Concanavalin A prevents phorbol-mediated redistribution of protein kinase C and β-adrenergic receptors in rat glioma C6 cells

Vol. 144, No. 3, 1987 May 14, 1987

BIOCHEMICAL

AND BIOPHYSICAL

CONCANAVALIN A PREVFNTS PHORBOL-MEDIATED PROTEIN KINASE

C AND 8-ADRENERGIC

Jitendra

Pate1

RESEARCH- COMMUNICATIONS Pages 1265-1272

REDISTRIBUTION

OF

RECEPTORS IN RAT GLIOMA C6 CELLS

and Shouki

Kassis

Unit on Neurochemistry, Biological Psychiatry Branch, NIMH, and the Membrane Biochemistry Section, Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, The National Institutes of Health, Bethesda, Maryland 20892 Received

March

11,

1987

Summary: Exposure of rat glioma C6 cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPk) caused an activation of protein kinase C wherein the enzyme rapidly became membrane-bound (TQ of 15 min). This translocation of protein kinase C from cytosol to membrane was followed by a sequestration of cell surface 8-adrenergic receptors and a loss of isoproterenol-stimulated adenylate cyclase activity. We had reported previously that prior exposure of rat glioma cells to concanavalin A prevents the TPA-mediated sequestration of receptors and desensitization of adenylate cyclase (Kassis et al., 1985). We now show that the concanavalin A treatment also prevents the translocation and activation of protein kinase C. These results are further evidence that in the TPA-treated cells, sequestration of 8-adrenergic receptors is mediated by membrane-bound protein kinase C.

It Ca+2-

is well-established

that

tumor-promoting

and phospholipid-dependent

protein

kinase

process

involves

the

translocation

of

plasma

membrane

(2,3).

Once

an active,

catalyzes

the

responses. tion of

of the

phosphorylation In

protein

a previous kinase

of paper,

C in rat

B-adrenergic-stimulated

agonist-mediated

in

desensitization

protein it

phorbol

protein

C (1). kinase

C from

C6 cells

the form,

and thus

was demonstrated

glioma

activate

the

The activation

membrane-bound substrates

esters

that

resulted

cytosol the

alters

the

adenylate

cyclase

by a mechanism

(4).

The former

desensitization

lhbbreviationa: TPA, 12-O-tetradecanoylphorbol 13-acetate; canavalin A; CGP-12177, 4-(3-tert-butylamino-2-hydroxylpropoxy)benzimidazole-2one hydrochloride; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

cellular activa-

desensitization distinct did ConA,

from not

in-

conacid.

0006-291X/87

1265

the

enzyme

TPAl-induced in

to

$1.50

Vol. 144, No. 3, 1987 volve

a change

cause

the

Upon in

in the

receptors

sucrose

functional

Treatment TPA prevented

of

the

both

report,

and ConA on the

for ceeds ness.

subcellular the

We further

the g-adrenergic from

centrifugation,

rat

glioma

the

the

C6 cells

activation

adenylate

sequestered

receptors

but

cyclase

activity.

receptors

adenylate

of

show that

individual

kinase

cyclase.

ConA blocks

to

their

did

appeared

and combined

C and the

kinase

C from

and the

reduction

the

translocation

exposure

(4).

In

effects

that cytosol

the

of

the

time

to membrane

in agonist of

to

of TPA

desensitization

We demonstrate

of protein 6 receptors

ConA prior

and desensitization

the

of protein

translocation

with

sequestration

we now characterize

sequestration

RESEARCH COMMUNICATIONS

fraction.

receptor

$-adrenergic-stimulated

of

segregate

gradient

membrane

AND BIOPHYSICAL

activity

to physically

density

a "lighter"

present

BIOCHEMICAL

course pre-

responsive-

protein

kinase

C.

MATRRIALS AND METHODS Materials - Concanavalin A was obtained Biochemicals for Cancer Research (Eden Praire, was obtained from Amersham Corp. Sources of scribed previously (4). Cell Culture - Rat glioma Cells were exposed to the (5). Eagle's medium buffered with 25 for the times indicated in the cells were incubated with ConA

from Sigma and phorbol ester from MN). [3H]CGP-12177 (38 Ci/mmol) all other reagents have been de-

C6 cells were cultured as described previously various agents in serum-free Dulbecco's modified nM Hepes (4). Cells were exposed to 100 nM TPA table and figure legends. Where indicated, the (0.25 mg/ml) for 45 min at 370C.

Assay of Protein Kinase C - Cytosolic and membrane-bound protein kinase C activity was assayed as described previously (4). Briefly, the cells were washed and scraped in phosphate-buffered saline and pelleted by centrifugation at 1000 x g for 10 min. The pellet was homogenized in Solution A and centrifuged at 100,000 x g for 1 h to obtain a cytosolic fraction (4). The pellet was suspended in Solution A and both fractions were adjusted to contain 1% Triton X-100, kept on ice for 30 min and centrifuged at 100,000 x g for 1 h. The supernatants were further purified on DEAE-cellulose columns as described previously (4) except EGTA was omitted from the buffer. Protein kinase C activity was determined as described previously (4) using a modification of the method of Kikkawa et al. (6). Binding of [~II]cGP-~~L~~ - Cells were lysed and crude membranes pared and assayed for specific binding of [3H]CGP-12177 as described Briefly, membranes were incubated in 0.5 ml of 20 ti Hepes (pH (7). mM NaCl, 5 mM MgC12, and 1.7 nM [3H]CGP-12177 for 1 h at 300C. Bound ligand was collected on glass fiber filters and quantified by liquid tion counting. Nonspecific binding was determined in the presence of propranolol. were

Other Methods - Assay performed as described

of adenylate previously

cyclase (4). 1266

and determination

were preelsewhere 7.5), 150 scintilla2 UM (+)-

of protein

Vol. 144, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS Kinetics resulted ing

of

Ester

in a decrease

increase

enzyme min

(Fig.

in cytosolic

ted

adenylate

was time

1).

cyclase also

protein

dependent

T+ of

exhibited

as described

was time

dependent

of

kinase The

with

cells

activity

Desensitization

- Exposure

activity.

The TPA-treated

desensitization 2).

Effects

in membrane-bound

activity

(Fig.

Phorbol

glioma

C activity

subcellular 15 min

C6 cells

to TPA

and a correspondtranslocation

of

and was maximal

a loss

of

previously and reached

was specific

rat

after

30

isoproterenol-stimula-

(4).

This

a maximum

at

as NaF-stimulated

the

TPA-induced about

activity

60 min

remained

m 1

I

I

10

20

30

Figure

1.

I

I

40 50 MINUTES

Phorbol-ester

1

I

I

60

70

80

induced

’ lo

subcellular

1

I

A

1

2

3

I

90

0

2

redistribution

HOURS of protein

kinase

Rat glioma C6 cells were exposed to 100 nM TPA for the indicated times. After the cells were washed, harvested and collected by centrifugation, cytosolic (0) and particulate (0) fractions were prepared and assayed for protein kinase C activity as described under "MATERIALS AND MHPHODS." Values are expressed as percent of activity in control cells (see Table II for typical values) and are from a representative experiment. Similar results were obtained in a second experiment. Figure

2.

Phorbol-ester induced desensitization adenylate cyclase and sequestration

of

of isoproterenol-stimulated -adrenergic receptors.

Rat glioma Cb cells were exposed to 100 nM TPA for the indicated times. Crude membranes were prepared and assayed for net isoproterenol-stimulated (A) and NaF-stimulated (0) adenylate cyclase activity and [3H]CGP-12177 binding (0) as described under "MATERIALS AND METHODS." Values are the mean of triplicate determinations which varied less than 5% and are expressed as percent of control cells. For the latter, adenylate cyclase basal, 253; isoproterenolactivities as were in pmol/lO min/mg protein: stimulated, 1205; and NaF-stimulated, 1040. Specific [3H]CGP-12177 binding was 242 fmollmg protein. Similar results were obtained in two additional experiments. 1267

C.

Vol.

144,

No.

unchanged. due to

BIOCHEMICAL

3, 1987

It

has been

shown

redistribution

of

membrane

fraction

to a lighter

that

sequestration

the

measured

with

Exposure

of

binding ing

the rat

sites,

lag

Effect ment

of

was

density

also

plasma

has been

agonists

caused

time

- Table

desensitization

receptors.

adenylate

It

could

a decrease

readily

in

1 shows

of

adenylate

ConA by itself

cyclase

in bind-

of desensitization

with

activity

the

effect

Effect

cyclase

had no effect

and caused

a small

of ConA pretreatment desensitization

Treatments

and sequestration

of

on isoproterenol-stimula-

increase

in [3H]CGP-12177

and receptor

TPA

+ + +

Rat glioma

C6 cells

37OC and then with

specific

for

sequestration [3H]CGP-12177

minlmg

protein

[3H]CGP-12177

553+30

132+2

727+15 -

247+4 -

697+36

248+4

with

were obtained

in three

and without 1 h. adenylate

as described

determinations

protein

187+3 -

100 nM TPA for

binding

fmol/mg

796+27 -

were incubated

and without

Bound

net isoproterenol-stimulated

-+ SD of triplicate sults

ester-induced

activity pmol/lO

and assayed

on phorbol

Isoproterenol-stimulated

+

a

of ConA pretreat-

TABLE1

ConA

be

[3H]CGP-12177

The decrease

course

found

(7-9).

sequestration.

the

desensitization

2).

ConA on TPA Action

on TPA-mediated

g-adrenergic ted

(Fig.

followed

(4).

[3H]CGP-12177

to TPA also

COMMUNICATIONS

a heavier

by $-adrenergic

due to receptor but

from

fraction

antagonist

C6 cells

dependent

substantial

induced

RESEARCH

the TPA-mediated

receptors

membrane

hydrophilic

BIOPHYSICAL

that

f3-adrenergic

presumably

was time

earlier

process

glioma

AND

in Figure

from a representative

additional

experiments. 1268

ConA for

5 min at

Membranes were prepared cyclase 2.

activity

and

Values

are the mean

experiment.

Similar

re-

Vol. 144, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL

TABLE Effect

of

ConA

II

pretreatment

on

translocation

of

Treatments ConA

TPA

RESEARCH COMMUNICATIONS

phorbol

ester-induced

protein

kinase

C

Protein

kinase

C activity

Cytosol

Membranes

-PS/DAG

+PS/DAG

cpmlminlmg

2720

a710

2080

7240

2970

47900

-I-

1540

14900

2 580

aa3o

+

1980

15800

1860

a365

glioma in

and

in

10% of

. on

both

localized its

protein the

1.

value

and

C activity

in

the

variation

without

ConA

membrane

fractions

the

(DAG)

represents The

and

Cytosol

diacylglycerol

treatment of

these

glioma in

C6

the

absence

and

as described

mean

of

between

two

and

TPA

de-

prepared

presence

under

separate

as

were of

phos-

“EXPERIMRNTAL

experiments,

two experiments

the

particulate

or

however,

processes

in

agreement

the

activation

bulk

prior

activity

C became

the

ConA,

and

fraction. both

with

cells,

cytosol

distribution kinase

prevented

Table

with

each

was

less

than

mean.

Prior

rat

and

Each

incubated

kinase

triplicate.

the

ins

to

(PS)

. ”

were

protein

serine

assayed

In

legend

for

PRGCRDURES

TPA

C6 cells

the

assayed

phatidyl

on

protein

17050

scribed

of

+PS/DhG

2900

Rat

bind

-PS/DAG

of

and

Prior

the

treatment and

translocation

the

protein

kinase ConA

When

the

cells

bulk

of

the

activity

of

the

protein

21t was observed previously that ConA pretreatment of [3H]CGP-12177 binding in cell lysates and the recovery This effect may be related to in crude membranes (7). reduce the amount of receptors appearing in the lighter tion of control cells (4).

1269

had

cells of

subsequent

earlier

with

II). the

the

with

treatment (Table

activated

blocked

with

effects

study

(4).

C activity no

is

significant

were

exposed was

ConA kinase

effect to

TPA,

recovered

in

completely C

to

the

par-

increased the amount of total receptors the ability of ConA to density membrane frac-

Vol. 144, No. 3, 1987 ticulate

BIOCHEMICAL

fraction.

no effect

ConA,

on activity

when

(data

added

not

AND BIOPHYSICAL directly

to

RESEARCH COMMUNICATIONS

the

protein

kinase

C assay,

had

shown).

DISCUSSION Desensitization transduction; The

represents

thus,

present

report

sitization

of others

tein

kinase

min,

about

occurred

extends

(2,3), C from

the

mechanisms

receptors

process. adenylate

event

plasma

membrane

the

that

such

may involve away

ester

and was not

appears

the

Thus,

until

are of

regulatory

1). 1 h.

the

fully

within

g-adreuergic

30

of

receptors

.4s it

receptors

pro-

was an even

functional

and catalytic

of

Sequestration

binding the

with

Desensitization

sequestration.

receptors

a redistribution

from

fraction.

of

desen-

In agreement a translocation

uncoupling

signal

interest.

caused

(Fig.

their

of

ester-induced

t3H]CGP-12177

to preceed

process of great

(4).

maximal

by loss

uncoupled

are

cyclase

was membrane-bound

rate

the

of phorbol

particulate

the TPA-mediated

cyclase

initial

finding

phorbol

as measured

Thus,

previously

to

activity

at a much slower

slower

that

in

involved

adenylate

cytosol

the

adaptation

previous

we found the

90% of

S-adrenergic

shown

underlying

b-adrenergic-stimulated

work

from

the

a primary

in

had been (4),

the

components

the

plane of

of

the

adenylate

cyclase3. The quent

of

phosphorylation

process vious in

translocation

of

of

uncoupling

substrate

for

protein

erthyrocytes

that

adrenergic

receptors

(10,ll).

also

sequestration.

was demonstrated

and phorbol to have

esters

several

kinase

C to

specific,

yet

unidentified

and sequestration

avian

no receptor

protein

kinase phorbol

of C is esters

Although

potential

Finally,

the S-adrenergic receptor

in

the

presence

S-adrenergic for

has been

protein

there

6-adrenergic of

was

receptor

protein receptor

kinase

shown

of g-

was observed, turkey

sites

It

the An ob-

phosphorylation

the

subse-

may trigger

itself. the

and its

receptors.

of

mammalian

phosphorylation

membrane

proteins,

desensitization

system the

plasma

induced

Phosphorylation in a cell-free

(12).

the

the

kinase

C

appears

C (13).

'It is important to emphasize that although phorbol esterand agonistmediated desensitization of rat glioma C6 cells have many similarities, the two processes involve distinct mechanisms. Thus, S-adrenergic receptor function is altered by the latter but not the former and ConA prevents desensitization by the former but not the latter (4). 1270

Vol. 144, No. 3, 1987

BIOCHEMICAL

Cona effectively adenylate

cyclase

ConA also

effectively

membrane

with

effect

of

phorbol in

a basis

receptor

In

that

these

for

the

for

anism

by which

membrane

the

is

kinase

direct

C assay.

This

to

cytoplasmic

binds

to membrane

direct surface tein

Kohout

and critical

with

the the

occupied

lecttn

of

for

these

the

the

membrane

additional The mechthe

to the

pro-

protein

ConA receptors the

kinase kinase

and the

interfere

C to

ConA to glycoconju-

protein

of

and

and

kinase

whereas

activity

(16).

thus

from

in

C translocation

of

organization

and

opposite

ConA when added

enzyme

ConA is

the

processes.

binding

4).

plasma

(14)

provide

Presumably,

esters

of

ref.

the

desensitization They

membrane

membrane.

(17)

kinase

of protein

the

plasma

may perturb

by the

C in

attachment

required

linkages

C

binding through

inner

with

C

membrane

binding

of

pro-

C.

Acknowledgements Trudy

of

caused

ester.

to

effect

dissociated

effect

by Ca2+ and phorbol

or indirect

kinase

the

kinase

1;

monocytes

to block

kinase

consistent

phospholipids

mediated

when

side

lectin

to phorbol

of

This

ConA on protein

protein

side

II).

of

(Table

human peripheral

no direct

is

protein

C rapidly

the

with

We found

binds

is

of

unknown.

extracellular

process

of

ConA interferes

on the

of

response

role

gates

the

ability

of

ConA treatment

kinase

effects

in

in

studies,

protein

cytosol.

(Table

desensitization receptors

translocation

observed

RESEARCH COMMUNICATIONS

ester-inediated 8-adrenergic

cells

these

the

sequestration

support

of

the

recently

ester;

We believe provide

phorbol

ester-treated

(15).

and appeared

tein

blocked

those

thymocytes

the

and sequestration

in phorbol

agreement rat

blocked

AND BIOPHYSICAL

for

- We thank

growing

review

of

the the

Molly

cells

Sullivan

and Dr.

Peter

for

expert

l-i. Fishman

technical for

his

assistance, suggestions

manuscript.

REFERENCES 1. 2. 3. 4. 5. 6.

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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