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Characterization of glycyrrhizin-binding protein kinase from the crude membrane fraction of rat liver
Vol. March
167,
No.
30,
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 876-882
1990
CHARACIERUATION
OF THE
CRUDE
GLYCYRRHIZDI-BINDING MEMBRANE FRACTION
Akira ISHIKAWAI, Shin-ichiro
OF
PROTEIN KINASE RAT LIVER
FROM
Ryunosuke KANAMARUI, Akira WAKUI’ KANNO and Kenzo OHTSUK12*
IDepartment
of Clinical Cancer Chemotherapy, Institute of Cancer Research, Tohoku University, Sendai 980, Japan
%epartment
of Bioscience, Kitasato University School of Hygienic Sciences, Sagamihara 228, Japan
Received
January
24,
1990
SUMMARY: Using GL-affinity column chromatography, a casein phosphorylating protein kinase was purified selectively from the crude membrane fraction of rat liver. The biochemical characteristics of the purified kinase (approximately Mr 210 kDa) are very similar to those reported for polypeptide-dependent protein kinase (kinase PI. Moreover, low doses of GL selectively inhibit phosphorylation of Mr 35-36 kDa polypeptides (which are cross-reacted with anti-lipocortins I and II) by the kinase h a. These results suggest that the anti-inflammatory activity of GL may involve the impairment of the physiological functions of lipocortins through their specific modification by the kinase at the cell membrane level. 0 1990 AcademicPress. Inc.
Glycyrrizin
(GL), is one of the aqueous extract from licorice root (Glycyrrkizu
The compound
is known for its anti-inflammatory
has anti-viral
activity
reported
against
that (i) GL inhibits
activity and protein
property in Chinese medicine (l), and
DNA and RNA viruses
vesicular stomatitis
phosphorylation
l’); and (ii) this compound their phosphorylating
several
by polypeptide-dependent
kinase
from
the crude
(iii) the inhibitory
protein
protein
we
kinase
kinase (kinase
binds directly to these kinases and results in the reduction fraction of rat liver, a GL-affinity
Here, we describe (i) the selevtive purification chromatography;
(2). Previously,
virus (VSV)-associated
abilities (3). In order to purify GL specific binding
from the crude membrane
radix).
membrane
(ii) some enzymatic
fraction
of rat liver
poylpeptides
(which
are cross-reacted
observation
suggests that the anti-inflammatory
protein
by GL-affinity
of the purified
effect of GL on phosphorylation
associated
protein kinases
column was constructed.
of a casein phosphorylating
properties
of
protein
column kinase; and
of the Mr 35-36 kDa membrane with
anti-lipocortins).
The latter
activity of GL may be implicated
in the
YTo whom correspondence should be addressed. Abbreviations used: GL, glycyrrhizin; kinase P, polypeptide-dependent protein kinase; DTT, dithiothreitol; PMSF, phenyl-methylsulfonyl fluoride; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; poly Arg, poly arginine; poly Lys, poly lysine. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
876
Vol.
167,
No.
3, 1990
BIOCHEMICAL
selective inhibition
of lipocortin
AND
BIOPHYSICAL
phosphorylation
RESEARCH
by GL-binding
COMMUNICATIONS
protein
kinase at the
cell membrane level. MATERIALS
AND METHODS
wrrhizin (GL): GL was kindly supplied by Minophagen Pharmaceutical Co., Ltd. (Tob’o). Crude protein kinase fraction from rat liver: SD rat liver (about 9 g wet weight) was homogenized in 100 ml of Buffer A [lo mM Tris-HCl (pH 7.6), 2 mM D’IT, 1 mM PMSF, 10 mM KU, 5 mM MgCl2 and 10 % glycerol]. The homogenate was centrifuged at 9,200 X g for 30 min at 4°C to remove the membrane of the liver cells. The protein kinase were extracted from the membrane pellet with Buffer A containing 0.1 % Nonidet P-40. The extracted kinase fraction was concentrated with solid ammonium sulfate (65 % saturation), then dialyzed overnight against Buffer B [20 mM Tris-HCl (pH 7.6), 2 mM DTT, 1 mM PMSF, 50 mM KC1 and 10 % glycerol]. The dialysate was used as a crude protein kinase fraction of rat liver. &ay of protein kinase activitv: The reaction mixture (0.1 ml) contained 40 mM Tris-HCl (pH 7.6), 4 mM DTT, 20 @I [~-~~l’] ATE (30 Ci/mmole), 5 mM Mg2+, 10 pg casein or the indicated phosphate acceptors and the indicated amount of purified or crude protein kinase. After incubation for the indicated period at 3O”C, protein phosphorylation by the kinases was determined, as previously reported (3,4). -ration of GL-affinitv column: Epoxy activated Sepharose 6B (pharmacia) was washed with distilled water, then suspended in 0.1 M Na2CO3. GL (10 mg per 1 ml) was added to the suspension. After shaking for 16 hrs at room temperture, the emlution was further washed with distilled water, followed successively by carbonate buffer 10.1 M Na2 CO3 and 0.1 M NaHC03 and 0.1 M acetic acid (5). The GL-Sepharose 6B complex was suspended in 1 M 2-amino-ethanol and left at room temperature for 16 hrs before final washes with distilled water, carbonate buffer and 0.1 M acetic acid. The washed materials were packed into a column (1.4 x 10 cm) and used for GL-Sepharose 6B affinity purification of GL-binding protein kinases. SDS-PAGE and autoradioeranhv: The [32P]-labelled polypeptide in the crude and partially purified kinases were analyzed by SDS-PAGE, according to a modification of method described by Laemmli (6), after incubation for 10 min at 30°C with [Y~~PIAV and 5 mM Mg2+, in the presence or absence of poly Arg as an activator of the kinase. RESLJLTS Purification
of nrotein kinases bv GL-affinitv column chromatoeraohv
In order to purify GL-binding liver, the fraction
was applied
protein kinase in the crude membrane fraction from rat on a GL-affinity
column,
previously
equilibrated
with
Buffer B containing 50 mM KCl. The column was eluted with a linear gradient between 50 mM and 1.5 M KCl. Protein kinase activity was detected between 0.6 M and 0.7 M KCl (Fig. ‘LA). The active fractions ammmium
were pooled
and immediately
concentrated
with solid
sulfate (65% saturation). This was further purified by DEAE-cellulose
chrmatography,
where two distinct
kinase activities
1B). !$ince the activity of the P-I kinase, which CAMP-dependent
manner,
column
(P-I and P-11) were detected (Fig.
highly phosphorylate
histone Hl in a
was less than 6% of total kinase activity
in the fraction
recovered from the column, the P-II kinase alone was further purified on Mono Q column (FPLC, Pharmacia)
previously
equilibrated
with Buffer B containing
column was eluted with a linear gradient between protein kinase, which phosphorylated Final purification
of the GL-binding
50 mM KCl.
The
0.1 M and 0.8 M KU. The activity of
casein, was eluted between 0.5 M and 0.58 M KCl. protein kinase was approximately 877
l,lOO-fold,
with
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FRACTION NUMBER Fie. [A] GL-affinity column chromatography of the crude membrane fraction of rat liver. The membrane fraction (about 10 mg protein) was applied on a GL-affinity cloumn (1.4 x 10 cm), previously equilibrated with Buffer A containing 50 mM. The column was eluted with a linear gradient between 50 mM and 1.0 M KCl. [BI Further purification of the active GL-affinity fraction (about 4 mg protein) on a DEAE-cellulose column (1.2 x 6 cm), previously equilibrated with Buffer A containing 50 mM KCl. Aliquots (10 ~1) of the indicated fractions were assayed for protein kinase activity (0) using casein as a phosphate acceptor. Absorbance at 280 run W.
an activity yield of about 8% of the crude membrane (HPLC, Tosoh, Tokyo) chromatography kinase to be approximately Characteristics of GL-binding Requirements summarized
showed
fraction. TSKgel G3COOSW column
the molecular
weight
of the purified
210 kDa. orotein kinase
for the activity of GL-binding
in Table 1. The kinase required
protein kinase (Mono Q fraction) are
divalent cations, such as Mg2+ and Mn2+.
The optimum concentrations of Mg2+ and Mn2+ were found to be approximately 2 mM, respectively. Ca2+, between 0.1 mM and 10 mM, inhibited effects CAMP or cGMP on phosphorylation Heparin,
an inhibitor
inhibited
the protein
kinase (A-kinasel polypeptides,
of casein by the kinase were detected.
of casein kinase II (4), and low doses (less than 50 pM) of GL phosphorylation.
was insensitive
However,
experiments
phosphorylation
of histone Hl by P-I
to high doses (over than 200 pM) of GL. Basic
such as poly Arg, stimulated
kinase. Confirmatory
5 mM and
the kinase activity. No
greatly phosphorylation
by SDS-PAGE follwed 878
of casein by the
by autoradiography
showed
Vol.
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No.
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1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table 1. Requirementsfor the activity of GL-binding protein kinase [32P]Phosphate-incorporationinto the TCA fraction (cpm)
Addiiions Complete Complete- Casein Complete- Mg2+ Complete- Mg2+ + Mn2+ Complete- Mg2+ + Ca2+ Complete + cAMP Complete + cGMP Complete + Poly Arg Complete + HistoneHl Complete - Casein + HistoneHl Complete + Heparin Complete + GL Complete + Poly Arg + GL
64,265 750 120 58,772 360 62,807 63,001 373,905 307,332 5,780 4,036 7,112 18,014
The complete reaction mixtures (0.1 ml) conteind 40 mM Tris-HCI (pH 7.6), 4 mM D’IT, 5 mM Mg2+, 20 w [p32P]ATP (500 cpm/pmol), 10 pg of casein (phosphate acceptor) and crude or purified protein kinase (Mono Q fraction, 0.2 pg). The mixtures were separately incubated for 15 min at 30°C in the presence or absence of the indicated chemicals. The concentrations used of Mn2+, Ca2+, CAMP, cGMP, histone Hl, heparin and GL were 2 mM, ‘I mM, 2 W, 2 m, 5 pg, 10 pg and 100 @4, respectively.
that (i) the purified
kinase phosphorylated
(ii) casein phosphorylation
by the kinase was stimulated greatly by poly Arg (lane 3 in
Fig. 2). These experimental
results suggest that (i) the enzymatic
protein kinase (P-II kinase) purified yeast (7) and
other
phosphorylation
casein (lane 2), but not histone (lane 41; and
mammalian
properties
of the
from rat liver are similar to those of kinase I’ from cells (8,9); and
of casein by the purified
kinase,
(ii) low
as previously
doses of GL inhibit reported
in kinase I’
from HeLa 53 cells (3). Polvneotides
coourified
To determine
with GL-bindinp
polypeptides
protein kinase
specifically phosphorylated
by the GL-binding
protein
kinase, the DEAE-cellulose fraction was directly analyzed by SDS-PAGE, followed by autoradiography after incubation with [y-32P]ATP and 5 mM Mg2+ in the presence or absence poly Arg (1 ug). Under these experimental
conditions, polypeptides
75 kDa and 35-36 kDa were detected as highly phosphorylated Fig. 2). Phosphorylation
of the Mr 35-36 kDa polypeptides
presence of poly Arg (lane 6), but significantly 35-36 kDa phosphorylating
polypeptides
inhibited
were identified
with Mr 7@
polypeptides
was enhanced greatly in the
by GL (lane 7 in Fig. 2). The Mr as lipocortins
I (35 kDa, Ip 6.5-
6.8) and II (36 kDa, Ip 7.4-7.81, in view of their molecular immunoprecipitated lipocortins
(lane 5 in
with anti-
(I and II).
Effect of CL on lioocortin
ohosohorvlation
The Mr 35-36 kDa phosphorylating could be completely
polypeptides
in the DEAE-cellulose
fraction
separated from the kinase by Mono Q column (FI’LC, Pharmacia)
879
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BIOCHEMICAL
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5
67
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BIOPHYSICAL
KDA
KDA
-97 - 67 -
RESEARCH
COMMUNICATIONS
1234567
97-, 674
43
43+
Casein -
- Lipocortin
-31 314
- Casei n
-21
21 -+
02
03 Fie. Phosphorylation of casein by purified GL-binding protein kinase and the Mr 3536 kDa polypeptides copurified with the kinase. Purified protein kinase (Mono Q fraction, 0.1 ugl was incubated with casein (phosphate acceptor1 in the presence or absence of poly Arg (lanes 1 through 4). Partially purified protein kinase (DEAEcellulose fraction, 0.2 ug) was also incubated in the absence of casein (lanes 5 through 7). 132PIphosphorylated polypeptides in these reaction mixtures were analyzed directly by SDS-PAGE, followed by autoradiography. Lane 1, purified protein ldnase alone; lane 2, casein was added; lane 3, both casein and poly Arg (1 pg) were added; lane 4, purified kina%? was incubated with histone Hl instead of casein; lane 5, DEAE-cellulose fraction in the absence of poly Arg; lane 6, poly Arg was added; and lane 7,50 f.tM GL were added.
&gJ. Selective inhibition of phosphorylation of the Mr 35-36 kDa polypeptides by GL. The GL-binding protein kinase (Mono Q fraction, 0.1 ug) was incubated for 10 min at 30°C with casein (lanes 2 through
4) or the Mr 35-36 kDa polypeptides
(lanes 4 through
7,0.2
ug), which were purified by Mono Q column chromatography, in the presence of [?I321’lATl’ and 5 mM Mg2+. After incubation, the mixtures were analyzed directly by SDSPAGE, followed by autoradiography. Lane 1, purified kinase alone; lanes 2 and 5, absence of GL; lanes 3 and 6, presence of 30 tt.M GL; and lanes 4 and 7,150 pM GL.
chromatography, of KC1 rather polypeptides various
because these polypeptides than the kinase.
inhibition
in
phosphorylation
of the Mr 35-36 kDa
Fig. 3, phosphorylation
by the kinase was reduced
of casein phosphorylation
that GL selectively polypeptides
Therefore,
(Mono Q fraction) by the kinase was carried out in the presence or absence of
doses of GL. As shown
polypeptides
were eluted with more lower concentrations
inhibits
(lipocortins
of the Mr 35-36 kDa
about 50% by 20 uM GL, whereas
required
similar
more than 200 @I GL. These results show
phosphorylation,
by the kinase, of the Mr 35-36 kDa
I and II), rather than of the Mr 70-75 kDa polypeptides
or
casein. DISCUSSION A GL-binding cGMP-
protein kinase, which highly phosphorylated
and Ca2+-independent
membrane fraction
of rat liver using a GL-affinity
the purified kinase (approximately
casein in a CAMP-,
manner, has been successfully purified
from the crude
column. The enzymatic properties of
Mr 210 kDa) were very similar to those of kinase Pf4,
7-91, hecause (il these two kinases could he extracted only from the membrane using suitable detergents
fraction,
such as NP-40; (ii) casein was the most effective phosphate
acceptor for these kinases; and (iii) protein
phosphorylation
880
by these
kinases in vitro
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
was enhanced greatly by basic polypeptides,
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
such as histone HI, poly Arg and poly Lys
(Table 1 and Fig. 2). These experimental
results suggest that a specific GL-binding
protein kinase may be identical to klnase P. This assumption is supported by our previous observation
that GL binds directly
to kinase P from HeLa S3 cells (3). However,
molecular
sire (Mr 210 kDa) of the purified
relatively
larger
the
kinase (Mono Q fraction)
of rat liver was
than those of the kinases from yeast (a heterodimer
of Mr 75 kDa
subunit; 7) and HeLa S3 cells (Mr 120 kDa; 4). It is interesting
to observe that (i) a GL-binding
Mr 70-75 kDa and 35-36 kDa polypeptides
protein kinase was copurified
by DEAE-cellulose
(lane 5 in Fig. 2); (ii) these polypeptides kDa polypeptides
of the Mr 35-36 kDa polypeptide one-tenth
was
phoshpholipasa
modification kinase
of the Mr 35-36 of phosphorylation
by the kinase was obtained by approximately of VSV-associated
of the concentration
20 l.tM GL,
protein kinase activity. However,
of GL that causes 50% inhibition
of
A2 activity (12).
Earlier reports concerning phospholipase
by the kinase h
phosphorylation
by the kinase (Figs. 2 and 3). The 50% inhibition
which caused a similar inhibition this dose
column chromatography
were highly phosphorylated
vitro: and (iii) low doses of GL selectively inhibited
with
the biological
A2 activity at the regulation by cAMPdependent
(C-kinase)
phosphorylation
(14)
and
significance and physiological of phospholipase
function of
A2 activity through
protein kinase (A-kinase) (13), Ca2+dependent EGF-receptor
protein
by these three kinases is insensitive
kinase
(15).
Since
their protein
protein
to GL, the anti-inflammatory
activity of the drug may be mediated by different mechanisms, which are controlled
by
GL sensitive protein kinases, such as kinase I’. This speculation
is supported
observations that (i) GL binds directly to the membrane-associated
Mr 210 kDa protein
kinase and selectively inhibits phosphorylation 3); and (ii) [3HJ-labeled GL also dire&y which were separated
of lipocortins
bound to lipocortins
from the kinase by Mono Q column
by our
by the kinase (Figs. 2 and (35-36 kDa polypeptides), chromatography
(data not
shown). These data suggest that the anti-inflammatory by the inhibition
of lipocortin
phosphorylation
activity
by GL-binding
of GL may be mediated protein kinase at the cell
membrane level. Further analytical studies are under way concerning significance of lipocortins inhibitory
modified
by GL-binding
mechanism of phospholipase
the physiological
protein kinase, and the details of the
A2 activity by GL.
REFERENCES 1. Finney, R.S.H. and Somers, G.F. (1959) J. Pharmacol. 10, 613- 620. 2. Pompei, R., Flore, O., Marccialis, M.A., Pani, A. and Loddo, B. (1979) Nature 281,689-680. 3. Ohtsuki, K. and Ishida, N. (1988) B&hem. Biophys. Res. Commun. 157,597-604. 4. Ikeuchi, T., Yokoyama, M. and Ohtsuki, K. (1988) Tohoku J.Exp.Med. 155,41-56. 5. Mannufactory’s information from Pharmacia Fine Chemicals. 6. Laemmli, U.L. (1970) Nature 227,680-685 881
Vol.
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No.
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BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
7. Yanagita, Y., Abdel-Ghany, M., Raden, D. and Racker, E. (1987) Proc. Natl. Acad. Sci. USA, 84,925-929. 8. Abdel-Ghany, M., Kole, H.K. and Racker, E. (1987) Proc. Natl. Acad Sci. USA, 84,8888-8892. 9. Abdel-Ghany, M., Kale, H.K., Abou, EL, Saad, M. and Racker, E. (1989) Proc. Natt. Acad. Sci. USA, 86,6072-6076. 10. Greke, V. and Weber, K. (1985) J. Biol. Chem., 260, 1688-1695. 11. Glenny, R. (1986) J. Biol. Chem., 261, 7247-7252. 12. Ohuchi, K., Kamada, Y., Levine, L. and Tsurufuji, S. (1981) kostaglandin and Medicine 7,457-463. 13. Hirata, F. (1981) J. Biol. Chem., 256, 7730-7733. 14. Khanna, N. C., Tokuda, M. and Waisman, D. M. (1986) Biochem. Biophys. Res. Commun., 141,547-554. 15. Fava, R. A. and Cohen, S. (1984) J. Biol. Chem., 259,2636-2645.
882
167,
No.
30,
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 876-882
1990
CHARACIERUATION
OF THE
CRUDE
GLYCYRRHIZDI-BINDING MEMBRANE FRACTION
Akira ISHIKAWAI, Shin-ichiro
OF
PROTEIN KINASE RAT LIVER
FROM
Ryunosuke KANAMARUI, Akira WAKUI’ KANNO and Kenzo OHTSUK12*
IDepartment
of Clinical Cancer Chemotherapy, Institute of Cancer Research, Tohoku University, Sendai 980, Japan
%epartment
of Bioscience, Kitasato University School of Hygienic Sciences, Sagamihara 228, Japan
Received
January
24,
1990
SUMMARY: Using GL-affinity column chromatography, a casein phosphorylating protein kinase was purified selectively from the crude membrane fraction of rat liver. The biochemical characteristics of the purified kinase (approximately Mr 210 kDa) are very similar to those reported for polypeptide-dependent protein kinase (kinase PI. Moreover, low doses of GL selectively inhibit phosphorylation of Mr 35-36 kDa polypeptides (which are cross-reacted with anti-lipocortins I and II) by the kinase h a. These results suggest that the anti-inflammatory activity of GL may involve the impairment of the physiological functions of lipocortins through their specific modification by the kinase at the cell membrane level. 0 1990 AcademicPress. Inc.
Glycyrrizin
(GL), is one of the aqueous extract from licorice root (Glycyrrkizu
The compound
is known for its anti-inflammatory
has anti-viral
activity
reported
against
that (i) GL inhibits
activity and protein
property in Chinese medicine (l), and
DNA and RNA viruses
vesicular stomatitis
phosphorylation
l’); and (ii) this compound their phosphorylating
several
by polypeptide-dependent
kinase
from
the crude
(iii) the inhibitory
protein
protein
we
kinase
kinase (kinase
binds directly to these kinases and results in the reduction fraction of rat liver, a GL-affinity
Here, we describe (i) the selevtive purification chromatography;
(2). Previously,
virus (VSV)-associated
abilities (3). In order to purify GL specific binding
from the crude membrane
radix).
membrane
(ii) some enzymatic
fraction
of rat liver
poylpeptides
(which
are cross-reacted
observation
suggests that the anti-inflammatory
protein
by GL-affinity
of the purified
effect of GL on phosphorylation
associated
protein kinases
column was constructed.
of a casein phosphorylating
properties
of
protein
column kinase; and
of the Mr 35-36 kDa membrane with
anti-lipocortins).
The latter
activity of GL may be implicated
in the
YTo whom correspondence should be addressed. Abbreviations used: GL, glycyrrhizin; kinase P, polypeptide-dependent protein kinase; DTT, dithiothreitol; PMSF, phenyl-methylsulfonyl fluoride; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; poly Arg, poly arginine; poly Lys, poly lysine. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
876
Vol.
167,
No.
3, 1990
BIOCHEMICAL
selective inhibition
of lipocortin
AND
BIOPHYSICAL
phosphorylation
RESEARCH
by GL-binding
COMMUNICATIONS
protein
kinase at the
cell membrane level. MATERIALS
AND METHODS
wrrhizin (GL): GL was kindly supplied by Minophagen Pharmaceutical Co., Ltd. (Tob’o). Crude protein kinase fraction from rat liver: SD rat liver (about 9 g wet weight) was homogenized in 100 ml of Buffer A [lo mM Tris-HCl (pH 7.6), 2 mM D’IT, 1 mM PMSF, 10 mM KU, 5 mM MgCl2 and 10 % glycerol]. The homogenate was centrifuged at 9,200 X g for 30 min at 4°C to remove the membrane of the liver cells. The protein kinase were extracted from the membrane pellet with Buffer A containing 0.1 % Nonidet P-40. The extracted kinase fraction was concentrated with solid ammonium sulfate (65 % saturation), then dialyzed overnight against Buffer B [20 mM Tris-HCl (pH 7.6), 2 mM DTT, 1 mM PMSF, 50 mM KC1 and 10 % glycerol]. The dialysate was used as a crude protein kinase fraction of rat liver. &ay of protein kinase activitv: The reaction mixture (0.1 ml) contained 40 mM Tris-HCl (pH 7.6), 4 mM DTT, 20 @I [~-~~l’] ATE (30 Ci/mmole), 5 mM Mg2+, 10 pg casein or the indicated phosphate acceptors and the indicated amount of purified or crude protein kinase. After incubation for the indicated period at 3O”C, protein phosphorylation by the kinases was determined, as previously reported (3,4). -ration of GL-affinitv column: Epoxy activated Sepharose 6B (pharmacia) was washed with distilled water, then suspended in 0.1 M Na2CO3. GL (10 mg per 1 ml) was added to the suspension. After shaking for 16 hrs at room temperture, the emlution was further washed with distilled water, followed successively by carbonate buffer 10.1 M Na2 CO3 and 0.1 M NaHC03 and 0.1 M acetic acid (5). The GL-Sepharose 6B complex was suspended in 1 M 2-amino-ethanol and left at room temperature for 16 hrs before final washes with distilled water, carbonate buffer and 0.1 M acetic acid. The washed materials were packed into a column (1.4 x 10 cm) and used for GL-Sepharose 6B affinity purification of GL-binding protein kinases. SDS-PAGE and autoradioeranhv: The [32P]-labelled polypeptide in the crude and partially purified kinases were analyzed by SDS-PAGE, according to a modification of method described by Laemmli (6), after incubation for 10 min at 30°C with [Y~~PIAV and 5 mM Mg2+, in the presence or absence of poly Arg as an activator of the kinase. RESLJLTS Purification
of nrotein kinases bv GL-affinitv column chromatoeraohv
In order to purify GL-binding liver, the fraction
was applied
protein kinase in the crude membrane fraction from rat on a GL-affinity
column,
previously
equilibrated
with
Buffer B containing 50 mM KCl. The column was eluted with a linear gradient between 50 mM and 1.5 M KCl. Protein kinase activity was detected between 0.6 M and 0.7 M KCl (Fig. ‘LA). The active fractions ammmium
were pooled
and immediately
concentrated
with solid
sulfate (65% saturation). This was further purified by DEAE-cellulose
chrmatography,
where two distinct
kinase activities
1B). !$ince the activity of the P-I kinase, which CAMP-dependent
manner,
column
(P-I and P-11) were detected (Fig.
highly phosphorylate
histone Hl in a
was less than 6% of total kinase activity
in the fraction
recovered from the column, the P-II kinase alone was further purified on Mono Q column (FPLC, Pharmacia)
previously
equilibrated
with Buffer B containing
column was eluted with a linear gradient between protein kinase, which phosphorylated Final purification
of the GL-binding
50 mM KCl.
The
0.1 M and 0.8 M KU. The activity of
casein, was eluted between 0.5 M and 0.58 M KCl. protein kinase was approximately 877
l,lOO-fold,
with
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FRACTION NUMBER Fie. [A] GL-affinity column chromatography of the crude membrane fraction of rat liver. The membrane fraction (about 10 mg protein) was applied on a GL-affinity cloumn (1.4 x 10 cm), previously equilibrated with Buffer A containing 50 mM. The column was eluted with a linear gradient between 50 mM and 1.0 M KCl. [BI Further purification of the active GL-affinity fraction (about 4 mg protein) on a DEAE-cellulose column (1.2 x 6 cm), previously equilibrated with Buffer A containing 50 mM KCl. Aliquots (10 ~1) of the indicated fractions were assayed for protein kinase activity (0) using casein as a phosphate acceptor. Absorbance at 280 run W.
an activity yield of about 8% of the crude membrane (HPLC, Tosoh, Tokyo) chromatography kinase to be approximately Characteristics of GL-binding Requirements summarized
showed
fraction. TSKgel G3COOSW column
the molecular
weight
of the purified
210 kDa. orotein kinase
for the activity of GL-binding
in Table 1. The kinase required
protein kinase (Mono Q fraction) are
divalent cations, such as Mg2+ and Mn2+.
The optimum concentrations of Mg2+ and Mn2+ were found to be approximately 2 mM, respectively. Ca2+, between 0.1 mM and 10 mM, inhibited effects CAMP or cGMP on phosphorylation Heparin,
an inhibitor
inhibited
the protein
kinase (A-kinasel polypeptides,
of casein by the kinase were detected.
of casein kinase II (4), and low doses (less than 50 pM) of GL phosphorylation.
was insensitive
However,
experiments
phosphorylation
of histone Hl by P-I
to high doses (over than 200 pM) of GL. Basic
such as poly Arg, stimulated
kinase. Confirmatory
5 mM and
the kinase activity. No
greatly phosphorylation
by SDS-PAGE follwed 878
of casein by the
by autoradiography
showed
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Table 1. Requirementsfor the activity of GL-binding protein kinase [32P]Phosphate-incorporationinto the TCA fraction (cpm)
Addiiions Complete Complete- Casein Complete- Mg2+ Complete- Mg2+ + Mn2+ Complete- Mg2+ + Ca2+ Complete + cAMP Complete + cGMP Complete + Poly Arg Complete + HistoneHl Complete - Casein + HistoneHl Complete + Heparin Complete + GL Complete + Poly Arg + GL
64,265 750 120 58,772 360 62,807 63,001 373,905 307,332 5,780 4,036 7,112 18,014
The complete reaction mixtures (0.1 ml) conteind 40 mM Tris-HCI (pH 7.6), 4 mM D’IT, 5 mM Mg2+, 20 w [p32P]ATP (500 cpm/pmol), 10 pg of casein (phosphate acceptor) and crude or purified protein kinase (Mono Q fraction, 0.2 pg). The mixtures were separately incubated for 15 min at 30°C in the presence or absence of the indicated chemicals. The concentrations used of Mn2+, Ca2+, CAMP, cGMP, histone Hl, heparin and GL were 2 mM, ‘I mM, 2 W, 2 m, 5 pg, 10 pg and 100 @4, respectively.
that (i) the purified
kinase phosphorylated
(ii) casein phosphorylation
by the kinase was stimulated greatly by poly Arg (lane 3 in
Fig. 2). These experimental
results suggest that (i) the enzymatic
protein kinase (P-II kinase) purified yeast (7) and
other
phosphorylation
casein (lane 2), but not histone (lane 41; and
mammalian
properties
of the
from rat liver are similar to those of kinase I’ from cells (8,9); and
of casein by the purified
kinase,
(ii) low
as previously
doses of GL inhibit reported
in kinase I’
from HeLa 53 cells (3). Polvneotides
coourified
To determine
with GL-bindinp
polypeptides
protein kinase
specifically phosphorylated
by the GL-binding
protein
kinase, the DEAE-cellulose fraction was directly analyzed by SDS-PAGE, followed by autoradiography after incubation with [y-32P]ATP and 5 mM Mg2+ in the presence or absence poly Arg (1 ug). Under these experimental
conditions, polypeptides
75 kDa and 35-36 kDa were detected as highly phosphorylated Fig. 2). Phosphorylation
of the Mr 35-36 kDa polypeptides
presence of poly Arg (lane 6), but significantly 35-36 kDa phosphorylating
polypeptides
inhibited
were identified
with Mr 7@
polypeptides
was enhanced greatly in the
by GL (lane 7 in Fig. 2). The Mr as lipocortins
I (35 kDa, Ip 6.5-
6.8) and II (36 kDa, Ip 7.4-7.81, in view of their molecular immunoprecipitated lipocortins
(lane 5 in
with anti-
(I and II).
Effect of CL on lioocortin
ohosohorvlation
The Mr 35-36 kDa phosphorylating could be completely
polypeptides
in the DEAE-cellulose
fraction
separated from the kinase by Mono Q column (FI’LC, Pharmacia)
879
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97-, 674
43
43+
Casein -
- Lipocortin
-31 314
- Casei n
-21
21 -+
02
03 Fie. Phosphorylation of casein by purified GL-binding protein kinase and the Mr 3536 kDa polypeptides copurified with the kinase. Purified protein kinase (Mono Q fraction, 0.1 ugl was incubated with casein (phosphate acceptor1 in the presence or absence of poly Arg (lanes 1 through 4). Partially purified protein kinase (DEAEcellulose fraction, 0.2 ug) was also incubated in the absence of casein (lanes 5 through 7). 132PIphosphorylated polypeptides in these reaction mixtures were analyzed directly by SDS-PAGE, followed by autoradiography. Lane 1, purified protein ldnase alone; lane 2, casein was added; lane 3, both casein and poly Arg (1 pg) were added; lane 4, purified kina%? was incubated with histone Hl instead of casein; lane 5, DEAE-cellulose fraction in the absence of poly Arg; lane 6, poly Arg was added; and lane 7,50 f.tM GL were added.
&gJ. Selective inhibition of phosphorylation of the Mr 35-36 kDa polypeptides by GL. The GL-binding protein kinase (Mono Q fraction, 0.1 ug) was incubated for 10 min at 30°C with casein (lanes 2 through
4) or the Mr 35-36 kDa polypeptides
(lanes 4 through
7,0.2
ug), which were purified by Mono Q column chromatography, in the presence of [?I321’lATl’ and 5 mM Mg2+. After incubation, the mixtures were analyzed directly by SDSPAGE, followed by autoradiography. Lane 1, purified kinase alone; lanes 2 and 5, absence of GL; lanes 3 and 6, presence of 30 tt.M GL; and lanes 4 and 7,150 pM GL.
chromatography, of KC1 rather polypeptides various
because these polypeptides than the kinase.
inhibition
in
phosphorylation
of the Mr 35-36 kDa
Fig. 3, phosphorylation
by the kinase was reduced
of casein phosphorylation
that GL selectively polypeptides
Therefore,
(Mono Q fraction) by the kinase was carried out in the presence or absence of
doses of GL. As shown
polypeptides
were eluted with more lower concentrations
inhibits
(lipocortins
of the Mr 35-36 kDa
about 50% by 20 uM GL, whereas
required
similar
more than 200 @I GL. These results show
phosphorylation,
by the kinase, of the Mr 35-36 kDa
I and II), rather than of the Mr 70-75 kDa polypeptides
or
casein. DISCUSSION A GL-binding cGMP-
protein kinase, which highly phosphorylated
and Ca2+-independent
membrane fraction
of rat liver using a GL-affinity
the purified kinase (approximately
casein in a CAMP-,
manner, has been successfully purified
from the crude
column. The enzymatic properties of
Mr 210 kDa) were very similar to those of kinase Pf4,
7-91, hecause (il these two kinases could he extracted only from the membrane using suitable detergents
fraction,
such as NP-40; (ii) casein was the most effective phosphate
acceptor for these kinases; and (iii) protein
phosphorylation
880
by these
kinases in vitro
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was enhanced greatly by basic polypeptides,
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such as histone HI, poly Arg and poly Lys
(Table 1 and Fig. 2). These experimental
results suggest that a specific GL-binding
protein kinase may be identical to klnase P. This assumption is supported by our previous observation
that GL binds directly
to kinase P from HeLa S3 cells (3). However,
molecular
sire (Mr 210 kDa) of the purified
relatively
larger
the
kinase (Mono Q fraction)
of rat liver was
than those of the kinases from yeast (a heterodimer
of Mr 75 kDa
subunit; 7) and HeLa S3 cells (Mr 120 kDa; 4). It is interesting
to observe that (i) a GL-binding
Mr 70-75 kDa and 35-36 kDa polypeptides
protein kinase was copurified
by DEAE-cellulose
(lane 5 in Fig. 2); (ii) these polypeptides kDa polypeptides
of the Mr 35-36 kDa polypeptide one-tenth
was
phoshpholipasa
modification kinase
of the Mr 35-36 of phosphorylation
by the kinase was obtained by approximately of VSV-associated
of the concentration
20 l.tM GL,
protein kinase activity. However,
of GL that causes 50% inhibition
of
A2 activity (12).
Earlier reports concerning phospholipase
by the kinase h
phosphorylation
by the kinase (Figs. 2 and 3). The 50% inhibition
which caused a similar inhibition this dose
column chromatography
were highly phosphorylated
vitro: and (iii) low doses of GL selectively inhibited
with
the biological
A2 activity at the regulation by cAMPdependent
(C-kinase)
phosphorylation
(14)
and
significance and physiological of phospholipase
function of
A2 activity through
protein kinase (A-kinase) (13), Ca2+dependent EGF-receptor
protein
by these three kinases is insensitive
kinase
(15).
Since
their protein
protein
to GL, the anti-inflammatory
activity of the drug may be mediated by different mechanisms, which are controlled
by
GL sensitive protein kinases, such as kinase I’. This speculation
is supported
observations that (i) GL binds directly to the membrane-associated
Mr 210 kDa protein
kinase and selectively inhibits phosphorylation 3); and (ii) [3HJ-labeled GL also dire&y which were separated
of lipocortins
bound to lipocortins
from the kinase by Mono Q column
by our
by the kinase (Figs. 2 and (35-36 kDa polypeptides), chromatography
(data not
shown). These data suggest that the anti-inflammatory by the inhibition
of lipocortin
phosphorylation
activity
by GL-binding
of GL may be mediated protein kinase at the cell
membrane level. Further analytical studies are under way concerning significance of lipocortins inhibitory
modified
by GL-binding
mechanism of phospholipase
the physiological
protein kinase, and the details of the
A2 activity by GL.
REFERENCES 1. Finney, R.S.H. and Somers, G.F. (1959) J. Pharmacol. 10, 613- 620. 2. Pompei, R., Flore, O., Marccialis, M.A., Pani, A. and Loddo, B. (1979) Nature 281,689-680. 3. Ohtsuki, K. and Ishida, N. (1988) B&hem. Biophys. Res. Commun. 157,597-604. 4. Ikeuchi, T., Yokoyama, M. and Ohtsuki, K. (1988) Tohoku J.Exp.Med. 155,41-56. 5. Mannufactory’s information from Pharmacia Fine Chemicals. 6. Laemmli, U.L. (1970) Nature 227,680-685 881
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7. Yanagita, Y., Abdel-Ghany, M., Raden, D. and Racker, E. (1987) Proc. Natl. Acad. Sci. USA, 84,925-929. 8. Abdel-Ghany, M., Kole, H.K. and Racker, E. (1987) Proc. Natl. Acad Sci. USA, 84,8888-8892. 9. Abdel-Ghany, M., Kale, H.K., Abou, EL, Saad, M. and Racker, E. (1989) Proc. Natt. Acad. Sci. USA, 86,6072-6076. 10. Greke, V. and Weber, K. (1985) J. Biol. Chem., 260, 1688-1695. 11. Glenny, R. (1986) J. Biol. Chem., 261, 7247-7252. 12. Ohuchi, K., Kamada, Y., Levine, L. and Tsurufuji, S. (1981) kostaglandin and Medicine 7,457-463. 13. Hirata, F. (1981) J. Biol. Chem., 256, 7730-7733. 14. Khanna, N. C., Tokuda, M. and Waisman, D. M. (1986) Biochem. Biophys. Res. Commun., 141,547-554. 15. Fava, R. A. and Cohen, S. (1984) J. Biol. Chem., 259,2636-2645.
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