Effect of calcium on cyclic AMP-dependent and cyclic GMP-dependent endogenous protein phosphorylation in mouse brain cytosol

Vol. 67, No. 2, 1975

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EFFECT OF CALCIUM ON CYCLIC AMP-DEPENDENT AND CYCLIC GMP-DEPENDENT ENDOGENOUS PROTEIN PHOSPHORYLATION IN MOUSE BRAIN CYTOSOL Alvin Department

of Psychiatry

M. Malkinson

and Dight

University

September

for

Human Genetics

of Minnesota

Minneapolis, Received

Institute

Minnesota

55455

25,1975

In the presence of Cat+ (>lmM), cyclic AMP (IOpM) inhibited, and cyclic GMP (10uM) stimulated, the phosphorylation of a protein in the brain cytosol fraction of A/Jax mice. Lower concentrations of cyclic AMP stimulated phosphorylation of this protein, while lower concentrations of cyclic GMP had no effect. This protein (called protein 49) co-migrated on SDS - polyacrylamide gels with the endogenously phosphorylated band of partially purified bovine heart protein kinase, an apparent molecular weight of 49,000. The F&;c~~~,im~'f',h~~ ;;y' -dependent effect of the two cyclic nucleotides is discussed. AMP (CAMP),

Cyclic

messengers kinase

enzymes

believed

become

state

Recent

the

kinase

kinase

of the

has been

Ca "

demonstrated

bovine

heart

rently

regulates

(10)

for

kinase using

and brain the

protein

cGMP may also

protein

and are

(5)

These and change

substrates

(6,7).

of a cGMP-dependent function

to inhibit

through

a pro-

CAMP-dependent

(9).

substrates protein

that

stimulation

occurrence

has been reported

activity

One of the subunit

suggests

CAMP-dependent

of CAMP (4).

hormonal

widespread

as second

in eukaryotes,

of endogenous

of the

step.

(l-3).

effects

following

of phosphorylation

(8)

and Cat+ serve

distributed

physiological

activated

kinase

protein

systems

ubiquitously

demonstration

protein tein

are

GMP (cGMP),

biological

to mediate

enzymes the

in several

cyclic

ability

Copyrikht a I9 75 by Academic Press. ITIC. All rights of reproduction in arz.v form reserved.

protein

kinase This

itself. purified (11)

protein tissues.

enzymes

autophosphorylation kinase

preparations

Autophosphorylation

of CAMP to dissociate 752

is the

the

protein

regulatory reaction from appakinase

Vol. 67, No. 2, 1975

holoenzyme with

[

32

rylated

BIOCHEMICAL

(12).

Incubation

of extracts

PI-ATP,

and subsequent

protein

substrates

phoresis,

showed

that

protein

region

on the

with

a protein

analog

of CAMP, which

finding, bovine

gel,

together heart

49 might

extent

of protein

nature

of this

effect

(13).

In the

presence

rylation

kinase

of Mg fractions,

protein

49 in both

tt

electro-

protein

substrate

to as protein

49, co-migrated

on

8-azido-CAMP,

a photo-affinity

with

CAMP-binding

molecular

regulatory

by the

mouse brain

(13).

cation

protein

had no effect

and particulate

of the that The

by CAMP, and the

divalent

i-t , CAMP decreased

This

suggested

subunit

, CAMP stimulated

sites.

weight

subunits,

present

49 phospho-

in particulate the

fractions.

of CAMP and cGMP on protein , using

phospho-

(SDS)-gel

of 49,000

was affected

but

of Zn

cytosol

the effects of Ca

+t

endogenously

This

CAMP-binding

was determined

In the presence

presence

(11)

tissues

(13).

49,000

49 phosphorylation

tions.

the

estimated

be a protein

in cytosol

describes

the

vertebrate

sulfate

weight

specifically

and brain

protein

bound

reacts

with

(10)

which

several

phosphorylated

molecular

referred

RESEARCH COMMUNICATIONS

of the

dodecyl

the .nost heavily

had an apparent

from

separation

by sodium

in each tissue

SDS-gels

AND BIOPHYSICAL

frac-

phosphorylation This

of

report

49 phosphorylation

cytosol.

METHODS: Whole brains were removed from strain A/Jax-Minn mice (about 6 weeks of age, 20 gm body weight). Cytosol fractions were obtained by differential centrifugation as previously described (13). The endogenous protein phosphorylation assay contained (lOOi.11, final volume): 40-1OOug of protein, 50mM 2(N-morp391 ino) ethanesulfonic acid (MES) (pH 6.8), 5mM CaC12, l.O-1.5pM [uPI ATP (5-20Ci/mmole), with or withou5210uM CAMP or 10~M cGMP. The reaction was initiated by addition of [y] ATP; incubation was for 3 min at 30°C. The reaction was terminated by addition of 50~1 of a "SDS stop solution" previously described (14), and the mixture was heated in a boiling water bath for 75~1 or 100~1 aliquots were 2 min to eliminate proteolytic activity. then subjected to electrophoresis on SDS - polyacrylamide slab gels The gels were then stained with Coomassie blue, destained and (15). dried, and autoradiographs made as described previously (14). Each autoradiograph was scanned using an Ortec 4300 microdensitometer and the rea under the protein 49 peak was used as a quantitative measure of 3'P incorporation. Protein chromatography

kinase after

from bovine the method

heart was purified of Miyamoto --et al 753

through D5$E (16). [yPI ATP

in

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BEEF HEART PROTEIN KINASE

MOUSE BRAIN CYTOSOL

CAMP

-

+

-

cGMP

-

-

+

Fig. 1. Effects of 10uM CAMP and 10uM cGMP on endogenous phosphorylation of mouse brain cytosol in the presence of 5mM CaC12. Following samples were subjected to SDS-polyacrylamide gel electroincubation, phoresis and autoradiography as described in Methods. The autoradiograph is shown. A partially purified39 reparation of bovine heart protein kinase was incubated with [vP] ATP in the presence of 5mM CaC12 (in the absence of cyclic nucleotides), and subjected to electrophoresis on the same gel for comparison.

was prepared according to the method of Post and Sen (17). content was determined by the method of Lowry --et al (18). cGMP were purchased from Boehringer-Mannheim.

Protein CAMP and

RESULTS: Figure

1 is an autoradiograph

and 1OuM cGMP on endogenous

and the on the

major gel

determined

phosphorylation

of 5mM Ca ++ .

in the presence

phosphorylated

showing

Only band

effects

(protein

to a molecular

using

markers

10uM CAMP

cytosol

fraction

were

phosphorylated,

49) migrated

at a position

weight

of about

of known molecular

754

of

of a brain

a few proteins

corresponding protein

the

49,000,

weight.

as The

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 2. Time course of the effects of 10uM CAMP and 10uM cGMP on the endogenous phosphorylation of protein 49 from mouse brain cytosol, in the presence of 5mM CaCl2. Autoradiography was done as described in Methods, except for the variation in incubation time. The density of the protein 49 band was quantitated by microdensitometry, and expressed in arbitrary units,

extent

of protein

about

16% of that

of the

49 phosphorylation phosphorylated

incubated

samples

15 min at room temperature and prior

that

protein.

5mM Ca tt

radiograph

while

after

band

and [y-

of the gel

cGMP stimulated 2 illustrates

or without

The inhibitory

addition

abolished at the

PI-ATP

which

the

49,000

of a partially 32

presence (Worthington;

the

purified

protein

solution

on the

heart

protein

49.

phosphorylation

gel,

with

protein

band on the

kinase auto-

In the

of protein

49,

49 phosphorylation.

the time

course

of protein

49 phosphorylation

10uM CAMP or 10~M cGMP, in the presence effect

for

was associated bovine

with

SDS-stop

radioactivity

in a single

co-migrated the

0.2mg/ml)

of the

position

resulted

of Ca tt , CAMP inhibited

Figure with

the

Incubation

presence

Pronase

to electrophoresis,

indicating

with

in the

with

of Ca++ was only tt of Mg . Treatment

in the presence

of CAMP and the

755

stimulatory

effect

of 5mM Ca of cGMP

++

.

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

‘:L-+ldp 4

6

0

Concentrotlon

a

j

IO

of CO++ ! mMi

Fig. 3. Effects of different concentrations of CaC12 on the endogenous phosphorylation of protein 49. Samples were incubated with or without 10nM CAMP or 10uM cGMP. 'tions were as described under incubation in conG concentrations Methods, except for the variation Ca

occurred

at all

The effect phorylation added

is

cyclic

protein

times

of varying

the

shown

from Ca

decreased

at higher

15 set

to 10 min.

concentration

on protein

Phosphorylation

in the

was maximal

betweenl-8mMCa

at all

protein

.

Cat'

Ca

t+

concentrations

level

49 phosabsence

Protein

of 49

at 1mM Ca

++

above

at all

for

Catt

cGMP stimulation

1mM. concenwas about

6mM. A dose-response 49 phosphorylation 0.5nM trations

in the

CAMP stimulated above

curve

for

presence

protein

10nM inhibited

the

effects

of CAMP and cGMP on protein

of 5mM Catt

is

49 phosphorylation this

reaction.

756

,

10uM CAMP inhibited

49 phosphorylation

The optimal

tt

of 10pM CAMP was maximal concentrations.

49 phosphorylation

tested.

tt

3.

in the presence

10i~M cGMP stimulated trations

in Figure

nucleotides

phosphorylation and then

incubation

shown in Figure while

This

CAMP concen-

concentration-depen-

4.

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-I j

T

1

-~~ e

I 7

-.--~I

1--L 5

6

Cyclic

Nucleotide

-I ‘l

( -Log

3

1~1,

Fig. 4. Effects of varying CAMP or cGMP concentrations on the endogenous phosphorylation of protein 49. Incubation conditions were as described under Methods, except for the variation in cyclic nucleotide concentration.

dence

of the

was only

direction

of the

CAMP effect

observed

in the presence

Mg++,

CAMP stimulated

protein

above

0.5uM

CAMP inhibited

(data

shown).

not

concentrations effect

on protein

cation,

has not

this

reaction

2uM in the

been observed. presence

lung

tissues

and kidney

presence

at all

tt

presence

of

above

1vM

concentrations

49 phosphorylation of Ca "

(Fig

at

4).

An inhibitory

presence

dose-dependent

1OmM

concentrations

effects

have been demonstrated

not

of

in the

by cGMP, in the Similar

of Ca (data

at all

protein

45 phosphorylation

and cGMP in the

presence

0.2mM CAMP); while

cGMP stimulated

above

49 phosphorylation

In the

49 phosphorylation

(up to and including

5mM Zn++,

of Ca

on protein ++ .

using

of any of CAMP mouse

shown).

DISCUSSION: There

is

a heterogeneity

kinase

enzymes

cyclic

nucleotide-divalent

described

in eukaryotic

in this

report

of cyclic tissues cation

nucleotide-dependent (18,19).

effects

and previously

757

It

on protein (13)

reflect

protein

is possible

that

the

49 phosphorylation this

heterogeneity.

Vol. 67, No. 2, 1975

The 49,000 contain

BIOCHEMICAL

molecular

different

to different

weight protein

no effect

kinase

nucleotide

of CAMP on the kinase

in the

ration,

CAMP inhibited

tested

in the presence

proteins

of 49,000

subunits

--

that

protein

kinase

nation

of the

presence

enzymes

by various

is

potential

for

pendent

protein

some of the

(as well

second

and

exami-

in the

be involved

effects

on cell

neurons by Ca

(21). tt

(24).

in some of the

and Cat+ A Cat+-de-

protein

phosphorylation

of kinases

of other

interdependent

(20)

The

Regulation

nucleotide-dependent as the

growth

(221,

of cGMP (23).

has been described

tt by CAMP, cGMP, and Ca

of these

activity

can be modulated

of cyclic

might

of different

procedures,

effects

of postsynaptic

autophosphorylation

substrates)

regulatory

in progress.

antagonistic

growth

seems to be required cGMP binding

may be other

of CAMP, cGMP, and Ca++ may be interrelated.

CAMP and cGMP have

of CAMP on cell

kinase

Separation

fractionation

brain

prepa-

there

protein

of CAMP and cGMP on their cations,

latter

was

concentrations

Alternatively, -- not

of

and there bovine

In this

phosphorylated.

effects

and on the membrane

the

weight

(13),

at all

of Ca++ (11).

of different

For example,

of Mg++ (11).

there

in the presence

of purified

autophosphorylation

are being

The biological

effects

presence

may

For example,

tissues

autophosphorylation

gel

each responding

conditions.

of various

molecular

effect

subunits,

49 phosphorylation

fractions

protein

on a SDS-polyacrylamide regulatory

of CAMP on protein

in particulate

Mg++

region

cation-cyclic

was no effect

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

protein

activities

messengers.

ACKNOWLEDGEMENTS: I would like to thank Lora Hogy for Dr. A. J. Gharrett for helpful discussions. supported by grants from the U. S. Public CA16228).

expert technical assistance and This work was partially Health Service (HL06314 and

REFERENCES: 1.

Rob&on, G. A., Butcher, R. W., and Sutherland, Cyclic AMP, Academic Press, New York. _____

758

E. W. (1971)

Vol. 67, No. 2, 1975

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22. 23. 24.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Goldberg, N. D., O'Dea, R. F., and Haddox, M. K. (1974) Adv. Cyclic Nut. Res. 3:155-224. Rasmussen, H., Goodman, D. A., and Tenenhouse, A. (197'2) CRC Crit. Rev. Biochem. 1:95-148. Kuo, J. F. and Greengard, P. (1969) Proc. Nat. Acad. Sci. 64:1349-1355. Soderling, T. R., Corbin, J. D., and Park, C. R. (1973) J. Biol. Chem. 248:1822-1829. Langan, T. A. (1969) J. Biol. Chem. 244:5763-5765. Rudolph, S. A. and Greengard, P. (1974) J. Biol. Chem. 243:5684-5687. Kuo, J. F. (1974) Proc. Nat. Acad. Sci. 71:4037-4041. Kuo, J. F. Krueger, B. K., Sanes, J. R., and Greengard, P. (1970) Biochim. Bfophys. Acta 212:79-91. Erlichman, J. R., Rubin, C. S., and Rosen, 0. M. (1974) Fed. Proc. 32:643. Maeno, H., Reyes, P. L., Ueda, T., Rudolph, S. A., and Greengard, P. (1974) Arch. Biochem. Biophys. 164:551-559. Erlichman, J. R., Rosenfeld, R., and Rosen, 0. M. (1974) J. Biol. Chem. 249:5000-5003. Malkinson, A. M., Krueger, B. K., Rudolph, S. A., Casnellie, J. E., Haley, B. K., and Greengard, P. (1975) Metabolism 24:331-341. DeLorenzo, R. J., and Greengard, P. (1973) Proc. Nat. Acad. Sci. 70:1831-1835. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochern. 10:2606-2617. Miyamoto, E., Kuo, J. F., and Greengard, P. (1969) J. Biol. Chem. 244:6395-6402. Post, R. W. and Sen, A. K. (1967) Meth. Enzymol. 10:773-776. Lowry, 0. H., Rosebrough, N. J., Parr, A. C., and Randall, R. J. (1951) J. Biol. Chem. 193:265-275. Corbin, J. D., Keely, S. L., and Park, C. R. (1975) J. Biol. Chem. 250:218-225. Goldberg, N. D., Haddox, M. K., Dunham, E., Lopez, C., and Hadden, J. W. (1974) In: Control of Proliferation in Animal Cells, eds B. Clarkson and R.za, Cold Spring HarborLaboratory, ppg. 609-625. Stone, T. W., Taylor, D. A., and Bloom, F. E. (1975) Science 187:845-846. Whitfield, J. F., Rixon, R. ii., MacManus, J. R., and Balk, S. D (1973) In Vitro 8:257-278. Schultz, G., Hardman, J. G., Hurwitz, L., and Sutherland, E. W. (1973) Fed. Proc. 32:773. Goldberg, N. D., Haddox, M. K., Nicol, S. E., Acott, T. S., Zeilig, C. E., and Glass, D. B. (in press) ICN UCLA Symposium ____ on Molecular and Cellular Biology, Vol z., C. F. Fox and K McMahon (Eds).

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