Isolation and characterization of two 70 kDa modulator-complexes from rabbit skeletal muscle

BIOCHEMICAL

Vol. 135, No. 2, 1986

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 367-373

March 13, 1986

IBOLATIONANDCBARACTERIZATION OF TWO70 kDa MODULATOR-CORPLEXEB FROMRABBIT BXELETALMUSCLE Jackie R.Vandenheede*, Carline Vanden

Afdeling Received

Biochemie,

January

21,

Abeele and Wilfried

Faculteit Geneeskunde, Katholieke Leuven, B-30&7 Belgium

Merlevede

Universiteit

1986

The activation as well as the inactivation of the ATP,Mg-dependent protein phosphatase has been shown to be totally dependent upon the presence of the modulator subunit. This modulator (inhibitor-21 is a heat stable protein and its isolation in pure form (32 kDa) always includes a boilin& step. The boiled modulator fractions are known to be inhibitory to the phosphatase activity. Unboiled rabbit skeletal muscle preparations do not contain “free modulator”, but two higher molecular weight complexes (70 kDa) can be isolated One complex is which have the 32 kDa modulator toeether with a 38 kDa protein. the already characterized inactive ATP,Mg-dependent phosphatase [FCM] while the second one, HI, although seemingly of identical composition, does not exhibit phosphatase activity when measured under the usual conditions. The RX-complex does not inhibit the phosphatase activity unless subjected to a boiling step which dissociates the modulator subunit. The unboiled [MXI exhibits the activation as well as the inactivation characteristics of the free modulator. m1986 Academic

Press,

Inc.

The heat stable modulator [Ml represents a very unit of

the inactive

ATP,Mg-dependent phosphatase [F,$lI

[Ml

FA unless exogeneous modulator is added (1).

by kinase FA is a necessary step in the activation

and the

heat-stable

protein

The phosphorylation

of

of the phosphatase (4-6)

ATP,Mg-dependent form (l-4,7).

ATP,Mg-dependent phosphatase does not result

modulator from the catalytic

(l-3).

is also responsible for the conversion of the ac-

tive phosphatase to the inactive of the

sub-

An inactive FCn) cannot be activated

phosphatase which does not have modulator bound (“free by kinase

regulatory

important

The activation

in the dissociation

subunit (5) and we have recently

of the

reported that un-

boiled muscle preparations do not contain free modulator (2). This would suggest that at all times, the modulator is a constituent of higher mol. wt. complexes.

It copurifies

constitutes gen particle

with the inactive

a regulatory which only

modulator activity

subunit,

ATP,Mg-dependent phosphatase, where it

and it is also present in the isolated glyco-

contains active

phosphatase (7).

is associated with heat-labile

kDa complex in sucrose density gradients

(2,7).

In both cases the proteins and migrates as a 70 Isolation

of pure modulator

*To whomcorrespondence may be adressed. 0006-291X/86 367

$1.50

Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 135, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL

always requires

a dissociating

potent inhibitor

of the phosphatase activity.

it as the heat-stable 70 kDa heat-labile

“inhibitor-2”

unit.

phosphatase to

This property originally

tunboiled)

its inactive

nase FA-mediated activation

is a

labeled

The present report characterizes

(10).

and is not

preparation reveals

The intact

step (8.9) and the boiled protein

modulator complex [Mxl which has little

dent phosphatase activity Boiling the

boiling

RESEARCH COMMUNICATIONS

inhibitory

the inhibitory

to

or no ATP,Mg-depen-

the active

capacity

a

phosphatase.

of its modulator sub-

RX-complex is however able to convert the active ATP,Mg-dependent form, and also stimulates the ki-

of a “free FC” enzyme.

MATERIALSANDMETHODS Materials and methods were as described in previous reports (1,5,6,12). The activity of the inactive ATP,Mg-dependent phosphatase was commonly measured after a 10 min preincubation at 30°C with 0.2 mI4ATP, 1 mM Mg2+ and the minimal amount of kinase FA required for full activation under the conditions used. The phosphatase activity unit is defined as the amount of enzyme which releases 1 nmol of [32Pl phosphate per min at 30°C from 32P-labeled phosphorylase a (2 me/ml). The assay time was 5 min unless mentioned otherwise. The preparation of “free PC” (1) had a specific activity of approximately 15,000 units/me when measured in the presence of optimal concentrations of modulator (1,5). In the absence of exogeneously added modulator, only 1,200 units of phosphorylase phosphatase were measured after activation by kinase FA and AP,Mg. The heat-stable modulator was purified to homogeneity from rabbit skeletal muscle as described in (6). The modulator activity was determined by measuring the increase in the kinase FA mediated activation of “free FC”(5.6): 5OmlJ“free FC“was usually preincubated at 30°C for 10 min with kinase FAATP,Mg and modulator and the phosphorylase phosphatase activity produced was measured in a 5 min assay. The active phosphatase catalytic subunit was isolated from rabbit skeletal muscle essentially as described in (11). It had a specific activity of about 50,000 U/w as phosphorylase phosphatase, and could be completely converted to the ATP,Mg-dependent form by preincubation with modulator. 10% Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate (SDBPA6E) was performed according to (13) using the Idea 8cientific mini-slab. Marker proteins were myosin (210 kDa), phosphorylase b (95 kDa), bovine serum albumine (68 kDa), ovalbumin (43 kDa) and a-chymotrypsinogen (27 kDa). (17 hrs, 2V, 48,000 rpm) were Sucrose density gradient centrifugations performed using a Beckman BW50.1 rotor with 14C-labeled ovalbumin as marker protein (43 kDa). Approximate mol. wts were calculated according to (16).

RESULTSAND DISCUSSION 1) )I

- n of Extracts

t ions were prepared from 1.5 kg of rabbit

skeletal

muscle as described

in (12), all buffers containing the proteinase inhibitors and EGTAthroughout the isolation procedure. The whole extract (pH 7.0) was adsorbed batchwise onto 25 g of

DEAE-SephadexA-50, equilibrated 368

in buffer

A

120 mMTris,

1 mM di-

Vol. 135, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS 0.4

8 %

a2 0.1

0

20

if Fig.

1.

60

FRACTION

M NaCl in buffer

proteins were then eluted

from the

0.2 M to 0.4 M NaCl in buffer the modulator

activity

A.

A before packing the column.

The

column with a 2x500 ml salt gradient from

(measured after

50% saturation).

(12)l and the resin was ex-

The ATP,Mg-dependent phosphatase activity boiling

coeluted and were pooled and concentrated (at

13

NUMBER

1 mMEGTA and proteinase inhibitors washed with 0.2

sulfate

0

60

Phenyl SephoroseU3 chromatography:gradient elution started at fraction one; ATP,lQ-dependentphosphataseactivity (01 and modulator activity to) measuredas stimulation of free FC-activation by kinase FA (5). protein concentrations were measuredby the absorption at 260 rxnand are indicated by the dotted line.

thiothreitol, tensively

LO

by

The precipitated

buffer A and the solution was made 1

M

the

individual

precipitation proteins

and

fractions)

with

awnonium

were redissolved in

in ammonium sulfate.

After

a clarifying

spin (10 min at 10,000 x g), the solution was adsorbed onto a column of phenylSepharose 48 (1.5~20 The proteins

were

cm) equilibrated

then

eluted with

containinS 1 M ammoniumsulfate elution

profile

tor activity

buffer A plus 1 M ammoniumsulfate.

a 2x200 ml linear gradient

to buffer

is shown in Fig. 1.

A

from buffer

containing 20% ethyleneglycol.

Two partially

A

The

overlapping peaks of modula-

were observed, only one of them corresponding to the ATP,Mg-depen-

dent phosphatase activity. separated from the activitybar (Fig.

in

1) the

conductivity

The vast majority of the other proteins were well fractions. After pooling as indicated by the solid

solution was dialyzed

of 0.1 M NaCl in buffer

until A.

the conductivity

The dialyzed

dropped below the

proteins were then passed

over a column of Blue Sepharose CL-68 (0.9~10 cm) onto a poly-L-lysine Sepharose column (0.9~10 cm). After applying the sample, the two columns were processed individually. The Blue Sepharose column was washed with 0.1 M NaCl in bound modulator

was then

ATP,Mg-dependent phosphatase

eluted with activity

0.2 M NaCl in

buffer

buffer

A.

was found in these fractions

A,

and the

Virtually

no

(Table I).

Roughly 40% of the total modulator activity present in the phenyl-Sepharose fraction was retained by the Blue Sepharose resin: this corresponds to the modulator

which

eluted ahead of the ATP,Mg-dependent phosphatase activity 369

in the

Vol. 135, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table purification

of the heat-labile

I

modulator-complexes

Fraction

protein (me)

DEAE-Sephadex A-50 Phenyl-Sepharose 45 Blue Sepharose CL-68 (0.2 M NaCl eluate fraction) Poly-L-lysine Sepharose 48

390 8.5 0.2-0.4

(') Phosphorylase and ATP,m

activity

Modulator’ activity (Units x 10V3)

0.8

phosphatase

seemed to be complexed in

known not the

to bind

to the

poly-L-lysine

NaCl in

buffer

by dialysis

muscle

Phosphatase( ’ ) activity (Units x 10m3)

55 60 0.2-l .o

62

34

measured after

activation

by kinase

FA

1).

The rest of the modulator ac-

the inactive

phosphatase as [FCM], which is

Blue Sepharose resin (4,12).

resin with

skeletal

300 180 10-50

phenyl-Sepharose chromatography step (Fie. tivity

from rabbit

a linear gradient

(2x100 ml) from 0.2 M to 0.4 M

A (not shown). The two fractions

against 10% polyethyleneglycol

It was recovered from

were concentrated separately

in buffer

A, and stored at -2OV in

the presence of 50%glycerol. The ATP,Mg-dependent phosphatase isolated known aspect

from the

protein staining contained the catalytic

one isolated

activity,

38 kDa and 32 kDa proteins

two

discussed in (6).

so that at 1:l

molar ratio

The modulator

showedthe

same

suggest the

fraction

by kinase the

which

FA nor identity

modulator

fraction

represents

the

activity

by metal

ions

of

the two

which binds 38 kDa component

mainly

which represent

the

The enzyme seemedto

of the

ratio

phosphatase activity

could raise the phosphatase unit

is expected,

is indicated by the Coomassie activity

by kinase FA was about 40,000 U/me.

was retained

by the

Blue

bands on SDS-PAGE(Fig.

presence of the 38 kDa catalytic

phosphorylase phosphatase Until

2A),

of modulator to catalytic

activation

no

than ATP,Mg-dependent phosphatase

with trypsin

protein staining

two

The preparation

of the two proteins on SDS-PAGE. The specific

enzyme after

in

(12) except that the 62 kDa

(14,15).

inhibition

A similar

measurements.

blue staining

of the inactive

partial

Limited proteolysis

based on activity brilliant

the

(Fig.

respectively

more modulator activity

times

which reflects

activity

according to

way differed

band seen in SDS-PAGE(5) was absent.

and modulator subunits

have about

in this

subunit.

However,

Sepharose

resin

which may no significant

2A),

could be brought out of the preparation,

with

or without

38 kDa

an additional

proteins is proven,

trypsin

not

treatment.

we shall label the

to the Blue Sepharose resin as HI where [Xl of the modulator complex under investigation. 370

Vol. 135, No. 2, 1986

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

lane

1

2

3

FRACTION NUMBER Fig.

2.

SDS-PAS8 of 2 jog of modulator purified as in (5) (lane FCM-enzyme (lane 2) and 4 JIB of [MXI (lane 3) at the final purification (see text). A:

B and C: Sucrose density gradient centrifugatfon: (8) or boiled [MX] (C) were put on the gradient bumin as ml. wt. marker.

1);

4 fig

stages of

2 U of "native" MXI together with 14C-oval-

2) Ch ) Fig. 28 shows the migration of the MX-complex in sucrose density gradient centrifugation.

The modulator activity,

measured as stimulation

of the kinase

FA mediated “free FC” activity (5), was determined after boiling the individual fractions of the gradient and was localized several fractions ahead of the marker protein Boiling the

14C-ovalbumin, indicating

MX-complex

prior

of the usual “free modulator” (Fig. Gel filtration for the wt. of protein identical

native 65,000

known kinetic

This is

dependency of

the

gave an apparent

The modulator

protein

and [FCMI migrates

obtained

after

illustrated

added [MXI or boiled [Mxl.

wt.

in

of 140,000

as a 120-140 boiling

kDa

EMXI was (B,9) in all

The unboiled MX-complex however differed

modulator in that it was not inhibitory kinase

mol.

according to the published reports

aspects.

of 70,000.

in the appearance

Free modulator is known to have a mol.

columns

or molecular

from the heat-stable activity.

AcA-34

sieving

the one purified

mol. wt.

resulted

2C) with a mol. wt. of 17,000.

MX-complex (not shown).

(2,3,B). to

using Ultrogel

in molecular

an approximate

to the centrifugation

Figure 3.

to the phosphatase

Fig. 3A shows the concentration

FA mediated activation

of “free FC” on exogeneously

The boiled MX-preparation shows the biphasic tstim371

Vol. 135, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

3.E @ 50 z 2 B=T” w 25 8 0I:0 8Y

5

10-2zlo

\ . 0

MX(ng)

2

Fig. 3. Determination

of the different

5

0'

10 50 loo MX(ng)

modulator

10

20

TIMEImin)

activities.

stimulation of the kinase FA-mediatedactivation of 50 mU of “free FC” 2.5 to 100 ng of native [MXI to) or boiled [Hxl (0) were addedin the activation mixture (20 ~1) of “free FC”, kinase FA and ATP,Mg as outlined in (5). Concentrations indicate the wount of protein present before the boiling step. A:

Inhibition of 50 mU of active phosphatasecatalytic subunit by different amountsof u&oiled to) and boiled (0) [XXI. The DlXI proteins were not preincubated with the phosphataseand a 2 min assay was performed. B:

C: Time-dependentinactivation of 50 mU of active phosphatasecatalytic subunit by 10 ng of unboiled to) or boiled (0) NXI. The [MXI and phosphatasewere preincubated at 3OW for different periods of time, before a 2 min assaywasperformed. ulation-inhibition)

curve,

typical

of

the free modulator

Cl),

whereas

the

na-

tive MX-complex does not produce the phosphatase inhibition at the higher concentrations used. Similarly, [Hxl did not inhibit the activity of the phosphatase catalytic

subunit, whereas identical

an instantaneous inhibition able to

convert the

(Fig. 3B).

concentrations

Surprisingly,

active phosphatase catalytic

of boiled [KU] caused

the native MX-complex was subunit to the ATP,Mg-depen-

dent enzyme form at the samerate as the boiled modulator (Fig. 3C). plest explanation

for these results

is to invoke two separate binding sites for

modulator on the phosphatase catalytic versible

activation/inactivation

subunit: one site which governs the re-

of the phosphatase, and a separate inhibitory

site which can only be occupied by “free modulator”, ical MX-complex.

In vivo this putative

served for the regulation lated) inhibitor-l. In this site similar identical to

The sim-

of the

hypothesis, the

inhibitor

not by the more physiologsite

may

be specifically

phosphatase by the heat-stable

X-protein

re-

(phosphory-

bind the modulator at a specific If [Xl would be to the “inhibitory site” on the phosphatase. the FC-protein, then we could envisage the MX-complex as an FCMmay

enzyme which has modulator bound at the inhibitory site, instead of at the usuThe portion of the modulator which binds to al activation/inactivation site. the activation/inactivation site on the phosphatase may be fully exposed in the native [MXI, so that the interconverting properties of the modulator can be measured without a dissociating

boiling

step. 372

Vol.

135,

No. 2, 1986

BIOCHEMICAL

[MX] differs be revealed is

Both

from the normal [FCM] in that

proteolysis

retained

by

characteristics

sites

between

in the presence

argue

This is however

cogen particles

which

ated with

them,

modulator

activity

ulator-complex described

but

the

its

potential

such as activation

of metal ions

that

highly

FC-protein [Xl

in the two

may

simply

unlikely

since

have a considerable no inactive

(15)

COMMUNICATIONS

activity

cannot

by kinase

FA (5)

and secondly

because

found to

form (7).

be identical

complexes.

represent

a denatured

preparations amount of active do contain

F,$-enzyme,

in a 70 kDa

were

RESEARCH

the Blue-Sepharose resin as is also the free modulator (8). be explained by the difference in the binding may again

the M- and

One could unit.

BIOPHYSICAL

by any of the known procedures

or limited it

AND

The properties

FC-catalytic

of rabbit

muscle gly-

phosphatase an

associ-

equal amount

of this

to the characteristics

of

70 kDa modof the [MX]

here (not shown).

ACKNOWLEDGEMENT8 JRV is a Senior Research Associate of the “Nationaal Fords voor Wetenschappel i jk Onderzoek”. This work was supported by the “Ponds voor Geneeskundig Vetenschappelijk Ondmmek” and by the “Onderzoeksfonds K.U.Leuven”. The authors are grateful to Mr L. Vanden Bosch for expert technical assistance.

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