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.

1123

Activation of rabbit skeletal muscle adenosine-3':5'-monophosphate-dependent protein kinase by agitation.

BIOCHEMICAL Vol. 76, No. 4, 1977 AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACTIVATION OF RABBIT SKELETAL MUSCLEADENOSINE-3':5'-MONOPHOSPHATE-DEPENDEN...
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