Evidence for phosphorylation of rat brain guanylate cyclase by cyclic AMP-dependent protein kinase

Vol. 101, No. 4,198l August

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31, 1981

Pages

1381-1387

EVIDENCE FOR PHOSPHORYLATION OF RAT BRAIN GUANYLATE CYCLASE BY CYCLIC AMP-DEPENDENT PROTEIN KINASE Jean

ZWILLER,

July

REVEL and

Paul

BASSET

Centre de Neurochimie du CNRS Pascal, 67084 Strasbourg Cedex - France

5, rue Blaise Received

Marie-Odile

1, 1981 SUMMARY

Direct phosphorylation of purified rat brain guanylate cyclase by cyclic AMP-dependent protein kinase is demonstrated. In the presence of [Y-~*P]ATP, 3*P was incorporated into the protein to the extent of 0.8 to 0.9 mol/mol of guanylate cyclase. The presence of 3*P in the guanylate cyclase molecule was demonstrated by gel-filtration and by autoradiography after gel electrophoresis. The phosphorylation was accompanied by an increase in enzyme activity, characterized by an increase of VM. These results suggest that the activity of guanylate cyclase may be regulated in viva by phosphorylation. INTRODUCTION A great known

number

to promote

therefore

mechanism candidates tion cyclase of its

cyclic

presumed

(cyclizing),

of agents

to activate

Enzymes

in serving

(2).

Therefore could

enzymatic

neurotransmitters

GMP accumulation

EC. 4.6.1.2.) (1).

including

a regulatory we examined

be directly

guanylate

in viva subject

in various

but

cyclase probably

to hormonal

control

function

after

the possibility

phosphorylated

tissues.

via

an indirect are known undergoing purified

are

Hormones

are

(GTP pyrophosphate,

that

resulting

and hormones

lyase

regulation to be better phosphorylaguanylate

in a significant

change

activity. EXPERIMENTAL

PROCEDURES

Rat brain soluble guanylate cyclase was purified to apparent homogeneity using chromatography on Blue Sepharose, precipitation by ammonium sulphate, preparative isoelectric focusing and gel-filtration (3). Guanylate cyclase activity was assayed as described previously (4), based on the formation of 132~1 cyclic GMP from [a- 3*Pl GTP. The reaction was carried out at 37'C for 10 min. The results were corrected for recovery of [%I cyclic GMP added to the incubation mixture. Protein was determined by the method of Lowry et al. (5).

0006-291X/81/161381-07$01.00/0 1381

Copyright 0 1981 by Academic Press, Inc. All rights of reproducrron in anv form resewed.

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Phosphorylation of purified guanylate cyclase was carried out, unless otherwise specified, in a final volume of 0.1 ml at 30°C for 60 min. Reaction mixtures contained 40 mMphosphate buffer, pH 6.8, 10 mMMgC12, lo UMcyclic AMP, 0.4 mM ~~-32~1 ATP (1 UCi), 10 - 30 ug purified guanylate cyclase and 10 ug of CAMP-dependentprotein kinase from bovine heart (Sigma Chemical Co). After the incubation, 50 nmol GTPwere added and incubation was continued for 10 min in order to avoid ATP binding at the substrate site of guanylate cyclase. Measurements of protein-bound phosphate were then processed according to Reimann et al. (6). For kinetic studies of phosphorylated guanylate cyclase, incubation was performed without radioactive ATP and without further incubation with GTP ; phosphate ions, which inhibit guanylate cyclase activity, were replaced by 40 mMTris-HCl buffer, pH 7.5. The mixtures were then dialysed overnight against 10 mMTris-HCl buffer, pH 7.5 containing 10 % glycerol, 10 mMP-mercaptoethanol and 1 mMMnC12before determining guanylate cyclase activity, in order to remove ATP which is known to be a strong inhibitor of the enzyme. For the samereason, the control experiment was performed in the presence of ATP, without protein kinase in the phosphorylation incubation step. Electrophoresis was performed as described (3) and the autoradiographic study was done using Fuji X-Ray film. Analytical isoelectric focusing was performed in slab gels (115 x 115 x 1.5 mm)of 1 % agarose containing 4 % of carrier ampholytes pH 4-9 and 10 % of glycerol. Sampleswere applied on the gel after a prerun of 30 min and the migration was carried on for 1.5 h at 7'C with a constant power of 8 W.

RESULTS Incorporation

of 32P into guanylate cyclase.

clase was subjected to phosphorylation AMP-dependent protein kinase, guanylate cyclase.

Whenpurified

under conditions

guanylate cy-

involving

cyclic

32 P from [y-32 P] ATP was incorporated

The time course for the phosphorylation

into

of purified

en-

zyme is shown in Figure 1. As can be seen, a plateau was reached after 60 min. incubation. protein 32

This maximumphosphorylation

kinase concentrations

P was significantly

were used (not shown). The protein bound

greater in the complete system, when compared with

those observed in the control cyclase (Figure 1). Subtracting control

was unchanged when higher

systems lacking only the kinase or only the the 32P radioactivity

incorporated

systems from that observed in the complete system, we found a

maximumincorporation

of 32 P into guanylate cyclase of between 0.8 and

0.9 mol/mol, assuming a molecular weight of 150 000 for (7,8).

in the

This result

suggests that only one phosphorylation

in the guanylate cyclase molecule.

1382

guanylate

cyclase

site is present

Vol. 101, No. 4,198l

Figure

BIOCHEMICAL

Samples [y-

dures"

32

of purified

guanylate

with

was released

under

soluble 0.1

in

N NaOH at O'C. for

relative

however,

has not

yet

Independent under

our

on a serine

to acid

or a histidine

conditions

been

guanylate

co-eluted

the peak

when all

are

results

No radio-

the protein

was pre-

of the radioactivity

phosphate

inconsistent (9).

are

or threonine

N NaOH. However,

The precise

for

consistent residue.

: lability with

Proce-

conditions.

by incubation

10 min at with Thus,

to alkali

the involvement site

the 0.1 and

of a

of phosphorylation,

determined. that

guanylate

cyclase

can be seen in Figure

of 32 P-labeled with

acyl

residue

evidence

These

protein

"Experimental

10 min at lOO*C in 1.4

remove protein-bound stability

But virtually

acid.

phosphorylated

in

acid

without

by phosphorylation

and acidic

was released

1OO'C in 5 % trichloroacetic

NaOH would

basic

trichloroacetic

by incubation

enzyme being

labeled

described

various

26 % of the radioactivity

lysine

RESEARCH COMMUNICATIONS

performed

cyclase

the conditions

incubated

became

incubated

compared to the incubation

P] ATP under

were

activity

only

BIOPHYSICAL

1 : Time course of phosphorylation and activation of purified guanylate cyclase. Purified enzyme was submitted to phosphorylation as described under "Experimental Procedures". At the time indicated, trichloroacetic acid-precipitable 3*P radioactivity was determined in the complete system ( l ), system minus guanylate cyclase (r) and minus protein kinase (m). Guanylate cyclase activity (0 - - - 0) is expressed as a percentage of increased

activity kinase.

with

AND

cyclase.

The single

2, which

1383

phosphorylated

shows a gel-filtration

peak of enzyme activity Further

of radioactivity.

is being

evidence

is

supported

was

N

Vol. 101, No. 4,198l

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t

+ fraotton

4

1

Number

03 Figure

2 : Gel filtration of 32 P-labeled guanylate cyclase. Purified guanylate cyclase was phosphorylated in the complete system including 0.1 mM [v-~~P.] ATP. After incubation, the mixture was subjected to gel-filtration on Ultrogel AcA 34 (2.9 x 60 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 10 % glycerol, 10 mM p-mercaptoethanol, 1 mM MnC12, 5 mM MgC12 and 0.1 mM EDTA. The column was eluted with the same buffer at a flow rate of 18 ml/h in fractions of 4 ml. Aliquots of 60 ul were assayed for guanylate cyclase activity and aliquots of 2 ml were withdrawn for 32P radioactivity measurement. o - o, guanylate cyclase activity ; o-e, J2P radioactivity. 32 P-labeled 3 : Autoradiographic profile of purified guanylate cy-

Figure

clase on gel electrophoresis. Before electrophoresis, samples of uanylate cyclase labeled by phosphorylation with 0.1 mM [y- 9 2P1 ATP were submitted to isoelectric focusing on agarose gel to separate guanylate cyclase from kinase. The fractions corresponding to guanylate cyclase were then submitted to analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis. 1) Coomassie brilliant blue staining ; 2) autoradiographic de-

tection.

by gel

electrophoresis

The mixtures isoelectric kinase.

which

the phosphorylation

incubation

focusing

on agarose

in order

to sodium

showed

purified Enzymatic

incubated

fractions

slabs

corresponding

dodecyl

sulphate

activity

by Coomassie

in absence

cyclase

of protein

blue

(Figure

submitted

3).

to

the protein cyclase

were

then

electrophoresis

correlated

well

with

the

staining.

was increased

1384

gel

which

guanylate

kinase.

were

to eliminate

polyacrylamide

of phosphorylated guanylate

step

cyclase

to guanylate

a 32 P-band

by autoradiography

enzyme detected

phosphorylated

guanylate

from

The agarose

submitted

32 P-labeled

of purified

The time

cyclase.

The activity

when compared course

for

of

to enzyme the activation

Vol. 101, No. 4,198l

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ANP

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Figure 5: Gel filtration of reticulocyte protein synthesis mixtures after Lysate mixtures (105 ~1) were incuincubation wfth (0) and without 10) EF-Tu. bated with [ Hlleucine for 10 minutes at 30' and applied to a column of Sephacryl 5200. In the experiment with EF-Tu, 329 ng of the factor were added. The arrows indicate the elution fraction number for the following molecular weight markers: 1, reticulocyte polysomes; 2, dimer of bovine serum albumin;

3, bovine serum albumin; 4, EF-Tu; 5, tRNA. Fraction number 29, the valley in the elution profile of the lysate mixture containing EF-Tu corresponds to a molecular weight of 8.9 x lo4 daltons (based on molecular weight standards) , a value slightly

in

excess

of

ternary complexes Methods.

that

the

cglculated

(7.3 x 10

of EF-Tu*GTP.aminoacyl-tRNA

The simplest

mechanism

the

factor

forms

complex

cannot

of protein acyl-tRNA were

synthesis for

incubated

Sephacryl Control

lysates

throughout

valley

formation

The results exhibited filtration The EF-Tu

this

ternary

to the conclusion

that

view

of tRNA available was obtained

and GTP, but

uniform

profile

ratio

indicating lysate,

A site.

this

subjected

of such an experiment

of EF-Tu

to gel

if

on the other

at approximately

lysates

filtration

on

in Figure

of alkali-labile little,

the

of amino-

hypothesis

are shown

5.

to total any,

hand,

is

The inhibition

the availability

To test

and then

lysates.

activity

in

and

CPM

accumulation

exhibited

the position

of

a striking of elution

complexes.

that

deficiency

eEF-Tu.

radioactivity

of EF-Tu*aminoacyl-tRNA*GTP

in inhibited

aminoacyl-tRNA

a reduction

EF-Tu

treated

see Materials

the inhibitory

with

from

a nearly

aminoacyl-tRNA*EF-TusGTP

at the ribosomal

with

and without

of alkali-labile

The finding

complex

result

for

details

complexes

for

effectively

would

with

the gel

aminoacyl-tRNA.

bind

complex

S-ZOO.

to account

a ternary

weight

For further

Accumulation

ternary

leads

molecular

daltons).

complexes

accumulate

the bacterial for

protein

by supplementing

factor

in lysates sequesters

synthesis. lysates

1392

calf

with

tRNA resulting

Support with

treated

for liver

EF-Tu in a

the validity tRNA prior

of to

BIOCHEMICAL

Vol. 101, No. 4,198l

vity

of guanylate

phorylation the

system,

guanylate

of guanylate

characterized

examples

as tyrosine

hydroxylase

Our results represent

that

phosphorylation cyclic

that

(12)

activation might

; (b)

cyclic

cyclic azide

guanylate

cyclase

activity

of guanylate

a mechanism

activation cesses,

for

could

phosphatase

in

involved

(13)

and in Effective

by phosphorylation

would

cyclase

terms

of protein

vivo.

The

dibutyryl

neuroblastoma the cyclic

GMP

increasing

the

physiological presumably

by dephosphorylation.

activity kinase

such

might

by : (a)

(14).

the

well

AMP dependent

in cultured

of the enzymatic in

in

by cyclic

supported

cells

to other

(11).

in potentiating

in hepatocytes

inactivating

be explained

is

conver-

enzyme activation

activity

cyclase

V~VO

in C-6 glial

cyclase

or inactivation

is analogous

phosphorylation

of the cyclase

GMP concentration

AMP is

to sodium

quire

occur

PI ATP into

by the

lipase

cyclase

of guanylate

also

response

regulation

form,

- mediated

guanylate

32

[v-

phos-

- 0.9 mol of phosphate/

or hormone-sensitive

mechanism

AMP increases

cells

(10)

a complete

was accompanied

to a more active

suggest

with from

0.8

which

of phosphorylation

a regulatory

possibility

of cyclase

of about

phosphorylation,

cyclase

RESEARCH COMMUNICATIONS

was transferred

to the extent

This

BIOPHYSICAL

By incubation

radioactivity

cyclase

mol of cyclase. sion

cyclase.

AND

during

various

reThus, pro-

or phosphoprotein

activities.

REFERENCES 1. Goldberg, N.D. and Haddox, M.K. (1977) Ann. Rev. Biochem. 46, 823-896. 2. Krebs, E.G. and Beavo, J.A. (1979) Ann. Rev. Biochem. 48, 923-959. 3. Zwiller, J., Basset, P. and Mandel, P. (1981) Biochim. Biophys. Acta 658, 64-75. 4. Goridis, C., Zwiller, J. and Reutter, W. (1977) Biochem. J. 164, 33-39. 5. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 6. Reimann, E.M., Walsh, D.A. and Krebs, E.G. (1971) J. Biol. Chem. 246, 1986-1995. 7. Garbers, D.L. (1979) J. Biol. Chem. 254, 240-243. 8. Braughler, J.M., Mittal, C.K. and Murad, F. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 219-222. 9. Weller, M. (1978) Biochim. Biophys. Acta, 509, 491-498. 10. Yamauchi, T. and Fujisawa, H. (1978) Biochem. Biophys. Res. Commun. 82, 514-517.

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11. Huttunen, J.K. and Steinberg, D. (1971) Biochim. Biophys. Acta 293, 411-427. 12. Zwiller, J., Goridis, C., Ciesielski-Treska, J. and Mandel, P. (1977) J. Neurochem. 29, 273-278. 13. Kon, C. and Breckenridge, B.M. (1979) J. Cyclic Nucleot. Res. 5, 31-41. 14. Earp, H.S. (1980) J. Biol. Chem. 255, 8979-8982.

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