The present inventors have also proposed that myosin light chain phosphatase present in smooth muscle and the myosin-binding subunit (Y. h. Chen.
al., FEBS Lett., 356, 51-55 (1994)) is the most suitable physiological substrate for Rho kinase (Example 2 (3), Example 5 and Example 6). Phosphorylation of chain phosphatase (including the myosin-binding subunit) suppresses the phosphatase activity (Examples 5 and 6), and results in Rho kinase in cells thought to endogenously express Rho kinase. It was found that myosin-binding subunit and myosin light chain were phosphorylated when the protein was expressed (Example 6).

Furthermore, the present inventors have determined that Rho kinase
Phosphorylating both the isolated myosin light chain and the myosin light chain of intact myosin in a TP-bound Rho protein-dependent manner (Example 7); the main phosphorylation site of myosin light chain by Rho kinase is myosin light Ser-19 phosphorylated by chain kinase (Example 8), phosphorylation of intact myosin myosin light chain increases MgATPase activity of myosin light chain (Example 9), protein kinase It has been found that a Rho kinase derivative whose activity is constantly activated promotes smooth muscle contraction (Example 12).

Therefore, without being bound by the following theory, it is considered that the Rho protein promotes smooth muscle contraction by the following mechanism. (1) The binding of active Rho protein to Rho kinase enhances the kinase activity of Rho kinase. (2) The myosin binding subunit of myosin light chain phosphatase is phosphorylated by the Rho kinase. (3) The phosphorylation suppresses the phosphatase activity of myosin light chain phosphatase and inhibits the dephosphorylation of myosin light chain. (4) As a result of suppression of dephosphorylation, myosin remains phosphorylated. (5) Myosin light chain is phosphorylated by the Rho kinase of (1). (6) From the phenomena (4) and (5), the polymerization of myosin and actin is promoted and the depolymerization is suppressed. (7) As a result, smooth muscle contraction is promoted and maintained. The model is shown in FIG.

As described above, the dominant negative Rho
Kinases are inhibitors of smooth muscle contraction and various circulatory diseases involving smooth muscle contraction (hypertension, vasospasm (cardiovascular and cerebral vasospasm), angina pectoris, myocardial infarction, and atherosclerosis obliterans) Etc.) as therapeutic agents.

The dominant negative Rho according to the invention
A kinase, when present in a cell, inhibits the action of an active Rho protein endogenously present in the cell. For example, as shown in Examples 15 and 16,
A GS comprising the amino acid sequence of positions 941 to 1075 of SEQ ID NO: 1, which is an example of a dominant negative Rho kinase
When the T fusion protein was microinjected into Swiss3T3 cells, the induction of stress fiber formation and focal adhesion formation of Swiss3T3 cells was inhibited. Induction of stress fiber formation and focal adhesion formation is one of the representative biological functions of activated Rho proteins (Ridley, A. & Hal
l, A., Cell 70, 389-399 (1992) and Ridley, A.
& Hall, A., EMBO J., 13, 2600-2610 (1994) and Examples 15 and 16). Thus, the dominant negative Rho kinase can inhibit the action of the active Rho protein endogenously present in the cell by causing it to be present in the cell.

The activated Rho protein induces cell adhesion or cell aggregation. Cell adhesion or cell aggregation includes, for example, platelet aggregation (Morii, N. et al.,
J. Biol. Chem., 29, 20921-20926 (1992)) and aggregation of leukocytes (lymphocytes) (Tominaga, T. et al., J. Cell
Biol., 120, 1529-1537 (1993) and Laudanna, C.et
al., Science 271, 981-983 (1996)).
According to Morii, N. et al. (1992), thrombin and p
gpIIb-IIIa complex-dependent platelet aggregation induced by horbol myristate acetate (PMA) was inhibited by botulinum extracellular enzyme C3 (hereinafter referred to as C3 extracellular enzyme). Also, Tominaga, T. et
According to al. (1993), PMA-induced lymphocyte function-related antigen (LFA-1) -dependent lymphocyte aggregation was
Inhibited by 3 extracellular enzymes. In addition, Laudin, supra.
According to a, C. et al. (1996), the formyl peptide (f
ormylpeptide; fMLP), interleukin 8 (IL8)
Alternatively, cell adhesion of lymphocytes and neutrophils stimulated by PMA was also inhibited by C3 extracellular enzyme. And
The C3 extracellular enzyme was due to inhibition of α4β1 lymphocyte and β2 integrin neutrophil adhesion. As described above, activated Rho protein induces cell adhesion and cell aggregation, and the cell adhesion and cell aggregation are induced by cell adhesion molecules (gpIIb of platelets).
-IIIa complex, LFA-1 and α4β1 of lymphocytes,
Neutrophil β2 integrin). All of these cell adhesion molecules are included in the integrin family (Hynes, R. et al., Cell 69, 11-25 (1992)). As described above, the activated Rho protein promotes integrin-mediated cell adhesion.

We have shown that dominant-negative Rho-kinase, as shown in the examples below,
It has been shown that the activity of Rho kinase or the activation of Rho kinase by activated Rho protein is inhibited, and that the formation of focal adhesion as well as the stress fiber of cells is thereby inhibited. According to the above, it is clear that dominant negative Rho kinase also inhibits integrin-related cell adhesion and cell aggregation. In addition, it is known that cells are activated by such cell adhesion and aggregation. Therefore, dominant-negative Rho-kinase is involved in platelet aggregation and activation involving integrins, aggregation / adhesion and activation of immunocompetent cells (T lymphocytes and B lymphocytes), inflammatory blood cells (neutrophils, eosinophils). It is clear that it inhibits adhesion / aggregation and activation of spheres, basophils and macrophages, and is useful for antiplatelet drugs, anti-inflammatory drugs, antiallergic drugs, It can be used as a therapeutic.

The inhibitor and therapeutic agent according to the present invention are used in the sense that they include the gene therapeutic agents described below.

The inhibitors and therapeutic agents according to the present invention are also administered orally or parenterally (eg, intramuscular, intravenous, subcutaneous, rectal, transdermal, nasal, etc.), preferably orally. It can be used in humans and non-human animals in various dosage forms suitable for oral or parenteral administration as medicaments.

Inhibitors and therapeutic agents are, for example, oral preparations such as tablets, capsules, granules, powders, pills, fine granules, and troches, and injections such as intravenous injections and intramuscular injections, depending on their use. , Rectal preparations, oily suppositories, water-soluble suppositories and the like. These various formulations, commonly used excipients, for example, bulking agents,
Conventional methods using binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, dissolution aids, preservatives, flavoring agents, soothing agents, stabilizers, etc. Can be manufactured. Examples of the non-toxic additives that can be used include lactose, fructose, glucose, starch, gelatin,
Magnesium carbonate, synthetic magnesium silicate, talc,
Examples include magnesium stearate, methylcellulose, carboxymethylcellulose or a salt thereof, gum arabic, polyethylene glycol, syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium sulfite, sodium phosphate and the like.

[0110] The content of the protein of the present invention in the drug varies depending on the dosage form, but is usually about 0.5% in the total composition.
The concentration is from 1 to about 50% by weight, preferably from about 1 to about 20% by weight. The dosage for various suppressions and treatments is appropriately determined in consideration of the usage, the age of the patient, gender, degree of symptoms, and the like, and is usually about 0.1 to about 500 m / day for an adult.
g, preferably about 0.5 to about 50 mg, which can be administered once or several times a day.

According to the present invention, dominant negative R
Including ho kinase in cells where tumors are forming, cells where the tumor may metastasize, cells with increased smooth muscle contraction, or cells with increased inflammation or autoimmunity A method for suppressing tumor formation or metastasis, a method for suppressing smooth muscle contraction, a method for suppressing inflammation and autoimmunity, and a method for suppressing platelet aggregation. In this case, the effective dose, administration method, administration form and the like can be in accordance with the above-mentioned inhibitors for tumor formation and the like.

The nucleotide sequence encoding dominant-negative Rho kinase can be obtained by transforming a target cell with the vector having the same or by using this sequence alone to suppress the formation or metastasis of tumor cells. It can be used in a form that suppresses the enhancement of contraction, or in a form that suppresses the enhancement of inflammation or autoimmunity.
That is, the nucleotide sequence can be used as a gene therapy agent for suppressing tumor formation or metastasis, a gene therapy agent for a circulatory disease, a gene therapy agent for an inflammatory disease or an autoimmune disease, or a gene therapy agent for inhibiting platelet aggregation.

[0113] According to the screening method the present invention, (1) the subject to substance screening, be present in the screening system comprising an active Rho protein, a protein according to the present invention having the activated Rho protein binding activity, and (2) Activated Rho
Activated Rho protein and activated Rh, comprising measuring the degree of inhibition of binding between the protein and the protein according to the present invention having activated Rho protein binding ability.
o A method for screening for a substance that inhibits binding to a protein according to the present invention having protein binding ability is provided.

Here, "measuring the degree of inhibition of binding"
As a method, a method of measuring the binding between the protein of the present invention and the recombinant GTPγS.GST-RhoA protein in a cell-free system using glutathione sepharose beads, a method of the present invention in an animal cell (cell line), Method for measuring the binding between Rho and Rho protein using immunoprecipitation and immunoblot
System (two hybridsystem) (M. Kawabata Experimental Medicine 13,2111-2120 (1995), ABVojetk et al. Cell 74,20)
5-214 (1993)). For example, the degree of inhibition of binding can be measured according to the method described in Example 1 or 4. Further, in the present specification, "measuring the degree of inhibition of binding" is used in a sense including measurement of the presence or absence of binding.

The screening system may be either a cell system or a cell-free system. Examples of the cell system include yeast cells, COS cells, Escherichia coli, insect cells, nematode cells, lymph cells, fibroblasts (3Y1 Cells, NIH / 3T3 cells, Rat1 cells, Balb / 3T3 cells, etc.), CHO
Cells, blood cells, tumor cells, smooth muscle cells, cardiomyocytes,
Examples include nerve cells, myeloid cells, glial cells, and astrocytes.

The substance to be screened is not particularly restricted but includes, for example, peptides, peptide analogs, microorganism cultures, organic compounds and the like.

According to the present invention, (1) the substance to be screened is present in a screening system containing the protein of the present invention having protein kinase activity or its derivative, and (2) the protein kinase activity is A method for screening a substance inhibiting the protein kinase activity of a protein or a derivative thereof according to the present invention having a protein kinase activity, comprising measuring the degree of inhibition of the protein kinase activity of the protein or a derivative thereof according to the present invention. Is done.

According to the present invention, further, (1) the substance to be screened is an active Rho protein, a protein according to the present invention having an active Rho protein binding ability and having a protein kinase activity. And (2) inhibition of the protein kinase activity of the protein or a derivative thereof according to the present invention having an active Rho protein binding ability and having a protein kinase activity, or an increase in the activity thereof. A method for screening for a substance that inhibits the protein kinase activity of a protein or a derivative thereof according to the present invention, which has an active Rho protein binding ability and has a protein kinase activity, which comprises measuring the degree of Provided.

As a method for measuring the "degree of inhibition of protein kinase activity" or the "degree of inhibition of enhancement of protein kinase activity", the autophosphorylation activity of the protein of the present invention or the activity of phosphorylating a suitable substrate can be used. Or a method for measuring the degree of enhancement of these activities in the presence of activated Rho protein. For example, protein kinase activity is measured according to the methods described in Examples 2, 5 to 9, 13, 19 and 20. Can be measured.

As a method for detecting phosphorylation of a substrate protein, for example, radioactive labeling (for example, [γ- ³² P] A
TP]) (Examples 2, 5 to 9, 13 to 1)
4), a method using an antibody against a specific site of a phosphorylated substrate (Examples 19 and 20), and the like. In order to detect the phosphorylation of a substrate protein by Rho kinase using an antibody against a specific site of the phosphorylated substrate, the site to be phosphorylated by Rho kinase is determined in advance.

The phosphorylation of MLC can be detected by immunoblotting and immunofluorescence staining of MLC phosphorylation by Rho kinase using an antibody against MLC in which Ser-19 is phosphorylated. Also, G described later
Phosphorylation of FAP was determined by Ser-13 and Ser-34.
Rh is phosphorylated using an antibody against GFAP.
Phosphorylation of GFAP by o-kinase can be detected by immunoblot and immunofluorescent staining.

In detecting phosphorylation of a substrate protein by Rho kinase using an antibody against a specific site of a phosphorylated substrate, ELISA can be used in addition to immunoblot and immunofluorescence staining.

The measurement of phosphorylation by ELISA is described, for example, in Yano, T. et al., Biochem. Biophys. Res. Commun.
175, 1144-1151 (1991). The method for detecting phosphorylation by an ELISA system using an antibody against a specific site of such a phosphorylated substrate does not require the use of radioisotopes and is extremely simple, so that a large number of samples can be safely processed. Especially good at doing. Therefore, a large amount (tens of thousands to several hundreds)
This is one of the most suitable methods for screening Rho kinase inhibitors from the compounds in (10).

In the present invention, "substance inhibiting the activity"
The term “substance that inhibits the enhancement of activity” refers to a substance that is evaluated by those skilled in the art as having an activity or inhibition of the enhancement of activity. For example, Examples 2, 5 to 9, 13, and 1
When the experiment is carried out under the same conditions as in 9 and 20, it means that it is evaluated that inhibition of the protein kinase activity or the enhancement of the activity is recognized.

As used herein, the term “degree of inhibition of protein kinase activity” or “degree of inhibition of enhancement of protein kinase activity” refers to the presence or absence of inhibition of protein kinase activity or enhancement of protein kinase activity. Shall be used in the sense including the measurement of

Further, as shown in Examples described later, “the degree of inhibition of protein kinase activity” can be measured using dominant active Rho kinase (Examples 7, 12 and 14). R like this
Ho kinase derivatives are derivatives whose protein kinase activity is constantly activated.

Further, as shown in the examples described later, the active Rho protein that has been subjected to post-translational modification is:
It enhances the protein kinase activity of Rho kinase more strongly than the active Rho protein that is not modified (Example 2, (4)). Therefore, the use of the activated Rho protein that has undergone post-translational modification enables the screening according to the present invention to be performed more clearly.

Examples of the substrate include non-physiological substrates (eg, myelin basic protein, S6 peptide, αPKC, histone, vinculin, talin, metavinculin, caldesmon, filamin, α-actinin, MAP-
4), and physiological substrates (eg, myosin, myosin light chain, myosin light chain phosphatase, one of its subunits, myosin binding subunit (MBS))
Is mentioned.

As shown in Examples 2 and 5 below, when a myosin-binding subunit is used as a substrate, phosphorylation is increased 5 to 15 times in the presence of the active Rho protein as compared to that in the absence of the active Rho protein. Is done. Therefore, when the myosin-binding subunit is used as a substrate in the presence of the activated Rho protein, the screening according to the present invention can be performed more clearly.

When a myosin light chain is used as a substrate as shown in Example 7 described later, the active Rh
In the presence of o-protein, the Km value of Rho kinase is reduced to about 1/5 compared to that in the absence. Therefore, when myosin or myosin light chain is used as a substrate in the presence of activated Rho protein, screening according to the present invention can be performed more clearly.

In the screening method according to the present invention, intermediate filaments (IF)
s) can be used as a substrate (Example 20). I
Fs is a major constituent protein of the cytoskeleton and nuclear membrane in most eukaryotic cells. IFs include, for example, glial fibrillary acidic protei
n: GFAP), vimentins (vimentins), neurofilaments (NFs), keratins (keratins), desmin (desmin), peripherin (peripherin), α-internexin (internexin), lamin (lamin), nestin (nestin) Etc. are included. They are classified as follows based on the homology of their amino acid sequences, the cell and tissue specificity of the expression, or the structure of the exon-intron of the gene encoding each subunit. [Type 1] acidic keratin (epithelial cells) [type 2] neutral and basic keratin (epithelial cells) [type 3] desmin (muscle cells), vimentin (mesenchymal cells and most cultured cells), GFAP (Glial cells), peripherin (peripheral neurons) [type 4] neurofilament (NF) subunits (NFL, NFM and NFH: neurons), α-internexin (neural cells) [type 5] lamin (all [Type 6] nestin (neural coat stem cell) In the present invention, the “intermediate filament” is used in the sense including the above.

[0132] Natural Rho kinase was used as an enzyme preparation.
When GFAP is used as the substrate protein, the phosphorylation of GFAP is increased about 300 times in the presence of the active Rho protein as compared to that in the absence (Example 20). Therefore, when the intermediate filament (particularly, GFAP) is used as a substrate in the presence of the activated Rho protein, the screening according to the present invention can be performed more clearly. The screening system and the subject of the screening system include those similar to the above-mentioned screening method.

According to the present invention, (1) a substance to be screened is present in a cell line in which the formation of stress fiber or focal adhesion is induced by the presence of dominant active Rho kinase,
And (2) a method of screening for a substance that inhibits the formation of stress fibers, comprising measuring the degree of inhibition of the formation of stress fibers or focal adhesion of the cell line.

Here, the “measurement of the degree of inhibition of the formation of stress fibers” and the “measurement of the degree of inhibition of the formation of focal adhesion” are performed by visualizing actin in cells with a fluorescently labeled probe. For example, the degree of formation inhibition can be measured according to the methods described in Examples 14 to 19.

In the present invention, "a substance inhibiting formation"
The term “substance” refers to a substance evaluated to be inhibited by a person skilled in the art. Shall mean the case where the evaluation is made. Further, in the present specification, “measuring the degree of inhibition of formation” is used in a sense including measurement of the presence or absence of formation.

Cells include, for example, yeast cells, COS cells,
Insect cells, nematode cells, lymphocytes, fibroblasts (3Y1
Cells, NIH / 3T3 cells, Rat1 cells, Balb /
3T3 cells, Swiss3T3 cells, etc.), CHO cells,
Examples include blood cells, tumor cells, smooth muscle cells, cardiomyocytes, nerve cells, myeloid cells, glial cells, and astrocytes.

The screening target includes the same ones as described above.

According to the invention, further (1) the object of screening is present in a cell line containing a protein according to the invention having protein kinase activity, and (2)
C-fos serum response factor (SRE) in said cell line
Measuring the extent of inhibition of the transcriptional activity of
A method for screening a substance that inhibits the transcriptional activity of os serum response factor (SRE) is provided.

As a method for measuring the “degree of inhibition of c-fosSRE transcriptional activity”, a fusion protein of c-fosSRE and an appropriate reporter protein (eg, luciferase) is expressed in a cell line (eg, NIH3T3 cells). And a method for measuring the degree of reporter gene activity. In this case, the nucleotide sequence encoding the fusion protein and the nucleotide sequence encoding the test sample (protein) may be co-transfected. For example, cf according to the method described in Example 19
The degree of inhibition of osSRE transcription activity can be measured.

In the present invention, the term “substance that inhibits transcriptional activity” refers to a substance which is evaluated by those skilled in the art as inhibiting the activity. It shall mean the case where the inhibition of transcription activity is evaluated.

As the cell line, the cell line described in “Screening method for substance that inhibits formation of stress fiber” can be used. Screening targets include those similar to those described above. In the present invention, the “screening method” is used in a sense including an assay.

As described above, it has been confirmed that the activated Rho protein is closely involved in tumor formation and metastasis, smooth muscle contraction, and aggregation or activation of platelets or leukocytes. Therefore, the above-described screening method can be used as a screening method for a substance that inhibits tumor formation or metastasis, a substance that inhibits smooth muscle contraction, and a substance that inhibits aggregation or activation of platelets or leukocytes.

Probes According to the present invention, (a) a base sequence containing a DNA sequence of at least 15 contiguous bases in the sequence of SEQ ID NO: 5 and a sequence complementary thereto, and (b) a base sequence of (a) And a base sequence selected from base sequences that hybridize under stringent conditions with the present invention.

The above sequence includes, for example, the base sequence of 930 to 2025 of SEQ ID NO: 5 and a sequence complementary thereto.

According to the present invention, (a) SEQ ID NO: 6
Selected from a base sequence containing a continuous DNA sequence of at least 15 bases in the above sequence, and a sequence complementary thereto, and a base sequence that hybridizes with the base sequence of (b) and (a) under stringent conditions. , A base sequence is provided.

The above sequence includes, for example, the base sequence at positions 646 to 667 or 536 to 555 (sequence in the intron) of SEQ ID NO: 6 and a sequence complementary thereto.

In the present invention, “stringent conditions” in the hybridization refers to a Southern hybridization method, a PCR method (Example 4,
8, 10, and 11) and the FISH method (Example 2)
It is defined as the hybridization condition of 1).

According to the present invention, there is provided a probe comprising the above-mentioned nucleotide sequence and a label. Detectable labels can be selected from those known to those of skill in the art, for example, interacting substances (eg, avidin and biotin), enzymes (eg, peroxidase, alkaline phosphatase),
Examples include radioisotopes (eg, ³² P and ³⁵ S), fluorescence (eg, FITC, europium), antigens (eg, digoxigenin), and the like.

The method of labeling the nucleotide sequence may be determined depending on the labeling molecule, and can be performed by, for example, nick translation, chemical (or photochemical) crosslinking, chemical synthesis of oligonucleotides, chelation, or the like.
The method for detecting the label may be determined depending on the label molecule. For example, the label can be detected using fluorescence, an enzyme or a ferritin-labeled antibody, avidin-FITC, β-galactosidase, colloidal gold, or the like. .

As mentioned above, the Rho kinase gene is
Malignant tumors, especially tumor neuroblastoma, retinoblastoma, lung cell carcinoma,
And rhabdomyosarcoma. Therefore, these diseases can be diagnosed (including, for example, presymptomatic diagnosis such as prenatal diagnosis) using the nucleotide sequence and the probe (hereinafter, may be simply referred to as “probe”) according to the present invention.

According to the present invention, there is also provided a diagnostic agent comprising the above-mentioned probe. Diagnosis can be performed by removing a gene sample (chromosomal or genomic DNA, poly (A) ⁺ RNA or mRNA, etc.) from a patient and measuring the degree of hybridization between the probe and the gene sample. Methods for measuring the degree of such hybridization include the FISH method, the Southern hybridization method, and the PCR method.

(1) FISH method When the FISH method is used, the above probe is hybridized with a sample in which chromosomes of cells (for example, lymphocytes) isolated from humans are fixed, and the Rho kinase gene on the chromosome is mapped. . In normal human cells, the Rho kinase gene maps only to the short arm p24 site (2p24) of a pair of chromosomes 2, as described in the Examples below.

On the other hand, among the patients with malignant tumors, those in which the signal of the Rho kinase gene is not detected (the Rho kinase gene is deleted) and those in which the signal of the Rho kinase gene is strongly detected (the Rho kinase gene is not detected) Amplified) or a patient in which the signal of the Rho kinase gene is detected at a site other than 2p24 (the Rho kinase gene is translocated). Such a mutation (deletion, amplification or translocation) of the Rho kinase gene is a change characteristic of any of the above diseases.
Therefore, the disease can be diagnosed by measuring the degree of hybridization by the FISH method or detecting the position of the Rho kinase gene on the chromosome.

The FISH method can be carried out, for example, under the conditions described in Example 21. Actually, the purpose of the experiment, the probe and the DNA to be hybridized
Hybridization is performed by selecting the optimal conditions within the range of these conditions according to the type of the above. The conditions of the FISH method are described in Heng HHQ et al., Proc. Natl. Acad. Sci. USA, 89,
9509-9513 (1992); Heng HHQ & Tsui LC, In situ
hybridization protocols: Methods in Molecular Biol
ogy. Choo KHA (ed) .Humana Press: New Jersey, pp.10
9-122 (1994) Choo, KHA, ed., Methods in Molecu
lar Biology: In situ hybridization protocoles. (Ch
oo, KHA, ed.). pp35-49 (1994) Humana Press, C
lifton, NJ., USA and Gerhard, DSet al., Proc. Na
USA. 78, 3755-3759 (1981).

(2) Southern hybridization method Southern hybridization (J. Sambrook et. Al.,
Molecular Cloning 2nd ed.Ch. 9 (1989) Cold Spring
When using Harbor Laboratory Press, New York, genomic DNA is isolated from cells (eg, lymphocytes) isolated from humans, digested with appropriate restriction enzymes, and then subjected to gel electrophoresis for digestion. Separated DNA fragments. Thereafter, the separated DNA fragment is transferred to a filter and hybridized with the probe on the filter. Among the above patients, there are patients in which hybridization is not observed or the amount of hybridization is small compared to healthy individuals (part or all of the Rho kinase gene is deleted, etc.). Patients with a greater amount of hybridization (Rh
okinase gene is amplified) or a patient whose DNA fragment size and number to which the probe hybridizes is different from that of a healthy person. In addition, Southern hybridization was performed using human / rodent somatic cel.
l By combining with a hybrid panel, the patient's R
The location of the ho kinase gene on the chromosome can be detected (Macera, MJ et al., Genomics 13,829-831 (199
2)). Thereby, the presence or absence of Rho kinase gene deletion or translocation in the patient can be detected.

Such a mutation (deletion, amplification, translocation, etc.) of the Rho kinase gene is a characteristic change in any of the above-mentioned diseases. Therefore, the disease can be diagnosed by measuring the pattern of the band that hybridizes with the probe by Southern hybridization.

When a DNA fragment is used as a probe, 2-6 × SSC (0.15 M sodium chloride solution,
0.015M sodium citrate solution), 65 ° C-70
Southern hybridization can be performed under the condition of ° C. In practice, hybridization is performed under optimal conditions according to the purpose of the experiment, the type of the probe and the DNA to be hybridized with the probe, and the like. The conditions for hybridization can be referred to “Gene manipulation manual” edited by Yasutaka Takagi, Kodansha, Tokyo, Japan (1982).

(3) PCR method A gene sample (chromosomal or genomic DNA, poly (A) ⁺ RNA or mRNA) is taken out from a patient and replaced with Southern hybridization by PCR (Saiki, R. et al.).
K. et. Al., Science 239 (1988) 487-491).
Diagnosis can also be performed by measuring the degree of amplification of the gene fragment. In the PCR method, a pair of primers (primer pairs) is used as a template for DNA or RNA
And the DNA fragment is amplified using polymerase or reverse transcriptase. The above probes can be paired and used as primers for PCR. By using such a primer pair and performing PCR using the extracted patient gene sample as a template, all or a part of the Rho kinase gene is amplified. By analyzing the amplified DNA by electrophoresis or nucleotide sequence analysis, it is possible to detect the mutation (deletion, amplification, recombination, translocation, base substitution, etc.) of the Rho kinase gene or the degree of mRNA expression. is there.

For example, when a gene sample of a patient whose Rho kinase gene is amplified is applied to the PCR method, the specific region is amplified more than in a healthy person. Therefore, amplification of the Rho kinase gene can be detected by checking the amount of the amplified fragment.

In addition, when poly (A) ⁺ RNA or mRNA of a gene sample of a patient whose Rho kinase mRNA expression level is enhanced is applied to the PCR method, the specific region is amplified more than in a healthy person. . Therefore, an increase in Rho kinase mRNA expression can be detected by examining the amount of the amplified fragment.

The PCR method is described, for example, in Takara LA taq kit
Using ver.2, the reaction can be performed 30 times at 95 ° C for 15 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 minutes. In practice, optimal conditions are selected according to the purpose of the experiment, the type of gene sample used as a probe or its template, etc.
I do. PCR conditions are described in Saiki, RK et. Al., Scie
nce, 239, 487-491 (1988).

Mutations in the Rho kinase gene are characteristic of malignant tumors, as described above, particularly tumor neuroblastoma, retinoblastoma, lung cell carcinoma, and rhabdomyosarcoma. Therefore, the above diagnostic agents can be used as diagnostic agents for these diseases.

According to the present invention, there is provided a method for detecting a mutation in the Rho kinase gene, comprising hybridizing the probe with a mammalian gene sample and measuring the degree of hybridization. The above detection method can be carried out in the same manner as in the use mode of the above diagnostic agent.

[0164]

The present invention will be described in detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Example 1 Purification of Activated Rho Protein Binding Protein
Ltd. and the pieces cut its identification (1) brain membranes extract prepared bovine brain gray matter a (200 g) in scissors, 3
00 ml of homogenization buffer (25 mM Tr
is / HCl, pH 7.5, 5 mM EGTA, 1 mM
Dithiothreitol (DTT), 10 mM MgCl
₂ , 10 μM (p-amidinophenyl) -methanesulfonyl fluoride, 1 mg / l leupeptin, 1
0% sucrose) to obtain a crude membrane fraction. The protein in the crude membrane fraction was extracted by adding a homogenization buffer containing 4M NaCl. After shaking at 4 ° C. for 1 hour, the membrane fraction was subjected to 20,000 × g for 1 hour,
Centrifuged at 4 ° C. The supernatant fraction was transferred to buffer A (20 m
M Tris / HCl, pH 7.5, 1 mM EDT
A, 1 mM DTT, 5 mM MgCl ₂ )
Dialyzed twice. Thereafter, solid ammonium sulfate was added so that the final concentration was 40% saturation. The precipitate precipitated with 0-40% ammonium sulfate was dissolved in 16 ml of buffer A, dialyzed again against buffer A three times, and used as a bovine brain membrane extract.

(2) Preparation of low molecular weight G protein affinity column Glutathione-S-transferase (hereinafter referred to as “GS
T ")-RhoA, GST-Rho
A ^Ala37 , GST-Rac1 and GST- ^HR
as is described in H. Shimizu et al., J. Biol. Chem., 269,
30407-30411 (1994), H. Shimizu et al., J. Biol. C
hem., 269, 22917-22920 (1994) and loaded with guanine nucleotides. GST-
The low molecular weight G protein (24 nmol each) was immobilized on a 1 ml glutathione-Sepharose column 4B and packed in the column.

(3) GST-low molecular weight G protein affinity column chromatography The bovine brain membrane extract was applied to a 1 ml glutathione-Sepharose column. The eluted fraction was subjected to 24 nmol GS
T, GDP, GST-RhoA, or GTPγS
Glutathione-Sepharose column containing GST-RhoA was loaded. GTPγS is an analog of GTP that is not hydrolyzed. The protein bound to the glutathione-Sepharose column was eluted with 10 ml of buffer A containing glutathione or 1% CHAPS (3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid), and the eluted fraction was 1 ml. Collected one by one. A part of the eluted fraction is used for UK Laemmli, Natur
e, 227, 680-685 (1970)
-PAGE was performed and silver stained. The results were as shown in FIG. A protein with a molecular weight of about 164 kDa (bovine Rho kinase) was eluted in 2-10 fractions.
Bovine Rho kinase is GTPγS · GST-RhoA
Is eluted only from the glutathione-Sepharose affinity column containing
It was not eluted from the GST-RhoA column. Bovine Rho kinase is GTPγS · GST-Rh
It was hardly eluted from oA ^Ala37 (RhoA mutant protein having an amino acid substitution in the effector region). Bovine Rho kinase is GTPγS · GST
It was not eluted from the affinity column of -Rac1 or GTPγS.GST-H-Ras. in this way,
It has been revealed that bovine Rho kinase specifically binds to activated RhoA via the effector region.

(4) Purification of bovine Rho kinase In order to purify bovine Rho kinase, GTPγS · G
The eluted fraction (fractions 3 to 10) from the glutathione-Sepharose affinity column containing ST-RhoA was diluted with the same amount of buffer A and loaded onto a Mono Q5 / 5 column equilibrated with buffer A. Column 1
After washing with 0 ml Buffer A, the protein was
Elution was performed with buffer A adjusted to a linear density gradient of a 0.5 M NaCl solution, and 0.5 ml of the eluted fraction was collected. The results were as shown in FIG. Bovine Rho kinase was recovered as a single peak in fractions 10-12 (FIG. 2, upper row). Part of the eluted fraction (8-14)
When SDS-PAGE was performed and silver staining was performed, the purity of the purified sample was about 95% (lower part in FIG. 2).

(5) Rho by overlay assay
Identification of protein binding proteins Manser et al. (E. Manser et al., J. Biol. Chem., 26
7, 16025-16028 (1992)), and an overlay assay was performed as a modification of the method described previously. The sample was subjected to 6% SDS-PAGE, and then blotted to a nitrocellulose membrane. Nitrocellulose membrane at 4 ° C
Buffer B containing 6 M guanidine hydrochloride for 5 minutes
(25 mM Hepes / NaOH, pH 7.0, 0.
5 mM MgCl ₂ , 0.05% Triton X-100)
After incubation in buffer B, it was incubated in buffer B for a further 3 minutes. After repeating this four times, 6M
Of buffer B containing guanidine hydrochloride. The nitrocellulose membrane was shaken for 10 minutes, and an equal volume of buffer B was added 5 times every 10 minutes. After immersing the nitrocellulose membrane in buffer B, 1% bovine serum albumin (BSA), 0.1% Triton X-100, 0.1%
5M was transferred into a phosphate buffer (PBS) containing MgCl _2, 5mM DTT. The nitrocellulose membrane was ^washed with 0.5 ml of GAP buffer (25 mM Hep) containing [ ³⁵ S] GTPγS · GST-RhoA or [ ³⁵ S] GTPγS · GST-RhoA ^Ala37.
es / NaOH, pH 7.0, 2.5 mM DTT, 5
mM MgCl ₂ , 0.05% Triton X-100, 1
(00 mM GTP) for 10 minutes. The nitrocellulose membrane is 25 mM Hepes / NaOH, pH 7.0.
0, 5 mM MgCl ₂ , 0.05% Triton X-10
After washing three times with PBS containing 0, the substrate was dried, exposed to an X-ray film, and subjected to autoradiography. still,[
³⁵ S] GTPγS is DuPont New Eng
Land Nuclear.

The results were as shown in FIG. Crude bovine Rho kinase membrane extract both purified bovine ^Rho-kinase, binds to [35 S] GTPγS · GST- RhoA, [35 S] GTPγS · GST-RhoA
^It did not bind to ^Ala37 . This suggested that the active RhoA protein was directly bound to bovine Rho kinase via the effector domain. On the other hand, bovine Rho kinase is GTPγS · GST-
It did not bind to Rac1.

Example 2 Bovine Rho Kinase Kinase
Activity test In order to examine whether bovine Rho kinase has phosphorylation activity, the following experiment was performed. The kinase activity test was carried out using purified bovine Rho kinase (10 ng protein amount) and 2 μM [γ- ³² P] ATP (600-800).
50 μl of kinase buffer (50 mM Tris / HCl, pH 7.5, 1 mM containing MBq / mmol)
EDTA, 5 mM MgCl ₂ , 0.06% CHA
PS) in the presence or absence of a substrate (myelin basic protein, S6 peptide, or a synthetic peptide [αPKC] containing serine synthesized based on a pseudo-substrate of protein kinase C, 40 μM each). . After incubation at 30 ° C for 10 minutes, the reaction solution was boiled in SDS sample buffer and subjected to SDS-PAGE to check for autophosphorylation. Radiolabeled bands were detected by autoradiography. The reaction solution was prepared using Whatman p81 for the kinase activity test.
Spotted on Payper. The incorporation of ³² P into the substrate was measured with a scintillation counter. The results were as shown below. Incidentally, [γ- ^{3 2} P] ATP
Was purchased from Amersham.

(1) The enhancement of the autophosphorylation activity of bovine Rho kinase by the active RhoA protein was as shown in FIG. The purified bovine Rho kinase showed autophosphorylation ability in vitro in the presence of [γ- ³² P] ATP. This autophosphorylation ability is based on GTPγS
-GST-RhoA (lane 3) was enhanced to about 2-fold by ^GTPγS.GST -RhoA ^Ala37.
(Lane 4) and the enhancement effect of GDPγS.GST-RhoA (Lane 2) were smaller than GTPγS.GST-RhoA. The concentrations of GST-RhoA used were 1 μM each.

(2) Bovine Rho kinase activity was enhanced by active RhoA protein when a non-physiological substrate was used, as shown in FIGS. 5 and 6.
Bovine Rho kinase is a myelin basic protein, S
When using 6 peptides, αPKC as a substrate,
These were phosphorylated in the absence of GST-Rho (FIG. 5).
And FIG. 6). Phosphorylation of myelin basic protein, S6 peptide, αPKC by bovine Rho kinase
GTPγS-GST-RhoA enhanced G
The enhancing effect of DP.GST-RhoA was very low (FIG. 5). Phosphorylation of S6 peptide by bovine Rho kinase was enhanced by GTPγS.GST-RhoA, but GTPγS.GST-H-Ras and GDP.G
ST-H-Ras has no promoting effect and GTPγS
・ GST-RhoA ^Ala37 , GTPγS ・ GST-
Rac1 and GDP.GST-Rac1 showed only a trace of a promoting effect (FIG. 6). Thus, of the three substrates, the S6 peptide was most suitable as a substrate for bovine Rho kinase. The concentration of GST-low molecular weight G protein used was 1 μM each.

(3) The results of a search for a physiological substrate protein that enhances bovine Rho kinase kinase activity using activated RhoA protein were as shown in FIG. Since the Rho protein is thought to be involved in cytoskeletal rearrangement, vinculin (vin), a cytoskeletal regulatory protein by bovine Rho kinase,
culin), talin (talin), metavinculin (metavinculin), caldesmon (ca)
ldesmon), filamin, vimentin, α-actinin (E.
A. Clark & JSBrugge, Science, 268, 233-239 (1
995)), MAP-4 (H. Aizawa et al., J. Biol. Che.
m., 265, 13849-13855 (1990)), the myosin binding subunit of myosin light chain phosphatase (YH Chen e
, FEBS Lett., 356, 51-55 (1994)) were studied according to the above conditions. However, the myosin-binding subunit of myosin light chain phosphatase was used in the form of a fusion protein between the C-terminal of rat myosin-binding subunit (amino acid sequence at positions 699 to 976) and maltose-binding protein. This fusion protein was prepared by purifying the protein expressed in Escherichia coli by a conventional method. As a result, bovine Rho kinase phosphorylated these substrates in the absence or presence of GST-RhoA. Vinculin, Tallinn, Metavinculin, Caldesmon, Filamine,
When vimentin, α-actinin or MAP-4 was used as a substrate, the degree of enhancement of phosphorylation in the presence of GTPγS · RhoA was low (data not shown). However, when the myosin binding subunit (50 nM) was used as a substrate, the degree of phosphorylation was significantly enhanced in the presence of GTPγS.GST-RhoA (FIG. 7). GT
The degree of enhancement of phosphorylation of the myosin subunit in the presence of PγS.GST-RhoA was about 15-fold as compared to that in the absence (FIG. 7). The GS used
The concentration of T-RhoA was 1 μM each.

(4) Influence of Post-Translational Modification of RhoA The post-translational modification of Ras protein is caused by yeast adenyl cyclase (H. Horiuchi et al., Mol. Cell. Biol., 1, 1).
2,4515-4520 (1992)) and Ras protein-dependent MA
P-kinase kinase kinase (B-Raf) (T. Itoh
, et al., J. Biol. Chem., 268, 3025-3028 (199
3)) is known to be important. Post-translational modification of the Rac protein is also important for the activation of NADPH oxidase (S. Ando et al., J. Biol. Chem., 26
7, 25709-25713 (1992)). Therefore, it was examined whether the post-translational modification of the RhoA protein affects the enhancement of the kinase activity of bovine Rho kinase. H. Horiu
chi et al., Mol.Cell.Biol., 12, 4515-4520 (199
2) and T. Itoh et al., J. Biol. Chem., 268, 3025.
Post-translationally modified Rho according to the method described in JT-3028 (1993).
A protein was made. Using this, the effect of bovine Rho kinase on kinase activity was examined according to the method described above. The results were as shown in FIG. The post-translationally modified GTPγS-binding RhoA protein enhanced the phosphorylation activity of the S6 peptide more than the unmodified form.

Example 3 Amino acids of bovine Rho kinase
Sequence and DNA sequence encoding the sequence (1) Determination of peptide fragment The purified bovine Rho kinase was subjected to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane. The band corresponding to bovine Rho kinase was digested with lysyl endopeptidase, Achromobacter protease I, and endoproteinase Asp-N (A. Iwamatsu, Electrophoresis, 13, 142-147 (1992)).
), The obtained peptide fragments were separated by C18 column chromatography, and the amino acid sequence was determined. 37
Species peptide fragments were obtained.

(2) cDNA cloning To clone a cDNA encoding bovine Rho kinase, a bovine brain cDNA library (1.
2 × 10 ⁶ independent plaques) (Clontech)
Was screened with a degenerate oligo probe corresponding to the partial amino acid sequence determined by purified bovine Rho kinase (the double underlined portion of the amino acid sequence shown in FIG. 9). Hybridization when screening libraries is described in J. Sambrook et.
al., Molecular Cloning: A Laboratory Manual: Cold
Spring Habor Laboratory, Cold Spring Harbor, NY
(1989). cDNA
In order to determine the nucleotide sequence of cDNA, the cDNA inserted into the positive clone of the isolated λgt10 phage was ligated with pBluescript II SK (-) (MA Al
ting-Mees & JM Short, Nucleic Acids Res., 17,
9494 (1989)) and sequenced using a DNA sequencer 373S from ABI.

(3) Sequence analysis The nucleotide sequence and deduced amino acid sequence of bovine Rho kinase cDNA were as shown in SEQ ID NO: 2 and SEQ ID NO: 1, respectively. The bovine Rho kinase predicted from the cDNA base sequence consists of 1388 amino acid residues, has a calculated molecular weight of 160,797 Da,
It was similar to the molecular weight of about 164 kDa measured by S-PAGE. All of the 37 peptide fragments determined were c
It is included in the bovine Rho kinase amino acid sequence deduced from the DNA base sequence, and these are indicated by underlining of the amino acid sequence in FIG. In the structure of bovine Rho kinase, a sequence consisting of N-terminal 260 amino acids (SEQ ID NO: 1)
Mitotic dystrophy kinase, one of serine threonine kinases (JD Brook et al., Cell, 68,
799-808 (1992), YH Fu et al., Science, 255, 12
56-1258 (1992), M. Madadevan et al., Science, 25.
5, 1253-1255 (1992)) and a characteristic sequence showing 72% homology to the kinase domain. In the center of the structure of bovine Rho kinase, a coiled coil region showing homology to the myosin rod (corresponding to the amino acid sequence of positions 438 to 1124 of SEQ ID NO: 1)
It was found that a zinc finger region (corresponding to the amino acid sequence of Nos. 1261-1315 of SEQ ID NO: 1) was present on the C-terminal side.

The kinase region, bovine Rho kinase, coiled-coil region, pleckstrin homology (P
The results of comparison between the H) region and those of the mitonic dystrophy kinase were as shown in FIG. Protein homology searches were performed with the BLAST program (SF Altschul et al., J. Mol. Bio
l., 215, 403-410 (1990)).

(4) Analysis of tissue-specific expression by antibody production and immunoblot analysis The partial amino acid sequence of bovine Rho kinase
In order to prepare a rabbit polyclonal antibody against 69 to 681 (KRQLQERFTDLEK), a rabbit is immunized according to a conventional method using a synthetic peptide (CKRQQLERRTDDLEK: corresponding to the amino acid sequence of SEQ ID NO: 3) as an antigen and bovine serum albumin as a carrier. And the serum was purified.

The immunoblot analysis of bovine Rho kinase was performed according to E. Harlow & D. Lame, Antibodies: A Laboratory.
Mannual: Cold Spring Harbor Laboratory, Cold Sprin
g Harbor, NY (1988). Protein concentration was determined using bovine serum albumin as a target.
M. Bradford, Anal.Biochem., 72, 248-254 (1976)
Was carried out according to the method described in (1). In addition, it was confirmed that the anti-bovine Rho kinase antibody produced in rabbits showed a cross-reactivity with rat Rho kinase.

The results were as shown in FIG.
Rat tissues were used to examine tissue-specific expression of bovine Rho kinase. Rho kinase expression was prominent in the cerebrum and cerebellum, weak in heart, spleen, thoracic line, lung and kidney, with little expression in skeletal muscle, liver and pancreas.

Example 4 Activated RhoA Protein and Pair
Binding to Recombinant Bovine Rho Kinase (1) Plasmid Construction Coiled bovine Rho kinase by in vitro translation
In order to obtain proteins in the coil region, pGEM-HA
-Rho-Kinase was constructed as follows. Among bovine Rho kinases, 421 to 1137 of SEQ ID NO: 1
2.2k coding for the part corresponding to the amino acid sequence of No.
b) from the bovine Rho kinase cDNA (Example 3 (2)) by PCR using primer 5′ATAA
GGATCCCTACTAAGTGACTCTCCAT
CTTG 3 'and 5' TATAGGATCCTTA
Amplification was performed using ACTGCCTATACTGGAACTATCC3 '. This amplified cDNA fragment was converted into pGEM-H
A was cloned into the BamHI site.

(2) In Vitro Translation pGEM-HA-Rho-Kinase was converted to a reticulocyte lysate system (Promega) conjugated to TNT T7 under the conditions described in the instruction manual.
In vitro translation was carried out using Co. a) to obtain a protein in the coiled-coil region of bovine Rho kinase. GST-low molecular weight G protein loaded with guanine nucleotides (0.75 nmol each) was immobilized on 31 μl of glutathione-Sepharose 4B beads and 3
Washing was performed with 10 μl (10-fold volume) of buffer A. The immobilized beads were added to 30 μl of the in vitro translation product mixture, to which bovine serum albumin was added to a final concentration of 1 mg / ml, and gently shaken at 4 ° C. for 1 hour. The beads were washed six times with 102 μl (3.3 volumes) of buffer A to bind the bound proteins to GST-
Elution was performed three times with 102 μl (3.3 times the volume) of buffer A containing 10 mM glutathione together with the low molecular weight G protein. The first eluate was subjected to SDS-PAGE, dried under vacuum, and then subjected to autoradiography. The result is shown in FIG.
Was as shown in FIG.

The protein in the coiled-coil region of bovine Rho kinase obtained by in vitro translation was GTPγS.
It bound to GST-RhoA affinity beads and was eluted with GTPγS · GST-RhoA by glutathione (lane 3). Meanwhile, GST (lane 1),
GDP GST-RhoA (lane 2), GTPγS
GST-RhoA ^Ala37 (lane 4), GTPγS
GST-Rac1 (lane 6) or GTPγS
-It did not bind to the affinity beads of GST-H-Ras (lane 8). Protein near the coiled-coil region of bovine Rho kinase (799 of SEQ ID NO: 1)
-1137), essentially the same binding pattern was observed (data omitted).
This means that GTPγS.GST-RhoA
This indicates that the protein is directly bound to the coiled-coil region of ho kinase. As described below (Example 11), the Rho binding region in human Rho kinase was determined using a two-hybrid system. As a result, the Rho binding region of bovine Rho kinase was 94% of SEQ ID NO: 1.
It was presumed to be the amino acid sequence of positions 3 to 1068 (FIG. 10).

Example 5 Chick with Bovine Rho Kinase
Phosphorylation of avian myosin binding subunit
Inhibition of myosin light chain phosphatase activity (1) Phosphorylation test of chicken myosin binding subunit by bovine Rho kinase Bovine Rho kinase phosphorylated chicken myosin binding subunit even when it was used. Chicken myosin binding subunit (Shimizu, H. etal., J.
Biol. Chem., 269, 30407-30411 (1994)), a fusion protein (MBS-C) of a C-terminal peptide fragment (amino acids 753 to 1004) and a maltose binding protein was described in Example 2 (3). It was prepared according to the method, and using this as a substrate, the degree of phosphorylation by bovine Rho kinase was measured according to the method described in Example 2 (3) (FIG. 1).
3). As a result, MBS-C by bovine Rho kinase
Phosphorylation was enhanced 5 times or more in the presence of GTPγS.GST-RhoA as compared to the control (lane 1 in the presence of GST) (lane 3). In contrast, GDP
GST-RhoA (lane 2), GTPγS · GST-
RhoA ^Ala37 (lane 4), GDP / GST-R
ac1 (lane 5), GTPγS.GST-Rac1
In (lane 6), no increase in phosphorylation was observed.
In addition, chicken myosin-binding subunit (Shimizu,
H. et al., J. Biol. Chem., 269, 30407-30411 (1994)
)) N-terminal peptide fragment (amino acids 1 to 721)
Was used as a substrate instead of MBS-C, bovine Rho kinase did not phosphorylate it (data not shown).

(2) Inhibition of chicken myosin light chain phosphatase activity by bovine Rho kinase Purification of native myosin light chain phosphatase from chicken gizzard was performed according to Shimizu, H. et al., J. Biol. Chem., 269,
30407-30411 (1994).
Phosphorylation of myosin light chain phosphatase was measured in the presence of various concentrations of bovine Rho kinase from native bovine brain (Experiment 1). In addition, the enzymatic activity of phosphorylated myosin light chain phosphatase in the presence of various concentrations of bovine Rho kinase was measured (Experiment 2).

As a result, the myosin binding subunit in myosin light chain phosphatase is phosphorylated depending on the concentration of bovine Rho kinase, and the enzymatic activity of myosin light chain phosphatase is suppressed by phosphorylation by bovine Rho kinase. It became clear that it would be. FIG.
No. 4 shows the concentration of bovine Rho kinase along the horizontal axis, combining the results of the above two independent experiments (Experiment 1 and Experiment 2). Experiment 1 and Experiment 2
The specific method is as shown below.

Phosphorylation of native myosin light chain phosphatase by bovine Rho kinase (Experiment 1) was performed in the presence of various amounts of bovine Rho kinase at 1 μM GTPγS · G.
In the presence or absence of ST-RhoA, 40 μl of a buffer (34 mM Tris / HC) containing purified myosin light chain phosphatase (1.0 μg protein).
1, pH 7.5, 34 mM KCl, 4.0 mM
MgCl ₂ , 1.625 mM EDTA, 1.2 mM
DTT, 1.3% sucrose, 0.38% CHAP
S, 10 μM [ ³⁵ S] ATPγS).
After incubation for 3 minutes, the reaction mixture was subjected to SDS-PAG.
E, the degree of phosphorylation of the myosin binding subunit was determined by autoradiography (Fuji BAS-
2000).

To examine the effect of phosphorylation on myosin light chain phosphatase activity (Experiment 2), myosin light chain phosphatase (1.0 μg protein) purified by the same method as described above was used without radiolabeling.
1 μM G in the presence or absence of μM ATPγS
In the presence or absence of TPγS · GST-RhoA,
It was phosphorylated by various concentrations of bovine Rho kinase.
The reaction was stopped by adding 5 μl of 46 mM EDTA. Then 30 mM containing 5 μl of radiolabeled myosin light chain
Tris / HCl, pH 7.5, 30 mM KCl,
0.5 mM DTT was added, and the reaction was started as a total of 50 μl reaction mixture (including 5 μM ³² P-myosin light chain). The reaction was performed at 30 ° C. for 6 minutes. After stopping the reaction, the amount of ³² P bound to the myosin light chain was determined.
Ishihara, H. et al., Biochem. Biophys. Res. Commu
n. 159, 871-877 (1989).

The results were as shown in FIG.
³⁵ S-thiophosphate was incorporated into the myosin binding subunit in a kinase concentration-dependent manner. On the other hand, in the presence of ATPγS, suppression of myosin light chain phosphatase activity was found in a bovine Rho kinase concentration-dependent manner, but this suppression was not observed in the absence of ATPγS. From the above results, when phosphorylated by bovine Rho kinase,
It was revealed that the enzyme activity of myosin light chain phosphatase was suppressed.

Example 6 Rh in NIH / 3T3 cells
o protein binding myosin binding subunit and myosin
Measurement of Enhanced Osin Light Chain Phosphorylation As described below, NIH / 3T3 cells
We examined whether the protein enhances the phosphorylation of the myosin-binding subunit. Manufacturer (St
ratagene), NIH / 3T3 cells were treated with p3'SS and pOPRSVI-HA-Rho.
A or pOPRSVI-HA-RhoA ^Val14 were stably transfected. These plasmids have the hemagglutinin (HA) -Rho under IPTG control.
A or HA-RhoA ^Val14 can be expressed. NIH / 3T3 cell line (parent strain, parental strain) cultured in a 35 mm dish and reached confluent
NIH / 3T3-RhoA-5, NIH / 3T3-Rh
oA-24, NIH / 3T3-RhoA ^Val14-7
And NIH / 3T3-RhoA ^Val14-25 ) were treated with 5 mM IPTG for 24 hours. For the last 12 hours, the cells were cultured in a serum-free medium, and then 9.2.
Labeled with 5 MBq of [ ³² P] -orthophosphoric acid for 2 hours. Thereafter, the cells labeled with ³² P were lysed and the myosin-binding subunit was immunoprecipitated. The washed immunoprecipitates were subjected to SDS-PAGE and autoradiographed.

According to the above method, RhoA or RhoA
^{When Val14} was ^{overexpressed} in NIH / 3T3 cells, AJ Ridley & A. Hall, Cell 70, 389-399.
(1992) and AJ Ridley & A. Hall, EMBO J., 13, 2
High levels of stress fiber and focal contact formation were observed, as described in 600-2610 (1994). The amount of myosin-binding subunit was almost the same in all NIH / 3T3 cell lines including the parent strain (data not shown), but myosin-binding subunit in NIH / 3T3 cells overexpressing RhoA or RhoA ^Val14. Phosphorylation degree of the parent strain NIH / 3T3
It was significantly higher than the degree of phosphorylation of the myosin-binding subunit in the cells (FIG. 15).

Next, the parent strain and RhoA or RhoA
The degree of phosphorylation of myosin light chain in NIH / 3T3 cells overexpressing ^Val14 was measured according to the method described below. IPTG treatment and serum removal operation
10% TCA was added to the NIH / 3T3 cell line performed in a 00 mm dish. To determine the extent of phosphorylation of the myosin light chain, the trichloroacetic acid (TCA) precipitate was subjected to glycerol-urea gel electrophoresis and phosphorylated (monophosphorylated (MLCP) and d).
The relative amounts of iphosphorylated (MLCP ₂ ) myosin light chain and non-phosphorylated myosin light chain were determined by immunoblotting (DA Taylor & JT Stull, J. Biol.
Chem. 263, 14456 (1988)). On this occasion,
Cells are harvested with 0.1 μM phosphatase inhibitor (calyculi
nA (CLA)) for 10 minutes increased the degree of phosphorylation of the myosin light chain (FIG. 16). NIH / 3T overexpressing RhoA or RhoA ^Val14
3 The degree of phosphorylation of the myosin light chain in the cells
It was clearly higher than the degree of phosphorylation of myosin light chain in IH / 3T3 cells (FIG. 16). Three different strains of Rho
NIH overexpressing A or RhoA ^Val14
Essentially identical results were obtained using / 3T3 cells.

Without being bound by the following theory,
The above results indicate that expression-induced RhoA or RhoA
^Val14 activates Rho kinase endogenously present in NIH / 3T3 cells, resulting in enhanced phosphorylation of the myosin binding subunit and inhibition of myosin light chain phosphatase activity, thereby inhibiting the myosin light chain. It is interpreted that dephosphorylation was suppressed.

Example 7 Bovine Rho Kinase and Its
Deletion mutant phosphorylation of myosin light chain In a cell-free system, it was examined whether the bovine Rho kinase mutant phosphorylates isolated myosin light chain. Specifically, the experiment was performed according to the following method. Myosin light chain (Hathaway, DR & Haeberle, JR Anal. Biochem.
135, 37-43 (1983)), myosin and myosin light chain kinase (Ikebe, M., & Hartshorne, DJJ Biol. Ch.
em. 260, 10027-10031 (1985)) was purified from frozen chicken gizzards. Purified bovine Rho kinase was purified from bovine brain (Example 1).

Further, a recombinant fusion protein (Rho kinase (CAT)) of the catalytic domain fragment of bovine Rho kinase and GST was prepared according to the method described below.
Bovine Rho kinase catalytic domain fragment (6 to SEQ ID NO: 1)
The cDNA fragment encoding the amino acid sequence of No. 553) was ligated to the plasmid pAcYM1-GST (Matsuura, Y. e.
tal., J. Gen. Virol. 68, 1233-1250 (1987))
Inserted into mH1 site. Using the obtained plasmid,
Matsuura, Y. et al., J. Gen. Virol. 68, 1233-1250
(1987) by utilizing the baculovirus system to obtain Sf9 cells (ATCC
CRL 1711) produced Rho kinase (CAT), which was purified.

The kinase reaction for bovine Rho kinase and its deletion mutants was performed in a 50 μl reaction mixture (5
0 mM Tris-HCl (pH 7.5), 2 mM E
DTA, 1 mM DTT, 7 mM MgCl ₂ , 0.1
5% CHAPS, 250 μM [γ- ³² P] ATP
[1-20 GBq / mmol], purified bovine Rho kinase [20 ng protein] or bovine Rho kinase (CAT) and the indicated amount of myosin light chain or myosin) (in the presence of 1 μM GTPγS · GST · RhoA,
Or in the absence). The kinase reaction for myosin light chain kinase was performed in a 50 μl reaction mixture (5
0 mM Tris-HCl (pH 7.5), 1 mM M
gCl ₂ , 85 mM KCl, 500 μM [γ-
³² P] ATP [0.5-5 GBq / mmol], purified myosin light chain kinase [50 ng protein] and the indicated amount of myosin light chain or myosin) (0.1 mM
(In the presence or absence of CaCl ₂ and 10 μg / ml calmodulin). 10 at 30 ° C
After incubation for minutes, the reaction mixture was boiled in SDS-sample buffer and subjected to SDS-PAGE. S
DS-PAGE is based on previously described methods (Laemmli, U.
K. Nature 227, 680-685 (1970)).
The radiolabeled bands were visualized by an image analyzer (Fuji).

As a result, bovine Rho kinase (CAT)
Phosphorylates myosin light chain (Fig. 1).
7). GTPγS · GST-RhoA is purified bovine Rho
Increased phosphorylation of myosin light chain by kinase,
GDP-GST-RhoA or GTPγS-GST-
RhoA ^Ala37 did not enhance (FIG. 17). By the way,
Rho ^Ala37 is structurally equivalent to Ras ^Ala35 . Ra
s ^Ala35 contains an amino acid substitution in the effector domain that prevents Ras ^Ala35 from binding its target (Satoh, T. et al., J. Biol. C
hem. 267, 24149-24152 (1992), McCormick, F., Curr.
Opin. Genet. Dev. 4, 71-76 (1994)). GTP
γS.GST-Rac1 also had no effect. Constitutively activated recombinant bovine Rho kinase (bovine Rho
Kinase (CAT)) also phosphorylated myosin light chain.
Under similar conditions, myosin light chain kinase phosphorylated myosin light chain in a Ca2 ⁺ -calmodulin-dependent manner (FIG. 17). Also, bovine Rho kinase is intact (in
tact) myosin light chain of GTPγS · GS
It was found to phosphorylate in a T-RhoA dependent manner (FIG. 17).

Up to about one mole of phosphate is present in one mole of isolated myosin light chain or intact myosin myosin light chain in the presence of GTPγS.GST-RhoA.
By purified bovine Rho kinase or bovine Rho
Incorporated by kinase (CAT) (data not shown). Incidentally, certain kinases such as myosin light chain kinase and protein kinase C are known to phosphorylate intact myosin in a stoichiometrical manner (Tan, JL et al., Annu. Rev. Biochem. 61). ,
721-759 (1992)).

The apparent affinity of the isolated myosin light chain for purified bovine Rho kinase was estimated by measuring the phosphorylation of various concentrations of myosin light chain (FIG. 18). The apparent Km values for myosin light chain in the presence or absence of GTPγS.GST-RhoA were 2.6 ± 0.4 and 12.6 ±, respectively.
1.6 μM and the molecular acti
vities) are 0.26 ± 0.03 and 0.15 ± 0.0
It was 2 sec ^-1 . Therefore, GTPγS · GST-
RhoA appeared to increase the affinity of bovine Rho kinase for myosin light chain and to produce the maximum rate of phosphorylation. The apparent Km value and molecular activity of bovine Rho kinase (CAT) were 0.91 ± 0.07 μM and 0.67 ± 0.0, respectively.
It was 9 sec ^-1 . Apparent Km value and molecular activity of myosin light chain kinase against myosin light chain (molecu
lar activity) is 5
2.1 ± 7.1 μM and 2.0 ± 0.36 sec ⁻¹
Met. The Km value of bovine Rho kinase for myosin light chain is lower than that of myosin light chain kinase. This indicated that bovine Rho kinase phosphorylated myosin at lower concentrations, but the molecular activity of bovine Rho kinase was lower than that of myosin light chain kinase. The molecular activity of purified bovine Rho kinase is
The reason for the lower activity of kinase (CAT) can be explained by the fact that the activity of purified bovine Rho kinase was inactivated during the purification process (data not shown).

Example 8 Bovine Rho Kinase and Its
Phosphorus in myosin light chain phosphorylated by deletion mutants
Determination of the oxidation site Myosin light chain is preferentially Ser-19, then Thr
-18 is phosphorylated by myosin light chain kinase.
(Ikebe, M. & Hartshorne, DJJ Biol. Chem. 26
0, 10027-10031 (1985)). And phosphorylation of Ser-19 is essential for activation of myosin ATPase by actin (Kamisoyama, H. et al., Biochemistry).
33, 840-847 (1994), Bresnick, AR et al., Bioche.
mistry 34, 12576-12583 (1995)). Myosin light chain is Se
r-1, Ser-2 and Thr-9 are phosphorylated by protein kinase C, and this phosphorylation by protein kinase C inhibits actin activation by myosin ATPase (Nishikawa, M. et a
l., J. Biol. Chem. 259, 8808-8814 (1984), Bengur,
AR et al., J. Biol. Chem. 262, 7613-7617 (1987)
And Ikebe, M. & Reardon, S. Biochemistry 29,
2713-2720 (1990)).

To determine the major phosphorylation site of myosin light chain by bovine Rho kinase, peptide mapping of myosin light chain phosphorylated in vitro by bovine Rho kinase, myosin light chain kinase or protein kinase C was performed. did. Mapping analysis of the phosphorylated peptide of myosin light chain was performed according to the method described (Naka, M. et al., Nature 306, 490-492 (1983)). As a result, the pattern of two-dimensional peptide mapping of myosin light chain phosphorylated by bovine Rho kinase was identical to that produced by myosin light chain kinase, but different from that produced by protein kinase C ( (FIG. 19).

Next, the analysis of phosphorylated amino acids was performed according to the method described (Hunter, T. & Sefton, BM, Proc.
Acad. Sci. USA 77, 1311-1315 (1980)). As a result, phosphorylation by bovine Rho kinase occurs mainly at serine residues and some threonine residues of myosin light chain, and phosphorylation by myosin light chain kinase results in serine residue of myosin light chain (Ser-19). It was found that this occurred only in (data omitted). Under these conditions, it was recalled that myosin light chain kinase preferentially phosphorylates myosin light chain Ser-19. Essentially the same results were obtained when bovine Rho kinase (CAT) was used instead of purified bovine Rho kinase.

The GST protein was fused with wild-type myosin light chain and myosin light chain in which Thr-18 and Ser-19 were substituted with alanine residues, and bovine Rho kinase and myosin light chain kinase linked these recombinant proteins to phosphorylation. We examined whether it could be oxidized. Vectors ( ^pGEX- myosin light chain and ^pGEX- myosin light chain ^{Ala18, Ala19} ) for expressing these recombinant proteins in Escherichia coli were constructed as follows. Primer 5'AATAGGATCCGATTTAACCG
CCACCATGTCG 3 'and 5' ATAAGGA
TCCTCAGTCATCTTTGTCTTTTCCGCT
Using C3 ', rat brain Quick clone cDN
A (Clontech) by polymerase chain reaction
0.55 kilobase pair cDN encoding myosin light chain
The A fragment was amplified. Thr- by alanine (Ala)
Replacement of 18 and Ser-19 was performed by the polymerase chain reaction (Higuchi, R. in PCR Technology (E
rlich, HA ed) pp. 61-70, Stockton Press, New Yo
rk (1989)). The cDNA fragment was ligated with BamH of pGEX-2T.
Cloned into the I site.

As a result, both bovine Rho kinase and myosin light chain kinase phosphorylated GST-myosin light chain, but GST or GST-myosin light chain.
^{Ala18 and Ala19} were not phosphorylated (FIG. 20). Protein kinase C contains GST-myosin light chain and GST-
Both myosin light chains ^{Ala18 and Ala19} were phosphorylated (data not shown). These results indicated that bovine Rho kinase phosphorylates myosin light chain mainly at Ser-19, and that Ser-19 is identical to the site phosphorylated by myosin light chain kinase.

Example 9 Actin-Activated MgATP Art
Zeassei bovine Rho kinase to consider whether to function equivalent to myosin light chain kinase in a cell-free system, was performed actin activated MgATP ATPase assay. Purified intact myosin was purified using bovine Rho kinase (CA).
Phosphorylation by T) resulted in 1 mole of phosphorylation per mole of million, and then the actin-activated MgATPase activity was measured. Specifically, it was carried out according to the method described below. The myosin ATPase assay is a method described previously (Ikebe, M. et.
al., Biochemistry 23, 5062-5068 (1984)) with some modifications. First, 0.1 mg / ml myosin was added to a 0.45 ml reaction mixture (50 mM) using bovine Rho kinase (CAT) (450 ng protein).
Tris-HCl (pH 7.5), 2.2 mM ED
TA, 1 mM DTT, 6 mM MgCl ₂ , 1 mM
EGTA, 85 mM KCl, 1 μM GTPγSG
ST-RhoA and 500 μM ATP [80-20
0MBq / mmol] at 30 ° C for 20 minutes. 0.1 mg / ml myosin was converted to 0.1 mg / ml by myosin light chain kinase (450 ng protein).
Phosphorylation was carried out under the same conditions except that mM, CaCl ₂ and 10 μg / ml calmodulin were present. Myosin ATPase reaction was performed using 0.45 ml of ATPase buffer (0.05 mg / ml phosphorylated myosin, 5
0 mM Tris-HCl (pH 7.5), 0.5 mM
DTT, 10 mM MgCl ₂ , 0.5 mM EG
TA, 85 mM KCl, and 1 mM ATP [80-
[200 MBq / mmol]) (in the presence or absence of 1 mg / ml F-actin) at 30 ° C for 30 minutes. Stop solution (1.3% charcoal, 0.12 M NaH ₂ P) was given at the indicated times for each 80 μl of reaction mixture.
O ₄ and 0.33M was added to the perchloric acid), and filtered. The inorganic phosphate released from [γ- ³² P] ATP is
It was measured by a scintillation counter. In addition, F-
Actin was purified from rabbit skeletal muscle (Spudich, J. et al.
A., & Watt, SJ Biol. Chem. 246, 4866-4871 (197
1)) and [γ- ³² P] ATP were purchased from Amersham.

The MgATPase activity of myosin phosphorylated by Rho kinase (CAT) increased, the increase was in an F-actin dependent manner, and the extent of the increase was similar to that by myosin light chain kinase ( (FIG. 21). The apparent Ka value against actin and the molecular activity of phosphorylated myosin were 0.56 ± 0.0, respectively.
5 μM and 0.18 ± 0.02 sec ⁻¹ .
These values were similar to those for myosin phosphorylated by myosin light chain kinase.
The reason why Rho kinase (CAT) was used in this experiment instead of natural purified Rho kinase is that the myosin AT
High concentrations of myosin are required to measure Pase activity, since no stoichiometrical phosphorylation of myosin is seen with native purified Rho kinase under these experimental conditions.

Example 10 Human Rho Kinase cD
NA Cloning Human brain mRNA (CLONTECH) 0.5 μg as a template
Using rScriptTM Preamplification System (BRL)
1st strand DNA was synthesized. 1/20 volume of this reaction solution
A primer (5-CAT TTT CAT
TTC TAG GAG ATG ATT ATT CTC TTG CTTTAA C-3, 5'-AA
A AAG CAC TTC TTC AGC ACA AAA ATG CAG AAT ATC AGC
G-3), PCR was performed using the TAKARA LA PCR Kit to obtain a partial fragment of the human Rho kinase cDNA (base sequence 1151 to 2476 described in SEQ ID NO: 4). This cD
Using the NA fragment as a probe, 1.0 × 10 ⁶ plaques were screened from the human brain λgt10 cDNA library.
Screening was performed according to J. Sambrook et.al., MolecularClon.
ing: A Laboratory Manual: Cold Spring Habor Labora
Tory, Cold SpringHarbor, NY (1989). As a result of comparison with the nucleotide sequence of bovine Rho kinase gene, two clones obtained, p164-20 (SEQ ID NO: 5)
938 to 3710) and the C-9 nucleotide sequence (the nucleotide sequence of 2898-4365 in SEQ ID NO: 5) were found to cover about 2 kbp of the translation region at the C-terminal end.

To clone about 1 kbp of the missing N-terminal, an additional 1.0 × human brain λgt10 cDNA library was used.
10 ⁶ were screened using clone p164-20 as a probe. As a result, it was found that one clone, N6, covered the N-terminal of about 1 kbp including the initiation codon (base sequence 1 to 929 described in SEQ ID NO: 5). p164-2
To obtain a cDNA fragment covering between 0 and N6,
Using ONTECH Human Brain QUICK-CloneTM cDNA as a template, primer 5-CCT TTG TCA TCT TCA ATGTCA TCG A
AA TTG-3 and 5-CGT GTA TGA AGA TGG ATG AAA CAG GCA
PCR was performed using TGG-3 and TAKARA LA-PCR kit (Takara Shuzo Co., Ltd.), and c corresponding to nucleotide sequences 734-1145.
The DNA fragment was amplified.

These four clones covering the translation region of the human Rho kinase gene were obtained from Stratagene's pBlu
scriptII SK (-) (MAAlting-Mees and JMShort,
Nucleic Acids Res., 17, 9494 (1989)). For nucleotide sequence determination, double-st manufactured by Pharmacia
delete mutan using randed Nested Deletion Kit
t was prepared and sequenced using an ABI 377 DNA sequencer. The human brain λgt10 cDNA library used was purchased from CLONTECH.

The nucleotide sequence and deduced amino acid sequence of human Rho kinase cDNA were as described in SEQ ID NO: 5 and SEQ ID NO: 4, respectively. The predicted protein of human Rho kinase was composed of 1388 amino acids and the predicted molecular weight was approximately 161 kDa. The amino acid sequence of human Rho kinase (SEQ ID NO: 4) is bovine (SEQ ID NO: 1),
Rat ROKα (Leung, T. et al., J. Biol. Chem.,
Rat ROK described in 270, 29051-29054 (1995)
The amino acid sequence of α lacks the N-terminal 84 amino acids, but the full-length amino acid sequence was subsequently registered in the database (EMBL Data Bank accession number U3848
1). Hereinafter, the amino acid sequence number of rat ROKα is shown to be high according to the sequence numbers described in this database.) And 95% identity. N
There was a kinase domain on the terminal side, a coiled coil domain containing a Rho binding region (Example 11) in the center, and a zinc finger-like motif on the C-terminal side. Human R
The kinase domain of ho kinase (90-
At the amino acid level, bovine Rho kinase (amino acid sequence 90 to 359 of SEQ ID NO: 1) and rat ROKα (amino acid sequence 88)
357) and 97% identity. Coiled-coil domain of human Rho kinase (438 of SEQ ID NO: 4)
~ 1241 amino acid sequence) at the amino acid level,
It showed 97% identity with bovine Rho kinase (amino acid sequence of positions 438 to 1124 of SEQ ID NO: 1) and 95% with rat ROKα (amino acid sequence of 436 to 1122). The zinc finger-like motif of human Rho kinase (amino acid sequence 1261 to 1315 of SEQ ID NO: 4) is expressed at the amino acid level with bovine Rho kinase (1 of SEQ ID NO: 1).
261-1315) and rat ROKα (amino acid sequences 1259-1313) and 98%
% Identity.

The amino acids 669 to 681 of the amino acid sequence of human Rho kinase (SEQ ID NO: 4) correspond to the amino acids 669 to 68 of the amino acid sequence of bovine Rho kinase (SEQ ID NO: 1).
It has the same sequence as No. 1 and has the anti-R
amino acid sequence (K
RQLQERFTDLEK). This indicates that human Rho kinase is also a synthetic peptide CKRQLQE
It was revealed that the antibody could be recognized by an anti-Rho kinase antibody prepared using RFTDLEK as an antigen (Example 3 (4)). From the above, it was concluded that human Rho kinase is a protein of the human counterpart of bovine Rho kinase.

On the other hand, the amino acid sequence (full length) of human Rho kinase (SEQ ID NO: 4) is the same as that of human p160 ^ROCK (Is
hizaki, T., et.al.EMBO J., 15, 1885-1893 (199
6)) showed 67% identity with that of Kinase domain of human Rho kinase (90-359 of SEQ ID NO: 4)
No. amino acid sequence) is the kinase domain of human p160 ^ROCK (Ishizaki, T., et. Al. EMBO J., 15, 1885).
-1893 (1996)).
Both showed a high identity of 92%. From this,
It was concluded that human Rho kinase is an isozyme of human p160 ^ROCK .

Embodiment 11 Two-Hybrid System
Rho Kinase and Activated Rho Protein Using a Cell
Detection of Quality Binding Using the yeast two-hybrid system, according to the method described below, the Rho kinase of human Rho kinase
The binding region was determined. A nucleotide sequence corresponding to amino acids 943 to 1068 of human Rho kinase was ligated to a primer (5-TTG CGG CCG CTA AAG ATC ATG AAA GAG CTG GAG AT
C-3 ', 5' TAG CGG CCG CAA CAT ATG TAGCTT TCT ATT CT
C-3 ') and amplified by PCR, followed by NotI of pVP16.
Into the Rho kinase-VP16-fusion protein expression vector (Vojtek, ABet. Al. C
ell, 74, 205-214 (1993)). LiCl method (Ito, H. et.
J. Bacteriol., 153, 163-168 (1983)), LexA-wild type H-Ras fusion protein, Le, respectively.
xA-wild-type Rho fusion protein, LexA-active Rho fusion protein, LexA-wild-type H-Ra
fusion protein and Rho kinase-VP16-fusion protein, LexA- wild-type Rho fusion protein and Rho kinase-VP16-fusion protein, LexA
-Activated Rho fusion protein and Rho kinase-VP
The 16-fusion protein was converted to yeast (S. cerevisiae) L40.
Strain (Mat a trp1 leu2 his3 ade2 LYS2: :( LexAop) 4-HIS3
URA3: :( LexAop) 8-LacZ) and expressed in a selective medium (Le
u-, Trp-, His-, 200 mM 3AT) and examined for histidine requirement. As a result, only the strain expressing the LexA-activated Rho fusion protein and the Rho kinase-VP16-fusion protein could survive on the selection medium (FIG. 23). This revealed that the amino acid sequence at positions 943 to 1068 of human Rho kinase or a partial sequence thereof is a Rho binding region.

The Rho binding region of human Rho kinase;
As a result of comparing the amino acid sequences of rat Rho kinase and the corresponding portion of bovine Rho kinase, human Rho kinase (amino acid sequences 943 to 1068) and rat RO were compared.
Kα (amino acid sequences 941 to 1066) showed 98% identity, and human Rho kinase and bovine Rho kinase (amino acid sequences 943 to 1068) showed 98% identity. On the other hand, in the Rho binding region of human Rho kinase (amino acid sequences 943 to 1068) and the corresponding portion of human p160 ^ROCK (amino acid sequences 910 to 1039), 53%
Identity was seen.

Example 12 Rho Kinase (CAT)
It is well known that Rho protein regulates phosphorylation of myosin light chain (MLC) and consequent smooth muscle contraction (Somlyo, AP & Somlyo, A. et al.
V. Nature 372, 231-236 (1994); Noda, M. et al. FE
BS Lett. 367, 246-250 (1995); Hirata, K. et al.
J. Biol. Chem. 267, 8719-8722 (1991); Gong, MC e
Natl. Acad. Sci. USA 93, 1340-1345 (19
96)). Rho protein enhances Ca ²⁺ sensitivity (Gon
g, MC et al. Proc. Natl. Acad. Sci. USA 93, 134
0-1345 (1996)) requires a diffusible cofactor (Gong,
MC et al. Proc. Natl. Acad. Sci. USA 93, 1340-13
45 (1996)). However, until now, the detailed mechanism of the enhancement of Ca ²⁺ sensitivity of smooth muscle contraction via Rho protein was unknown (Gong, MC et al. Pro.
c. Natl. Acad. Sci. USA 93, 1340-1345 (1996)).

On the other hand, the present inventors have proposed that Rho kinase, a novel target protein of Rho, is activated by Rho protein, and the activated Rho kinase phosphorylates the myosin binding subunit (MBS) of myosin light chain phosphatase. It was found that the phosphorylation of the myosin light chain increased as a result (Examples 1 to 6). The present inventors have also found that Rho kinase directly phosphorylates myosin light chain (Examples 7 to 9). These data are
It has been shown that Rho kinase not only serves as a target protein downstream of the Rho protein, but also plays a pivotal role in controlling smooth muscle contraction.

The present inventors have determined that Rho kinase is
The following experiment was performed to confirm that the protein is a Rho target protein necessary for enhancing Ca ²⁺ sensitivity of vascular smooth muscle contraction by a protein. (1) Induction of contraction of rabbit portal vein blood vessels treated with Triton X-100 by Rho kinase First, the present inventors prepared recombinant Rho kinase catalytic domain (Rho kinase (CAT)) (Example 7) using Triton X
The effect of exogenous administration into the cytoplasm of rabbit portal vein mesenteric vessels (Triton X-100-permiabilized RPV) whose permeability was enhanced using -100 was examined. As a result, Rho kinase (CAT) administered into the cytoplasm contracts blood vessels, and at the same time, Triton X-100-permiabilized R
Monophosphorylatio of myosin light chain in PV
n) increased. This effect was observed even when the Ca ²⁺ concentration in the cytoplasm was substantially zero (due to the buffering action of 10 mM EGTA). Details are described below.

In Triton X-100-permiabilized RPV, the permeability of which was significantly enhanced using 0.5% Triton X-100,
High molecular weight compounds, such as proteins, can cross cell membranes without coupling to receptor G protein signaling. Taking advantage of this, 0.5% Triton X-100
Rho kinase (C) is exogenously introduced into the cytoplasm of rabbit portal vein smooth muscle (RPV) whose permeability has been significantly enhanced using
AT) (molecular weight about 80 kDa) was introduced. Specifically, the experiment was performed according to the method described below.

A small piece (width: 50 to 100 μm, length: 0.5 to 1 m) of a portal vein media sample of a rabbit (2 to 2.5 kg)
m) was cut out, connected to an isometric tension transducer (UL-2GR manufactured by Minebea), and placed on a well on a bubble plate (Kobayashi, S. et al.).
et al. J. Biol. Chem. 264, 17997-18004 (1989)).
After recording the contraction caused by 118 mM K ⁺ , the specimens were incubated in a relaxation solution and then 0.5%
Treat with Triton X-100 for 20 minutes at 25 ° C or 5000
Treated with IU / ml α-toxin for 60-75 minutes at 25 ° C. The solutions used above have been described in detail in the past (Persechini, AK et al. J. Biol. Chem. 261,
6293-6299 (1986)). Calmodulin (0.5 μM)
Was added to all the reaction solutions used in the experiments utilizing chemical hyperpermeability. Rho kinase (CAT) was prepared according to the method described in Example 7.

As a result, in pCa6.5 (FIGS. 24a and 24b) and pCa << 8.0 (FIG. 24c), Rho kinase (CAT) induced remarkable contraction. These contractions were completely restored by washing the Rho kinase (FIG. 24b). In contrast, microc
Shrinkage induced with ystin-LR did not recover after washing (FIG. 24b). On the other hand, no contraction by Rho kinase (CAT) was observed in an intact blood vessel specimen or a blood vessel specimen whose permeability was enhanced using α-toxin (data not shown). Based on the above results, Triton X
Through the large hole created by the -100 treatment, it was shown that Rho kinase (CAT) was exogenously introduced into the smooth muscle cytoplasm, thereby contracting the blood vessels.
Also, since the action of the introduced Rho kinase (CAT) is reversible, this contraction probably reflects a physiological phenomenon, is not coupled to receptor-type G proteins and is not dependent on cytoplasmic Ca ²⁺ concentration Was suggested.

(2) Triton X-100 using Rho kinase
Of the Rho-kinase (CAT) introduced into the cytoplasm enhances the Ca ²⁺ sensitivity of the contractile apparatus of Triton X-100-permiabilized RPV. To see if they are (potentiates)
P in the presence of 7.5 nM Rho kinase (CAT)
The relationship between Ca and tension was examined. The results showed that this concentration of Rho kinase (CAT) did not normally induce a tension response at pCa << 8, but increased pCa stimulated the contractile apparatus.

That is, Rho kinase (CAT) significantly increased the maximal tension response as compared with the control (p <ANOVA analysis).
0.05) (Fig. 25a, open circles). To determine the EC ₅₀ values, each indicating the pCa value required to induce 50% of maximum tension as an indicator of Ca ²⁺ sensitivity,
The data shown in FIG. 25a was obtained under non-reaction conditions (pCa <8.
0 and the maximum reaction conditions (500 nM okadaic acid
Or 7.5 nM Rho kinase (CAT)
Were renormalized by assuming values at pCa4.5) in the presence or absence of 0% and 100%, respectively. Calculated EC ₅₀ value in the presence of Rho kinase (CAT) (0.299 ± 0.045 μM, n
= 4) and control value (0.376 ± 0.046, n = 4)
Okadaic ac, a potent inhibitor of type 1 and 2A protein phosphatases
id (OA) (Bialojan, C. et al. Nature 298, 81-95 (19
88); Takai, A. et al. FEBS Lett. 217, 81-95 (198
8)) Value in the presence (0.212 ± 0.013 μM, n =
4) was statistically significantly different from the control (FIG. 2).
5a, solid circle, p <0.05). From the above results, Rho
Kinase (CAT) enhances the sensitivity of contraction independent of intracellular Ca ²⁺ concentration, suggesting that the mechanism of this increased sensitivity is not solely due to the inhibition of myosin light chain phosphatase as by OA. .

Phosphorylation of myosin light chain via the Ca ²⁺ -calmodulin-myosin light chain kinase (MLCK) pathway results in a smooth activation of myosin-actin interaction and subsequent myosin ATPase activation. Plays a major role in muscle contraction (Ka
mm, KE & Stull, JT Annu. Rev. Pharmacol. Tox
ical. 25, 593-603 (1985); Hartshorne et al. in Ph.
ysiology of the gastrointestinal Tract (ed Johnso
n, LR) 423-482 (Raven Press, New York (1987);
Sellers, JR & Adelstein, RS in The Enzyme Vo
l.18 (eds Boyer, P. & Erevs, EG) 381-418 (Acade
mic Press, San Diego, CA (1987)). To investigate the involvement of the Ca ²⁺ -calmodulin-myosin light chain kinase (MLCK) pathway in Rho kinase-induced contraction, we studied the Rho kinase (C
The effect of wortmannin (WM) on the tension formation induced by the cumulative administration of AT) was examined (FIG. 25b).

As a result, the physiological Ca ²⁺ concentration p
In Ca6.5, tension formation showed a Rho kinase dose-dependent, but the dose-dependent curve shifted downward in the presence of 10 μM WM. In contrast to the results with pCa6.5, the tension formation induced by Rho kinase (CAT) at pCa << 8.0 was 10 μM.
There was no significant change in the presence of WM (no significant difference in ANOVA analysis).

Incidentally, WM is known to inhibit Ca ²⁺ -dependent MLCK (Nakanishi, S. et al.
Biol. Chem. 267, 2157-2163 (1992)). Also, Rho
C in pCa6.5 in the absence of kinase (CAT)
Contraction by a ²⁺ was completely inhibited by WM treatment at 10 μM (data not shown). In view of the above, the reason that WM alters the enhanced Ca ²⁺ sensitivity induced by Rho kinase in pCa6.5 is probably due to the cytoplasmic C
By inhibiting the contraction itself induced by a ²⁺ , it is not likely that WM is inhibiting the Rho kinase-mediated pathway. Taken together the data above, including the statistical analysis result, contraction induced by Rho-kinase in Ca ²⁺ absence Ca ²⁺
-Calmodulin-Myosin light chain kinase (MLCK)
It is thought to be based on a mechanism independent of the pathway.

(3) Triton X-10 by Rho kinase
Enhanced phosphorylation of myosin light chain in rabbit portal vein blood vessels treated with R. 0 Are Rh in phosphorylation of myosin light chain.
The effect of o-kinase (CAT) was examined using an anti-myosin light chain polyclonal antibody. Specifically, the experiment was performed according to the following method.

0.255 μM of Rho kinase (CA
T) and / or a fringe-like rabbit portal vein vascular specimen with enhanced permeability by Triton X-100 after treatment with 10 μM wortmannin (WM) containing 10% TCA and 10 mM DTT rapidly The contraction reaction was stopped by placing in acetone frozen slurry. TCA
After removing, the sample was treated with urea sample buffer (20 mM Tris, 22 mM glycine (pH 8.6), 8 M urea,
10 mM DTT, 10% sucrose, 0.1% bromphenol
blue), subjected to glycerol-urea polyacrylamide gel electrophoresis, and subjected to immunoblotting using an anti-myosin light chain antibody (Pe
rsechini, AK et al. J. Biol. Chem. 261, 6293-62
99 (1986)).

As a result, as shown in lane 1-3 in FIG. 26a, at pCa << 8.0, monophosphorylation of myosin light chain was observed in the presence of Rho kinase (CAT) (lane 2).
And it was insensitive to 10 μM WM (lane 3). On the other hand, pCa6.5 (lane 4
At -6), Rho kinase (CAT) significantly enhanced the monophosphorylation of myosin light chain (lane 5) and it was sensitive to 10 μM WM (lane 6). These results were consistent with the effect of Rho kinase (CAT) on the contractile response (FIG. 26b). Thus, Rho kinase (CAT) is probably Ca2 ⁺ -calmodulin-M
It was concluded that increased phosphorylation levels of myosin light chain mediated by an LCK-independent pathway resulted in enhanced contractile responses.

(4) Proof that Rho-kinase is not present in rabbit portal vein blood vessels treated with Triton X-100 By the way, Rho protein is a diffusible coupling factor (diffusible) in order to cause an increase in Ca ²⁺ sensitivity. c
ofactor) (Gong, MC et al. Proc. Nat
l. Acad. Sci. USA 93, 1340-1345 (1996)). That is, Tr
When permeability is enhanced by iton X-100 treatment, Rho
Downstream molecules involved in the signal transduction pathway via
^The RPV (extensively Triton) which has enhanced the permeability by such Triton X-100, because the diffusive coupling factor necessary for causing ²⁺ increased sensitivity leaks from the cells. X-100-permeabilized RP
V), it is speculated that Rho cannot exert a contractile effect (Gong, MC et al. Proc. Natl. Acad. Sci. USA 9
3, 1340-1345 (1996)).

Therefore, the present inventors examined extensively Triton X-100-permeabilized in order to examine whether Rho kinase is the above-mentioned diffusible coupling factor essential for enhancement of Ca ²⁺ sensitivity by Rho protein. R
It was examined whether Rho kinase was present in PV.
Specifically, the experiment was performed according to the method described below.

Rabbit portal vein blood vessels whose permeability was enhanced by 0.5% Triton X-100 and rabbit portal vein blood vessels not permeabilized (ie, intact) were extracted with extraction buffer (50 mM Tris -HCl (pH 7.2), 100
mM NaCl, 2 mM EGTA, 1 mM EDT
A, 1 mM DTT, 0.1 μM p-amidinophenyl me
thansulfonyl fluoride hydrochloride, 10μg / m
l leupeptin, 1 mM benzamidine). 30 mL of each extract at 43000 rpm
For 4 minutes at 4 ° C., and the supernatant was subjected to SDS-PAGE and immunoblotted. For use in immunoblot analysis, a fusion protein of the coiled-coil region of bovine Rho kinase (amino acids 421 to 701 of SEQ ID NO: 1) and GST (Rho kinase (COI
L)) was prepared according to the method described in Example 13, and rabbits were immunized with the same as an antigen according to a conventional method to prepare a polyclonal antibody. The immunoblot analysis using the anti-Rho kinase (COIL) antibody and the anti-20kDa myosin light chain antibody (provided by Dr. JT Stull) was performed by the method described previously (Harlow, E. & Lan).
e, D. Antibodies: A Laboratory Mannual. (Cold Spri
ng Harbor Laboratory Press, Cold Spring Harbor, NY
(1988)). The stained proteins were visualized by ECL (Amersham) and subjected to densitometry for quantification.

As a result, as shown in FIG. 27, the present inventors have made extensive use of Triton X-100-permeabilized RPV.
Found that endogenous Rho kinase was lost. As shown in FIG. 27a (lanes 1 and 2), the amount of Rho kinase in extensively Triton X-100-permeabilized RPV was significantly reduced compared to that of intact tissue. Extensively
The amounts of myosin light chains (lanes 3, 4) and myosin heavy chains (lanes 5, 6) in Triton X-100-permeabilized RPV were almost comparable to those in intact tissues. FIG. 27b summarizes the data shown in FIG. 27a, and each value is expressed as the mean ± SD from three experiments.

[0234] From these results, it can be seen that when the permeability is strongly enhanced by Triton X-100 treatment, cytoplasmic proteins including Rho kinase are lost, but cytoskeletal proteins such as myosin light chain are stable. Was confirmed to be present. As described above, unless Rho kinase (CAT) is introduced exogenously into the cytoplasm,
The reason that iton X-100-permeabilized RPV does not shrink
This is probably because ho kinase is one of the above diffusible coupling factors.

Thus, the inventors have shown evidence that Rho kinase induces a contractile response under physiological conditions, which is accompanied by phosphorylation of the myosin light chain. From this, the present inventors, Rho kinase,
It was concluded that Rho is a downstream target protein involved in enhancing Ca ²⁺ sensitivity in smooth muscle contraction by smooth muscle contraction and plays a crucial role in smooth muscle contraction via phosphorylation of myosin light chain.

Example 13 Deletion Modification of Bovine Rho Kinase
Production of heteroproteins and characterization in vitro Rho kinase is composed of a catalytic domain (CAT), a coiled coil domain (COIL), a Rho binding domain (RB) and a plextrin homology domain (PH). We made four fragments containing each region (FIG. 28) as GST-fusion proteins.
First, in order to obtain a fragment corresponding to the Rho binding region (RB), cDNA encoding Rho kinase (amino acid sequence of amino acids 941 to 1075 of SEQ ID NO: 1) was converted into pGEX- according to the method described in Example 4. Inserted into the 2T BamH1 site, the resulting plasmid was expressed in E. coli,
A fusion protein (Rho kinase (RB)) of Rho kinase (amino acid sequence of positions 941 to 1075 of SEQ ID NO: 1) and GST was purified.

The binding of Rho to Rho kinase was determined by an overlay assay according to the method described in Example 1. 0.25 μg of purified Rho-kinase or 2.5 μg of Rho-kinase (RB) (amino acids 941 to 1075 of SEQ ID NO: 1) were separated by SDS-polyacrylamide gel electrophoresis (12%). After transfer to a cellulose membrane, [ ³⁵ S] GTPγS · GST-RhoA or [
^[35 S] GTPγS · GST-RhoA ^Binding to ^Ala37 was detected. Radiolabeled bands were visualized by an image analyzer.

As a result, GTPγS · GST-RhoA
Is Rho kinase (RB) (941-10 of SEQ ID NO: 1)
75th amino acid sequence)), but GTPγS
GST-RhoA ^Ala37 bound weakly to it (FIG. 29). By the way, RhoA ^Ala37 is H-Ras
Structurally equivalent to ^Ala35 , H-Ras ^Ala35 is a mutation in the effector binding domain (threonine to alanine substitution) (C. Nobes & A. Hall, Cur.
r. Opin. Genet. Dev. 4, 77 (1994), Y. Takai et al.,
Trends. Biochem. Sci. 2O, 227 (1995), T. Satoh e.
t al., J. Biol. Chem. 267, 24149 (1992), and F.
McCormick, Curr. Opin. Genet. Dev. 4, 71 (199
Four)).

[0239] Also, the bovine Rho-kinase coiled-
Coil region (amino acid sequence from 421 to 701 of SEQ ID NO: 1) or PH region of Rho kinase (SEQ ID NO: 1
CDNAs encoding the amino acid sequences of Nos. 1125 to 1388 of pGEX-2T were inserted into the BamH1 site of pGEX-2T.
OIL), pGEX-GST-Rho kinase (P
H) was prepared. These are expressed in E. coli and purified to obtain Rho kinase (COIL) and Rh
o-kinase (PH) was prepared.

Next, as described in Example 7, recombinant bovine Rh lacking the C-terminal half containing the Rho binding site.
o kinase catalytic domain (CAT) (6-5 of SEQ ID NO: 1)
A fusion protein (Rho kinase (CAT) with GST) was prepared and purified (FIG. 2).
8). Specifically, Rho kinase (6 to SEQ ID NO: 1)
CDNA encoding the amino acid sequence of 553)
Inserted into the BamHI site of cYM1-GST, pAcY
M1-GST-Rho kinase (CAT) (SEQ ID NO: 1
No. 6-553). Rho kinase (CAT) (amino acid sequence from positions 6 to 553 of SEQ ID NO: 1) is a baculovirus system (Y. Matsuura et a).
l., J. Gen. Virol., 68, 1233 (1987)) and purified from Sf9 cells by a glutathione sepharose column. ATP of bovine Rho kinase catalytic region (amino acid sequence of Nos. 6 to 553 of SEQ ID NO: 1)
A mutant in which the lysine at position 121 constituting the binding site has been substituted with glycine according to a conventional method (Rho kinase (CAT-
KD) was purified by expressing the baculovirus strain in Sf9 cells according to the same method as for Rho kinase (CAT) (FIG. 28). Natural bovine Rho kinase was purified according to the method described in Example 1.

Next, these recombinant and natural Rho
The kinase activity of the kinase was checked using myosin light chain (MLC) as a substrate. The kinase reaction of Rho kinase was performed in a 50 μl reaction mixture (40 mM Tris / HC).
1 (pH 7.5), 2 mM EDTA, 1 mM DTT,
6.5 mM MgCl ₂ , 0.1% CHAPS, 0.
1 μM calclin A, 100 μM [γ- ³²
P] ATP [0.5-20 GBq / mol], 4 μg
1.5 μM GTPγS in myosin light chain and 20 ng of native Rho kinase or 8 ng of Rho kinase (CAT) or Rho kinase (CAT-KD).
-It carried out according to the method of Example 7 except having performed in the presence or absence of GST-RhoA.

As a result, a result equivalent to that of Example 7 was obtained. Natural bovine Rho kinase is GTPγS · GST-R
It showed kinase activity on myosin light chain in a ho-dependent manner, whereas GST-Rho kinase (CAT)
Kinase activity was shown independent of the presence of GST-RhoA (FIG. 30). Natural Rho kinase and GTPγS.GST in the presence of GTPγS.GST-RhoA
-Rho kinase (CA in the absence of RhoA)
The molecular activity of T) was 0.32 ± 0.02 / s, respectively.
ec and 0.71 ± 0.02 / sec. This indicates that Rho kinase (CAT) is constitutively activated. On the other hand, a Rho kinase catalytic region having a mutation in the ATP binding site (Rho kinase (CAT
-KD)) showed no kinase activity (data not shown).

Rho kinase (RB) is dose-dependently
GTPγS-GST-RhoA inhibited the activation of native Rho kinase enhanced by, but found that it did not affect the kinase activity of Rho kinase (CAT) (FIG. 31). Rho Kinase (RB) IC
₅₀ was 0.7 μM. On the other hand, Rho kinase (C
AT-KD), did not affect Rho kinase (data not shown).

Example 14 Rh with Staurosporine
Inhibition of okinase The formation of stress fibers induced by Rho is known to be inhibited by protein kinase inhibitors such as staurosporin (CD Nobes & A. Hall, Cell 81, 53 (1995). )
). Staurosporine dose-dependently inhibited the kinase activity of Rho kinase (CAT) (FIG. 31). Staurosporine had an IC ₅₀ of 4 nM. This value is the value described for protein kinase C (T. Tamaoki
et al., Biochem. Biophys. Res. Commum. 135, 397
(1986)). Essentially similar results were obtained when using native Rho kinase stimulated with GTPγS.GST-RhoA instead of Rho kinase (CAT) (FIG. 31).

Example 15 Stress Fiber Form of Cell
Effect of Rho Kinase Deletion Mutant Microinjection on Growth
The effect of Rho kinase (CAT) and Rho kinase (CAT-KD) on stress fiber formation in Swiss3T3 cells was examined. Swiss 3
T3 cells were obtained from Dulbecco's supplemented with 10% fetal bovine serum.
Cultured in 's modified Eagle's medium (DMEM). Cells were seeded on 12 mm glass coverslips at a density of 8-10 × 10 ³ cells. After 4 days of culture, by culturing for 24 hours in DMEM medium without serum,
Cells were serum starved. The recombinant protein was microinjected into the cytoplasm of the cells along with the marker protein (1 mg / ml rabbit IgG). After microinjection, cells were incubated at 37 ° C. for 30 minutes. Previously described methods (Ridley, A. & Hal
l, A., Cell 70, 389 (1992); Ridley, A. & Hall, A.,
According to EMBO J., 13, 2600 (1994)), intracellular actin is converted to TRITC-labeled phalloidin.
Visualized by

As a result, as described previously (AJ Ridley and A. Hall, Cell 70, 389 (1992))
And AJ Ridley & A. Hall, EMBO J. 13, 2600 (19
94))) Confluent serum-starved Swiss 3T3 cells had very small amounts of stress fibers, which were visualized by phalloidin (Fig. 32a). Previously described method (AJ Ridley &
A. Hall, Cell 70, 389 (1992), and AJ Ridley
& A. Hall, EMBO J. 13, 2600 (1994)), when cells were stimulated with LPA (200 ng / ml), new stress fibers appeared and their numbers and diameters increased (FIG. 32B). Rho kinase (CAT)
(0.5 mg / ml) microinjection
It also induced the formation of stress fibers (FIG. 32).
E). Such an effect was not observed for Rho kinase (CAT-KD). Injected Rho kinase (C
AT) often caused large aggregate formation of actin filaments associated with stress fibers in the central part of the cells (FIGS. 32E, 32F). It is not clear why such hub-like actin filaments are present, but this may be due to the high contractility of the stress fibers induced by the injected Rho kinase (CAT).

On the other hand, C3 transferase (C3), an extracellular enzyme of Clostridium botulinum, which inhibits Rho by ADP-ribosylation (K. Aktories et al., Ibid. 15).
8, 209 (1989), and A. Sekine et al., J. Biol. C
hem. 264, 8602 (1989)) (80 μg / ml) was microinjected into cells as previously described (HF Paterson et al., J. Cell Bio.
l. 111, 1001 (1990)), cells rounded within 30 minutes (data not shown). Injected C3 inhibited LPA-induced stress fiber formation (FIG. 32).
C), but it did not inhibit the formation of stress fibers induced by Rho kinase (CAT) (FIG. 3).
2F). Coinjection of C3 with Rho kinase (CAT) prevented the cells from rounding (data not shown). Cells stimulated with LPA in the presence of 50 nM staurosporin were described previously (Nobes, C. & Hall, A. Cell 81, 53 (1995) and Ridle
y, P. et al., Mol. Cell, Biol. 15, 1110 (1995)) resulted in a randomly arraneged actin filament (FIG. 32D). However, stress fibers did not occur in cells injected with Rho kinase (CAT) in the presence of staurosporine (FIG. 32G).

Example 16 Influence on Focal Adhesion of Cells
Microinjection of Rho kinase deletion mutant
It was studied the effect of Rho kinase (CAT) and Rho kinase on focal adhesion formation in effect Swiss 3T3 cells down (CAT-KD). Cell culture, LP
The A concentration, microinjection conditions, and amount of protein injected were the same as described in Example 15. For cell staining, the method described previously (Ridley,
A. & Hall, A., Cell 70, 389 (1992); Ridley, A. & H
All, A., EMBO J., 13, 2600 (1994)), intracellular vinculin was visualized with an anti-vinculin antibody. The nucleus is bis-benzim
ide).

As a result, as described previously (AJ Ridley & A. Hall, Cell 70,389 (1992) and AJ Ridley & A. Hall, EMBO J. 13, 2600 (1992)
4)), very little focal adhesion was detected by anti-vinculin antibody in confluent serum-starved Swiss 3T3 cells (FIG. 33A). L cells
When stimulated with PA, as previously observed (AJ
Ridley & A. Hall, Cell 70, 389 (1992), and A.
J. Ridley & A. Hall, EMBO J. 13, 2600 (1994)),
New focal adhesions containing vinculin appeared and increased in number (FIG. 33B). Rho kinase (CA
Microinjection of T) induced the formation of focal adhesions (FIG. 33E). The present inventors confirmed by double immunofluorescence analysis that stress fibers newly synthesized by the injected Rho kinase (CAT) were linked to focal adhesion (FIG. 33H). Microinjection of C3 inhibited the formation of focal adhesion induced by LPA (FIG. 33C), but it did not inhibit the formation of focal adhesion induced by Rho kinase (CAT) (FIG. 33F, FIG. 31).
G).

Staurosporine (50 nM) inhibited both focal adhesion formation induced by LPA and Rho kinase (CAT) (FIGS. 33d and 33g). Constitutively activated PKN catalytic domain or M
Neither stress fiber formation nor focal adhesion formation was induced by BS injection (data not shown). These infusions also include Rho kinase (CAT)
Had no effect on the formation of stress fibers and the formation of focal adhesions induced by ATP (data not shown).

Example 17 Rho kinase (RB),
Rho kinase (PH), Rho kinase (CAT-K
D) Stress injection by microinjection
Inhibition of Bar and Focal Adhesion Formation We next turn to Rho kinase (RB), Rho kinase (PH), Rho kinase (CAT-KD) and Rho kinase (LPH) in stress fibers and focal adhesion formation induced by LPA. COI
The effect of the microinjection of L) was examined. Rho kinase (RB) or Rho kinase (P
Injection of H) inhibited LPA-induced formation of stress fibers (FIGS. 34C, D) and focal adhesions (FIGS. 34G, H). About 30% of cells injected with Rho kinase (CAT-KD) contain LP
A stress fiber (FIG. 34A) and focal adhesion (FIG. 34E) did not form in the presence of A. Rh
o-kinase (COIL) had no effect (FIG. 34).
B, F). None of Rho kinase (CAT-KD), Rho kinase (COIL), Rho kinase (RB) or Rho kinase (PH) inhibited Rho kinase (CAT) -induced stress fiber and focal adhesion formation. This means that Rho kinase (CAT-KD), Rho kinase (RB) or Rho kinase (PH) inhibits the function of endogenous Rho kinase, but that Rho kinase (CAT) is exogenously injected in excess. No function was shown to be inhibited.

Example 18 Deletion mutant of Rho kinase
Changes in Cell Morphology by Introduction of cDNA Encoding Swiss 3T3 cells are not suitable for injecting plasmid into the cell nucleus. The effect of Rho kinase on cell morphology was examined by microinjection. First,
Rho kinase (CAT), Rho kinase (CAT
-KD), Rho kinase (COIL), Rho kinase (RB), pEF-BOS-myc mammalian expression plasmid for expressing Rho kinase (PH) was prepared. MDCK cells were cultured in minimal essential medium supplemented with 10% fetal calf serum. Cells at a concentration of 2 × 10 ³
Seed on a 1 mm glass coverslip and cultured for 1 day. Various plasmids are described (A. Ridley
et al. Cell 70, 401 (1992)). After microinjection,
Cells were cultured at 37 ° C. for 3 hours. Actin, Ridley,
A. & Hall, A. Cell 70, 389 (1992) and Ridley, A.
& Hall, A. EMBO J. 13, 2600 (1994)) and stained with TRITC-labeled phalloidin.

First, the cDNA encoding the constitutively activated Rho mutant (Rho ^Val14 ) was cloned into MDCK.
Microinjection into cells was performed as previously described (Nobes, C. & Hall, A. Cell 81, 53).
(1995) and Ridley, A. et al. Mol. Cell. Biol. 15,
1110 (1995)), formation of stress fibers (Fig. 35)
A) and focal adhesion (data not shown) were induced. Stress fiber and focal bonding are Rh
cDNA encoding o-kinase (CAT) was formed in injected cells (FIG. 35B). This effect was not observed with cDNAs encoding Rho kinase (CAT-KD), constitutively activated PKN or MBS.

Rho kinase (CAT-KD), Rho
^{Coinjection of} cDNA encoding kinase (RB) or Rho kinase (PH) with Rho ^{Val14 inhibited} Rho ^Val14- induced ^formation of stress fibers (FIGS. 35C, D, E) and focal adhesion (data not shown). Was done. R
The effect of ho kinase (CAT-KD) was lower than that of Rho kinase (RB) or Rho kinase (PH). On the other hand, Rho kinase (COIL) did not show this effect (data omitted).

As described in Examples 15-18, we found that injection of Rho kinase (CAT) induced the formation of stress fibers and focal adhesions, and that Rho kinase (CAT-KD), Rho kinase
It was shown that kinase (RB) or Rho kinase (PH) ^inhibits LPA or Rho ^Val14- induced formation of stress fibers and focal adhesions. From these facts, it was found that R
Ho-kinase (CAT) acts as a dominant-active form, and Rho-kinase (CAT)
-KD), Rho kinase (RB) and Rho kinase (PH) were found to act as dominant negative bodies.

Rho kinase (CAT-KD) did not affect the kinase activity of Rho kinase in vitro under our experimental conditions (Example 1).
3), but higher enzyme concentrations may inhibit the interaction between Rho kinase and substrate. This is because such a phenomenon has been described for other kinases (Kolch, W. et al., Nature 349, 426 (199
1)). From this, Rho kinase (CAT-K
D) Inhibited Rho kinase function in cells (Examples 17-18), but in vitro R
The one that did not affect the kinase activity of the ho kinase (Example 13) was the Rho kinase used (CAT-
This is probably because the concentration of KD) was low.

[0257] On the other hand, since the PH region is considered to localize a protein containing the same to a specific intracellular region, Rho kinase (PH) becomes Rh in the cell.
It is thought to inhibit the proper localization of okinase. Although native Rho kinase is partially present at the site of cell-cell adhesion, Rho kinase (C
AT) was present in the cytoplasmic region in MDCK cells (data not shown), suggesting this possibility.

By the way, Rho kinase (RB)
In addition to inhibiting the binding of the ho protein to Rho kinase, other Rho proteins, such as PKN,
It may also inhibit binding to the target protein. In contrast, Rho kinase (CAT-KD) and Rho kinase
Ho kinase (PH) may act as a more specific inhibitor for Rho kinase.

In any case, Swiss 3T3 cells or MD
In CK cells, Rho kinase is downstream of Rho,
Controlling the formation of stress fibers and focal adhesions, and Rho kinase (CAT-KD), R
It has been shown that ho kinase (RB) and Rho kinase (PH) inhibit the above physiological actions of Rho kinase.

The binding of actin filaments to myosin is promoted by phosphorylation of the myosin light chain and has been postulated to be one of the late stages of stress fiber formation (J. Kolega et al., Bioimaging l, 136 (1993),
And KA Giuliano & DL Taylor, Curr. Opin. Ce
ll Biol. 7, 4 (1995)). Rho is the myosin light chain (M
We have shown that Rho kinase, which phosphorylates LC) and myosin binding subunit (MBS), activates myosin light chain phosphorylation (Example 2 and Examples 5-9). . This phosphorylation is responsible for the binding of actin to myosin and their contractility (contract
(Example 9 and Example 1)
2). In fact, the present inventors have confirmed that myosin binds to actin filaments induced by Rho kinase (CAT) injected in the present invention (data not shown). These observations suggest that Rho-stimulated actin and myosin contractility drives the formation of stress fibers and focal adhesions (M. Chrza
nowska-Wodnicka & K. Burridge, J. Cell Biol. 133,
1403-1415 (1996)).

It is thought that Rho kinase induces focal adhesion formation by promoting the assembly of integrin with various proteins (vinculin, FAK, paxillin and α-actinin) constituting the focal adhesion site.

Example 19 Cell Bone by Rho Kinase
Case reconstitution and c-fos serum response element (SRE)
Transcriptional activation We have now constitutively activated Rho kinase (Rho kinase (CAT); Examples 7-9, 12-
18) expression was found to increase the level of phosphorylation of myosin light chain (MLC) and the regulated reconstitution of cytoskeletal proteins including actin, vinculin and MLC in intact cells.

In this example, the activity of Rho kinase was
Although actin stress fibers, which are necessary for maintaining focal adhesion including vinculin, do not require focal adhesion, microinjection of Rho kinase (CAT) into intact cells results in organized actin stress fibers. That focal adhesion is induced even under conditions where Rho kinase (CAT) is destroyed by c-fos
It stimulates the transcriptional activation of serum response element (c-fos SRE).

[0264] These results indicate that Rho kinase was
Have different roles in the pathways diverging from downstream (MLC phosphorylation leading to stress fiber formation, focal adhesion formation and gene expression). Detailed experimental methods and results are shown below.

[Materials and Methods] (1) Materials and Compounds GST-Rho kinase (CAT) and GST-Rh
o-kinase (CAT-KD) was produced in Sf9 cells using the baculovirus system and purified on a glutathione Sepharose column (Examples 7 and 1).
3). Anti-vinculin antibody, tetrametyl rhodamine iso
Thiocyanate (TRITC) labeled phalloidin and anti-ML
Antibody C was purchased from Sigma. Anti-MLC-pS19 monoclonal antibody was obtained from Mitsuo Ikebe (University of Massachusetts). Calyculin A
A) and cytochalasin D
Purchased from Pure Chemical Industries, Ltd. (Osaka, Japan). [γ- ³² ATP] was purchased from Amersham Corp.

(2) Plasmid construct C3 transferase and cD of Rho ^Ile41
NA was determined using the BamH of pGEX-2T according to Example 13.
After cloning into one site and producing, it was purified from E. coli. GST-C3 and GST-Rho obtained
^Ile41 uses Ba for microinjection.
r-Sagi, D. (1995). Methods Enzymol. 255, 436-442;
According to Ridley A. et al. (1992). Cell 70: 389-399, the cells were cut with thronbin to remove GST, purified, and concentrated. pEF-BOS-HA-Rh
o ^Val14 is Mizushima, S. & Nagata, S. (1990).
Plasmid p described in Nucleic. Acid Res., 18: 5322
It was constructed by inserting a Rho ^Val14 cDNA linked to a hemagglutinin (HA) tag into EF-BOS. Rho kinase (CAT), Rho kinase (CAT-KD) and Rho kinase (COIL)
Was inserted into the BamH1 site of the plasmid for mammalian expression of pEF-BOS-myc (Example 13).

The pEF-BOS-myc MLC was constructed as follows. 0.6 kb cd encoding MLC
Rat brain Quick clone cDNA by PCR of NA fragment
(Clontech) Primer 5'-AAT AGG ATC CGA TTT A
AC CGC CAC CAT GTC G -3 'and 5'-ATA AGG ATC CTC
Amplification was performed using AGT CAT CTT TGT CTT TCG CTC -3 ′.
The obtained cDNA fragment was cloned into the BamH1 site of the pEF-BOC-myc vector. For gene expression analysis, two copies of SRE. L (5'- GGTACC TCG A
GC ATG TAC TGT ATG TCC ATA TTA GGA CAT CTA TGT ACT
GTA TGT CCATAT TAG GAC ATC TGG ATC C-3 ') pG
It was inserted into KpnI and BamH1 of VB-tk-luciferase and SRE. L-luciferase was obtained. pGV
B-tk-luciferase contains the thymidine kinase promoter. A cDNA fragment encoding β-galactosidase of Escherichia coli was inserted into the NotI site of pME18S containing the SRα promoter.
S-lacZ was obtained.

(3) Transfection into COS cells COS cells were prepared from Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS).
s medium: DMEM). Transfection of the plasmid into COS cells was performed using the standard DEAE-dextran method (Lopata MA et al. (1984). Nucleic
Acid Res. 12: 5707-5717). Cells are seeded on a 60 mm dish at a cell density of 5 × 10 ⁵ cells / dish in DMEM containing 10% FBS,
Cultured overnight. Transfection 2
An hour before, it was replaced with fresh DMEM containing 10% FBS. Prepare a DEAE-dextran / DNA mixture,
The dish was added to the dish while slowly penetrating. Two hours later, cells were treated with 10% DMSO / phosphate buffered saline (PBS). Cells were grown in DMEM with 10% FBS for 1 day and then in DMEM for 1 day. In some experiments, cells were treated with caliculin A (0.
(1 μM) for 10 minutes. Cells are 10% (w / v)
Was treated with trichloacetic acid (TCA). The resulting precipitate was subjected to immunoblot analysis.

(4) Microinjection and immunofluorescence analysis REF52 cells were cultured in DMEM containing 10% FBS. Cells were seeded on 12 mm glass coverslips at a density of 1-3 × 10 ³ cells / coverslip,
Cultured for 2 days. Swiss 3T3 cells were cultured in DMEM containing 10% FBS. Cells were seeded on 12 mm glass coverslips at a density of 8-10 × 10 ³ cells / coverslip. After 4 days of culture, cells were cultured in DMEM for 24 hours under serum starvation. The recombinant protein was a marker protein (rabbit IgG, 1 mg / m
l) was microinjected into the cytoplasm of the cells. Cells were pretreated with 0.1 μM cytochalasin D 30 minutes before microinjection. After the injection, the cells were cultured at 37 ° C. for 20 minutes.

Actin, vinculin, MLC and phosphorylated MLC were prepared as described previously (Example 15).
-18), TRITC-labeled phalloidin, an antibody against vinculin, an antibody against MLC, and an antibody against phosphorylated MLC, respectively.

(5) Transfection into NIH3T3 cells NIH3T3 cells are DM containing 10% bovine serum (CS).
Cultured in EM. Transfection of the plasmid into NIH3T3 cells was performed using the standard calcium phosphate method (Ch
en, C. & Okayama, H. (1987). Mol. Cell. Biol. 7:
2745-2752). Cells are plated on 35 mm dishes at a density of 6 × 10 ⁴ cells / dish at 10% C
The cells were seeded in DMEM containing S and cultured overnight. Two hours before transfection, add 10% medium
DMEM containing CS and 10 mM Hepes (pH 7.
Replaced in 5). A calcium phosphate / DNA mixture was prepared and added to the dish while gently penetrating the dish. After 16 hours, cells were washed twice with PBS for 10 minutes each and grown in DMEM for 36 hours.

(6) Luciferase assay After transfection, NIH3T3 cells were transferred to PBS.
3 times with 200 μl of lysis buffer (25 m
M Tris / PO ₄ (pH7.8), 2 mM DTT, 10% glycerol, 1% Tri
ton X-100) for 15 minutes at room temperature. Cell lysates were centrifuged at 13,000 xg for 5 minutes at 4 ° C. The supernatant was used for the enzyme assay. For the luciferase assay, the supernatant was mixed with 440 μl of the reaction mixture (20 mM Tricine / NaOH (pH 7.8), 1.07 mM (MgCo ₃ ) ₄
Mg (OH) ₂ / 5H ₂ O, 2.67 mM MgSO ₄ , 0.1 mM EDTA, 33 mM DT
T, 270 μM coenzyme A, 530 μM 5'-ATP / 2Na, 477 μM D
-lucuferin potassium salt) at room temperature.

The luciferase activity was measured with a Lumiphotometer (Model TD-4000, Labo Science, Japan) and expressed as that standardized with β-galactosidase activity. For the β-galactosidase assay,
The supernatant was added to 100 μl of the reaction solution (60 mM Na ₂ HPO ₄ / 12H ₂ O, 4
0 mM NaH ₂ PO ₄ / 2H ₂ O, 10 mM KCl, 1 mM MgCl ₂ , 48 mM ₂ -mercaptoethanol, 330 μg / ml o-nitrophenyl β-D
-Galactopyranoside) at 37 ° C. The reaction was stopped by the addition of 50 μl of 1 M Na ₂ CO ₃ and measured by absorption at 415 nm.

(7) Others SDS-PAGE is described in Laemmli, UK (1970). Nature 2
According to 27, 680-685, immunoblot analysis was performed by Harlow &
Lame (1988). Antibodies: A Laboratory Manual. Col
d Spring Harbor Laboratory Press, Cold Spring Harb
or, NY, respectively.

[Results] (1) Expression of Constitutively Activated Rho Kinase and Kinase-Inactivated Rho Kinase The present inventors have determined that the catalytic region of Rho kinase (CAT) is constitutively activated. (Examples 12 to 1)
8). In order to express various types of Rho kinase in animal cells, myc-tagged full-length Rho kinase is expressed.
Kinase, Rho kinase (CAT), and the catalytic region (CAT-KD) in which the ATP binding site was mutated were pEF-BO
It was cloned into a plasmid for expressing S-myc (Example 18). Full-length Rho kinase, Rho kinase (CA
Plasmids encoding T) and Rho kinase (CAT-KD) were transfected into COS7 cells. Various types of Rho kinase were immunoprecipitated with anti-myc antibodies from lysates of cells transfected with these plasmids. Immunoprecipitates were subjected to immunoblot analysis. The various types of Rho kinase are all COS7
It was expressed intracellularly (data not shown).

(2) Detection of phosphorylated myosin in COS7 cells We have determined that Rho kinase phosphorylates myosin phosphatase, thereby inhibiting myosin phosphatase, and that Rho kinase also inhibits myosin light chain (MLC ) Was demonstrated to directly phosphorylate Ser-19 in vitro (Example 2, Examples 5-9, Example 1).
3). These data suggest that Rho kinase enhances the level of phosphorylation of MLC, thereby enhancing the interaction of intact actin with myosin and promoting stress fiber formation in intact cells. Indeed, microinjection of Rho kinase (CAT) into serum-starved Swiss 3T3 cells induced stress fiber formation (Examples 15-17). To investigate whether Rho kinase enhances MLC phosphorylation in vivo, a monoclonal antibody against MLC phosphorylated with Ser-19 (anti-MLC-pS19 antibody) was used. The specificity of this antibody was examined by immunoblot analysis. Equal amounts of MLC containing non-phosphorylated and phosphorylated forms at various rates were loaded on the gel. MLC phosphorylation by Rho kinase in vitro was specifically detected in a dose-dependent manner by the anti-MLC-pS19 antibody (FIG. 36).

The endogenous M expressed in COS7 cells
Since the amount of LC is not sufficient to analyze the effect of Rho kinase on MLC phosphorylation levels, pEF-BOS-
myc-MLC was co-transfected into COS7 cells with a plasmid containing Rho or Rho kinase cDNA. The level of MLC phosphorylation was lower in serum starved COS7 cells expressing myc-MLC alone. Calycu is a cell phosphatase inhibitor
Treatment with lin A increased the level of phosphorylated MLC (FIG. 37). This indicates that phosphatase activity is involved in regulating MLC phosphorylation levels in resting COS7 cells. ^{Expression of} Rho ^Val14 or Rho kinase (CAT) increased MLC phosphorylation, but not Rho kinase (CAT-KD). Full-length Rho-kinase slightly increased MLC phosphorylation (this weak activity of full-length Rho-kinase was attributed to the inactivation of full-length Rho-kinase in unstimulated, ie, non-Rho-activated, cells). Example 2) (FIG. 38). The above results
Figure 4 shows that constitutively activated Rho kinase enhances the level of MLC phosphorylation in intact cells.

(3) Reconstitution of cytoskeletal protein by Rho kinase Rho kinase inhibited MLC phosphorylation in vitro (Examples 5 to 9 and Example 13) and in vivo (Example 12)
And control according to Example 19 (2)), the present inventors have developed C3 transferase (hereinafter referred to as C3), GTPγ
^{S.Rho Ile41} ( ^{Asn- of} wild-type Rho protein)
41 is a mutant Rho protein substituted with Ile-41,
3 not ADP-ribosylated (not inactivated) by 3; Paterson, HF et al. (1990). J. Cell B
iol. 111: 1001-1007) or Rho kinase (CA
The distribution of phosphorylated or non-phosphorylated myosin in REF52 cells microinjected with T) was examined by immunoblotting. REF52 cells grown in 10% fetal bovine serum (FBS) contain large amounts of actin stress fiber and vinculin (vinculi).
n) It had contained focal adhesion and showed a fibrous regular pattern of myosin and stress fibers (FIG. 38). Phosphorylated myosin showed a similar pattern to myosin, suggesting that most myosins are present on stress fibers and are phosphorylated under these conditions. Microinjection of C3 into REF52 cells disrupted stress fibers and focal adhesion to some extent, disrupted the fibrous pattern of myosin, and reduced staining for phosphorylated myosin. ^{Coinjection of GTPγS.Rho Ile41} or Rho kinase (CAT) with C3 ^neglected the effect of C3 alone. Thus, Rho kinase is thought to mediate signals from Rho proteins and maintain cytoskeletal organization. In the case of cells injected with Rho kinase (CAT), C3
Stress fibers, whether present or absent, often adhered to each other at certain points.

(4) Relationship between stress fiber and focal adhesion Nobes & Hall proposed that Rho induces the formation of actin stress fiber and focal adhesion by an independent pathway (Nobes, C. & Hall, A. et al. (1994) .Curr.
Opin. Genet. Dev., 4: 77-81). Meanwhile, Chrzanowska-
Wodnicka & Burridge proposed that contraction of actin stress fibers triggered by myosin phosphorylation leads to focal adhesion formation (Chrzanowska-Wo
dnicka, M. & Burridge, K. (1996) .J. Cell Biol., 13
3: 1391-1402). We show that Rho kinase increases the level of MLC phosphorylation in vitro and in vivo (Examples 5-9, 12-13, 1).
9 (2)-(3)) and found that constitutively activated Rho kinase (Rho kinase (CAT)) enhances the formation of actin stress fiber and focal adhesion in Swiss 3T3 cells (Examples). 15-18, Example 19 (2)-(3)).

To determine whether the signaling pathway of stress fiber formation and focal adhesion diverges downstream of Rho kinase, we examined the stress fiber and focal adhesion formation by Rho kinase (CAT) on cytochalasin D. Examined in the presence. This cytochalasin D is actin polymerization
Is specifically inhibited. Cytochalasin D of REF52 cells
Treatment broke the stress fibers but did not break the focal bond (FIG. 39). Microinjection of C3 into REF52 cells resulted in the loss of stress fibers and focal adhesion (FIG. 3).
8 and 39). The above results suggest that actin stress fibers are not necessarily required to maintain focal adhesion, and that Rho is required to maintain both.

Rho kinase (CAT) alone or Rh
Microinjection of o-kinase (CAT) together with C3 in the presence of cytochalasin D induced a mass of actin and apparently normal focal adhesion with a small amount of short actin fibers (FIG. 40). This indicates that Rho kinase inhibits the effect of C3 and that Rho kinase is probably involved in maintaining focal adhesion.

We further investigated whether Rho kinase newly formed focal adhesions in Swiss 3T3 cells in a manner independent of actin stress fibers. Serum-starved Swiss 3T3 cells have little stress fiber or focal adhesion, but as shown in Examples 15-18, Rh
Microinjection of o-kinase (CAT) into cells induces stress fibers and focal adhesion (FIG. 40). In the presence of Cytochalasin D, R
Microinjection of ho kinase (CAT) failed to induce stress fiber formation, but induced focal adhesion formation (although focal adhesion was not
smaller in the absence of cytochalasin D).

(5) c-fos serum by Rho kinase
Transcriptional activation of response element (SRE) It has been reported that Rho induces transcriptional activation of c-fos SRE mediated by serum response factor (SRF) (Hill, CS et al. (1995). Cell 81: 1159-11
70). To determine whether Rho-kinase mediates Rho-induced transcriptional activation, we examined the transcriptional activation of c-fos SRE by Rho-kinase. SRE. Fused to the coding sequence of the firefly luciferase gene. SRE. The L fusion gene (SRE.L-luciferase) was used as a reporter. SRE. L is a derivative of c-fos SRE, which contains a binding site for SRF intact, but lacks a ternary complex factor binding site (Hill, CS et al. (1995) supra). )). To standardize the transcription efficiency, pME18S-lacZ was used as an internal control. SRE.L-luciferase and pME18S-lacZ to pEF-BO
When NIH3T3 cells were co-transfected with S-myc-CAT, luciferase activity was increased (FIG. 41). The extent was similar to the luciferase activity observed in cells co-transfected with pEF-BOS-HA-Rho ^V14 (FIG. 41). pEF-BOS-myc-CAT-KD
Is Rho ^Val14 or Rho kinase (CAT)
, But not pEF-BOS-myc-COIL (FIG. 42).

Rho kinase (CAT) induced S
Rho kinase for RE activation (CAT-KD)
^Was weaker than the inhibitory activity against that induced by Rho ^Val14 . The above results indicate that Rho
This suggests that the kinase is involved in Rho-regulated SRE activation.

PK as one of Rho target proteins
N (Amano, M. et al. (1996). Science 271: 648-65.
0) and PRK2 (Pal
mer, RH et al. (1994). FEBS Lett. 356: 5-8)
Identified as one of the Rho target proteins (Quil
liam, LA et al. (1996). J. Biol. Chem. 271: 287.
71-28776). PRK2 is SR with activated Rho
E potentiates (potentiates) the transcriptional activation of E, but its activity is weak (Quilliam, LA et al. (1996). J. Biol.
Chem. 271: 28771-28776). We have noted that Rho kinase (CAT) very strongly activates SRE, as described above, and that Rho kinase (CAT-K
D) is Rho kinase (CAT) or Rho
It was shown to inhibit SRE activation by ^Val14 . This indicates that SRE activation by activated Rho is mediated primarily by Rho kinase (activated by activated Rho), but not by PRK2.

Example 20 Rho kinase and fissure
Glial fibrillary acidic protein by kinase (CF kinase)
Phosphorylation sites and phosphorylation-specific antibodies at the same site of a protein are useful for analyzing the spatiotemporal distribution of phosphorylated proteins of a research target in vivo. In this example, the temporal and spatial distribution of site-specific phosphorylation of glial fibrillary acidic protein (GFAP) was analyzed in vivo. As a result, Rho kinase that binds to GTP-linked Rho, which is the active form of low molecular weight GTPase Rho,
hr-7, Ser-13, and Ser-34 were specifically phosphorylated. The kinase activity of Rho kinase to GFAP seen here was dramatically enhanced by GTP-bound RhoA.
Furthermore, phosphorylation of GFAP by Rho kinase almost completely inhibited GFAP filament formation in vitro. From the above results, it was inferred that Rho kinase is a strong candidate for CF kinase. Detailed experimental methods and results are shown below.

[Materials and Methods] (1) Materials Recombinant human GFAP was prepared in E. coli by the method already described (Sekimata, M. et al. (1996) J. Cell. Biol. 13
2: 635-641). Mouse monoclonal antibodies (YC10, KT13, and KT34, respectively) that specifically react with the phosphorylation sites of Ser-8, Ser-13, and Ser-34 of GFAP were prepared by the method described above (Sekimata, M. et al., Supra). al. (1996), Yano, T.
et al. (1991) Biochem. Biophys. Res. Commun. 175:
1144-1151). Monoclonal antibody MO389, which recognizes both phosphorylated and non-phosphorylated GFAP, was also prepared by the method described (Sekimata, M. et al., Supra). GST-RhoA was loaded with guanidine nucleotides after purification (Shimizu,
K. et al. (1994) J. Biol. Chem. 269: 22917-2292
0). Rho kinase was purified from bovine brain (Example 1). A GST fusion protein with the catalytic domain of Rho kinase, a constitutively activated GST-Rho kinase (Rh
okinase (CAT)) was purified from Sf9 cells by the method described above (Examples 7 to 9, 12 to 19). cAMP-dependent protein kinase (A kinase) is
o et al. (Beavo, JA et al. (1974) Methods Enzym
ol. 38: 299-308). cdc2 kinase is
The method of Kusubata et al. (Kusubata, M. et al.
(1992) J. Biol. Chem. 267: 20937-20942). Protein concentration was determined by the Bradford method (Bradford, M .;
M. (1976) Anul. Biochem. 72: 248-254), using bovine serum albumin as a standard protein.

(2) Phosphoric acid reaction of GFAP The phosphorylation reaction by Rho kinase is GST or GDP.
20 μl of 25 mM Tris-HCl (pH 7.5) containing GST-RhoA or GSTγS · GST-RhoA (1 μM each), 0.2% CHAPS, 4 mM
MgCl ₂ , 3.6 mM EDTA, 100 μM [γ- ³² P] ATP (5 μCi), 0.
30 minutes in a reaction solution containing 1 μM calyculin A, 130 μg / ml GFAP, and 0.5 μg / ml purified Rho kinase.
I went in. Rho kinase (CAT), A kinase, cd
For phosphorylation of c2 kinase, 100 μl of the reaction solution (2
5 mM Tris-HCl (pH 7.5), 0.4 mM MgCl ₂ , 100 μM ATP, 0.1
8.5 μg / μM calyculin A, 130 μg / ml GFAP)
Performed at 25 ° C. for 30 minutes in the presence of either ml GST-Rho kinase, or 5 μg / ml A kinase, or 0.5 μg / ml cdc2 kinase. The reaction is Laemmli '
The mixture was stopped by adding s sample buffer and boiling.

(3) Immunoblotting After SDS-PAGE, the protein was subjected to 25 mM Tris, 192 mM glycine,
It was transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) in a solution containing 20% methanol and 0.03% SDS. T with 5% skim milk (Difco)
BS-T (20 mM Tris / HCl (pH7.6), 137 mM NaCl, 0.1% Tw
After blocking the membrane with een-20)), the membrane was subjected to monoclonal antibody MO389, YC10, KT13, or KT34 (0.1 μg / m each).
Incubated for 1.5 hours in TBS-T containing l). Immunoreactive bands were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) and ECL western blotting detection kit (Amersham).

(4) Fragmentation of phosphorylated GFAP GFAP (130 μg) was replaced with GST-Rho kinase (8.5 μg)
And [γ- ³² P] ATP (50 μCi) for 120 minutes at 25 ° C
Was incubated in the above reaction solution (1 ml). Phosphorylated GFAP was precipitated with 10% trichloroacetic acid and
100 μl of 5 with 5 g of lysyl endopeptidase (Wako)
The digestion was carried out in 0 mM Tris / HCl (pH 8.0), 4M urea at 30 ° C. for 2 hours. Reversed phase HPLC of phosphorylated GFAP head domain
And separated by 1/50 (w) according to the method of Tsujimura et al. (26).
/ W) treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone. The resulting peptide was purified with 5% (v / v) 2-propanol / acetone tolyl containing 0.1% trifluoroacetic acid.
Separation by HPLC using a Zorbax C8 (0.46 × 25 cm) column equilibrated with (7: 3). Elution was performed with a linear gradient of 5-50% 2-propanol / acetone tolyl for 60 minutes,
Subsequently, 50-80% of 2-propanol / acet
elution speed with linear gradient of ontrile for 10 minutes
The test was performed at 0.8 ml / min.

(5) Other operations The immunofluorescence microscope was performed according to the method described (Sekimata, M. et al.
(1996) J. Cell. Biol. 132: 635-641). Amino acid sequence analysis was performed on an ABI476A gas phase sequencer. Two-dimensional phosphorylated amino acid analysis was performed by the method described (Boyle, W. et al. (1991) Methods Enzymol. 201, 110).
-149). Electron microscopy was performed according to the method described (Inagaki, M. et al. (1990) J. Biol. Chem. 265,
4722-4729).

[Results and Discussion] Recently, the present inventors have developed pig GFAP.
The four monoclonal antibodies YC10 (Ya10) against four different phosphorylated peptides corresponding to the partial amino acid sequences of
no, T. et al. (1991) Biochem. Biophys. Res. Commu
n. 175: 1144-1151), KT13, KT34, and MO389 (supra)
Sekimata, M. et al. (1996)).

Each of YC10, KT13 and KT34 was Ser-
8. Ser-13 and Ser-34 react with phosphorylated GFAP.
MO389 reacts with both phosphorylated and non-phosphorylated GFAP, staining filamentous structures in mitotic and mitotic cells.

[0294] MO389 immunostained (in green) glial fibrous intricatemesh in the whole cytoplasm of metaphase and late mitosis cells, while YC10 stained the filament structure found in metaphase cytoplasm (green). However, that of late cells did not stain (FIG. 43A, Reference Photo A). On the other hand, the staining activity (green) of KT13 and KT34 was specifically observed between the daughter nuclei (red) of late cells (division cleft) (FIG. 43A, reference photograph A).

KT13 by confocal laser scanning microscope
An immunocytochemical study using (FIG. 43B, reference photograph B) and KT34 (data not shown) revealed that Ser-13 and Ser-34 phosphorylated GFAP (green) was present in the fissure. Andr
eassen et al. (Andreassen, PRet al. (1991) J. Cell
Sci. 99, 523-534).
Instead of a disc-like structure like sc, a ring-like structure (ring-lik
e structure).

In order to search for CF kinase involved in cleavage groove-specific phosphorylation as described above, it was examined whether Rho kinase purified from bovine brain could phosphorylate GFAP in vitro. As a result, it was revealed that Rho kinase phosphorylates GFAP in a GST-RhoA-dependent manner (FIG. 4).
4A). GDP-bound GST-RhoA increased the phosphorylation of GFAP by Rho kinase 13-fold, whereas GTPγS-bound GST-RhoA
One-fold enhancement (FIG. 44A).

Then, the phosphorylation site of GFAP by Rho kinase was examined using the above-mentioned anti-phosphorylated GFAP antibody. After the phosphorylation reaction, the sample was separated by SDS-PAGE and YC
Immunoblot with 10, KT13, KT34, or MO389. As shown in FIG. 44B, Rho kinase was Ser-13
And Ser-34 was phosphorylated in a GTPγS.GST-RhoA-dependent manner, but Ser-8 was not.

The present inventors have proposed that a fusion protein of GST, which is a constitutively active GST-Rho kinase fusion protein, and the catalytic domain of Rho kinase was replaced with a recombinant baculovirus by Sf9.
The cells were prepared by infecting cells and used. As a result of analysis with anti-phosphorylated GFAP antibody, GST-Rho kinase also phosphorylated Ser-13 and Ser-34 of GFAP, but Ser-
8 was not phosphorylated (FIG. 44C). These results
The catalytic properties of GST-Rho kinase have been shown to be similar to that of native Rho kinase activated by GTPγS.GST-RhoA. In contrast, cAMP-dependent protein kinase phosphorylates all three Ser residues,
dc2 kinase slightly phosphorylated Ser-8 only (FIG. 44C).

In order to confirm the phosphorylation site of GFAP by Rho kinase, GFAP (130 μg) was phosphorylated in the presence of [γ- ³² P] ATP to about 2.7 mol phosphoric acid / 1 mol GFAP (FIG. 45A). ). Radioactive GFAP was digested with lysyl endopeptidase to produce a 6.5 kDa fragment consisting essentially of the N-terminal head domain. Tricine-SDS-PAGE
Analysis (Schagger, H. & von Jagow, G. (1987) Anal. Bi
ochem. 166: 368-379) revealed that all radioactivity of GFAP was retained in this fragment (Figure 4).
5B). The phosphorylated head domain was separated by reverse phase HPLC, and after trypsin digestion, subjected to reverse phase HPLC again. As shown in FIG. 46A, three peaks R1 to R3 were obtained. As a result of phosphorylated amino acid analysis, phosphothreonine was detected in R1 and phosphoserine was detected in both R2 and R3 (FIG. 46B). As a result of amino acid sequence analysis, R1 was Thr-
7, R2 was a peptide containing Ser-34 and R3 was a peptide containing Ser-13 (FIG. 46A). Treatment of R2 and R3 with ethanethiol (this treatment specifically converts fosserin to S-ethylcysteine) indicated that phosphorus was localized at Ser-34 and Ser-13, respectively (data Omitted). These results indicate that the phosphorylation site of GFAP by Rho kinase is Th
r-7, Ser-13 and Ser-34 were revealed. It has already been reported that Thr-7 is also phosphorylated in the fissure using the rabbit polyclonal antibody that recognizes phosphorylated Thr-7 (Matsuoka, Y. et al. EMBO J.
11: 2895-2902).

[0300] Therefore, the present inventors have proposed that GF by Rho kinase
The effect of phosphorylation of AP on the filament formation activity of GFAP was investigated. Soluble GFAP was preincubated for 30 minutes in the presence and absence of GST-Rho kinase, and the samples were subjected to the polymerization conditions (25 mM imidazole-HC
l, pH 6.75, 100 mM NaCl, 37 ° C) for another hour. Next, NaCl- and pH-dependent filament formation of GFAP was examined by centrifugation (FIG. 47A) and electron microscopy (FIG. 47B). As shown in FIG. 47, phosphorylation of GFAP by Rho kinase almost completely inhibited GFAP filament formation. These results
Thr-7, Ser-13 and Ser-3 of GFAP during cytokinesis
This suggests that phosphorylation of 4 may induce fragmentation of glial fibrils in the dividing furrow.

With the present invention, the inventors have obtained evidence that GFAP can serve as a very suitable substrate for Rho kinase in a GTP-Rho-dependent manner. GFAP is the third Rho kinase selective substrate found after MBS and myosin (Examples 2, 6-9, 12-19). Phosphorylated GFAP lost its ability to form filaments in vitro. In vitro GF by Rho kinase
The phosphorylation sites of AP were Thr-7, Ser-13 and Ser-34, which are located at the cleavage groove site during cytokinesis.
It was identical to the phosphorylation site by CF kinase. From the above, the present inventors speculate that Rho kinase is CF kinase itself. If so, Rho kinase may play an important role in the reorganization of fissure-specific IFs during cytokinesis.

The present inventors have also determined that Rho kinases
Vimentin, an IF, was also phosphorylated in a GTP / Rho-dependent manner and was found to inhibit filament formation in vitro (data not shown).

Example 21 Human Rho Kinase Gene
Determination of position on chromosome (1) FISH analysis To know the position of human Rho-kinase gene on chromosome,
FISH analysis was performed. 10 lymphocytes isolated from human blood
Α-fetal bovine serum, phytohemagglutinin (PHA) supplemented
The cells were cultured in a minimum essential medium (MEM) at 37 ° C for 68 to 72 hours.
The cultured lymphocytes were treated with BrdU (0.18 mg / ml Sigma) and synchronized. Synchronized lymphocytes were incubated in serum-free medium for 3 hours.
Wash twice, and add α-MEM containing thymidine (2.5 mg / ml Sigma)
The cells were cultured at 7 ° C for 6 hours. The cells were collected to prepare a slide.
Human Rho kinase cDNA clone 164-3C (SEQ ID NO: 5)
930-2025) from BRL BioNick labeling ki
biotinylated using t (Heng, HHQ et. al. (19
92) Proc. Natl. Acad. Sci. USA 89: 9509-9513). FIS
H detection method is described in Heng, HHQ et al. (1992) and Hen
g, HHQ and Tuji, L.-C. (1993) Chromosoma 102: 3
Followed 25-332.

The slide was heat-treated at 55 ° C. for 1 hour. After RNase treatment, slides were run in 2X SSC containing 70% formamide.
Denatured for 2 minutes and dehydrated with ethanol at 70 ° C. Probes are incubated in hybridization solution (50%
Formamide, 10% dextran sulfate). The probe was placed on a denatured chromosome slide and placed at 37 ° C. overnight. Slides into 50% formamide, 2
After washing three times with × SSC for 3 minutes, 2 × SSC was washed three times at 43 ° C. for 3 minutes, and a signal was detected. The FISH signal and DAPI band pattern were separately photographed and superimposed to determine their chromosomal location (Hen
g, HHQ and Tuji, L.-C. (1994) In: Choo, KHA, e
d. Methods in Molecular Biology: In situ hybridiza
tion protocols. (KHA Choo, ed) pp35-49).

As a result of FISH analysis, signals were detected on a set of chromosomes in 89 chromosomes out of 100 chromosomes of dividing cells. Based on the DAPI band, the signal was found to be on the short arm of chromosome 2. Detailed chromosomal location was determined by combining the results of the ten photographs. The human Rho kinase gene was found to be located on p24 of chromosome 2.

(2) Panel analysis using PCR In order to obtain primers specific to human Rho kinase,
Primers 5'-AAG AGCTCC AGG ATC AGC TC-3 '(base sequence of 2579-2598 of SEQ ID NO: 5) and 5'-CCA AGTTTG GTC T
Using TT TCT TCA CA-3 '(complementary strand of the nucleotide sequence of No. 2659-2681 of SEQ ID NO: 5), 100 ng of human genomic DNA (CLON
PCR was performed using TECH Human Genomic DNA) as a template. As a result, the intron of the human Rho kinase genomic gene (42 to
A 741 bp DNA fragment (SEQ ID NO: 6) containing the 679 base sequence was amplified.

[0307] The amplified DNA fragment was digested with pBluscript II SK.
^- subcloned into (Stratagene, Inc.) to determine the partial nucleotide sequence. Based on this base sequence, a primer specific for human Rho kinase (5'-ATA CAA CAG AAA TGC GTA
5′-TCA TTA AAC TAT TCA TTT GC-3 ′ (complementary strand of the base sequence from 646 to 667 of SEQ ID NO: 6) (SEQ ID NO: 6)
536-555).

To determine the location of the human Rho kinase gene on the chromosome, BIOS Monochromosomal Hydro
Analysis was performed using brid Panel PCRable DNAs. D
PCR was performed by using 25 ng of NAs as a template. PCR, Tak
Using ara LA PCR kit Ver.2, 98 ° C for 10 seconds, 55 ° C for 30 seconds,
One minute at 72 ° C. was repeated 30 times. The reaction solution was subjected to 8% polyacrylamide gel electrophoresis. As a result, template D having chromosome 2
Only from NA, about 132 bp of human Rho kinase specific D
The NA fragment was amplified.

[0309]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1388 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: protein Origin: Biological sequence: bovine Sequence Met Ser Arg Pro Pro Pro Thr Gly Lys Met Pro Gly Ala Pro Glu Ala 1 5 10 15 Val Ser Gly Asp Gly Ala Gly Ala Ser Arg Gln Arg Lys Leu Glu Ala 20 25 30 Leu Ile Arg Asp Pro Arg Ser Pro Ile Asn Val Glu Ser Leu Leu Asp 35 40 45 Gly Leu Asn Pro Leu Val Leu Asp Leu Asp Phe Pro Ala Leu Arg Lys 50 55 60 Asn Lys Asn Ile Asp Asn Phe Leu Asn Arg Tyr Glu Lys Ile Val Lys 65 70 75 80 Lys Ile Arg Gly Leu Gln Met Lys Ala Glu Asp Tyr Asp Val Val Lys 85 90 95 Val Ile Gly Arg Gly Ala Phe Gly Glu Val Gln Leu Val Arg His Lys 100 105 110 Ala Ser Gln Lys Val Tyr Ala Met Lys Leu Leu Ser Lys Phe Glu Met 115 120 125 Ile Lys Arg Ser Asp Ser Ala Phe Phe Trp Glu Glu Arg Asp Ile Met 130 135 140 Ala Phe Ala Asn Ser Pro Trp Val Val Gln Leu Phe Cys Ala Phe Gln 145 150 155 160 Asp Asp Lys Tyr Leu Tyr Met Val Met Glu Tyr Met Pro Gly Gly Asp 165 170 175 Leu Val Asn Leu Met Ser Asn Tyr Asp Val Pro Glu Lys Trp Ala Lys 180 185 190 Phe Tyr Thr Ala Glu Val Val Leu Ala Leu Asp Ala Ile His Ser Met 195 200 205 Gly Leu Ile His Arg Asp Val Lys Pro Asp Asn Met Leu Leu Asp Lys 210 215 220 His Gly His Leu Lys Leu Ala Asp Phe Gly Thr Cys Met Lys Met Asp 225 230 235 240 Glu Thr Gly Met Val His Cys Asp Thr Ala Val Gly Thr Pro Asp Tyr 245 250 255 Ile Ser Pro Glu Val Leu Lys Ser Gln Gly Gly Asp Gly Tyr Tyr Gly 260 265 270 Arg Glu Cys Asp Trp Trp Ser Val Gly Val Phe Leu Phe Glu Met Leu 275 280 285 Val Gly Asp Thr Pro Phe Tyr Ala Asp Ser Leu Val Gly Thr Tyr Ser 290 295 300 Lys Ile Met Asp His Lys Asn Ser Leu Cys Phe Pro Glu Asp Ala Glu 305 310 315 320 Ile Ser Lys His Ala Lys Asn Leu Ile Cys Ala Phe Leu Thr Asp Arg 325 330 335 Glu Val Arg Leu Gly Arg Asn Gly Val Glu Glu Ile Lys Gln His Pro 340 345 350 Phe Phe Lys Asn Asp Gln Trp Asn Trp Asp Asn Ile Arg Glu Thr Ala 355 360 365 Ala Pro Val Val Pro Glu Leu Ser Ser Asp Ile Asp Ser Ser Asn Phe 370 375 380 Asp Asp Ile Glu Asp Asp Lys Gly Asp Val Glu Thr Phe Pro Ile Pro 385 390 395 400 Lys Ala Phe Val Gly Asn Gln Leu Pro Phe Ile Gly Phe Thr Tyr Tyr 405 410 415 Arg Glu Asn Leu Leu Leu Leu Ser Asp Ser Pro Ser Cys Lys Glu Asn Asp 420 425 430 Ser Ile Gln Ser Arg Lys Asn Glu Glu Ser Gln Glu Ile Gln Lys Lys 435 440 445 445 Leu Tyr Thr Leu Glu Glu His Leu Ser Thr Glu Ile Gln Ala Lys Glu 450 455 460 Glu Leu Glu Gln Lys Cys Lys Ser Val Asn Thr Arg Leu Glu Lys Val 465 470 475 480 Ala Lys Glu Leu Glu Glu Glu Ile Thr Leu Arg Lys Asn Val Glu Ser 485 490 495 Thr Leu Arg Gln Leu Glu Arg Glu Lys Ala Leu Leu Gln His Lys Asn 500 505 510 Ala Glu Tyr Gln Arg Lys Ala Asp His Glu Ala Asp Lys Lys Arg Asn 515 520 525 Leu Glu Asn Asp Val Asn Ser Leu Lys Asp Gln Leu Glu Asp Leu Lys 530 535 540 Lys Arg Asn Gln Asn Ser Gln Ile Ser Thr Glu Lys Val Asn Gln Leu 545 550 555 560 Gln Arg Gln Leu Asp Glu Thr Asn Ala Leu Leu Arg Thr Glu Ser Asp 565 570 575 Thr Ala Ala Ala Arg Leu Arg Lys Thr Gln Ala Glu Ser Ser Lys Gln Ile 580 585 590 Gln Gln Leu Glu Ser Asn Asn Arg Asp Leu Gln Asp Lys Asn Cys Leu 595 600 605 Leu Glu Thr Ala Lys Leu Lys Leu Glu Lys Glu Phe Ile Asn Leu Gln 610 615 620 Ser Val Leu Glu Ser Glu Arg Arg Asp Arg Thr His Gly Ser Glu Ile 625 630 635 640 640 Ile Asn Asp Leu Gln Gly Arg Ile Ser Gly Leu Glu Glu Asp Val Lys 645 650 655 Asn Gly Lys Ile Leu Leu Ala Lys Val Glu Leu Glu Lys Arg Gln Leu 660 665 670 Gln Glu Arg Phe Thr Asp Leu Glu Lys Glu Lys Asn Asn Met Glu Ile 675 680 685 Asp Met Thr Tyr Gln Leu Lys Val Ile Gln Gln Ser Leu Glu Gln Glu 690 695 700 Glu Thr Glu His Lys Ala Thr Lys Ala Arg Leu Ala Asp Lys Asn Lys 705 710 715 715 720 Ile Tyr Glu Ser Ile Glu Glu Ala Lys Ser Glu Ala Met Lys Glu Met 725 730 735 Glu Lys Lys Leu Ser Glu Glu Arg Thr Leu Lys Gln Lys Val Glu Asn 740 745 750 Leu Leu Leu Glu Ala Glu Lys Arg Cys Ser Ile Leu Asp Cys Asp Leu 755 760 765 Lys Gln Ser Gln Gln Lys Ile Asn Glu Leu Leu Lys Gln Lys Asp Val 770 775 780 Leu Asn Glu Asp Val Arg Asn Leu Thr Leu Lys Ile Glu Gln Gl u Thr 785 790 795 800 Gln Lys Arg Cys Leu Thr Gln Asn Asp Leu Lys Met Gln Thr Gln Gln 805 810 815 Val Asn Thr Leu Lys Met Ser Glu Lys Gln Leu Lys Gln Glu Asn Asn 820 825 830 His Leu Leu Glu Met Lys Met Ser Leu Glu Lys Gln Asn Ala Glu Leu 835 840 845 Arg Lys Glu Arg Gln Asp Ala Asp Gly Gln Met Lys Glu Leu Gln Asp 850 855 860 Gln Leu Glu Ala Glu Gln Tyr Phe Ser Thr Leu Tyr Lys Thr Gln Val 865 870870 875 880 Arg Glu Leu Lys Glu Glu Cys Glu Glu Lys Thr Lys Leu Cys Lys Glu 885 890 895 Leu Gln Gln Lys Lys Gln Glu Leu Gln Asp Glu Arg Asp Ser Leu Ala 900 905 910 910 Ala Gln Leu Glu Ile Thr Leu Thr Lys Ala Asp Ser Glu Gln Leu Ala 915 920 925 Arg Ser Ile Ala Glu Glu Gln Tyr Ser Asp Leu Glu Lys Glu Lys Ile 930 935 940 Met Lys Glu Leu Glu Ile Lys Glu Met Met Ala Arg His Lys Gln Glu 945 950 955 960 Leu Thr Glu Lys Asp Ala Thr Ile Ala Ser Leu Glu Glu Thr Asn Arg 965 970 975 Thr Leu Thr Ser Asp Val Ala Asn Leu Ala Asn Glu Lys Glu Glu Leu 980 985 990 Asn Asn Lys Leu Lys Glu Ala Gln Glu Gln Leu Ser Arg Leu Ly s Asp 995 1000 1005 Glu Glu Ile Ser Ala Ala Ala Ile Lys Ala Gln Phe Glu Lys Gln Leu 1010 1015 1020 Leu Thr Glu Arg Thr Leu Lys Thr Gln Ala Val Asn Lys Leu Ala Glu 1025 1030 1035 1040 Ile Met Asn Arg Lys Glu Pro Val Lys Arg Gly Asn Asp Thr Asp Val 1045 1050 1055 Arg Arg Lys Glu Lys Glu Asn Arg Lys Leu His Met Glu Leu Lys Ser 1060 1065 1070 Glu Arg Glu Lys Leu Thr Gln Gln Met Ile Lys Tyr Gln Lys Glu Leu 1075 1080 1085 Asn Glu Met Gln Ala Gln Ile Ala Glu Glu Ser Gln Ile Arg Ile Glu 1090 1095 1100 Leu Gln Met Thr Leu Asp Ser Lys Asp Ser Asp Ile Glu Gln Leu Arg 1105 1110 1115 1120 Ser Gln Leu Gln Ala Leu His Ile Gly Leu Asp Ser Ser Ser Ile Gly 1125 1130 1135 Ser Gly Pro Gly Asp Thr Glu Ala Asp Asp Gly Phe Pro Glu Ser Arg 1140 1145 1150 Leu Glu Gly Trp Leu Ser Leu Pro Val Arg Asn Asn Thr Lys Lys Phe 1155 1160 1165 Gly Trp Val Lys Lys Tyr Val Ile Val Ser Ser Lys Lys Ile Leu Phe 1170 1175 1180 Tyr Asp Ser Glu Gln Asp Lys Glu Gln Ser Asn Pro Tyr Met Val Leu 1185 1190 1195 1200 Asp Ile Asp Lys Leu Phe His Val Arg Pro Val Thr Gln Thr Asp Val 1205 1210 1215 Tyr Arg Ala Asp Ala Lys Glu Ile Pro Arg Ile Phe Gln Ile Leu Tyr 1220 1225 1230 Ala Asn Glu Gly Glu Ser Lys Lys Glu Gln Glu Phe Pro Val Glu Pro 1235 1240 1245 Val Gly Glu Lys Ser Asn Tyr Ile Cys His Lys Gly His Glu Phe Ile 1250 1255 1260 Pro Thr Leu Tyr His Phe Pro Thr Asn Cys Glu Ala Cys Met Lys Pro 1265 1270 1275 1280 Leu Trp His Met Phe Lys Pro Pro Pro Ala Leu Glu Cys Arg Arg Cys 1285 1290 1295 His Ile Lys Cys His Lys Asp His Met Asp Lys Lys Glu Glu Ile Ile 1300 1305 1310 Ala Pro Cys Lys Val Tyr Tyr Asp Ile Ser Ser Ala Lys Asn Leu Leu 1315 1320 1325 Leu Leu Ala Asn Ser Thr Glu Glu Gln Gln Lys Trp Val Ser Arg Leu 1330 1335 1340 Val Lys Lys Ile Pro Lys Lys Pro Pro Ala Pro Asp Pro Phe Ala Arg 1345 1350 1355 1360 Ser Ser Pro Arg Thr Ser Met Lys Ile Gln Gln Asn Gln Ser Ile Arg 1365 1370 1375 Arg Pro Ser Arg Gln Leu Ala Pro Asn Lys Pro Ser 1380 1385

SEQ ID NO: 2 Sequence length: 5053 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA Origin: Biological name: Bovine Sequence ATG AGC CGG CCC CCG CCG ACG GGG AAG ATG CCC GGC GCC CCC GAG GCC 48 Met Ser Arg Pro Pro Pro Thr Gly Lys Met Pro Gly Ala Pro Glu Ala 1 5 10 15 GTG TCG GGG GAC GGC GCG GGC GCG AGC CGC CAG AGG AAG CTG GAA GCG 96 Val Ser Gly Asp Gly Ala Gly Ala Ser Arg Gln Arg Lys Leu Glu Ala 20 25 30 CTG ATC CGA GAC CCT CGT TCG CCC ATC AAC GTG GAG AGC TTG CTG GAT 144 Leu Ile Arg Asp Pro Arg Ser Pro Ile Asn Val Glu Ser Leu Leu Asp 35 40 45 GGC TTA AAT CCT TTG GTC CTT GAT TTG GAT TTT CCT GCT TTG AGG AAA 192 Gly Leu Asn Pro Leu Val Leu Asp Leu Asp Phe Pro Ala Leu Arg Lys 50 55 60 AAC AAA AAT ATA GAT AAT TTC TTA AAT AGA TAT GAG AAA ATT GTG AAA 240 Asn Lys Asn Ile Asp Asn Phe Leu Asn Arg Tyr Glu Lys Ile Val Lys 65 70 75 80 AAA ATT AGA GGT TTA CAG ATG AAG GCA GAA GAC TAC GAT GTT GTA AAA 288 Lys Ile Arg Gly L eu Gln Met Lys Ala Glu Asp Tyr Asp Val Val Lys 85 90 95 GTT ATC GGA AGA GGT GCT TTT GGT GAA GTC CAG TTG GTT CGT CAT AAG 336 Val Ile Gly Arg Gly Ala Phe Gly Glu Val Gln Leu Val Arg His Lys 100 105 110 GCA TCA CAG AAA GTT TAT GCT ATG AAG CTT CTT AGT AAG TTT GAA ATG 384 Ala Ser Gln Lys Val Tyr Ala Met Lys Leu Leu Ser Lys Phe Glu Met 115 120 125 ATA AAA AGA TCA GAT TCT GCT TTT TTC TGG GAG GAA AGA GAT ATT ATG 432 Ile Lys Arg Ser Asp Ser Ala Phe Phe Trp Glu Glu Arg Asp Ile Met 130 135 140 GCC TTT GCC AAC AGT CCC TGG GTG GTT CAG CTC TTT TGT GCC TTT CAA 480 Ala Phe Ala Asn Ser Pro Trp Val Val Gln Leu Phe Cys Ala Phe Gln 145 150 155 160 GAT GAT AAG TAT CTG TAC ATG GTA ATG GAG TAC ATG CCT GGT GGA GAC 528 Asp Asp Lys Tyr Leu Tyr Met Val Met Glu Tyr Met Pro Gly Gly Asp 165 170 175 CTT GTA AAC CTT ATG AGT AAC TAT GAT GTA CCT GAA AAA TGG GCC AAA 576 Leu Val Asn Leu Met Ser Asn Tyr Asp Val Pro Glu Lys Trp Ala Lys 180 185 190 TTT TAT ACT GCT GAA GTT GTT CTT GCT TTG GAT GCC ATA CAC TCC ATG 624 Phe Tyr Thr Ala Glu Val Val Leu Ala Leu Asp Ala Ile His Ser Met 195 200 205 GGT TTA ATT CAC AGA GAT GTG AAG CCT GAC AAC ATG CTC TTG GAT AAA 672 Gly Leu Ile His Arg Asp Val Lys Pro Asp Asn Met Leu Leu Asp Lys 210 215 220 CAT GGG CAT CTA AAA TTA GCA GAT TTT GGC ACA TGT ATG AAG ATG GAT 720 His Gly His Leu Lys Leu Ala Asp Phe Gly Thr Cys Met Lys Met Asp 225 230 235 240 GAA ACA GGC ATG GTG CAT TGT GAT ACA GCA GTT GGA ACA CCC GAT TAT 768 Glu Thr Gly Met Val His Cys Asp Thr Ala Val Gly Thr Pro Asp Tyr 245 250 255 ATA TCA CCC GAG GTC CTG AAA TCA CAA GGG GGT GAT GGT TAC TAT GGG 816 Ile Ser Pro Glu Val Leu Lys Ser Gln Gly Gly Asp Gly Tyr Tyr Gly 260 265 270 270 CGA GAA TGT GAT TGG TGG TCC GTG GGT GTT TTC CTT TTT GAA ATG CTG 864 Arg Glu Cys Asp Trp Trp Ser Val Gly Val Phe Leu Phe Glu Met Leu 275 280 285 GTG GGG GAT ACT CCA TTT TAT GCA GAT TCA CTT GTA GGA ACA TAT AGC 912 Val Gly Asp Thr Pro Phe Tyr Ala Asp Ser Leu Val Gly Thr Tyr Ser 290 295 300 AAA ATT ATG GAT CAT AAA AAC TCA CTA TGT TTC CCT GAA GAT GCA GAA960 Lys Ile Met Asp His Lys Asn Ser Leu Cys Phe Pro Glu Asp Ala Glu 305 310 315 320 ATT TCT AAA CAT GCG AAG AAT CTC ATC TGT GCC TTC TTA ACA GAT AGG 1008 Ile Ser Lys His Ala Lys Asn Leu Ile Cys Ala Phe Leu Thr Asp Arg 325 330 335 GAG GTA CGC CTT GGA AGA AAC GGG GTA GAA GAA ATC AAA CAA CAT CCT 1056 340 345 350 TTC TTT AAG AAT GAT CAG TGG AAT TGG GAT AAC ATA AGA GAG ACT GCA 1104 Phe Phe Lys Asn Asp Gln Trp Asn Trp Asp Asn Ile Arg Glu Thr Ala 355 360 365 GCT CCT GTG GTA CCT GAA CTC AGC AGT GAC ATA GAC AGC AGC AAT TTT 1152 Ala Pro Val Val Pro Glu Leu Ser Ser Asp Ile Asp Ser Ser Asn Phe 370 375 380 GAT GAC ATT GAA GAT GAT AAA GGA GAT GTA GAA ACC TTC CCA ATT CCC 1200 Asp Asp Ile Glu Asp Asp Lys Gly Asp Val Glu Thr Phe Pro Ile Pro 385 390 395 400 400 AAG GCT TTT GTG GGA AAT CAG CTA CCT TTT ATA GGA TTT ACC TAC TAC 1248 Lys Ala Phe Val Gly Asn Gln Leu Pro Phe Ile Gly Phe Thr Tyr Tyr 405 410 415 AGA GAA AAT TTG CTA CTA AGT GAC TCT CCA TCT TGT AAA GAA AAT GAC 1296 Arg Glu Asn Leu Leu Leu Ser Asp Ser P ro Ser Cys Lys Glu Asn Asp 420 425 430 TCA ATT CAA TCA AGG AAG AAT GAA GAG AGT CAA GAG ATT CAG AAA AAA 1344 Ser Ile Gln Ser Arg Lys Asn Glu Glu Ser Gln Glu Ile Gln Lys Lys 435 440 445 445 CTG TAC ACA TTA GAA GAA CAC CTT AGC ACT GAG ATT CAG GCC AAA GAG 1392 Leu Tyr Thr Leu Glu Glu His Leu Ser Thr Glu Ile Gln Ala Lys Glu 450 455 460 GAA CTA GAA CAG AAG TGC AAG TCT GTT AAT ACT CGC TTA GAG AAA GTG 1440 Glu Leu Glu Gln Lys Cys Lys Ser Val Asn Thr Arg Leu Glu Lys Val 465 470 475 480 GCA AAG GAG TTA GAA GAA GAG ATT ACC TTA AGG AAA AAT GTG GAA TCA 1488 Ala Lys Glu Leu Glu Glu Glu Ile Thr Leu Arg Lys Asn Val Glu Ser 485 490 495 ACA TTA AGA CAA TTA GAA AGA GAA AAA GCA CTT CTT CAG CAC AAA AAT 1536 Thr Leu Arg Gln Leu Glu Arg Glu Lys Ala Leu Leu Gln His Lys Asn 500 505 510 GCA GAA TAT CAG CGG AAA GCT GAT CAT GAA GCA GAC AAG AAG CGA AAT 1584 Ala Glu Tyr Gln Arg Lys Ala Asp His Glu Ala Asp Lys Lys Arg Asn 515 520 525 TTG GAG AAT GAT GTT AAC AGT TTA AAA GAT CAG CTT GAA GAT TTG AAA 1632 Leu Glu Asn Asp Va l Asn Ser Leu Lys Asp Gln Leu Glu Asp Leu Lys 530 535 540 AAA AGA AAT CAG AAC TCT CAG ATA TCC ACT GAG AAA GTG AAT CAA CTC 1680 Lys Arg Asn Gln Asn Ser Gln Ile Ser Thr Glu Lys Val Asn Gln Leu 545 550 555 560 CAG AGA CAA CTG GAT GAA ACC AAT GCT TTG CTG CGA ACA GAA TCT GAT 1728 Gln Arg Gln Leu Asp Glu Thr Asn Ala Leu Leu Arg Thr Glu Ser Asp 565 570 575 ACT GCA GCC CGG TTA AGG AAA ACA CAG GCA GAA AGT TCA AAA CAG ATT 1776 Thr Ala Ala Arg Leu Arg Lys Thr Gln Ala Glu Ser Ser Lys Gln Ile 580 585 590 CAG CAG CTG GAA TCT AAC AAT AGA GAT CTA CAA GAC AAA AAT TGC CTG 1824 Gln Gln Leu Glu Ser Asn Asn Arg Asp Leu Gln Asp Lys Asn Cys Leu 595 600 605 CTG GAG ACT GCC AAG TTA AAA CTT GAA AAG GAA TTT ATC AAT CTT CAG 1872 Leu Glu Thr Ala Lys Leu Lys Leu Glu Lys Glu Phe Ile Asn Leu Gln 610 615 620 620 TCA GTT CTA GA TCT GAA AGG AGG GAC CGA ACC CAT GGA TCA GAG ATT 1920 Ser Val Leu Glu Ser Glu Arg Arg Asp Arg Thr His Gly Ser Glu Ile 625 630 635 640 ATT AAT GAT TTA CAA GGT AGA ATA TCT GGC CTA GAA GAA GAT GTA AAG 1968 Ile Asn Asp Leu Gln Gly Arg Ile Ser Gly Leu Glu Glu Asp Val Lys 645 650 655 AAT GGT AAA ATC TTA TTA GCA AAA GTA GAG CTG GAG AAG AGA CAA CTA 2016 Asn Gly Lys Ile Leu Leu Ala Lys Val Glu Leu Glu Lys Arg Gln Leu 660 665 670 CAG GAG AGA TTT ACT GAT TTG GAA AAG GAA AAG AAC AAC ATG GAA ATA 2064 Gln Glu Arg Phe Thr Asp Leu Glu Lys Glu Lys Asn Asn Met Glu Ile 675 680 685 GAT ATG ACA TAC CAA CTA AAA ATA CAG CAA AGT TTA GAA CAA GAA 2112 Asp Met Thr Tyr Gln Leu Lys Val Ile Gln Gln Ser Leu Glu Gln Glu 690 695 700 GAA ACT GAA CAT AAG GCT ACA AAA GCA CGG CTT GCA GAT AAA AAC AAG 2160 Glu Thr Glu His Lys Ala Thr Lys Ala Arg Leu Ala Asp Lys Asn Lys 705 710 715 720 ATT TAT GAA TCC ATT GAA GAA GCT AAA TCA GAA GCC ATG AAA GAA ATG 2208 Ile Tyr Glu Ser Ile Glu Glu Ala Lys Ser Glu Ala Met Lys Glu Met 725 730 735 GAG AAA AAG CTC TCG GAG GAA AGA ACT TTA AAA CAG AAA GTA GAG AAC 2256 Glu Lys Lys Leu Ser Glu Glu Arg Thr Leu Lys Gln Lys Val Glu Asn 740 745 750 TTG TTG CTG GAG GCT GAG AAA AGA TGC TCT ATA T TA GAC TGT GAC CTC 2304 Leu Leu Leu Glu Ala Glu Lys Arg Cys Ser Ile Leu Asp Cys Asp Leu 755 760 765 AAA CAG TCA CAG CAG AAA ATA AAT GAA CTC CTC AAA CAG AAA GAT GTG 2352 Lys Gln Ser Gln Gln Lys Ile Asn Glu Leu Leu Lys Gln Lys Asp Val 770 775 780 780 CTA AAT GAG GAT GTT AGA AAC TTG ACA TTA AAA ATA GAA CAG GAA ACT 2400 Leu Asn Glu Asp Val Arg Asn Leu Thr Leu Lys Ile Glu Gln Glu Thr 785 790 795 800 CAG AAG CGC TGC CTT ACA CAA AAT GAC TTG AAG ATG CAA ACA CAG CAA 2448 805 810 815 815 GTT AAC ACA CTA AAA ATG TCA GAA AAG CAG TTA AAG CAA GAG AAT AAT 2496 Val Asn Thr Leu Lys Met Ser Glu Lys Gln Leu Lys Gln Glu Asn Asn 820 825 830 CAT CTC CTA GAA ATG AAA ATG AGC TTG GAA AAA CAG AAT GCT GAA CTT 2544 His Leu Leu Glu Met Lys Met Ser Leu Glu Lys Gln Asn Ala Glu Leu 835 840 840 845 CGA AAA GAA CGT CAA GAT GCA GAT GGA CAG ATG AAA GAG CTC CAG GAT 2592 Arg Lys Glu Arg Gln Asp Ala Asp Gly Gln Met Lys Glu Leu Gln Asp 850 855 860 CAG CTT GAA GCA GAG CAG TAT TTC TCA ACC CTC TAT AAA ACA CAG GTT 2640 Gln Leu Glu Ala Glu G ln Tyr Phe Ser Thr Leu Tyr Lys Thr Gln Val 865 870 875 880 880 AGG GAA CTT AAG GAA GAA TGT GAA GAA AAG ACC AAA CTT TGT AAA GAA 2688 Arg Glu Leu Lys Glu Glu Cys Glu Glu Lys Thr Lys Leu Cys Lys Glu 885 890 895 TTA CAG CAG AAG AAG CAG GAA TTA CAG GAT GAA AGG GAC TCC TTG GCT 2736 Leu Gln Gln Lys Lys Gln Glu Leu Gln Asp Glu Arg Asp Ser Leu Ala 900 905 910 GCT CAA CTG GAG ATT ACC TTA ACC AAA GCA GAT TCT GAG CAA CTG GCT 2784 Ala Gln Leu Glu Ile Thr Leu Thr Lys Ala Asp Ser Glu Gln Leu Ala 915 920 925 CGT TCA ATT GCT GAG GAA CAG TAT TCT GAT TTG GAA AAA GAG AAG ATC 2832 Arg Ser Ile Ala Glu Glu Gln Tyr Ser Asp Leu Glu Lys Glu Lys Ile 930 935 940 ATG AAA GAG CTG GAG ATC AAA GAG ATG ATG GCT CGA CAC AAA CAG GAA 2880 Met Lys Glu Leu Glu Ile Lys Glu Met Met Ala Arg His Lys Gln Glu 945 950 955 960 CTC ACC GAA AAA GAT GCT ACT ATT GCG TCT CTT GAA GAA ACT AAT AGG 2928 Leu Thr Glu Lys Asp Ala Thr Ile Ala Ser Leu Glu Glu Thr Asn Arg 965 970 975 ACA CTA ACT AGT GAT GTT GCC AAT CTT GCA AAT GAG AAA GAA GAA TTA 297 6 Thr Leu Thr Ser Asp Val Ala Asn Leu Ala Asn Glu Lys Glu Glu Leu 980 985 990 AAT AAC AAA CTG AAG GAA GCC CAA GAG CAA CTA TCA AGG TTG AAA GAT 3024 Asn Asn Lys Leu Lys Glu Ala Gln Glu Gln Leu Ser Arg Leu Lys Asp 995 1000 1005 GAA GAA ATA AGT GCA GCA GCT ATT AAA GCA CAA TTT GAG AAG CAG CTG 3072 Glu Glu Ile Ser Ala Ala Ala Ile Lys Ala Gln Phe Glu Lys Gln Leu 1010 1015 1020 TTA ACA GAG AGG ACA CTC AAA ACT CAA GCT GTG AAT AAG TTG GCT GAG 3120 Leu Thr Glu Arg Thr Leu Lys Thr Gln Ala Val Asn Lys Leu Ala Glu 1025 1030 1035 1040 ATC ATG AAT CGA AAG GAA CCT GTT AAG CGT GGT AAT GAC ACA GAT GTG 3168 Ile Met Asn Arg Lys Glu Pro Val Lys Arg Gly Asn Asp Thr Asp Val 1045 1050 1055 CGG AGA AAA GAA AAG GAG AAT AGA AAG CTA CAT ATG GAA CTT AAA TCT 3216 Arg Arg Lys Glu Lys Glu Asn Arg Lys Leu His Met Glu Leu Lys Ser 1060 1065 1070 GAA CGC GAA AAA CTG ACC CAG CAG ATG ATC AAG TAT CAG AAA GAA CTG 3264 Glu Arg Glu Lys Leu Thr Gln Gln Met Ile Lys Tyr Gln Lys Glu Leu 1075 1080 1085 AAT GAA ATG CAG GCT CAA ATA GC T GAA GAG AGT CAG ATT CGA ATT GAA 3312 Asn Glu Met Gln Ala Gln Ile Ala Glu Glu Ser Gln Ile Arg Ile Glu 1090 1095 1100 CTA CAG ATG ACA CTG GAC AGT AAG GAC AGT GAC ATT GAG CAG CTG CGC 3360 Leu Gln Met Thr Leu Asp Ser Lys Asp Ser Asp Ile Glu Gln Leu Arg 1105 1110 1115 1120 TCC CAG CTC CAG GCC TTG CAC ATT GGT TTG GAT AGT TCC AGT ATA GGC 3408 Ser Gln Leu Gln Ala Leu His Ile Gly Leu Asp Ser Ser Ser Ile Gly 1125 1130 1135 AGT GGA CCA GGG GAT ACT GAA GCT GAT GAC GGT TTT CCA GAA TCA AGA 3456 Ser Gly Pro Gly Asp Thr Glu Ala Asp Asp Gly Phe Pro Glu Ser Arg 1140 1145 1150 CTA GAA GGA TGG CTT TCA TTG CCT GTG CGA AAC AAC ACT AAG AAA TTT 3504 Leu Glu Gly Trp Leu Ser Leu Pro Val Arg Asn Asn Thr Lys Lys Phe 1155 1160 1165 GGA TGG GTT AAA AAG TAT GTG ATT GTA AGC AGT AAG AAG ATC CTT TTC 3552 Gly Trp Val Lys Lys Tyr Val Ile Val Ser Ser Lys Lys Ile Leu Phe 1170 1175 1180 TAT GAC AGT GAG CAA GAT AAA GAA CAA TCT AAT CCT TAC ATG GTT TTA 3600 Tyr Asp Ser Glu Gln Asp Lys Glu Gln Ser Asn Pro Tyr Met Val Leu 118 5 1190 1195 1200 GAT ATA GAC AAG TTA TTT CAT GTC CGA CCA GTT ACA CAG ACA GAT GTA 3648 Asp Ile Asp Lys Leu Phe His Val Arg Pro Val Thr Gln Thr Asp Val 1205 1210 1215 TAT AGA GCA GAT GCT AAA GAA ATT CCA AGG ATA TTC CAG ATT CTG TAT 3696 Tyr Arg Ala Asp Ala Lys Glu Ile Pro Arg Ile Phe Gln Ile Leu Tyr 1220 1225 1230 GCC AAC GAA GGA GAA AGT AAG AAG GAA CAA GAA TTT CCA GTG GAG CCA 3744 Ala Asn Glu Gly Glu Ser Lys Lys Glu Gln Glu Phe Pro Val Glu Pro 1235 1240 1245 GTG GGA GAA AAA TCA AAT TAT ATT TGC CAC AAG GGA CAT GAA TTT ATT 3792 Val Gly Glu Lys Ser Asn Tyr Ile Cys His Lys Gly His Glu Phe Ile 1250 1255 1260 CCT ACT CTG TAT CAT TTC CCA ACC AAC TGT GAG GCA TGT ATG AAG CCA 3840 Pro Thr Leu Tyr His Phe Pro Thr Asn Cys Glu Ala Cys Met Lys Pro 1265 1270 1275 1280 TTG TGG CAC ATG TTT AAA CCC CCT CCT GCT TTA GAG TGC CGT CGC TGT 3888 Leu Trp His Met Phe Lys Pro Pro Pro Ala Leu Glu Cys Arg Arg Cys 1285 1290 1295 CAT ATT AAA TGT CAT AAA GAT CAC ATG GAC AAA AAG GAG GAA ATT ATA 3936 His Ile Lys Cys His Lys Asp His Met Asp Lys Lys Glu Glu Ile Ile 1300 1305 1310 GCG CCT TGC AAA GTG TAT TAT GAT ATT TCA TCG GCA AAG AAT CTA TTG 3984 Ala Pro Cys Lys Val Tyr Tyr Asp Ile Ser Ser Ala Lys Asn Leu Leu 1315 1320 1325 TTA TTG GCA AAT TCT ACA GAA GAG CAG CAA AAG TGG GTT AGT CGG TTA 4032 Leu Leu Ala Asn Ser Thr Glu Glu Gln Gln Lys Trp Val Ser Arg Leu 1330 1335 1340 GTG AAA AAA ATA CCT AAA AAG CCT CCA GCT CCA GAC CCT TTT GCA CGG 4080 Val Lys Lys Ile Pro Lys Lys Pro Pro Ala Pro Asp Pro Phe Ala Arg 1345 1350 1355 1360 TCA TCT CCT AGA ACG TCA ATG AAA ATA CAA CAA AAC CAG TCT ATT CGA 4128 Ser Ser Pro Arg Thr Ser Met Lys Ile Gln Gln Asn Gln Ser Ile Arg 1365 1370 1375 CGG CCA AGT CGA CAA CTT GCT CCA AAC AAA CCA AGC 4164 Arg Pro Ser Arg Gln Leu Ala Pro Asn Lys Pro Ser 1380 1385 TAACTGCCTT CTGTGAATGC AGTCATTATT TAAGGTGAAT ATATTCATTCT AGTTGAAAATATGAAGATCATATTAGAAATGAAGA CACATATATA TTCCCTCTAT TCCTGCAATA TAAATTCTAA ATCTTGAATA GGTTTTCTGG 4344 GCTCCTTTGG AGCAACAAG T TGAACCAACA GTGATTGGTT AATAGAATAA GAATATCATG 4404 TGCAACTCTT CCAGACTTAT TCCATAAAGC TCTCCTAGCA TCACTCACAC TACATTGCAT 4464 AAAGGATTTA GAAGAGTTAC AGAAATCATC TTTTCAGCTT CAACAGAGAG ATTTCACCAG 4524 CACATTTGCC AGAAGAATCT GGGAATGGAT TCCACTACAG TGATAGAGAC TGCGTCTTTA 4584 AGAAGTGACC ATTGTAGTGT GTGTGTGAAC ACACACACAC ACATACACAC ACACACACAC 4644 ACACACATAG TACTGTAATA CTGCAAGAGG GTTTTTTAAC TTCCCACTTT ATTTTTTTAT 4704 AAACATTAAT CAGATATCAT TACTTACTGC AGTTGTAACT ATGCACTTGT ATAAAGCCAT 4764 AATGTTGGAG TTTATATCAC TCATTGTGTG TACCTGCTGG AAGCTGCATG TTCATGTTTA 4824 AGCAGTTATT GTAACAAGAA GTTTGAAGTT AATTATATCA GTTTCTTAAT GCTTTGTAAT 4884 AGGCAATTTT ACCCATTTTG AATGCCTTAA TTTAATTTTT TTCAAGGTAT CCACCCTTTC 4944 CTGTATTTAA AACAAAAAAA AAAGTATTTG CCAGCTCTTA GGATGCAAAT TTGCTTTGCA 5004 GAAGAAAATT AGTGCACTAT TTTTACACAT AGTAGTTATC ATTGTCGGC 5053

SEQ ID NO: 3 Sequence length: 14 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Origin: Biological name: bovine sequence Cys Lys Arg Gln Leu Gln Glu Arg Phe Thr Asp Leu Glu Lys 1 5 10

SEQ ID NO: 4 Sequence length: 1388 Sequence type: amino acid Number of chains: single-stranded Topology: linear Sequence type: protein Origin: Biological name: Human sequence Met Ser Arg Pro Pro Pro Thr Gly Lys Met Pro Gly Ala Pro Glu Thr 1 5 10 15 Ala Pro Gly Asp Gly Ala Gly Ala Ser Arg Gln Arg Lys Leu Glu Ala 20 25 30 Leu Ile Arg Asp Pro Arg Ser Pro Ile Asn Val Glu Ser Leu Leu Asp 35 40 45 Gly Leu Asn Ser Leu Val Leu Asp Leu Asp Phe Pro Ala Leu Arg Lys 50 55 60 Asn Lys Asn Ile Asp Asn Phe Leu Asn Arg Tyr Glu Lys Ile Val Lys 65 70 75 80 Lys Ile Lys Gly Leu Gln Met Lys Ala Glu Asp Tyr Asp Val Val Lys 85 90 95 Val Ile Gly Arg Gly Ala Phe Gly Glu Val Gln Leu Val Arg His Lys 100 105 110 Ala Ser Gln Lys Val Tyr Ala Met Lys Leu Leu Ser Lys Phe Glu Met 115 120 125 Ile Lys Arg Ser Asp Ser Ala Phe Phe Trp Glu Glu Arg Asp Ile Met 130 135 140 Ala Phe Ala Asn Ser Pro Trp Val Val Gln Leu Phe Tyr Ala Phe Gln 145 150 155 160 Asp Asp Arg Tyr Leu Tyr Met Val M et Glu Tyr Met Pro Gly Gly Asp 165 170 175 Leu Val Asn Leu Met Ser Asn Tyr Asp Val Pro Glu Lys Trp Ala Lys 180 185 190 Phe Tyr Thr Ala Glu Val Val Leu Ala Leu Asp Ala Ile His Ser Met 195 200 205 Gly Leu Ile His Arg Asp Val Lys Pro Asp Asn Met Leu Leu Asp Lys 210 215 220 His Gly His Leu Lys Leu Ala Asp Phe Gly Thr Cys Met Lys Met Asp 225 230 235 240 Glu Thr Gly Met Val His Cys Asp Thr Ala Val Gly Thr Pro Asp Tyr 245 250 255 Ile Ser Pro Glu Val Leu Lys Ser Gln Gly Gly Asp Gly Phe Tyr Gly 260 265 270 Arg Glu Cys Asp Trp Trp Ser V Val Gly Val Phe Leu Tyr Glu Met Leu 275 280 285 Val Gly Asp Thr Pro Phe Tyr Ala Asp Ser Leu Val Gly Thr Tyr Ser 290 295 300 Lys Ile Met Asp His Lys Asn Ser Leu Cys Phe Pro Glu Asp Ala Glu 305 310 315 320 Ile Ser Lys His Ala Lys Asn Leu Ile Cys Ala Phe Leu Thr Asp Arg 325 330 335 Glu Val Arg Leu Gly Arg Asn Gly Val Glu Glu Ile Arg Gln His Pro 340 345 350 Phe Phe Lys Asn Asp Gln Trp His Trp Asp Asn Ile Arg Glu Thr Ala 355 360 365 Ala Pro Val Val Pro Glu Leu Ser Ser A sp Ile Asp Ser Ser Asn Phe 370 375 380 Asp Asp Ile Glu Asp Asp Lys Gly Asp Val Glu Thr Phe Pro Ile Pro 385 390 395 400 Lys Ala Phe Val Gly Asn Gln Leu Pro Phe Ile Gly Phe Thr Tyr Tyr 405 410 415 Arg Glu Asn Leu Leu Leu Ser Asp Ser Pro Ser Cys Arg Glu Asn Asp 420 425 430 Ser Ile Gln Ser Arg Lys Asn Glu Glu Ser Gln Glu Ile Gln Lys Lys 435 440 445 Leu Tyr Thr Leu Glu Glu His Leu Ser Asn Glu Met Gln Ala Lys Glu 450 455 460 Glu Leu Glu Gln Lys Cys Lys Ser Val Asn Thr Arg Leu Glu Lys Thr 465 470 475 480 Ala Lys Glu Leu Glu Glu Glu Ile Thr Leu Arg Lys Ser Val Glu Ser 485 490 495 Ala Leu Arg Gln Leu Glu Arg Glu Lys Ala Leu Leu Gln His Lys Asn 500 505 510 Ala Glu Tyr Gln Arg Lys Ala Asp His Glu Ala Asp Lys Lys Arg Asn 515 520 525 Leu Glu Asn Asp Val Asn Ser Leu Lys Asp Gln Leu Glu Asp Leu Lys 530 535 540 Lys Arg Asn Gln Asn Ser Gln Ile Ser Thr Glu Lys Val Asn Gln Leu 545 550 555 560 Gln Arg Gln Leu Asp Glu Thr Asn Ala Leu Leu Arg Thr Glu Ser Asp 565 570 575 Thr Ala Ala Arg Leu Arg Lys Thr GlnAla Glu Ser Ser Lys Gln Ile 580 585 590 Gln Gln Leu Glu Ser Asn Asn Arg Asp Leu Gln Asp Lys Asn Cys Leu 595 600 605 Leu Glu Thr Ala Lys Leu Lys Leu Glu Lys Glu Phe Ile Asn Leu Gln 610 615 620 620 Ser Ala Leu Glu Ser Glu Arg Arg Asp Arg Thr His Gly Ser Glu Ile 625 630 635 640 Ile Asn Asp Leu Gln Gly Arg Ile Cys Gly Leu Glu Glu Asp Leu Lys 645 650 655 Asn Gly Lys Ile Leu Leu Ala Lys Val Glu Leu Glu Lys Arg Gln Leu 660 665 670 Gln Glu Arg Phe Thr Asp Leu Glu Lys Glu Lys Ser Asn Met Glu Ile 675 680 685 Asp Met Thr Tyr Gln Leu Lys Val Ile Gln Gln Ser Leu Glu Gln Glu 690 695 700 Glu Ala Glu His Lys Ala Thr Lys Ala Arg Leu Ala Asp Lys Asn Lys 705 710 715 720 Ile Tyr Glu Ser Ile Glu Glu Ala Lys Ser Glu Ala Met Lys Glu Met 725 730 735 Glu Lys Lys Leu Leu Glu Glu Arg Thr Leu Lys Gln Lys Val Glu Asn 740 745 750 Leu Leu Leu Glu Ala Glu Lys Arg Cys Ser Leu Leu Asp Cys Asp Leu 755 760 765 Lys Gln Ser Gln Gln Lys Ile Asn Glu Leu Leu Lys Gln Lys Asp Val 770 775 775 780 Leu Asn Glu Asp Val Arg Asn Leu Thr LeuLys Ile Glu Gln Glu Thr 785 790 795 800 Gln Lys Arg Cys Leu Thr Gln Asn Asp Leu Lys Met Gln Thr Gln Gln 805 810 815 Val Asn Thr Leu Lys Met Ser Glu Lys Gln Leu Lys Gln Glu Asn Asn 820 825 830 His Leu Met Glu Met Lys Met Asn Leu Glu Lys Gln Asn Ala Glu Leu 835 840 845 Arg Lys Glu Arg Gln Asp Ala Asp Gly Gln Met Lys Glu Leu Gln Asp 850 855 860 Gln Leu Glu Ala Glu Gln Tyr Phe Ser Thr Leu Tyr Lys Thr Gln Val 865 870 875 880 Arg Glu Leu Lys Glu Glu Cys Glu Glu Lys Thr Lys Leu Gly Lys Glu 885 890 895 Leu Gln Gln Lys Lys Gln Glu Glu Leu Gln Asp Glu Arg Asp Ser Leu Ala 900 905 910 Ala Gln Leu Glu Ile Thr Leu Thr Lys Ala Asp Ser Glu Gln Leu Ala 915 920 925 Arg Ser Ile Ala Glu Glu Gln Tyr Ser Asp Leu Glu Lys Glu Lys Ile 930 935 940 Met Lys Glu Leu Glu Ile Lys Glu Met Met Ala Arg His Lys Gln Glu 945 950 955 960 Leu Thr Glu Lys Asp Ala Thr Ile Ala Ser Leu Glu Glu Thr Asn Arg 965 970 975 Thr Leu Thr Ser Asp Val Ala Asn Leu Ala Asn Glu Lys Glu Glu Leu 980 985 990 Asn Asn Lys Leu Lys Asp Val Gln Glu Gln Leu Ser Arg Leu Lys Asp 995 1000 1005 Glu Glu Ile Ser Ala Ala Ala Ile Lys Ala Gln Phe Glu Lys Gln Leu 1010 1015 1020 Leu Thr Glu Arg Thr Leu Lys Thr Gln Ala Val Asn Lys Leu Ala Glu 1025 1030 1035 1040 Ile Met Asn Arg Lys Glu Pro Val Lys Arg Gly Asn Asp Thr Asp Val 1045 1050 1055 Arg Arg Lys Glu Lys Glu Asn Arg Lys Leu His Met Glu Leu Lys Ser 1060 1065 1070 Glu Arg Glu Lys Leu Thr Gln Gln Met Ile Lys Tyr Gln Lys Glu Leu 1075 1080 1085 Asn Glu Met Gln Ala Gln Ile Ala Glu Glu Ser Gln Ile Arg Ile Glu 1090 1095 1100 Leu Gln Met Thr Leu Asp Ser Lys Asp Ser Asp Ile Glu Gln Leu Arg 1105 1110 1115 1120 Ser Gln Leu Gln Ala Leu His Ile Gly Leu Asp Ser Ser Ser Ile Gly 1125 1130 1135 Ser Gly Pro Gly Asp Ala Glu Ala Asp Asp Gly Phe Pro Glu Ser Arg 1140 1145 1150 Leu Glu Gly Trp Leu Ser Leu Pro Val Arg Asn Asn Thr Lys Lys Phe 1155 1160 1165 Gly Trp Val Lys Lys Tyr Val Ile Val Ser Ser Lys Lys Ile Leu Phe 1170 1175 1180 Tyr Asp Ser Glu Gln Asp Lys Glu Gln Ser Asn Pro Tyr Met Val Leu 1185 1190 1195 120 0 Asp Ile Asp Lys Leu Phe His Val Arg Pro Val Thr Gln Thr Asp Val 1205 1210 1215 Tyr Arg Ala Asp Ala Lys Glu Ile Pro Arg Ile Phe Gln Ile Leu Tyr 1220 1225 1230 Ala Asn Glu Gly Glu Ser Lys Lys Glu Gln Glu Phe Pro Val Glu Pro 1235 1240 1245 Val Gly Glu Lys Ser Asn Tyr Ile Cys His Lys Gly His Glu Phe Ile 1250 1255 1260 Pro Thr Leu Tyr His Phe Pro Thr Asn Cys Glu Ala Cys Met Lys Pro 1265 1270 1275 1280 Leu Trp His Met Phe Lys Pro Pro Ala Leu Glu Cys Arg Arg Cys 1285 1290 1295 His Ile Lys Cys His Lys Asp His Met Asp Lys Lys Glu Glu Ile Ile 1300 1305 1310 Ala Pro Cys Lys Val Tyr Tyr Asp Ile Ser Thr Ala Lys Asn Leu Leu 1315 1320 1325 Leu Leu Ala Asn Ser Thr Glu Glu Gln Gln Lys Trp Val Ser Arg Leu 1330 1335 1340 Val Lys Lys Ile Pro Lys Lys Pro Pro Ala Pro Asp Pro Phe Ala Arg 1345 1350 1355 1360 Ser Ser Pro Arg Thr Ser Met Lys Ile Gln Gln Asn Gln Ser Ile Arg 1365 1370 1375 Arg Pro Ser Arg Gln Leu Ala Pro Asn Lys Pro Ser 1380 1385

SEQ ID NO: 5 Sequence length: 4363 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA Origin: Biological name: Human sequence ATG AGC CGG CCC CCG CCG ACG GGG AAA ATG CCC GGC GCC CCC GAG ACC 48 Met Ser Arg Pro Pro Pro Thr Gly Lys Met Pro Gly Ala Pro Glu Thr 1 5 10 15 GCG CCG GGG GAC GGG GCA GGC GCG AGC CGC CAG AGG AAG CTG GAG GCG 96 Ala Pro Gly Asp Gly Ala Gly Ala Ser Arg Gln Arg Lys Leu Glu Ala 20 25 30 CTG ATC CGA GAC CCT CGC TCC CCC ATC AAC GTG GAG AGC TTG CTG GAT 144 Leu Ile Arg Asp Pro Arg Ser Pro Ile Asn Val Glu Ser Leu Leu Asp 35 40 45 GGC TTA AAT TCC TTG GTC CTT GAT TTA GAT TTT CCT GCT TTG AGG AAA 192 Gly Leu Asn Ser Leu Val Leu Asp Leu Asp Phe Pro Ala Leu Arg Lys 50 55 60 AAC AAG AAC ATA GAT AAT TTC TTA AAT AGA TAT GAG AAA ATT GTG AAA 240 Asn Lys Asn Ile Asp Asn Phe Leu Asn Arg Tyr Glu Lys Ile Val Lys 65 70 75 80 AAA ATC AAA GGT CTA CAG ATG AAG GCA GAA GAC TAT GAT GTT GTA AAA 288 Lys Ile Lys Gly Le u Gln Met Lys Ala Glu Asp Tyr Asp Val Val Lys 85 90 95 GTT ATT GGA AGA GGT GCT TTT GGT GAA GTG CAG TTG GTT CGT CAC AAG 336 Val Ile Gly Arg Gly Ala Phe Gly Glu Val Gln Leu Val Arg His Lys 100 105 110 GCA TCG CAG AAG GTT TAT GCT ATG AAG CTT CTT AGT AAG TTT GAA ATG 384 Ala Ser Gln Lys Val Tyr Ala Met Lys Leu Leu Ser Lys Phe Glu Met 115 120 125 ATA AAA AGA TCA GAT TCT GCC TTT TTT TGG GAA GAA AGA GAT ATT ATG 432 Ile Lys Arg Ser Asp Ser Ala Phe Phe Trp Glu Glu Arg Asp Ile Met 130 135 140 GCC TTT GCC AAT AGC CCC TGG GTG GTT CAG CTT TTT TAT GCC TTT CAA 480 Ala Phe Ala Asn Ser Pro Trp Val Val Gln Leu Phe Tyr Ala Phe Gln 145 150 155 160 GAT GAT AGG TAT CTG TAC ATG GTA ATG GAG TAC ATG CCT GGT GGA GAC 528 Asp Asp Asp Arg Tyr Leu Tyr Met Val Met Glu Tyr Met Pro Gly Gly Asp 165 170 175 CTT GTA AAC CTT ATG AGT AAT TAT GAT GTG CCT GAA AAA TGG GCC AAA 576 Leu Val Asn Leu Met Ser Asn Tyr Asp Val Pro Glu Lys Trp Ala Lys 180 185 190 TTT TAC ACT GCT GAA GTT GTT CTT GCT CTG GAT GCA ATA CAC TCC ATG 624 Phe Tyr T hr Ala Glu Val Val Leu Ala Leu Asp Ala Ile His Ser Met 195 200 205 GGT TTA ATA CAC AGA GAT GTG AAG CCT GAC AAC ATG CTC TTG GAT AAA 672 Gly Leu Ile His Arg Asp Val Lys Pro Asp Asn Met Leu Leu Asp Lys 210 215 220 CAT GGA CAT CTA AAA TTA GCA GAT TTT GGC ACG TGT ATG AAG ATG GAT 720 His Gly His Leu Lys Leu Ala Asp Phe Gly Thr Cys Met Lys Met Asp 225 230 235 240 GAA ACA GGC ATG GTA CAT TGT GAT ACA GCA GTT GGA ACA CCG GAT TAT 768 Glu Thr Gly Met Val His Cys Asp Thr Ala Val Gly Thr Pro Asp Tyr 245 250 255 ATA TCA CCT GAG GTT CTG AAA TCA CAA GGG GGT GAT GGT TTC TAT GGG 816 Ile Ser Pro Glu Val Leu Lys Ser Gln Gly Gly Asp Gly Phe Tyr Gly 260 265 270 270 CGA GAA TGT GAT TGG TGG TCT GTA GGT GTT TTC CTT TAT GAG ATG CTA 864 Arg Glu Cys Asp Trp Trp Ser Val Gly Val Phe Leu Tyr Glu Met Leu 275 280 285 285 GTG GGG GAT ACT CCA TTT TAT GCG GAT TCA CTT GTA GGA ACA TAT AGC 912 Val Gly Asp Thr Pro Phe Tyr Ala Asp Ser Leu Val Gly Thr Tyr Ser 290 295 300 AAA ATT ATG GAT CAT AAG AAT TCA CTG TGT TTC CCT GAA GAT GCA GAA960 Lys Ile Met Asp His Lys Asn Ser Leu Cys Phe Pro Glu Asp Ala Glu 305 310 315 320 ATT TCC AAA CAT GCA AAG AAT CTC ATC TGT GCT TTC TTA ACA GAT AGG 1008 Ile Ser Lys His Ala Lys Asn Leu Ile Cys Ala Phe Leu Thr Asp Arg 325 330 335 GAG GTA CGA CTT GGG AGA AAT GGG GTG GAA GAA ATC AGA CAG CAT CCT 1056 Glu Val Arg Leu Gly Arg Asn Gly Val Glu Glu Ile Arg Gln His Pro 340 345 350 350 TTC TTT AAG AAT GAT CAG TGG CAT TGG GAT AAC ATA AGA GAA ACG GCA 1104 Phe Phe Lys Asn Asp Gln Trp His Trp Asp Asn Ile Arg Glu Thr Ala 355 360 365 GCT CCT GTA GTA CCT GAA CTC AGC AGT GAC ATA GAC AGC AGC AAT TTC 1152 Ala Pro Val Val Pro Glu Leu Ser Ser Asp Ile Asp Ser Ser Asn Phe 370 375 380 GAT GAC ATT GAA GAT GAC AAA GGA GAT GTA GAA ACC TTC CCA ATT CCT 1200 Asp Asp Ile Glu Asp Asp Lys Gly Asp Val Glu Thr Phe Pro Ile Pro 385 390 395 400 AAA GCT TTT GTT GGA AAT CAG CTG CCT TTC ATC GGA TTT ACC TAC TAT 1248 Lys Ala Phe Val Gly Asn Gln Leu Pro Phe Ile Gly Phe Thr Tyr Tyr 405 410 415 AGA GAA AAT TTA TTA TTA AGT GAC TCT CCA TC T TGT AGA GAA AAT GAT 1296 Arg Glu Asn Leu Leu Leu Ser Asp Ser Pro Ser Cys Arg Glu Asn Asp 420 425 430 TCC ATA CAA TCA AGG AAA AAT GAA GAA AGT CAA GAG ATT CAG AAA AAA 1344 Ser Ile Gln Ser Arg Lys Asn Glu Glu Ser Gln Glu Ile Gln Lys Lys 435 440 445 CTG TAT ACA TTA GAA GAA CAT CTT AGC AAT GAG ATG CAA GCC AAA GAG 1392 Leu Tyr Thr Leu Glu Glu His Leu Ser Asn Glu Met Gln Ala Lys Glu 450 455 460 GAA CTG GAA CAG AAG TGC AAA TCT GTT AAT ACT CGC CTA GAA AAA ACA 1440 Glu Leu Glu Gln Lys Cys Lys Ser Val Asn Thr Arg Leu Glu Lys Thr 465 470 475 475 480 GCA AAG GAG CTA GAA GAG GAG ATT ACC TTA CGG AAA AGT GTG GAA TCA 1488 Ala Lys Glu Leu Glu Glu Glu Ile Thr Leu Arg Lys Ser Val Glu Ser 485 490 495 GCA TTA AGA CAG TTA GAA AGA GAA AAG GCG CTT CTT CAG CAC AAA AAT 1536 Ala Leu Arg Gln Leu Glu Arg Glu Lys Ala Leu Leu Gln His Lys Asn 500 505 510 GCA GAA TAT CAG AGG AAA GCT GAT CAT GAA GCA GAC AAA AAA CGA AAT 1584 Ala Glu Tyr Gln Arg Lys Ala Asp His Glu Ala Asp Lys Lys Arg Asn 515 520 525 TTG GAA AAT GAT GTT AA C AGC TTA AAA GAT CAA CTT GAA GAT TTG AAA 1632 Leu Glu Asn Asp Val Asn Ser Leu Lys Asp Gln Leu Glu Asp Leu Lys 530 535 540 AAA AGA AAT CAA AAC TCT CAA ATA TCC ACT GAG AAA GTG AAT CAA CTC 1680 Lys Arg Asn Gln Asn Ser Gln Ile Ser Thr Glu Lys Val Asn Gln Leu 545 550 555 560 CAG AGA CAA CTG GAT GAA ACC AAT GCT TTA CTG CGA ACA GAG TCT GAT 1728 Gln Arg Gln Leu Asp Glu Thr Asn Ala Leu Leu Arg Thr Glu Ser Asp 565 570 575 ACT GCA GCC CGG TTA AGG AAA ACC CAG GCA GAA AGT TCA AAA CAG ATT 1776 Thr Ala Ala Arg Leu Arg Lys Thr Gln Ala Glu Ser Ser Lys Gln Ile 580 585 590 590 CAG CAG CTG GAA TCT AAC AAT AGA GAT CTA CAA GAT AAA AAC TGC CTG 1824 Gln Gln Leu Glu Ser Asn Asn Arg Asp Leu Gln Asp Lys Asn Cys Leu 595 600 605 CTG GAG ACT GCC AAG TTA AAA CTT GAA AAG GAA TTT ATC AAT CTT CAG 1872 Leu Glu Thr Ala Lys Leu Lys Leu Glu Lys Glu Phe Ile Asn Leu Gln 610 615 620 TCA GCT CTA GAA TCT GAA AGG AGG GAT CGA ACC CAT GGA TCA GAG ATA 1920 Ser Ala Leu Glu Ser Glu Arg Arg Asp Arg Thr His Gly Ser Glu Ile 625 630 635 640 ATT AAT GAT TTA CAA GGT AGA ATA TGT GGC CTA GAA GAA GAT TTA AAG 1968 Ile Asn Asp Leu Gln Gly Arg Ile Cys Gly Leu Glu Glu Asp Leu Lys 645 650 655 AAC GGC AAA ATC TTA CTA GCG AAA GTA GAA CTG GAG AAG AGA CAA CTT 2016 Asn Gly Lys Ile Leu Leu Ala Lys Val Glu Leu Glu Lys Arg Gln Leu 660 665 670 CAG GAG AGA TTT ACT GAT TTG GAA AAG GAA AAA AGC AAC ATG GAA ATA 2064 Gln Glu Arg Phe Thr Asp Leu Glu Lys Glu Lys Ser Asn Met Glu Ile 675 680 685 GAT ATG ACA TAC CAA CTA AAA GTT ATA CAG CAG AGC CTA GAA CAA GAA 2112 Asp Met Thr Tyr Gln Leu Lys Val Ile Gln Gln Ser Leu Glu Gln Glu 690 695 700 GAA GCT GAA CAT AAG GCC ACA AAG GCA CGA CTA GCA GAT AAA AAT AAG 2160 Glu Ala Glu His Lys Ala Thr Lys Ala Arg Leu Ala Asp Lys Asn Lys 705 710 715 720 ATC TAT GAG TCC ATC GAA GAA GCC AAA TCA GAA GCC ATG AAA GAA ATG 2208 Ile Tyr Glu Ser Ile Glu Glu Ala Lys Ser Glu Ala Met Lys Glu Met 725 730 735 GAG AAG AAG CTC TTG GAG GAA AGA ACT TTA AAA CAG AAA GTG GAG AAC 2256 Glu Lys Lys Leu Leu Glu Glu Arg Thr Leu Lys Gln Lys Val Glu Asn 740 745 750 CTA TTG CTA GAA GCT GAG AAA AGA TGT TCT CTA TTA GAC TGT GAC CTC 2304 Leu Leu Leu Glu Ala Glu Lys Arg Cys Ser Leu Leu Asp Cys Asp Leu 755 760 765 AAA CAG TCA CAG CAG AAA ATA AAT GAG CTC CTT AAA CAG AAA GAT GTG 2352 Lys Gln Ser Gln Gln Lys Ile Asn Glu Leu Leu Lys Gln Lys Asp Val 770 775 780 CTA AAT GAG GAT GTT AGA AAC CTG ACA TTA AAA ATA GAG CAA GAA ACT 2400 Leu Asn Glu Asp Val Arg Asn Leu Thr Leu Lys Ile Glu Gln Glu Thr 785 790 795 800 CAG AAG CGC TGC CTT ACA CAA AAT GAC CTG AAG ATG CAA ACA CAA CAG 2448 Gln Lys Arg Cys Leu Thr Gln Asn Asp Leu Lys Met Gln Thr Gln Gln 805 810 815 815 GTT AAC ACA CTA AAA ATG TCA GAA AAG CAG TTA AAG CAA GAA AAT AAC 2496 Val Asn Thr Leu Lys Met Ser Glu Lys Gln Leu Lys Gln Glu Asn Asn 820 825 830 CAT CTC ATG GAA ATG AAA ATG AAC TTG GAA AAA CAA AAT GCT GAA CTT 2544 His Leu Met Glu Met Lys Met Asn Leu Glu Lys Gln Asn Ala Glu Leu 835 840 845 CGA AAA GAA CGT CAG GAT GCA GAT GGG CAA ATG AAA GAG CTC CAG GAT 2592 Arg Lys Glu Arg Gln Asp Ala Asp Gly G ln Met Lys Glu Leu Gln Asp 850 855 860 CAG CTC GAA GCA GAA CAG TAT TTC TCA ACC CTT TAT AAA ACA CAA GTT 2640 Gln Leu Glu Ala Glu Gln Tyr Phe Ser Thr Leu Tyr Lys Thr Gln Val 865 870 875 880 880 AGG GAG CTT AAA GAA GAA TGT GAA GAA AAG ACC AAA CTT GGT AAA GAA 2688 Arg Glu Leu Lys Glu Glu Cys Glu Glu Lys Thr Lys Leu Gly Lys Glu 885 890 895 895 TTG CAG CAG AAG AAA CAG GAA TTA CAG GAT GAA CGG GAC TCT TTG GCT 2736 Leu Gln Gln Lys Lys Gln Glu Leu Gln Asp Glu Arg Asp Ser Leu Ala 900 905 910 GCC CAA CTG GAG ATC ACC TTG ACC AAA GCA GAT TCT GAG CAA CTG GCT 2784 Ala Gln Leu Glu Ile Thr Leu Thr Lys Ala Asp Ser Glu Gln Leu Ala 915 920 925 CGT TCA ATT GCT GAA GAA CAA TAT TCT GAT TTG GAA AAA GAG AAG ATC 2832 Arg Ser Ile Ala Glu Glu Gln Tyr Ser Asp Leu Glu Lys Glu Lys Ile 930 935 940 940 ATG AAA GAG CTG GAG ATC AAA GAG ATG ATG GCT AGA CAC AAA CAG GAA 2880 Met Lys Glu Leu Glu Ile Lys Glu Met Met Ala Arg His Lys Gln Glu 945 950 955 960 CTT ACG GAA AAA GAT GCT ACA ATT GCT TCT CTT GAG GAA ACT AAT AGG 2928 Leu Thr Glu Ly s Asp Ala Thr Ile Ala Ser Leu Glu Glu Thr Asn Arg 965 970 975 ACA CTA ACT AGT GAT GTT GCC AAT CTT GCA AAT GAG AAA GAA GAA TTA 2976 Thr Leu Thr Ser Asp Val Ala Asn Leu Ala Asn Glu Lys Glu Glu Leu 980 985 990 AAT AAC AAA TTG AAA GAT GTT CAA GAG CAA CTG TCA AGA TTG AAA GAT 3024 Asn Asn Lys Leu Lys Asp Val Gln Glu Gln Leu Ser Arg Leu Lys Asp 995 1000 1005 GAA GAA ATA AGC GCA GCA GCT ATT AAA GCA CAG TTT GAG AAG CAG CTA 3072 Glu Glu Ile Ser Ala Ala Ala Ile Lys Ala Gln Phe Glu Lys Gln Leu 1010 1015 1020 TTA ACA GAA AGA ACA CTC AAA ACT CAA GCT GTG AAT AAG TTG GCT GAG 3120 Leu Thr Glu Arg Thr Leu Lys Thr Gln Ala Val Asn Lys Leu Ala Glu 1025 1030 1035 1040 ATC ATG AAT CGA AAA GAA CCT GTC AAG CGT GGT AAT GAC ACA GAT GTG 3168 Ile Met Asn Arg Lys Glu Pro Val Lys Arg Gly Asn Asp Thr Asp Val 1045 1050 1055 CGG AGA AAA GAG AAG GAG AAT AGA AAG CTA CAT ATG GAG CTT AAA TCT 3216 Arg Arg Lys Glu Lys Glu Asn Arg Lys Leu His Met Glu Leu Lys Ser 1060 1065 1070 GAA CGT GAG AAA TTG ACC CAG CAG ATG ATC AAG TAT CAG AAA GAA CTG 3264 Glu Arg Glu Lys Leu Thr Gln Gln Met Ile Lys Tyr Gln Lys Glu Leu 1075 1080 1085 AAT GAA ATG CAG GCA CAA ATA GCT GAA GAG AGC CAG ATT CGA ATT GAA 3312 Asn Glu Met Gln Ala Gln Ile Glu Glu Ser Gln Ile Arg Ile Glu 1090 1095 1100 CTG CAG ATG ACA TTG GAC AGT AAA GAC AGT GAC ATT GAG CAG CTG CGG 3360 Leu Gln Met Thr Leu Asp Ser Lys Asp Ser Asp Ile Glu Gln Leu Arg 1105 1110 1115 1120 TCA CAA CTC CAA GCC TTG CAT ATT GGT CTG GAT AGT TCC AGT ATA GGC 3408 Ser Gln Leu Gln Ala Leu His Ile Gly Leu Asp Ser Ser Ser Ile Gly 1125 1130 1135 AGT GGA CCA GGG GAT GCT GAG GCA GAT GAT GGG TTT CCA GAA TCA AGA 3456 Ser Gly Pro Gly Asp Ala Glu Ala Asp Asp Gly Phe Pro Glu Ser Arg 1140 1145 1150 TTA GAA GGA TGG CTT TCA TTG CCT GTA CGA AAC AAC ACT AAG AAA TTT 3504 Leu Glu Gly Trp Leu Ser Leu Pro Val Arg Asn Asn Thr Lys Lys Phe 1155 1160 1165 GGA TGG GTT AAA AAG TAT GTG ATT GTA AGC AGT AAG AAG ATT CTT TTC 3552 Gly Trp Val Lys Lys Tyr Val Ile Val Ser Ser Lys Lys Ile Leu Phe 1170 1175 1180 TAT GAC AGT GAA CAA GAT AAA GAA CAA TCC AAT CCT TAC ATG GTT TTA 3600 Tyr Asp Ser Glu Gln Asp Lys Glu Gln Ser Asn Pro Tyr Met Val Leu 1185 1190 1195 1200 GAT ATA GAC AAG TTA TTT CAT GTC CGA CCA GTT ACA CAG ACA GAT GTG 3648 Asp Ile Asp Lys Leu Phe His Val Arg Pro Val Thr Gln Thr Asp Val 1205 1210 1215 TAT AGA GCA GAT GCT AAA GAA ATT CCA AGG ATA TTC CAG ATT CTG TAT 3696 Tyr Arg Ala Asp Ala Lys Glu Ile Pro Arg Ile Phe Gln Ile Leu Tyr 1220 1225 1230 GCC AAT GAA GGA GAA AGT AAG AAG GAA CAA GAA TTT CCA GTG GAG CCA 3744 Ala Asn Glu Gly Glu Ser Lys Lys Glu Gln Glu Phe Pro Val Glu Pro 1235 1240 1245 GTT GGA GAA AAA TCT AAT TAT ATT TGC CAC AAG GGA CAT GAG TTT ATT 3792 Val Gly Glu Lys Ser Asn Tyr Ile Cys His Lys Gly His Glu Phe Ile 1250 1255 1260 CCT ACT CTT TAT CAT TTC CCA ACC AAC TGT GAG GCT TGT ATG AAG CCC 3840 Pro Thr Leu Tyr His Phe Pro Thr Asn Cys Glu Ala Cys Met Lys Pro 1265 1270 1275 1280 CTG TGG CAC ATG TTT AAG CCT CCT CCT GCT TTG GAG TGC CGC CGT TGC 3888 Leu Trp His Met Phe Lys Pro Pro Pro Ala Leu Glu Cys Arg Arg Cys 1285 1290 1295 CAT ATT AAG TGT CAT AAA GAT CAT ATG GAC AAA AAG GAG GAG ATT ATA 3936 His Ile Lys Cys His Lys Asp His Met Asp Lys Lys Glu Glu Ile Ile 1300 1305 1310 GCA CCT TGC AAA GTA TAT TAT GAT ATT TCA ACG GCA AAG AAT CTG TTA 3984 Ala Pro Cys Lys Val Tyr Tyr Asp Ile Ser Thr Ala Lys Asn Leu Leu 1315 1320 1325 TTA CTA GCA AAT TCT ACA GAA GAG CAG CAG AAG TGG GTT AGT CGG TTG 4032 Leu Leu Ala Asn Ser Thr Glu Glu Gln Gln Lys Trp Val Ser Arg Leu 1330 1335 1340 GTG AAA AAG ATA CCT AAA AAG CCC CCA GCT CCA GAC CCT TTT GCC CGA 4080 Val Lys Lys Ile Pro Lys Lys Pro Pro Ala Pro Asp Pro Phe Ala Arg 1345 1350 1355 1360 TCA TCT CCT AGA ACT TCA ATG AAG ATA CAG CAA AAC CAG TCT ATT AGA 4128 Ser Ser Pro Arg Thr Ser Met Lys Ile Gln Gln Asn Gln Ser Ile Arg 1365 1370 1375 CGG CCA AGT CGA CAG CTT GCC CCA AAC AAA CCT AGC TAA CTGCCTTCTA 4177 Arg Pro Ser Arg Gln Leu Ala Pro Asn Lys Pro Ser 1380 1385 TGAAAGCAGT CATTATTCAA GGTGATCGTA TTCTTCCAGT GAAAACAAGA CTGAAATATG 4237 ATGACCCCAT GGTACCC GGA TCCTCGAATC TTTTGCTTTT TACCCTGGAA GAAATACTCA 4297 TAAGCCACCT CTGTAATCGG ATCCCCGGGT ACCGAAATAC TCATAAGCCA CCTCTGTAAT 4357 CGGATC 4363

SEQ ID NO: 6 Sequence length: 741 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Genomic DNA Sequence characteristics Characteristic symbol: intron Location: Sequence AAGAGCTCCA GGATCAGCTC GAAGCAGAAC AGTATTTCTC AGTAAGTTCA AGACACATTA 60 ATTTGCAAAT AGCAAACAGA GTTTTGGTTG GGATATTTTA TTTATACCAT ACAACTCATA 120 TAAAAAGAGC AGCTGGGCAA AAGAATGTAG ATAGTTTAAG AGAAATTTAT TATCATTGCT 180 AGAAAAATAA TTCTAAGCAC AAAACATACC TGGTGAAGCT GATTAACAGC ATTGTTTTCA 240 GGAAGAAAAT ATTTTCATAA TGCAAGAAAT AAAATTGTAA TAAAATTATC ATTTTAATAG 300 TTGTGCTTTT TTTGAGATCA GTTTTAAAAA TATGAAGTAG AAACTCCTGT TTTATTGATT 360 TAAGCCAATA ACATTTTTTT GTCTAAATTG TTAATGTTTT TCCTTGCCTC CTTATTCATT 420 TTACCTATCT GTGCTATTAA ATGAGAAATA TCACTGTGAT ATCCAGTTGA AAATCATGCT 480 TCTTAATTAT TACCTGAACT TGTATTAGTT AATTGATGAC TAGAAATAGG AAAGTTCATT 540 AAACTATTCA TTTGCTCGGG GACTTAGCAG TAAGAAAATA TTTGTATGTG TTCATTCTCC 600 TTTTTGTCTG AATAGCTTTT GAATGTAGTATGATGATGATAGATAGATTA 60 GTTGTATTTT GCTATTTAGA CCCTTTATAA AACACAAGTT AGGGAGCTTA AAGAAGAATG 720 TGAAGAAAAG ACCAAACTTG G 741

[Brief description of the drawings]

FIG. 1 is an electrophoretic photograph showing the results of purification of a protein that specifically binds to an active Rho protein. The crude membrane fraction was subjected to GST (lane 1), GDP / GST-Rh
oA (lane 2) or GTPγS · GST-Rho
A (lane 3) containing Glutathione-Sepharose column. To increase the purity of bovine Rho kinase, the crude membrane fraction was applied to Glutathione Sepharose containing GTPγS.GST-RhoA, and bovine Rho kinase was eluted by adding 1% CHAPS (lane 4).

FIG. 2 is a diagram showing the results of purification of bovine Rho kinase by Mono Q column chromatography, and an electrophoresis photograph. The CHAPS-eluted fraction is
On a Q column, Rho kinase was eluted with a linear NaCl gradient. The results are representative of three independent experiments.

FIG. 3 is an electrophoretic photograph showing the binding between bovine Rho kinase and activated RhoA. Lane 1 and Lane 3
Indicates a membrane extract, and lanes 2 and 4 indicate SDS-PA.
Shown are nitrocellulose membranes each containing purified Rho kinase separated by GE. Lane 1 and Lane 2
Shows [ ³⁵ S] GTPγS.GST-RhoA, and lanes 3 and 4 show [ ³⁵ S] GTPγS.GST-R.
The lane using hoA ^Ala37 as a probe is shown. The arrow indicates the position of Rho kinase on SDS-PAGE. The results are representative of three independent experiments.

FIG. 4 is an electrophoretic photograph showing the autophosphorylation ability of bovine Rho kinase. Rho kinase was autophosphorylated in the presence of the following proteins (1 μM each): Lane 1: GST, Lane 2: GDP · GST-RhoA, Lane 3: GTPγS · GST-RhoA, Lane 4: G
TPγS · GST-RhoA ^Ala37 . The arrow is Rh
The position of o-kinase on SDS-PAGE is indicated.
The results are representative of three independent experiments.

FIG. 5: Phosphorylation of myelin basic protein, S6 peptide, αPKC (40 μM each) by bovine Rho kinase under the presence of either GST, GDP / GST-RhoA, or GTPγS / GST-RhoA. It is the figure which showed the degree.

FIG. 6. S6 peptide (40
μM) was phosphorylated by GTPγS · GST-RhoA.
(1 μM) shows that it promotes.

FIG. 7. Phosphorylation of rat myosin binding subunit by bovine Rho kinase was determined by GTPγS.GST-R.
It is an electrophoresis photograph which showed that hoA promoted. Lane 1: GST, Lane 2: GDP-GST-Rho
A, Lane 3: GTPγS · GST-RhoA. Arrows indicate SDS-P of myosin binding subunit protein.
Indicates the position on the AGE.

FIG. 8 shows phosphorylation of S6 peptide by bovine Rho kinase. Black square: GTPγS-Rho
A (post-translationally modified), open square: GDP-RhoA (post-translationally modified), filled circle: GTPγS-GST-RhoA
(Non-translationally modified type), open circle: GDP-GST-RhoA
(Untranslated, modified).

FIG. 9 shows a deduced amino acid sequence of bovine Rho kinase. The amino acid sequence determined by partial peptide sequence analysis of Rho kinase purified from bovine brain gray matter is underlined. The amino acid sequence of the probe used for Rho kinase cDNA cloning is double underlined.

FIG. 10 is a diagram comparing the domain structures of bovine Rho kinase and mitonic dystrophy kinase.

FIG. 11 is an electrophoretic photograph showing the tissue distribution of bovine Rho kinase expression. Lane 1: 1% CHAPS eluate from GST-RhoA affinity column, lane 2: cerebrum, lane 3: cerebellum, lane 4: heart, lane 5: skeletal muscle, lane 6: spleen, lane 7: lung, lane 8: Liver, lane 9: kidney, lane 10: pancreas. The arrow indicates the position of Rho kinase on SDS-PAGE.

FIG. 12. Protein in the coiled-coil region of bovine Rho kinase synthesized by in vitro translation and activated R
5 is an electrophoretic photograph showing binding to ho. Lane 1:
GST, lane 2: GDP · GST-RhoA, lane 3: GTPγS · GST-RhoA, lane 4: GTP
γS · GST-RhoA ^Ala37 , lane 5: GDP
GST-Rac1, lane 6: GTPγS GST-
Rac1, lane 7: GDP · GST-H-Ras, lane 8: GTPγS · GST-H-Ras.

FIG. 13 shows that phosphorylation of chicken myosin binding subunit by bovine Rho kinase was determined by GTPγS · GST-
It is an electrophoresis photograph which showed that RhoA promoted.
Lane 1: GST, Lane 2: GDP-GST-Rho
A, Lane 3: GTPγS · GST-RhoA, Lane 4: GTPγS · GST-RhoA ^Ala37 , Lane 5: GDP · GST-Rac1, Lane 6: GTPγS
-GST-Rac1. The numbers above the lanes indicate the degree of phosphorylation as a relative value when GST (lane 1) is 1.0.

FIG. 14. Bovine Rho kinase concentration-dependent chicken
FIG. 3 shows thiophosphorylation of myosin binding subunit and inhibition of myosin light chain phosphatase activity. Black and white circles are GTPγS · GST-RhoA, respectively.
FIG. 9 shows incorporation of ³⁵ S-thiophosphate into myosin binding subunit in the presence or absence. The black square and the white square are GTPγS · GST-RhoA, respectively.
Fig. 4 shows the enzymatic activity of myosin light chain phosphatase in the presence or absence of Rho kinase phosphorylated in the presence of ATPγS. Diamonds indicate the enzymatic activity of myosin light chain phosphatase in the absence of ATPγS (ie, when using unphosphorylated Rho kinase).

FIG. 15 is an electrophoresis photograph showing the degree of phosphorylation of a myosin-binding subunit in each NIH / 3T3 cell line overexpressing RhoA or RhoA ^Val14 . The numbers above the lanes indicate the degree of phosphorylation, GST
(Lane 1) is represented by a relative value when 1.0 is set.

FIG. 16 shows the degree of phosphorylation of myosin light chain in the NIH / 3T3 cell line overexpressing RhoA or RhoA ^Val14 .

FIG. 17 is an electrophoresis photograph showing phosphorylation of myosin light chain by bovine Rho kinase. The isolated myosin light chain (0.5 μg of protein) was analyzed using GST (lane 1), GDP • GST-RhoA (lane 2), GTP
γS • GST-RhoA (lane 3), GTPγS • G
ST-RhoA ^Ala37 (lane 4), GDP / GST-
Rac1 (lane 5) or GTPγS.GST-Ra
Bovine Rho purified in the presence of c1 (lane 6)
Phosphorylation by kinase (20 ng protein) or GST-bovine Rho kinase (protein 5
ng) (lane 7) or phosphorylation by myosin light chain kinase in the absence (lane 8) or presence (lane 9) of Ca ²⁺ and calmodulin. Intact myosin (5 μg protein) was added to GTPγS · GST-
Phosphorylation was carried out in the absence (lane 10) or presence (lane 11) of RhoA. The phosphorylated myosin light chain was separated by SDS-PAGE and visualized by an image analyzer. The results are representative of three independent experiments.

FIG. 18 shows phosphorylation of myosin light chain by bovine Rho kinase. Various amounts of myosin light chain
It was phosphorylated by ho kinase. "MLC" represents myosin light chain, "Rho-Kinase" represents Rho kinase,
"MLC Kinase" means Myonsi light chain kinase, respectively (hereinafter the same). In the left figure, the black circle is GTPγ
In the presence of S-GST-RhoA, circles indicate GTPγS-GS
Fig. 3 shows phosphorylation by Rho kinase in the absence of T-RhoA. Solid triangles indicate phosphorylation by GST-Rho kinase. In the right figure, black squares and squares indicate phosphorylation by myosin light chain kinase in the presence and absence of Ca ²⁺ and calmodulin.

FIG. 19 is an electrophoresis photograph showing mapping analysis of a phosphorylated peptide of myosin light chain. The myosin light chain (0.5 μg protein) is converted to Rho kinase (Rho
-Kinase), myosin light chain kinase (MLC Kinase)
Alternatively, it was phosphorylated by protein kinase C (PKC). The phosphorylated myosin light chain was digested with trypsin and each sample was loaded on a silica gel plate. Phosphorylated peptides were separated by electrophoresis (horizontal direction) and chromatography (vertical direction), and then visualized by an image analyzer. The star indicates the origin.

FIG. 20 is an electrophoresis photograph showing phosphorylation of a recombinant myosin light chain. Myosin light chain (MLC), GST, G
ST-myosin light chain (GST-MLC) or GST-
Myosin light chain ^{Ala18 Ala19} (2 μM each) as shown, purified Rho kinase (20 ng protein), G
It was phosphorylated by ST-Rho kinase (10 ng protein) or myosin light chain kinase (10 ng protein). Lanes 1-4: Myosin light chain was phosphorylated with purified Rho kinase. Lanes 5-8: Myosin light chain was phosphorylated by GST-Rho kinase. Lanes 9-12: Myosin light chain was phosphorylated by myosin light chain kinase. Lanes 1, 5, and 9: myosin light chain, lanes 2, 6, and 10: GST, lanes 3, 7, and 11: GST-myosin light chain, lanes 4, 8, and 12: GST-myosin light chain
^{Ala18, Ala19} . Results are representative of three independent experiments.

FIG. 21 shows the effect of bovine Rho kinase phosphorylation of myosin on MgATPase activity activated by actin. The vertical axis represents the phosphorylation rate of the myosin head. Myosin to GST ・ R
Incubation with ho kinase (solid squares), myosin light chain kinase (solid diamonds), or in the absence of kinase (solid circles). After incubation,
ATPase activity was measured in the presence of various concentrations of F-actin. The values shown are the mean ± SEM of three replicate experiments. E. FIG.
It is.

FIG. 22. Rho protein, Rho kinase (Rho kinase)
-Model showing the regulation of myosin light chain by -Kinase) and myosin light chain phosphatase. C
at: catalytic subunit of myosin light chain phosphatase, Myosin: myosin, MBS: myosin binding subunit.

FIG. 23. R by yeast two hybrid system
5 is a photograph showing detection of binding between a ho protein and a human Rho kinase protein.

FIG. 24 is a view showing that Rho kinase contracts rabbit portal vein blood vessels whose permeability has been enhanced by Triton X-100. Rho kinase (CAT) was introduced exogenously into rabbit portal vein vascular smooth muscle preparations. pCa6.5
(A and b) or Trit with pCa << 8.0 (c)
Figure 4 shows a representative record of tension responses induced in a rabbit portal vein specimen whose permeability was enhanced by on X-100.
All of these results are representative of 3-5 independent experiments. The contractile effect of Rho kinase (CAT) was completely reversed by washing Rho kinase (CAT) (b). Further, in c, the administration dose of the vehicle is changed to the total amount of the chamber (2).
(00 μl).

FIG. 25 shows the sensitivity of contractile sensitization of rabbit portal vein blood vessels whose permeability was enhanced by Triton X-100.
y) is enhanced by Rho kinase (potentiate
s). a: Relationship between pCa and tension in the presence of 7.5 nM Rho kinase (CAT) (open circles) or 500 nM OA (solid circles). Below pCa 7.0, the degree of tension produced by 7.5 nM Rho kinase (CAT) is based on basal
Level. The relationship between the Ca ²⁺ and the tension of the Steady-state was determined in the presence of Rho kinase (CAT) (open circles) or O
It was obtained by increasing the cytoplasmic Ca ²⁺ concentration in the presence (solid circle) or absence (open triangle / dashed line: control) of A. To demonstrate increased sensitivity to contraction in the presence of Rho kinase (CAT) or OA, each tension response reduced tension at control pCa4.5 by 1
The relative values are standardized as shown. The vertical axis indicates the relative value of the tension, and the horizontal axis indicates the Ca ²⁺ concentration in pCa. Each value is the mean ± standard deviation obtained from four experiments. b: Effect of wortmannin (WM), a potent inhibitor of myosin light chain kinase (MLCK), on the dose-response curve of Rho kinase (CAT) in rabbit portal vein vessels with enhanced permeability by Triton X-100 . The practice is at pCa 6.5, and the dashed line shows the effect at pCa << 8.0 (buffered with 10 mM EGTA), respectively. Steady sta
te tension reaction in the presence of 10 μM WM (solid circles indicate pC
a6.5, closed triangles at pCa << 8.0 or in the absence (open circles at pCa6.5, open triangles at pCa <<
8.0), the cumm of Rho kinase (CAT)
Obtained by ulative addition. The tension response is pCa4.5
The maximum tension response in the above was shown as a relative value with 1. The vertical axis indicates the relative value of tension, and the horizontal axis indicates the molar concentration (μM) of Rho kinase (CAT). Each value is 5-8
It is the average value ± standard deviation obtained from the experiment. ANO
VA analysis was used to determine statistical significance.
A P value of 0.05 or less was considered "significant"(**; p <0.01, NS; no significant difference).

FIG. 26. Rho kinase is Ca ²⁺ independent and Wo
FIG. 9 is an electrophoretic photograph and a diagram showing that in a rtmannin-insensitive manner, phosphorylation of myosin light chain in rabbit portal vein blood vessels whose permeability was enhanced by Triton X-100 is shown. a: A myosin light chain (MLC-P0) and a monophosphorylated myosin light chain (MLC-P1) which have not undergone phosphorylation in rabbit portal vein blood vessels whose permeability has been enhanced by Triton X-100 are glycerol. -Separation by urea polyacrylamide gel electrophoresis followed by immunoblot using an anti-myosin light chain antibody. Control (lane 1, p
In contrast to Ca <<8.0; lane 2, pCa6.5), 0.255 μM Rho kinase (CAT) had 1
In the absence (lanes 2, 5) and presence (lanes 3, 6) of 0 μM WM, pCa << 8.0 (10 m
MEGTA; lanes 1-3) and pCa6.5 (lanes 4-6) increased myosin light chain monophosphorylation. These results are representative of four experiments. b: Quantification of the effect of Rho kinase (CAT) on the degree of phosphorylation of myosin light chain (open bars) and contractile response (hatched bars) in the presence or absence of 10 μM WM comparison. The white bar is the total ML
The percentage of MLC monophosphorylated relative to C is expressed as the mean ± SD of four experiments. Total MLC phosphorylation values were compared by Student's test (** P <0.01, * P <0.05). The number under each column is the same as each lane in a. Abbreviations in a are as follows: G10, 10m
Relaxation solution containing MEGTA; 6.5, pCa6.5; W
M, 10 mM wortmannin, RK, 0.255 Rho kinase (CAT).

FIG. 27 is an electrophoretogram and a diagram showing the absence of Rho kinase in a blood vessel specimen whose permeability was enhanced by Triton X-100. a: Intact rabbit portal vein vascular specimen (without increased permeability; lanes 1, 3, 5) and rabbit portal vein vascular specimen whose permeability was enhanced by Triton X-100 (lane 2,
To compare the tissue distribution of Rho kinase in 4, 6), anti-Rho kinase antibody (lane A) and anti-2
Immunoblot analysis using a 0 kDa myosin light chain antibody was performed. As a control, CBB staining of each extract was shown (lane C). 5% SDS-PA for each extract
By GE (lanes A and B) or 15% SDS
Each was analyzed by PAGE (lane C).
"RK" indicates Rho kinase, and "MLC" indicates myosin light chain. These results are representative of three independent experiments. b: Densitometric analysis of the results shown in a. Rh in intact tissues (lanes 3, 5) and in skinned tissues (lanes 4, 6)
FIG. 9 is data showing the quantitative quantitative value of o-kinase immunostaining as a ratio to that of myosin light chain. Data were expressed as mean ± standard deviation.

FIG. 28 is a diagram schematically showing the structures of various Rho kinases used in the experiment. Rho kinase, Rho kinase (CAT), Rho kinase (CAT-KD),
Rho kinase (COIL), Rho kinase (R
B) and a schematic diagram of Rho kinase (PH).

FIG. 29 is an electrophoretic photograph showing the binding between Rho protein and Rho kinase by overlay assay. Purified Rho kinase (lanes 1, 3) transferred to a nitrocellulose membrane after SDS-PAGE, Rh
o Kinase (RB) (lanes 2, 4) was converted to [ ³⁵ S] GTP
γS · GST-RhoA (lanes 1 and 2) or [
^The results of overlay assay using ³⁵ S] ^GTPγS.GST -RhoA ^Ala37 (lanes 3, 4) as probes are shown. The results are representative of three independent experiments.

FIG. 30 shows that myosin light chain is GTPγS · GST-Rho.
FIG. 3 shows the results of phosphorylation with natural Rho kinase or Rho kinase (CAT) in the presence or absence of A (1.5 μM). Data are means ± SEM of three independent experiments.

FIG. 31 shows the effects of dominant negative mutants and staurosporine on phosphorylation of myosin light chain. a: Myosin light chain was prepared at various concentrations of Rho kinase (R
Together with B), phosphorylation was carried out by Rho kinase (CAT) (□) or by natural Rho kinase in the presence of GTPγS · GST-RhoA (。). b: Myosin light chain with various concentrations of staurosporine by Rho kinase (CAT) (□) or in the presence of GTPγS.GST-RhoA
It was phosphorylated by ho kinase (○). Data are means ± sSEM of three independent experiments.

FIG. 32 is a photograph (photograph of the form of an organism) showing reconstitution of actin by Rho kinase. Vehicle (vehicl
e) Stimulation (a), LP without microinjection
A (200 ng) for 15 minutes (b), C3 enzyme (8
0 μg / ml) by microinjection and LPA
(C), treatment with staurosporine (100 nM), treatment with LPA after 15 minutes (d), Rho kinase (CA)
T) (0.5 mg / ml) alone by microinjection (e), Rho kinase (CAT) and C3 by microinjection (f), 15 min after treatment with staurosporine, Rho kinase (CAT) by microinjection (g) 3) shows actin filaments formed in confluent serum-starved Swiss3T3 cells.

FIG. 33: Focal adhesion by Rho kinase (Foca
1 is a photograph (photograph of the form of an organism) showing the formation of adhesion. The localization of vinculin is shown. Experimental conditions other than the photograph h are the same as those in FIG. Photo h shows Swi microinjected Rho kinase (CAT).
FIG. 4 shows the localization of actin filaments and vinculin in ss 3T3 cells. Bar marks indicate microinjection locations, and bar lines indicate 20 μm.

FIG. 34 is a photograph (photograph of the form of an organism) showing the effect of various Rho kinases on LPA-induced reconstitution of actin filaments and formation of focal adhesion. Confluent serum-starved Swiss3T3 cells receive 2 mg / ml Rho kinase (CAT-KD) (A, E), 5 mg / ml R
ho kinase (COIL) (B, F), 5 mg / ml Rho kinase (RB) (C, G), 5 mg / ml R
Ho kinase (PH) (D, H) was stimulated with 200 ng / ml LPA after microinjection. The localization of actin filaments and vinculin is shown. Bar marks indicate microinjection locations, and bar lines indicate 20 μm.

FIG. 35 is a photograph (photograph of the form of an organism) showing actin reconstitution in MDCK cells. pEF-BOS
^-HA -RhoA ^Val14 (0.1 mg / ml) + p
EF-BOS-myc (1 mg / ml) (A), pE
F-BOS-myc-Rho kinase (CAT) (0.
1 mg / ml) + pEF-BOS-myc (1 mg / m
1) (B), pEF-BOS-HA-RhoA
^Val14 + pEF-BOS-myc-Rho kinase (CAT-KD) (1 mg / ml) (C), pEF-
BOS-HA-RhoA ^Val14 + pEF-BOS-
myc-Rho kinase (RB) (1 mg / ml)
(D), pEF-BOS-HA-RhoA ^Val14
+ PEF-BOS-myc-Rho kinase (PH)
(1 mg / ml) Actin filaments in MDCK cells microinjected with (E). Bar marks indicate microinjection locations, and bar lines indicate 20 μm.

FIG. 36 is an electrophoretic photograph showing the specificity of the anti-MLC-pS19 antibody. MLC containing the indicated amount of phosphorylated MLC phosphorylated by Rho kinase (25 pmo
l) was subjected to SDS-PAGE. Anti-MLC-pS19
Immunoblot analysis was performed using the antibody (top) or anti-MLC antibody (bottom). The results shown are representative of three independent experiments.

FIG. 37 is an electrophoretic photograph showing detection of phosphorylated MLC in COS7 cells expressing Rho or Rho kinase. COS7 cells were transformed into pEF-BOS-myc-MLC
Together with pEF-BOS-myc (lane 1), pEF-BOS-HA-Rho
^Val14 (lane 2), pEF-BOS-myc-Rho kinase (lane 3), pEF-BOS-myc-Rho kinase (CAT) (lane 4), pEF-BOS-myc-Rho kinase (CAT-KD) (lane 5) or pEF-BOS- treated with calyculin A (0.1 μM)
Cotransfected with myc (lane 6). 10% T
The precipitate obtained from the cells by the CA treatment was subjected to SDS-PAGE.
And the relative amount of phosphorylated MLC was determined by immunoblot analysis. The results shown are representative of three independent experiments.

FIG. 38. RE into which Rho kinase was injected
It is a micrograph which showed distribution of actin (actin), vinculin (vinculin), MLC (MLCp), and phosphorylated MLC in F52 cell. 1
C3 (0.1 mg / ml) was added to REF52 cells in 0% FBS.
(Eh), C3 and Rho ^Ile41 (0.4 mg / m
l) (il) or C3 and Rho kinase (C
AT) (0.6 mg / ml) (mp) was microinjected, incubated for 20 minutes and fixed. Control non-injected cells are shown in ad. Actin is phalloidin (a, e, i and m)
And vinculin (b, f, j and n), MLC
(C, g, k and o) and phosphorylated MLC (d,
h, l and p) were stained with antibodies against each.
The scale bar represents 20 μm.

FIG. 39. Actin and vinculin (v) in REF52 cells treated with Cytochalasin D
4 is a photomicrograph showing the distribution of (inculin). 1
REF52 cells in 0% FBS were replaced with cytochalasin D
(0.1 μM) for 30 minutes (c, d) and then C
3 (e, f), Rho kinase (CAT) (g, h) or C3 and Rho kinase (CAT) (i, j) were injected. After a 20 minute incubation, cells were fixed and stained with phalloidin (a, c, e, g and i) or anti-vinculin antibodies (b, d, f, h and j), respectively. Control untreated cells are shown in a and b. The scale bar represents 20 μm.

FIG. 40 is a micrograph showing the distribution of actin and vinculin in Swiss 3T3 cells treated with Cytochalasin D. Serum-starved Swiss 3T3 cells were transferred to Cytochalasin D
(C, d) and then Rho kinase (CAT)
Injection (e, f) or Cytochalasin D
Rho kinase (CAT) was injected without treatment. After a 20 minute incubation, cells were fixed and phalloidin (a, c, e and g)
Alternatively, the cells were stained with anti-vinculin antibodies (b, d, f, and h). Control untreated cells are shown in a and b. The scale bar represents 20 μm.

FIG. 41 shows the activation of SRE by Rho and Rho kinase. NIH3T3 cells contain SR
E. FIG. L-luciferase (1 μg) and pME18S-lacZ
(3 μg) were co-transfected with the ^indicated amounts of pEF-BOS-myc-Rho kinase (WT), pEF-BOS-myc-Rho kinase (CAT) or pEF-BOS-HA-Rho ^Val14 . Luciferase activity was assayed as described in the examples. Data are mean ± SEM (trip
licate).

FIG. 42. FIG. 2 shows the effects of Rho and Rho kinase on L transcription activation. NIH3T3
Cells are given SRE. L-luciferase (1 μg), pME1
8S-lacZ (3 μg) and pEF-BOS-HA-Rho ^Val14
(0.6 μg) or pEF-BOS-myc-Rho kinase (C
AT) (0.6 μg) and pEF-BOS-myc (3.4 μg)
g), pEF-BOS-myc-Rho kinase (COIL)
(3.4 μg) or pEF-BOS-myc-Rho kinase (C
AT-KD) (3.4 μg). Luciferase activity was assayed as described in the examples. Data are mean ± SEM (triplica
te).

FIG. 43. Anti-GFAP antibody (MO389) and anti-phosphorylated GFAP antibody (YC10, KT13 and KT3)
4 is a photomicrograph showing the immunoreactivity of 4) U251 human astrocytoma cells. A: U251 mitotic cells (metaphase: metaphase cells, anaphase: late cells)
389, YC10, KT13 or KT34). The chromosomes were stained with propidiumiodide. The bar indicates 10 μm. B: Confocal images of nine consecutive planes of late mitotic U251 cells stained with KT13 and propidium iodine, taken with a confocal laser scanning microscope.

FIG. 44 is an electrophoretic photograph showing phosphorylation of GFAP by Rho kinase. A: GFAP was converted to GST (lanes 1 and 4), GDP
ST-RhoA (lanes 2, 5) or GTPγS
Phosphorylated by Rho kinase in the presence of either GST-RhoA (lanes 3, 6) (Example 2)
0). Samples (10 μl each) were subjected to 10% PAGE.
The gel was stained with Coomassie blue (CBB) (lanes 1-3) and autoradiographed (Autor
(lane 4-6). B: GFAP was phosphorylated in the presence of ³² P-labeled ATP in the same manner as A, analyzed by SDS-PAGE, and
9. Immunoblot with YC10, KT13 or KT34. Chemifluorescence was detected after 1 hour exposure. C: GFAP was converted to GST-Rho kinase (GST-Rh
o-kinase), buffer (control),
Incubation was carried out together with A kinase (A-kinase) or cdc2 kinase (cdc2 kinase) under the conditions shown in Example 20. Samples (2 μl each) were analyzed by immunoblot with MO389, YC10, KT13 or KT34. Chemifluorescence was detected after 5 seconds exposure.

FIG. 45 shows a diagram and an electrophoresis photograph showing phosphorylation of GFAP head domain (GFAP-head) by GST-Rho kinase. A: GFAP (130 μg) was phosphorylated with GST-Rho kinase for 120 minutes according to the method described in Example 20. A portion (5 μl each) of the reaction solution was taken out at the indicated time and added to Laemmli's sample buffer. After SDS-PAGE, the radioactivity incorporated into GFAP was analyzed using an image analyzer (Fujix BAS2).
000). B: After the phosphorylation reaction according to A above, GFAP was precipitated with trichloroacetic acid, and the lysyl endopeptidase undigested (lane 1) and digested (lane 2) samples were extracted from Trici.
Ne-SDS-PAGE was performed. After electrophoresis, the gel (16.5% acrylamide) was subjected to autoradiography. The position of the molecular weight marker (kDa) is shown on the left.

FIG. 46. T of GFAP by GST-Rho kinase
It is the figure which showed the phosphorylation of hr-7, Ser-13, and Ser-34, and an electrophoresis photograph. A: The 6.5 kDa phosphorylated fragment from GFAP was digested with trypsin and reverse phase HP was digested as described in the Examples.
LC was applied. Three radioactivity peaks (R1-R3)
Was analyzed by a gas-phase sequencer. R1 determined
(5-11 residues), R2 (37-41 residues) and R3
The amino acid sequence of (13-29 residues) is shown on the right. Ser-38 of human GFAP is Se of porcine GFAP.
In this example, human GFAP
It should be pointed out that Ser-38 of No. was designated as Ser-34. B: A part of R1, R2 and R3 obtained in the above A was subjected to two-dimensional phosphorylated amino acid analysis.

FIG. 47. GST-Rho kinase (GST-Rho-
Kinase) shows an electrophoretic photograph and a micrograph showing the inhibition of filament formation from soluble GFAP. A: GFAP (170 μg / ml) was added to GST-Rho kinase (8.5 μg) in the presence (Rho-K) or absence (control) for 30 minutes at 50 ° C. at 25 ° C.
1 of 25 mM Tris-HCl (pH 7.5), 0.4
mM MgCl ₂ , 100 μM ATP, 0.1 μM
Pre-incubated in calyculin A. Next, the reaction mixture was diluted with 25 mM imidazole-H.
Incubate at 37 ° C. in Cl (pH 6.75) and 100 mM NaCl for an additional hour,
Centrifuged at 2 ° C for 0 minutes. The supernatant (s) and the precipitate (p) were electrophoresed on a 10% acrylamide gel. Gel is Co
Stained with massie blue. B: After the polymerization reaction described in A above, a part of each sample was negatively stained and observed with an electron microscope.
The bar indicates 200 nm.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/78 C12P 21/02 C C12N 1/19 C12Q 1/68 A 1/21 G01N 33/573 Z 5/10 A61K 37/02 AAR C12P 21/02 ABN C12Q 1/68 AED G01N 33/573 C12N 5/00 B (72) Inventor Makoto Kobayashi 1-2-10-106 Kitakotoshiba, Ube City, Yamaguchi Prefecture

Claims (61)

[Claims] 1. A human-derived protein or a derivative thereof having the following characteristics: (1) having an active Rho protein binding ability;
(3) Activated Rho having protein kinase activity
Binding to a protein enhances protein kinase activity, and (4) the gene is located on human chromosome 2p24. 2. A protein or a derivative thereof comprising the following amino acid sequence: (1) the amino acid sequence of SEQ ID NO: 4, or (2) one or more amino acid sequences added and / or inserted, and / or one or more An amino acid sequence of SEQ ID NO: 4 in which amino acids have been substituted and / or deleted, wherein the active form is Rho.
An amino acid sequence having protein binding ability and having protein kinase activity. 3. The amino acid sequence of positions 1 to 89 of SEQ ID NO: 4 or a partial sequence thereof, and / or the amino acid sequence of positions 360 to 942 or a partial sequence thereof, and / or 10
The protein according to claim 2, wherein the amino acid sequence at positions 69 to 1388 or a partial sequence thereof has been deleted. 4. The protein according to claim 2, which has the amino acid sequence of positions 90 to 359 and the amino acid sequence of positions 943 to 1068 of SEQ ID NO: 4. 5. A human-derived protein or a derivative thereof having an active Rho protein binding ability and having no protein kinase activity. 6. The protein according to claim 5, which comprises the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid sequences have been added and / or inserted, and / or one or more amino acids have been substituted and / or deleted. . 7. The protein according to claim 6, wherein the amino acid sequence of Nos. 90 to 359 of SEQ ID NO: 4 or a region containing a part thereof having protein kinase activity has been deleted. 8. The Lys at position 121 in SEQ ID NO: 4 is Gly
The protein according to claim 6, which is substituted with 9. Nos. 421 to 1137, 438 of SEQ ID NO: 4.
# 1124, # 799-1137 or # 943-1
The protein according to claim 6, comprising the amino acid sequence of position 068. 10. A protein comprising the amino acid sequence of SEQ ID NO: 4 wherein one or more amino acid sequences are added and / or inserted, and / or one or more amino acids are substituted and / or deleted, and / or a derivative thereof. A protein or a derivative thereof, wherein the amino acid sequence inhibits the function of an activated Rho protein and / or Rho kinase; 11. The protein according to claim 10, wherein the amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of positions 941 to 1075 or 943 to 1068 of SEQ ID NO: 4. 12. The amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of positions 1125 to 1388 of SEQ ID NO: 4.
The protein according to claim 10. 13. The amino acid sequence of SEQ ID NO: 4 comprising 12
The protein according to claim 10, comprising the amino acid sequence of Nos. 6 to 553 of SEQ ID NO: 4 in which Lys at position 1 has been substituted with Gly. 14. A human-derived protein having protein kinase activity and not having the ability to bind to active Rho protein, or a derivative thereof. 15. The protein according to claim 14, which comprises the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid sequences have been added and / or inserted, and / or one or more amino acids have been substituted and / or deleted. . 16. A protein comprising the amino acid sequence of SEQ ID NO: 4 wherein one or more amino acid sequences are added and / or inserted, and / or one or more amino acids are substituted and / or deleted, or a derivative thereof, A protein and a derivative thereof, wherein the kinase activity is constitutively activated in the amino sequence. 17. The method according to claim 15, wherein the amino acid sequence at positions 943 to 1068 of SEQ ID NO: 4 or a region containing a part thereof having an active Rho protein binding ability has been deleted.
7. The protein according to 6. 18. The nucleotide sequence of SEQ ID NO: 4 from No. 90 to 359 or 6 to
Claim 15 or 1 consisting of the amino acid sequence at position 553.
7. The protein according to 6. (19) a base sequence encoding the protein according to any one of (1) to (4) and (14) to (18); [20] a base sequence encoding the protein according to any one of [5] to [9]; A base sequence encoding the protein according to any one of claims 10 to 13. 22. The base sequence according to claim 19, which has a part or all of the DNA sequence of SEQ ID NO: 5. 23. A part of the base sequence is 1 to 4 of SEQ ID NO: 5.
No. 167, 1263-1431, 1312-3372
No., 2395-3411, 2821-225, 2
No. 68-1077, No. 2827-3204, No. 2821
The base sequence according to claim 22, which is a DNA sequence of No. 3225 or No. 3373-4164. 24. A vector comprising the nucleotide sequence according to any one of claims 19 to 23. 25. A vector comprising the nucleotide sequence according to claim 20 or 21. 26. The vector according to claim 24, which is selected from the group consisting of a plasmid vector, a virus vector, and a liposome vector. 27. A host cell transformed with the vector according to any one of claims 24 to 26 (however, in the case of a human cell, it is limited to a cell isolated from a human). 28. An Escherichia coli, yeast, insect cell, Sf9 cell,
28. The host cell according to claim 27, which is selected from the group consisting of COS cells, lymph cells, fibroblasts, CHO cells, blood cells, and tumor cells. 29. The method according to claim 1, comprising culturing the host cell according to claim 27 or 28, and isolating the protein according to claim 1 from the culture. 19. The method for producing a protein according to any one of 18. (30) A screening target comprising an active Rho protein and a protein or a derivative thereof according to any one of (1) to (9), and (2) an activity of: An active Rho protein, comprising measuring the degree of inhibition of binding between the type Rho protein and the protein or a derivative thereof according to any one of claims 1 to 9, and any one of claims 1 to 9. A method for screening a substance that inhibits binding to the protein or a derivative thereof according to claim 1. 31. The screening method according to claim 30, wherein the screening system is a cell line or a cell-free system. 32. The screening method according to claim 30, wherein the screening system is a yeast two-hybrid system. 33. (1) The subject of screening is present in a screening system containing the protein or its derivative according to any one of claims 1 to 4 and 14 to 18, and (2) 19. The method according to any one of claims 1-4 and 14-18, comprising determining the degree of inhibition of protein kinase activity of the protein or derivative thereof according to any one of claims 4 and 14-18. A method for screening a substance that inhibits the activity of protein kinase of a protein or a derivative thereof. (1) A substance to be screened is present in a screening system containing an active Rho protein and the protein or a derivative thereof according to any one of claims 1 to 4, and 2) Claims 1-4
5. The protein or derivative thereof according to any one of claims 1 to 4, which comprises measuring the degree of inhibition of protein kinase activity or enhancement of the activity of the protein or derivative thereof according to any one of claims 1 to 4. A method for screening for a substance that inhibits the activity of protein kinase or its enhancement. 35. An active form R to be present in a screening system
The screening method according to claim 34, wherein the ho protein is a post-translationally modified protein. 36. The degree of inhibition of the activity or enhancement of protein kinase activity is determined by measuring the level of myelin basic protein, S6
Peptide, αPKC, vinculin, talin, metavinculin, caldesmon, filamin, vimentin, α
The screening according to any one of claims 33 to 35, wherein the measurement is performed using a substrate selected from the group consisting of actinin, MAP-4, myosin light chain phosphatase, and myosin binding subunit of myosin light chain phosphatase. Law. 37. The screening method according to any one of claims 33 to 35, wherein the degree of inhibition of protein kinase activity or enhanced activity is measured using myosin light chain as a substrate. 38. The method according to claim 37, wherein the degree of inhibition of protein kinase activity or enhanced activity is measured using an antibody against myosin light chain in which Ser-19 is phosphorylated.
2. The screening method according to 1. 39. The activity of protein kinase or the degree of inhibition of the enhancement of activity is determined by measuring acidic keratin, neutral and basic keratin, desmin, vimentin, GFAP, peripherin, neurofilament subunit, α-internexin, lamin, Measured using an intermediate filament selected from the group consisting of nestin as a substrate,
The screening method according to any one of claims 33 to 35. 40. The screening method according to claim 39, wherein the degree of inhibition of protein kinase activity or enhanced activity is measured using an antibody against a phosphorylated intermediate filament. 41. The intermediate filament is GFAP.
A screening method according to claim 39. 42. The degree of inhibition of protein kinase activity or enhanced activity is determined by measuring Ser-13 or Ser-34.
42. The screening method according to claim 41, wherein the measurement is carried out using an antibody against GFAP phosphorylated. (43) A cell in which the formation of a stress fiber or focal adhesion is induced by the presence of the protein or a derivative thereof according to any one of (14) to (18) as a substance to be screened. A method of screening for a substance that inhibits the formation of stress fibers or focal adhesion, which comprises measuring the degree of inhibition of the formation of stress fibers or focal adhesion in the cell line, and (2) measuring the degree of inhibition of the formation of stress fibers or focal adhesion in the cell line. 44. (1) The subject of screening is present in a cell line containing the protein or its derivative according to any one of claims 1 to 4 and 14 to 18, and (2) in the cell line. c-fos serum response factor (S
Measuring the degree of inhibition of the transcriptional activity of RE).
A screening method for a substance that inhibits the transcriptional activity of c-fos serum response factor (SRE). 45. The screening method according to any one of claims 30 to 44, which is a method for screening a substance that inhibits tumor formation or metastasis. 46. The screening method according to claim 30, which is a method for screening a smooth muscle contraction inhibitor. 47. A method for screening a substance that suppresses the aggregation or activation of platelets or leukocytes.
45. The screening method according to any one of -44. (48) a smooth muscle contraction inhibitor comprising the protein or derivative thereof according to any one of (5) to (13), or the base sequence of (20) or (21) or the vector of (25); . (49) a treatment for a circulatory disease comprising the protein or a derivative thereof according to any one of (5) to (13), the base sequence of (20) or (21), or the vector of (25); Agent. 50. The treatment according to claim 25, wherein the cardiovascular disease is selected from hypertension, vasospasm (cardiovascular and cerebral vasospasm), angina, myocardial infarction and obstructive atherosclerosis. Agent. (51) a method for suppressing tumor formation or metastasis, comprising the protein or a derivative thereof according to any one of (5) to (13), the base sequence of (20) or (21), or the vector of (25); Agent. 52. An inflammatory disease or an autologous disease comprising the protein according to any one of claims 5 to 13 or a derivative thereof, or the base sequence according to claim 20 or 21 or the vector according to claim 25. Immunological disease therapeutic agent. 53. A platelet aggregation inhibitor comprising the protein according to any one of claims 5 to 13 or a derivative thereof, or the base sequence according to claim 20 or 21 or the vector according to claim 25. 54. A nucleotide sequence selected from the following: (a) a nucleotide sequence comprising a DNA sequence of at least 15 consecutive nucleotides in the sequence of SEQ ID NO: 5, a sequence complementary thereto, and (b) (a) A) a base sequence which hybridizes with the base sequence under stringent conditions. 55. The nucleotide sequence according to claim 54, which is selected from the group consisting of the nucleotide sequence of positions 930 to 2025 of SEQ ID NO: 5 and its complementary sequence. 56. A base sequence selected from the following: (a) a base sequence containing a DNA sequence of at least 15 consecutive bases in the sequence of SEQ ID NO: 6, a sequence complementary thereto, and (b) (a) A) a base sequence which hybridizes with the base sequence under stringent conditions. 57. SEQ ID NO: 6 646-667 and 5
The base sequence according to claim 56, wherein the base sequence is selected from the group consisting of the base sequences of Nos. 36 to 555 and their complementary sequences. 58. A probe comprising the nucleotide sequence according to claim 54 or 56 and a label. 59. The probe of claim 58, wherein the label is selected from the group consisting of an enzyme, a radioisotope, fluorescence, and an antigen. A diagnostic agent comprising the base sequence according to any one of claims 54 to 57 or the probe according to claim 58 or 59. 61. The method according to claim 6, which is used for diagnosing a malignant tumor.
The diagnostic agent according to 0.