Alteration of adenylate cyclase activity by phosphorylation and dephosphorylation

Vol. 112, No. 1, 1983 April

BIOCHEMICAL

RESEARCH COMMUNICATIONS

AND BIOPHYSICAL

Pages 250-256

15, 1983

ALTERAWON

OF ADENYLATE CYCLASE ACTIVITY

BY PHOSPHORYLATION AND DEPHOSPHORYLATION Tetsu Second

Received

Akiyama,

Emiko Gotho

and Hiroshi

Department of Biochemistry, Nozawa-1, Setagaya-ku,

February

28,

Ogawara

Meiji College of Pharmacy, Tokyo 154, Japan

1983

Incubation of S49 cell membranes at 0°C resulted in a loss of adenylate cyclase activity, but addition of ATP and ATP regenerating system prevented the decrease of the activity. A non-phosphorylating analogue of ATP, adenyl-5'-yl imidodiphosphate, was less effective than ATP. Treatment of solubilized adenylate cyclase with calf intestine alkaline phosphatase caused the decrease of the activity. Membranes from cyc- S49 mutant cells, which are devoid of guanine nucleotidebinding protein, yielded the same results as membranes from S49 cells, indicating that the catalytic component is involved in the alteration of the enzyme activity by these treatments. These results suggest that phosphorylation and dephosphorylation of the catalytic component may regulate adenylate cyclase activity.

Protein process

phosphorylation of cellular

activity

are

observed

the

case of adenylate

which

fluoride

play

a role

and molybdate

To examine volved tion

in the

ATP, ATP regenerating treatment.

Furthermore,

system, using

under

protein and Mgzt, cyc-

0006-291X/83 $1.50 Cop.vright :G 1983 b-vAcademic Press. Inc. All rights of reproduction in any form reserved.

activity

cyclase,

membranes

by an endogenous

that

that

kinase

suggesting the

250

In

regulation

stimulation

inhibitory reported

of

properties ATP-dependent

the phosphorylation

by ATP may

and dephosphorylation we studied

the

effect

conditions

where

can occur:

i.e.

and the dephosphorylation

S49 mutant

(1,2).

(6).

phosphorylation

of adenylate cell

proposed

and suggested

that

proteins

and dephosphorylation

They recently

cyclase

in the regulatory

and nonenzymatic

to phosphatase

(7-9).

adenylate

the possibility regulation

of enzymatic

et a1.(5)

share

role

have been some reports

is related

cyclase

of S49 mouse lymphoma

phosphorylation

there

Richards

in elevating

an important

by phosphorylation

by molybdate

of adenylate

to play

and a number

cyclase, (3-6).

cyclase

activation

known

to be regulated

by phosphorylation adenylate

is

cells

(lo-13),

are

in-

of incuba-

ATP-dependent incubation

with

by phosphatase we sought

to identi-

Vol

112, PJo. 1, 1983

f y which

BIOCHEMICAL

component

of the

of the adenylate

enzyme activity

presented

AND BIOPHYSICAL

in this

by these

cyclase

system

RESEARCH COMMUNICATIONS

is

involved

The results

treatments.

in the alteration

of these

experiments

are

report. MATERIALS

AND METHODS

Cell culture: 549 wild type and cyc- mutant cells were grown in Dulbecco's modified Eagle's basal medium supplemented with 10% heat-inactivated horse serum in suspension culture. Preparation of plasma membrane was according to Courtneidge et a1.(14). Adenylate cyclase activity was measured by the method of Salomon et a1.(15). Plasma membranes were incubated with ATP as described in the legend to Fig. 1. Phosphatase treatment of solubilized adenylate cyclase: Adenylate cyclase was so-ubilized from plasma membranes (2.3 mg protein/ml) with 1% Triton NlOl in solution A (20 mM Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid]-NaOH pH 7.11, 1 mM dithiothreitol, 5 mM MgC12). After incubation for 1 h at O"C, insoluble material was removed by centrifugation at 100,000 x g for 30 min. Extracts were incubated for various times at 0°C with agarose or calf intestine alkaline phosphcltase immobilized on agarose (final concentration: 0.76 units/100 111 reaction mixture). Agarose or immobilized alkaline phosphatase was removed by centrifugation and the adenylate cyclase activity of the supernatant was measured. 32P-labeled plasma membranes were prepared by incubation with 20 mM Pipes [1,4pi.)erazinediethanesulphonic acid]-NaOH pH 7.2, 4 mM MgC12, and [,32P]ATP (7 uM, 63 mCi/pmole) for 20 min at 30°C. 32P-labeled plasma membranes collected by centrifugation at 100,000 x g for 30 min were solubilized and treated with agarose or immobilized alkaline phosphatase as described above. Radioactivity not removed fr)m proteins was measured according to the procedure of Cassel et a1.(16). Immobilized alkaline phosphatase was obtained from Sigma. RESULTS Plasma

membranes

without

ATP,

shown

in Fig.

ing

system

manner.

prepared

l(a),

resulted

did

not

To examine

ljzed

calf

centrifugation. by the treatment

this

activity

the

adenylate

of adenylate

analogue

phosphorylation, immobilized

in a loss

membranes

without

cyclase

activity

incubated

with

(Table

cyclase

adenylate

cyclase

solubilized

with

intestine

alkaline

As shown with

in Fig.

phosphatase,

alkaline

2(a),

alkaline adenylate

phosphatase. 251

various

with

times.

or As

ATP and ATP regeneratin a time-dependent

were

incubated

with

a

imidodiphosphate],

1).

adenylate

and immobilized

incubated

ATP and ATP regenerating

membranes

that

cyclase

were

of ATP, AppNHp [adenyl-5'-yl decreased

possibility

membranes

When plasma

loss.

cells

and MgC12 at 0°C for

of plasma

exhibit

cyclase

system

incubation

plasma

nc'n-phosphorylating adenylate

S49 mouse lymphoma

the ATP regenerating

By contrast,

system

from

activity Triton

NlOl

and after

By contrast,

changed was reacted

incubation

phosphatase cyclase

is

were

activity adenylate

the separated

by dewith solubiby

was decreased cyclase

incu-

Vol. 112, No. 1, 1983 bated

with

agarose

phosphate

from

alone

with

[y32P]ATP

which

from

examined

cyc-

the

and the

variant

alteration

treatment

in the

cells

presence

was similar

by this

which

alkaline

to S49 adenylate

decrease

of the activity

proteins

(Fig.

treatment

described

lack

guanine

cyclase

cyclase.

in Fig.

Phosphatase

3(a),

32P manner.

involved

in the

al-

above,

we prepared

plasma

mem-

protein incubation

cyclase

and

with

activity

response

resulted

was removed

ATP

was assayed

to incubation

treatment

phosphate

alka-

is

by the

the

membrane

immobilized

nucleotide-binding

l(b),

and concurrently

plasma

of

system

Adenylate

in Fig.

with

removal

in a time-dependent

activity

phosphatase.

the

the

As shown

cyclase

treatment

of hy~:~+. As shown

2(b),

was measured.

of adenylate

with

reaction,

and treated

of adenylate

by the

To verify

by this

was solubilized

component

RESEARCH COMMUNICATIONS

of activity.

proteins

was removed

of the activity

brane

no decrease

membrane

of proteins

To identify teration

AND BIOPHYSICAL

and 32P radioactivity

phosphatase

radioactivity

showed

solubilized

phosphorylated line

BIOCHEMICAL

with

ATP

in the

from

the reacted

3(b)).

DISCUSSION In this

report

phorylation

are

purification present, level

we have studied involved

of the studies

are

alteration

adenylate

with

impossible. of the

in the

possibility

regulation

cyclase

purified

that

phosphorylation

of adenylate

system

molecules

Therefore activity

the

cyclase

is difficult

by incubation

this

analysis

enzyme

of plasma

activity.

to achieve

and detailed

we studied

and dephos-

for

the

at the molecular

system

membrane

As the

by investigating

with

the

ATP and phospha-

tase. While ing

incubation

of plasma

membrane

resulted

in a loss

of adenylate

system

ATP regenerating

system,

A non-phosphorylating 1).

Though

with

Richards

and Mg2+ prevented

analogue et al.

reported

ATP, ATP regenerating

system,

we did

not

observe

prepared

from other

cell

lines

(rat

cells)

by various

methods

normal yielded '52

ATP and ATP regenerat-

activity,

incubation

of activity

was not as effective

that

this

without

such a loss

incubation 2+ and Mg results

cyclase,

3Yl

cyclase

of ATP, AppNHp,

adenylate

formed

of S49 cells

stimulation. 3Yl cells similar

of rat

liver

in a 4-7

fold

Membrane and adenovirus results

(data

with (Fig.

ATP,

l(a)).

as ATP (Table plasma

membrane

activation

of

fractions type not

12-transshown).

Vol. 112, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

Incubation

Time

RESEARCH COMMUNICATIONS

(hours)

Fig. 1. Effect of incubation with ATP and ATP regenerating system on adenylate cyclase activity. Plasma membranes of S49 cells (a), or cyccells (b) (1.5 mg protein/ml, final concentration) were incubated with 50 mM Hepes-NaOH pH 7.4, 1 mM ATP, 10 mg/ml phosphocreatine, 5 mg/ml phosphocreatine kinase, and 5 mM MgC12 (o), or with 50 mM Hepes-NaOH pH 7.4 and 5 mM MgC12 (A) at O'C and then diluted with 6 volumes of 20 mM Hepes-NaOH pH 7.4, and centrifuged at 100,000 x g for 30 min. The obtained membrane pellets were used for the assay of adenylate cyclase activity. 100% is 10.5 pmol/mg protein/min in (a) and 5.1 pmol/mg protein/min in (b).

('ne possible front adenylate without

interpretation cyclase

of these

by an endogenous

ATP and ATP regenerating

system

Table Incubation Condition

ATP (1 mM), AppNHp -

of

of

S49 cell

ATP regenerating

system system

is as follows:

phosphatase resulted

activity in the

Removal during

decrease

of phosphate

the

incubation

in adenylate

1 membranes adenylate pmol/mg

incubation

ATP regenerating

(1 mM),

plasma

results

with

ATP or

AppNHp

cyclase activity protein/min (X of initial

9.5

(96)

3.9

(39)

3.0

(30)

activity)

S49 cell plasma membranes (1.5 mg protein/ml, final concentration) were Incubated with 50 mM Hepes-NaOH pH 7.4, 5 mM MgC12, and other components for 20 h ~lt 0°C. Membranes were recovered from incubation and assayed for adenylate cyclase ~lctivity as described in Materials and Methods. ATP regenerating system consists of phosphocreatine and phosphocreatine kinase (final 10 mg/ml and 5 mg/ml, -espectively). The initial adenylate cyclase activity of the membrane preparation was 9.9 pmol/mg protein/min.

BIOCHEMICAL

Vol. 112, No. 1, 1983

(a)

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(b)

549

P

P

o- -A---r-\

CYC-

p-

100'

A

-A

\

P 0

0 \ \

O\ 0

-------0

O-------O-

50

0

30

60

0

Incubation

Time

30

60

(minutes)

Fig. 2. Effect of alkaline phosphatase treatment on adenylate cyclase activity. Adenylate cyclase was solubilized from plasma membranes of S49 cells (a), or cyccells (b) (2.3 tng protein/ml) with 1% Triton NlOl as described in Materials and Methods. The solubilized adenylate cyclase (80 m protein) was reacted with 0.76 units of immobilized calf intestine alkaline phosphatase (0) or with agarose (L) at 0°C in a total volume of 100 ~1. After centrifugation the obtained supernatant was assayed for adenylate cyclase activity in the presence of 50 fl guanyl-5'-yl imidodiphosphate (a) or 5 mM MnCl (b). 100% is 12.4 pmol/mg protein/min in (a) and 7.3 pmol/mg protein/min in (b 5 .

cyclase by

activity.

an

and

When

endogenous

prevented This

was

phosphatase. the

In

memebrane: membrane.

observed

in

removal It

of is

kinase of

order

ATP

competed

adenylate

to

by exclude

regenerating

the

In

this

system,

1 with

the

suggest

that

the

the

were

added,

endogenous

phosphorylation

phosphatase

activity

activity.

effect an

system

with

cyclase

phospholipids,

paralle

results

and

supported

e.g.

plasma

These

protein a loss

model

ATP

of

effect we

dephosphorylation of

used

decrease

phosphatase

the

dephosphorylation activity

on

adenylate of

cyclase adenylate

of of

by

the

adenylate

added

other

components

solubilized cyclase

proteins cyclase

immobilized

from

activity (Fig.

can

of

was

2(a),

3(a)).

be changed

by

phosphate. known

components: and

a catalytic

the

component

that

adenylate

a hormone

receptor,

component involved

cyclase

in

systems

a guanine

(17,

18).

the

alteration

consist

of

at

nucleotide-binding

We employed of 254

cycadenylate

least

three

separable

regulatory mutant cyclase

S49

cells

activity

component, to by

identify the

Vol. 112, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(b)

0

30

60

CYC-

30

0

Incubation

Time

60

(minutes)

Fig. 3. Time course of dephosphorylation of 32P-labeled solubilized membrane proteins. Plasma membranes (2.3 mg protein/ml) were phosphorylated with [r32P]ATP and solubilized with 1% Triton NlOl as described in Materials and Methods. The solubilized adenylate cyclase (80 a protein) was incubated with 0.76 units of immobilized calf intestine alkaline phosphatase (0) or with agarose (A) at 0°C in a total volume of 100 ~1. At the indicated times samples were centrifuged and the supernatant was used for the determination of 2P radloactivity associated with proteins. (a) S49 (100% = 2101 cpm), (b) cyc(100% = 1902 cpm).

treatment

described

defective

guanine

to

stimulation

of

the

Using

of

these

the

vance

activity

of

hormone

The

by

the

the

assumed

to

be

indicate

of

protein

kinase

can

cyclase

remain

to

fication

and

of

and

protein

enhance be

or

measured

type

lymphoma

fluoride in

S49

(Fig.

component

site

of

action

the

activity

of of

and

of

Mn *+

cells

the

treatment.

The

catalytic

same

3(b)).

component, the

(10-13).

the

2(b),

cyc-

respond

activity

exhibit

l(b),

a

to

the

of

to

has

unable

presence shown

cells

is ion

the

was

cells

and

nucleotide-binding

has

no

rele-

the

catalytic

results

component

of

may

be

dephosphorylation. kinase

the

mouse

component

guanine

and

decreased Final

determined.

characterization

S49

component

catalytic

the

and the

be

wild

that

phosphorylation

identity

can

solubilized

receptors

of

nucleotides

catalytic

cyclase

experiments

gulated

mutant regulatory

alone

cells,

adenylate

was

cyc-

guanine

component mutant

component these

hormones,

as

to

The

nucleotide-binding

catalytic

response As

above.

of

the

whether

phosphorylation

activity proof catalytic

255

of of

the

by

a purified

dephosphorylated hypothesis

component

and

adenylate will

the

require protein

purikinase

re-

Vol. 112, No. 1, 1983

involved.

Analysis

of several specific

BIOCHEMICAL

of protein

memebrane-associated membrane

of adenylate

proteins

cyclase,

AND BIOPHYSICAL

kinases

of the plasma

protein (unpublished

characterization

kinases data). of these

RESEARCH COMMUNICATIONS

membrane

revealed

and each of which In relation protein

the presence

phosphorylated

to the phosphorylation kinases

is

under

investi-

gation. ACKNOWLEDGEMENTS We thank Y. Osawa, K. Tanaka, and A. Takayama for helpful technical assistance. This work is supported by the Institute of Microbial Chemistry and a Grant-in-aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Krebs, E. G. and Beavo, J. A. (1979) Ann. Rev. Biochem. 48, 923-959 2. Cohen, P. (1982) Nature 296, 613-620 3. Constantopoulos, A. and Najjar, V. A. (1973) Biochem. Biophys. Res. Commun. 53, 794-799 A., Judge, J., Rauner, R. and Najjar, V. A. (1973) 4. Layne, P., Constantopoulos, Biochem. Biophys. Res. Commun. 2, 800-805 5. Richards, J. M. and Swislocki, N. I. (1979) J. Biol. Chem. 254, 6857-6860 6. Richards, J. M., Tierney, J. M. and Swislocki, N. I. (1981) J. Biol. Chem. 256, 8889-8891 7. Hollander, V. P. (1971) The Enzymes, pp. 450-498, Academic Press, New York 8. Dipietro, D. L. and Zengerle, F. S. (1967) J. Biol. Chem. 242, 3391-3396 9. Ikawa, T., Nishizawa, K. and Miwa, T. (1964) Nature 203, 939-940 10. Bourne, H. R., Coffino, P. and Tompkins, G. M. (1975)cience 187, 750-752 11. Ross, E. M. and Gilman, A. G. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 37153719 12. Ross, E. M., Howlett, A. C., Ferguson, K. M. and Gilman, A. G. (1978) J. Biol. Chem. 253, 6401-6412 13. Ferguson, K. M., Northup, J. K. and Gilman, A. G. (1982) Fed. Proc. fi, 1407 14. Courtneidge, S. A., Levinson, A. D. and Bishop, A. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3783-3787 15. Salmon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 58, 541-548 16. Cassel, D. and Glaser, L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2231-2235 17. Rodbell, M. (1980) Nature 284, 17-22 18. Ross, E. M. and Gilman, A. G. (1980) Ann. Rev. Biochem. 49-, 533-564

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