Vat.

176,

No.

May

15, 1991

BIOCHEMICAL

3, 199’1

AND

ACID

BLOCKS

MEMBRANE

FUSION

MYOBLASTS

Hye

Sun

Kim,

Department

Chin

of

Ha

of

IN

Chung

Molecular

College

Received

March

30,

SUMMARY:

Biology

Natural

embryonic

myoblasts bipolar

from

could be including

tropomyosin cell

certain,

may

It

removal

050

EMBRYONIC

for

Doo

Ceil

Bong

Ha

Differentiation, University,

Korea

also

of on

drug

protein(s)

from

of

the

inhibition

by

the

that

regulate

the

the

differentiation

culture

muscle

fusion

the and

medium.

was

treated

to

significantly

myoblast

the

It proteins

proteins of

in

of

specific acid

many

increase

chick

change

okadaic

state

of

dose-dependent,

the

of

When

that

mediated

were

induction

kinase.

fusion

morphological

effects

the

the

suggest

membrane

induced

phosphorylation

be

block

These

creatine

results

unknown

and

National

to

shape.

effect

the

acid

SRC

Seoul

found

culture.

and

These

okadaic

CHICK

Kang

15 l-742,

was

spherical upon

lysates,

increased.

fusion

by

phosphorylation

of

0 1991 Academic

process.

Inc.

marked

A fusion

of

Concurrent

event

such

synthesized

the

morphological

as

actin,

(5-7). be

dramatically

IThis work Foundation whom

The

was and all

myosin,

activities altered

correspondence

an

both

protein

as

by of

the

grants Education. should

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in aq form reserved.

a large

number

kinases

myogenic

be

Korea

addressed.

1044

of

have

(1.2). specific

A and

C have

that myoblast

been proceeds

and

are

suggested

mediating

differentiation

the

kinase

in

Science

is

muscle

creatine

myoblasts role

cells

myotubes

and

important

from

muscle

multinucleated

cultured

play of

skeletal

tropomyosin

using

may

supported Ministry

into changes,

Studies

phosphorylation

fusion

of

myoblasts

(l-4).

protein

in

mononucleated with

proteins

~To

in to

no

however,

and

Sciences,

acid

reversed

showed,

OF

1044-l

1991

Okadaic

cells

to

COMMUNICATIONS

CULTURE’

2, Man-Sik

Seoul

Press,

RESEARCH

Pages

OKADAIC

the

BIOPHYSICAL

Engineering

shown (7).

Vol.

176,

No.

However, still

the

mechanisms

of

AND

the

acid

powerful

addition,

it

marked

is

kinase

of

has

found

been in

okadaic

studying

the

in

As

COMMUNICATIONS

myoblast

fusion

differentiation, and

in

culture.

Culture

obtained

from

of

Tropomyosin

and

muscle

(13,14).

purified

were

okadaic

are

that

is

muscle

Wako

kinase

were

Antibodies

were

prepared

Myoblasts

from

breast

In

produces

proteins

(1 1).

useful

probe

for

by

protein

the

role

of

protein

the

effect

of

okadaic

specific

from

proteins

Gibco.

Chemical

alkaline

creatine

is

in

chick

METHODS

from

with

cells

mediated

examined

and

[9,10).

cell

extremely

purchased

acid

conjugated

W

AND

many

an

(8),

vitro

intact

elucidate

we

synthesis

reagents

NEN;

anti-rabbit

to

acid

in

to

of

as

attempt

2A

drug

processes

an

fusion

the

introduced

fatty

and

states

MATERIALS Materials:

of

cellular

myogenic

myoblasts

treatment

been

(12).

myoblast

embryonic

has

38-carbon 1

phosphorylation

of

in

on

the

of

phosphatases

that

regulation

phosphorylation

[y-32PlATP

Co.

(Osaka,

Promega.

adult

chick

pectoralis

albino

rabbits

with

from

by

injecting

Japan);

from

phosphatase

purified

was

the

proteins.

Cell

Culture:

were

prepared

as

concentration horse

of

with

set

gels

in

50

mM

After

anti-creatine alkaline

ATP 37

%

4%

SDS.

the

resulting

40

pg

for

of

30 The

myoblasts

buffer

(pH

the

sulfate

and

cells

were

medium 1%

medium

disrupted 5

mM 10%

lysate (17),

then

with

was

by

in

reacted

at

a

10%

changed

extract.

containing

(SDS)

plated

containing

cell

and

embryos

antibiotic/antimycotic

culture

were

7.5) of

dodecyl

Assay:

mixtures and

embryo

chick

sonication MgClz

proteins

for and

0.5

polyacrylamide were

transferred

anti-tropomyosin

with anti-rabbit

IgG

or

conjugated

with

(18).

Phosphorylation reaction

the

2%

antiserum

phosphatase

and

seeding,

membranes

kinase

extract

electrophoresis

sodium

nitrocellulose

essential

cell

Cultured

12-day-old The

Eagle’s

containing

Tris-HCI the

containing

onto

the

of

(15.16). in

embryo

but

Analysis:

EDTA.

cells/ml

after

medium

muscle

previously

10s chick

day

same

lmmunoblot

mM

X

10% One

the

described 5

serum,

solution.

at

RESEARCH

involvements

derivative

protein

acid

phosphorylation

acid

a polyether

inhibitor

increases

Therefore,

30

BIOPHYSICAL

unclear. Okadaic

a

BIOCHEMICAL

3, 1991

assays

1.5

&i

the

in

a final

min. samples

gels

The

containing

were

lysates the

reaction were

then

dried

and

were of

performed

IT-32PlATP volume

was

of

terminated

electrophoresed exposed

1045

to

X-ray

by

(6,000 50

~1.

After

by

the

addition

in

7-14%

films.

incubating

the

Ci/mmole),

1 mM

the

gradient

incubation of

50

~1 of

gels,

and

Vol.

176,

No.

BIOCHEMICAL

3. 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS To

examine

myoblasts

if

that

had

concentrations after

of

the

extents

addition,

it

50

plates

of were

studies

all

the

culture

dishes

Fia.

1. had

were

to

then

fixed,

were

treated

under

Fig.

1,

with

higher

became (data

not

fusion

amounts the

shown).

Since

used

the

at

48

okadaic

to

acid.

and higher

detached

the

In

spherical

concentrations

were

cells

concentration

Effect for acid

stained,

of 24

Okadaic

hr

were

and

further

and

examined

Acid treated

cultured under 1046

on

Myoblast

with for

0 (A),

from treated in

the

next

a microscope.

Fusion. 1 (6). 48

hr.

hr

inhibited

became

At but

increasing

was of

cells

spherical

we

with

a microscope

myoblast

of

the

all

the at

further

indicated.

cultured

okadaic

hr

differentiation,

dose-dependent.

proliferate,

Dose-response

(D)

24

shape

also

cells

otherwise

been

in

bipolar

was

myogenic

examined

cells

the

nearly

that

nM

the

that

able

were

shown

nM,

still

for

and

change

unless

25

drug,

treated

appeared

influences

cultured

As

morphological

than

nM

the

as

acid

been

treatment.

greater

this

okadaic

The 10

(C) The

cells and cells

25

Vol.

176,

No.

3,

We

then

elimination

tested

of

been

treated

within

24

with hr

examine

whether

and cells

appears

likely been

Fig.

7

also

committed

of

okadaic were

acid

cultured

in then

at freed the treated

the

absence with

of 25

Fusion

by

hr.

acid of by

longer

had fusion

that

the

appears

to

myoblasts.

were

shown

acid treated

in

axes

were

the

Okadaic

Acid

2C

capable

cells

of

that

of

and

observed drug okadaic

24

hr

and at

and acid

1047

observed and

72

observed hr

at

(8). at

observed

(A.6)

treated

with 48

hr

Myoblasts 48

hr at

25 (A). were

(Cl. 72

These hr

and

Thus,

Is Reversible

were

is with

Figs.

unfused.

affect

To

okadaic

hr

As

that

indicating

okadaic

48

the

membrane

remained

Myoblasts

period

the nM

2B),

upon

(D).

it have

fusion.

(C,D).

drug

and

their

cells

membrane

culture

of

24

no

myoblasts

resume

for

along

can

Myoblast

then

next

proliferating

The

fusion

cultured

the

reversed

proliferation

myoblast

aligned

Stage-specific

then

allows

cells

acid

for

2A

COMMUNICATIONS

be

to

Thus,

but

already

okadaic

found

reversible.

for

the

medium.

were

of

RESEARCH

can

(Figs.

the

had

Inhibition

hr

inhibition

while

Differentiation

culture

24

incubated

that

effect

the

formation

the

that

BIOPHYSICAL

drug-withdrawal was

further

myotubes

were

for

stage-specific,

forming

already

drug

the

AND

inhibitory

from

myotube

differentiation

the

the

acid

change block

2D,

the

after

reversibly

drug

if

okadaic

morphological

the

BIOCHEMICAL

1991

and nM They

also cells

Vol.

BIOCHEMICAL

176, No. 3, 1991

A

number

of

differentiation muscle with in

of

specific okadaic

Fig.

3,

the

acid-inhibited the

appears

of muscle

drug

sizes

has

been

influences

the

of

and

the

if the

muscle

specific

proteins.

and

tropomyosin

to those

Fig.

4

synthesis cells

in

affecting

of

treated

As shown

in untreated

without

increase

intact

cells

shows

the

okadaic

cells.

Thus,

the

expression

a

the

Therefore,

c

state drug

many

suggest

b

phosphorylation

(11,191.

that of

result

the

phosphorylation

states

This

kDa.

to

protein

phosphorylation

20-50

the

examined

fusion

shown

lysates

myoblasts.

increases

membrane

from

morphological

we

kinase identical

the

proteins.

in cell

also

cultured

almost

to block

acid

proteins

creatine

that

be uncoupled

to accumulate

of

were

specific

Okadaic

the

levels

cells

drug

many

are able

can

Therefore,

(3,4).

RESEARCH COMMUNICATIONS

demonstrated

myoblasts

proteins acid

have

reports

cultured

AND BIOPHYSICAL

d

of

the

treatment

proteins,

that

we

the

state

of

tested

if

lysate

of

significantly

particularly inhibition

with of

the

membrane

e

A

04

6 Fio.

3.

Effect

Tropomyosin 1 (cl,

10

of (B).

(d)

25

for

the

indicates

the

bands

Fio.

Effect

Cultured

Acid

Myoblasts

and

cultured

4,

Okadaic

of

(e)

48

Okadaic

Acid

Phosphorylation

with

I?-szP]ATP

in indicates

the

absence the

drug.

proteins

hr

After

Creatine

were

treated

the

treatment,

to

immunoblot

creatine

kinase

(A)

Protein

were

was

and with

for

performed

and

with

by of

hr.

and

incubating

(b)

100

increased

phosphorylation.

nM

and

0 (lane

the

in

48

(A)

b),

cells

were Lane

tropomyosin

presence

1048

Kinase

analysis.

phosphorylation

cultured

assay (a)

24

of

subjected

on

Myoblasts

prepared.

bracket

and

Syntheses for

the

purified

were

The

of

hr

of the

Myoblasts.

the

cultured

nM

next

on

a

(B).

the

Lysate

their

of

lysates the

okadaic

lysate acid,

Vol.

176,

No.

BIOCHEMICAL

3, 1991

fusion

may

be

certain

critical

associated but

with

unknown

AND

the

cell

BIOPHYSICAL

alteration

in

protein(s)

that

RESEARCH

the

COMMUNICATIONS

phosphorylation

regulate

the

state

fusion

of

process.

DISCUSSION The

present

preventing

myoblast Of

proteins. myoblasts

and been

cells

to

change

that

relate

result

to

in Okadaic

was

phosphorylation 12-o-tetradecanoyl has

also

hence

block

myoblast

likely

to

the

(g-12),

be

increasing

the

activation

of

of

has,

20

protein

been

assumed

to

and

Interestingly,

the of the

cultured

myoblasts

identity

of

the

fusion

begins

to

(20).

It

of

and

ret-11

by

NIH3T3

okadaic

(21).

acid

which

may

the

an

protein

may

in

turn

suggested

to

the

fusion

competent

state

of

kDa

upon

the is 1049

totally

by

TPA-induced the

regulatory

phosphorylation interaction

and

reorganization

membrane

treatment

are C

actions

The

inhibitory

2A kinase

their

at

plasma

protein

protein

myosin-actin

a 20

C, and

1 and

of

phosphorylation

normal

kinase

phosphorylation

(12).

This

protein ester

protein

exert

protein(s)

the

phorbol

phosphatases activity

same

in

The of

the

prevent

of

4).

activator in

(6,23,24).

the

increase

protein

been

redistribution phosphorylation

the

increase

increases chain

as

(Fig.

reverse

has

light

Typically,

change

raf

such

acid,

fusion

morphology,

Since that

C

okadaic

proliferation

membrane

of

specific

shape.

of

a significant

an

of

of

morphological

lysates

(6,22).

with

for

(TPA),

TPA

interfere

cytoskeleton

although

and

axes

of

produce

enzymes

treatment spherical

rate

myoblast

induce

kinase

myosin

therefore,

lysate

myoblast

phosphorylation

kDa

consequently

the

acid

muscle

the

capable

fusion.

of

fusion

major

okadaic

site

the

membrane

to

of

the

inhibition

to

reported

induction

induces

phorbol-13-acetate

been

the

oncogenes

found

state

is

to as

also

in of

acid

on

bipolar

the

alteration

acid

shape

drug

that

okadaic

morphology

certain

prevention

that,

their

the

possible

the

the

their

with is

finding

along

that

affecting

bipolar

align

transformed it

the

form

reported

Therefore, be

was

then

demonstrate

without

interest

myoblasts

decline

have

fusion

tend

cultured

has

studies

of

components

protein of

unknown.

also okadaic

(6).

increased acid

However,

in

(Fig. much

4)

Vol.

176,

No.

studies

BIOCHEMICAL

3, 1991

are

necessary

acid-mediated

to

inhibition

support

are

BIOPHYSICAL

the

myoblast

We

and

clarify

of

Acknowledgment:

AND

RESEARCH

mechanism

COMMUNICATIONS

underlying

the

okadaic

fusion.

grateful

to

Dr.

Keiji

Tanaka

for

his

invaluable

criticism. REFERENCES

1. Okazaki,

K.

and

Holtzer,

H.

(1966)

Proc.

Natl.

Acad.

Sci.

USA

56,

52,

52-65.

(1983)

Cell

1484-1488. 2.

O’Neill,

3.

Nguyen,

M.C.

and

Stockdale,

F.E.

, Medford,

H.T.

A.M.

(1972)

and

J.

Cell

Biol.

Nadal-Ginard,

B.

34,

281-293. 4.

Endo,

T.

and

5. Toutant, 6.

Rapuano,

8.

and

M.

7. Adamo, Cell

Nadal-Ginard,

M.

Ross,

S., Biol.

Tachibana,

A.F.

(1987)

and C.,

Cell, Biol.

J.

(1989)

Prives,

Nervi,

49,

Dev. C.,

Ceci,

515-526. 124,

370-378.

Dev.

R. and

Biol.

134,

Molinaro,

71-278.

M.

(1989)

J.

153-158.

K., J.,

103,

A.

Caporale, 108,

Clardy,

B. (1987)

Sobel,

Scaufer,

P.J.,

Gopichand,

Y.

Tsukitani,

and

Y.,

Schmitz,

Ki kuchi,

F.

J.

H.,

(1981)

Van

J.

Engan,

Am.

D.,

Chem.

Sot.

2649-2471.

9.

Bialojan,

10.

Cohen,

C.

and

11.

Haystead, Cohen,

P. and

12.

Cohen,

P.,

P.

Takai,

A.

(1988)

(1989)

Annu.

Rev.

T.A.J.,

Sim,

A.T.R.,

Hardie,

D.G.

Holmes,

F.B.

Biochem.

J.

Biochem. Carling, (1989)

and

256,

58,

453-508.

D.,

Honnor,

Nature

337,

Tsukitani,

Y.

283-290. R.C.,

Tsukitani,

Y.,

78-81.

(1989)

Trends

Biochem.

Sci.

15,

98-102. 13.

Smillie,

14.

Eppenberger,

L.

242, 15.

D.B.,

Methods

Enzymol.

Dawson,

Boland,

165-l

16.Kwak, (1989) 18.

(1982) H.M..

D.M.

85,

and

234-240.

Kaplan,

N.O.

(1967)

J.

Biol.

Chem.

204-209.

Ha, 585,

17.

B.

K.B.,

Lee,

Y.S.,

Exp.

Cell

Res.

Laemmli,

U.

Blake,

K.

M.D.,

(1984) 19.

Redpath, Konigsberg,

Martonosi, Suh,

A.

(1970)

(1979)

Biochim.

Biophys.

Acta

and I.R.,

227,

K.H., Proud,

C.S.,

Ha,

D.B.

and

Chung,

C.H.

680-685.

Russell-Jones,

136,

G.J.

and

Gotschlich,

E.C.

175-179. C.G.

Sollmann,

Chung,

501-507.

Nature

Biochem.

N.T.

S.W.,

183,

Johnstone,

Anal.

20.

R. and

87.

(1989)

P.A.

Biochem.

and

Mixter,

J. L.O.

262,

69-75.

(1978)

Dev,

Biol.

63,

1 l-26. 21. 22. 23.

Sakai.

R.,

T.

Nagao,

and

Cohen,

R.,

Nature

266,

Naka, 306,

24.

M.,

Ikeda, M.

Kitani, (1989)

Pacifici,

M.,

H.,

Fujiki,

H.,

Proc.

Natl.

Acad.

Rubinstein,

N.,

Takaku,

F.,

Sci.

Biehl,

J.

Rapp,

USA

86,

and

Holtzer,

U.,

Sugimura,

9946-9950. H.

(1977)

538-540. Nishikawa,

M.,

Adelstein,

R. S.

and

Hidaka,

H.

(1983)

Nature

490-492.

Nishikawa, 14069-l

I.,

M.,

Hidaka,

H.

and

Adelstein,

4072. 1050

R.S.

(1983)

J.

Biol.

Chem.

258,