cAMP-dependent protein kinase from Mucor rouxii : Physical evidence of a ternary complex holoenzyme-cAMP

BIOCHEMICAL

Vol. 101, No. 2,198l

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BIOPHYSICAL

RESEARCH

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July 30, 1981

663-671

CAMP-DEPENDENT PROTEIN KINASE FROM MUCOR ROUXII: PHYSICAL EVIDENCE OF A TERNARY COMPLEX HOLOENZYME-CAMP R. L. Pastori,

N. Kerner,

S. Moreno

and S. Passeron

Programa de Regulaci6n Hormonal y Metabclica, Departamento de Quimica BiolBgica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina Received

June

22,

1981

SUMMARY: Gel electrophoresis and sucrose density gradient centrifugation techniques permitted the visualization for the first time of the ternary complex fomied by the binding of CAMP to Mucor rouxii CAMP-dependent protein kinase holoenzyme. The addition of 0.5 M NaCl or histone plus ATP-Mg++, together with CAMP, dissociates the holoenzyme into free regulatory (R) and catalytic (C) subunits. At 4"C, CAMP bound to the holoenryme is readily exchangeable with unlabeled CAMP (half life 2.5 min), while the nucleotide bound to the R subunit has a very slow exchange rate (half life 210 min). The amount of CAMP bound to R subunit is approximately twice the amount bound to holoenzyme at saturation. CAMP activated many eucaryotic subunit aid

Although

These

enzymes

are

initial

established.

4

C) that

CAMP,-

R2cAMP

interact

Several

in the dissociation

with

of two

each other

+

4

2c active

and final

the detailed

the formation

of CAMP on

(2,3):

reactants

have been demonstrated,

the effect

known to be formed

holoenzyme

the

supporting

seem to mediate

R and catalytic

CAMP as follows

inactive

step

(1).

(regulatory

R2C2 +

firmly

kinases

processes

types

with

protein

mechanism

authors

for

have presented

of a ternary process

products

complex

(4-ll),

but

of the overall the

process

kinetic

has not

evidence

holoenzyme-CAMP such a complex

reaction been

strongly

as an obligatory has never

been

isolated. In the

present

study

of CAMP to CAMP-dependent reported

the

isolated

from

partial extracts

we isolate protein

purification of the

a ternary kinase

from

complex, Mucor

and characterization fungus

(12,

663

formed rouxii.

by the

binding

We have already

of this

enzyme

13) 0006-291X/81/140663-09$01.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

8lOCHEMlCAL

Vol. 101, No. 2,198l

MATERIALS

AND

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AND METHODS

Unless otherwise indicated, experimental details were as described in preceding papers (12, 14) or as provided in the text and figure legends. Yeast cells of Mucor rouxii (NRRL 1894) were used throughout. 13H IcAMP (specific activity 38 Ci/mmol) and [32P lortophosphate for the preparation of [y -32~ ] ATP (15) were obtained from the Radiochemical Centre, Amersham. Protein kinase assay. The enzymatic activity was assayed as previously described (13) using 1.5 mg/ml histone (Type II S, Sigma) or 0.25 mg/ml kemptide (Sigma) as substrates. When kemptide was used as substrate,the [32P lphosphopeptide was measured as described by Glass et al (16).,At these saturating concentrations, kemptide was approximately 30tinles better substrate than histone. One unit of protein kinase activity was defined as the amount catalyzing the transfer of one pmol of [32P I into histone per minute under the conditions of the standard assay. CAMP-binding assay. [ 3H IcAMP-binding was assayed as previously described (13) by the membrane filtration technique(l7) using Schleicher & Schull BA-85 membrane filters. Polyacrylamide gel electrophoresis. Electrophoresis in 5% polyacrylamide gels was performed at 4°C according to the procedure of Davis (18). Following electrophoresis, some gels were stained with Coomassie Brilliant Blue. Other gels were cut into 2 mm slices and processed as described in the legend of Fig. 3. Protein assay. Protein was measured by the method of Bradford (19) using bovine serum albumin as standard. CAMP-dependent protein kinase preparation. The enzyme was prepared as previously described (13) up to the DEAE-cellulose step, except that the buffer used throughout the whole preparation was 20 mM Tris-HCl pH 7.4, 4 mM EGTA, 3 mM benzamidine (Buffer I). Aliquots of 4 mM EDTA, 2 mM mercaptoethanol, 300~1 of the preparation (5 mg protein/ml) were loaded onto six 4.5 ml linear sucrose gradients (5-20%) in buffer I and centrifuged for 16 hours at 40,000 rpm in a SW 55 Ti rotor. 25 fractions of each gradient were collected. The peak of protein kinase activity sedimenting at 8.8 S was concentrated through DEAE-cellulose, and stored in aliquots at -20°C. This preparation, analyzed by sucrose gradient centrifugation, did not contain detectable amounts of free catalytic or regulatory subunits, nor CAMP phosphodiesterase activity. This final preparation had a specific protein kinase activity of approximately 1,700 units/mg and the stimulation by 1~rM CAMP ranged from 7-11 fold. Preparation of the regulatory subunit - [ 3H IcAMP complex. The R- i3H IcAMP complex was prepared according to the following procedure. Fractions containing [ 3H I cAMP-binding activity from six gradients similar to that of Fig. 1C were pooled, dialyzed and concentrated through DEAE-cellulose. Two aliquots of the isolated complex were incubated at 4°C with or without 300 nM 1 3H IcAMP. The [3H IcAMP bound in both cases was similar, indicating that the complex remained saturated.

RESULTS

AND DISCUSSION

Visualization

of the

As previously wide

range

dissociated

ternarj

reported

complex (12,

of enzyme concentration by preincubation

0.5E? NaCl was included

with

on sucrose

13)

the protein (50-2000

micromolar

in the mixture.

664

gradient

centrifugation

kinase

units/ml)

of Mucor was not

concentrations

rouxii,

in a

completely

of CAMP, unless

BIOCHEMICAL

Vol. 101, No. 2,19Bl

TABLE

I:

Binding

of

AND

BIOPHYSICAL

CAMP to protein nondissociating

[ 3H ] CAMP AM

kinase conditions

NaCT 0.5 M

RESEARCH

under

[3H

dissociating

and

I CAMP bound (cpm)

0.025

3000

0.050

4800

0.140

6500

0.280

6900 +

2.8

COMMUNICATIONS

14000

10 units of the holoenzyme preparation were incubated 60 min at 4°C with the additions described in the Table. [3H ]cAMP bound was measured as described in Materials and Methods. (The values shown are average of triplicates in single experiments).

Sucrose

gradient

interaction tion

centrifugation

of holoenzyme

of catalytic

with

CAMP. Fig.

and binding

activities

enzyme was preincubated

with

Table

(0.13j~M)

I)

and

depicted

[ 3H IcAMP

in Fig.

same position indicating omitted the

that

could

(data

not

bound

subunit

These to the

when the

and 0.5

CAMP

bound

column.

activity

activity

1C).

after These

showed

results

dialysis indicate

that

protein.

665

the the

in the

CAMP-binding activity

nucleotide

sedimentation

behaviour

with

2 PM [3H Iregulatory

and chromatography CAMP is

but

catalytic

to the

CAMP,

CAMP was

cyclic

dissociated

that

profile

bound

If

however,

with

The 1 3H IcAMP associated

exhaustive

the

the same profile,

IC shows the

was completely

(see

sedimented

observed;

seem to indicate

kinase

When the

gradient,

was formed.

cosedimenting

Fig.

of sedimenta-

by a peak of protein

haloenzyme-CAMP

holoenzyme.

(Fig.

was not lost

DEAE-cellulose

results

protein

M NaCl

The catalytic

by assay,

profile

the

of [3H ]cAMP

in the

CAMP was no longer

investigate

holoenzyme.

was mantained

catalytic

be detected

shown).

weakly

obtained

the

gradient,

peak of protein

activity

is

the

complex

the

concentrations

accompanied

control,

a ternary

1A shows of a control

saturating

1B was obtained.

as in the

from

was used to further

strongly

through bound

to

BIOCHEMICAL

Vol. lOl,No.2,1981

AND

alk oh wr

BIOPHYSICAL

cvt C

RESEARCH

cer

COMMUNICATIONS

cvtC

OLO-

Fig. 1. Sucrose density yrduienc cencrifugation of protein kinase. Preincubations and sucrose gradients were performed in buffer I, with the additions indicated in each case. In cases A, B and C, 250 units of protein kinase activity were preincubated at 30°C for 1 min in a final volume of 0.2 ml. Additions: (A) none; (B) 2 )JM [3H IcAMP in the preincubation mixture and 0.13,uM r3H IcAMP in the gradient; (C) 5 AM [3H IcAMP and 0.5 M NaCl in the preincubation mixture, 0.13,uM 13H IcAMP and 0.5 M NaCl in the gradient. In case (D) 8 units of protein kinase activity were preincubated 1 min at 30°C in the complete incubation mixture of protein kinase. The gradient was prepared with the foIlowing additions: 0.75 mg/ml histone, 0.05 mM ATP, 10 mM MgC12 and 0.15~~M CAMP. This small amount of enzyme had to be used in order to prevent precipitation with histone. Aliquots were assayed for protein kinase activity using histone as substrate for gradients A-C and kemptide for gradient D ( l ). In gradiegts B and C aliquots from each fraction were filtered directly to measure the [ H ICAMP bound ( -o). In gradient A activity after 60 min incubation at aliquots were ass yed for CAMP binding 4°C with 140 nM I 9 H ICAMP ( -o).

Under

the

was completely

conditions

in the dissociated

enzyme concentration less

than

indicate the

of the

used

the

state

protein

of the

ternary

complex

of the

enzyme (data is

kinase

assay,

the

as can be seen in Fig.

in the experiment

20% dissociation that

standard

not

the

not active

holoenzyme

1D. At the

figure,

CAMP alone

shown).

These

produced

results

phosphorylating

form

of

enzyme.

Dissociation

rate

experiments

As can be seen enzyme

decreased

in Fig.

rapidly,

2A, the amount with

a half

life

666

of I 3H IcAMP bound of 2.5 min.

This

to the holndecrease

is the

Vol. 101, No. 2,198l

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AND

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RESEARCH

trne (mm)

COMMUNICATIONS

time (hours)

Fig. 2. Dissociation rate at 4°C of the holoenzyme and R subunit. (A) 500 units of protein kinase activity preincubated 60 min at 4°C with 60 nM [3H ]CAMP were incubated in buffer I with 540 nM unlabeled CAMP in a final volume of 1.3 ml. (B) An aliquot of R subunit preparation (250,000 cpm of [3H ]cAMP bound), in a final volume of 1.3 ml was incubated in buffer I with 0.1 mM unlabeled CAMP. Aliquots of 75~1 we e removed from each sample at the time by the membrane points indicated and the amount of [ 5 H ]cAMP bound determined filtration method. 100% represents 14,000 cpm in both cases.

result

of the exchange

unlabeled did

CAMP in the

not

produce

show a slow (half for

the

of the

of Table

obtained binding

are not

given

regulatory

from

28 clearly subunit

the one depicted exchange

since

the experiments

the

CAMP used

in Fig.

of isotopic

unexpected,

from

conditions 5).

binding from

to holoenzyme

I showthatmaximum

state

(line

of CAMP bound

binding

at 140 nM CAMP. Under

undissociated

dissociating

the

amount

was attained

a greater

subunit

rates

of total

to the

different

with

the

of

same

commented

in the

section.

Results

ly doubled

bound

in the

complex

the amount The data

quite

had been obtained

ultracentrifugation Comparison

enzyme.

of [ 3H IcAMP

The differences

results

in the ternary

since

210 min),

and regulatory

qualitative

the

of the

of approximately

holoenzyme

mixture,

exchange

holoenzyme.

[ 3H IcAMP

reaction

dissociation

isotopic

life

kinase

of bound

the

capacity

(Fig. (see

These

capacity

1B).

these

When the 1C) the

amount

results

indicate

that

than

comparison measured

the

holoenzyme.

of gradients in gradient

667

of [3H ] CAMP to protein conditions,

incubation

Fig.

and to R

the

was performed

of CAMP bound the Similar

1A and lC, 1C was again

enzyme was in

was approximate-

regulatory

subunit

conclusions since

under

the total

approximately

has

were area twice

of

BIOCHEMICAL

Vol. 101, No. 2,19Bl

the

one of gradient

dissociating tration the

influence were

CAMP-binding gel

5) cannot

described always

in the

CAMP bound

be ascribed

by several

diluted

RESEARCH COMMUNICATIONS

authors

20 times

to the

(20-22),

immediately

and regulatory

to protein high

salt

concen-

due to the before

subunit

under

fact

that

filtration.

analyzed

by polyacrylamide

electrophoresis

showed

kinase

a single

showed

submitted

(Fig.

3A).

a band migrating

A gel

with

enzyme was dissociated

with

a saturating

showed

same position

the catalytic

has been

formed

in the

phoretic

process

must be born different

the

both

run

stained

in parallel,

of Fig.

These

question

results

that

abscence

and dilution set-ups.

disappeared

if

preincubated

1B) and submitted

to

activity

in

migrated amount

a ternary

dye.

comigracomplex

The presence in the

CAMP resist but

conditions

The peak of ['H

of

Free [ 3H l-

holoenzyme.

of [ 3H IcAMP bound

Blue

that

ultracentrifugation,

diffusion

experimental

this

a

Coomassie

slices,

indicate and the

with

of half

the tracking

of why did after

in Fig.

counting

nucleotide

in the

kinase

and a significative

peak accompanying even

with

3B. Catalytic

holoenzyme

gel

migrating

band completely

(as

scintillation

and was lost

in mind

under

control

complex,

raises

profile

the cyclic

as a sharp

activity

NaCl and CAMP. Protein

activity.

between

and CAMP-binding

of i3H IcAMP

by direct

ted with

reservoir,

the

as the

r3H ] CAMP, estimated

CAMP migrated

with

in a 5% polyacrylamide

same Rf. This

the

concentration

electrophoresis

[ 3H IcAMP

to electrophoresis

peak of catalytic

same Rf of 0.17

the

(line

to holoenzyme

Protein

the

BIOPHYSICAL

1A. The increase

conditions

samples

AND

of

upper

the

electro-

of course

it

are

dramatically

l-label

did

this

complex

not

. represent mitted

['H

IcAMP

bound

to electrophoresis

of Rf 0.46

and 0.60

migrated (Fig.

probably

due to proteolytic

to occur

in different

value activity

to free

for

the

catalytic

in the gel

3C).

subunit,

as a broad

The broadness

attack

tissues

(20,

subunit, eluates,

regulatory

of the 23-25). since

area

moiety

Unfortunately

assay

two principal

radioactive

regulatory

the

668

with

of the

the efforts

sensitizing

since

done

peaks

area already

by using

was reported

we cannot to detect

sub-

give catalytic

kemptide

as

an Rf

Vol. 101, No. 2,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

02

g

0

i

COMMUNICATIONS

60

5

I

0

20

ul slue

60

mmber

Fig. 3. Polyacrylamide gel electrophoresis of holoenzyme and R subunit. Gels of protein kinase preincubated 1 min at A and B were loaded with 500 unit 3 30°C without (A) or with 150 nM [ H ] CAMP (B). Gel C was loaded with 200,ul of R - [3H ] CAMP subunit preparation (200,000 cpm). After completion of electrophoresis (approximately 4 h), gels were cut into 2 mn slices and the complete slice (A) or half ot it (B) were incubated with continuous shaking for 14 h at 4°C in 200.1~1 of 10 mM Tris-HCl buffer pH 7.4, containing 4 mM mercaptoethanol, 50% glycerol and 1 mg/ml bovine serum albumin. Protein kinase activity was assayed in an aliquot of the eluates, using histone as substrate l ). CAMP-binding activity of gel A was mgasured by incubating an (aliquot of the eluates, 60 min at 4°C with 150 nM [ H ]cAMP ( o ). [3H ]cAMP bound to protein in gels B and C was estimated by dissolving the half slice (B) or the whole slice (C) in 0.3 ml of H202 30%, 30 min at 100°C and counting the radioactivity in a scintillation mixture containing Triton X-100: toluene ( 30:70 ).

substrate

and

negative

result

already

reported

the

catalytic

for

the

from

very

specific

be

explained

by

Another

possibility

can (13). subunit

catalytic

entering

high

of subunit

the

gel

the of

at

the

activity the

for lability is

Mucor

enzyme

other

sources

pH used.

669

ATP,

was

were of

that

unsuccesful.

the the

catalytic

activity

isoelectric

point

sufficiently

(26)),so

high as

This

to

prevent

(as

of

reported the

protein

BIOCHEMICAL

Vol. 101, No. 2,1981

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Conclusions The results gation

obtained

in the

ternary

present

complex CAMP is

that

one of these

the

capable

system,

study

provide

entities

of histone

lyticsubunitfrom

the

is other

and sucrose

physical

evidence

In addition,

of binding

at least,

presence

electrophoresis

holoenzyme-CAMP.

that

In our

by gel

our

to two different the

factors

results

entities,

necessary

the ionic

and/or

ATP-Mgtt

are

to release

complex.

The role

of each of these

factors

of a

indicating

reinforce

forming

such as increased

centrifu-

the existence

kinetic

thus

holoenzyme,

for

density

the

idea

ternary

complex,

strength,

or

active is

cata-

under

study. Acknowledgements: This work has been supported by grants from the Consejo National de Investigaciones Cientificas y Tecnicas, (CONICET), Secretaria de Estado de Ciencia y Tecnologia and ComisiBn National de Energia AtBmica. N.K. is a post-graduate Fellow and S.P. and S.M. are Career Investigators of CONICET. The authors wish to express their gratitude to Dr. C.E. Cardini for his constant and enthusiastic support.

REFERENCES

::

Krebs, Builder,

E.G. and Beavo, J.A. S.E., Beavo, J.A.,

(1979) Annu. Rev. Biochem. 48, and Krebs, E.G. (1980) J. B3.

923-959. Chem. 255,

2350-2354. 3. 4.

Corbin, J.D., Sugden, P.H., West, L., Flockhart, D.A., Lincoln, T.M. and MC. Carthy, 0. (1978) J. Biol. Chem. 253, 3997-4003. Builder, S.E., Beavo, J.A. and Krebs, E.GTl980) J. Biol. Chem. 3,

3514-3519. 5. 6. 7. 8. 9.

Ogez, J.R. and Segel, I.H. (1976) J. Biol. Chem. 251, 4551-4556. Boeynaems, J.M. and Dumont, J.E. (1977) Mol. CellXndocrinol. 7, 275-295. Tsuzuki, J. and Kiger, J.A., Jr. (1978) Biochemistry II, 2961-2970. R.L. and Huang, Ch. (1980) Chau, V., Huang, L.C., Romero, G., Biltonen, Biochemistry 19, 924-928. Granot, J., MTdvan, A.S. and Kaiser, E.T. (1980) Arch. Biochem. Biophys.

205; 1-17.

12.

%i'istrong, R.N. and Kaiser, E.T. (1978) Biochemistry 17, 2840-2845. Huang, L.C., Froehlich, H.C., Charlton, J.P. and Huang, Ch. (1977) Fed. Proc. 3&, 690. Moreno, S., Paveto, C. and Passeron, S. (1976) Acta Physiol. Latin. 6,

13.

Moreno,

10. 11.

343-348. S. and Passeron,

S. (1980)

Arch.

Biochem.

Biophys.

199,

321-

330. 14. 15.

Galvagno, M.A., Moreno, S., Cantore, Biophys. Res. Cotmnun. @, 779-785. Chang, K., Marcus, N. and Cuatrecasas,

M.L.

and Passeron,

P. (1974)

J. Biol.

S. (1979)

Biochem.

Chem. 249,

6854-6865. 16.

Glass, D.B., Masarachia, R.A., Anal. Biochem. 87, 566-575.

Ferramisco,

670

J.R.

and Kemp, B.E.

(1978)

Vol. 101, No. 2,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

17. 18. 19. 20. 21.

Gilman, A.G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404~v7. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Sudgen, P.H. and Corbin, J.D. (1976) mochem. J. 159, 423-437. D$skeland, S.O., Ueland, P.M. and Haga, H.S. (197TBiochem. J. l&,

22. 23.

0greid;D. and Ddskeland, Potter, R.L., Stafford, l9J1, 174-180. Rannels, S. and Corbin, Potter, R.L. and Taylor, Gagelmar-q,M., Reed, J. Proc. Natl. Acad. Sci.

2 26.

653-665.

S.O. (1980) FEBS Lett. 121, 340-344. P. and Taylor, S. (1978) Arch. Biochem.

J.D. (1979) J. Biol. Chem. 254, S. (1980) 255, 9706-9712. Kubler, U.S.A.,

D., 17,

671

Pyerin, W. and Kinzel, 2492-2496.

Biophys.

8605-8610. V. (1980)