Vol.
174,
January
No.
2, 1991
BIOCHEMICAL
BIOPHYSICALRESEARCH
AND
COMMUNICATIONS
31, 1991
ISOLATION
Pages
OF HUMAN BRAIN PROTEIN KINASE C: EVIDENCE FOR KINASE C FRAGMENT MODULATING G PROTEIN-GTPase ACTIVITY Claude
*Centre France
de Recherche et +Centre
Received
Christopher
SAUVAGE*+,
November
D. CASH' and Michel
Delalande, 10 rue des Cart-i&es, de Neurochimie du CNRS, 5 Strasbourg cedex, France 29,
593-599
CATALYTIC
MAITRE'
92500 Rueil-Malmaison, rue Blaise Pascal, 67084
1990
SUMMARY: Protein kinase C from human brain was isolated and characterized. A protein kinase M like kinase of molecular weight 63 kDa was also partially purified and identified by its immunological properties similar to those of kinase C. The kinase M like kinase activity, devoid of Cazc and phospholipids dependency , was also characterized by its inhibition profile by several Since this kinase phosphorylates a G protein (M.W. 36 kDa) and ligands. decreases its GTPase activity which could be restored by alkaline concluded that this kinase M like kinase could interact phosphatase, it is with G protein mediated events of neuronal responses. G 1991 Acadrmlc Press, Inc.
Protein
kinase
transduction domain
in
of
A
role
kinase
proteinase,
active
absence
kinase
M
(7,8).
platelets
with
activation
of protein
several
regulated.
altering
derived here the
role
from that
and
in the
as the
human
brain kinase
activity
membranes and not
associated
after
could
constitute
with
treatment
of
Thus
the
a mechanism
by
are here
C and kinase
GTPase et kinase a
is
or
we report
kinase
protein
M)
in brain,
material,
(Sauvage
Ca*'
exocytosis
(10).
particularly
protein
the
kinase
like
esters
starting
signal
(4,6,7).
observed
M in attenuating
M
with
(protein
phorbol
cell,
of both
of kinase
protein
GTPase
(9)
of
The catalytic
digestion
cofactors
has been
systems
and characterization
(1,2,3,4,5).
in processes
C by proteolysis
human brain
the
suggested
C
mechanisms
fragment
and lipids
M accumulation
kinase
several
following
catalytic
been
phospholipase
Using
have addressed
document
Kinase
be cleaved This
has
in
and tissues
of Ca2'
transduction
purification
protein
C can
in the
for
role
of cells
calpain.
secretion
which
an important
a variety
protein
dependent fully
C plays
human
activity
al.
brain
the
M. We of
submitted).
C, is
up
capable membrane
a G We of G
Vol.
BIOCHEMICAL
174, No. 2, 1991
protein. after protein
This
study
specific mediated
provide
clues
AND BIOPHYSICAL to
proteolysis,
may
transduction
signals. MATERIALS
mechanisms
induce
long
RESEARCH COMMUNICATIONS
by which lasting
protein responses
kinase to the
C, G
AND METHODS
1. Chemicals Ci/mmol) were from Amersham. [y-3L~]~T~ (3000 Ci/mmol) and [r- 32P]GTP (10 I. cniorpromazine, Quercetin, staurosporine, Sphingosine, H7, polyvinylpyrrolidone M.W. 40,000, histone type IIIS, insoluble alkaline phosphatase were obtained from Sigma. Tamoxifen was a gift from ICI-Pharma. Polyclonal antibodies against kinase C was a generous gift from Dr K.P. Huang (NIH,U.S.A.). Rabbit anti goat IgG was purchased from Biosys. Matrix gels (Red A) was from Amicon. 2. Purification of PKM and PKC PKM was partially purified from human brain by modifications of the procedure of Takai et al. (11) and Junco et al. (12). Human brain was obtained after a 24 hours postmortem delay. The tissue was homogeneized in four volumes of a medium containing 1 mM phenylmethylsulfonyl fluoride, 2 mM EGTA, 5mM ,& mercaptoethanol and 50 mM Tris-HCl pH 7.65 (buffer A). After centrifugation, the supernatant was applied to a Red-A Matrix gel (Amicon) equilibrated with buffer A supplemented with 100 mM NaCl. The enzyme was directly eluted onto a DE52 column attached to Red-A Matrix gel using buffer A containing 5% polyvinylpyrrolidone. In the next chromatography (DE52 step), the enzyme was eluted with a gradient of 0 to 600 mM NaCl in buffer A supplemented with 10% glycerol. The active fractions, containing both protein kinase C and kinase M were applied to a phosphatidylserine Affigel 10 column (13). The protein kinase M activity passed through this column and was further purified by a phenyl Sepharose chromatography according to Junco et al. (12) followed by an hydroxyapatite step, as described by Takai et al. (11). Protein kinase C activity was eluted from the phosphatidylserine column by the method of Schatzman et al. (13) and was further subjected to hydroxyapatite chromatography according to Azhar et al. (14). 3. Determination of PKC and PKM activities PKC activity was determined as described by Louis et al. (15). PKM activity was assayed as for PKC activity but in the absence of Ca*+ and lipids (phosphatidylserine and diolein) and in the presence of 2 mM EGTA. 4. Characterization of PKC and PKM activities from human brain PKC and PKM activities were characterized first by determining the different Ki for various known inhibitors of PKC, as indicated by Junco et al. (12) and for PKC by measuring K,.values for Ca", ATP and phospholipids. In addition, immunological characterization (immunoblot) was carried out as described by Huang and Huang (16) using a specific polyclonal antibody directed against rat brain protein kinase C. 5. Measurement of 32P incorporation catalvsed bv kinase M into G protein from human brain The GTP-binding protein of molecular weight 36,000 Da (G,e), isolated and characterized from human brain (Sauvage et al submitted) was phosphorylated by kinase M using the procedure of Katada et al. (17) except that the incubation medium contained no Ca*' and no lipids but 2 mM EGTA. The kinetics of the phosphorvlation reaction was followed from 0 to 30 minutes. The radioactive'banhs were separated on SDS-PAGE (18). The gel was dried, the bands were excised and the radioactivity was determined. 6. Decrease The influence
of GTPase activitv of phosphorylation
followino induced 594
phosphorvlation bv kinase M by kinase M on the GTPase activity
Vol.
174,
No.
BlOCHEMlCALANDBlOPHYSlCALRESEARCH
2, 1991
was followed by measuring the Zki?Ri et al. (19). The kinetics of GTPase activity phosphorylation by kinase M. G incubated with protein kinase M (a&t the absence of ATP (20 PM) and with at different time and the kinetics of and compared to the control incubated (no ATP). 7. Recovery of GTPase After phosphorylation activity (for conditions for 90 minutes against and 2 mM mM NaCl phosphorylated G s phosphatase, attac 4 ed alkaline phosphatase.
hydrolysis
COMMUNICATIONS
of
[y-32P]GTP
as described
by
followed during progressive was from human brain (about 1.5 pg) was 1 g of protein) in the presence or in were taken [r- 3!! P]GTP (10 nM). Aliquots the [T-~*P]GTP hydrolysis was followed under non phosphorylating conditions
activitv followinq treatment with alkaline ohosphatase for 20 minutes in presence of kinase M like of G,, see paragraph 6), the incubation medium was dialysed 20 mM Tris HCl pH 8.0, 1 ml4 EDTA, 0.1 % Tween 20, 100 p-mercaptoethanol in order to eliminate ATP. The to hydrolysis by insoluble alkaline was submitted to beaded agarose. A control was performed using boiled
RESULTS Compared
to
retained
by
(without
two
Red-A
measuring
a single factor
the
peak is
largely
specific
activity
dependent
on the
first matrix
enzyme
has
chromatographic
been
gel
of
purified
the
presence
A
has
activity),
underestimated
B
been
where
directly
the
final
kinase
about
1582
fold.
Thus,
to the
initial
regards
purified
enzyme (Km app.=
was 1.21
M.W.
B 205
1386
E
activity
on DE52 support
C preparation
+0.36
D
enzyme
eluted
the
with
of Ca*'
steps
this
eluted
as
purification
homogenate. nmol/min/mg
PM) and
The and was
phosphatidyl-
F
kDa
kDa 97 kDa
116
66 kDa
45 kDa
29 kDa
Fig. (lanes lane lane lane lane lane lane
1. A: B: C: D: E: F:
-
SDS-PAGE of samples from stages of purification A,B,C) and protein kinase M like activity (lanes DEAE Sepharose pool Phosphatidyl serine affinity column pool Hydroxyapatite pool (purified kinase C) non retained fraction (pool) from phosphatidyl phenyi-sepharose pool Hydroxyapatite pool (purified kinase M activity)
595
of protein D,E,F).
serine
affinity
kinase
column
C
Vol.
174,
No.
2,
1991
BIOCHEMICALAND
Table
I.
BIOPHYSICAL
Inhibition by several
ligand
parameters ligands
(Ki)
Chlorpromazine
(fl)
Staurosporine
(nM)
38
NI
Tamoxifen
(Km app.
= 30 +5 PM).
= 3 20.9
The final
Polyacrylamide protein
gel
band
Endogenous after
two
2.3
nmol/min/mg)
enzyme
for
Various
ATP is
in
Staurosporine both
Quercetin
effect
the
protein
derived
of kinase
major kDa;
only
PKC activity,
inhibition
rat
protein
obtained
value
is
greatly
The
activity after
SDS gel
affinity
of
this
PM.
these
activity also
kinase
activity
bands
1).
characterize
the
a single
M (specific
protein Fig.
revealed
the the
fully
kinase from
two
also
app.
1).
to
to
were
against
case
preparation
and 60
Da (Fig.
compared
ATP (Km
at -70°C.
materiel
& 5,000
fold
for
stored
C and kinase
inhibited
Antiserum
when
purified
this
to
affinity
on kinase
the
on protein
showed
tested
inhibited
and chlorpromazine
the inhibitors reaction with
the
were order
kinases,
final
rise
+1.5
6
in
63 kOa
6.5
inhibitors
substrate,
the
(M.W.=
NI
of the
about
gived
electrophoresis
16
was stable
step,
However
NI
with
mass of 80,000
purified
a
90.3
The enzyme
preparation
first
underestimated.
869
incubated to starting
pg/ml).
of molecular
the
9.2
(PM)
electrophoresis
PKM was
NI
4
The enzymes were for 5 min. prior [1-32P]~~~. NI. not inhibited.
serine
3.1
(PM)
COMMUNICATIONS
PKM
PKW
(PM)
Sphingosine
PKC and
PKC
H7 (d-f) Quercetin
of
RESEARCH
while
of
protein
of
both
strong
two
protein
histone
activities
H7 inhibited
kinase
inhibitors
M using
(table the
C activity
kinases
I).
activity
of
was stronger.
C and
of protein
Hl as
kinase
M. Tamoxifen C,
with
no
M activity.
brain M like
protein protein
kinase
kinase kinase,
C.
As 596
C was
used
presumably
shown
on the
in order a
to
proteolytic
immunoblot
identify product
(Fig.
2) the
Vol.
174,
No.
protein
BIOCHEMICALAND
2, 1991
kinase
C preparation
and a faint
band
like
preparation
kinase
visible
at
reacts
with
exhibited
at 63 kDa.
Thus,
a proteolytic
one major
An immunoblot
revealed
80 kDa.
BIOPHYSICALRESEARCH
it
could
product
reactive
carried
a major
COMMUNICATIONS
out
band
on the
(M.W.
protein
concluded
of kinase
that
C having
the
kDa)
kinase
band of 63 kDa and a minor
be
80
band
antiserum
a molecular
M was
cross
mass
around
63 kDa. As
described
purified
in
from
36,000
Da.
purified
previous
crude
human
This
about
preparative submitted). protein et al.
a
GTP 50
report brain
binding
fold
kinase
by
reported
several
induced
The present
Of G36 inhibited
66 kDa,
t
al.
by
results
SDS-PAGE, steps
electroelution
decrease
catalysed
and
(Sauvage of G,,
in GTPase
demonstrate
we have
of molecular
in
phosphorylation
a strong
submitted),
a G protein
chromatographic
the
by phosphorylation A
116 97 kDa kDa
that
et
homogenous
followed
activity
submitted).
membranes
protein,
electrophoresis We have
(Sauvage
that
by kinase
mass
has been a
final et
by kinase activity
the
M like (Sauvage
GTPase
M like
al.
activity could
kinase,
B
c
.50
-(
m
45 kDa. .
29 kDab
0
2
0 3
0
10
20
30 lime
40 (nin
50
60
70
)
Fig. 7, Immunoblot of purified human brain protein kinase C and protein kinase M. lane A: protein kinase C (phosphatidylserine Affigel 10 step) lane B: protein kinase M (hydroxyapatite step) Proteins were separated by SDS-PAGE, followed by electrophoretic transfer to Immobilon PVDF. Membranes were incubated with anti-rat brain PKC antibody (16). The immunoreactive bands were detected by autoradiography after incubation with lz51-protein A. Fig. 3. Effect of phosphorylation by PKM on GTPase activity of human brain G protein (Gj6). purified as described in Sauva e et G proteins, al. (submitted) were incubated with PKM, [y-32~]~~~ and 20 PM ATP (0 3 or without ATP (0). GTPase activity was followed by measuring hydrolysis of [y-32~]~~~. Restoration of GTPase activitv after incubation with alkaline ohosohatase (A), control incubation carr;ed out with boiled alkaline phosphatese showed no change.
597
Vol.
174,
No.
2, 1991
be completely the
BIOCHEMICAL
restored
presence
AND
by incubation
of alkaline
BIOPHYSICAL
of the
phosphatase
(Fig.
RESEARCH
phosphorylated
COMMUNICATIONS
G,,
preparation
in
3).
DISCUSSION The purification hours
postmortem)
about rat
gave
one third brain
band
of
to about in
in the
present
extract.
This
study
purified
affected
resembled
the
(63
than
kDa)
Takai
et al.
mass of
(11)
64 kDa
possess
a similar
We have
previously
kinase
C)
from
This
ribosylated
by
phosphorylation
G
partial.
and
rat
kinase
molecular
M
M
the
activity
598
rat
from
from
brain
could
rat
(Sauvage (Gs6) Gia
(4).
molecular
of
In the
brain. (but
not
et
al.
was ADP-
or G,a.
This
present
paper,
32P incorporation
into
phosphorylation reached
(12). higher
human brain
either
of
closely brain
human
kDa
C but
50 kDa)
that
36
activity.
has
kinase
M with
same origin
kinetics
The
(about
from
representing
present
homogenate
was a little
brain
mass
in GTPase
used brain
result
from
of kinase
of the
thus
(16).
C
rat
brain
This M
different
this
kinase
of fresh
kinase
kinase
slighly
incubation, GTPase
M like
kinase
by H7 and quercetin,
kinase
Thus,
of
a decrease
Nevertheless,
for
M from
G protein
induces
minutes
brain
in presence
against
enzymes
purification
that
toxin,
G36.
human
active
or tamoxifen.
kinase
brain.
pertussis
study
protein
protein
in crude
was inhibited
kinase
protein,
this
lo-20
this
mass,
a
we have extented
C activity
the
reported
a
one band on immunoblot
found
reported
molecular
the
was fully
The antiserum
study
for
bovine
fresh
and phospholipids.
contained
which
sphingosine
reported have
on Ca"
was
from
a single
C inhibited
brain
but
kinase
mass of
phosphorylates
submitted).
After
only
profile
molecular that
C,
present
inhibition
24
activity
purified
revealed
kinase
human
by staurosporine,
However,
preparation
protein
specific
enzyme
and protease-degraded
in the
the
the
approximately
(12).
inhibits native
whose
kDa and was dependent
revealed
both
for
of phospholipids.
antibody
and recognize
enzyme
to kinase
in absence
(obtained
preparation
reported
experiments,
related
EGTA and
not
activity
same extent
our
human brain
enzyme
of mammalian the
immunologically
activity
an
mass 77-80
inhibitors
shown
C from
The purified
of molecular
enzyme
of
the
(20,21).
Classical
As
of kinase
of its
G,,
was
only
maximum decrease.
Vol.
174,
No.
Subsequent GTPase
BIOCHEMICAL
2, 1991
treatment
activity.
with
Hence,
AND
akaline
BIOPHYSICAL
phosphatase
a reversible
RESEARCH
completely
phosphorylation
COMMUNICATIONS
restored process
the
initial
of G protein
was
observed. Thus,
the
regulation
phosphorylation
here
have
Most
kinase
uniquely
M and
to show that
PKC action.
GTPase
may be implicated
a G protein. protein
of
the
However,
not
in long
such
term
triggered
due
cellular
responses
a phosphorylation
by protein
proteolytic the
activity
kinase cleavage
physiological
event
C. Direct product
relevance
is
evidence
to
reversible mediated
catalysed is
of PKC may be of
this
by by
a
presented a way for
phenomenon
shall
to be determined. ACKNOWLEDGMENTS
for helpful The authors thank Prof. A. N. Malviya Huang (NIH) for kindly the manuscript and Dr. K-P. protein kinase C antibody.
discussions providing
and review of anti-rat brain
REFERENCES 1. Carpenter, D., Jackson, T. and Hanley, M.R. (1987) Nature 325, 107-108. 2. Nishizuka, Y. (1988) Nature 334, 661-665. Y. (1984) Nature 308, 693-698. 3. Nishizuka, 4. Inoue, M., Kishimoto, A., Takai, Y. and Nishizuka, Y. (1977) J. Biol Chem. 252, 7610-7616. 5. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276. 6. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S.I., Tominaga, M., Kuroda, T. and Nishizuka, Y. (1989) J. Biol. Chem. 264, 4088-4092. 7. Kishimoto, A., Kajikawa, N., Shiota, M. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 1156-1164. 8. Pontremoli, S., Michetti, M., Melloni, E., Sparatore, B., Salamino, F. and Horecker, B. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3705-3707. 9. Tapley, P.M. and Murray, A.W. (1984) Biochem. Biophys. Res. Comm. 122, 158-164. P.M. and Murray, A.W. (1984) Biochem. Biophys. Res. Comm. 118, 10. Tapley,
835-841. 11. Takai, Y.,
Kishimoto, A., Inoue, M. and Nishizuka, Y. Chem. 252, 7603-7609. Junco, M., Diaz-Guerra, M. and Bosca, L. (1990) FEBS ::: Schatzman, R.C., Raynor, R.L., Fritz, R. B. and Kuo, Biochem. J. 209, 435-443. 14. Azhar, S., Butte, J. and Reaven, E. (1987) Biochemistry 15. Louis, J.C., Magal, E. and Yavin, E. (1988) J. Biol.
(1977) J. Lett. J.F.
Biol.
263,169-171. (1983)
26, 7047-7057. Chem. 263, 19282-
19285. Huang, K.P. and Huang, F.L. (1986) J. Biol. Chem. 261, 14781-14787. Katada, T., Gilman, A.G., Watanabe, Y., Bauer, S. and Jakobs K.H. (1985) Eur. J. Biochem. 151, 431-437. 18. Laemmli, U.K. (1970) Nature 227, 680-685. A., Yamashita, T., Kawata, M., Yamamoto, M., Ikeda, K., 19. Kikuchi, Tanimoto, T. and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904. Uchida, T. and Filburn, C.R. (1984) J. Biol. Chem. 259, 12311-12314. E: Woodgett, J.R. and Hunter, T. (1987) J. Biol. Chem. 262, 4836-4843.
E:
599
174,
January
No.
2, 1991
BIOCHEMICAL
BIOPHYSICALRESEARCH
AND
COMMUNICATIONS
31, 1991
ISOLATION
Pages
OF HUMAN BRAIN PROTEIN KINASE C: EVIDENCE FOR KINASE C FRAGMENT MODULATING G PROTEIN-GTPase ACTIVITY Claude
*Centre France
de Recherche et +Centre
Received
Christopher
SAUVAGE*+,
November
D. CASH' and Michel
Delalande, 10 rue des Cart-i&es, de Neurochimie du CNRS, 5 Strasbourg cedex, France 29,
593-599
CATALYTIC
MAITRE'
92500 Rueil-Malmaison, rue Blaise Pascal, 67084
1990
SUMMARY: Protein kinase C from human brain was isolated and characterized. A protein kinase M like kinase of molecular weight 63 kDa was also partially purified and identified by its immunological properties similar to those of kinase C. The kinase M like kinase activity, devoid of Cazc and phospholipids dependency , was also characterized by its inhibition profile by several Since this kinase phosphorylates a G protein (M.W. 36 kDa) and ligands. decreases its GTPase activity which could be restored by alkaline concluded that this kinase M like kinase could interact phosphatase, it is with G protein mediated events of neuronal responses. G 1991 Acadrmlc Press, Inc.
Protein
kinase
transduction domain
in
of
A
role
kinase
proteinase,
active
absence
kinase
M
(7,8).
platelets
with
activation
of protein
several
regulated.
altering
derived here the
role
from that
and
in the
as the
human
brain kinase
activity
membranes and not
associated
after
could
constitute
with
treatment
of
Thus
the
a mechanism
by
are here
C and kinase
GTPase et kinase a
is
or
we report
kinase
protein
M)
in brain,
material,
(Sauvage
Ca*'
exocytosis
(10).
particularly
protein
the
kinase
like
esters
starting
signal
(4,6,7).
observed
M in attenuating
M
with
(protein
phorbol
cell,
of both
of kinase
protein
GTPase
(9)
of
The catalytic
digestion
cofactors
has been
systems
and characterization
(1,2,3,4,5).
in processes
C by proteolysis
human brain
the
suggested
C
mechanisms
fragment
and lipids
M accumulation
kinase
several
following
catalytic
been
phospholipase
Using
have addressed
document
Kinase
be cleaved This
has
in
and tissues
of Ca2'
transduction
purification
protein
C can
in the
for
role
of cells
calpain.
secretion
which
an important
a variety
protein
dependent fully
C plays
human
activity
al.
brain
the
M. We of
submitted).
C, is
up
capable membrane
a G We of G
Vol.
BIOCHEMICAL
174, No. 2, 1991
protein. after protein
This
study
specific mediated
provide
clues
AND BIOPHYSICAL to
proteolysis,
may
transduction
signals. MATERIALS
mechanisms
induce
long
RESEARCH COMMUNICATIONS
by which lasting
protein responses
kinase to the
C, G
AND METHODS
1. Chemicals Ci/mmol) were from Amersham. [y-3L~]~T~ (3000 Ci/mmol) and [r- 32P]GTP (10 I. cniorpromazine, Quercetin, staurosporine, Sphingosine, H7, polyvinylpyrrolidone M.W. 40,000, histone type IIIS, insoluble alkaline phosphatase were obtained from Sigma. Tamoxifen was a gift from ICI-Pharma. Polyclonal antibodies against kinase C was a generous gift from Dr K.P. Huang (NIH,U.S.A.). Rabbit anti goat IgG was purchased from Biosys. Matrix gels (Red A) was from Amicon. 2. Purification of PKM and PKC PKM was partially purified from human brain by modifications of the procedure of Takai et al. (11) and Junco et al. (12). Human brain was obtained after a 24 hours postmortem delay. The tissue was homogeneized in four volumes of a medium containing 1 mM phenylmethylsulfonyl fluoride, 2 mM EGTA, 5mM ,& mercaptoethanol and 50 mM Tris-HCl pH 7.65 (buffer A). After centrifugation, the supernatant was applied to a Red-A Matrix gel (Amicon) equilibrated with buffer A supplemented with 100 mM NaCl. The enzyme was directly eluted onto a DE52 column attached to Red-A Matrix gel using buffer A containing 5% polyvinylpyrrolidone. In the next chromatography (DE52 step), the enzyme was eluted with a gradient of 0 to 600 mM NaCl in buffer A supplemented with 10% glycerol. The active fractions, containing both protein kinase C and kinase M were applied to a phosphatidylserine Affigel 10 column (13). The protein kinase M activity passed through this column and was further purified by a phenyl Sepharose chromatography according to Junco et al. (12) followed by an hydroxyapatite step, as described by Takai et al. (11). Protein kinase C activity was eluted from the phosphatidylserine column by the method of Schatzman et al. (13) and was further subjected to hydroxyapatite chromatography according to Azhar et al. (14). 3. Determination of PKC and PKM activities PKC activity was determined as described by Louis et al. (15). PKM activity was assayed as for PKC activity but in the absence of Ca*+ and lipids (phosphatidylserine and diolein) and in the presence of 2 mM EGTA. 4. Characterization of PKC and PKM activities from human brain PKC and PKM activities were characterized first by determining the different Ki for various known inhibitors of PKC, as indicated by Junco et al. (12) and for PKC by measuring K,.values for Ca", ATP and phospholipids. In addition, immunological characterization (immunoblot) was carried out as described by Huang and Huang (16) using a specific polyclonal antibody directed against rat brain protein kinase C. 5. Measurement of 32P incorporation catalvsed bv kinase M into G protein from human brain The GTP-binding protein of molecular weight 36,000 Da (G,e), isolated and characterized from human brain (Sauvage et al submitted) was phosphorylated by kinase M using the procedure of Katada et al. (17) except that the incubation medium contained no Ca*' and no lipids but 2 mM EGTA. The kinetics of the phosphorvlation reaction was followed from 0 to 30 minutes. The radioactive'banhs were separated on SDS-PAGE (18). The gel was dried, the bands were excised and the radioactivity was determined. 6. Decrease The influence
of GTPase activitv of phosphorylation
followino induced 594
phosphorvlation bv kinase M by kinase M on the GTPase activity
Vol.
174,
No.
BlOCHEMlCALANDBlOPHYSlCALRESEARCH
2, 1991
was followed by measuring the Zki?Ri et al. (19). The kinetics of GTPase activity phosphorylation by kinase M. G incubated with protein kinase M (a&t the absence of ATP (20 PM) and with at different time and the kinetics of and compared to the control incubated (no ATP). 7. Recovery of GTPase After phosphorylation activity (for conditions for 90 minutes against and 2 mM mM NaCl phosphorylated G s phosphatase, attac 4 ed alkaline phosphatase.
hydrolysis
COMMUNICATIONS
of
[y-32P]GTP
as described
by
followed during progressive was from human brain (about 1.5 pg) was 1 g of protein) in the presence or in were taken [r- 3!! P]GTP (10 nM). Aliquots the [T-~*P]GTP hydrolysis was followed under non phosphorylating conditions
activitv followinq treatment with alkaline ohosphatase for 20 minutes in presence of kinase M like of G,, see paragraph 6), the incubation medium was dialysed 20 mM Tris HCl pH 8.0, 1 ml4 EDTA, 0.1 % Tween 20, 100 p-mercaptoethanol in order to eliminate ATP. The to hydrolysis by insoluble alkaline was submitted to beaded agarose. A control was performed using boiled
RESULTS Compared
to
retained
by
(without
two
Red-A
measuring
a single factor
the
peak is
largely
specific
activity
dependent
on the
first matrix
enzyme
has
chromatographic
been
gel
of
purified
the
presence
A
has
activity),
underestimated
B
been
where
directly
the
final
kinase
about
1582
fold.
Thus,
to the
initial
regards
purified
enzyme (Km app.=
was 1.21
M.W.
B 205
1386
E
activity
on DE52 support
C preparation
+0.36
D
enzyme
eluted
the
with
of Ca*'
steps
this
eluted
as
purification
homogenate. nmol/min/mg
PM) and
The and was
phosphatidyl-
F
kDa
kDa 97 kDa
116
66 kDa
45 kDa
29 kDa
Fig. (lanes lane lane lane lane lane lane
1. A: B: C: D: E: F:
-
SDS-PAGE of samples from stages of purification A,B,C) and protein kinase M like activity (lanes DEAE Sepharose pool Phosphatidyl serine affinity column pool Hydroxyapatite pool (purified kinase C) non retained fraction (pool) from phosphatidyl phenyi-sepharose pool Hydroxyapatite pool (purified kinase M activity)
595
of protein D,E,F).
serine
affinity
kinase
column
C
Vol.
174,
No.
2,
1991
BIOCHEMICALAND
Table
I.
BIOPHYSICAL
Inhibition by several
ligand
parameters ligands
(Ki)
Chlorpromazine
(fl)
Staurosporine
(nM)
38
NI
Tamoxifen
(Km app.
= 30 +5 PM).
= 3 20.9
The final
Polyacrylamide protein
gel
band
Endogenous after
two
2.3
nmol/min/mg)
enzyme
for
Various
ATP is
in
Staurosporine both
Quercetin
effect
the
protein
derived
of kinase
major kDa;
only
PKC activity,
inhibition
rat
protein
obtained
value
is
greatly
The
activity after
SDS gel
affinity
of
this
PM.
these
activity also
kinase
activity
bands
1).
characterize
the
a single
M (specific
protein Fig.
revealed
the the
fully
kinase from
two
also
app.
1).
to
to
were
against
case
preparation
and 60
Da (Fig.
compared
ATP (Km
at -70°C.
materiel
& 5,000
fold
for
stored
C and kinase
inhibited
Antiserum
when
purified
this
to
affinity
on kinase
the
on protein
showed
tested
inhibited
and chlorpromazine
the inhibitors reaction with
the
were order
kinases,
final
rise
+1.5
6
in
63 kOa
6.5
inhibitors
substrate,
the
(M.W.=
NI
of the
about
gived
electrophoresis
16
was stable
step,
However
NI
with
mass of 80,000
purified
a
90.3
The enzyme
preparation
first
underestimated.
869
incubated to starting
pg/ml).
of molecular
the
9.2
(PM)
electrophoresis
PKM was
NI
4
The enzymes were for 5 min. prior [1-32P]~~~. NI. not inhibited.
serine
3.1
(PM)
COMMUNICATIONS
PKM
PKW
(PM)
Sphingosine
PKC and
PKC
H7 (d-f) Quercetin
of
RESEARCH
while
of
protein
of
both
strong
two
protein
histone
activities
H7 inhibited
kinase
inhibitors
M using
(table the
C activity
kinases
I).
activity
of
was stronger.
C and
of protein
Hl as
kinase
M. Tamoxifen C,
with
no
M activity.
brain M like
protein protein
kinase
kinase kinase,
C.
As 596
C was
used
presumably
shown
on the
in order a
to
proteolytic
immunoblot
identify product
(Fig.
2) the
Vol.
174,
No.
protein
BIOCHEMICALAND
2, 1991
kinase
C preparation
and a faint
band
like
preparation
kinase
visible
at
reacts
with
exhibited
at 63 kDa.
Thus,
a proteolytic
one major
An immunoblot
revealed
80 kDa.
BIOPHYSICALRESEARCH
it
could
product
reactive
carried
a major
COMMUNICATIONS
out
band
on the
(M.W.
protein
concluded
of kinase
that
C having
the
kDa)
kinase
band of 63 kDa and a minor
be
80
band
antiserum
a molecular
M was
cross
mass
around
63 kDa. As
described
purified
in
from
36,000
Da.
purified
previous
crude
human
This
about
preparative submitted). protein et al.
a
GTP 50
report brain
binding
fold
kinase
by
reported
several
induced
The present
Of G36 inhibited
66 kDa,
t
al.
by
results
SDS-PAGE, steps
electroelution
decrease
catalysed
and
(Sauvage of G,,
in GTPase
demonstrate
we have
of molecular
in
phosphorylation
a strong
submitted),
a G protein
chromatographic
the
by phosphorylation A
116 97 kDa kDa
that
et
homogenous
followed
activity
submitted).
membranes
protein,
electrophoresis We have
(Sauvage
that
by kinase
mass
has been a
final et
by kinase activity
the
M like (Sauvage
GTPase
M like
al.
activity could
kinase,
B
c
.50
-(
m
45 kDa. .
29 kDab
0
2
0 3
0
10
20
30 lime
40 (nin
50
60
70
)
Fig. 7, Immunoblot of purified human brain protein kinase C and protein kinase M. lane A: protein kinase C (phosphatidylserine Affigel 10 step) lane B: protein kinase M (hydroxyapatite step) Proteins were separated by SDS-PAGE, followed by electrophoretic transfer to Immobilon PVDF. Membranes were incubated with anti-rat brain PKC antibody (16). The immunoreactive bands were detected by autoradiography after incubation with lz51-protein A. Fig. 3. Effect of phosphorylation by PKM on GTPase activity of human brain G protein (Gj6). purified as described in Sauva e et G proteins, al. (submitted) were incubated with PKM, [y-32~]~~~ and 20 PM ATP (0 3 or without ATP (0). GTPase activity was followed by measuring hydrolysis of [y-32~]~~~. Restoration of GTPase activitv after incubation with alkaline ohosohatase (A), control incubation carr;ed out with boiled alkaline phosphatese showed no change.
597
Vol.
174,
No.
2, 1991
be completely the
BIOCHEMICAL
restored
presence
AND
by incubation
of alkaline
BIOPHYSICAL
of the
phosphatase
(Fig.
RESEARCH
phosphorylated
COMMUNICATIONS
G,,
preparation
in
3).
DISCUSSION The purification hours
postmortem)
about rat
gave
one third brain
band
of
to about in
in the
present
extract.
This
study
purified
affected
resembled
the
(63
than
kDa)
Takai
et al.
mass of
(11)
64 kDa
possess
a similar
We have
previously
kinase
C)
from
This
ribosylated
by
phosphorylation
G
partial.
and
rat
kinase
molecular
M
M
the
activity
598
rat
from
from
brain
could
rat
(Sauvage (Gs6) Gia
(4).
molecular
of
In the
brain. (but
not
et
al.
was ADP-
or G,a.
This
present
paper,
32P incorporation
into
phosphorylation reached
(12). higher
human brain
either
of
closely brain
human
kDa
C but
50 kDa)
that
36
activity.
has
kinase
M with
same origin
kinetics
The
(about
from
representing
present
homogenate
was a little
brain
mass
in GTPase
used brain
result
from
of kinase
of the
thus
(16).
C
rat
brain
This M
different
this
kinase
of fresh
kinase
kinase
slighly
incubation, GTPase
M like
kinase
by H7 and quercetin,
kinase
Thus,
of
a decrease
Nevertheless,
for
M from
G protein
induces
minutes
brain
in presence
against
enzymes
purification
that
toxin,
G36.
human
active
or tamoxifen.
kinase
brain.
pertussis
study
protein
protein
in crude
was inhibited
kinase
protein,
this
lo-20
this
mass,
a
we have extented
C activity
the
reported
a
one band on immunoblot
found
reported
molecular
the
was fully
The antiserum
study
for
bovine
fresh
and phospholipids.
contained
which
sphingosine
reported have
on Ca"
was
from
a single
C inhibited
brain
but
kinase
mass of
phosphorylates
submitted).
After
only
profile
molecular that
C,
present
inhibition
24
activity
purified
revealed
kinase
human
by staurosporine,
However,
preparation
protein
specific
enzyme
and protease-degraded
in the
the
the
approximately
(12).
inhibits native
whose
kDa and was dependent
revealed
both
for
of phospholipids.
antibody
and recognize
enzyme
to kinase
in absence
(obtained
preparation
reported
experiments,
related
EGTA and
not
activity
same extent
our
human brain
enzyme
of mammalian the
immunologically
activity
an
mass 77-80
inhibitors
shown
C from
The purified
of molecular
enzyme
of
the
(20,21).
Classical
As
of kinase
of its
G,,
was
only
maximum decrease.
Vol.
174,
No.
Subsequent GTPase
BIOCHEMICAL
2, 1991
treatment
activity.
with
Hence,
AND
akaline
BIOPHYSICAL
phosphatase
a reversible
RESEARCH
completely
phosphorylation
COMMUNICATIONS
restored process
the
initial
of G protein
was
observed. Thus,
the
regulation
phosphorylation
here
have
Most
kinase
uniquely
M and
to show that
PKC action.
GTPase
may be implicated
a G protein. protein
of
the
However,
not
in long
such
term
triggered
due
cellular
responses
a phosphorylation
by protein
proteolytic the
activity
kinase cleavage
physiological
event
C. Direct product
relevance
is
evidence
to
reversible mediated
catalysed is
of PKC may be of
this
by by
a
presented a way for
phenomenon
shall
to be determined. ACKNOWLEDGMENTS
for helpful The authors thank Prof. A. N. Malviya Huang (NIH) for kindly the manuscript and Dr. K-P. protein kinase C antibody.
discussions providing
and review of anti-rat brain
REFERENCES 1. Carpenter, D., Jackson, T. and Hanley, M.R. (1987) Nature 325, 107-108. 2. Nishizuka, Y. (1988) Nature 334, 661-665. Y. (1984) Nature 308, 693-698. 3. Nishizuka, 4. Inoue, M., Kishimoto, A., Takai, Y. and Nishizuka, Y. (1977) J. Biol Chem. 252, 7610-7616. 5. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276. 6. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S.I., Tominaga, M., Kuroda, T. and Nishizuka, Y. (1989) J. Biol. Chem. 264, 4088-4092. 7. Kishimoto, A., Kajikawa, N., Shiota, M. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 1156-1164. 8. Pontremoli, S., Michetti, M., Melloni, E., Sparatore, B., Salamino, F. and Horecker, B. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3705-3707. 9. Tapley, P.M. and Murray, A.W. (1984) Biochem. Biophys. Res. Comm. 122, 158-164. P.M. and Murray, A.W. (1984) Biochem. Biophys. Res. Comm. 118, 10. Tapley,
835-841. 11. Takai, Y.,
Kishimoto, A., Inoue, M. and Nishizuka, Y. Chem. 252, 7603-7609. Junco, M., Diaz-Guerra, M. and Bosca, L. (1990) FEBS ::: Schatzman, R.C., Raynor, R.L., Fritz, R. B. and Kuo, Biochem. J. 209, 435-443. 14. Azhar, S., Butte, J. and Reaven, E. (1987) Biochemistry 15. Louis, J.C., Magal, E. and Yavin, E. (1988) J. Biol.
(1977) J. Lett. J.F.
Biol.
263,169-171. (1983)
26, 7047-7057. Chem. 263, 19282-
19285. Huang, K.P. and Huang, F.L. (1986) J. Biol. Chem. 261, 14781-14787. Katada, T., Gilman, A.G., Watanabe, Y., Bauer, S. and Jakobs K.H. (1985) Eur. J. Biochem. 151, 431-437. 18. Laemmli, U.K. (1970) Nature 227, 680-685. A., Yamashita, T., Kawata, M., Yamamoto, M., Ikeda, K., 19. Kikuchi, Tanimoto, T. and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904. Uchida, T. and Filburn, C.R. (1984) J. Biol. Chem. 259, 12311-12314. E: Woodgett, J.R. and Hunter, T. (1987) J. Biol. Chem. 262, 4836-4843.
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