Protein kinase C phosphorylates both the light chains and the head portion of the heavy chains of brain myosin

Vol. 169, No. 3, 1990 June 29, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1191-l 197

BIOCHEMICAL

PROTEIN KINASE C PHOSPHORYLATES BOTH THE LIGHT CHAINS AND THE HEAD PORTION OF THE HEAVY CHAINS OF BRAIN MYOSIN Noribumi

Ikedat'z,

Departments

Muguruma*,

of tBiochemistry and $Obstetrics Medical School, Saga 849,

May 23,

t

and Sueo Matsumura

and Gynecology, Japan

*Department of Animal Science, of Agriculture, Kyushu University, Fukuoka

Faculty Received

Yasuda t , Michio

Seiji

812,

Saga

Japan

1990

SUMMARY: Protein kinase C phosphorylated both the 19/21-kDa regulatory light chains and heavy chains of bovine brain myosin. The major phosphorylation sites of the light chains were on their threonyl residues, while those for myosin light chain kinase were on their seryl residues. Whereas several non-muscle regular myosins have been reported to be phosphorylated by different types of protein kinases at the non-helical small segments at the tail ends of the heavy chains, the phosphorylation sites for protein kinase C were localized on the head portion of the heavy chains of brain myosin. The possible role of phosphorylation of brain myosin by protein kinase C in the regulation of motility of neural cells is discussed. Q 1990 Academic Press, Inc.

Phosphorylation non-muscle the

of

myosins (l-3).

C has been

Phosphorylation

protein

the

ATPase

activity

The

heavy

chains

sources

are

also

including

protein

been

is

known

properties

are

myosins kinase

remains (see

C (17).

role

unknown clearly

Refs. It

MLCK, myosin

non-helical

the

heavy a role of (l-3).

14-16).

may be likely light

several

chain

is

known

and

chain

to

muscle to

smooth

stimulate

muscle

myosin

from

various types

for

pieces

regulating

the

chain

The brain

of

Ca'+/calmodulinsome of of

the

phosphorylation

heavy

the kinase

different sites

and

stabilize

by protein

and

tail

in

the

smooth

myosins

The phosphorylation

different 12,

of of

(l-3,7,8)

the

to play

the

II

of

chains

by

kinase

(9). to

stability, myosins

Abbreviations:

II

localized Whereas

myosins

filament

phosphorylated casein

kinase

(l-3,10-13).

non-muscle

same light

(4-6).

chains

various

myosins

suppress

protein

vertebrate

(MLCK)

of the

to

have

and

kinase

MLCK

myosins amoeboidal

of the

chains

by kinases

dependent

light

reported

pre-activated non-muscle

regulatory

by myosin light chain 2+ Mg -ATPase activity

actin-activated

filaments

the

of

ATPase

some

activity

phosphorylation

contains

myosin(s)

of whose

from those of muscle and other The brain contains a high level of therefore kinase;

that

protein

SDS, sodium

kinase dodecyl

C also sulfate.

0006-291X/90 1191

the

heavy

$1.50

Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

169, No. 3, 1990 a role

plays

system. protein

in

In the

BIOCHEMICAL myosin/actin-based

present

kinase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

study,

contractile the phosphorylation

processes profile

the

in of brain

nervous myosin

by

C was characterized. MATERIALS

AND METHODS -

Bovine brain myosin was purified as described previously (16). Protein kinase C was purified from rat or bovine brains as described by Woodgett and Hunter (18). Brain myosin (0.5-1.0 mg/ml) was phosphorylated by protein kinase C (15-30 pg/ml) at 30% for various periods in a reaction mixture containing 30 mM Tris-HCl (pH 7.51, 120-300 mM KCl, 5 mM MgCl 0.5 mM3saCl , of phosphatidylserine, 25 ,,g/ml of diolein and 0. 2' mM [uP]A?P 125 ,,g/gl (6 x 10 cpm/nmol). The incubation mixtures were subjected to SDS-polyacrylamide gel electrophoresis (19) and then to autoradiography for analyzing the phosphorylated myosin subunits. For the analysis of the phosphorylated amino acid residues, the phosphorylated myosin light chains and heavy chains were cut out from the gels and digested with trypsin (4) and then subjected to acid hydrolysis and high voltage electrophoresis, as described paper previously (20). RESULTS -__ The time C is

shown

w

course

in Fig.

of the 1.

phosphorylation

The phosphorylation

of brain increased

myosin gradually

by protein

kinase

and reached

Tfmetmln)

Fig. 1. Phosphorylation of brain myosin by protein kinase C. Brain myosin and protein kinase C ( o ), b@in myosin alone ( l ), or protein kinase C alone ! n ) were incubated with [yP]ATP for the indicated times, as described in "MATERIALS AND METHODS". The radioactivity incorporated into protein was determined as described previously (7). Fig. 2. SDS-polyacrylamide gel electrophoresis and autoradiography of brain myosin phosphorylated by protein kinase C. Brain myosin was incubated with (lanes l-6) or without protein kinase C (lanes 7 and 8) as in Fig. 1 and subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiotimes were: lane 1, 120 min; lane 2, 0 min; lane 3, washy . The incubation 20 min; lane 4, 50 min; lane 5, 80 min; lane 6, 120 min; lane 7, 50 min; lane Blue-stained gel; lanes 2-9, 0, 120 min; lane 9, 50 min. Lane 1, Coomassie Lane 9, protein kinase C alone. Molecular mass values are autoradiogram. indicated on the left side of the gel. HC, heavy chain; LC, light chain; PK-C, protein kinase C; F, dye front.

1192

a

Vol.

BIOCHEMICAL

169, No. 3, 1990

AND BIOPHYSICAL

1

2

RESEARCH COMMUNICATIONS

3

4

5

6

Pi

P&r

4236-

P-Thr Orig

0

,=-em

0

3

-I-

4

Fig. 3. Identification of phosphorylated amino acid residues in the heavy chain and light chains. Brain myosin phosphorylated for 60 min as in Fig. 1 by protein kinase C was resolved into the heavy chain and light chains as in Fig. 2. The HC, LCl and LC2 regions were cut into small pieces and hydrolyzed as described in "MATERIALS AND METHODS". Lane 1, HC; lane 2, LC 1; lane 3, LC 2, Pi, inorganic phosphate; P-Ser, phosphoserine; P-Thr, phosphothreonine; Orig, origin. Fig. 4. Identification of phosphorylated chymotryptic fragments of brain myosinI Brain myosin was phosphorylated for 60 min by protein kinase C and then digested with a l/500 protein concentration of chymotrypsin for various times as described previously (15,27). The digests were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The times of digestion were: lanes 1 and 4, 3 min; lanes 2 and 5, 10 min; lanes 3 and 6, 30 min. Lanes l-3, Coomassie Blue-stain; lanes 4-6, autoradiogram. HC, heavy chain; 140, 140-kDa polypeptide; 66, 6%kDa polypeptide; PK-C, protein kinase C.

plateau

at about

mol/mol of about

50 min,

the

19-

and 21 kDa and with of the

heavy

radioactivity

chain

kinase

about

to

the

identify

C, the

phosphorylated

hydrolysis

and

residues

on the threonine

To identify of brain

myosin,

subjected 4).

heavy

0.6

chain,

about

acid

was with

(Fig.

2.0

as

myosin,

about

light

2.6

chains

The levels

2).

mol/mol

residues

chain high

Fig.

and

myosins, estimated

and

of that

from

phosphorylated

light

voltage

the

chains

by protein

were

subjected

to

electrophoresis

3, the

primarily

light

chain

21-kDa

and

paper phosphorylated

amino

acid

and

chain

were

19-kDa

light

respectively. of the brain

to SDS-polyacrylamide

The ZOO-kDa heavy

chains

mol/mol

amino

and threonine, the location 32 P-labeled

phosphorylation

was associated

each subunit.

to in

of

heavy

were

heavy

then

As shown

serine,

(Fig.

was

the

chains into

autoradiography.

then

light

extent

phosphate

incorporated

In order acid

the

myosin.

phosphorylation of

where

The incorporated

chain

phosphorylation

myosin gel

was digested electrophoresis

was cleaved 1193

into

site

on the heavy with and

polypeptides

chains

chymotrypsin

and

autoradiography of about

140

Vol.

BIOCHEMICAL

169, No. 3, 1990

PPt I

A

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

B

Sup I

A

I

B

of phosphorylated papain fragments of brain myosin. Fig. 5. Identification kinase C was digested Brain myosin phosphorylated for 60 min with protein with a l/500 protein concentration of papain for various times, and the 0.1 M KCl-soluble and 0.1 M KCl-insoluble fragments were separated as described previously (15,27). The precipitates (Ppt) and supernatants (Sup) were The subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. times of digestion were: lanes 1, 3 min; lanes 2, 10 min and lanes 3, 30 min. (A) Coomassie Blue-stain; (B) autoradiogram.

kDa and 65~70 derived

from

which

kDa. the

approximately

corresponded

to nearly

approximately latter

25-kDa

65%70-kDa

portion

of

associated this

The former

(11,lS).

both

the

one phosphorylation

site site

phosphorylation

was digested

with

into

segment

the were

25-kDa

polypeptide,

derived

fragments

in

from

(11,15).

that

of the

to

be derived

70-

myosin

the head

myosin

tail,

head

(11,15).

from

the

phosphate and

to be chain, and

found

to

polypeptides.

myosin

an The

N-terminal

was

140-kDa

of brain

shown

of the heavy

the

of

radioactive

which

was able

to

of about

25 kDa and

Fig.

the

in the

portion

stretch

shown

The

was previously

be From

contained

at least

the location of for protein kinase C. To determine 32 on the 140-kDa polypeptide, P-labeled brain myosin

papain,

As shown

whole segment

approximately

was apparent

the

(11,151.

C-terminal

were

head

the head

120-kDa the

polypeptides

the it

polypeptide

C-terminal

with

result,

140-kDa

5,

radioactive

addition

head

The insoluble

cleave

the

to

portion

the of

120-kDa 1194

the

70-

and

140-kDa

segment

phosphate

brain tail

tail

polypeptide

polypeptide about

120 kDa

was associated

90-kDa

myosin

of

as

polypeptide low did

with that

salt-soluble not

contain

an

Vol.

169,

No.

3, 1990

appreciable

BIOCHEMICAL

level

that

the

head

portion

of

radioactivity.

phosphorylation

BIOPHYSICAL

From

sites

of brain

AND

for

RESEARCH

these

protein

COMMUNICATIONS

results,

kinase

it

C were

was

concluded

localized

on

the

of

the

myosin.

DISCUSSION We have 19/21-kDa that

the

previously

light

chains

primary

sites

threonyl

residues

the

chains, (21) myosin

kinase

muscle

myosin

light

have

light

chain

of brain

muscle

also

that

myosin

some threonyl

were

Ca2+-independent

by protein

protein

proteolytically

activated nature

of

form

the

The primary

or

that

the

19/21-kDa

an

(20).

The

protein

kinase

of

endogenous

our

of

platelet for of

brain

the

myosin in

the

structure to resemble with

respect report

we

light

chains

of

contaminating

kinase

C (23).

contaminating

parts

previous

endogenous protein

of

those

appears

In

of by

of

myosin

C.

sites

site

secondary

myosin,

the

residue

like

of platelet

are

(4-61,

primary

sites 3),

residues

kinase

myosin

(Fig.

kinase

phosphorylated

different

the

phosphorylation

than

the

on a seryl

a non-muscle

rather

at

muscle

residues

myosin,

myosin

that

located

located

(5.6).

we demonstrated

phosphorylation

Whereas

the

residues

report,

indicating

are

C was

(21),

In this

smooth

threonyl

phospholylated

shown

precise

chain on their

chains

sites

kinases

by protein light

of smooth

protein

seryl

C-catalyzed

chains,

(22).

smooth

brain

same light for

were

to the

the

(20).

kinase

myosin

chains

of the

myosin

protein

thymus

light

that

MLCK phosphorylates

as demonstrated

and

phosphorylation platelet

for

by these

light

that

of brain

of

phosphorylation myosin

shown

could

be

Nevertheless,

protein

kinase

a the

remains

unknown. The

phosphorylation

dependent of

the

protein

located

chains

study

shows

in

first

the

report

contains

II of

the

to in

casein

localized (10-12) for

the

heavy

chains

of of

segments

the

at the

myosins

of brain

heavy

of

kinase.

ends The C are

This

is

vertebrate It

kinase and

tail

(13). kinase

myosin.

chains

protein

non-muscle

Ca2+/calmodulin-

by protein

a protein

other

and

macrophage

chains the

for

location of

II

on small and

heavy

portion site

kinase

phosphorylation

of the

head

determine

the

were

sites

head portion

that

for

brain

the

a phosphorylation

interest sites

kinase

heavy

present

site

the

myosin

would

be

of

C phosphorylation

vascular

smooth

muscle

myosins. Phosphorylation reported preliminary protein activity, (unpublished

to modulate experiments kinase but

C had

of

smooth

its

actin-activated indicated

slightly

muscle that

myosin by protein kinase C has 2+ activity (4-6,24). Mg -ATPase phosphorylation the

stimulated

no significant effect as reported observations),

of brain

actin-activated

on superprecipitation for myosin from the

1195

of thymus

been Our

myosin with Mg2+ -ATPase actomyosin (22).

Vol.

BIOCHEMICAL

169, No. 3, 1990 Several are

protein, myosin

has

and actin cone

proteins known also

to

been

are

involved

filopodia

and

phosphorylation cones

including

and

assumed

localized in

adult

to

kinase alter

brain

protein

C may also

growth

cones

as well

-ACKNOWLEDGMENT:-

axonal

growth

the

growth

cones,

The

has

been

as of other

properties

We thank

is

also

with

described in

in the

to in

the

undergo growth signals

Phosphorylation

(25). long-term

this

myosin

of growth

extracellular potentiation

report

regulation

membrane-rimmed

that

known

enriched to

systems

correlated

a role

suggesting

responses

transducing

evidence play

is

Recently,

(25).

and movements

GAP-43

C which

a neuron's

growth-associated

cones

of tension

(26).

signal

(25).

mechanochemical

in

production

RESEARCH COMMUNICATIONS

an axonal

and GAP-43,

lamellipodia

some synapses kinase

in

the

intracellular in

actin

enriched

by protein is

by altering of GAP-43

be

AND BIOPHYSICAL

of motility

structures

at

suggests

that of

by affecting

the some

of myosin.

Miss

Tomoko Inoue

for

her

secretarial

assistance.

REFERENCES -___1. Warrick, H.M., and Spudich, J.A. (1987) Annu. Rev. Cell Biol. 3, 379-421. 2. Korn, E.D., and Hammer, J.A.111. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 23-45. B. (1988) Int. J. Biochem. 20, 559-568. 3. Kuznicki, J., and Barylko, 4. Nishikawa, M., Hidaka, H., and Adelstein, R.S. (1983) J. Biol. Chem. 258, 14069-14072. 5. Nishikawa, M., Sellers, J.R.. Adelstein, R.S., and Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814. 6. Ikebe, M., Hartshorne, D.J., and Elzinga, M. (1987) J. Biol. Chem. 262, 9569-9573. 7. Matsumura, S., Murakami, N., Yasuda, S., and Kumon, A. (1982) Biochem. Biophys. Res. Commun. 108, 1595-1600. 8. Murakami, N., Matsumura, S., and Kumon, A. (1984) J. Biochem. 95, 651-660. 9. Rieker, J.P., Swanljung-Collins, H., Montibeller, J., and Collins, J.H. (1987) FEBS Lett. 212, 154-158. 10. Matsumura, S., Murakami, N., Yasuda, S., Tashiro, Y., and Kumon, A. (1985) Bull. Jpn. Neurochem. Sot. 24, 592-594; (1986) Neurochem. Res. 11, 1804. 11. Barylko, B., Tooth, P., and Kendrick-Jones, J. (1986) Eur. J. Biochem. 158, 271-282. 12. Murakami, N., Healy-Louie, G., and Elzinga, M. (1990) J. Biol. Chem. 265, 1041-1047. 13. Trotter, J.A., Nixon, C.S., and Johnson, M.A. (1985) J. Biol. Chem. 260, 14374-14378. 14. Burridge, K., and Bray, D. (1975) J. Mol. Biol. 99, 1-14. 15. Matsumura, S., Kumon, A., and Chiba, T. (1985) J. Biol. Chem. 260, 1959-1966. 16. Matsumura, S., Takashima, T., Ohmori, H., and Kumon, A. (1988) J. Biochem. 103, 237-246. 17. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 13341-13348. 18. Woodgett, J.R., and Hunter, T. (1987) J. Biol. Chem. 262, 4836-4843. 19. Laemmli, U.K. (1970) Nature 227, 680-685. 1196

Vol.

169, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

20. Matsumura, S., Murakami, N., Yasuda, S., and Kumon, A. (1982) Biochem. Biophys. Res. Commun. 109, 683-688. 21. Kawamoto, S., Bengur. A.R., Sellers, J.R., and Adelstein, R.S. (1989) J. Biol. Chem. 264, 2258-2265. 22. Carroll, A.G., and Wagner, P. (1989) J. Must. Res. Cell Motil. 10, 379-384. 23. Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., and Nishizuka, Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550. 24. delanerolle, P., and Nishikawa, M. (1988) J. Biol. Chem. 263, 9071-9074. 25. Skene, J.H.P. (1989) Annu. Rev. Neurosci. 12, 127-156. 26. Bridgman, P.C., and Dailey, M.E. (1989) J. Cell Biol. 108. 95-109. 27. Matsumura, S., Takashima, T., Ohmori, H., Yasuda, S., and Kumon, A. (1987) Life Sci. Adv. (Biochem.) 6, 125-131.

1197