- Email: [email protected]
Effect of chlorpromazine on the smg GDS action
Vol.
182,
February
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
14, 1992
EFFECT
OF CHLORPROMAZINE
Xmg Department
ON THE smg GDS ACTION1
Sik Kim2, Akira Kikuchi, of
Biochemistry, Medicine,
Kobe
1446-1453
and Yoshimi Takai 3
Kobe University 650, Japan
School
of
Received January 13, 1992 S~mmarg: A stimulatory GDP/GTP exchange protein for smg p21 (smg GDS) stimulated the binding of guanosine 5'-(3-O-thio)triphosphate (GTPYS) to smg p21B. Chlorpromazine (CPZ) inhibited the smg GDS action in a manner competitive with smg GDS and in a manner noncompetitive with smg p21B. In spite of the inhibitory effect of CPZ on the smg GDS action, it counteracted the inhibition of the smg GDS action by acidic phospholipids. These results suggest that CPZ interacts with smg p21B, smg GDS, or both, and thereby inhibits the smg GDS action, and that CPZ also interacts with the acidic phospholipids and thereby counteracts their inhibitory effect on the smg GDS action. 0 1992 Academic Press, Inc.
The smg p21 family, consisting of A and B members, belongs to a ras p2l/ras p21-like small G protein superfamily (1,2). smg p21A is identical to the raplA and Krev-1 proteins and smg p21B is identical
to the
modified
with
raplB
protein
a geranylgeranyl
(3-5).
smg p21B is
moiety
at
its
post-translationally C-terminal cysteine
1 This investigation was supported by grants-in-aid for Scientific Research and Cancer Research from the Ministry of Education, Science and Culture, Japan (1990) and a grant-in-aid for Abnormalities in Hormone Receptor Mechanisms from the Ministry of Health and Welfare, Japan (1990), and by grants from the Human Frontier Science Program (1990), the Yamanouchi Foundation for Research on Metabolic Disease (1990) and the Research Program on Cell Calcium Signal in Cardiovascular System (1990). 2 Present address: Department of Psychiatry, College of I-ledicine, Seoul National CYniversity,Seoul 110, Korea. 3 To whom all correspondence should be addressed. Abbreviations used are: G protein, GTP-binding protein; GDS, GDP dissociation stimulator; PA, phosphatidic acid; PI, phosphatidylinositol; PIP, phosphatidylinositol-4-monophosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PS, phosphatidylserine; CPZ, chlorpromazine; GTPyS, guanosine 5'-(3-O-thio)triphosphate. ooo6-291x/92$1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1446
Vol.
182, No. 3, 1992
residue
BIOCHEMICAL
and has a polybasic
teine residue (6). p21B are essential
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
region
upstream
of the prenylated
cys-
The post-translational modifications of smg for its binding to membranes, and the polybasic
region of smg p21B may interact with acidic polar head groups of membrane phospholipids (7). smg GDS stoichiometrically forms a complex with smg ~21 and stimulates its GD,P/GTP exchange reaction the binding of smg p21 to membranes (8) . smg GDS also regulates modifications of smg p21B are neces(9) * The post-translational sary for its interaction with smg GDS (7). The smg GDS action to stimulate the GDP/GTP exchange reaction of smg p21B is inhibited by acidic phospholipids p21B is phosphorylated which is cysteine
located residue
hibitory lated
effect of
form
smg
such as PA, PI, PIP, PIP2, and PS (9). smg by protein kinase A at its Ser17' residue,
between (10,ll).
the polybasic The acidic
region and the prenylated phospholipids show a less
in-
on the smg GDS-induced activation of the phosphoryp21B than on that of the non-phosphorylated form
of smg p21B (12).
it is likely that the phosphorylation Therefore, of smg p21B reduces the positive charge of its C-terminal region, inhibits the interaction with the acidic phospholipids, and thereby reverses
the
inhibition
of the
smg GDS action
by the acidic
phos-
pholipids. It is possible from these lipid-interacting drugs affect
observations that membrane phosphothe smg GDS action. CPZ is a repre-
sentative drug of cationic amphiphilic drugs which interact membrane phospholipids. The interactions induce cellular changes (13,14) and alterations of many steps metabolism (15-17). Furthermore, CPZ inhibits activity
in a manner competitive
with
Materials
of phospholipid the protein kinase
phospholipids
paper, we have examined whether the cationic cluding CPZ affect the smg GDS action.
with shape
(18).
amphiphilic
C
In this drugs
in-
and Methods
Materials and Chemicals -smg p21B was purified from human platelet membranes (19). sm GDS was purified from an overexpressing E. coli strain (20). [ 95 SlGTP'yS (44.4 TBq/mmol) was purchased from Du Pont-New England Nuclear. CPZ, propranolol, and dibucaine were from Sigma. Amitriptyline and baclofen were from Research Biochemical Inc. Phenobarbital was from Wako Pure Chemical Industry. PA, PI, and PS were from Serdary Research Laboratory. Nitrocellulose filters BA-85 (pore size, 0.45 pm) were from Schleicher & Schuell. Assay for the smg GDS Action -The smg GDS action to stimulate the binding of [35 SJGTPYS to smg p21B was assayed as described (8) except that smg GDS was first incubated for 5 min at 25OC with various concentrations of a drug and/or a phospholipid in 30 pl of 8.3 mM Tris/HCl at pH 7.5, 0.17 mM EDTA, and 0.33 mM 1447
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The concentrations of smg GDS and smg p21B used in dithiothreitol. the standard assay were 30 nM and 20 nM, respectively, and the incubation time in the standard assay was 20 min. Under these condithe assay was within linear range. tions,
CPZ inhibited [35S]GTPyS
to
the
smg GDS action
to
smg p21B in a dose-dependent
stimulate manner
the binding (Fig.
of
1).
The IC50 value of CPZ on the smg GDS action was about 2 x 10-4 M. CPZ inhibited the smg GDS action in a manner competitive with smg GDS and in a manner noncompetitive with smg p21B (Fig. 2, A and CPZ did not affect the basal veB) . Under the same conditions, locity
of the
shown). Although
GDP/GTP exchange
reaction
CPZ inhibits
smg GDS action,
the
of smg p21B (data it
not
counteracted
the
PA-induced
inhibition of the smg GDS action in a dose-related manPA inhibited the smg GDS action in a dose-dependent ner (Fig. 3). manner in the absence of CPZ. However, PA did not inhibit the smg GDS action
in the
presence
of
1 x 10B3 M, 2 x 10s4 M, or 1 x 10B4 M
CPZ. Other acidic phospholipids, smg GDS action (data not shown). PS-induced inhibition Amitriptyline, smg GDS action.
such as PI and PS, inhibited the CPZ also counteracted the PI- and
of the smg GDS action (data not shown). propranolol, and dibucaine also inhibited
Baclofen
or phenobarbital
did
not
show the
the in-
#'ia. 1, Effect of CPZ on the smg 009 action. The smg GDSaction was assayed in the presence of various concentrations of CPZ. The smg GDSaction was expressed as percentage relative to the activity in the absence of CPZ. The results shown are means f S.E. of six independent experiments. 1448
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
,.OT--
7
1.0
,0.8 8 e D 5 0.6
Sl.5 B 0 5 $ 1.0
;,.4 a I L
c iii a -0.5
0.2
0
b
0
20
40 60 80 smg GDS (nM)
1M
1D
0E 0
50 loo smg p21 B (nM)
150
Kinetic study of the smg GDS action in the presence-gf CPZ. The smg GDS action was assayed in the presence of 2 x 10 M CPZ. A, with various concentrations of smg GDS in the presence of the fixed concentration of smg p21B (20 nM). B, with various concentrations of smg p21B in the presence of the fixed concentration of smg GDS (30 nM). in the absence of CO), in the presence of CPZ. cpz; (e), The results shown are representative of three independent experiments.
hibitory nolol,
effect and
dibucaine
smg GDS action. this
counteracting
(Table
1).
Furthermore,
counteracted However, activity
amitriptyline, PA-induced inhibition
the
baclofen
or
phenobarbital
(Table
2).
20
30 PA @g/ml)
I
did
I
I
40
50
propraof not
show
Fia. 3, Effect of CPZ on the PA-induced inhibition of the smg GDS action. The smg GDS action was assayed in the presence of various concentrations of CPZ and/or PA. The smg GDS action was expressed as percentage relative to the activity in the absence of CPZ and P in the absence of CPZ; (e), in the (0 )I presence of 1 x 10 -2. M CPZ; (A), in he presence of 1 x 10s5 M -5 1 x 10 CPZ; (A), in the presence of M CPZ; (O), in he presence of 2 x 10s4 M CPZ; ( n ), in the presence of 1 x 10 -5 M CPZ. The results shown are representative of three independent experiments. 1449
the
Vol.
182,
No.
BIOCHEMICAL
3, 1992
Table
1.
Effect
of
AND
various
BIOPHYSICAL
drugs
on
Drugs
RESEARCH
the
smg
COMMUNICATIONS
GDS action
smg GDS action
(9)
CPZ
29 f 7.6
(N=6)
Amitriptyline
30 f
6.0
(N=4)
Propranolol
56 + 9.6
(N=6)
Dibucaine
37 f 2.3
(N=6)
Baclofen
100 + 6.7
(N=6)
Phenobarbital
134 k 8.7
(N=4)
The smg GDS action was assayed in various drugs. The smg GDS action was ative to the activity in the absence of shown are means It: S.E. of the indicated
Table
2.
Effect
of
bition
various of the
Drugs
None
drugs
smg
the presence expressed as the drugs. number (N)
on the GDS action
PA-induced
-PA
+PA
(%)
(%)
100
of 1 x 10m3 M percentage relThe results of experiments.
29 f
7.6
(N=6)
CPZ
92 + 5.4
88 f
7.9
(N=6)
Amitriptyline
89 f 7.1
98 f
7.4
(N=4)
Propranolol
98 + 10
91 f 3.4
(N=4)
Dibucaine
95 f 2.7
105 f
9.8
(N=3)
Baclofen
99 f 13
35 f
11
(N=4)
103 f 14
36 f
10
(N=4)
Phenobarbital
inhi-
The smg GDS action was assayed in the presence of 1 x 10q4 M various drugs and/or 10 pg/ml PA. The smg GDS action was expressed as percentage relative to the activity in the absence of CPZ and PA. The results shown are means f S.E. of the indicated number (N) of experiments.
1450
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Discussion
We have shown here that CPZ inhibits the smg GDS action to stimulate the binding of [35 SIGTPYS to smg p21B without affecting the basal velocity of its GDP/GTP exchange reaction. CPZ is a cationic amphiphilic drug, mechanism of the inhibition previous
results
geranylgeranyl p21B interacts
show that
moiety in its C-terminal region through with smg GDS (6,7). The smg GDS action
is of
inhibited by the smg p21B reverses
the
smg GDS action
ative tion
charge of the
and this property may be related to the of the smg GDS action (14,21-24). Our smg p21B has a polybasic region and a
acidic phospholipids, and the the acidic phospholipid-induced (9,12).
Therefore,
acidic
phospholipids
of the interaction
of
smg GDS with
itive charge of CPZ contributes smg GDS action. We have also inhibition of the
it
to
shown here that smg GDS action.
its
phosphorylation inhibition
is possible contributes smg p21B,
inhibitory
which smg for smg p21B
and that effect
CPZ counteracts We have already
of
that the negto the inhibithe
pos-
on the
the PA-induced demonstrated
that PA itself inhibits the smg GDS action in a manner competitive with smg GDS or smg p21B (9). However, the effect of PA and CPZ is neither synergistic nor additive on the smg GDS action. Instead, CPZ counteracts This
effect
lifies hibition
of
the
PA-induced
CPZ might
inhibition
be due to its
of the positive
smg GDS action.
charge,
which
the negative charge of PA and counteracts the PA-induced of the smg GDS action. In addition to a electrostatic
nulinin-
teraction, it is possible that CPZ forms a complex with smg GDS or smg p21B through the hydrophobic interaction, and that this complex formation hinders the access of PA to smg GDS or smg p21B. Therethe PA-induced inhibition of the smg GDS action can not be fore, fully
reversed
by CPZ.
However,
this
interaction
minor role compared with the electrostatic fect of CPZ on the PA induced-inhibition cause baclofen
with
charge
does not
action
(14,25). Other cationic
no net
counteract
charge the
have a
interaction in the efof the smg GDS action, be-
or phenobarbital
PA-induced
seems to
inhibition
with
a negative
of the
smg GDS
amphiphilic drugs, such as amitriptyline, proalso inhibit the smg GDS action and counpranolol, and dibucaine, teract the PA-induced inhibition of the smg GDS action. These observations suggest that the effect of CPZ might be common characCPZ also counteracts teristics of the cationic amphiphilic drugs. The effect the PI- or PS-induced inhibition of the smg GDS action. of CPZ on PA also seems common to the acidic phospholipids. The 1451
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
significance of the effect of the cationic amphiphilic smg GDS action is not known, but these drugs may serve tool
to
clarify
the
mode of activation
drugs on the as a useful
of smg p21 by smg GDS.
Acknowledgment We are grateful
to J.
Yamaguchi
for
her skillful
secretarial
assistance.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y ., and Takai, Y. (1988) J. Biol. Chem. 263, 18968-18971. Matsui, Y., Kikuchi, A., Kawata, M., Kondo, J., Teranishi, Y ., and Takai, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1010-1016. Pizon, V., Chardin, P., Lerosey, I., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204. Pizon, V., Lerosey, I., Chardin, P., and Tavitian, A. (1988) Nucleic Acids Res. 16, 7719. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84. Kawata, M., Farnsworth, C.C., Yoshida, Y., Gelb, M.H., Glomset, J.A., and Takai, Y. (1990) Proc. Natl. Acad. Sci. USA 87, 8960-8964. Hiroyoshi, M., Kaibuchi, K., Kawamura, S., Hata, Y., and Takai, Y. (1991) J. Biol. Chem. 266, 2962-2969. Yamamoto, T., Kaibuchi, K., Mizuno, T., Hiroyoshi, M., Shirataki, H., and Takai, Y. (1990) J. Biol. Chem. 265, 16626-16634. Kawamura, S., Kaibuchi, K., Hiroyoshi, M., Fujioka, H., Mizuno, T., and Takai, Y. (1991) Jpn. J. Cancer ReS. 82, 758761. Kawata, M., Kikuchi, A., Hoshijima, M., Yamamoto, K., Hashimoto, E., Yamamoto, H., and Takai, Y. (1989) J. Biol. Chem. 264, 15688-15695. Hata, Y., Kaibuchi, K., Hiroyoshi, M., Kawamura, S., Shirataki, H., and Takai, Y. (1991) J. Biol. Chem. 266, 65716577. Itoh, T., Kaibuchi, K., Sasaki, T., and Takai, Y. (1991) Biochem. Biophys. Res. Commun. 177, 1319-1324. Butikoper, P., Lin, Z.W., Kuypers, F.A., Scott, M.D., Wagner, G.M., Chiu, D.T., and Lubin, B. (1989) Blood 73, 1699-1704. ROSSO, J., Zachowski, A., and Devaux, P.F. (1988) Biochim. Biophys. Acta 942, 271-279. Abdel-Latif, A.A. Metabolism of phosphoinositides. In A. Lajtha (Ed.), (1983) Handbook of Neurochemistry Vol.3, Plenum Press, New York pp. 91-131. Kodavanti, U., and Mehendale, H. (1990) Pharmacol. Rev. 42, 327-354. Koul, O., and Hauser, G. (1987) Arch. Biochem. Biophys. 253, 453-461. Mori, T., Takai, Y., Minakuchi, R., Yu, B., and Nishizuka, Y. (1980) J. Biol. Chem. 255, 8378-8380. Ohmori, T., Kikuchi, A., Yamamoto, K., Kim, S., and Takai, Y. (1989) J. Biol. Chem. 264, 1877-1881. 1452
Vol.
20. 21. 22. 23. 24. 25.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Kaibuchi, K., Mizuno, T., Fujioka, H., Yamamoto, T., Kishi, Fukumoto, Y., Hori, Y., and Takai, Y. (1991) Mol. Cell. K B&. 11, 2691-2697. Barthel, D., Zschoernig, O., Lange, K., Lenk, R., and Arnold, K. (1988) Biochim. Biophys. Acta. 945, 361-366. Leli, U., and Hauser, G. (1987) Biochim. Biophys. Acta. 918, 126-135. Ogiso, T., Imai, S., Hozumi, R., Kurobe, M., and Kato, Y. (1976) Pharmacol. Bull. 24, 479-486. Yamaguchi, T., Watanabe, S., and Kimoto, E. (1985) Biochim. Biophys. Acta. 820, 157-164. Ahuja, S., Baclofen, In K. Florey (Ed. ), (1985) Analytical Profiles of Drug Substances, Vol. 14, Academic Press, Orlando, PP. 527-548.
1453
182,
February
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
14, 1992
EFFECT
OF CHLORPROMAZINE
Xmg Department
ON THE smg GDS ACTION1
Sik Kim2, Akira Kikuchi, of
Biochemistry, Medicine,
Kobe
1446-1453
and Yoshimi Takai 3
Kobe University 650, Japan
School
of
Received January 13, 1992 S~mmarg: A stimulatory GDP/GTP exchange protein for smg p21 (smg GDS) stimulated the binding of guanosine 5'-(3-O-thio)triphosphate (GTPYS) to smg p21B. Chlorpromazine (CPZ) inhibited the smg GDS action in a manner competitive with smg GDS and in a manner noncompetitive with smg p21B. In spite of the inhibitory effect of CPZ on the smg GDS action, it counteracted the inhibition of the smg GDS action by acidic phospholipids. These results suggest that CPZ interacts with smg p21B, smg GDS, or both, and thereby inhibits the smg GDS action, and that CPZ also interacts with the acidic phospholipids and thereby counteracts their inhibitory effect on the smg GDS action. 0 1992 Academic Press, Inc.
The smg p21 family, consisting of A and B members, belongs to a ras p2l/ras p21-like small G protein superfamily (1,2). smg p21A is identical to the raplA and Krev-1 proteins and smg p21B is identical
to the
modified
with
raplB
protein
a geranylgeranyl
(3-5).
smg p21B is
moiety
at
its
post-translationally C-terminal cysteine
1 This investigation was supported by grants-in-aid for Scientific Research and Cancer Research from the Ministry of Education, Science and Culture, Japan (1990) and a grant-in-aid for Abnormalities in Hormone Receptor Mechanisms from the Ministry of Health and Welfare, Japan (1990), and by grants from the Human Frontier Science Program (1990), the Yamanouchi Foundation for Research on Metabolic Disease (1990) and the Research Program on Cell Calcium Signal in Cardiovascular System (1990). 2 Present address: Department of Psychiatry, College of I-ledicine, Seoul National CYniversity,Seoul 110, Korea. 3 To whom all correspondence should be addressed. Abbreviations used are: G protein, GTP-binding protein; GDS, GDP dissociation stimulator; PA, phosphatidic acid; PI, phosphatidylinositol; PIP, phosphatidylinositol-4-monophosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PS, phosphatidylserine; CPZ, chlorpromazine; GTPyS, guanosine 5'-(3-O-thio)triphosphate. ooo6-291x/92$1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1446
Vol.
182, No. 3, 1992
residue
BIOCHEMICAL
and has a polybasic
teine residue (6). p21B are essential
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
region
upstream
of the prenylated
cys-
The post-translational modifications of smg for its binding to membranes, and the polybasic
region of smg p21B may interact with acidic polar head groups of membrane phospholipids (7). smg GDS stoichiometrically forms a complex with smg ~21 and stimulates its GD,P/GTP exchange reaction the binding of smg p21 to membranes (8) . smg GDS also regulates modifications of smg p21B are neces(9) * The post-translational sary for its interaction with smg GDS (7). The smg GDS action to stimulate the GDP/GTP exchange reaction of smg p21B is inhibited by acidic phospholipids p21B is phosphorylated which is cysteine
located residue
hibitory lated
effect of
form
smg
such as PA, PI, PIP, PIP2, and PS (9). smg by protein kinase A at its Ser17' residue,
between (10,ll).
the polybasic The acidic
region and the prenylated phospholipids show a less
in-
on the smg GDS-induced activation of the phosphoryp21B than on that of the non-phosphorylated form
of smg p21B (12).
it is likely that the phosphorylation Therefore, of smg p21B reduces the positive charge of its C-terminal region, inhibits the interaction with the acidic phospholipids, and thereby reverses
the
inhibition
of the
smg GDS action
by the acidic
phos-
pholipids. It is possible from these lipid-interacting drugs affect
observations that membrane phosphothe smg GDS action. CPZ is a repre-
sentative drug of cationic amphiphilic drugs which interact membrane phospholipids. The interactions induce cellular changes (13,14) and alterations of many steps metabolism (15-17). Furthermore, CPZ inhibits activity
in a manner competitive
with
Materials
of phospholipid the protein kinase
phospholipids
paper, we have examined whether the cationic cluding CPZ affect the smg GDS action.
with shape
(18).
amphiphilic
C
In this drugs
in-
and Methods
Materials and Chemicals -smg p21B was purified from human platelet membranes (19). sm GDS was purified from an overexpressing E. coli strain (20). [ 95 SlGTP'yS (44.4 TBq/mmol) was purchased from Du Pont-New England Nuclear. CPZ, propranolol, and dibucaine were from Sigma. Amitriptyline and baclofen were from Research Biochemical Inc. Phenobarbital was from Wako Pure Chemical Industry. PA, PI, and PS were from Serdary Research Laboratory. Nitrocellulose filters BA-85 (pore size, 0.45 pm) were from Schleicher & Schuell. Assay for the smg GDS Action -The smg GDS action to stimulate the binding of [35 SJGTPYS to smg p21B was assayed as described (8) except that smg GDS was first incubated for 5 min at 25OC with various concentrations of a drug and/or a phospholipid in 30 pl of 8.3 mM Tris/HCl at pH 7.5, 0.17 mM EDTA, and 0.33 mM 1447
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The concentrations of smg GDS and smg p21B used in dithiothreitol. the standard assay were 30 nM and 20 nM, respectively, and the incubation time in the standard assay was 20 min. Under these condithe assay was within linear range. tions,
CPZ inhibited [35S]GTPyS
to
the
smg GDS action
to
smg p21B in a dose-dependent
stimulate manner
the binding (Fig.
of
1).
The IC50 value of CPZ on the smg GDS action was about 2 x 10-4 M. CPZ inhibited the smg GDS action in a manner competitive with smg GDS and in a manner noncompetitive with smg p21B (Fig. 2, A and CPZ did not affect the basal veB) . Under the same conditions, locity
of the
shown). Although
GDP/GTP exchange
reaction
CPZ inhibits
smg GDS action,
the
of smg p21B (data it
not
counteracted
the
PA-induced
inhibition of the smg GDS action in a dose-related manPA inhibited the smg GDS action in a dose-dependent ner (Fig. 3). manner in the absence of CPZ. However, PA did not inhibit the smg GDS action
in the
presence
of
1 x 10B3 M, 2 x 10s4 M, or 1 x 10B4 M
CPZ. Other acidic phospholipids, smg GDS action (data not shown). PS-induced inhibition Amitriptyline, smg GDS action.
such as PI and PS, inhibited the CPZ also counteracted the PI- and
of the smg GDS action (data not shown). propranolol, and dibucaine also inhibited
Baclofen
or phenobarbital
did
not
show the
the in-
#'ia. 1, Effect of CPZ on the smg 009 action. The smg GDSaction was assayed in the presence of various concentrations of CPZ. The smg GDSaction was expressed as percentage relative to the activity in the absence of CPZ. The results shown are means f S.E. of six independent experiments. 1448
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
,.OT--
7
1.0
,0.8 8 e D 5 0.6
Sl.5 B 0 5 $ 1.0
;,.4 a I L
c iii a -0.5
0.2
0
b
0
20
40 60 80 smg GDS (nM)
1M
1D
0E 0
50 loo smg p21 B (nM)
150
Kinetic study of the smg GDS action in the presence-gf CPZ. The smg GDS action was assayed in the presence of 2 x 10 M CPZ. A, with various concentrations of smg GDS in the presence of the fixed concentration of smg p21B (20 nM). B, with various concentrations of smg p21B in the presence of the fixed concentration of smg GDS (30 nM). in the absence of CO), in the presence of CPZ. cpz; (e), The results shown are representative of three independent experiments.
hibitory nolol,
effect and
dibucaine
smg GDS action. this
counteracting
(Table
1).
Furthermore,
counteracted However, activity
amitriptyline, PA-induced inhibition
the
baclofen
or
phenobarbital
(Table
2).
20
30 PA @g/ml)
I
did
I
I
40
50
propraof not
show
Fia. 3, Effect of CPZ on the PA-induced inhibition of the smg GDS action. The smg GDS action was assayed in the presence of various concentrations of CPZ and/or PA. The smg GDS action was expressed as percentage relative to the activity in the absence of CPZ and P in the absence of CPZ; (e), in the (0 )I presence of 1 x 10 -2. M CPZ; (A), in he presence of 1 x 10s5 M -5 1 x 10 CPZ; (A), in the presence of M CPZ; (O), in he presence of 2 x 10s4 M CPZ; ( n ), in the presence of 1 x 10 -5 M CPZ. The results shown are representative of three independent experiments. 1449
the
Vol.
182,
No.
BIOCHEMICAL
3, 1992
Table
1.
Effect
of
AND
various
BIOPHYSICAL
drugs
on
Drugs
RESEARCH
the
smg
COMMUNICATIONS
GDS action
smg GDS action
(9)
CPZ
29 f 7.6
(N=6)
Amitriptyline
30 f
6.0
(N=4)
Propranolol
56 + 9.6
(N=6)
Dibucaine
37 f 2.3
(N=6)
Baclofen
100 + 6.7
(N=6)
Phenobarbital
134 k 8.7
(N=4)
The smg GDS action was assayed in various drugs. The smg GDS action was ative to the activity in the absence of shown are means It: S.E. of the indicated
Table
2.
Effect
of
bition
various of the
Drugs
None
drugs
smg
the presence expressed as the drugs. number (N)
on the GDS action
PA-induced
-PA
+PA
(%)
(%)
100
of 1 x 10m3 M percentage relThe results of experiments.
29 f
7.6
(N=6)
CPZ
92 + 5.4
88 f
7.9
(N=6)
Amitriptyline
89 f 7.1
98 f
7.4
(N=4)
Propranolol
98 + 10
91 f 3.4
(N=4)
Dibucaine
95 f 2.7
105 f
9.8
(N=3)
Baclofen
99 f 13
35 f
11
(N=4)
103 f 14
36 f
10
(N=4)
Phenobarbital
inhi-
The smg GDS action was assayed in the presence of 1 x 10q4 M various drugs and/or 10 pg/ml PA. The smg GDS action was expressed as percentage relative to the activity in the absence of CPZ and PA. The results shown are means f S.E. of the indicated number (N) of experiments.
1450
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Discussion
We have shown here that CPZ inhibits the smg GDS action to stimulate the binding of [35 SIGTPYS to smg p21B without affecting the basal velocity of its GDP/GTP exchange reaction. CPZ is a cationic amphiphilic drug, mechanism of the inhibition previous
results
geranylgeranyl p21B interacts
show that
moiety in its C-terminal region through with smg GDS (6,7). The smg GDS action
is of
inhibited by the smg p21B reverses
the
smg GDS action
ative tion
charge of the
and this property may be related to the of the smg GDS action (14,21-24). Our smg p21B has a polybasic region and a
acidic phospholipids, and the the acidic phospholipid-induced (9,12).
Therefore,
acidic
phospholipids
of the interaction
of
smg GDS with
itive charge of CPZ contributes smg GDS action. We have also inhibition of the
it
to
shown here that smg GDS action.
its
phosphorylation inhibition
is possible contributes smg p21B,
inhibitory
which smg for smg p21B
and that effect
CPZ counteracts We have already
of
that the negto the inhibithe
pos-
on the
the PA-induced demonstrated
that PA itself inhibits the smg GDS action in a manner competitive with smg GDS or smg p21B (9). However, the effect of PA and CPZ is neither synergistic nor additive on the smg GDS action. Instead, CPZ counteracts This
effect
lifies hibition
of
the
PA-induced
CPZ might
inhibition
be due to its
of the positive
smg GDS action.
charge,
which
the negative charge of PA and counteracts the PA-induced of the smg GDS action. In addition to a electrostatic
nulinin-
teraction, it is possible that CPZ forms a complex with smg GDS or smg p21B through the hydrophobic interaction, and that this complex formation hinders the access of PA to smg GDS or smg p21B. Therethe PA-induced inhibition of the smg GDS action can not be fore, fully
reversed
by CPZ.
However,
this
interaction
minor role compared with the electrostatic fect of CPZ on the PA induced-inhibition cause baclofen
with
charge
does not
action
(14,25). Other cationic
no net
counteract
charge the
have a
interaction in the efof the smg GDS action, be-
or phenobarbital
PA-induced
seems to
inhibition
with
a negative
of the
smg GDS
amphiphilic drugs, such as amitriptyline, proalso inhibit the smg GDS action and counpranolol, and dibucaine, teract the PA-induced inhibition of the smg GDS action. These observations suggest that the effect of CPZ might be common characCPZ also counteracts teristics of the cationic amphiphilic drugs. The effect the PI- or PS-induced inhibition of the smg GDS action. of CPZ on PA also seems common to the acidic phospholipids. The 1451
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
significance of the effect of the cationic amphiphilic smg GDS action is not known, but these drugs may serve tool
to
clarify
the
mode of activation
drugs on the as a useful
of smg p21 by smg GDS.
Acknowledgment We are grateful
to J.
Yamaguchi
for
her skillful
secretarial
assistance.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y ., and Takai, Y. (1988) J. Biol. Chem. 263, 18968-18971. Matsui, Y., Kikuchi, A., Kawata, M., Kondo, J., Teranishi, Y ., and Takai, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1010-1016. Pizon, V., Chardin, P., Lerosey, I., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204. Pizon, V., Lerosey, I., Chardin, P., and Tavitian, A. (1988) Nucleic Acids Res. 16, 7719. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84. Kawata, M., Farnsworth, C.C., Yoshida, Y., Gelb, M.H., Glomset, J.A., and Takai, Y. (1990) Proc. Natl. Acad. Sci. USA 87, 8960-8964. Hiroyoshi, M., Kaibuchi, K., Kawamura, S., Hata, Y., and Takai, Y. (1991) J. Biol. Chem. 266, 2962-2969. Yamamoto, T., Kaibuchi, K., Mizuno, T., Hiroyoshi, M., Shirataki, H., and Takai, Y. (1990) J. Biol. Chem. 265, 16626-16634. Kawamura, S., Kaibuchi, K., Hiroyoshi, M., Fujioka, H., Mizuno, T., and Takai, Y. (1991) Jpn. J. Cancer ReS. 82, 758761. Kawata, M., Kikuchi, A., Hoshijima, M., Yamamoto, K., Hashimoto, E., Yamamoto, H., and Takai, Y. (1989) J. Biol. Chem. 264, 15688-15695. Hata, Y., Kaibuchi, K., Hiroyoshi, M., Kawamura, S., Shirataki, H., and Takai, Y. (1991) J. Biol. Chem. 266, 65716577. Itoh, T., Kaibuchi, K., Sasaki, T., and Takai, Y. (1991) Biochem. Biophys. Res. Commun. 177, 1319-1324. Butikoper, P., Lin, Z.W., Kuypers, F.A., Scott, M.D., Wagner, G.M., Chiu, D.T., and Lubin, B. (1989) Blood 73, 1699-1704. ROSSO, J., Zachowski, A., and Devaux, P.F. (1988) Biochim. Biophys. Acta 942, 271-279. Abdel-Latif, A.A. Metabolism of phosphoinositides. In A. Lajtha (Ed.), (1983) Handbook of Neurochemistry Vol.3, Plenum Press, New York pp. 91-131. Kodavanti, U., and Mehendale, H. (1990) Pharmacol. Rev. 42, 327-354. Koul, O., and Hauser, G. (1987) Arch. Biochem. Biophys. 253, 453-461. Mori, T., Takai, Y., Minakuchi, R., Yu, B., and Nishizuka, Y. (1980) J. Biol. Chem. 255, 8378-8380. Ohmori, T., Kikuchi, A., Yamamoto, K., Kim, S., and Takai, Y. (1989) J. Biol. Chem. 264, 1877-1881. 1452
Vol.
20. 21. 22. 23. 24. 25.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Kaibuchi, K., Mizuno, T., Fujioka, H., Yamamoto, T., Kishi, Fukumoto, Y., Hori, Y., and Takai, Y. (1991) Mol. Cell. K B&. 11, 2691-2697. Barthel, D., Zschoernig, O., Lange, K., Lenk, R., and Arnold, K. (1988) Biochim. Biophys. Acta. 945, 361-366. Leli, U., and Hauser, G. (1987) Biochim. Biophys. Acta. 918, 126-135. Ogiso, T., Imai, S., Hozumi, R., Kurobe, M., and Kato, Y. (1976) Pharmacol. Bull. 24, 479-486. Yamaguchi, T., Watanabe, S., and Kimoto, E. (1985) Biochim. Biophys. Acta. 820, 157-164. Ahuja, S., Baclofen, In K. Florey (Ed. ), (1985) Analytical Profiles of Drug Substances, Vol. 14, Academic Press, Orlando, PP. 527-548.
1453