BIOCHEMICAL
Vol. 76, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACTIVATION OF RABBIT SKELETAL MUSCLEADENOSINE-3':5'-MONOPHOSPHATE-DEPENDENTPROTEIN KINASE BY AGITATIONX Brigitte
Weinhold
Lehrstuhl fiir Arbeitsgruppe Ruhr-UniversitZt Received
and Nikolaus
Amrhein
Pflanzenphysiologie fiir Hormonphysiologie der Pflanzen Bochum, D 4630 Bochum,FRG
May 2,1977
SUMMARY Protein kinase activity in a preparation from rabbit skeletal muscle (Wastila,W.B.,Stull,J.T.,Ma er,S.E.,and Walsh,D.A.(1971)J.Biol.Chem. 246,1996-2003. ‘5 was increased appr. 15-fold after a 30 to 4Eec agitation of the incubation mixture on a Vortex mixer in either the presence or absence of the protein substrate,histone.Saturating concentrations of adenosine-3':5'-monophosphate (CAMP) stimulated the activity appr. 23-fold. 0.1% Triton X-100,present during the agitation,completely prevented agitation-induced activation,but left CAMP-dependent activation unaffected. Adenosine-3':5'-monophosphate kinases
[EC.2.7.1.37]have
similar
subunits.While catalytic
of the y-phosphate threenyl Highly
residues
the holoenzyme subunit
and specific
tissues
activation
by the cyclic kinases
(R) effects
(C),which protein
the release
catalyzes
(see reviews
methods for
nucleotide heart
was supported
1 The
1,2).
are based on the purified
skeletal
muscle(3,4).
by the Deutsche Forschungs-
abbreviations used are:cAMP,adenosine-3':5'-monophosphate; DEAE-,diethylaminoethyl-;EDTA,ethylenedi~inotetraacetic
Copyright 0 1977 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
or
the assay of CAMP in
of partially or rabbit
of
the transfer
groups of seryl
have been developed,which
from bovine
x This investigation gemeinschaft.
(RC) is inactive,binding
of ATP to the hydroxyl
biological protein
subunit
of substrate
sensitive
protein
been shown to be composed of two dis-
of CAMP to the regulatory the active
(CAMP)'-dependent
acid.
1116 ISSN
0006-291
X
Vol. 76, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Activation
of CAMP-dependent protein
with
substrate
their
from various
proteins
sources
protamine)cause
(5-7).
resulting
protein
kinasc
In this
communication
that
protein
from rabbit
The substrate
for
kinases
proteins
(his-tone,
of the CAMP-dependent protein
in a concomitant
increase
of CAMP-independent
activity.
kinase
we wish to report activity
skeletal kinase
on the novel
in a partially
muscle can be greatly
absence of CAMP and histone the protein
by interaction
has been reported
the dissociation
kinase
kinases
purified
preparation
stimulated
in the
simply by mechanical
in the test
observation
agitation
of
buffer.
lWTERIALS AND IVETHCDS The protein kinase preparation used in this study was prepared from rabbit skeletal muscle essentially as described by Wastila et al.(8)with adjustment of the buffer and ion exchange column volumes to the smaller scale of the preparations(500 g muscle for the first,30C 0m for the second preparation).DEAEcellulose DE-52(Whatman,Maidstone)was used for ion-exchange chromatography.Protein kinase activity was assayed according to Kuo and Greengard(3) using histone(Type III-S,lysine-rich,from calf thymus,Sigma,St.Louis) as substrate. All assays were carried cut in 15 ml conical test tubes.y-[32P]-ATP was synthesized according to Penefsky(9) with a specific activity of 20 Ci/mmol. Inorganic [32P]-phosphate was obtained from the Radiochemical Centre,Amersham.Biochemical reagents were from Boehringer,Mannheim,except for the protein kinase inhibitor from beef heart, which was from Sigma.Triton X-100,analytical grade,was from Serva,Heidelberg, and all other chemicals in analytical grade from Merck,Darmstadt. RESULTS AND DISCUSSION Fractions
eluted
step of the partial skeletal containing
muscle(8)
from the DEAE-cellulose
column in the final
purification
kinase
with
of protein
30 mM potassium
2 mM EDTA, showed low protein
absence of cAMP,but was stimulated of CAMPwere included to slightly
kinase
buffer, activity
15 to 20 fold,when
in the incubation
enhanced background
phosphate
from rabbit
activity,
1117
mixture(Figure
in the 10
pmoles
1). Due
in the absence of ad-
BIOCHEMICAL
Vol. 76, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
+F Fraction
Figure
1.
in Figure
fraction
High activities
with
in the subsequent
of the kinase
in our work with
of the kinase three
the kinase
the individual
the assays in this
laboratory.These
onents
or four
activity
fractions.
concomitant
preparation
experimentator
back to differences
of the incubation
activity
by added CAMPwere repeatedly
found to vary with traced
kinase
in the absence of CAMPwith
poor stimulation
finally
protein
stimulation
1)relative
by CAMPwas higher
encountered
30
Elution profile of protein kinase from DEAE-cellulose column.Protein kinase from rabbit skeletal muscle was adsorbed to a DEAE-cellulose column, equilibrated with 5 mM potassium phosphate buffer, pH 7.0,containing 2 mM EDTA,and,after washing, eluted with 30 mM potassium phosphate buffer,pH 7.0, again containing 2 mM EDTA(see ref.8 for details). 6 ml fractions were collected, and 10 ~1 of each fraction were tested for protein kinase activity in the absence and presence of 10 pmoles CAMP. Protein content of the fractions was determined according to (12).See Table I for details of kinase assay.
ded cAMP,in the first (No.19
20 Number
mixture:the
1118
puzzling
and were who performed
variations
in the mixing
were
of the comp-
more rigorously
the mixing
BIOCHEMICAL
Vol. 76, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Activation
of protein
kinsse
by cANP and agitation
Incubation mixtures contained the following components (in the IO ci; 0.5 M sodium acetate buffer, pH 6.0, order of addition): cAHP standard. :G ~1 O.? M Mg-acetate , 70 1~1 H20 or appropriate 10 1~1histone (2 mg/ml), 10 1.11of fraction 19(Fig.l),containing 53 kg protein determined according to (12),and IO $1 y-[32P]ATP(500 pnoles,0.5 &Ci). Incubation was for 5 min at 30°C,and phosphorylated histone was isolated according to (3). Agitation experiments (right column) were carried out in the absence of cAMP,and test tubes were agitated on a Vortex mixer for the periods indicated prior to the addition of ATP. Kinase activit in the absence of CAMP and without agitation was 1.2 pmoles "52P incorporated per min into histone. One pmole of incorporated ALP represents about 2200 cpm.Reactions were linear for at least 15 min. CAMP
pmoles/assay
relative kinase activity
tube
agitation period (set)
0
1
0
5
6
13
II.5
5 IO
20
16
50 100
23 21
1OOG
23
relative kinase activity
15 jc 40 60 120
was done the higher
was the kinase
cAMP.When incubation
mixtures
periods
on a Vortex
of agitation
mixer,prior
which the reaction
that
the kinase
activity
fold
by a 30 to 40 set agitation,
fold
stimulation
obtained
with
tion
to be fully from another
reproducible
to increasing to the addition
was started(3),
be maximally
it
was found
stimulated
15 to
as compared to an appr. saturating
CAMP in the assay mixture(Table1). found
in the absence of
were subjected
of the ATP,with
could
activity
concentrations
The agitation with
rabbit. 1119
a protein
effect kinase
23 of was
prepara-
16
BIOCHEMICAL
Vol. 76, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II Factors
affecting
agitation-induced
activation
of protein
kinase
Conditions of the assay are described in the legend of Table I. Incubation mixtures were completed with all components prior to the addition of ATP, with which the reaction was started. The term "incubation mixture" refers to the complete mixture without ATP. BEperiment relative kinase activity control(-CAMP,-agitation) 50 set agitation of incubation mixture 50 set agitation of incubation mixture containing 100 pmoles CAMP 50 set agitation of incubation mixture without his-tone 50 set agitation of incubation mixture without kinase 50 set agitation of incubation mixture containing 0.1% Triton X-100 50 set agitation of incubation mixture containing 0.1/aO' Triton X-100 + 100 pmoles CAMP 5C sec.agitation of incubation mixture without histone,followed by 4 min incubation with 40 pg of protein kinase inhibitor,The incubation mixture was then completed by addition of histone and the kinase reaction started by addition of ATP.
When protein
kinase,which
was subsequently activity that
subunits. stable free
agitation
1
0.4 25
6.4
observed
inhibitor,which
subunit
of protein
1120
in the presence
II),which
indicated
the same effect,i.e.
the regulatory
by experiments
kinase
by agitation,
of cAlYIPthe total
and CAMPproduce
This was corroborated
catalytic
16
assayed in the presence
of the holoenzyme into
protein
26
CAMP concentrations(Table
mechanical
dissociation
15
had been activated
did not exceed the activity
of saturating
1
and catalytic
in which a heat-
is known to bind to the kinase
(lO),was
edded to the
BIOCHEMICAL
Vol. 76, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE III Relative activation of protein in the fractions of the eluate
kinase by CAMP and by agitation from the DEAE-cellulose column Fraction
Protein kinase activity in fractions 19-25(Fig.l) was determined either in the presence of 100 pmoles CAMP or after a 50 set agitation of the incubation mixture prior to the addition of ATP. Conditions of the assay were as described in the legends of Fig.1 and Table I.
agitation-activated inhibition subunit
buffer
vation aerobic Inclusion
of
the agitation tion (Table
alone
alone was without as well
0.17
0.20 0.19
to the assay- (Table indicates
agitation
that
as anaerobic Trition
(Table
and subsequent
by agitation, II).Background
conditions
(Table
but left
II).The
that of
degree of actiand under
(data not shown).
assay totally the activation
activity
in acetate-
II).Agitation
in the incubation
X-100
of the
in the same activation
of histone
effect
is independent
at 4'C and at room temperature
0.1%
II):
the catalytic
of the kinase
resulted
in the presence
was similar
0.21 0.22
by agitation
of histone,since
was obtained
0.3-l
by the agitation.
of the kinase
and Mg-acetate
histone
prior
had been released
No.
1s 20 21 22 24 24 25
of the kinase activity
Activation presence
kinase
ratio of agitation-induced activity to CAMP-induced activity 0.69
of the kinase
mixture
prevented
during
the activa-
by CAMPunaffected in the presence
cf
0.1% Trition
X-100 and in the absence of added CAMPwas reduced,
thus leading
to a higher
relative
stimulation
1121
of the kinase
ac-
BIOCHEMICAL
Vol. 76, No. 4, 1977
tivity gure
bjj CAMP (Table
II).When
of the eluate
1)
checked for
by agitation
is was found that
the relative
tion
than that
No.19,Figure tion
l).This
dependent species
that
is preferentially
behaviour
of the two kinases
rabbit
skeletal
their
activation
of the test as a surface pletely
that
by casein
solution tension suggested
for
agent,such
to include activation
reproducible
background
for
in
two CAMP-
only one kinase
assay for
from
example,
High surface the agitation as Triton activation.
C.l% Triton
activities
pro-
combined with
have been isolated
the agitation-induced
kinase
was obtained
that
or histone(l1).
reducing
never
by agitation.Dissimilar
may be required
dard protein
in the earlier
at least
muscle has been described,
suppresses
therefore,
that
might be
agitation
indicate
dissociated
of agita-
one,but
that
muscle contains
kinases(ll),may
by agita-
the effect
CAMP concentrations,
rabbit
protein
that
of the kinase
III),
the peak fraction
is not a direct
of saturating
the knowledge
achieved
with
hand, the fact
activation
the presence
column were
which is more concentrated
the other
duced the full
(No.20-25,Fi-
as bjr CAMP (Table
activations achieved
activity
by a factor,
fractions.On
fractions
a s well
might indicate
on the kinase
mediated
the other
RESEARCH COMMUNICATIONS
from the DEAE-cellulose
activation
were smaller
AND BIOPHYSICAL
X-100
for tension effect,
X-lOO,comIt
is,
in the stan-
CAMP to ensure low,
in the absence of added CAMP.
References 1. Krebs,E.G. (1972) Curr.Topics Cell Regul.2,99-133. 2. Langan,T.A. (1973) Advan.Cyclic Nucleotide Res.&99-153. 3. Kuo,J.F.,and Greengard,P. (1972) Advan.Cyclic Nucleotide Res. &41-50. 4. Mayer,S.E.,Stull,J.T.,Wastila,W.B.,and Thompson,B. (1974) Methods Enzymol.38,66-73.
1122
Vol. 76, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Miyamoto,E.,Petzold,G.L.,Kuo,J.F.,and Greengard,P. (1973) J.Biol.Chem.248,179-189. 6. Murray,A.W.,Froscio,M.,aKemp,B.E. (19'72) Biochem.J.129, 995-I 002. 8. Wastila,W.B.,Stull,J.T.,Mayer,S.E.,and Walsh,D.A. (1979) J.Biol.Chem.246,1996-2003. 9. Penefsky,H.S. (1967) Metms En2ym01.10,703-704. IO. Ashby,C.D.,and Walsh,D.A.(1973),J.BiorChem.248,1255-1261. 11. Reimann,E.M.,Walsh,D.A.,and Krebs,E.G.(1971)miol.Chem. 246,1986-1995.
12.
Kalckar,H.M.(1947)J.Biol.Chem.~,461-475.
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