Properties of protein kinase c subspecies in human platelets

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]387-]395

Vol. 155, No. 3,1988 September 30,1988

PROPERTIESOF PROTEINKINASE C SUBSPECIESIN HUMANPLATELETS* Masanori

Tsukuda,t Yoshinori Asaoka,§ Kazuo Sekiguchi,$ Ushio Kikkawa, and Yasutomi Nishizuka

Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan

Received August 16, 1988 SUMMARY: Protein kinase C (PKC) from human platelets was resolved into two fractions by hydroxyapatite column chromatography. One of the enzymes was indistinguishable from the brain type III PKC having e-sequence in its kinetic and immunological properties. The other enzyme was kinetically different from any of the brain PKC subspecies so far isolated, although it resembled the brain type II PKC having 8-sequence. With HI histone as substrate, this platelet enzyme was not very sensitive to Ca 2+, and activated partly by phosphatidylserine plus diacylglycerol or by free arachidonic acid. Both platele£ enzymes could phosphorylate the P47 protein in vitro, but the enzyme physiologically responsible for the P47 protein phosphorylation in the activated platelets remains to be identified. © 1988 Academic

Press,

Inc.

Biochemical existence 8I,

and molecular

of several

cloning

subspecies

8II, and y, that are related

analysis

of protein

reveals

the

kinase C (PKC),

to, yet clearly distinct

e, from

* This work was supported in part by research grants from the Scientific Research Fund of Ministry of Education, Science and Culture, Japan (19851988); Muscular Dystrophy Association (1986-1988); Yamanouchi Foundation for Research on Metabolic Disorders (1986-1988); Merck Sharp & Dohme Research Laboratories (1986-1988); Biotechnology Laboratories of Takeda Chemical Industries (1986-1988); Ajinomoto Central Research Laboratories (1986-1988); Meiji Institute of Health Sciences (1986-1988); and New Lead Research Laboratories of Sankyo Company (1988). The data are taken in part from the dissertation that will be submitted by M. Tsukuda to Kobe University School of Medicine in partial fulfillment of the requirement for the degree of Doctor of Medical Science. t Present address: Department of Orthopedic Surgery, Kobe University School of medicine, Kobe 650, Japan. § Present address: Department of Internal Medicine (Ist Division), Kobe University School of medicine, Kobe 650, Japan. $ Present address: Second Tokushima Institute of New Drug Research, Otsuka Pharmaceutical Co.,Ltd., Tokushima 771-01, Japan. Abbreviations used are: EGTA, ethylene glycol bis(B-aminoethyl ether)N,N,N',N'-tetraacetic acid; SDS, sodium dodecyl sulfate. 0006-291)(/88 $1.50 1387

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 155, No. 3, 1988

one another

BIOCHEM1CAE AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(i).

It has been shown that mRNAs of 8I- and 8II-

PKC are derived

from a single RNA transcript

splicing

(2).

resolved

into three major

hydroxyapatite and 8II)-, expressed cord,

and s-sequence,

Recently,

ubiquitously

In platelets,

substrate

of PKC

appears

reactions

of platelets

isolated

immunological cells,

tissues,

of PKC,

an approximate

is a specific

(9,10).

(11,12).

against

(10,13).

of a Igtll cDNA library cell line

(14).

trisphosphate

(16), or some other

not related

to the structure

communication

will describe

human platelets, P47 protein

the amino acid sequence

Recently,

by

(15),

from

Although

functional

of any known proteins the PKC subspecies

the

inositol

of the P47 protein

and in vitro phosphorylation

by these enzyme

this

constructed

to be lipocortin

5'-phosphatase

of this

and release

cDNA clones of the P47 protein

a human promyelocytic

(17,18),

molecular

The P47 protein was highly and an antiserum

P47 protein was once thought

proteins

(8).

and major

with aggregation

by Haslam and coworkers

screening

and type

6, E, and

The phosphorylation

to be associated

protein was prepared

HL-60

P47,

from human platelets,

this group

cDNA clones

a protein having designated

protein

Type I PKC is

tissue and spinal

from the rat brain cDNA library

weight of 47,000,

purified

(2,4,6).

nervous

8(8I

in many tissues and cell types

three additional

~, have been isolated

protein

(2-5), which have y-,

respectively

in the central

can be

type I, II, and III, on a

type II PKC is found in several

III PKC is present (7).

fractions,

column chromatography

exclusively

whereas

by alternative

PKC from the rat and rabbit brains

(16).

expressed

is

This in

of the platelet

subspecies.

EXPERIMENTAL

PROCEDURES

Protein purification --Washed human platelets are prepared by the method of Baenziger and Majerus (19). All subsequent procedures were carried out at 0-4°C. Platelets (2 x 1010 cells) were disrupted by sonication in i0 ml of 20 ___~ Tris-HCl at pH 7.5, containing 0.25 M sucrose, i0 ~_ EGTA, 2 ~_~ EDTA, 1 mM phenylmethylsulfonyl fluoride, and i0 pg/ml leupeptin. The homogenate was centrifuged for 60 min at i00,000 x g. The supernatant was applied to a TSK DEAE-5PW column (Toyo Soda, Tokyo, 0.75 x 7.5 cm) which was connected to a Pharmacia FPLC system and equilibrated with 20 ___~ Tris-HCl at pH 7.5, containing 0.5 mM EGTA, 0.5 ___~ EDTA, and i0 ~ 2-mercaptoethanol (Buffer A). The column was washed with Buffer A, and proteins were eluted by application of a 20-ml linear concentration gradient of NaCI (0 to 400 ~_~) prepared in Buffer A. Fractions of 1 ml each were collected and aliquots of each fraction were 1388

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH.COMMUNICATIONS

employed for PKC assay and detection of P47 protein as described below. PKC and P47 protein were eluted separately from the column in fractions 9 to 12 and 14 to 18, respectively. T h e P47 protein fraction was dialyzed against Buffer A and used for the analysis of phosphorylation. PKC fraction obtained from t~he column was applied to a packed hydroxyapatite column (KOKEN Co. Ltd., Tokyo, type S, 0.78 x i0 cm) connected to the FPLC system and equilibrated with 20 mM potassium phosphate buffer at pH 7.5, containing 0.5 ~ EGTA, 0.5 ~ EDTA, 10% glycerol, and i0 mM 2-mercaptoethanol (Buffer B). The column was washed with Buffer B, and PKC was eluted by application of a linear concentration gradient of potassium phosphate buffer at pH 7.5 (20 to 215 mM) prepared in Buffer B. Fractions of 1 ml each were collected. Rat brain type I, II, and III PKC were purified as described (20). PKC assay PKC was assayed by measuring the incorporation of 32pi into HI histone from [7-32p]ATP in the reaction mixture (0.25 ml) containing 20 ~_~ Tris-HCl at pH 7.5, 10 9_~ [Y32p]ATP, 5 ~_~ magnesium acetate, and 200 ug/ml HI histone under the conditions specified earlier (4) in the presence of various concentrations of CaCl 2, phosphatidylserine, diolein, and arachidonic acid as indicated in each experiment. P47 pr0tein phosphorylation --P47 protein purified partially by TSK DEAE-5PW column as described above (35 ug protein) was incubated with PKC subspecies for i0 min at 30°C. The reaction mixture (0.i ml) contained 20 ___~ Tris-HCl at pH 7.5, 10 ~M [y-32P]ATP, 5 mM magnesium acetate, 0.3 __mM CaCI2, 0.2 ~___ EGTA, 12 ug/ml phosphatidylserine, 1.2 ug/ml diolein, P47 protein, and PKC. The radioactive P47 protein was separated by SDS-polyacrylamide gel electrophoresis. The gel was dried on a filter paper and exposed to an X-ray film. The relative intensity of ~ the radioactive protein bands visualized by autoradiography was determined by densitometric tracing at 430 nm as described (9). Immunoblot analysis----Immunoblot analysis was carried out as described (7). Briefly, PKC fraction to be tested was subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose filters. The filter was reacted with subspecies-specific antibodies, and stained by the avidin-biotin peroxidase complex method. The antibodies against PKC subspecies, were raised against synthetic oligopeptides which are parts of the deduced amino acid sequences of PKC (7). The antibody CKpClS-a reacts equally with ~-, 8I-, BII-, and, y-PKC, and the antibody CKpVlS-a reacts with 8(8I and 8II)-PKC, specifically. RESULTS The human platelet

PKC and P47 protein were separated

TSK DEAE-5PW column equipped with high performance matography.

The PKC recovered

two major peaks when subjected matography

(Fig.

IA). I

by a

liquid chro-

from the column was resolved to hydroxyapatite

into

column chro-

The first and second peaks,

a and b,

I A minor protein kinase was found at the fractions 52 through 58, between the positions of peak a and b. This minor protein kinase was dependent on Ca 2+ and phospholipid, but the identity of this enzyme remains to be clarified. 1389

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

~ T

f

7

A peak a 3 0.2

peak 2

b

-

& 0.i

p

0

U

o~ Z H

6

Z

4

03 © m #4

B

m H

type II 0.2

type

--

O O r~ 0.i

0

0 2O

4O FRACTION

60

80

NUMBER

Fig. i. Hydroxyapatitecolumn chromatography of PKC from human platelets and rat brain. PKC from the supernatant of human platelets and rat brain were subjected to hydroxyapatite column chromatography and assayed as described under "EXPERIMENTAL PROCEDURES." A, PKC from human platelets; B, PKC from rat brain. ( O - - Q ) , protein kinase activity in'the presence of 8 ~g/ml phosphatidylserine, 0.8 pg/ml diolein, and 0.3 mM CaCI2; (0---0), protein kinase activity in the presence of 0.5 ~___ EGTA instead of phosphatidylserine, diolein, and CaCI2; (--), potassium phosphate.

were eluted at the positions II (fractions through 70),

corresponding

to the rat brain type

41 through 50) and type III PKC respectively

(Fig.

IB).

(fractions

Immunoblot analysis

62 indi-

cated that both platelet enzyme peak a and b react with the antibody,

CKpClS-a,

and the immunoreactive enzymes showed an

approximate m o l e c u l a r

weight of 80-kD

(Fig.

was raised against a synthetic peptide, which is a part of the conserved portion of the rat brain PKC

(8).

This antibody

FARKGALRQKNVHEVKNHKF,

region C I in the amino-terminal

(8-PKC),

brain type I, II, and III PKC equally. found in 6-, e-, and ~-PKC

2A).

and thus reacted with the This sequence is not

On the other hand,

the platelet

enzyme peak a, but not peak b reacted with the antibody, a, which was raised against a synthetic peptide, 1390

CKpVIS-

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

B

116 986645-

1 2

3

4

5

1

2

3

4

5

Fig. 2- Immunoblot analysis of PKC fraction from human platelets and rat brain. PKC fractions of hydroxyapatite column chromatography were subjected to immunoblot analysis as described under "EXPERIMENTAL PROCEDURES." A, B; immunoblot analysis by polyclonal antibodies, CKpC18-a and CKpV18-a, respectively. !, 2; peak a and b PKC from human platelet, respectively. ~, 4, 5; type I, If, and Ill PKC from rat brain, respectively. Arrow indicates the position of PKC. Molecular weight markers used are shown in kilodaltons: 116-kD, B-galactosidase; 98-kD, phosphorylase b: 68-kD, bovine serum albumin; 45-kD, ovalbumin.

PAAGPPPSEGEESTVR, region

a sequence

Vl of the

rat brain

Enzymological acceptor shows type

revealed,

distinctly II PKC.

responded (Fig. for

The

only

3A).

peak

criteria

so far

3B).

the brain

examined

analogous

glycerol. inhibitory.

Higher

a responded

concentrations

required of

the

the p l a t e l e t

on Ca 2+, and

shown

the p l a t e l e t identical

in all

It is w o r t h

noting

to free

specific

the p l a t e l e t

on Ca 2+ nor

In c o n t r a c t ,

3D).

to the b r a i n of

peak

the b r a i n

II PKC are

III PKC w e r e

3C and

2B). 2

and d i a c y l g l y c e r o l

hand,

peak

activation

not d e p e n d

to d e p e n d

On

the other

(Fig.

enzyme from

type

type

PKC

as a p h o s p h a t e

of the brain

(Fig.

in a m a n n e r

did

appear

in the v a r i a b l e

81I)

properties

to p h o s p h o l i p i d

acid

This

(8I and

the p l a t e l e t

not

enzyme

acid

that

did

the p l a t e l e t

4).

HI h i s t o n e

enzyme

that

(Fig.

with

kinetic

(Fig.

b and

II

however,

partially

present

type

different

The p r o p e r t i e s

comparison

enzyme

studies

uniquely

arachidonic type

enzyme

phospholipid free enzyme

fatty peak

I PKC

by a r a c h i d o n i c or d i a c y l -

acid a,

was

like

the

2 The platelet peak a contained proteins having approximate molecular weight of 45- and 30-kD, which reacted with the antibodies CKpC18-a and CKpVIB-a, respectively. These proteins do not seems to be proteolytic fragments of PKC. The identities of these immunoreactive proteins are not known. 1391

Vol. 155, No. 3, 1988

150

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

B

-

C

D

E~ I00 u

I

o

0 50

0

6

5

4

3

6

5

4

CaCI 2

(-log M)

F__~g. ~. Effects of phosphatidylserine, diolein, and various concentrations of Ca2+ on PKC activity from human platelets and rat brain. PKC fractions of hydroxyapatite column chromatography were dialyzed against Buffer A containing 10% glycerol and assayed in the presence of 8 ug/ml phosphatidylserine, 0.8 ~g/ml diolein, and various concentrations of CaCI2 as described under "EXPERIMENTAL PROCEDURES." Where indicated with arrows, 0.5 mM EGTA was added instead of CaCI2. Results were normalized to the maximal activity obtained in the presence of phosphatidylserine, diolein, and 0.3 mM CaCI2. A, peak a PKC from human platelets; B, type II PKC from rat brain; C, peak PKC from human platelets; D, type Ill PKC from rat brain. (Q---O), in the presence of phosphatidylserine and diolein; ( 0 ~ ) , in the presence of phosphatidylserine alone;(A--A), in the presence of diolein alone; and (~--~), in the absence of phosphatidylserine and diolein.

brain

type

trations The brain

the of

activated

isolated

P47

obtained

was

acid.

subspecies

all

capable

protein,

glycerol,

PKC,

PKC

absolute the

III

of a r a c h i d o n i c

were

genous

II a n d

P47

and with

HI

rates.

although Table

phosphorylation

Ca 2+.

human

of p h o s p h o r y l a t i n g

protein,

reaction

protein

from

by h i g h e r

The

platelets

it

is d i f f i c u l t

I summarizes

kinetics

historic as a p h o s p h a t e

and

the p l a t e l e t

the

on p h o s p h o l i p i d ,

reaction

concen-

are

rat

endoto c o m p a r e

dependency diacyl-

similar

to

those

acceptor.

DISCUSSION

The encoded present tional

results

presented

by s - s e q u e n c e in p l a t e l e t s . subspecies

above

is a m a j o r

show

which

type

subspecies

In a d d i t i o n

is f o u n d

that

to

this

elutes

1392

of

III the

PKC

is

enzyme

subspecies,

from

which

an a d d i -

the h y d r o x y a p a t i t e

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i

I

I

I

I

I

I

I

B

A

_

i00

type III --

II

oo

0i 0

{

1

1

1

i00

200

300

400

ARACHIDONIC

i00

200

ACID

(uM)

300

400

Fi~. 4. Effects of arachidonic acid on PKC activity from human platelets and rat brain. PKC fractions of hydroxyapatite column chromatography were assayed in the presence of 0.3 mM CaC12 and various concentrations of arachidonic acid as described under "EXPERIMENTAL PROCEDURES." Results were normalized to the maximal activity obtained in the presence of 8 vg/ml phosphatidylserine, 0.8 vg/ml diolein, and 0.3 mM CaC12. A, PKC from human platelets; B, PKC from rat brain. (A--A), type I PKC from rat brain; (O ~), peak a PKC from human platelets and type II PKC from rat brain; (O---O), peak b PKC from human platelets and type Ill PKC from rat brain.

column and

in

the

same

BII-sequence.

different although

from it

is

the

fraction This

as

the

platelet

brain

type

recognized

by

II the

brain

type

II

enzyme

shows

PKC

briefly

as

antibody

PKC

having

propertles

which

outlined is

BI-

clearly above,

reactive

with

Table I Phosphorylation of P47 protein by PKC subspecies

Human platelets

Complete system a (-) phosphatidylserine (-) diolein (÷) EGTA b a

b

Rat brain

Peak a

Peak b

Type

I

Type

II

Type

III

(%)

<%)

(%)

(%)

(%)

100 62 83 56

100 22 83 24

100 21 81 9

100 16 83 87

100 22 82 20

P47 protein was phosphorylated by each enzyme subspecies in the presence of Ca2÷, phosphatidylserine, and diolein under the conditions specified under "EXPERIMENTAL PROCEDURES." Results are normalized to the maximal phosphorylation rate in the presence of Ca2+, phosphatidylserine, and diolein. EGTA (0.5 raM) was added instead of Ca 2+, phosphatidylserine, and diolein.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the amino-terminal portion of this brain PKC subspecies.

The

relation of this enzyme to 6-, e-, and ~-PKC is not clear.

At

present, it may not be ruled out that the platelet enzyme peak is a mixture of some PKC subspecies. The phosphorylation of P47 protein by this newly found platelet enzyme proceeds without added Ca 2+, and depends on phospholipid and diacylglycerol only partly.

However, in

general, the in vitro dependency of PKC on Ca 2+, and various lipids varies markedly with the phosphate acceptor employed (21), and, in the most extreme case, no activator is needed with protamine as a substrate (22).

Another problem is that, i__n_n

vitro, most protein kinases phosphorylate many proteins, only some of which are physiological substrates.

The possibilities

that the PKC subspecies respond differently to the activators i_nn vivo, and that some of the substrate preferences of these enzymes will result from the location of the substrates as well as the enzyme molecules in various intracellular compartments, greatly complicate the search for the true physiological target proteins for each subspecies.

It remains to be identified which

PKC subspecies described above is responsible for the P47 protein phosphorylation in intact platelets. ACKNOWLEDGMENT: We are grateful to Sachiko Nishiyama and Yoko Goto for their skillful secretarial assistance.

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