Calyculin A and okadaic acid: Inhibitors of protein phosphatase activity

Vol. 159, No. 3, 1989 March 31, 1989

Calyculin

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Pages 871-877

A and Okadaic

Acid:

Inhibitors

of Protein

Phosphatase

activity

H. Ishihara' B. L. Martin2 , D. L. Brautigan2, H. Karaki3, H. Ozaki3, Y. Kato‘4 , N. Fusetani4, S. Watabe4, K. Hashimoto4, D. Uemura' and D. J.Hartshome 1 1Muscle Biol.

Group, Shantz Bldg.,

2Biochem. Sect. Box G, Div. Biol.

Univ. of Arizona,

Tucson, AZ 85721

& Med., Brown Univ.,Providence,

R.I.

02912

3Dept. of Vet. Pharmacol., Fat. of Ag.,The Univ. of Tokyo, Tokyo 113, Japan 4Dept. of Marine Biochem., Fat. of Ag., 5Fac. of Lib. Arts,

The Univ. of Tokyo, Tokyo 113, Japan

Shizuska Univ.,

Ohya, Shizuoka, 422, Japan

Received February 9, 1989 Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-l, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compoundswith IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had XC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-l enzyme. 0 1989 Academic Press, Inc.

Phosphorylation regulatory well

processes in smooth muscle (1).

characterized

Although

and dephosphorylation

several

the identity

reaction

phosphatases have been isolated is not established.

is

myosin

are

The phosphorylation

of the phosphatase responsible

myosin in-vivo, preparations

(1) but the reverse

of

for

reaction

is not well

because,

is

documented.

from smooth muscle (2-8) the dephosphorylation

Comparison of different

made more difficult

important

in

of

phosphatase

general,

a common

functional classification has not been applied. In this study we use the classification of Ingebritsen and Cohen (9) which separates type-l and Abbreviations: OA, Okadaic acid; CL-A, calyculin A; LC20, 20,000-dalton myosin light chain; MLCK, myosin light chain kinase; EGTA, ethylene glycol bis @-aminoethyl ether)-N,-N,N:-Nttetraacetic acid; PMSF, phenylmethylsulfonyl fluoride. 0006-291X789 $1.50 871

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 159, No. 3, 1989

type-2

enzymes

based

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on substrate

specificity

and

effects

of

inhibitor-l

and inhibitor-2. Recently, of

a valuable

phosphatases,

(10)

termed

intact

namely okadaic

(11)

and

phosphorylation been

linked

cytotoxic induces this and if

the

acid

to

the

contraction

skinned

were

to

which

compound

smooth

muscle

of myosin

of

also

and skinned if

CL-A

phosphatases MATERIALS

fibers

also

the

from

sea

fibers

and These

(20).

role

sponges of

increases effects

(14-18). from

inhibits are

of

contraction

activity isolated

study

induces

B (13).

phosphatase

A (CL-A)

the

isolated

This

activity

intact

for

compound

.

inhibition

determine

available

cytotoxic (12)

calyculin in

so to determine

became

(OA)

and ATPase

compound,

study

tool

have Another

sea sponges

(19),

Our objectives phosphatase

in

activity,

affected.

AND METHODS

Okadaic acid and calyculin A were isolated as described by Tachibana (10) and Kato et al. (19), respectively. Fresh chicken gizzards were used for the preparation of myosin (21), LC20 was isolated from gizzard myosin (23). myosin B (12) and MLCK (22). The catalytic subunit of Calmodulin was prepared from bull testes (22). type-l phosphatase were isolated as described by Brautigan et al. (24) with the following modifications: 1) EDTA and EGTA were excluded from all 2) poly(lysine)-agarose was washed with a column volume of buffer buffers; plus 50 mM NaCl, and eluted with a 10 volume linear gradient from 50 mM to 400 mM NaCl; 3) Ultrogel-AcA44 was substituted for G-75 Sephadex; and 4) the final step was chromatography on a Mono-Q FPLC column (Pharmacia) with elution by a linear gradient (80 ml) from 50 mM to 400 mM NaCl. The catalytic subunit of type-2A phosphatase was isolated with the same protocol except that protein was not digested with trypsin and was not chromatographed on Mono-Q FPLC. The two enzymes were separated by chromatography on poly(lysine)-agarose (type-2A eluted before type-l). Criteria used to distinguish type-l and 2A phosphatases were: a) activity with phosphorylase a; b) greater sensitivity of type-l to inhibitor-2; and c) reactivity to anti-type-l antibodies. Phosphotyrosine protein phosphatase was prepared from human placenta cytoskeleton (25). The endogenous phosphatase of myosin B was partially purified (as the catalytic subunit) as follows: myosin B was added with stirring to an equal volume of acetone, at +20 "C, and after 10 min. was centrifuged at 10,OOOg for 10 min. The pellet was suspended in 0.5 M KCl, 20 mM Tris-HCl (pH 7.0), 0.1% 2-mercaptoethanol, 1 r&l benzamide, 1 mM PMSF and 2 mM EGTA, homogenized with a polytron and dialyzed against this buffer, plus 0.01% Brij-35. The solution was clarified by centrifugation at 10,OOOg for 10 min and the supernatant used for assays. The following alkaline phosphatases were assayed: bovine liver (Sigma, P-5760) E. coli (Sigma P-4377), bovine intestinal mucosa (Sigma P-5521), bovine kidney (Sigma P-6028). Acid phosphatases were from bovine semen (Sigma P-28917) and potato (Sigma P-3752 and Calbiochem 524528). Assay conditions were 50 mM Tris-maleate (pH 7.0) 1 mM dithiothreitol, 0.5 mM MgC12 and 10 mM p-nitrophenyl phosphate. The reaction was started by the addition of substrate and for the acid phosphatases stopped by the addition of NaOH to 100 mM. For the alkaline phosphatase HCl was added to 50 mM and then NaOH to 100 mM. The absorbance at 410 nm was measured. The protein phosphatase substrates were: 32P-labeled phosphorylase a myosin 1.5 to 2.0 mol P/mol myosin, (27); and, 32P(26); 32P-labeled labeled light chains (0.6 to 1.0 mol P/mol light chain) prepared by phosphorylation with MLCK (5 pg/ml) and calmodulin (5 pg/ml) followed by exhaustive dialysis to remove [Y-~~P) ATP. et al.

872

Vol. 159, No. 3, 1989

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Protein phosphatase assays were carried out in 50 mM KCl, 20 mM Trismaleate (pH 7.0), 3 mM MgC12, 1 mM dithiothreitol, 2 mM EGTA, 1 mM theophylline and 1 mg/ml bovine serum albumin with LC20 (0.3 mg/ml) or myosin (1 mg/ml) or phosphorylase a (0.3 mg/ml). Type-l and 2A catalytic subunits were used at 6 and 2 pg/ml, respectively. The myosin B phosphatase was used at about 80 and 140 pg/ml with myosin and phosphorylase a, respectively. Potato acid phosphatase was used at 0.1 units/ml. Reaction at 25 'C was started by addition of substrate. Aliquots (25 ~1) were withdrawn at different times and added to 100 ~1 10% trichloroacetic acid in Beckman airfuge tubes. Followin centrifugation, 30,OOOg for 2 min, 100 ~1 of supernatant was removed and 32 P determined by Cerenkov counting. For any experiment the released Pi was less than 15% of the label in the substrate. Phosphatase activities were calculated from initial linear rates. Other procedures as described previously (27). RESULTS AND DISCDSSION The

initial

activity.

objective

The

and

type-2A

CL-A

and

hundred

effects

are

of

more

potent

for

potency

for

inhibition

(IC50)

of the

for

CL-A,

1.6

respectively. 0.5 al.

and

1 nM.

(15)

who

catalytic Using was type-l

catalytic

respectively,

Fig.

2.0

Using

the

type-2A

These

values

for

subunit and with

values

as were

IC50

whereas

1 and

OA,

obtained 500

about for

50%

as substrates)

330

and

500

are

both

between

by Hescheler

nM with

OA for

nM, et the

respectively.

As shown approximately catalytic

a similar in Fig

pattern

2A the

2 and subunit

of

inhibition

IC50 values

60 nM, the

for IC50

CL-A

for

the

and OA,

values

acid of the catalytic 1. Inhibition by calyculin A and okadaic type-l (A) and type-2A (B). subunits of protein phosphatases Substrates: Procedures given in Materials and Methods. phosphorylated myosin (O,@) and phosphorylated LC20 (a#). @en calyculin A (CL-A); solid symbols, okadaic acid (OA). symbols, Error bars were Each point represents mean from four experiments. omitted.

873

a

CL-A has

required

values

those

type-l

OA is

type-l,

for

the

substrate,

type-2A

to

of

as substrates.

LC20 and myosin

with of

subunits

activity.

respectively; well

phosphatase

and LC20

concentrations

(with

and type-l,

and OA. the

The

subunit

agree

of type-2A CL-A

nM,

IC50

phosphorylase-a

observed

compared

enzyme

inhibited

catalytic

myosin

phosphatase

enzyme.

and

CL-A

OA on the of

type-l

if

1, using

type-2A

each

calculated

subunit

Fig.

inhibitors

a similar are:

in

potent

determine

CL-A and

compared

OA are fold

was to

were

Vol. 159, No. 3, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

1-‘\\1 t i

‘\ + \ \ ‘* --o--o

II

IO

9

5

OA or CL-:

Fig.

both

1 nM.

substrates

it

alteration

Since

is

was not that

and

CL-A

CL-A

(to

was not

on protein that

The

OA did

4, 5-trisphosphate The distinctive the

help crude

and Methods)

2Al

in

and the of inhibition the

complex.

phosphatase and its activity CL-A and OA, as shown in Fig of

0.3

than

nM. CL-A

obtained

OA also (IC50 and

the

it for

and it

was reported

effects None of

by OA (to

LC20 by potato

acid

In addition, It

was

on 2C.

and alkaline

phosphatase B was used

the

10 PM) or phosphatase

found

previously

IC50 = 100 nM), Later

it

phosphatase,

with

of OA these

OA had no effect

enzyme,

effect acid

Myosin

non-linear

to

was

and also

inositol

1,

phosphatases. two catalytic

activity as the

subunits

associated source

with

of protein

against phosphorylated myosin was assayed with 3. CL-A inhibited activity with an IC5D value

inhibited 20 nM).

major

due

were

inhibited

phosphotyrosyl

patterns

are

the phosphatase.

screened

activity.

phosphatase

CL-A

with

substrate.

polymolecular

inhibit

.dentifying

actomyosin

were as were

no

different

inhibitor.

of

10 PM) with not

OA and

three

Previously

OA or CL-A.

(aortic

with

data

Ki values.

phosphatase

-

IC5U

these

phosphate

phosphotyrosine

that

of

dephosphorylation

OA inhibits

of

phosphatases

by either

similar

interaction

non-competitive

and acid

effected

2B(calcineurin, (17)

plots

p-nitrophenyl

1 PM).

are

effects

than

apparent

(see Materials

also

could

or Dixon

alkaline

phosphatases

the

rather

as a mixed

using

values

that

to derive

OA acts

Several

IC50

substrate,

reciprocal

possible

(16)

the

unlikely

of the

Double

shown

M I6

2. Inhibition by calyculin A and okadaic acid of the catalytic subunits of protein phosphatase type-1 (A) and type-2A (B) using Procedures given in Materials and phosphorylase a as substrate. Open symbols, calyculin A (CL-A); solid symbols, okadaic Methods. Each point represents mean f S.E. from four acid (OA). experiments.

about

(15)

(-l&j

phosphatase Using

IC50

values

suggest

that

the

there

is

activity

but

phosphorylase-a, were

0.4

and

was less

a similar 15

nM for

effective pattern

was

CL-A

and

OA,

in myosin

B is

the

could

be

respectively. These type-l

results

enzyme.

However,

dominant also 874

the

phosphatase possibility

that

it

Vol.

159,

No.

BIOCHEMICAL

3, 1989

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

. Fig.

the

3.

Inhibition by calyculin A and okadaic acid of the phosphatase of myosin B (actomyosin). Procedures Materials and Methods. Phosphorylated myosin used as Open symbols, calyculin A (CL-A); solid symbols, okadaic Each point represents mean ? S.E. from four experiments.

holoenzyme

OA than

the

of

subunit

the

catalytic

possibilities partially

purified

phosphorylated the

respectively,

partially

The

IC50

summarized the

Table

1.

each

suggests

CL-A

is

Inhibition

of

dominant also

effective

Protein acid

OA

Myosin

2.0

500

In

2.0

60

0.5

Phosphatases (I$0 values

in

and

0.7

200 and 0.7

B

nM, nM.

myosin

B phosphatase

enzyme

in myosin

B.

substrate

are

each

myosin OA in

by a)

Calyculin

All

phosphatase in skinned eliciting

Mvosin

A and

Okadaic

Subunit Myosin

B

in myosin and intact contraction

Catalytic

B

CL-A

OA

CL-A

values

0.7

70

0.7

200

range

0.3

20

__-

__

- 1.0

0.4

15

__-

--

OA

are

and

be expected than

CL-A

330

a Values

the

Subunits

1.6

a

the

would

LC20

Phos.

of

myosin

70

were

endogenous

phosphatase

Tvoe-2A

Tyne-1

CL-A

that

more

Catalytic

Substrate

subunit

protein

This

enzyme.

since

fibers,

for

were

was using

(intact)

subunit

to OA as the

assayed

two

1.

data

type-l

catalytic

and

control

CL-A

to

these

B phosphatase

Methods)

and

sensitive

between

myosin

the

OA

catalytic

to be less

the

and

isolated

purified

in Table

of For

for

sensitivity values

The above B is

values the

reported

To distinguish

subunit

substrate.

IC50

a similar

was

Materials

as

and for

the

retains

this

of 2A (15).

(see LC20

phosphatase Thus

since

type-2A

catalytic

endogenous given in substrate. acid (OA).

nM.

875

OA

BIOCHEMICAL

Vol. 159, No. 3, 1989 This

(28). more

conclusion,

direct

major the in

myosin

subunit muscle

in

is

not that

towards

also is

known, in the

but

in cardiac

that

the

dephosphorylation

of

consideration

phosphatase is

is

effective

Since

inhibited

only

CAMP usually

inhibitor-l,

via

effects.

Obviously,

identify future

these

by inhibitor-l

induces this

scenario, other

and clarify

of

would

their

represented is

and exists is

an earlier the

dominant

responsible its

smooth

be of

for

regulation (32)

becomes

muscle

the

could is

former kinase.

involvement

with be

the

involved

an important

an

Type-l

and the protein

inconsistent action

the

process.

CAMP-dependent

factors

modes

This

subunit

contrast,

and inhibitor-2

regulatory

30).

the

as phosphatase

In

then

by the

relaxation

is

composition

contraction-relaxation

when phosphorylated

enzyme

by

(31).

myosin,

the

confirmed

(29,

a possibility

enzyme

muscle

in

subunit

phosphatase muscle

type-l

muscle

preparations.

type-l

be

a myosin-binding

a similar is

must

the

cardiac of

this

type-2A

smooth

that

and

myosin

myosin

and

proposed

composed

Assuming important

tentative

Whether

(30).

contaminant

is

skeletal

type-lM,

suggested

activity

was

phosphatase

a persistent report

It

termed

catalytic smooth

however,

evidence.

phosphatase,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

observed and area

to for

research. ACKNOWLEDGMENTS

Work supported by NIH grants HL 23615 and HL 20984 (to D.J.H.) and DK 31374 (to D.L.B.) and a Grant-in-aid for Scientific Research from the Ministry of Education, Culture and Science, Japan (to H.K.). IUWERENCES 1 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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