Phorbol esters, but not insulin, promote depletion of cytosolic protein kinase C in rat adipocytes

Vol. 135, No. 3. 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1119-1125

March 28, 1986

PHORBOL ESTERS, Brendan

BUT NOT INSULIN, PROMOTE DEPLETION OF CYTOSOLIC PROTEIN KINASE C IN RAT ADIPOCYTES

P. Glynn,

Julie

W. Colliton,

Endocrine-Metabolism Division, Dartmouth Medical Received

December

30,

Joan M. McDermott,

and Lee A. Witters

Departments of Medicine and Biochemistry, School, Hanover, NH 03756

1985

insulinomimetic effects The tumor-promoting phorbol esters have Employing two different assay systems, we have in several tissues. este band insulin on the activity and compared the effects of phorbol intracellular distribution of the Ga and phospholipid dependent protein kinase (protein kinase C) in isolated rat adipocytes. Phorbol ester leads to a prompt depletion of kinase activity from the cytosolic fraction and appearance of activity in membrane extracts; neither of these effects is mimicked by insulin. These results, taken together important divergences between the actions of with other data, emphasize these agonists and suggest that changes in protein kinase C activity or intracellular distribution are not a necessary concomitant of the cascade of insulin action. @ 1986 Academic Press, Inc.

Phorbol effects hexose

esters

have been

in a variety

of tissues.

transport

[l],

hormone-stimulated

adenylate [4]

It

believed

generally

binding

of phorbol

protein

kinase

esters

lead

membrane Phorbol from

(protein

like

the hydrolysis

possible

role

to the kinase

that

insulin-like

effects

are

of the kinase

both

kinase

phospholipase on adipocytes

of

S6 protein initiated

with

occurring

C, which [9].

kinase through

of tissues,

its

action

[2,7,8]. generated

the kinase

and phospholipid

This

the

to the

activation

activate

generates

[5].

phorbol

the cytosol

diacylglycerols

C in insulin

S6

(PL)-dependent

from

associated

Ca*

inhibition of ribosomal

In a number

of phosphatidylinositol,

of protein

(21,

+ phospholipid

C) [6].

the naturally

Km for

of

stimulation

effects

Ca*

presumably

apparent

observations

[3],

the stimulation

of a ribosomal

these

to a translocation

esters,

the

cyclase

that

ester

include

of lipogenesis

and activation

compartment,

lowering

These

activation

phosphorylation is

shown to have many insulinomimetic

[6]. also

The

derives

diacylglycerol,

has lead

by

to the

from has suggestion

0006-291X/86 1119

All

Copyright 0 1986 rights qf reprodwcrlon

$1.50

by Academic Press, Inc. in any form reserved.

Vol.

135,

No. 3, 1986

that

insulin

BIOCHEMICAL

might

diacylglycerol.

activate It

this

would

to the translocation

AND

then

BIOPHYSICAL

lipase

RESEARCH

leading

be predicted

to the

that

of the kinase

from

the

fractionation

of protein

insulin

cytosol

COMMUNICATIONS

production might

of also

lead

to the membranous

fraction. Employing

both

cytosol

and membranes

permits

detection

and a new peptide

of kinase

compared

the effects

activity

and compartmentation

of these

investigations

activate

the kinase

activity

of phorbol

or to cause

assay in crude

ester

that its

for

C activity

rat

insulin

that

we have

on protein

adipocytes.

from

kinase

C

The results

has no effect

depletion

in

the kinase

extracts,

and insulin

in isolated

indicate

kinase

either

to

the cytosolic

compartment. Materials

and Methods

Adipocytes were prepared from male Sprague-Dawley rats (130-15Og) as in [lo] and incubated in Krebs-Ringer bicarbonate buffer cont$.ning 10 mM glucose and 3.5% bovine serum albumin for 30 minutes at 37 C. Insulin (100 ulJ/ml) or phorbol 12-myristate 13-acetate (PMA;l uM) were then added; control cells were incubated in a concentration of DMSO (0.01%) equivalent to that with PMA addition. After exposure, aliquots of the cell suspension were centrifuged in a bench top centrifuge (1000 rpm) for 30 seconds, and the infranatants were removed. The cells were washed twice with 7 ml of buffer A (sodium betaglycerophosphate (100 mM; pH 7.5), EDTA (2 mM), EGTA (0.5 mM), dithiothreitol (2 mM) and sucrose (0.25M)) at 4’C and homogenized in the same buffer (lg/O.75 ml) using an Dltraturrax homogenizer at top spee$ for 30 seconds. The homogenates Infranatants were were centifuged at 105,000 x g at 4 C for 60 minutes. removed and assayed for kinase activity immediately; pellets were extracted in buffer A containing 1% (v/v) NP40, bg homogenization with a glass rod and gentle rotation for 30 minutes at 4 C followed by were equalized for protein centrifugation at 105,000 x g. All extracts concentration prior to kinase assays; protein concentration was determined by the method of Bradford [ll]. In other experiments, cytosol and membrane extracts were prepared and partially purified by DEAR-cellulose chromatography as in [12]. Protein kinase C activity in extracts was determined by assay using either histone III-S or the N-bromosuccinimide-cleavage COOH-terminal All assays were performed in duplicate histone Hl peptide, as in [lo]. and kept within linear rates with respect to protein concentration and time. N-bromosuccinimide (NBS) cleavage of phosphorylated histone III-S SDS polyacrylamide gel electrophoresis was was performed as in [lo]. carried out by the method of Laemmlli [ 131. Results

and Discussion Difficulties

crude

extracts

in the detection have led

of protein

many investigators 1120

kinase

to employ

C activity preliminary

in DE-

Vol.

135,

No. 3, 1986

cellulose

chromatography

measurement

of kinase

prepared the

BIOCHEMICAL

after

elution a prompt

small

decrease activity

associated

Hl kinase

quantitative

versus

The technique with

Furthermore,

it

activity whole for

from cells

histone

not

be argued

the columns

and that

the expression

this

technique

extracts

without

substrate

for

for

step

assays

with

by N-bromosuccinimide

cytosol

that

leads

also

activity

as a Cai-t + PL in membrane

seen,

although

assured

digestion

cumbersome,

might

remove

kinase

activity.

samples.

recoveries

after

certain

treatments co-factors

This

extracts.

of phosphorylated

us to apply

in

Hl is

Control

crude

not

a suitable

As determined

histone

Hl,

basal

against purified expressed per total extracts treatment

PMA

Kinase experiment

C Activity. (one of

1 :

DE-cellulose C activity

Insulin

Fractionation in

histone

this

of

Protein

representative

III-S

with

cytosolic

Protein was

three)

and membrane extracts

by DE-cellulos&chromatography, as pmol of Ca and PL-stimulated volume of DE-eluate, in cytosol (stippled bars) was determined of the adipocytes.

1121

as

‘Js

Methods. P incorporation (solid bars) after 10 minutes

of

necessary

has led

activity

Histone

unfractionated

of kinase

various

of kinase

purification. these

is

Boo-

Figure

to

in the membrane

replicate

identical

the measurement

preliminary

to PMA is

of multiple

are not

of altered

a second

insulin,

An increase

chromatography

performance

could

by step

observed.

of DE-cellulose the

1).

in response

not

and

purified

C, measured

Hl (Fig.

were

adipocytes

partially

kinase

to

Extracts

rat

PMA, but

of the diminished

was consistently

particularly

protein

prior

Hl (121.

extracts

COMMUNICATIONS

extracts

of isolated

columns.

activity

recovery

histone

treatment

cytosolic

RESEARCH

and membrane

and membrane

in

BIOPHYSICAL

against

DE-cellulose

stimulated

fraction

activity

fraction

from

of cytosol

PMA or insulin

cytosolic

AND

kinase measured

partially Activity, per

minute

and membrane of

agonist

Vol.

135,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

32-

12

Figure

3

4

5

2:

N-bromosuccinimide Digestion of Phosphorylat3$ HistonedIII-S. Histone III-S was phosphorylated in the presence of P-ATP& with the catalytic subunit of the CAMP-dependent protein kinase (lane 2), purified protein kinase C (lanes 1 and 3) and adipgyte extracts either in the absence (lane 4) or presence (lane 5) of Ca /PL. Following incubation, the protein was precipitated with 25% TCA, washed, and redissolved in IN acetic acid. N-bromosuccinimide (NBS) digestion of samples 2-5 was then performed, as in [lo]; NBS was omitted from sample 1. These reactions were terminated by snap-freezing and the products lyophilised prior to preparation of samples for SDS gel electrophoresis (18% acrylamide) and radioautography. The indicated molecular weights, determined as in [lo], are those of intact histone Hl (32K), the COOHterminal NBS peptide (22K) and the NH2-terminal NBS peptide (14K).

histone

Hl

at the

kinase

NH*-terminal

primary

site

kinase

(lane

2).

in the

peptide,

as is

should

incorporation

adipocyte

would

that

not

kinase

(Fig.

2, lane

Ca++ plus is

This

primarily

peptide

that

stimulated

kinase

the addition

protein

values

C (lanes ++

protein

(lane

since

subtraction

estimate

3 and 5).

5).

C activity

(the

NBS-

This in

may

crude

of basal

of protein

kinase

be valid.

Hl peptide C activity

described in the

is easily

assay

the use of the NBS-generated of protein

measured 1122

in crude

klnase

C [lo].

extracts

It

+ PL diminishes

kinase Hl,

the

stimulated

of Ca

NBS peptide

histone

is

at the COOH-terminal

protein

to detect

Ca*/PL

4).

directed

phospholipid

directed

the NH*-terminal

employing

is

of Hl by the CAMP-dependent

purified noted

We have recently terminal

NBS-peptide

for

inability

extracts from

extracts

same extract

into

our

adipocyte

In contrast,

be additionally

explain

in

of phosphorylation

activity

values

activity

COOHProtein

and the

peptide

C)

Vol.

135,

is

No. 3, 1986

specific

BIOCHEMICAL

to

dependent

distinguish

kinase,

the

two

of

the

AND

BIOPHYSICAL

activities major

of

kinase

RESEARCH

this

kinase

activities

COMMUNICATIONS

and

in

the

CAMP-

a number

of

tissues. Employing insulin

on

this

protein

This

these

appearance clearly

the

persists

a kinase

stimulated

It

is

kinase

increase

or

both

Ca *

at

kinase that

is

the

+t

Ca

Ongoing

able

by

or

However,

with

document

the

are

is

membrane-

insulin

(data

contain

inhibitors

diacylglycerol

investigations

time

that

Total

extracts

, PL,

to

fractions

unaltered

membrane

of

values.

membrane

of

detectable

phospholipid.

activity

possible

been

rat

depletion

10 minutes.

yet

PMA and

in

earliest

least

the

of

rapid

the

not

and

effects

compartmentation

at

in

the

extremely

we have

concentrations

basal

an

for

activity

by

peptide

shown).

to

observable

conditions, of

associated

3),

depletion

examined and

leads

(Fig.

extraction

have

C activity

exposure

kinase

point.

we

kinase

PMA

adipocytes. cytosolic

assay,

not of

that

exploring

this

problem. In cytosolic (as

measured

any

event,

protein at

insulin, kinase

V max

unlike

PMA,

C activity

conditions

0

I

or with

2

3

Time

does

cause

activation

respect

4

not

5

6

of to

7

9

depletion

a cytosolic Ca ++

added

8

the

and

of form

PL),

,o

(min)

Figure 3: Effects of PMA and Insulin on Cytosolic Protein Kinase C Activity. Protein kinase C activity, measured as the increment in kdnase activity against the NBS-cleavage peptide with the addition of Ca and PL, was measured in cytosolic fractions prepared from adipocytes after exposure to PMA (closed squares), insulin (open circles), or no additions (closed circles). Zero time indicates the time of homogenization, which begins 1.5 minutes after agonist addition. Results in this representative exzx;r$yent are plotted as 4, control; control values were constant at 174 P incorporated per minute per mg extract protein.

1123

Vol.

135,

No. 3, 1986

Table

BIOCHEMICAL

1.

Cytosol

AND

Protein Kinase Treatment

BIOPHYSICAL

C Activity of Adipocytes

Protein

Activity

20.2

+ 3.7

7.1

+ 1.3

19.3

f. 3.5

3.9

+ 0.3

+ PS

Ca++

f

4.3

6.2

Insulin

26.6

-+ 4.1

4.0

+ 0.6 activity

Insulin

+ 0.8

26.4

kinase

PMA or

with

Control

protein

COMMUNICATIONS

Increment

Ca++

Cytosolic

After

Kinase

Condition

PMA

RESEARCH

against

PS

0.1 + 0.4

the

NBS-cleavage

was measured in the presence of Ca** alone (1 mM) and in the presence of Ca+* + phosphatidylserine, (PS: 80 ug/ml) with the addition of 3-5 ug of cytosolic protein, prepared as in Methods, after 2 minutes exposure of intact cells to agonist. Data (+SEM) shown peptide

represents

duplicate

assays

,":~p::",::~~~do~sa~~~~c~~es; extract.

measured

over

3) and in (Table insulin

a time

five

1).

These

with

include

cascade

do have

between

the

effects

observations of insulin

actions

we must kinase

of these

conclude

and intracellular

properties,

the

to a growing compounds 1141,

protein a role

that

C.

metabolism

support

(Fig.

of maximum PMA effect

can be added

cannot

exposure

insulinomimetic

on glycogen

[15]

5 separgte

of insulin

on protein

investigations

carboxylase

from

was 5 minutes at 30 C. Results per minute per total volume of

assays,

effect esters

opposite

These

[15,16].

condition

at the time

two separate

phorbol

divergences

of acetyl-CoA

in the

experiments

of the present

specific

each

up to 10 minutes

has no discernable Although

results

course

separate

Thus,

for

assay time P incorporation

for

list

of

and insulin. regulation

phosphorylation protein

kinase

action.

Acknowledgements This Institutes Janet

work was supported in part by a grant from the National The manuscript was ably prepared of Health (AM35712).

by

W. Fatherley.

References t11

Lee,

L-S.

and Weinstein,

I.B.

(1979)

460.

1124

J.

Cell.

Physiol.

99,

451-

C

Vol.

135,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Skogland, G., Hannson, A. and Ingelman-Sundberg, M. (1985) Eur. J. Biochem. 148, 407-412. A.R. and Houslay, M.D. 131 Heyworth, C.M., Whetton, A.D., Kinsella, (1984) FEBS Lett. 170, 38-42. Trevillyan, J.M., Perisic, O., Traugh, J.A. and Byus, C.V. (1985) [41 J. Biol. Chem. 260, 3041-3044. J. and Rosen, O.M. (1985) Proc. Natl. Tabarini, D., Heinreich, t51 Acad. Sci. USA 82, 4369-4373. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and 161 Nishiauka, Y. (1982) J. Biol. Chem. 257, 7847-7851. Kraft, A.S. and Anderson, W.B. (1983) Nature 301, 621-623. [71 Kraft, A.S., Anderson, W.B., Cooper, H.L. and Sando, J.J. (1982) [81 J. Bfol. Chem. 257, 13193-13196. Honeyman, T.W., Strohsnitter, W., Scheid, C.R. and Schimmel, J. [91 (1983) Biochem. J. 212, 489-498. J.W., McDermott, J.M. and Witters, L.A. 1101 Glynn, B.P., Colliton, (1985) Biochem. J. 231, 489-492. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. illI W.L. and Anderson, W.B. (1985) Nature 315, 233-235. 1121 Farrar, [I31 Laemmli, U.K. (1980) Nature 227, 680-685. Roach, P.J. and Goldman, M. (1983) Proc. Natl. Acad. Sci. USA 80, t141

[21

7170-7172. [151

[I61

Witters, L.A., Colliton, J.W., McDermott, J.M., Zhang, F. and Glynn, B.P. (1986) in: Mechanisms of Insulin Action (Belfrage, P ., Donner, J. and Stralfors, P. eds) Elsevier, Amsterdam, in press. Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F. and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315.

1125