Vol.

167,

March

No.

16,

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1990

767-771

INVOLVEMENT OF PROTEIN KINASE(S) IN THE INTRACELLULAR SIGNAL TRANSDUCTION PATHWAYS FOR ACTIVATION AND ADAPTATION OF ADENYLATE CYCLASE IN DICTYOSTELIUM DISCOIDEUM MXSAKAZU 1 Department

*

KOU KUBOTX*

Botany,

Faculty of Science, Kyoto 606 Japan

of

Laboratory

Received

OYAMAl'*'+,

of Shosha

January

31.

Biology, 216i,

Himeji Himeji

Hyogo

and

KOJI

OKAMOTO'

Kyoto

Institute 671-22

University,

of Japan

Technology,

1990

Binding of cyclic AMP (cA>lP) to cell surface receptors induces activation and adaptation of adenylate cyclase while 2,3dimercapto-1-propanoi (BXL) acts only on the activation pathriay. Here we show that an inhibitor of protein kinase (K252a) inhibits the c.WP-induced activation of the cyclase but not (rather enhances1 the BAL-induced activation. These results suggest that protein kinase is involved in transduction of the actix-ation signal that phosphorylation might take and place between the receptor and the action site of BAL. Since adaptation causes cessation of the activation, the enhancement of the BAL-induced c.QlP accumulation by K252a might imply that K252a also blocks transduction of the adaptation signal. 0 1990 Academic Press, Inc.

Extracellular slime

c.4MP

mould,

the

Dictrostelium

specific and

and

Kubota

receptors

reagents

such

activation B-IL

and

is

transduction +After to M. Technology,

on

as

(5)

useful

to for

of

Oyama

the

cell

(7)

but

adenylate showed or

not

distinguish these

April 1, 199U, Oyama, Laboratory Shosha 2167,

the

the

Binding

of

surface

that BAL

CAMP

for

specificaiiy

activation Oyama

bond-breaking stimulate

pathway. involved

to two

Recently,

disulfide

mechanisms

cellular

activates (2)

cyclase.

adaptation the

txo

in

pathways

dithiothreitol

pathway

a pheromone (11.

transduction

adaptation (6)

as

discoideum

signal

intraceliular (3,41

works

the

Therefore, in

signal

pathways. all

correspondence should of Biology, Himeji Himeji Hyogo 671-22 Japan.

addressed be Institute of

The abbreviations used are: BAL, 2,3-dimercapto-1-propanol; c:-\MP , 3 ' : 5'-cyclic adenosine monophosphate; dcA?1P, deosyadenosine 3' :5'-cyclic monophosphate. 0006-291X/90 767

2'-

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

167,

No.

inhibits

K253a

IY,9,101. ha\.-e

BIOCHEMICAL

2, 1990

protein

Iqhile

side

cellular

due

components

lesser

fact,

the

inhibition

known

action

of

of

the

has signal

Dictyostelium,

it

competing

with

of

non-specific as

is

protein

.\TP

prorein

kinase

interactions

phospholipids,

this

with

G!52a/,,might

a neutral

kinase

been

has

been

modifies

the

of of

substance.

acti\,ity

is

intracellular

signal

important

In

the

.

021x-

the

In

phosphorylation

is

(11,12,13) of

cyclase

the

and

receptors

actions

of

BAL

is

involved

that and/or

(11,15,16,17).

phosphorylation

for

organisms.

that

receptors

transduction

mechanism

many

characterisics

specific

whether

an in

adenylate

the

investigated

as

reported

of

phosphorylation activation

known

transduction

modification

advantage

COMMUNICATIONS

K252a.

Phosphorylation

in

to

since

by

RESEARCH

inhibitors

such

effect

side

transmembrane involved

known

probably

have

BIOPHYSICAL

kinase

prex-iously

effects

charged

AND

Taking and

Ii252a, in

we

the

two

pathways.

METHODS Cellular slime mould, Dictvostelium discoideum NC4 amoebae shake-cultured with Escherichia coli B/r. Amoebae were were washed and developed on a nitrocellulose filter. Cells at the stage were collected and shake-cultured at 1.5x10' aggregation cells/ml in 20 mM phosphate buffer (pH6.4) containing 10 mM KC1 with or without Ii252a or staurosporine (Ryowa Hakko). K252a and staurosporine were dissolT.;ed in dimethylsulfoside and l/300 added to the ceil suspension. volume was After 20-30 min of shaking, cells were stimulated with l/50 volume of dcAMP (final 10 uM) or BAL (final 3 mM). 60-100 ul of the cell suspension was into a microtest-tube containing HCIO1. sampled The amount of CAMP in the cell lysate was assayed with an Amersham CAMP assay kit.

RESULTS

and

dc.ilP

DISCUSSION ,

an

surface

and

without

disturbing

analogue

eTvokes

is

ceased

at

is

also

Induced

Z min by

BAL

(7)

(Fig.

either is

after

ceased

binds

the

1-i)

to bp

BAL

itself

adaptation

receptors

of

adenglate

the

c.UP or

to

768

on

celi

cyclase accumulation

to

adaptation

accumulation

of

\

Involvement of protein kinase(s) in the intracellular signal transduction pathways for activation and adaptation of adenylate cyclase in Dictyostelium discoideum.

Binding of cyclic AMP (cAMP) to cell surface receptors induces activation and adaptation of adenylate cyclase while 2,3-dimercapto-1-propanol (BAL) ac...
293KB Sizes 0 Downloads 0 Views