Effect of phosphorylation by different protein kinases on the behaviour of glycogen synthase as a substrate for hepatic synthase phosphatases

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 3, 1986

Pages 1033-]039

September 30, 1986

EFFECT OF PHOSPHORYLATION BY DIFFERENT PROTEIN KINASES ON THE BEHAVIOUR OF GLYCOGEN SYNTHASE AS A SUBSTRATE FOR HEPATIC SYNTHASE PHOSPHATASES

+

Mathieu Bollen*, Maria Plana ÷, Emilio Itarte ,and Willy Stalmans*

* A f d e l i n g Biochemie, F a c u l t e i t Geneeekunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium +Departament de Bioqufmica, Facultat de Ci&ncies, Universitat Autbnoma de Barcelona, Bellaterra (Barcelona), Spain

Received August 4, 1986

Glycogen synthase a from skeletal muscle was phosphorylated in vitro and then used as substrate for the two major synthase phosphatases from liver. Synthase phosphorylated by cAMP-dependent protein kinase (1.4-1.7 P/subunit) was preferentially activated by the cytosolic S-component; in contrast, progressive phosphorylation by casein kinase-1 (0.9-6.5 P/subunit) yielded substrates that were always better dephosphorylated and activated by the glycogen-bound G-component. We have previously isolated from dog liver several types of synthase b that differ by their need for the S- and/or G-component for prompt activation. After additional phosphorylation by a mixture of synthase kinases the activation of these enzyme preparations required the presence of both components. © 1986A~ademic Press, Inc.

Glycogen synthase is inactivated by phosphorylation. The enzyme from skeletal muscle can be phosphorylated in vitro on at least 9 different serine residues by a host of protein kinases that each recognize one or several sites (I). Reactivation of a different

muscle glycogen synthase is catalyzed by protein phosphatases;

site-preference has

been noted between phosphatases of type-1 and

type-2 (2). Hepatic glycogen t i o n by

a l s o s u b je c t t o m u l t i p l e s i t e phosphoryla-

the same p r o t e i n kinases ( 3 - 5 ) . However, much less i s known about the

phosphorylatable sites. f i e d from

synthase i s

In our

g l u c a g o n - t r e a t e d dogs.

l a b o r a t o r y l i v e r synthase b i s r o u t i n e l y p u r i Several types

o f l i v e r synthase b have been

i d e n t i f i e d on the b a s i s o f d i f f e r e n c e s i n k i n e t i c p r o p e r t i e s , i n chromatographi c behaviour,

and i n

d i f f e r e n c e s between

substrate quality

f o r synthase

phosphatases ( 6 - 8 ) . The

these enzymes were a t t r i b u t e d t o t h e i r s t a t e o f phosphory-

lation, but a proteolytic modification could not be excluded. 0006-291X186 $1.50

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Copyr~ht © 1986 by Academ~" Press, lnc. Aft righ~ ~' reproduction in any ,~rm reservel

Vol. 139, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The activity tion between

of synthase

two major

protein phosphatases, i.e. a soluble S-component and a

glycogen-bound Z-component to the

class of

they are

(6,8). Although

"type-l" (2)

both synthase

phosphatases belong

or "ATP,Mg-dependent" (9) protein phosphatases,

functionally distinct

involve exclusively

phosphatase in the liver stems from a coopera-

entities, and

all known regulatory mechanisms

the Z-component (10). Some preparations of liver synthase

b were preferentially activated by the S-component, and other preparations were significantly activated by the Z-component (6,7). However, both components were required for

the activation

of the

common type

of liver

synthase b,

which

appeared to be maximally phosphorylated. The aim of the present work was to investigate whether the ability of the S- and

Z-component to dephosphorylate and activate a particular preparation of

synthase b is affected by the phosphorylation state of that substrate.

MATERIALS AND METHODS The sources of relevant commercial products have been reported (7). Different types of liver synthase b were isolated from glucagon-treated dogs (7,10). Glycogen synthase a was purified from rabbit muscle (11). One unit of glycogen synthase converts I ~mol of substrate per min (7). Casein kinase-1 (12), casein kinase-2 (13) and cAMP-dependent protein kinase (14) were purified from rat liver cytosol. One unit incorporates I nmol of phosphate per min into casein (casein kinases) or into histone at 30°C. The subcellular fractions containing either the S- or the G-component of synthase phosphatase were prepared from the livers of fed rats (15). Muscle glycogen synthase (I-2 U/ml) was incubated in the presence of 0.5 mM EZTA, 8 mM magnesium acetate, 0.10-0.15 mM ATP with or without [F-32p]ATP (200-1000 cpm/pmol), buffer A (25 mM glycerol 2-phosphate, pH 7.0, 0.5 mM EDTA and I mM dithiothreitol), and either casein kinase-1 (I-8 U/ml) or cAMP-dependent protein kinase (1.8 U/ml). After incubation for 90 min at 30°C, bovine serum albumin was added to a concentration of 2 mg/ml, and the reaction was stopped by addition of an (NH4)2SO 4 solution (pH 8.0) till 75% saturation. The precipitated proteins were collected by centrifugation, resuspended in a minimal volume of buffer B (0.25 M sucrose, 50 mM imidazole, pH 7.4, and 0.5 mM dithiothreitol), and dialyzed against the same buffer. The extent of phosphorylation was determined after thin-layer chromatography (16). Synthase b from liver (2 U/ml) was further phosphorylated by incubation for 60 min at 30°C as described above, in the presence of casein kinase-1 (4 U/ml) plus casein kinase-2 (I U/ml) and cAMP-dependent protein kinase (0.6 U/ml). In the control experiment no protein kinases were added. The incubations were terminated by the addition of 50 mM NaF and 5 mM EDTA, and the preparations were equilibrated with buffer B by drop-dialysis (17) prior to incubation with synthase phosphatases. Activation and ~ dephosphorylation experiments were done in the presence of 5 mM magnesium acetate and 3 mM AMP (8). The cell fractions containing the Sand G-component were always present at a relative concentration of 2%, as compared to the livers from which they were prepared.

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

RESULTS AND DISCUSSION Synthase ~ from liver Four preparations of purified hepatic glycogen synthase b were preineubated under phosphorylation conditions in the absence or presence of a cocktail of protein kinases.

Subsequently, the

synthase phosphatases

rate of

was followed.

activation of these substrates by

Fig. IA illustrates the case of a type of

hepatic synthase b that could already be activated at a substantial rate by the diluted S-component kinases the

or G-component

alone. After

incubation with

the protein

substrate became completely dependent on the presence of both com-

ponents for its activation (Fis. IS). Similar results were obtained after phosphorylation of

3 other

requirement for out, however,

preparations of

synthase b,

which showed a different

the S- and G-component (not illustrated). It should be pointed that the

lag in

the activation

of additionally phosphorylated

synthase b (Fig. IS) was not always observed. That these phorylated during fold decrease

preparations of

glycogen synthase were supplementarily phos-

the incubation

with protein kinases was inferred from a 2-?

in the enzyme activity (not illustrated), as measured at 0.25 mM

UDP-glueose and activity ratio

I mM

glucose 6-phosphate

as measured

(at 5 mM UDP-glucose in the

g.

// "

0.2

//

0.! "•

o¢! i o

FiR.

classical way

A.

0.3

U9

in the

(18). No effect was observed on the

A

3o

/

0 -------"

45'

6'0

0

•

15

30

45

60

1. E f f e c t o f a d d i t i o n a l p h o s p h o r y l a t t o n o f hepatic synthase b on the r a t e

of i t s a c t i v a t i o n by synthase phosphatases. Liver synthase b was preincubated in the absence (A) or presence (B) of casein kinase-1, casein kinase-2 and cAMP-dependent protein kinase under phosphorylation conditions, as described in the experimental section. Subsequently the activation of either synthase preparation was followed during incubation in the presence of the S-fraction (S), or the G-fraction (g), or both (S+G). 1035

Vol. 139, No. 3, 1986

absence or

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

presence of

10 mM

glucose 6-phosphate),

probably because

of the

already very low ratio (<0.05) before incubation with the kinases. These data teristics of state of

provide strong evidence that the different activation charac-

various types

of hepatic

synthase b

(6,7) are related to their

phosphorylation, rather than to some proteolytic modification. Appar-

ently, maximally phosphorylated liver synthase can only be activated by diluted liver preparations are present.

when both

Glycogen synthase

relevant since

it represents

glucagon-treated livers thase phosphatase vivo and

the cytosolic and the glycogen-bound phosphatase with such the major

(6). Moreover,

activity corresponds

in isolated

hepatocytes from

characteristics is part of

the synthase

with this to the

physiologically isolated

from

substrate the measured syn-

actual glycogenic

capacity in

normal, diabetic, and adrenalectomized

starved animals (15,19).

Synthase from skeletal muscle Muscle glycogen synthase, phosphorylated by cAMP-dependent protein kinase, was dephosphorylated

much more rapidly by the S-component than by the G-compo-

nent (20). The same

picture applies

to the rate of reactivation of this sub-

strate (Fig. 2). In contrast, glycogen synthase that had been phosphorylated by casein kinase-1

was better

activated and

dephosphorylated by the G-component

0.3 S._~+G Q

o 0.2 v A G____~---A

/

0,1

i

0 0

I

i

7

14

2'I

218

Time (rain)

Fi~. 2. Activation of muscle glycogen synthase by hepatic synthaae phosphatases after phosphomylatlon by cAMP-dependent protein klnase. The data are the means of 2 experiments. Muscle glycogen synthase a was phosphorylated until incorporation of 1.4 and 1.7 P/subunit respectively. The reactivation of the enzyme (0.25 U/ml) was then followed as in Fig. I. 1036

Vol. 139, No. 3, 1 9 8 6

BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

o6A

B.

0.5

E 0.4 v 0.3

301

I

o

lO1~ ! / o

7

14

2'1

2~

0i

3;

0

. / 7

14

21

~ 28

L 35

Time (min) Fig.

3. Activation (A) and dephosphorylatton (B) of muscle 8lycosen synthase by

hepatic synthase phosphatases a f t e r phosphot~lation by casein kinase-l. The d a t a a r e t h e means ± S.E.M. o f 3 e x p e r i m e n t s . Muscle g l y c o g e n s y n t h a s e a was p h o s p h o r y l a t e d u n t i l i n c o r p o r a t i o n o f 5.7 ± 0 . 5 P/ s u b u n i t . The p h o s p h o r y l a t e d enzyme (1.1 ± 0 . 2 U/ml) was t h e n i n c u b a t e d as i n F i g . 1.

(Fig. 3). Regardless of the kinase that was used for the phosphorylation of the synthase (Fig. 2 and 3), the effect of the combined S- and G-component could be approximately accounted

for by

addition of

the separate action of each phos-

phatase. Fig. 4

illustrates the

influence of the extent of phosphorylation by

casein kinase-1. The rate of activation of glycogen synthase by either phosphatase decreased sharply until 3 phosphates had been incorporated per subunit. On the other hand, the relative contributions of the S- and G-component to the activation of rated into the rate

the synthase

were independent of the amount of phosphate incorpo-

the synthase by casein kinase-1. When both components were present, of activation

is remarkable

corresponded again closely to arithmetic addition.

It

that muscle glycogen synthase, even when phosphorylated to 6.5 P

/subunit, did not show the cooperative activation characteristics that are typical for heavily phosphorylated liver synthase b. This may be due to structural differences between

muscle and

liver synthase or to differences in the amount

or location of the phosphorylated residues. Our results with in vitro labelled glycogen synthase from muscle indicate that the ity for

two components of hepatic synthase phosphatase have a distinct affindifferent phosphorylated

sites in

1037

glycogen synthase.

From the known

Vol 139, No 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

@

120

E

80

A

E

x:

40

>, o9

~

0

©--$

c

i

i

i

2

4

6

mol phosphate / mol subunit

F i g . 4. Influence of the extent of phosphorylatlon of muscle 81ycoBen synthase

by casetn ktnase-1 on i t s a c t i v a t i o n by synthase phosphatases, Muscle glycogen synthase a was phosphorylated by casein kinase-1 to the indicated stoeehiometry. The enzyme preparations (0.4 U/ml) were then incubated for 7 min with the S-fraction, or the G-fraction, or both, and the amount of synthase a formed was determined. Symbols as in Fig. 1.

site-specificity of (1,14,21) we

the cAMP-dependent

conclude tentatively

be specifically

recognized by

protein kinase

and of casein kinase-1

that phosphorylated

residues of site I may

the S-component.

Our future aim is to identify

directly the sites in glycogen synthase that are preferentially dephosphorylated by the S- and G-components.

ACKNOWLEDGEMENTS This work was supported by the Belgian Fonds voor Geneeskundig ~etenschappelijk Onderzoek (Grant 3.0051.82), by the Flemish Commissariaat-Generaal voor de Internatlonale Culturele Samenwerkln8, and by the Spanish ComisiOn Asesora de InvestlgaciOn Cientffica y T4cnJca (Grant 2565/83).

REFERENCES

1. Kuret, J . , Woodgett, J.R. & Cohen, P. (1985) Eur. J. Biochem. 151, 39-48. 2. I n g e b r i t s e n , T.S. & Cohen, P. (1983) Science 221, 331-338. 3. Camici, M., Ahmad, Z., DePaoli-Roach, A.A. & Roach, P.J. (1984) J. B i o l . Chem. 259, 2466-2473. 4. Imazu, M., S t r i c k l a n d , W.G., Chrisman, T.D. & E x t o n , J.H. (1984) J. B i o l . Chem. 259, 1813-1821. 5. Imazu, M., S t r i c k l a n d , W.G. & Extort, J.H. (1984) Biochim. Biophys. Aeta 789, 285-293. 6. Doper4, F., Vanstapel, F. & Stalmans, W. (1980) Eur. J. Biochem. 104, 137146. 7. Doper4, P. & Btalmans, W. (1982) Arch. I n t . P h y s i o l . Biochim. 90, B113Bl14. 1038

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8. Mvumbi, L., Doper~, P. & Btalmans, W. (1983) Biochem. J. 212, 407-416. 9. Merlevede, W. (1985) Adv. Prot. Phosphatases I, 1-18. 10. Stalmans, W., Bollen, M. & Mvumbi, L. (1986) Diabetes/Metabolism Rev. 4, in press. 11. Takeda, Y., Brewer, H.B., Jr. &Larner, J. (1975) J. Biol. Chem. 250, 89438950. 12. Itarte, E., Plana, M., Guaseh, M.D. & Martos, C. (1983) Bioehem. Biophys. Res. Comlnun. 117, 631-636. 13. Itarte, E., Mor, M.A., Balavert, A., Pena, J.M., Bertomeu, J.F. & Guinovart, J.J. (1981) Biochim. Biophys. Acta 658, 334-347. 14. Vila, J., Balavert, A., Itarte, E. & Guinovart, J.J. (1982) Arch. Bioehem. Biophys. 218, I-7. 15. Bollen, M. & Stalmans, W. (1984) Bioehem. J. 217, 427-434. 16. Huang, K.-P. & Robinson, J.C. (1976) Anal. Biochem. 72, 593-599. 17. Marusyk, R. & Sergeant, A. (1980) Anal. Bioehem. 105, 403-404. 18. Guinovart, J.J., Salavert, A., Massagu4, J., Ciudad, C.J., Balsas, E. & Itarte, E. (1979) FEBB Lett. 106, 284-288. 19. Bollen, M., Gevers, G. & Stalmans, W. (1983) Bioehem. J. 214, 539-545. 20. Bollen, M., Doper4, F., Goris, J., Merlevede, W. & Stalmans, W. (1984) Eur. J. Biochem. 144, 57-63. 21. Embi, N., Parker, P.J. & Cohen, P. (1981) Eur. J. Bioehem. 115, 405-413.

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