Kinetic properties of glycogen synthase from skeletal muscle after phosphorylation by glycogen synthase kinase 4

Vol.

134,

February

No. 3, 1986 13,

BIOCHEMICAL

AND

BIOPHYSICAL

COMMUNICATIONS Pages

1986

KINETIC PROPERTIES OF GLYCOGEN SYNTHASE AFTER PHOSPHORYLATION BY GLYCOGEN Donna F.BrownI, 1 Hamilton

Received

RESEARCH

Mohammed

FROM SKELETAL MUSCLE SYNTHASE KINASE 4

and Erwin

Hegazyq

1129-1135

M. Reimann 2*

College, Clinton, NY 13323, and 2Departmentof Biochemistry, Medical College of Ohio, C.S. 10008, Toledo, OH 43699

December

20, 1985

Summary:Glycogen synthase I (EC 2.4.I.ll)from rat and from rabbit skeletal muscle was phosphorylated in vitro by glycogen synthase kinase 4 (EC 2.7.1.37)to the extent of 0.8 phosphates/subuiiitx both phosphorylated enzymes,the activity ratio (activity without glucose 6-P divided by activity with 8 mM glucose 6-P) was0.8 when determined with low concentrations of glycogen synthase and/orshortincubation times. However, the activity ratio was 0.5 with high enzyme concentrations and longer incubation times. It was found that the lower activity ratios result largely from UDP inhibition of activity measured in the absence of glucose 6-P. Inhibition by UDP was much less pronounced for glycogen synthase I, indicating that a major consequence of phosphorylation by glycogen synthase kinase 4is an increased sensitivity to UDP inhibition. 0 1986 Academic Press. Inc.

Glycogen different

serine

synthase

kinases

phosphorylated

synthase

from

residues

(l-

for

these

3).

skeletal

has been

Phosphorylation

decreases the activity

(activity

measured

separates which

the

in the

phosphoserines

can be separated

determined

presence

synthase

into

phase chromatography

glycogen serine

7 is

kinase 4 (GSK-4)(5),

and

kinase

in the absence

of 8 m M Glc-6-P)

In vitro,

protein

by phosphorylase

measured

of glycogen

by reversed

synthase

at ten

of 9 different

(l- 3).

by the CAMP-dependent

several other kinases (3).

activity

specificity

kinase (4) and glycogen

sites phosphorylated

ratio

muscle can be phosphorylated

The phosphorylation

sites

by phosphorylase

is one of several

rabbit

kinase

(6, 7) and

or GSK-4

in -- vitro

of Glc-6-P

(4, 5,8, 9). 6 separate

divided

Tryptic groups

(10). The peptide

by the

digestion of peptides containing

*To whom correspondence should be addressed. This work was supported in part by a grant (AM 19231) from the National Institutes of Health. Abbreviations: GSI, dephospho-form of glycogen synthase; GSD:2, glycogen synthase phosphorylated by GSK-4; GSK-4, glycogen synthase kinase 4; AO.5,concentration of Glc6-P required for 50% of maximal activation; SO.5, concentration of UDP-Glc required for 50% of maximal activity; HPLC, high-performance liquid chromatography; activity ratio, rate of glycogen synthesis in presence of 4.5 m M UDP-Glc and 0 Glc-6-P divided by the rate of glycogen synthesisinthe presence of4.5 mM UDP-Glc and 8 mM Glc-6-P. 0006-291X/86 1129

$1.50

Copyright 0 I986 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

134,

No. 3, 1986

Ser 7 (peptide regulation

2) is of special

by insulin

Recently with

BIOCHEMICAL

digesting

to

interest

several

32P-labeled

glycogen

synthase

were

synthase

had slightly

different

chromatographic

glycogen

synthase.

We were

therefore

difference

would

the

synthase.

The present

study reports

synthase

phosphorylated

by GSK-4,

glycogen

synthase

by GSK-4

markedly

by trypsin containing

interested properties

increases

to

similar

kinetic

a kinase

Ser7

we determined

of glycogen glycogen

in rabbit

that

rat

structural

for rat and rabbit

phosphorylates

the sensitivitytoinhibition

glycogen from

this

forms

by

and rabbit

rabbit

whether

phosphorylated

properties

which

obtained

The rat from

muscle

than the counterpart

in knowing of

maps

(12). Ser7

properties

In the course of these studies

(5).

is subject

rabbitskeletal

phosphopeptide

the peptide

kinetic

state

I from ratandfrom

synthase

enzymes

affect

However

COMMUNICATIONS

the phosphorylation

including

of glycogen

very similar.

RESEARCH

(10-11).

characteristics

forms

BIOPHYSICAL

in that

and catecholamines

we compared

respect

AND

muscle

phosphorylation

by U DP.

Materials and Methods: Minor modifications of published methods were used for purification of GSI from rat and rabbit skeletal muscle (13), for purification of GSK-4 from rabbit skeletal muscle (14), and for phosphorylation of GSI from rat and rabbit skeletal muscle by GSK-4 (13). GSI from rat and rabbit skeletal muscle was phosphorylated by GSK-4tothe extent of 0.76 and 0.83 phosphates per subunit, respectively. HPLC mapping of tryptic phosphopeptides (10) showed that only peptide 2 was labeled, consistent with phosphorylation at Ser 7 in rabbit glycogen synthase. Because serine 7 in rabbit muscle glycogen synthase is commonly referred to as site 2, and since the exact site ofphosphorylationin peptide 2hasnotbeen determinedforratglycogen synthase, we refer to our phosphorylated enzymes as GSD:2s and the site phosphorylated by GSK-4 as site 2. Glycogen synthase activity was measured as described by Thomas et al. (15) using enzyme concentrations of0.13to 5.4 ug/ml. Product formation is given in terms of mol For kinetic experiments, UDP[ f 4CJglucose incorporated from UDP-Glc into glycogen. Glc (obtained from ICN) was lyophilized to remove ethanol and stock solutions of unlabeled U DP-Glc were added to achieve a specific activity of 0.22 to 5.7 cpm/pmole. The activity ratio was determined on 60 ul aliquots as described previously (16). Kinetic characterization of glycogen synthase was carried out under the same reaction conditions When Glc-6-P was varied, the UDP-Glc concentration used to determine activity ratio. Kinetic constants were determined by fitting the data to either the was 40 pM. Michaelis-Menten equation orthe Hill equation by nonlinearregression analysis(l7). Results

and Discussion:In order to compare

and rabbit for

skeletal

Glc-6-P

determined

absence

(concentration for

phosphorylation

muscle after

phosphorylation

of Glc-6-P

required

GSIs and GSD:2s (Table

both resulted

of Glc-6-P)

the properties

in 2to

4-fold

increases

and in the AO.5 for

Glc-6-P.

1130

of glycogen

by GSK-4,the for

1).

both

in the Km for In addition,

from

SO.5 for U DP-Glc

50% of maximal For

synthase

rat

UDP-Glc

and AO.5

activation)

and rabbit

rat

were

enzymes,

(measured

in the

we used the Vmax values

Vol. 134. No. 3. 1986

8lOCHEMlCALAND8lOPHYSlCALRESEARCH

Table 1. Effect of Phosphorylation

Enzyme Rabbit

by GSK-4 on Kinetic Properties

of Glycogen Synthase

A0.5 for Glc-6-Pa

So.5 for UDP-Glcb

Vmax (-I+)'

2.5 uM

240 uM

NAd

2.0 pM

150 uM

N.D.

9.2 uM

450 uM

0.91

8.6 pM

590 uM

0.89

GSI

Rat GSI Rabbit

COMMUNICATIONS

GSD:Z

Rat GSD:2

a Measured in the presence of 40 uM UDP-Glc b Measured in the absence of Glc-6-P ' Extrapolated activity ratio at saturating concentrations of UDP-Glc, determined when UDP-Glc was varied in the presence and absence of Glc-6-P. d Not determined

determined

when UDP-Glc

an activity

ratio

ratio

was near

differences

was varied in the presence

at saturating unity

concentrations

(Table

1).

were noted between

For

glycogen

rabbit

GSD:2.

However,

laboratories

(4, 5, 10, 11).

concentrations wondered

0.5, which

were

if this

difference

values and the experimentally expected, (panel

A).

decreased ratio

the activity However, from

ratio

kinetic

is similar longer

account

determined for rabbit

of GSD:2 depends

0.54 during on length

ratio

ratio

times

than for

ratios.

muscle GSI(0.95)

activity reported

and

major

muscle. for

ratio

for

by other

higher

kinetic

enzyme

experiments,

between

is constant

conditions,

under

no

we

our calculated

The data in Fig. 1 show that,

the 40 min of incubation of incubation

activity

of 0.84 is predicted

for the discrepancy

activity

skeletal

the observed

incubation

under the same incubation

0.78to

ratio

to the activity

assay of activity might

determined,

from rat and rabbit

1 an activity

to calculate

This extrapolated

parameters

assay conditions

Since

used for

all

Table

under standard

GSD:2 was approximately

of UDP-Glc.

synthase

From the SD.5 and Vmax valuesin

and absence of Glc-6-P

during

the activity (panel

conditions

as

the incubation ratio

for

GSD:2

B). Thus, the activity where

the activity

ratio

of GSI does not. The time-dependent rate of product

formation

decrease which

in activity

occursin

ratio

for GSD:2 results

the absence of Glc-6-P 1131

(Fig.

from the nonlinear 1, panel

B).

This

Vol. 134, No. 3, 1986

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

900

-

6OOm 3oos/p I IO

I 20

I 30

I 40

I 50

IO

20

30

1 40

I 50

MINUTES FJg&. Effectofincubationtime and enzyme concentration onthe activityratio ofrabbit muscle GSI (panel A) and ofrabbit muscle GSD:Z(panels B-D). In each panel,therate of product formation in the presence of 4.5 m M UDP-Glc and 8 m M Glc-6-P (upperline)is compared to the rate of product formation with 4.5 mM UDP-Glc and 0 Glc-6-P (lower line). The relative enzyme concentrations for panels A, 8, C and D are 130, 130,5, and 1 respectively. The activity ratios at40 min are 0.94,0.54,0.74 and 0.78 respectively.

nonlinear

rate

product

inhibition

Glc-6-P,

increased

the activity

of0.78

which

were

We wished absence

assay,

on the

ratio

was obtained obtained

formation

we determined activity

ratio.

at 40 min from

after40

Dilution

min of incubation concentration with

for GSD:P in the absence

When Glc-6-P

effect

formed

depends

of decreasing

of GSD:2

a value of 0.54 (panel

values and are consistent

the

the

to

in the absence of

of the enzyme of product

sensitivity

on the

the

assay

by a factor

B) to a value

of 26 of 0.74

of GSD:2 was further decreased 5-fold, an activity

as the enzyme

also to exclude

of Glc-6-P.

instability

Since the amount

When the concentration

with our calculated of product

of both.

in the

of GSD:2

C).

for GSD:2 could be due to an increased

in the absence of Glcd-P,

of enzyme

concentration

ratio

formation

or a combination

amount

(panel

of product

possibility

that

(panel

The higher

was reduced

of Glc-6-P

results

activity

ratios

are in good agreement

the hypothesis

that the nonlinear

rate

from productinhibition.

GSD:2 is unstable

was added to a control(0

1132

D).

Glc-6-P)

to incubation assay after

in the 40 min

Vol.

134,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

62500

1000

50000

800

37500

600

U

400

D p

P M

COMMUNICATIONS

P

hi

0 L ;

25000 I2500

12.5

25

37.5

50

62.5

MINUTES Fig& Comparison of the rate of product formation for rat muscle GSD:Linthe presence of 4.5 mM UDP-Glc and 800 uM Glc-6-P (upper line) or 4.5 m M UDP-Glc and 0 Glc-6-P (lower line). At 40 minutes (arrow), Glc-6-P was added to the 0 Glc-6-P incubation to achieve a final concentration of 800 pM. The concentration of UDP is calculated assuming 1 mol of U DP released per mol glucose incorporated into glycogen.

of reaction,the beginning

rate

increased

of the incubation

absence of Glc-6-P

these conditions whereas

(Fig. 2). This result

is not due to enzyme

The sensitivity U DP to reaction

immediatelytothatseen

containing

50% inhibition

Thus phosphorylation to UDP inhibition.

product

inhibition

200 uM UDP-Glc

of GSI required

only 8 uM UDP was required

demonstrates

_

0.6

r

0.2-

was tested

by adding

(Fig. 3).

Under

of either

rat or rabbit of glycogen

and U DP in skeletal

GSD:2. synthase

muscle have been

RAT

b

?&

20

in the

or 30 pM U DP (rat),

sensitivity

of U DP-Glc

directly

and no Glc-6-P

for 50% inhibition

RABBIT I.0

was added atthe

that the nonlinearity

50 uM U DP (rabbit)

by GSK-4 significantlyincreasesthe The concentrations

Glc-6-P

instability.

of GSI and GSD:2to mixtures

when

40

60

60

,;

12.5

25

37.5

50

62.5

UDP OJM) Figa Inhibition of GSI (upper lines) and GSD:E (lower lines) by UDP in the absence of Glc-6-P. Theconcentration of UDP-Glcinthese experiments was 200 pM. 1133

Vol.

134,

No. 3, 1986

BIOCHEMICAL

reported

as 30 pM (18) and 17 umol/kg

glycogen

synthase

prevailing activity

concentrations ratios

UDP (data

(4.5

inhibition

(Fig.

is responsible

for inhibition In preliminary

UDP-Glc.

Although

expected

apparent mechanism

UDP-Glc.

was clear

this

for a competitive

by UDP should experiments

of UDP-Glc

by the

used to determine

50% inhibition

of those formed

that

ratio

increased

25 uM

of GSD:P.

in the activity

sensitivity

synthesisinthe

to

absence

UDP

of Glc-

of GSD:E. muscle with

the effect to establish

could overcome

requires

glycogen respect

synthase

to the substrate

of UDP at varied the inhibition

the inhibition

(20),

levels

of

pattern,

it

by UDP, as would be

inhibitor.

the inhibitory

effect

m M) and did not identify

show that phosphorylation

inhibition

significant

to produce

be competitive

are needed

vivo,

inhibition

for skeletal

we tested

Therefore,in

measurable

the range

low activity

In 1976, Roach and Larner(21)showed

UDP (0.5

COMMUNICATIONS

conditions

of glycogen

reported

more experiments

that high levels

vivo increased

standard

we conclude

for the nonlinearrate

the kinetic

the kinetics

the

undergo

and 0 Glcd-P),

From

6-P, and consequentlyforthe From

Under

of UDP are within 2).

RESEARCH

wet weight(19),respectively.

and 200 uM UDP is required

concentrations

experiments

BIOPHYSICAL

at site 2islikelyto

of UDP.

m M U LIP-Glc

not shown)

These higher ratio

phosphorylated

AND

that

of UDP.

Thatstudytested

the phosphorylation

of site 2 resultsin

by U DP and furtherthatthisinhibition

phosphorylation

a marked

occurs

sites. increase

at physiological

of glycogen

synthasek

only one concentration The studies in sensitivity concentrations

reported

of here

to inhibition of U DP.

References

1.

Picton, C., Aitken, A.,Bilham,T.and Cohen, P. (1982) Eur. J. Biochem. 124,37-45. Sheorain, VS., Corbin, J.D. and Soderling T.R. (1985)J. Biol. Chem. 260, 1567-1572. 32: Kuret, J., Woodget, J.R. and Cohen, P. (1985) Eur. J. Biochem. 151, 39-48. 4. Soderling, T.R., Sheorain, V.S., and Ericsson, L.H. (1979) FEBS Letters, 106, 181184. D., Aitken, A., Donella-Deana, A., Hemmings, B.A., and 5. Cohen, P., Yellowlees, Parker, P.J. (1982) Eur. J. Biochem., 124, 21-35. Embi, N., Parker, P.J., and Cohen, P. (1981) Eur. J. Biochem., 115,405-413. ;: Juhl, H., Sheorain, V.S., Schworer, C-M., Jett, M.R. and Soderling, T.R. (1983) Arch. Biochem. Biophys., 222, 518-526. 8. Embi, N., Rylatt, D.B. and Cohen, P. (1979) Eur. J. Biochem., 100, 339-347. 9. DePaoli-Roach, A.A., Roach, P.J. and Larner, J. (1979) J. of Biol. Chem., 254,4212-

4219.

10. 11. 12.

Sheorain, VS., Juhl, H., Bass, M., and Soderling T.R. (1984) J. Biol. Chem. 259, 7024-7030. Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem., 124, 47-55. Hegazy, M., Brown, D.,Schlender, K., and Reimann, E. (1985) Fed. Proc.,44, 1073. 1134

Vol.

134,

No.

3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Schlender, K.K., Beebe, S.J., Willey, J., Lutz, S.A. and Reimann, E.M. (1980) Biochim. Biophys. Acta, 615, 324-340. W.G., Chrisman, T.D. and Exton, J.H. (1984) J. Biol. Chem. 14. Imazu, M., Strickland, 259, 1813-1821. 15. Thomas,J.A.,Schlender, K.K.,and Larner, J.A.(1968) Anal. Biochem., 25,486-499. .16. Brown, D.F., Reimann, E.M., and Schlender, K.K. (1980) Biochim. Biophys. Acta, 612,352-360. Cleland, W.W.(1967) Adv. Enzymol. 29,1-32. ii: Piras, R., Staneloni, R. (1969) Biochemistry 8, 2153-2160. 19. Weber, G., Burt, M.E., Jackson, R.C., Prajda, N., Lui, M.S. and Takeda, E. (1983) Cancer Res. 43, 1019-1023. 20. Salsas, E. and Larner, J.(1975)J. Biol. Chetn., 250, 3471-3475. 21. Roach, P.J. and Larner, J.(1976)J. Biol. Chem., 251, 1920-1925. 13.

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