Vol.

177,

No.

June

28,

1991

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1033-1040

Pages

NADPH-OXIDASE ACTIVATION

P. Sharma,

A.T.

Department

of

BY PROTEIN KINASE C-ISOTYPES

Evans,

P.J.

Parker*

Pharmacognosy,

University

of London,

The School

29-39

Cancer

Lincoln's Received April

Research

Inn

of

Brunswick

London WClN lAX, *Imperial

and F.J.

Evans1

Pharmacy

Square

U.K. Fund Laboratories

London WC2, U.K

Fields,

23, 1991

The cell free activation of NADPH-Oxidase in membranes Abstract of mouse peritoneal macrophages by purified PKC-isotypes was investigated. Unstimulated iqtrinsic activity of PKC-isotypes showed little dependence on Ca for activation of the oxidase. In the presence of TPA, the activation of the oxidase2yas greatly enhanced, and a-, and Y-subtypes were strongly Ca dependent in this system and E-subtypes were active both in the and abserfjc-'of'free Ca2+ ions. presence The results suggest S+ levels certain PKC-isotypes that at resting Ca can activate NADPH-oxidase. B1991Academic Press,Inc.

Protein

kinase

superoxide

the

oxidase

of

analysis of

C (PKC) has been linked (02-)-generating

phagocytes

multiple

subspecies

initially

four

(3-5).

More recently

identified Phagocytes phagocytosis,

cDNA

produce or

having

clones,

and designated

protein

four

and

new members of

this

as PKC 6-,

E -,

substantial

amounts

upon stimulation

as a

related

PI,-

by a variety

y-

NADPH-

family

structure: were isolated

family

t-

of

and biochemical

C exists

closely

p,-,

a-,

enzyme

cloning

kinase a

phosphorylation

burst

Molecular

that

the

respiratory

(l-2).

have revealed

to

have been

and

of

q-

O2 of

(6-9). during

agents

(10).

1 To whomcorrespondence should be addressed. 0006-291X/91

1033

All

Copyright 0 1991 rights of reproduction

$1.50

bv Academic Rress. Inc. in any form reserved.

Vol.

177,

No.

Since

BIOCHEMICAL

3, 1991

the

oxidase

biochemical is

might

occur

to

NADPH

oxidase

(14).

The

distinct

roles

purified

for

the we

demonstrate

the

relative

requirements

and

the

activation the

of

activation in

of

cells

were

the

the

study

In

the

of study

NADPH-oxidase

a cell of

importance

proteins'

for

of

the

of

'activation

isotypes.

retention

by PKC

components

a potential

C isotypes

NADPH-

of

post-receptor

(ll-12),

kinase

examine

COMMUNICATIONS

pathways

of

has

various

kinase

Ca2+ -independent

used

therefore

RESEARCH

different

phosphorylation

complex

protein

several

phosphorylation

via

here,

presented

activation

agonist

direct or

of

involve

on the

via

BIOPHYSICAL

sequence

likely

depending

steps,

AND

free

by

system

and

co-factor

selective

characterised

recently

subtypes.

METHODS AND MATERIALS Preparation

of

Macrophage

Starch-elicited of

the

with

peritoneal peritoneal

10

serum,

ml 2%

After were

of

by washing

with of

log

in

in

miY WC12 sucrose.

t

Cells on

centrifugation

ice.

medium.

cells,

20g body

with

10%

Cells (30

min

peritoneal

5 mM EGTA in

weight)

foetal

gentamycin

w/ml

lavage

calf

(Flow

(400

x g ,

were

then

at cells

PBS (Sigma,

Labs., x 2)

the

enriched

by

3OOC).

Following

were

recovered

Poole).

Oxidase

cold 2

50

G25 column

ice-cold

starch

suspended

supplemented

by aseptic (e

by centrifugation

non-adherent

Preparation

15OW)

washing

CD1 mice

male

medium

on a Sephadex

elution

-

of

and

resuspended

incubation

lo8

RPM1 1640 glutamine

Irvine). cells

cavity

obtained

elicited

mouse

40 mM Tris-Cl,

mM NaN3,

pH 7.5,

2 mM PMSF,

were

lysed

Nuclei (1100

peritoneal

g

containing

cells

x

and

mins)

1034

and

(3 x 10s

and unbroken

were

10 mM EDTA,

2 mM Leupeptin

by sonication

10

macrophages

were the

post

10%

1

w/v

pulses

of

removed

by

nuclear

Vol.

177,

No.

3, 1991

supernatant

BIOCHEMICAL

layered

(10%/40%,

w/v) layer

supernatant

stored

bound

oxidase

and also

The

assay

determined reduction of

8.0)

2

by

a

reference

RESULTS

2

NaN3,

The

x g for

the

The

2h and

The

the

membrane-

40% sucrose

mM

PMSF,

the

rate

of

assay

of

assay

mix

C;

5.1

~1

layer

of

100

production

SOD-inhibitable in

a final

buffer

1 mM MgC12, and

2

cytosolic

an additional

addition

-

50 mM Nap04

mM EGTA,

50

O2

contained

2 mM leupeptin

and

contained

mM

(pH 0.1

M

DTT,

50

factor.

300U

The

The reaction

SOD.

PM NADPH co-factor.

AND DISCUSSION of

partially

and

as measured the

subsequent

were

by 02-

assaying

the &

fl-

transfer

and

and

was

off

was

a

limited of

y-

E- were

reached

below

the time

PKC-isotypes

assayed

with

3

threshold

for

duration

was

-ATP

rate

within

oxidase.

the

[ 32-P-y]

of

The maximal

production

suggesting

CY-,

NADPH-oxidase

compared.

and dephosphorylation of

and

membrane-bound

levelling

non-linearity

activity

purified

PKC isotypes

phosphorylation

by

from

PM cytochrome

protein

by

activity

assay

60 mins.

factor).

kinetic

20% glycerol,

by TPA-stimulated

mins

100,000

initial

C.

80

pl,

The activation

of

the

continuous

cuvette

started

g for

gradient

(11).

cytochrome

membrane

Pl

was

essentially

700

mM

recovered

was

containing

NaCl,

was

sucrose

100,000

at

COMMUNICATIONS

Oxidase

of

volume

at

(cytosolic

-7O'C

RESEARCH

discontinuous

-7OOC

activity

of

BIOPHYSICAL

recentrifuged

at

at

a

centrifuged

was

stored

Activation

onto

and

10% sucrose

AND

to

peptide

of Specific

standardised histone

IIIS,

substrates

(15.16). Linear

with

observed used

increments

dose-dependent

(0.01

the - 1.0

in

concentrations u/ml)

of

(Table 1035

1).

the CX ,

activity

oxidase Pr 6-

and and

were

Y-isotypes E- were

not

Vol.

177,

No.

TABLE

3,

BIOCHEMICAL

1991

1.

Dose-dependent

isotypes

in

purified

by the

20°C

in

0.02%

AND

the

activation

presence

of

method

50% glycerol Triton buffer

activity

in

activity,

the

range

prior

to

0.01

- 1.0

use.

stored

pH 7.5, were

diluted

in

to

an

U (nmoles/min/ml)

give

of

shown

as

kinase

nmol

of

Enzyme

Activity

(VI

0.234

+ 0.03

0.288

2 0.05

0.324

2 0.051

0.108

?1 0.05

0.144

2 0.05

0.126

c 0.018

,8+ ca*+

0.216

+ 0.051

0.270

? 0.285

0.306

f 0.020

8-

0.126

+ 0.025

0.144

2 0.036

0.144

+ 0,051

yt

Ca2+ Ca 2+

0.216

+ 0.051

0.270

2 0.025

0.288

i 0.060

y-

Ca2+

0.108

+ 0.06

0.162

t 0.02

0.162

?- 0.025

of

0.1

to

assay

low

0.1

the

quantities u/ml

response

of

of

by

isotypes

stimulation

in

these the

of Ca'+-ions.

presence

This

physiological

each

the

of

the

isozyme

the

as

in

activity

of

by

the

The due by

than

compared

oxidase

be

mediated

greater

observed

In

used. to

complex

showed Ca 2+

was

activation

oxidase

activity

available.

isozymes

Ca2' -dependency substrate,

with

consistent

of

suggests

phosphorylation

Activation

O2 -

1.0

~1+ Ca*+ a- Ca 2+

linearity

-

2 mM EDTA,

present)

is

at

0.01

subsequent

with

for a-,

standard

the fi-

the

to PKC.

2-fold absence

activation and

y-

is

kinaseihistone

(20).

Unstimulated

intrinsic

PKC-isotypes

were

oxidase

and

were

Enzyme

due

assays

(20)

isotypes

Activity

PKC-

by

S.D.

investigated

of the

COMMUNICATIONS

PKC-isotypes

Parker

The

Units

specific

TPA.

10% glycerol

only of

-1

20 mM Tris-Cl,

1mM DTT.

(except

produced/min

mL and

containing

RESEARCH

of NADPH-oxidase

5 ng.

of Marais

x-100,

similar

BIOPHYSICAL

(Table

2).

not

dependent

However

PKC-isotypes on

Ca

purified 1036

2+

for

basal

revealed activation

activity

that

the

of

the

of

PKC-

Vol.

177,

No.

3, 1991

TABLE 2. of

BIOCHEMICAL

Unstimulated

intrinsic

NADPH-oxidase.

minute

Activity

measured

by

fl-

and

Y-

6-

and

c- isozymes.

peptides

for

pseudosubstrate

(100

PM)

EGTA (10 mM)

18

Y

54

f

36

72

?r

36

6

144

f

36

126

t 18

E

126

+_ 36

162

f

lower this

is

in

all

cases

than

the

in

line

with

the

basal

The basal

assays.

times

more

effective the

absolutely

ester

stimulation activation

(100

/AM),

value

of

since

to

that

of

the

the

phagocytes

may

the

reference phorbol be due presently

ester to

the

presence

of

isotypes 1037

using

of

the PKC

the

Ca 2+ ions an

may

EC50 be

of

02-

by

complex

oxidase to

of TPA,

utilise

production

by TPA

for

This

TPA

phorbol

with

which

of idase

free

observed

of

E-

1).

responsible

phosphorylation

available

(Fig.

was

stimulated

and

suggesting

studies

their

presence

itself

cellular

in

NADPH-ox

va lue system

In

to

6-

the of

a dose-dependent response was -1 lo-20 ngml TPA for all isotypes. with

y-

standardised

In

activity

this

kinase

observed.

were

intrinsic

in

and

in E- was

and

of

111s. the

the

no response of

they

histone

enhanced

6-

activity

induced

observed

of p-

36

ester

activity

01-,

specific

and not

phorbol

activity

than

comparable

PKC isotypes compared

all

a -,

f

produced

by

111s for

Histone

36

substrates

that

is

* 18

alone

in

into

PKC-isotypes

54

although

importance

/min

of

per

B

response,

greater

Unit

S.D.

produced

18

4.0

5-fold

as pmol 02-

t

kinase

however,

activation

36

standard

peptide

mediated

f 18

and

not

COMMUNICATIONS

36

activity

were

RESEARCH

a

was

to

shown

Ca2+ ions

Isotype

2.0

is

and specific

isozyme

BIOPHYSICAL

PKC-isotypes

unit of enzyme 32 -P incorporation

per

isotypes

AND

a

similar

Vol.

177,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

(i)

COMMUNICATIONS

(ii)

0.4

. z w rl> ? .4 -c

0.3

0.2

‘0”

';t E c

0.1

0

3

100

10

0

1000 ng.ml

FIGURE

1.

isotypes Ca2+

Activation in

ions.

presence

O2

was determined reduction

5.2

sodium

the

contained

Triton

of

C (21000

0 2 - using

( l -

0)

molar

(ii)

of

g/ml

P.S.

by calculating

extinction

coefficient

the

text

10 mM EGTA or

and/or

expressed

of

PKC-isotype

in

10 mM MgC$,

M

100 assay

For

detailed

M ATP,

free

kinetic

C.

buffer

PKC-

TPA-stimulated

TPA

in

0.02%

the of

initial

cytochrome

SD. (0-n)

However,

increase

are

absence

cytochrome

100

1000

@ -PKC:

(x-x)Y-PKC;

(A-A)

E-PKC

6 -PKC.

independent

ain

O2 -

concentrations.

activation

the

M-l .cm -l)

0 ) a -PKC;

were

with

Results

( o-

degree.

100

by

by a continuous

phosphate

together

X-100.

and

of

an additional

mM CaC12

rate

(i)

100

TPA

NADPH-oxidase

the

SOD-inhibitable studies

of

10

I

-1

in

the

and

E-

active is

also

of

showed

production The

weakly

presence

in

an

10 mM EGTA, active

response

in

the

observed

absence witih

Cxof

Ca

o-subtypes 1038

2+

.

ca2+-

dose-dependent

to

Ca2+ -dependent

the

increasing and

y-

The but

TPA subtypes

Ca2+ -dependent it

also

shows

a

Vol.

177,

No.

3, 1991

BIOCHEMICAL

significant

ca*+

demonstrated

that

was

able

was

to

-independent

stimulate

02by

resiniferatoxin,

probe

phosphorylation In

this

a

potent only

certain

we have

isotypes

can

calcium

was

stimulates in

the

presence

demonstrated

activate

at

of

NADPH-oxidase

(19),

and

demonstrated

(141,

individual

with

restricted

identify

not.

isotype

Using

(18),

substrate

cell

free

been

(14). Ca 2+ -

minimal in

cell

implicated

free in

specific

selective

specificity

selective

reconstituted

has

the

protein PKC-isotype

diterpene it

in

calcium

NADPH-oxidase

PKC

This

@-isozyme

that

activation

has

agent

excluded.

of

purified

activation

Rx-kinase

the

Although

mediated

Rx-kinase,

(10).

systems.

phosphorylation

have

pro-inflammatory

when

only

we

termed

assays

weakly

and

communication

levels

cell-free

COMMUNICATIONS

Recently kinase,

in

also

assays

RESEARCH

activity.

Rx (171, related

BIOPHYSICAL

a calcium-inhibited

stimulated

phorbol

AND

esters

may be possible

phosphorylation

to

in

this

system.

ACKNOWLEDGMENTS We are

grateful

and to

the

to

Olivier

Dr. A.

Medical

Research

for

Council

preparation for

of

a project

PKC-6

grant.

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Rossi,

T.L.,

RESEARCH

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A.,

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