A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin

Vol. 184, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH-COMMUNICATIONS Pages lZ04-1211

May 15, 1992

A NOVEL REGULATORY MUSCLE

E F F E C T OF M Y O S I N LIGHT CHAIN KINASE F R O M

ON THE ATP-DEPENDENT

Kazuhiro

Kohama Ryoki

I, Tsuyoshi

Ishikawa,

Department

INTERACTION B E T W E E N

Okagaki 2, Kohichi

Teruo

of Pharmacology, of Medicine,

*National

Shimmen

Institute

3, and

Gunma

Gunma

ACTIN A/~D M Y O S I N

Hayakawa, Akihiro

University

SMOOTH

Yuan

Lin,

Inoue*

School

371, Japan

for Physiological

Sciences,

Aiehi 444, Japan

Received March 30, 1992

SUMMARY: The actin-binding activity of myosin light chain kinase (MLCK) from smooth muscle was studied with special reference to the ATP-dependent interaction between actin and myosin. MLCK in the presence of calmodulin endowed sensitivity to Ca 2+ on the movement of actin filaments on phosphorylated myosin from smooth muscle that was fixed on a coverslip. This regulatory effect was not attributable to the kinase activity of MLCK but could be explained by its actin-binding activity. The importance of the actin-binding activity was further substantiated by results of an experiment with Nitellopsis actin-cables in which MLCK regulated the interaction under conditions where MLCK was exclusively associated with the actin-cables. © 1992 Academic Press,

MLCK between

property

CaM,

is a

actin

properties,

and

Inc.

key

and

namely,

myosin

is characterized

resultant

of the latter property been

demonstrated

that

in

by

regulates

smooth

myosin-binding

by the subsequent with

protein

and

activation

actin-binding of MLCK

of the interaction

MLCK

by

interaction

has

activities

two

(2). The

to unphosphorylated in the presence (3).

However,

of the actin-myosin

of interference

ITo whom correspondence should 2 Present address: Department of 3 University Medical College, New Present address: Department of Institute of Technology, Hyogo

ATP-dependent

(i).

of myosin

in the regulation because

muscle

the binding

phosphorylation

the

the

former myosin

of Ca 2+ and

the

involvement

interaction

activating

distinct

effect

has

not

of

the

be addressed. Cell Biology and Anatomy, Cornell York, NY 10021. Life Science, Faculty of Science, Himeji 678-12, Japan.

ABBREVIATIONS: CaM, calmodulin; MLCK, myosin light chain kinase; EGTA, polyethyleneglycol-bis- (13-aminoethylether)-N,N,N',N'-tetra-acetic acid; PAGE, acrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TFP, t r i f l u o p e r a zine dihydrochloride.

0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of:reproduction in any form reserved.

1204

Vol. 184, No. 3, 1992

former

property.

vitro (4, 5) have

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

We

report

enabled

here

that

motility

us to clarify the

assays

role of the

of the latter

interaction

in the

fn

regulatory

mechanism.

MATERIALS

AND

METHODS

MLCK was purified from bovine stomach smooth muscle by modifying the method of Kuwayama et al. (6) and used as MLCK unless otherwise mentioned. Chicken MLCK was prepared from gizzard smooth muscle by the method of Adelstein and Klee (7) with slight modifications (8). Smooth muscle myosin, free of phosphatase activity, was prepared from bovine stomach by modifying the method of Ebashi (9), as described elsewhere (i0). The myosin was incubated with chicken MLCK in the presence of CaM (Sigma, P-2277) and Ca 2+ for i0 min at 25"C as described previously (8) and then mixed with MgCl 2 to a final concentration of i0 mM. The mixture was centrifuged at i0,000 x g for i0 min. The supernatant, containing chicken MLCK and CaM, was removed. The precipitate was dissolved in 0.5 M KCI that contained 2 mM NaHCO 3 and used as phosphorylated myosin. Urea-PAGE (Ii) of the phosphorylated myosin indicated that 100% of Ser 19 of the 20-kDa light chain was phosphorylated (12). Actin (13) and skeletal muscle myosin (14) were purified from chicken breast muscle as described elsewhere. The purities of MLCK, myosin, and actin were checked routinely by SDS-PAGE (15). The effect of MLCK on the ATP-dependent interaction between actin and phosphorylated myosin was analyzed by the myosin-coated surface assay (16), as m o d i f i e d f o r s m o o t h m u s c l e m y o s i n (8, 17). C o v e r s l i p s w e r e c o a t e d w i t h t h e p h o s p h o r y l a t e d myosin. Actin filaments w e r e labelled with r h o d a m i n e - p h a l l o i d i n ( M o l e c u l a r P r o b e s R-415). T h e f l u o r e s c e n t a c t i n f i l a m e n t s (6 nM) m i x ed w i t h s p e c i f i e d c o n c e n t r a t i o n s of MLCK w e r e m o u n t e d o n t h e c o v e r s l i p s i n 0.12 tim CaM, 50 mM KC1, 30 mM Tris-HC1 (pH 7.5), 4 mM MgC12, 1 mM ATP, 25 mM d i t h i o t h r e i t o l , a n d 0.1 mM EGTA-Ca b u f f e r . G l u c o s e o x i d a s e (0.2 m g / m l ; S i g m a G-2133), c a t a l a s e (0.04 m g / m l ; Sigma C-10) a n d g l u c o s e (4.5 m g / m l ) w e r e a d d e d t o t h e a b o v e s o l u t i o n t o p r e v e n t p h o t o b l e a c h i n g of r h o d a m i n e . The mounted coverslips were examined under a fluorescence microscope equipped with a v i d e o c a m e r a a n d r e c o r d e r , as d e s c r i b e d p r e v i o u s l y (8). A c t i n - l i n k e d p r o p e r t i e s of t h e r e g u l a t o r y e f f e c t of MLCK w e r e e x a m i n e d w i t h a Nitella-based m o t i l i t y a s s a y (5, 18). A c t i n - c a b l e s in i n t e r n o d a l c e l l s of Nitellopsfs o b u t u s a w e r e e x p o s e d b y i n t r a c e l l u l a r p e r f u s i o n a n d s u c h c e l l s a r e r e f e r r e d t o a s c o n t r o l cells. MLCK (6 pM) w a s i n t r o d u c e d i n t o a c o n t r o l cell a n d a l l o w e d t o b i n d t o t h e a c t i n c a b l e s as d e s c r i b e d f o r n a t i v e t r o p o m y o s i n b y S h i m m e n a n d Yano (19). T h e f r e e , u n b o u n d MLCK in t h e cells, if a n y , w a s removed by subsequent perfusion. L a t e x b e a d s (2 pm in d i a m e t e r ) w e r e c o a t e d w i t h s k e l e t a l m y o s i n a n d mixed w i t h a s o l u t i o n of 1 mM ATP, 6 mH MgC12, 5 mM EGTA, 200 mM s o r b i t o l a n d 30 mM PIPES, pH 7.0, as d e s c r i b e d elsew~here (18, 19). An a l i q u o t of t h e m i x t u r e w a s i n j e c t e d i n t o a c o n t r o l cell as well as i n t o a cell t r e a t e d w i t h MLCK. T h e i n j e c t e d c e l l s w e r e e x a m i n e d w i t h a phase-contrast microscope connected to a videocamera and recorder, as described previously (18, 19). Actin-activated ATPase activity was determined as follows. ATP (0.5 mM) was hydrolyzed at 37°C for i0 min by 20 lig/ml skeletal myosin in a solution of 40 lig/ml actin, 0.5 mM ATP, 60 mM KCI, i0 mM MgCI2, 2 mM EGTA and MLCK at various concentrations. The liberated phosphate was quantitated by the malachite green method (20). Concentrations of Ca 2+ were calculated using a value of 2.5 x lO -7 M -I as the apparent binding constant of EGTA to Ca 2÷ at pH 7.5 (21, 22). Protein concentrations were determined by the method of Bradford with bovine serum albumin as standard (23). 1205

Vol. 184, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS The ATP-dependent surface

that

1A, t h e

mean velocity

When the 10

had

actin

llM C a 2+

MLCK

effect

circles),

nM.

effect.

of

(data

18

with

were

o f MLCK.

at

stimulatory

coated

of actin filaments phosphorylated

mixed the

The

MLCK w a s

in

concentration and

CaM

was

the

the

the

observed

was

Fig. 17).

presence

of

increases in the

concentration

velocity

in (8,

results

in

with

o f MLCK w a s

on a glass

As shown

previous

increased

velocity

increases

inhibitory,

our

MLCK a n d

velocity

maximum

Further

When the

with

was observed

myosin.

w a s 0.58 l l m / s e c , c o n f i r m i n g

filaments

(closed

concentrations of

been

movement

in

the

presence

obscured

sufficiently reduced

to

the

high,

the

0.05 l ~ m / s e c

not shown). In the

presence

o f MLCK o n t h e with

increases

movement

velocity in the

of the

circles

movement

concentration

i n F i g . 1A), t h e

was minimal.

o f MLCK.

The

stimulatory velocity

effect

decreased

MLCK a t

16

nM

abolished

the

examined

at

concentrations

of

completely.

The

effect

MLCK a n d shown

o f EGTA ( o p e n

of

CaM t h a t

in Fig.

( C a 2+ < 1 riM). increased

with

C a 2+ g i v i n g

C a 2+ o n caused

1B, t h e

the

filaments

Slow m o v e m e n t

half-maximal

was

maximal stimulation

actin

increases

velocity

was

did

not

detected

in the

concentrations

velocity

was in the

(see

move

presence

at

in

the

1 l~M C a 2+, a n d

o f Ca 2+. micromolar

A

1.0

of movement

The

the

1A). of

As EGTA

velocity

concentration

range.

B 1.5"

/\

/ e / ° ~ ° " E 10-

~o5

5

/

-

o >

o >

Q 0

Fig.

10

0.5-

o( 20

30

f:,/ ,

/

I

I

I

6

5

4

pCa 2+

M LC K (riM)

Fig.1. Velocities of A T P - d e p e n d e n t m o v e m e n t of a c t i n f i l a m e n t s o n a g l a s s s u r f a c e c o a t e d w i t h p h o s p h o r y l a t e d m y o s i n from s m o o t h muscle. F l u o r e s c e n t a c t i n f i l a m e n t s w e r e m o u n t e d o n a c o v e r s l i p c o a t e d w i t h t h e m y o s i n (8). The m o v e m e n t of t h e a c t i n f i l a m e n t s was o b s e r v e d u n d e r a f l u o r e s c e n c e m i c r o s c o p e i n t h e p r e s e n c e of 0.12 llM CaM, MLCK a n d 1 mM Mg-ATP. Mean v e l o c i t i e s ( o r d i n a t e ) of 10 a c t i n f i l a m e n t s w e r e p l o t t e d a g a i n s t t h e c o n c e n t r a t i o n s of MLCK o r Ca 2+ ( a b s c i s s a ) . A, E f f e c t s of MLCK a t v a r i o u s c o n c e n t r a t i o n s . I , c o n t r o l ; @, 10 llM Ca 2+ ; O, 0.1 mM EGTA (Ca 2+ < 1 nM). B, E f f e c t s of Ca 2+ a t v a r i o u s c o n c e n t r a t i o n s . T h e c o n c e n t r a t i o n of MLCK was fixed a t 18 nM. pCa 2+ = -log Ca2+(M). 1206

of

Vol. 184, No. 3, 1992

Since

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

coverslips

coated

repeatedly, w e examined and i0 n M M L C K

circles).

However,

velocity

moved,

was

myosin

can

abolished

the

When

TFP,

an

inhibitor

Since

phosphatase

CaM

Ca 2+ was (open

moved

of C a M

of 50 IIM in the presence

completely.

used

results in Fig. IA

of i0 I~M Ca 2+, the filaments

llm/sec. W h e n

be

of the effect of 0.12 IIM

confirming

in the presence

of 0.63

included at a final concentration movement

phosphorylated

by using a single preparation on a coverslip.

absent, the filaments hardly

mean

with

the reversible nature

at a

(24), was

of Ca 2+ and

activity

was

CaM,

absent

from the myosin preparation (i0), there was no possibility that this reversible effect of CaM,

MLCK

and

Ca 2+ on the m o v e m e n t

cycle of dephosphorylation

and

phosphorylation

was

brought

about

by

the

of the myosin that coated the

surface of coverslips. One might argue that, since the stimulatory effect of M L C K in the

presence

further

of CaM

and

phosphorylated

by

increases in the velocity. myosin that had been

Ca 2+, the

the

phosphorylated

kinase

activity

phosphorylated

the

movement

additional phosphorylation at the "standard"

actin

activity of the higher than

filaments

is

sites, i.e.,

in the myosin-coated not

modified

by

Thus, the stimulatory effect is not mediated

the

of K u w a y a m a

by

This stimulatory effect is quite compatible with et al. (6), that

activation

of the

ATP-dependent

of the kinase activity of

(see Discussion). The inhibitory effect of M L C K

further

examined

with

a

is quite novel. Therefore, this effect was

Nitella-based

monitoring the A T P - d e p e n d e n t

assay,

which

interaction between

produces

barely modifies the activity (26); and

the absence

of CaM,

because

CaM

may

the

(4).

phosphorylation

(ii) the assay was

modify

method

actin and myosin

simplicity (i) skeletal muscle myosin was used, because myosin

resultant

at the "standard"

However,

interaction between actin and myosin is independent MLCK

with

be

once smooth muscle myosin has been phosphorylated

sites (8).

the kinase activity of MLCK. the proposal

of

should

at multiple sites was m u c h

Ser 19 of the 20-kDa myosin light chain (25). assay,

MLCK,

Indeed, the actin-activated ATPase

that of the myosin that had been phosphorylated

surface

of

was observed

myosin

effect

interaction. As s h o w n in Fig. 2A, skeletal myosin m o v e d

1.85 l~m/sec along the actin-cables of control cells. However,

of this

conducted

of M L C K

at a m e a n when

for For

on

in the

velocity of the cables

were allowed to bind MLCK, the velocity was reduced to 0.14 l~m/sec (Fig. 2B). As described

in M A T E R I A L S

the cables was

removed

AND

METHODS,

free M L C K

by repeated washing.

was attributable exclusively to M L C K

that failed to bind

to

Thus, the reduction in velocity

that was

bound

to actin, demonstrating

the actin-linked nature of the inhibition of the movement. The Nitella-based assay is of limited utility in evaluating the inhibitory effect of M L C K

because

experimental conditions.

of difficulties encountered Therefore,

w e pursued 1207

in creating

appropriate

the role of the actin-binding

Vol. 184, No. 3, 1992

rA

No.

o

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o

1.o

2.o

/Jm/sec

q

50

0---ff

QoP

Q 110

o

w

0

!

1 10 MLCK (nM)

210 Jum/sec

100

F.Fig.2. H i s t o g r a m of t h e v e l o c i t y of t h e A T P - d e p e n d e n t m o v e m e n t of s k e l e t a l muscle myosin along actin-cables. I n t e r n o d a l cells, t h e a c t i n - c a b l e s of w h i c h were exposed by internal perfusion, were used as c o n t r o l cells. The control cells w e r e f u r t h e r p e r f u s e d w i t h MLCK to allow t h e i r a c t i n - c a b l e s t o b i n d MLCK. A f t e r r e m o v a l of e x c e s s , f r e e MLCK t h a t w a s n o t b o u n d b y t h e c a b l e s , t h e c e l l s w e r e u s e d a s M L C K - t r e a t e d cells. Small l a t e x b e a d s c o a t e d w i t h s k e l e t a l m u s c l e m y o s i n w e r e i n j e c t e d w i t h Mg-ATP i n t h e p r e s e n c e of EGTA i n t o b o t h c o n t r o l a n d M L C K - t r e a t e d cells. T h e m o v e m e n t of t h e b e a d s w a s observed with a phase contrast microscope. A, C o n t r o l cells; B, M L C K - t r e a t e d cells. Arrows indicate mean velocities. O r d i n a t e , n u m b e r of b e a d s ; a b s c i s s a , velocity. Fig.3. E f f e c t s of MLCK o n t h e a c t i n - a c t i v a t e d A T P a s e a c t i v i t y of s k e l e t a l muscle myosin. ATP w a s h y d r o l y z e d b y s k e l e t a l m u s c l e m y o s i n i n t h e p r e s e n c e of a e t i n a n d MLCK a t v a r i o u s c o n c e n t r a t i o n s . The relative rate ( o r d i n a t e ) of l i b e r a t i o n of p h o s p h a t e f r o m ATP w a s p l o t t e d a g a i n s t t h e c o n c e n t r a t i o n of HLCK ( a b s c i s s a ) . Q, MLCK f r o m b o v i n e s t o m a c h ; O, MLCK from chicken gizzard.

activity

of MLCK

myosin,

which

myosin

by

interaction

increases

measuring

is understood

in the

(27). As shown concentration

is in accord

with

measurement

of ATPase

that

is quite

compatible

of EGTA

(Fig. IA, open

the

actin-myosin We

made

with

actin-activated

in Fig. 3, the ATPase

activity

in the

of the

Nitella-based was

attempt

assay.

in EGTA,

from

the

surface

Thus,

the inhibitory

the

for the modulations associated with actin.

1208

in various region

decreased

range.

the

This since

inhibitory

assay effect

of skeletal

of the

Furthermore,

out

monitored

to discuss

micromolar

carried

that obtained

was

activity

measure

of MLCK

circles).

ATPase

fundamental

activity

interaction an

the

to be the most

in the was

actinwith result the effect

presence

evident

when

ways.

of MLCK

that

is responsible

Since caldesmon of smooth

muscle

Vol. 184, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b i n d s to a c t i n f i l a m e n t s t o modify t h e A T P - d e p e n d e n t i n t e r a c t i o n of a c t i n a n d myosin

(28),

the

amino

acid

sequence

of

chicken

gizzard

c o m p a r e d w i t h t h a t of c h i c k e n g i z z a r d c a l d e s m o n (30).

M L C K (29)

The s e q u e n c e b e t w e e n

L y s 42 a n d Ala 80 i n t h e N - t e r m i n a l p o r t i o n of c h i c k e n g i z z a r d 38.5%

was

h o m o l o g o u s w i t h t h e C - t e r m i n a l 37 amino a c i d s

MLCK (29) w a s

of c a l d e s m o n f r o m t h e

same s o u r c e (Lys 695 - Ala73i), w h i c h i n c l u d e s t h e c o m p l e t e a c t i n / C a M - b i n d i n g r e g i o n of c a l d e s m o n (30).

The homology i s q u i t e

compatible with the

l i n k e d n a t u r e of t h e e f f e c t of MLCK o n t h e a c t i n - m y o s i n i n t e r a c t i o n .

actin-

Although

the e x p e r i m e n t s for which r e s u l t s are shown in Figs. 1 and 2 were performed w i t h MLCK from b o v i n e stomach, c o m p a t i b l e d a t a w e r e o b t a i n e d w i t h MLCK from chicken

gizzard

comparison.

(not

shown),

which

may

provide

F u r t h e r m o r e , t h e amino acid s e q u e n c e

some

rationale

of t h e

for

this

b o v i n e MLCK t h a t

w a s d e d u c e d from t h e p r e l i m i n a r y s e q u e n c e of i t s cDNA is h i g h l y h o m o l o g o u s w i t h t h a t of c h i c k e n MLCK ( K o b a y a s h i e t al., 1992, i n p r e p a r a t i o n ) .

DISCUSSION

The present

study

revealed that

MLCK b i n d s to a c t i n t o

regulate the

A T P - d e p e n d e n t i n t e r a c t i o n b e t w e e n a c t i n a n d m y o s i n , a n d t h a t CaM e n d o w s t h e i n t e r a c t i o n with s e n s i t i v i t y to

Ca 2+.

These

novel effects

of MLCK w e r e

not

m e d i a t e d v i a t h e k i n a s e a c t i v i t y of MLCK as s h o w n (i) b y t h e r e v e r s i b i l i t y of t h e e f f e c t of MLCK (see t e x t ) a n d (ii) b y

the

use

of s k e l e t a l m u s c l e m y o s i n

(Fig. 2 a n d Fig. 3), b u t t h e y w e r e a t t r i b u t a b l e to t h e a c t i n - b i n d i n g a c t i v i t y of MLCK. Ebashi

and

his

colleagues

proposed

that

an

s y s t e m f o r t h e a c t i n - m y o s i n i n t e r a c t i o n is p r e s e n t

actin-linked

regulatory

in smooth muscle

(31), i n

w h i c h MLCK o r a n MLCK-like p r o t e i n is i n v o l v e d (6).

The s t i m u l a t o r y e f f e c t of

MLCK i n o u r p r e s e n t

is q u i t e

study

(Fig. 1A, c l o s e d c i r c l e s )

compatible with

t h e i r p r o p o s a l . T h e i r s t u d i e s w e r e b a s e d o n s u p e r p r e c i p i t a t i o n of a c t o m y o s i n . In other words, they used unphosphorylated

m y o s i n w h i c h is e a s i l y p h o s p h o -

r y l a t e d b y MLCK i n t h e p r e s e n c e of CaM a n d Ca 2+.

T h e r e f o r e , i t was d i f f i c u l t

f o r t h e m t o r u l e o u t t h e i n v o l v e m e n t of t h e k i n a s e a c t i v i t y of MLCK. a f f i n i t y of MLCK f o r b o t h a c t i n a n d u n p h o s p h o r y l a t e d

The high

m y o s i n (2, 32) m a k e s i t

d i f f i c u l t t o a t t r i b u t e t h e role of MLCK e n t i r e l y to t h e a c t i n - b i n d i n g a c t i v i t y if superprecipitation

is

employed.

Our

results

from

the

surface

assay

with

p h o s p h o r y l a t e d m y o s i n c l e a r l y r u l e o u t t h e i n v o l v e m e n t of t h e k i n a s e a c t i v i t y of MLCK i n t h i s s t i m u l a t o r y e f f e c t . As is well k n o w n , c o m p l e t e r e g u l a t i o n b y Ca 2+ of s m o o t h m u s c l e c a n n o t b e a c h i e v e d i n t h e a b s e n c e of t r o p o m y o s i n (33). exogenous t r o p o m y o s i n was

not added

In the

to p r e p a r a t i o n s .

examined as a next step in our studies.

1209

present

experiment,

This issue

must be

Vol. 184, No. 3, 1992

The revealed

the

MLCK

ATPase

inhibitory by

in systems

this that

the

(34). studies

The

ratio

are required

on

the

unexpected.

myosin,

it must

of actin

be

result

to 1 on

a molar

to be 200:1

(32).

effect

of MLCK

might

by

i000-i00

to

actin-myosin Since

reported

65-kDa

studies

MLCK

been

has

is supported

contains Our

of

quite

skeletal

(Fig. 2) was

inhibitory

possibility

was

involve

molecule. activity

muscle

effect

study,

cells, this ratio that

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

protein

are, however, before

we

following which

have

an in

effective basis.

the

allocate

a physiological

of

inhibition

of

muscle

it is possible

reporting

stage.

property

relevance.

inhibitory

as

demonstrated

In smooth

Therefore,

communication

abolishes

interaction, be

intrinsic

physiological

still at a preliminary can

it can

This

that

effect Further

smooth of

MLCK

detailed

role to this effect.

ACKNOWLEDGMENTS: We thank Prof. S. Ebashi, National Institute for Physiological Sciences, for helpful discussions throughout our research. This work was supported in part by grants from the Yamanouchi Foundation for Research on Metabolic Disorders, the Chiyoda Mutual Life Foundation, the Japan-China Medical Association, Bayer Yakuhin Ltd., and the Uehara Memorial Foundation, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Y. L. was on leave of absence from the Department of Pharmacology, Dalian Medical College, Dalian, P. R. of China, and was supported by a fellowship from the Uehara Memorial Foundation.

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