Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin

Vol. 97, No. I, 1980

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

November 17, 1980

Pages 309-317

PHOSPHOLIPID TURNOVER AS A POSSIBLE TRANSMEMBRANE SIGNAL FOR PROTEIN PHOSPHORYLATION DURING HUMAN PLATELET ACTIVATION BY THROMBIN* Y a s u h i r o K a w a h a r a %, Y o s h i m i Takai, Ryoji Minakuchi, K i m i h i k o Sano and Y a s u t o m i N i s h i z u k a

Department of Biochemistry Kobe University School of Medicine, Kobe 650, Japan Received October 3,1980 2+ H u m a n p l a t e l e t s c o n t a i n a large amount of Ca -activated, p h o s p h o l i p i d - d e p e n d e n t p r o t e i n kinase (protein kinase C). The activation of this enzyme is initiated by u n s a t u r a t e d d i a c y l g l y c e r o l w h i c h results from the t h r o m b i n - i n d u c e d p h o s p h a t i d y l i n o s i t o l hydrolysis. P r o t e i n kinase C p r e f e r e n t i a l l y p h o s p h o r y l a t e s in v i t r o a p o l y p e p t i d e having a m o l e c u l a r w e i g h t of about 40,000 (40K protein). This p r o t e i n is l a b e l l e d rapidly in p l a t e l e t s s t i m u l a t e d by t h r o m b i n as well as by exogenous p h o s p h o l i p a s e C, and d i a c y l g l y c e r o l f o r m a t i o n always accompanies 40K p r o t e i n p h o s p h o r y l a t i o n . The p h o s p h o r y l a t i o n of 40K p r o t e i n in vivo induced by t h r o m b i n is s e l e c t i v e l y inhibited by c h l o r p r o m a z i n e and dibucaine, w h i c h are p o t e n t inhibitors for p r o t e i n kinase C. Thus, p h o s p h a t i d y l i n o s i t o l turnover p r o v o k e d by thrombin seems to serve as a t r a n s m e m b r a n e signal for p r o t e i n p h o s p h o r y l a t i o n during p l a t e l e t activation. SUMMARY:

Since the s t i m u l a t i o n to hormonal clarify tually Ref.

control

of platelets

of c e l l u l a r

processes,

the r e g u l a t o r y m e c h a n i s m s leading to a g g r e g a t i o n

i).

Among

these events

r y l a t i o n of some e n d o g e n o u s upon s t i m u l a t i o n

by t h r o m b i n

by t h r o m b i n may be analogous attempts

of several

and release attention

have been m a d e to

intracellular

reaction

(for a review see

has been paid to the p h o s p h o -

p r o t e i n s w h i c h may be o b s e r v e d (2-5).

events even-

In particular,

specifically

two p r o t e i n s h a v i n g

*/ This investigation has been supported in part by research grants from the Scientific Research Fund of Ministry of Education, Science and Culture, Japan (19791980), the Intractable Diseases Division, Public Health Bureau, the Ministry of Health and Welfare, Japan (1979-1980), a Grant-in-Aid of New Drug Development from the Ministry of Health and Welfare, Japan (1979-1980), the Yamanouchi Foundation for Research on Metabolic Disorders (1977-1980) and the Foundation for the Promotion of Research on Medicinal Resources, Japan (1977-1980). %__/ On leave from the Department of Internal Medicine (Ist Division), Kobe University School of Medicine, Kobe 650, Japan° i/ The abbreviations used are: protein kinase A, cyclic AMP-dependent protein kinase; protein kinase G, cyclic GMP-dependent protein kinase; SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(8-aminoethylether)N,N,N',N'-tetraacetic acid.

0006-291X/80/210309-09501.00/0 309

Copyright © 1980 by Academic Press, Inc. All rights o f reproduction in any form reserved.

Vol. 97, No. 1, 1980

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

molecular weights

of about 40,000

(40K protein)

have been shown to be heavily phosphorylated stimulated by thrombin, posed to be intimately the phosphorylation

and 20,000

in platelets which are

and the phosphorylation

of 40K protein is pro-

related to release reaction

(6) and Bell and Majerus

(7) have shown recently

is a product of phosphatidylinositol Preceding reports

Ca2+-activated,

Rittenhouse-Simmons that diacylglycerol

from this laboratory have shown that the

tissues

protein kinase

(protein kinase

is activated by unsaturated

glycerol which may result from phosphatidylinositol This communication describes

for the phospho-

turnover which is provoked by

phospholipid-dependent

C) found in many mammalian

Although

to be catalyzed by

(4), the enzyme responsible

rylation of 40K protein has not yet been defined.

thrombin.

(2-5).

of 20K protein has been proposed

myosin light chain kinase

(20K protein)

diacyl-

hydrolysis

that the phosphorylation

(8-11).

of 40K protein

is catalyzed by protein kinase C, and that this enzyme may be directly involved in the transmembrane

control of protein phosphorylation

during

platelet activation. EXPERIMENTAL PROCEDURES Washed human platelets were prepared by the method of Baenziger and Majerus (12). Phospholipid was prepared f r o m b o v i n e brain as described previously (8). C ~½ f thymus HI histone was prepared as described earlier (13). [¥- P]ATP was prepared by the method of Glynn and Chappell (14). Bovine thrombin and Clostridium perfringens phospholipase C were the products of Mochida Pharmaceutical Co. and Sigma, respectively. Other chemicals were obtained from commercial sources. Protein kinases C, A and G!/ were assayed by measuring the incorporation of 32p i into HI histone from [y-32P]ATP. Other details are specified in the legends to Figures. Labelling of platelets with [3H]arachidonic acid and quantitation of diacylglycerol produced were carried out as described by Rittenhouse-Simmons (6). Labelling of platelets with 32p i was carried out as described by Lyons et al. (2). The platelet proteins labelled with 32pi were analyzed by SDS-polyacrylamide slab gel electrophoresis under the conditions described by Laemmli (15). The separating gel consisted of a 5 to 18% linear acrylamide gradient, and the stacking gel contained 3% acrylamide. After electrophoresis each slab gel was stained with Coomassie brilliant blue, dried on a filter paper and exposed to Kodak Royal X-Omat film to prepare autoradiogram. The relative intensity of each band was quantitated by densitometric tracing of the autoradiogram at 430 nm using a Shimadzu dual-wavelength chromatogram scanner, Model CS-910. Protein was determined by the method of Lowry et al. (16).

310

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

RESULTS When protein shows

platelets

kinases

the

was

A.

protein

Protein

detected

12 t h r o u g h G-150

under

column

and

employed

of b r a i n

protein

pholipid.

conditions. further

for t h e p r e s e n t which

C previously

in a r e v e r s i b l e

manner

indispensable

20

L ×

E

I

~ /

~

5

HI h i s t o n e

active

kinase

filtration

studies. ~/

were

for t h i s

1

as p h o s -

C

(Fractions

on a Sephadex The

enzyme

(8,9,17,18).

by a s s o c i a t i o n

Among

showed

from those The

with membrane

activation.

were

and no p e a k

indistinguishable

described

1

than protein

in a c t i v i t y ,

Protein

to gel

Fig.

EGTA,

A and C which

With

20 t i m e s m o r e low

containing

fraction.

kinases

column.

extremely

properties

kinase

Ca 2+ w a s

C was

G was

subjected

and kinetic

activated

kinase

medium

in s o l u b l e

of p r o t e i n

(DE-52)

comparable

22) w a s

in a b u f f e r

mostly

pattern

kinase

physical

was

recovered

on a D E A E - c e l l u l o s e

acceptor

kinase

were

DISCUSSION

disrupted

chromatographic

developed phate

were

AND

enzyme phos-

various

1

-

1.5

~= c~

-i.0~

"-" \, -o.5~

..j'

',. o

a_ 0

20

40

60

I o 8Q

FRACTION NUMBER

FiK. i. DE-S2 column chromatography of platelet protein kinases. Platelets (5 x i010) were disrupted by sonication in i0 ml of 20 mM Tris/HC1 at pH 7.5 containing 0.25 M sucrose, i0 mM 2-mercaptoethanol, 2 mM EDTA, 5 mM EGTA, 0.01% leupeptin and--2 ~phenylmeth---ylsulfonylfluoride. The I00,000 x g supernatant (I0 ml, 35 mg of protein) was subjected to a DE-S2 column (5 x 1.4 cm) equilibrated with 20 ~_Tris/HCl at pH 7.5 containing i0 ~_ 2-mercaptoethanol, 2 mM EDTA, 2 mM EGTA and 0.001% leupeptin. Elution was carried out with a 96-ml-linear concentration gradient of NaCI (0 to 0.4 M) in the same buffer. Protein kinases were assayed in the presence of 0.5 mM CaCI2, 20 ~g of phospholipid, 0.6 ~g of diolein, 1 ~_MMcyclic AMP and 0.I mM EGT-Aas indicated under the conditions described earlier (I0) (¢ ~), with--CaCl2, phospholipid and diolein; (o o), with cyclic AMP; (; A), with EGTA; (......), optical density at 280 nm. 2--/ The enzyme preparation thus obtained was free of other protein kinases, calmodulin, interfering enzymes and endogenous phosphate acceptor proteins.

311

Vol. 97, No. I, 1980

phespholipids a small

enzyme

tested

amount

affinity

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phosphatidylserine

of u n s a t u r a t e d

of enzyme

phosphatidylinositol

rated

fatty

(19),

it is p o s s i b l e

mostly that

phosphatidylinositol turn

serves

kinase

thrombin

to produce

as an i n i t i a t o r

of this d i v a l e n t

tissues

acid,

induces

increased

the

is c o m p o s e d

cation. of u n s a t u -

particularly

at p o s i t i o n

the

hydrolysis

unsaturated

specific

diacylglycerol,

for the selective

activation

which

2 of

in

of p r o t e i n

C.

The e x p e r i m e n t the p r e v i o u s

given

produced,

given

observations

phosphorylated results

arachidonic

In addition,

as for Ca 2+, and a l l o w e d

range

in m a m m a l i a n

effective.

markedly

as well

at the m i c r o m o l a r

Since

acid,

diacylglycerol

for p h o s p h o l i p i d

fully active

was m o s t

in Fig. by Lyons

in p l a t e l e t s in Fig.

2A shows

upon

that,

et al.

(2),

stimulation

40K p r o t e i n

by thrombin.

3A show that d i a c y l g l y c e r o l

and that this r e a c t i o n

appeared

in c o n f i r m a t i o n

was

was r a p i d l y Quantitative

even m o r e

to be i m m e d i a t e l y

of

rapidly

followed

!~¸ i¸¸i¸

i!i!ili ~

0

lO

20

40

60

0

60

120

140

240

TIME IN SECONDS FiK. 2. Stimulation of 40K protein phosphorylation by thrombin and phospholipase C. Samples of platelets which were labelled with 32p i were stimulated by thrombin (0.04 unit/l x 108 platelets) or by phospholipase C (0.3 unit/l x 108 platelets) at 37°C.- Radioactive proteins were analyzed as described under "EXPERIMENTAL PROCEDURES". A, with thrombin; B, with phospholipase C.

312

Vol. 97, No. 1, 1980

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

i

I

I

B

,,--I X8

g

P

3(3

~6

i/I

"

D.4

20 D

iC

//

~,2

0

I

I

1

20

40

60

0

L

60

I

o

o~ 0.2 ~=

..o"

I,

120 180

1,,

240

0

o

TIME IN SECONDS

F i g . 5. P a r a l l e l s t i m u l a t i o n o f d i a c y l g l y c e r o l f o r m a t i o n and 40K p r o t e i n p h o s p h o r y l a t i o n by t h r o m b i n and p h o s p h o I i p a s e C. Samples o f p l a t e l e t s which were l a b e l l e d w i t h [ 3 H ] a r a c h i d o n i c a c i d or 32p i were s t i m u I a t e d by t h r o m b i n or phosphol i p a s e C as d e s c r i b e d i n t h e legend t o F i g . 2. In some e x p e r i m e n t s samples o f p l a t e l e t s were p r e i n c u b a t e d w i t h 10 p_MMprostaglandin E1 f o r 5 min a t 37°C and t h e n s t i m u l a t e d by t h r o m b i n . D i a c y l g l y c e r o l f o r m a t i o n and 40K p r o t e i n p h o s p h o r y l a t i o n were q u a n t i t a t e d a s d e s c r i b e d under "EXPERIMENTAL PROCEDURES". Backgrounds f o r d i a c y l g l y c e r o l f o r m a t i o n and 40K p r o t e i n p h o s p h o r y l a t i o n , which were o b t a i n e d in t h e a b s e n c e o f t h r o m b i n and p h o s p h o l i p a s e C were about 250 cpm and 0 . 5 0 D , respectively. These v a l u e s were s u b t r a c t e d from each p o i n t . A, w i t h t h r o m b i n ; B, w i t h p h o s p h o l i p a s e C. (e i ) and (m), d i a c y l g l y c e r o l f o r m a t i o n in t h e a b s e n c e and p r e s e n c e o f p r o s t a g i a n d i n El, r e s p e c t i v e l y ; (o . . . . . o) and ( a ) , 40K p r o t e i n p h o s p h o r y l a t i o n i n t h e a b s e n c e and p r e s e n c e o f p r o s t a g l a n d i n El, r e s p e c t i v e I y .

by 40K protein phosphorylation. glycerol w a ~ p r o d u c e d

formation

then the exogenous

platelet activation.

to phosphatidic

(20,21).

addition of phospholipase

In fact, bacterial

Fig.

acid

(data not shown).

is a signal for 40K protein phospho-

phospholipase

to mimic thrombin action and induce irreversible reaction

and that

of this lipid was presumably due to further

and also to conversion

If such diacylglycerol rylation,

that this diacyl-

in the expense of phosphatidylinositol,

the rapid disappearance degradation

It was suggested

C may induce C has been shown

aggregation

and release

2B shows that 40K protein was indeed phospho-

rylated upon treatment with Clostridium

perfringens

phospholipase

C,

and Fig.

3B shows that the reaction was roughly parallel with diacyl-

glycerol

formation.

So far, in all experiments,

tion always accompanied

diacylglycerol

40K protein phosphorylation,

and both reactions

were equally

inhibited to same extent by prostaglandin

of thrombin,

as also shown in Fig.

3A.

313

forma-

El, an antagonist

Vol. 97, No. I, 1980

The protein When

the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

experiments kinase crude

described

C was

indeed

extract

in

able

was

Fig. to

20K

designed

phosphorylate

incubated

0.5

4 were

with

Ca 2+

40K and

to

show

protein

radioactive

that in vitro. ATP,

LIOK

0

B 0.5

~

o 1.o

0,5

i

l

r

l

13

42

i00

180

MOLECULARWEIGHT (x 10-3)

Fig. 4. Phosphorylation of 40K protein by exogenous protein kinase C in vitro and in platelets in vivo induced by thrombin. A, In vitro phosphorylation with endogenous protein kinase. The isolated platelets (i x 1010) suspended in 2 ml of 20 mM Tris/HCl at pH 7.5 containing 0.25 M sucrose were disrupted by sonication. The sonicate (100 ~g of protein) was incubated with 0.2 mM CaC12 or 0.2 ~ _ E G T A in the reaction mixture (0.i ml) containing 2 ~mol of Tris/HCl at pH 7.5, 0.5 ~mol of magnesium acetate and 5 nmol of [y-32p]ATP (l x lO 6 cpm/nmol). The incubation was carried out for 30 sec at 30°C. The radioactive proteins were analyzed as described under "EXPERIMENTAL PROCEDURES". (-), with CaCl2; ~...... ), with EGTA. B, In vitro phosphorylation by exogenous protein kinase C. The sonicate was incubated under the conditions described above except that the purified platelet protein kinase C (2 ~g of protein) was exogenously added. (-), with CaCI2; (...... ), with EGTA. C, In vivo phosphorylation. In vivo phosphorylation was carried out with or without thrombin for 30 sec as described in the legend to Fig. 2. ( --), with thrombin; (...... ), without thrombin.

314

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

40K p r o t e i n w a s p h o s p h o r y l a t e d extent

(Fig.

ly e n h a n c e d a n d this

4A).

enhancement

stimulation port

interacting

lipid, enzyme.

hibited

laboratory

protein

although Neither

these

above

drugs

drugs

(Fig.

in Fig.

5.

I

I

1.0

2.0

and

The

A preceding

re-

phospholipid-

specifically phosphosite of

to these drugs.

the p h o s p h o r y l a t i o n profoundly

4B),

in v i v o u p o n

the c a t a l y t i c

k i n a s e A nor G is s u s c e p t i b l e

as shown

significant-

C by c o m p e t i n g w i t h

do not i n t e r a c t w i t h

by t h r o m b i n w a s

C

several

and d i b u c a i n e

kinase

these observations

induced

4C).

that

was

to some

of EGTA.--3/

enhanced

(Fig.

has d e s c r i b e d

of p r o t e i n

protein

with

by t h e s e

(22)

kinase

in the p r e s e n c e

such as c h l o r p r o m a z i n e

the a c t i v a t i o n

in v i v o

as d e s c r i b e d

kinase

of this p r o t e i n

of e x o g e n o u s

not e v i d e n t

protein

of this p r o t e i n was r e m a r k a b l y

drugs

In c o n s i s t e n t tein

was

by t h r o m b i n

f r o m this

inhibit

The p h o s p h o r y l a t i o n

by the a d d i t i o n

phosphorylation

by e n d o g e n o u s

of 40K pro-

selectively

in-

ioo

~

5o

N 25 .::r

o

0

CHLORPROMAZINEOR DIBUCAINE (raM)

Fig. 5. Inhibition by chlorpromazine and dibucaine of phosphorylation of 40K protein in vivo induced by thrombin. In vivo phosphorylation was carried out with thrombin for 30 sec as described in the legend to Fig. 2. except that the incubation mixture contained chlorpromazine or dibucaine as indicated. (o.....o), with chlorpromazine; (e --e), with dibucaine. 3--/ When the crude extract which was heated briefly (90°C for 2 min) to inactivate endogenous protein kinase was employed, 40K protein was more clearly shown to serve as phosphate acceptor for protein kinase C.

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

The results presented above suggest strongly that protein kinase C is activated in a manner directed by thrombin,

and is responsible

for the phosphorylation of at least 40K protein which has been proposed to be directly related to release reaction.

Although the experi-

mental support described above has resulted from a limited number of studies with human platelets,

it is possible that phosphatidylinositol

turnover provoked by a variety of extracellular messengers may be a signal for transmembrane control of protein phosphorylation.

Acknowledgement Authors are grateful to Dr. T. Fujii and A. Tamura, Kyoto College of Pharmacy, for their generous gifts of phosphatidylserine. Skillful secretarial assistance of Mrs. S. Nishiyama and Miss K. Yamasaki are greatly acknowledged.

REFERENCES i. Haslam, R.J., Davidson, M.M.L., Davies, T., Lynham, J.A. and McClenaghan, M.D. (1978) Adv. Cyclic Nucleotide Res. 9, 533-552 2. Lyons, R.M., Stanford, N. and Majerus, P.W. (1975) J. Clin. Invest. 56, 924-936 3. Haslam, R.J. and Lynham, J.A. (1977) Biochem. Biophys. Res. Commun. 77, 714-722 4. Daniel, J.L., Holmsen, H. and Adelstein, R.S. (1977) Thrombos. Haemostas. 38, 984-989 5. Wallace, W.C. and Bensusan, H.B. (1980) J. Biol. Chem. 255, 19321937 6. Rittenhouse-Simmons, S. (1979) J. Clin. Invest. 63, 580-587 7. Bell, R.L. and Majerus, P.W. (1980) J. Biol. Chem. 255, 1790-1792 8. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695 9. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., Nishizuka, Y., Tamura, A. and Fujii, T. (1979) J. Biochem. 86, 575578 i0. Takaip Y., Kishimoto, A., Kikkawa, U., Mori, T. and Nishizuka, Y. (1979) Biochem. Biophys. Res. Commun. 91, 1218-1224 ii. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276 12. Baenziger, N.L. and Ma3erus , P.W. (1974) Methods Enzymol. 31, 149155 13. Hashimoto, E., Takeda, M., Nishizuka, Y., Hamana, K. and Iwai, K. (1976) J. Biol. Chem. 251, 6287-6293 14. Glynn, I.M. and Chappell, J.B. (1964) Biochem. J. 90, 147-149 15. Laemmli, U.K. (1970) Nature 227, 680-685 16. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 17. Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A. and Nishizuka, Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550 18. Inoue, M., Kishimoto, A., Takai, Y. and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616

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19. Holub, B.J., Kuksis, A. and Thompson, W. (1970) J. Lipid Res. ii, 558-564 20. Schick, P.K. and Yu, B.P. (1974) J. Clin. Invest. 54, 1032-1039 21. Chap, H.J., Zwaal, R.F.A. and Van Deenen, L.L.M. (1977) Biochim. Biophys. Acta 467, 146-164 22. Mori, T., Takai, Y., Minakuchi, R., Yu, B. and Nishizuka, Y. (1980) J. Biol. Chem. in press

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