Vol.

177,

No.

June

28,

1991

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1155-1160

OKADAIC ACID STIMULATES THE ATP-DEPENDENT INTERACTION BETWEEN ACTIN AND MYOSIN OF SMOOTH MUSCLE VIA A DIRECT EFFECT ON MYOSIN .** Kohichi Hayakawa*'**, Tsuyoshi Okagaki**', Toshiaki Dobashi Katsuyoshi Kaneko*92 and Kazuhiro Kohama*:3 Akio Sakanishi**, *Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371, Japan **Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 376, Japan Received

May 17,

1991

SUMMARY: The direct effect of okadaic acid (OA) on the ATP-dependent interaction between actin and myosin of smooth muscle was examined not only by the conventional measurement of ATPase activity but also by application of in vitro motility assay developed recently. The motility was effectively enhanced by PM levels of OA. Measurements of the activities of myosin confirmed that the myosin mediated this effect. The result of this study, which was carried out in the absence of protein phosphatase, are not compatible with the recent reports that the stimulatory effect of OA on smooth muscle contraction is attributable to its inhibitory effect on the activity of the protein phosphatase. Q1‘391 Academic mess,Inc.

Contraction interaction

between actin and myosin. The interaction

phosphorylated the

cells

of smooth muscle cells is induced by the ATP-dependent

state of the myosin, as reviewed in reference

depends

dephosphorylation and the latter

is dependent

on

activities.

the

balance

between

upon the

1. The state of

phosphorylation

and

The former is mediated by the CaM-MLCK system,

by protein phosphatase.

OA, a toxin isolated from the black sponge Halichondria okadai, can enter smooth muscle cells and stimulates their

contraction

(2-7).

The stimulatory

' Present address: Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021. 2 Present address: Department of Pharmacology, Research Center, Taisho Pharmaceutical Co., Ltd., Saltama 330, Japan. 3 To whom correspondence

should

be addressed.

ABBREVIATIONS: CaM, calmodulin; MLCK, myosin light chain kinase; OA, okadaic acid; DTT, dithiothreitol; EGTA, ethyleneglycol-bis-@-aminoethyether)-N,N,N',N'tetraacetic acid. 0006-291X/91 1155

$1.50

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

177,

No.

effect

has

BIOCHEMICAL

3, 1991

been

phosphatase

attributed

activity

that

results

a review,

see

ref 3).

(for

explanation

is that

the

activity

in

a crude

required

to stimulate

We recently

fully

but

concentration observation

similar

effect

in the

one

is

of OA on protein phosphorylation

difficulty

in

to inhibit

loo-times

such

a

phosphatase

lower

than

that

actomyosin

that

has

(5, 6). crude,

activity. not

OA is

of actomyosin

to the concentration

step

on purified

complications

its effect

cells

COMMUNICATIONS

In this

preparation,

present

in

is markedly that

with

compatible

native

the

stimulates above

the

myosin

assay

enhanced

medium.

by OA at a

contraction

mentioned

is

(9). This

explanation

of

of OA.

As the first

preparations

or

activity

is also not

the effect

of the

in preparing

whether

effect

of OA required

no phosphatase

the ATPase

inhibitory

However,

the contraction

phosphorylated

However,

its

homogenate

RESEARCH

in an increase

concentration

succeeded

BIOPHYSICAL

a potent

(5-7)

of myosin

MLCK activity

to

AND

that

defining

actin

and myosin

originate

of native through

towards

from

actomyosin

myosin

the site of action

the (cf.

ref.

in

an assay

presence 4). Here

of OA, we examined system

free

unidentified we report

that

from

the

proteins

in

OA exerts

itself. MATERIALS

AND METHODS

A muscle mince was prepared from the smooth muscle of bovine stomach. The mince was washed 3 times with 4 volumes of 1 mM NaHCO3 to remove phosphatase (9). Myosin was purified from the mince by the method of Ebashi The preparation of myosin still contained a very low level of (10). which was removed completely by the method of phosphatase activity, Nakamura and Nonomura (11). MLCK (lz), tropomyosin (13) and actin (14) were prepared from chicken gizzard, chicken gizzard and breast muscle, respectively. After polymerization by dialysis against 50 mM KC1 and 20 mM Tris-HCl (pH 7.5), actin was used for the assay of actin-activated ATPase activity of myosin, as well as for the in vitro motility assay. The purity of these proteins was usually checked with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (15). OA was donated by Dr. T. Ono (Fujisawa Pharmaceutical Co., Osaka). CaM of bovine brain was purchased from Sigma(St. Louis, MO, USA). The myosin (0.5-l mg/ml) was incubated in a mixture of 20 pg/ml MLCK, 2 pg/ml CaM, 50 mM KCl, 10 mM MgC12, 0.2 mM CaC12, 30 mM imidazole-HCl (pH 7.6) and 5 mM ATP at 25-C for 20 min. The incubated myosin was allowed to stand for 3 hr in ice or dialyzed briefly against 10 mM MgC12 in 20 mM TrisHCl (pH 7.6), and then it was subjected to centrifugation at 10,000 x g for 10 min. The precipitate was used as phosphorylated myosin, as assayed by urea-glycerol polyacrylamide gel electrophoresis(l6). ATPase activity of the phosphorylated myosin was determined by the method of Youngberg (17) or by the malachite green method (18). In vitro 1156

Vol.

177,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

motility assay (for a review, see ref. 19) was carried out as described previously (13,20,21). In brief, phosphorylated myosin was fixed on a coverslip, on which fluorescent actin filaments were then mounted, and finally the ATPdependent movement was monitored with a fluorescence microscope. Protein COn~ntrati0n.S were determined by the method of Bradford (22) with bovine serum albumin as a standard. RESULTS Figure

la shows the actin-activated

myosin with various

concentrations

phosphatase activity, presence

was

of OA.

phosphorylated

of Ca2+. After

phosphorylated

ATPase activity

removal

of phosphorylated

Smooth muscle myosin, free

from

by the CaM - MLCK system in the

of the

phosphorylation

system,

myosin was subjected to measurements of activity.

increased with increases in the concentration

the

The activity

of OA (Fig. la).

The maximum

extent of the increase was 1.2%fold + 0.07 (n=6). A half-maximal increase was observed

in the presence of pM

levels

comparable with these observed to activate Such pM concentrations phosphatase activity be noted that

are definitely

the contraction

higher

than

8

7

data do not,

those that

8

however,

5

Effect oP Ca-ATPase

okadaic

8

7

rule

6

the It must

out the

5

-log(OAW)

-log(OA(W)

F&.& and the

inhibit

necessarily

9

are

of smooth muscle.

of the crude homogenate of smooth muscle (5, 6).

the present

9

of OA. These concentrations

acid

(OA) on the a&in-activated

ATPase

activity

(a)

mxyin. -9TPase activities n the ordinates were 33.3 nmol min mg myosin for (a) mg-l myosin for (b). Assay conditions: (a) 50 mM KCI, 4 activity

(b)

of

phosphorylated

mM MgCl3, 0.1 mM EGTA, 1 mM ATP, 0.03 mg/ml myosin, 0.06 mg/ml actin, 30 mM imidazole-HCl (pH 7.6); (b) 50 mM KCl, 4 mM CaCl3, 1 mM ATP, 0.03 mg/ml myosin, 30 mM imidazole-HCl (pH 7.6). Bars refer to s.e.m. (n=6). 1157

Vol.

177,

No.

possibility

that

review,

phosphatases

in

of

the

direct which

and

and

phosphatase

effect

presence

of

sensitivity

of the

OA in activities

of

resultant

in

that

smooth

muscle

involvement

Experiments

myosin.

a

of protein possible

to OA is present a

(for

viva

it is theoretically

with

myosin,

of 8 PM

The

increase

of the

measurement

visualization

activities with

confirmed

(13,

with

of the

complications

that

to

raise

in

the

test

the

was

1.25-fold,

of ATPase

from

myosin.

2b),

the which

activity

a

the

When

is in good la).

this

was

were in

between

Figure

on

2a,

carried

increased

the

of kinase mounted

assay

agreement Since

assay

presence

As shown

velocity

(Fig.

motility sliding

filaments

of 0.60 ? 0.13 pm/set. OA (Fig.

vitro

in

ATP-dependent

Actin

21).

phosphorylated

at a velocity

pm/set.

OA was

direct

without

coated

moved

inhibition

of

COMMUNICATIONS

to be performed.

myosin

coverslips

with

allows

actin

low

RESEARCH

of action

Therefore,

(7).

phosphorylation

remain

The

with

BIOPHYSICAL

is a site

of OA for

identical

association

possibility

the

Kd values

not

AND

phosphatase

phosphatase

regulation

(Fig.2),

8).

are

specific

actin

protein

see ref.

a protein

BIOCHEMICAL

3, 1991

out

with actin

the

result

filaments

(pm/s)

w Effect5 of OA as examined with an in vitro motility assay. Fluorescent actin filaments (0.25 pg/ml) were allowed to move on a surface coated with phosphorylated myosin in 50 mM KCl, 4 mM MgC12, 2 mM ATP, 25 mM DTT and 30 mM imidazole-HCl (pH 7.6) in the presence of an enzymatic system that was induced to prevent fading of the fluorescence (X3). The analyses were carried out in the absence (a and b) and presence (c and d) of 0.04 pg/ml tropomyosin. a and c, In the absence of OA. b and d, In the presence of El pM indicated by arrows were 0.60 ? 0.13 for a, 0.75 f 0.16 for OA. Mean velocities b, 1.10 ? 0.13 for c, 1.28 ? 0.11 for d (m ? s.e.m., n=30). 1158

in

to 0.75 + 0.18

C

Velocity

the

in

Vol.

177,

the

No.

3, 1991

smooth

muscle

similar

experiments

effect

of OA.

(compare

As reported

lb

effects

enhanced

2~).

with

(21),

myosin

effects

the extent

the

greater

extent

when

low. above,

it was

in the experiment

and 1.00~fold

of ATP was not enhanced

+_ 0.02 (n=6),

significant

when

examined

hydrolyzes

ATP,

the

supports

the suggestion

At present, perform expect action

myosin

radioactive

experiments that

that

the

due

site

is amplified

the

of action

(P

of 1 actinof

is statistically

< 0.01).

or not actin

of

(m +s.e.m.,

difference

Since

of

ATP

myosin further

of OA.

Therefore,

of OA is on the

was

effects

? 0.07

is the site of action

whether

activity

in the presence

concentration

OA is not available.

to determine

to exerts

to 0.1 mM, the

1.25fold The

by

in the presence

reduced

t test

to

ATPase

out

of

increased

by OA. The stimulatory

Student’s

difference

activity

was

measured

respectively.

by

high velocity

OA appears

the

carried

8 pM OA in 1 mM ATP and in 0.1 mM ATP were n=12)

ATPase

Thus,

to a much

was

out

relatively

of 8 pM OA, the

was

described

concentration

carried

the stimulatory

was

The activity

As

ATPase activity

velocity

OA on the

myosin.

mM ATP. When the

to confirm

of actin.

of the increase

la), as shown

we

2d). of

in the absence

COMMUNICATIONS

tropomyosin,

Upon the addition

to 1.28 pm/sec.(Fig. the

RESEARCH

of tropomyosin

through

(Fig.

activated

Fig.

BIOPHYSICAL

associated

previously

shows

phosphorylated OA, although

are

AND

in the presence

increased

Figure

actin

cells

Fig. 2a with

was further

its

BIOCHEMICAL

binds

myosin

we are unable

to

OA.

However,

we

and that

its

molecule

by actin. DISCUSSION

This dependent effect the

report

clearly

interaction

demonstrates between

of OA on myosin. complication

skeletal

that

muscle

phosphorylated.

present

study

and myosin

muscle

originates

from

is

Micromolar

of myosin

actin

Skeletal

myosin

activity

that the enhancement

(not

on smooth

in

levels

shown), muscle

an

is attributable

myosin

provides

phosphorylation active

form

of OA enhanced an observation myosin. 1159

by OA of the ATP-

that

Reports

a system of

whether the

to the free

myosin, or

are now

from

because not

actin-activated is quite

direct

it

is

ATPase

compatible

with

accumulating

that

Vol.

177,

No.

OA modifies effects

BIOCHEMICAL

3, 1991

a

variety

of biological

have been suggested

phosphorylation

AND

BIOPHYSICAL

processes,

to modify

as reviewed

processes However,

and dephosphorylation.

RESEARCH

involved this

COMMUNICATIONS

in ref.

23.

in the

cycle

study

warns

Its of

against

such an interpretation. Another

issue to be stressed

has been examined assay directly

measures

mechano-chemical will suggest

for the first

coupling

sliding

in this report time with an in velocity

on myosin

between actin,

a new pharmacological

is that the effect of the toxin

method

myosin

vitro

motility

assay.

The

and allows analysis

of the

and ATP. Thus, this

report

for analyzing

their

coupling.

ACKNOWLEDGMENTS: This work was supported in part by grants from the Yamanouchi Foundation for Research on Metabolic Disorders, the Chiyoda Mutual Life Foundation and the Japan-China Medical Association, and by Grants-in-Aids for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Kendrick-Jones, J. and Scholey, J. M. (1981) J. Muscle Res. Cell Motil. 2, 347-372 2. Shibata, S., Ishida, Y., Ohizumi, Y., Habor, J., Tsukitani, Y. and Kikuchi, H. (1982) J. Pharmacol. Exp. Ther. 223, 135- 143 3. Ozaki, H., Kohama, K., Nonomura, Y., Shibata, S. and Karaki, H. (1987) Naunyn-Schmiedeberg's

Arch

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4. Ozaki, H., Ishihara, H., Kohama, K., Nonomura, Y., Shibata, S. and Karaki, H. (1987) J. Pharmacol. Exp. Ther. 243, 1167- 1173 5. Takai, A., Bialojan, C., Troschka, M. and Ruegg, J. C. (1987) FEBS Lett. 217,

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6. Biaiojan, C., Ruegg, J. C. and Takai, A. (1988) J. Physiol. 398, 81-95 7. Bialojan, C. and Takai, A. (1988) Biochem. J. 256, 283-290 8. Takai, A. (1988) J. Muscle Res. Cell Motil. 9, 563-565 9. Hayakawa, K., Dobashi, T., Sakanishi, A., Iwai, T. and Kohama, K. (1990) Rep. Prog. Polymer Phys. Japan 33, 619-620 10. Ebashi, S. (1976) J. Biochem. 79, 229-231 11. Nakamura, S. and Nonomura, Y. (1984) J. Biochem. 96, 575-578 12. Adelstein, R. S. and Klee, C. B. (1981) J. Biol. Chem. 256, 7501-7509 13. Kohama, K. (1980) J. Biochem. 87, 997-999 14. Laemmli, U. K. (1970) Nature 227, 680-685 15. Pirrie, W. T. and Perry, S. V. (1970) Biochem. J. 119, 31-38 16. Youngberg, G. E. and Youngberg, M. J. (1930) J. Lab. Clin. Med. 6. 158-166 17. Kodama, T., Fukui, K. and Kometani, K. (1986) J. Biochem. 99, 146511472 18. Warrick, H. M. and Spudich, J. A. (1987) Ann. Rev. Cell Biol. 3, 379-421 19. Kishino, A. and Yanagida, T. (1988) Nature 334, 74-76 20. Okagaki, T., Higashi-Fujime, S. and Kohama, K. (1989) J. Biochem. 160, Q55957 21. 22. 23.

Okagaki, T., Higashi-Fujime, S., Ishikawa, R. Takano-Ohmuro, H. and Kohama, K. (1991) J. Biochem. 109, in press Bradford, M. (1978) Anal. Biochem. 72, 248-254 Cohen, P., Holmes, C. F. B. and Tsukitani, Y. (1990) Trend. Biochem. Scj. 15,

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