Histone H1-stimulated phosphorylase phosphatase from rabbit skeletal muscle

BIOCHEMICAL

Vol. 117, No. 2, 1983 December 16, 1983

HISTONE HI-STIMULATED

PHOSPHORYLASE PHOSPHATASE FROM RABBIT SKELETAL MUSCLE

Ronald

L. Mellgren

Department

Received

October

12,

RESEARCH COMMUNICATIONS Pages 501-508

AND BIOPHYSICAL

and Keith

of Pharmacology Medical College Toledo, Ohio

K. Schlender

and Therapeutics of Ohio 43699

1983

A major rabbit skeletal muscle phosphorylase phosphatase activity which is markedly stimulated by histone Hl has been resolved from inhibitor-sensitive phosphorylase phosphatase (type-l phosphatase), glycogen synthase kinase 3activated phosphatase, phosphatase heat-stable inhibitor proteins, and alkaline phosphatase activity by various purification techniques. Evidence is presented that this phosphatase is a high-molecular weight form of a type-2 phosphatase. Our data suggest that this phosphatase may be regulated by histone Hl, protamine or analogous polycationic compounds. Cohen major

and his

colleagues

phosphorylase

type-2

phosphatases

inhibitor Type-2

proteins

inhibited rabbit

by the renal

heat-stable

(1).

phosphatases

cortical

activated

molecular

weight

sensitive

from

Hl (5). activity

by histone form

phosphatase

Hl.

of type-2

(4).

This

preparation

inhibition

phosphatase

appears

are not that

was activated we found

we report muscle

by ATP.

reported

Subsequently,

skeletal

and

by heat-stable

We recently

communication,

in rabbit

to

of the

as type-l

by 0.1 mM ATP but

proteins.

In this

tissues

insensitive

same source

subunits

can be inhibited

to inhibition

inhibitor

the

the catalytic mammalian

and are relatively

phosphorylase

phosphatase

markedly

Type-l

heat-stable

was histone

phosphorylase

in various

are

protein

activator

phosphatases

(2,3)

phosphatases

have classified

that extracts

a by a

that

the

a major is

to be a high

phosphatase.

MATERIALS

AND METHODS

Materials: DEAE-Cellulose 52 (Whatman), DEAE-Sepharose CL-6B (Pharmacia), bovine liver catalase, cadaverine dihydrochloride, putrescine dihydrochloride, spermine tetrahydrochloride, spermidine trihydrochloride, protamine base, and disodium ATP were obtained from Sigma. Bio-Gel A-1.5m was purchased from Bio Rad. Mixed histones were obtained from Aldrich. Calf thyrnus histones H2b and H4, and horse spleen ferritin were obtained from Boehringer Mannheim. Buffers: Column chromatography was routinely performed utilizing buffer A: mmidazole-HCl, 0.2 mM EGTA, 2 mM dithiothreitol, pH 7.4. Buffer B was the same as buffer A, except that it contained 5 mM EGTA.

501

0006-291 X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol. 117, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Enzymes and other proteins: Phosphorylase 2 was prepared from crystallized phosphorylase b and y-j/P-ATP (6). Rabbit muscle protein phosphatase inhibitor-l was prepared to apparent homogeneity by a modification of the procedure in (7). Core histones were prepared from mixed histone as previously described (5). Histone Hl was prepared from calf thymus by the method of Sanders (8). Rabbit muscle protein phosphatase-1 catalytic subunit (type I phosphatase) was purified to near-homogeneity by a procedure which will be described in detail elsewhere (R. Mellgren, manuscript in preparation). The purification involved batch chromatography on DEAE-cellulose (0.1-0.4 M NaCl fraction) followed by Bio-Gel A-1.5m, Phenyl-Sepharose and Sephadex G-200 chromatography steps. The catalytic subunit had a molecular weight of 31,000. It could be inhibited >90% by inhibitor-l, but not by 0.1 mM ATP. Hl-stimulated phosphatase was obtained as a side-product in this protocol. In contrast to phosphatase-1 catalytic subunit, which was eluted from the Phenyl-Sepharose column by a combined decreasing ammonium sulfate (1 M to 0 M) Hl-stimulated phosphatase remained and increasing dioxane (0 to 10%) gradient, This urea fraction was extensively on the column and was eluted with 4 M urea. dialyzed and stored at -20°C in the presence of buffer A containing 50% glycerol, 10 mM benzamidine, 1 pg/ml pepstatin A and 10 pg/ml leupeptin. Glycogen synthase kinase 3 was a generous gift from Dr. T. Soderling of Vanderbilt University. Analytical methods: -Phosphorylase phosphatase was assayed as previously described (6). When histone Hl was added. it was oresent at a concentration of 7.2 pg/ml unless otherwise stated. A unit of phosphorylase phosphatase produces one nMole of phosphorylase b dimer per minute. Heat-stable phosphatase inhibitor was assayed as previously described (9). A unit of inhibitor activity produces a 10% inhibition of 1 milliunit of phosphatase-1 catalytic subunit. RESULTS Preparation 10,000 major

of a histone

x g supernatant regions

from

peaks

I,

II,

phosphatase inhibitory (apparent

(Fig.

and III

phosphorylase III

phosphatase

was well

activity.

The concentrated activity

peak

III

had a Stokes (not

be partially

resolved

fraction

approximately from

a major

as a 5.5 nm form

the same degree DEAE-cellulose 502

to as

in the inhibited

while

The Hl-stimulated phosphatase radius

of 5.5 nm

shown). from

chromatography

contained

chromatographed

phosphatase

heat-stable

by Phenyl-Sepharose

urea

1A).

chromatography

could

activity

which

1B) and displayed

Hl as the

A-1.5m

phosphatase

Methods).

the

phosphatase

be referred

was fully

(Fig.

from

will

three

at 120 mM, 170 mM,

Hl was included

several-fold

III

on Bio-Gel

phosphatase

peaks

of a

revealed

eluting

I phosphatase

separated

The peak

Mr 260,000)

activity

peak

was activated

activity

activity

When histone

assay,

Hl-inhibited

(Fig.

These

Chromatography

on DEAE-cellulose

phosphatase

1A).

phosphatase

phosphatase: muscle

respectively.

The Hl-stimulated

phosphatase

rabbit

of phosphorylase

and 260 mM chloride

peak

Hl-stimulated

(see

Hl-stimulated on Bio-Gel

of stimulation

chromatography.

A-1.5m by histone The urea

Vol. 117, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

3.0 F * .Z c 3

0.6

i

60

2.5

20

80

Fraction

30

40

Fraction

Number

50

60

Number

Figure 1. A. DEAE-Cellulose chromatography of a rabbit muscle low-speed supernatant. Ten grams of fresh rabbit skeletal muscle was homogenized in 20 ml of ice-cold buffer B containing 250 mM sucrose and centrifuged for 20 min at 10,000 x g at 4°C. A 15 ml sample was applied to a 1.6 x 10 cm column of DEAE-cellulose equilibrated in buffer 6 and washed successively with one column volume of buffer B and three column volumes of buffer B containing 0.10 M NaCl. A linear gradient of 0.10-0.60 M NaCl in 200 ml of buffer B was applied and 2 ml fractions were collected. The fractions were assayed for phosphorylase phosphatase activity in the absence (a) or presence (0) of 7.2 pg/ml of histone Hl. Aliquots of the fractions were heated at 100°C for 5 min, centrifuged to remove denatured protein, and assayed for phosphatase inhibitor activity (A). B. Bio-Gel A-1.5m chromatography of Phenyl-Sepharose purified HI-stimulated phosphatase. A 0.5 ml sample of the concentrated urea fraction from Phenyl-Sepharose chromatography of a rabbit muscle phosphatase preparation (see Methods) was applied to a Sephadex G-25 column in buffer A containing 0.10 M NaCl to remove the glycerol. The sample in a volume of ca. 0.7 ml was applied to a 1.0 x 45 cm column of Bio-Gel A-1.5-n equilibrated 5th buffer A containing 0.10 M NaCl. Fractions of 0.64 ml were collected and assayed for phosphorylase phosphatase activity in the absence (m) or presence (0) of 7.2 ug/ml of histone Hl. Protein concentrations (V) were determined by the method of Bradford (17). The marker proteins ferritin (F, 5.85 nm Stokes radius) and catalase (C, 5.15 nm Stokes radius) were chromatographed separately.

fraction The of

contained most

the

used

active urea

fraction

the

The

Hl-stimulated at

5"C,

activity

the

time.

same

Hl

for

heat-stable

pooled.

Thus,

less

activity

.

activity

than in

with

where

from noted

this

inhibitor

activity.

Bio-Gel

chromatography

pooled

fraction

stable

during

was

below.

phosphatase

measured

fractions

Except

described

losing

phosphatase

phosphatase

were

studies

However,

detectable

Hl-stimulated

for

storage

histone

no

the

10% of

its

absence

ageing

the

Ageing

of

was original of

histone

preparation a phosphatase 503

relatively activity Hl

after

decreased

became preparation

more

14 by

dependent while

days.

81%

over on

bound

to

Vol. 117, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

0.6

0.5

,’

0.4

; 5 I

0.3

.g

0.2

‘0 i 0

0.1 . AA 0

10

20

30

40

Fraction

50

60

Number

Figure 2. DEAE-Sepharose chromatography of an aged preparation of rabbit muscle phosphatase. A rabbit skeletal muscle extract, prepared as described in Figure 1, was applied to a DEAE-cellulose column and washed with 3 to 4 bed volumes of Buffer A containing 0.1 M NaCl. The column was left at 2-5'C for 7 days, and then eluted with Buffer A containing 0.4 M NaCl. The pooled protein peak was concentrated by dialysis against a 20% solution of polyethylene glycol (Mr 20,000) in buffer A and chromatographed on a Bio-Gel A-1.5m column. The tubes eluting at ca. 5.5 nm Stokes radius were pooled, dialyzed against buffer A containing 50% glycerol, and stored at -20°C. A 60 ml sample was dialyzed against buffer A for several hours to remove glycerol. The dialyzed sample containing 34 units of Hl-stimulatable phosphatase was applied to a 2.6 x 10 cm column of DEAE-Sepharose equilibrated in buffer A. Phosphatase activities were eluted with a linear gradient of 0 to 0.50 M NaCl in 500 ml of buffer A. Fractions of 5.5 ml were collected and assayed for phosphorylase phosphatase activity in the presence of histone Hl (WI. Samples of the fractions were incubated with 1 mM Mg-GTP and glycogen synthase kinase 3 at 30°C for 30 min. These samples were then assayed for phosphorylase phosphatase in the absence of added histone Hl (A). The fractions were also assayed for alkaline phosphatase activity (0) by the method of ii et al. (18). There was no detectable phosphorylase phosphatase activity iii-any of the fractions nor in the sample applied to the column, when assayed without preincubation with GTP and synthase kinase, or in the absence of histone Hl.

DEAE-cellulose of

resulted

in

this

preparation

on

glycogen

synthase

kinase

phosphatase its

high

activities

Hl-stimulated

31.

The

the

on histone

phosphatase enzyme

sample

used

21.

This

gel

filtration

maximum in

the

aged

this

experiment

504

Hl-dependent

phosphatase (not

activity

Chromatography

(10,111,

phosphatase

Hl-stimulated

had

phosphatase.

resolved

3-modulated

weight of

Hl-dependent

DEAE-Sepharose

(Fig.

molecular

Characterization

a totally

activity and

from

alkaline

preparation

retained

shown).

phosphatase: at had

4-8 aged

The pg

histone for

several

Hi/ml

(Fig. days.

Vol. 117, No. 2, 1983

BIOCHEMICAL

0

AND BIOPHYSICAL

4

8

12

Histone

Figure

3.

muscle rabbit

HI-stimulated phosphatase muscle phosphatase-1 (A).

Thus,

the

extent

experiments

of histone

of activation

presented

inhibited could

The effect

be completely

whether

the phosphatase

histone

Hl.

In the

Hl concentration (0)

1.

and

rabbit

was assayed

muscle

alone

of histone

of Mn2+ effectively

Bio-Gel

converted

A-1.5m

phosphatase

was activated by inhibitor-l.

protamine

as well

either

very

phosphatase In

contrast,

low concentrations

the ability

of inhibitor-l

or had no significant activities purified

by polyamines rabbit

of histone to inhibit

muscle

Hl (Fig. this 505

form

phosphatase.

of 0.2 by

be activated

and the

by

polyamines

of elsewhere

was inhibited Hl did

M

was not

has been reported

Histone

of

of the

and it

Inhibition

phosphatase-1 3).

presence

could

histones

effect.

30% to

as determined

by ATP,

Other

ug/ml

Hl activation

in the

phosphatase

I).

obtained

of 4.0

histone

weight

inhibited

The Hl-stimulated Hl (Table

were

it

Ca2+ or Mg2+.

to a 2.3 nm form

Hl,

was not

was activated

with

the phosphatase

in the

However,

in the presence

reversed

of

observed

inhibitor-l/ml.

low molecular

by histone

than

phosphatase

observed

phosphatase This

as histone

inhibitory

phosphorylase (12,131.

the

chromatography.

inhibited

were

and thawing

of rabbit subunit

The same results

or

was not

The same concentrations

mercaptoethanol

activities catalytic

Hl the phosphatase

Activation

Freezing

purified

Hl was greater

50% by 1 to 5 mM Mn2+.

the phosphatase.

on the

the

by 0.1 mM ATP.

absence

20

The Hl-stimulated

of purified

inhibited

16

pg/ml

by histone

in Fig.

by 100 units

Hl,

RESEARCH COMMUNICATIONS

not

by

influence

Vol. 117, No. 2, 1983 Table I: mulated

BIOCHEMICAL

AND BIOPHYSICAL

The Effect of Various Histones, Phosphorylase Phosphatase.

Addition

RESEARCH COMMUNICATIONS

Protamine,

Concentration w/ml

and Polyamines Relative

None

on the

Phosphatase Activity (100)

Histone

Hl

4

Histone

H2b

670

4:

10';

404

;i

Core Histones

4 40

103 65

Mixed Histone

4 40

163 86

Protamine

4 50

222 30

Histone

H4

Spermine

4 101 (0.5 mM)

65 51

7; (0.5 mM)

!?I

Cadaverine

4 51 (0.5 mM)

103 86

Putrescine

4 44 (0.5 mM)

94 84

Spermidine

DISCUSSION The rabbit laboratories several

(4,5)

phosphatase

to characterize which

phosphatase

is

stimulated

did

activation

by histone

heat-stable

inhibitor.

inhibitor-l,

reverse

in earlier

studies

impure

preparation

which

activities.

here

inhibition

and by the

This

Hl.

by high

resolution

appear

to be the result by the

studies

activity

in rabbit

by low

Thus,

of reversal

observation

Hl-stimulated

were

of inhibitor-l.

activity.

phosphatase-1

of the

may have contained

Hl-stimulated

concentrations

inhibitor

of purified

our

by Mn 2+ , inhibited

heat-stable

was confirmed

from

present

phosphatase

slightly

inhibited

not

the

The histone

was activated

not contain Hl did

Therefore,

phosphorylase

by histone

of ATP, and not

The phosphatase

employed

the major

described

concentrations

does not

phosphatase

was a relatively

different

designed muscle

renal

catalytic

that

the of a

histone

subunit

phosphatase

from

Hl by

Vol. 117, No. 2, 1983

glycogen

synthase

BlOCHEMlCAt

kinase

inhibitor-2

complex

phosphatase

is

a type-2

et al.

other

phosphatases

type-2

phosphatase

Hl,

in rabbit

muscle

Hl appeared

phosphatase histone

phosphatase phosphatase-1

characterized

but

The latter

enzyme muscle

phosphatases

phosphorylase

appears

enzyme.

exist

which

(Fig.

(Fig.

to be very

may represent

activity

rabbit

amount

Hl was a potent

present

studies, renal

preparation

a small

a new

of histone

In our earlier

this

classic

of the

in the presence

an inhibitor-sensitive

cortex

and find

that

of type-2 inhibitor

of purified

3). study,

phosphatase

Thus,

and it

1A).

activate

as the

classification

phosphatase

reinvestigated

than

may be a mixture

exact

At any rate,

independent

an Hl-stimulated

the

to be established,

Histone

subunit

The latter

Thus,

revealed

preparation.

In a simultaneous

skeletal

is

(15).

(1).

of latent

catalytic

scheme of

radius

had the

:

the Hl-stimulated

same Stokes

It

to dramatically

in the

that

(14).

to activate

Hl appears

suggest

was larger

We have since

(4).

results

phosphatase

in the classification

phosphatase.

extracts

(a type-l

phosphatase

remains

amount

RESEARCH COMMUNICATIONS

The Hl-stimulated

phosphatases

protein

a substantial

These

of Lee -et --al

phosphatase

of type-2

histone

(1).

and type-2

Hl-stimulated

11).

phosphatase

phosphatase

H holoenzyme

of type-l

form

3-activated

- ref.

Ingebritsen,

AND BIOPHYSICAL

DiSalvo

from

similar

bovine

muscle

(16)

vascular

in properties

in two different

may be regulated

et al.

have smooth

to the tissues

by polycationic

muscle.

rabbit

latent

macromolecules.

ACKNOWLEDGEMENTS This work was supported by USPHS grants AM 28277 and AM 14723. The authors thank Scott Jakes for purifying the histone Hl and core histones used in this study, The excellent technical assistance of Susan Wilson, Maura Mericle, and Tere Lampathakis is also acknowledged. REFERENCES 1.

2. 3. 4. 5. 6.

Ingebritsen, 9-15. Brandt, H., Res. Commun. Huang, F.L., Wilson, S.E.,

T.S.,

Foulkes,

J.G.,

and Cohen,

P. (1980)

Lee, E.Y.C., and Killilea, S.D. (1975) 63, 950-956. and Glinsmann, W.H. (1976) FEBS Lett. Mellgren, R.L., and Schlender, K.K.

FEBS Lett.

Biochem.

119,

Biophys.

6J, 326-329. (1982) FEBS Lett.

146, 331-334. Wilson, S.E., Mellgren, R.L., and Schlender, K.K. (1983) Biochem. Biophys. Res. Commun. - in press. Killilea, S-D., Aylward, J.H., Mellgren, R-L., and Lee, E.Y.C. (1978) Arch. Biochem. Biophys. 191, 638-646.

507

Vol. 117, No. 2, 1983

i: 9. 10. 11. 12. 13. ::: 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Foulkes, J.G., and Cohen, P. (19791 Eur. J. Biochem. 97, 251-256. Sanders, C. (1977) Mellgren, R.L., 8, 27-37. Vandenheede, J.R., Yang, S.D., Goris, J., and Merlevede, W. (1980) 23 Biol. Chem. 255, 11768-11774. Hemmings, B.A., Resink, T.J., and Cohen, P. (1982) FEBS Lett. 150, 319-324. Killilea, S.D., Mellgren, R.L., Aylward, J.H., and Lee, E.Y.C. (1978) Biochem. Biophys. Res. Commun. g, 1040-1046. Nakai, C., and Glinsmann, W. (1977) Mol. Cell. Biochem. fi, 141-143. Ingebritsen, T.S., and Cohen, P. (1983) Science 221, 331-338. Lee, E.Y.C., Mellgren, R.L., Killilea, S.D., and Aylward, J.H. (1978) FEBS Symposia 42, 327-346. DiSalvo, J., Waelkens, E., Gifford, D., Goris, J., and Merlevede, W. (1983) Biochem. Biophys. Res. Commun. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Li, H.-C., Hsiao, K.-J., and Sampathkumar, S. (1979) J. Biol. Chem. 254, 3368-3374.

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