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Inhibition of the calcium- and phospholipid-dependent protein kinase activity from mouse brain cytosol by quercetin
Vol. 117, No. 2, 1983 December
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 444-447
16, 1983
INHIBITION OF THE CALCIUM- AND PHOSPHOLIPID-DEPENDENT PROTEIN KINASE ACTIVITY FROM MOUSE BRAIN CYTOSOL BY QUERCETIN M. Gschwendt,
F. Horn,
W. Kittstein
and F. Marks
Deutsches Krebsforschungszentrum, Institut fur Im Neuenheimer Feld 280, D - 6900 Heidelberg, Received
October
19,
Biochemie, F.R.G.
1983
The flavonoid quercetin is a potent inhibitor of calciumand phospholipid-dependent protein kinase (Ca,PL-PK) activity from mouse brain. Halfmaximal inhibition of the kinase occurs at about 10 !JM. If the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is used instead of calcium as a stimulating factor of the kinase enzyme activity is still inhibited by quercetin. The kinase inhibitor, however, does not interfere with the binding of TPA to its receptor. The flavonoid example
quercetin
cyclooxygenase
decarboxylase
is
known to inhibit
and 15-lipoxygenase
and xanthinoxidase
(2),
CAMP and cGMP phosphodiesterase (11)
and CAMP-independent
inhibits (17),
D-glucose
exhibits our
(18)
cells mutagenic
studies,
thought
it
(19).
activity appears
to
to be initiated receptor.
be identical
the
with
well
but
that
it
as well
(21)
and most
inhibit
tumor
Ca,PL-PK
by TPA (23).
Ca,PL-PK not
interfere
systems
of lactate
and proliferation
as an antioxidant with (22).
quercetin
respect
most
of this inhibits
from mouse brain
it to
is
compounds
found
binding
of
(20),
Promotion
promoting
The activity
the
(3,4),
quercetin
has been
with
hexokinase
reductase
as growth
promotion
activity
histidin-
aldolase
transport
of tumor
that
for
Furthermore,
interestingly
C (23-25).
We now report
does
(7-lo),
can function
The TPA receptor
as the TPA stimulated
tively,
It
bound
(12-15). (16),
by the binding
TPA to a specific stimulated
ATPase
synthesis
enzymes,
hyaluronidase,
mitochondrial
kinases
and Ca2+ (9,lO)
in vitro
(l),
(5,6),
protein
DNA, RNA and protein
malignant
various
like
likely kinase
the very
Ca
2+
to is as
effec-
of TPA to its
re-
ceptor. MATERIALS
AND METHODS
Materials: TPA was kindly supplied by Prof. E. Hecker, German Cancer Resejrch Center, Heidelberg. 20 Ci/mnole, and Y~~P-ATP, spec. activity [20- H(N)]-TPA, spec. activity 9 Ci/mmole, were from New England Nuclear. Phosphatidylserine, histone III-S and quercetin (3,3',4',5,7-pentahydroxyflavone) were from Sigma, MUnchen. Rutin (quercetin-3-rutinoxid) was from Serva, Heidelberg. Animals:
Female
NMRI mice
(age
7 to 8 weeks)
0006-291X/83 $1.50 Copyright @ 1983 by Academic Press, inc. All rights of reproduction in any form reserved.
were
used
in all
experiments.
Vol. 117, No. 2. 1983
Preparation as described
BIOCHEMICAL
of cytosol, previously
Determination
AND BIOPHYSICAL
PK assay (26,27).
of protein:
and 'H-TPA
was carried
out
RESEARCH COMMUNICATIONS
binding
assay:
according
were
to Lowry
performed
(28).
RESULTS Protein 32P-ATP
kinase and histone
monstrate
that
pholipid
activity
III-S
could
(Fig.
1).
a flavonoid residue
quercetin. maximal As shown
in position
PK activity.
2, quercetin
PK activity
100 PM quercetin, receptor
of the
(26,27)
able
inhibition
(compare
however,
earlier
10 uM. but with
effective
was required
was also Fig.
did
in the mouse brain
described
activity a
than
to obtain
half-
1).
The extent
stimulated
Ca,PL-PK
as quercetin
1 de-
phos2+ Ca .
at about
to be much less
concentration
using
to replace
the
occurred structure
3, was found higher
(Fig.
in Fig.
TPA was able
inhibition
the same basic
was measured shown in Table 2+ by Ca and the
both
of 100 UM inhibited
with
inhibition
The results
and that
Halfmaximal
A loo-fold
cytosol
be stimulated
(PS)
at a concentration
completely Rutin,
of mouse brain
as substrates.
PK activity
phosphatidylserine
Quercetin
sugar
(PK)
not
the
was similar 1 and 2).
influence
cytosol (not
to inhibit
TPA stimulated
to that
of the
At a concentration the binding
as measured
Ca 2+
of
of TPA to its
by the
filter
assay
shown). DISCUSSION
A great vonoid
variety
special
interest:
kinases
(12-15)
Our data activity. PK C might ing
of biological
quercetin
(l-22). quercetin
Recently,
Table
like 1.
promoter
quercetin
several
be identical
compounds
respect
with
Stimulation
The
assays
to inhibit also
a potent
strongly
the specific Based of
binding
PK-activity
by
Ca*+
site
finding, PS,
Ca '+
tumor
the
PK C has been
or
TPA (10b7M)
-PS
+PS
+PS
+Ca'+
+Ca2+
+TPA
324
498
708
2698
2243
445
protein
for
+ps
EGTA.
of
of Ca,PL-PK
-Ca*+
10m3M
fla-
were
the
-PS
contained
the
that
-Ca'+
protein
without
inhibitor
indicated
on this
with
two reports
CAMP-independent
(22).
is
TPA (23-25).
studies,
action
reports
PK-activity cpmx10-3/mg
have been observed to our
was found
and tumor
now show that
effects
With
Ca,PLpromot-
Vol. 117, No. 2, 1983
: ~~!l?l 0 BIOCHEMICAL
q k.
AND BIOPHYSKAL
--% \ ‘A\ .
\
\
\
RESEARCH COMMUNICATIONS
‘.
\
Fig.
l:Inhibition quercetin
The
results
Fig.
2:
The
results
from
from
suggested
to play that
duces not
the
PL-PK and
activity rutin
assys
without
control
control
a key role
in (A-;;').
TPA stimulated with different that
be of great
in tumor
activity
TPA binding, from
Ca
for
(see
Table
2t
(see
1)
brain
were
by
subtracted.
Since
as a potent studies
very
most
likely
subtracted.
context
promotion,
intriguing. of Ca,PL-PK
1) were
In this
of tumor
quercetin
further
mouse
Table
promotion.
the TPA binding
quercetin value
of
mouse brain in the presence of of the TPA stimulated enzyme
without
is
cytosol
Ca
an inhibitor
of Ca,PL-PK
the
tumor
10-d 10” Inhibitor(M)
assays
quercetin,
activity
We believe for
of Ca, (O-0)
interfere
at a site could
10-S
Ca,PL-PK activity in cytosol of either Ca2+ or TP and inhibition activity by 5x10- e M quercetin.
servation blocks
10-e
quercetin effectively acts
our
ob-
completely also but on the
redoes kinase
site. inhibitor on the
of Ca,PL-PK significance
activity of PK C
promotion. REFERENCES
1. Kingston, W.P. (1983) British. J. Pharmacol. 80, 515 P. 2. Gabor, F1. (1972) The Anti-inflammatory Actions of Flavonoids, Akademiai Kiadb, Budapest. 3. Graziani, Y. (1977) Biochim. Biophys. Acta 460, 364-373. 4. Bustamante, E., and Pedersen, P.L. (1977) Proc. Natl. Acad. Sci. USA 74, 3735-3739. 5. Beretz, A., Anton, R., and Stoclet, J.C. (1978) Experientia 34, 10541055. 6. Ruckstuhl, M., Beretz, A., Anton, R., and Landry, Y. (1979) Biochem. Pharmacol. 28, 535-538. E. (1974) Biochim. Biophys. Acta 333, 180-186. 7. Lang, D.R., and Racker, 8. Kuriki, Y., and Racker, E. (1976) Biochemistry 15, 4951-4956. 446
Vol. 117, No. 2, 1983
9. Fewtrell,
C.M.S.,
BIOCHEMICAL
and Gomperts,
AND BIOPHYSICAL
B.D.
(1977)
RESEARCH COMMUNICATIONS
Biochim.
Biophys.
Acta
469, 52-60. 10. Shoshan, V., and MacLennan, D.H. (1981) J. Biol. Chem. 256, 887-892. 11. Varma, S.D., Mikum, I., and Kinoshita, J.H. (1975) Science 188, 1215-1216. 12. Glossmann, H., Presek, P., and Eigenbrodt, E. (1981) Naunyn-Schmiedeberg's Arch. Pharmacol. 317, 100-102. 13. Graziani, Y., Chayoth, R., Karny, N., Feldman, B., and Levy, J. (1982) Biochim. Biophys. Acta 714, 415-421. 14. Cachet, J., Feige, J.J., Pirollet, F., Keramidas, M., and Chambaz, E. M. (1982) Biochem. Pharmacol. 31, 1357-1361. R.L. (1983) Eur. J. Biochem. 15. Graziani, Y. Erikson, E., and Erikson, 135, 583-589. R. (1979) Biochem. Pharmacol. 28, 397-403. 16. Graziani, Y., and Chayoth, 17. Belt, J.A., Thomas, J.A., Buchsbaum, R.N., and Racker, E. (1979) Biochemistry 18, 3506-3511. and Gee, J.B.L. (1974) Biochem. Pharmacol. 23, 178118. Khandwala, A.S., 1786. 19. Snolinna, E.M., Buchsbaum, R.N., and Racker, E. (1975) Cancer Res. 35, 1865-1872. Photobiol. 38, 363-367. 20. Takahama, U. (1983) Photochem. 21. Bjeldanes, L.F., and Chang, G.W. (1977) Science 197, 577-578. 22. Nishino, H., Iwashima, A., Fujiki, H., Nagao, M., and Sugimura, T. (1982) Mutation Res. 97, 406. 23. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. 24. Niedel, and Vandenbark, G.R. (1983) Proc. Natl. Acad. J.E., Kuhn, L.J., Sci. USA 80, 36-40. R.K. (1983) Cancer Res. 25. Ashendel, C.L., Staller, J.M., and Boutwell,
43, 4333-4337.
Gschwendt, M., and Kittstein, W. (1983) Cancer Letters Gschwendt, M., Horn, F., Kittstein, W., Fiirstenberger, F. (1983) FEBS Letters, in press. 28. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, J.Biol.Chem. 193, 265-275.
E:
447
19, 159-164. G., and Marks,
R.J. (1951)
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 444-447
16, 1983
INHIBITION OF THE CALCIUM- AND PHOSPHOLIPID-DEPENDENT PROTEIN KINASE ACTIVITY FROM MOUSE BRAIN CYTOSOL BY QUERCETIN M. Gschwendt,
F. Horn,
W. Kittstein
and F. Marks
Deutsches Krebsforschungszentrum, Institut fur Im Neuenheimer Feld 280, D - 6900 Heidelberg, Received
October
19,
Biochemie, F.R.G.
1983
The flavonoid quercetin is a potent inhibitor of calciumand phospholipid-dependent protein kinase (Ca,PL-PK) activity from mouse brain. Halfmaximal inhibition of the kinase occurs at about 10 !JM. If the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is used instead of calcium as a stimulating factor of the kinase enzyme activity is still inhibited by quercetin. The kinase inhibitor, however, does not interfere with the binding of TPA to its receptor. The flavonoid example
quercetin
cyclooxygenase
decarboxylase
is
known to inhibit
and 15-lipoxygenase
and xanthinoxidase
(2),
CAMP and cGMP phosphodiesterase (11)
and CAMP-independent
inhibits (17),
D-glucose
exhibits our
(18)
cells mutagenic
studies,
thought
it
(19).
activity appears
to
to be initiated receptor.
be identical
the
with
well
but
that
it
as well
(21)
and most
inhibit
tumor
Ca,PL-PK
by TPA (23).
Ca,PL-PK not
interfere
systems
of lactate
and proliferation
as an antioxidant with (22).
quercetin
respect
most
of this inhibits
from mouse brain
it to
is
compounds
found
binding
of
(20),
Promotion
promoting
The activity
the
(3,4),
quercetin
has been
with
hexokinase
reductase
as growth
promotion
activity
histidin-
aldolase
transport
of tumor
that
for
Furthermore,
interestingly
C (23-25).
We now report
does
(7-lo),
can function
The TPA receptor
as the TPA stimulated
tively,
It
bound
(12-15). (16),
by the binding
TPA to a specific stimulated
ATPase
synthesis
enzymes,
hyaluronidase,
mitochondrial
kinases
and Ca2+ (9,lO)
in vitro
(l),
(5,6),
protein
DNA, RNA and protein
malignant
various
like
likely kinase
the very
Ca
2+
to is as
effec-
of TPA to its
re-
ceptor. MATERIALS
AND METHODS
Materials: TPA was kindly supplied by Prof. E. Hecker, German Cancer Resejrch Center, Heidelberg. 20 Ci/mnole, and Y~~P-ATP, spec. activity [20- H(N)]-TPA, spec. activity 9 Ci/mmole, were from New England Nuclear. Phosphatidylserine, histone III-S and quercetin (3,3',4',5,7-pentahydroxyflavone) were from Sigma, MUnchen. Rutin (quercetin-3-rutinoxid) was from Serva, Heidelberg. Animals:
Female
NMRI mice
(age
7 to 8 weeks)
0006-291X/83 $1.50 Copyright @ 1983 by Academic Press, inc. All rights of reproduction in any form reserved.
were
used
in all
experiments.
Vol. 117, No. 2. 1983
Preparation as described
BIOCHEMICAL
of cytosol, previously
Determination
AND BIOPHYSICAL
PK assay (26,27).
of protein:
and 'H-TPA
was carried
out
RESEARCH COMMUNICATIONS
binding
assay:
according
were
to Lowry
performed
(28).
RESULTS Protein 32P-ATP
kinase and histone
monstrate
that
pholipid
activity
III-S
could
(Fig.
1).
a flavonoid residue
quercetin. maximal As shown
in position
PK activity.
2, quercetin
PK activity
100 PM quercetin, receptor
of the
(26,27)
able
inhibition
(compare
however,
earlier
10 uM. but with
effective
was required
was also Fig.
did
in the mouse brain
described
activity a
than
to obtain
half-
1).
The extent
stimulated
Ca,PL-PK
as quercetin
1 de-
phos2+ Ca .
at about
to be much less
concentration
using
to replace
the
occurred structure
3, was found higher
(Fig.
in Fig.
TPA was able
inhibition
the same basic
was measured shown in Table 2+ by Ca and the
both
of 100 UM inhibited
with
inhibition
The results
and that
Halfmaximal
A loo-fold
cytosol
be stimulated
(PS)
at a concentration
completely Rutin,
of mouse brain
as substrates.
PK activity
phosphatidylserine
Quercetin
sugar
(PK)
not
the
was similar 1 and 2).
influence
cytosol (not
to inhibit
TPA stimulated
to that
of the
At a concentration the binding
as measured
Ca 2+
of
of TPA to its
by the
filter
assay
shown). DISCUSSION
A great vonoid
variety
special
interest:
kinases
(12-15)
Our data activity. PK C might ing
of biological
quercetin
(l-22). quercetin
Recently,
Table
like 1.
promoter
quercetin
several
be identical
compounds
respect
with
Stimulation
The
assays
to inhibit also
a potent
strongly
the specific Based of
binding
PK-activity
by
Ca*+
site
finding, PS,
Ca '+
tumor
the
PK C has been
or
TPA (10b7M)
-PS
+PS
+PS
+Ca'+
+Ca2+
+TPA
324
498
708
2698
2243
445
protein
for
+ps
EGTA.
of
of Ca,PL-PK
-Ca*+
10m3M
fla-
were
the
-PS
contained
the
that
-Ca'+
protein
without
inhibitor
indicated
on this
with
two reports
CAMP-independent
(22).
is
TPA (23-25).
studies,
action
reports
PK-activity cpmx10-3/mg
have been observed to our
was found
and tumor
now show that
effects
With
Ca,PLpromot-
Vol. 117, No. 2, 1983
: ~~!l?l 0 BIOCHEMICAL
q k.
AND BIOPHYSKAL
--% \ ‘A\ .
\
\
\
RESEARCH COMMUNICATIONS
‘.
\
Fig.
l:Inhibition quercetin
The
results
Fig.
2:
The
results
from
from
suggested
to play that
duces not
the
PL-PK and
activity rutin
assys
without
control
control
a key role
in (A-;;').
TPA stimulated with different that
be of great
in tumor
activity
TPA binding, from
Ca
for
(see
Table
2t
(see
1)
brain
were
by
subtracted.
Since
as a potent studies
very
most
likely
subtracted.
context
promotion,
intriguing. of Ca,PL-PK
1) were
In this
of tumor
quercetin
further
mouse
Table
promotion.
the TPA binding
quercetin value
of
mouse brain in the presence of of the TPA stimulated enzyme
without
is
cytosol
Ca
an inhibitor
of Ca,PL-PK
the
tumor
10-d 10” Inhibitor(M)
assays
quercetin,
activity
We believe for
of Ca, (O-0)
interfere
at a site could
10-S
Ca,PL-PK activity in cytosol of either Ca2+ or TP and inhibition activity by 5x10- e M quercetin.
servation blocks
10-e
quercetin effectively acts
our
ob-
completely also but on the
redoes kinase
site. inhibitor on the
of Ca,PL-PK significance
activity of PK C
promotion. REFERENCES
1. Kingston, W.P. (1983) British. J. Pharmacol. 80, 515 P. 2. Gabor, F1. (1972) The Anti-inflammatory Actions of Flavonoids, Akademiai Kiadb, Budapest. 3. Graziani, Y. (1977) Biochim. Biophys. Acta 460, 364-373. 4. Bustamante, E., and Pedersen, P.L. (1977) Proc. Natl. Acad. Sci. USA 74, 3735-3739. 5. Beretz, A., Anton, R., and Stoclet, J.C. (1978) Experientia 34, 10541055. 6. Ruckstuhl, M., Beretz, A., Anton, R., and Landry, Y. (1979) Biochem. Pharmacol. 28, 535-538. E. (1974) Biochim. Biophys. Acta 333, 180-186. 7. Lang, D.R., and Racker, 8. Kuriki, Y., and Racker, E. (1976) Biochemistry 15, 4951-4956. 446
Vol. 117, No. 2, 1983
9. Fewtrell,
C.M.S.,
BIOCHEMICAL
and Gomperts,
AND BIOPHYSICAL
B.D.
(1977)
RESEARCH COMMUNICATIONS
Biochim.
Biophys.
Acta
469, 52-60. 10. Shoshan, V., and MacLennan, D.H. (1981) J. Biol. Chem. 256, 887-892. 11. Varma, S.D., Mikum, I., and Kinoshita, J.H. (1975) Science 188, 1215-1216. 12. Glossmann, H., Presek, P., and Eigenbrodt, E. (1981) Naunyn-Schmiedeberg's Arch. Pharmacol. 317, 100-102. 13. Graziani, Y., Chayoth, R., Karny, N., Feldman, B., and Levy, J. (1982) Biochim. Biophys. Acta 714, 415-421. 14. Cachet, J., Feige, J.J., Pirollet, F., Keramidas, M., and Chambaz, E. M. (1982) Biochem. Pharmacol. 31, 1357-1361. R.L. (1983) Eur. J. Biochem. 15. Graziani, Y. Erikson, E., and Erikson, 135, 583-589. R. (1979) Biochem. Pharmacol. 28, 397-403. 16. Graziani, Y., and Chayoth, 17. Belt, J.A., Thomas, J.A., Buchsbaum, R.N., and Racker, E. (1979) Biochemistry 18, 3506-3511. and Gee, J.B.L. (1974) Biochem. Pharmacol. 23, 178118. Khandwala, A.S., 1786. 19. Snolinna, E.M., Buchsbaum, R.N., and Racker, E. (1975) Cancer Res. 35, 1865-1872. Photobiol. 38, 363-367. 20. Takahama, U. (1983) Photochem. 21. Bjeldanes, L.F., and Chang, G.W. (1977) Science 197, 577-578. 22. Nishino, H., Iwashima, A., Fujiki, H., Nagao, M., and Sugimura, T. (1982) Mutation Res. 97, 406. 23. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. 24. Niedel, and Vandenbark, G.R. (1983) Proc. Natl. Acad. J.E., Kuhn, L.J., Sci. USA 80, 36-40. R.K. (1983) Cancer Res. 25. Ashendel, C.L., Staller, J.M., and Boutwell,
43, 4333-4337.
Gschwendt, M., and Kittstein, W. (1983) Cancer Letters Gschwendt, M., Horn, F., Kittstein, W., Fiirstenberger, F. (1983) FEBS Letters, in press. 28. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, J.Biol.Chem. 193, 265-275.
E:
447
19, 159-164. G., and Marks,
R.J. (1951)