Protein kinase and cyclic AMP levels in differentiating myoblasts are altered by extracellular calcium

Vol.

97, No.

November

2, 1980 28,

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AND

BIOPHYSICAL

RESEARCH

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Pages 384-390

1980

PROTEIN KINASE AND CYCLIC AMP LEVELS IN DIFFERENTIATING MYOBLASTS ARE ALTERED BY EXTRACELLULAR G. E. Morris School

Received

September

of Biological Falmer,

and Nguyen

thi

CALCIUM

dn

Sciences, University of Sussex, BRIGHTON, BNl 9QG, U.K.

8,198O

SUMMARY Protein kinase specific activities and cyclic AMP levels show a similar pattern of response, when the Ca*+ concentration is altered in the culture medium of differentiating chick skeletal muscle cells; an increase at intermediate Ca*+ concentrations (0.05-0.2mM), followed by a decrease at higher concentrations (2mM). Effects of Ca*’ on protein kinase appear to be on cyclic AMP-independent enzymes in both nucleus and cytoplasm, and are quite the reverse of Ca*+ effects on the muscle-specific enzyme, creatine kinase. INTRODUCTION Addition

of different

an inhibition

at intermediate

concentrations tion

(2-3mM),

kineses

concentrations

myoblasts

but one possibility act

AMP levels

known

to

that

since

exist

creatine

(1).

is

as intermediates, are

(0.05-0.2mM),

of muscle-specific

by differentiating

unknown,

of Ca 2+ to Ca 2+ -depleted

amounts

(2)

cyclic

AMP and/or

interrelationships

(MM-CPK)

cytoplasmic

kineses

accumula-

Ca 2+ effects

of these

between

and some protein

in

but not at higher

kinase

The mechanism

medium results

is

protein

Ca

are Ca

2+ 2+

and cyclic -dependent

(3).

The object externa

1 Ca 2+ of intracellular

independent analysis

of the present

protein

kineses

of the mechanism

study cyclic

was to determine AMP levels

in cytoplasm of Ca

2+

effects

the

response

and the levels

and nucleus,

as a first

to

of cyclic step

AMP-

in an

on myogenesis.

MATERIALS AND METHODS Cell culture. Cultures were prepared from 12-day embryonic chick thigh muscle by mechanical dissociation (4) and grown as described previously (1). Total Ca*+ levels in Ca*+-depleted medium (containing dialysed serum and 0006-291X/80/220384-07$01.00/0 Copyright U 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

384

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embryo extract) were 15-2OpM as prepared and 25-3OpM at the end of 3 days incubation with cell cultures, as determined on a Unicam SP 9oA atomic All cultures were grown for 24h in standard, 2+ absorbance spectrophotometer. to medium with the appropriate amount of Ca high Ca2+ medium before changing added. Protein kinase assays. Cell pellets were lysed by freezing and thawing in 0.2ml of double glass-distilled water and centrifuged at 2,000g for 10 min. The supernatants were centrifu ed again at 10,OOOg for 10 min. 20~1 of volume of lOOp1 of assay buffer supernatant were assayed at 30 8 C in a final (60mM sodium phosphate buffer pH 6.4; 0.5mM EGTA; 1OmM Magnesium acetate; 2OmM NaF; 60mg/ml casein, pretreated according to (5); 1uCi Y-[~~P]ATP (16-19 Ci/mmole; Radiochemical Centre, Amersham, Bucks, U.K.); 0.2mM ATP). Reactions were terminated by cooling in iced-water and adding excess 10% trichloroacetic acid with 50mM Na phosphate. Samples were filtered and washed four times with trichloroacetic acid and once with ethanol, Nuclear protein phosphorylation previously, with endogenous nuclear

assays were performed as deecribed protein as substrate (6).

Cyclic AMP assays. Cells were rinsed very rapidly with ice-cold 0.9% in a 4OC cold-room in a small NaCl - 1mM EDTA, frozen --in situ and harvested volume of 0.9% Nacl - 1mM EDNA. An aliquot of the resulting cell lysate was taken for protein estimation (7) and the remainder was boiled for 3 min. and centrifuged for 20 min. at 2,000g. Cyclic AMP in the supernatant was determined with an assay kit.(Radiochemical Centre) which depends on competition with [3H] cyclic AMP for cyclic AMP-binding protein. Cyclic AMPphosphodiesterase (Sigma) destroyed over 95% of the cyclic AMP competitor activity in the samples and internal cyclic AMP standards added to samples produced the predicted results in the assay. RESULTS Fig, is

linear

Fig.

lA shows for

the

1B shows

that

in the assay

for

incorporation extract

unusually

ca

2+

possible protein were

cyclic

(saturation

and later

2 shows the

dependence

of cyclic

accumulation

which this

that

Figs.

in the cell

involvement

made during

any endogenous

period

a further

linearity

of protein

20 min.

cyclic

cyclic

of cell

suggests kinase

extract that

the

enzyme in the

AMP in the extract with

extracts

is not

AMP is most unlikely

AMP levels

in these

cells

kinase

levels

at

are

not

4 and 5)). of cytoplasmic

culture

medium.

AMP and protein

occurs

by cytoplasmic

to the amount This

the amount

used , given

for

proportional

reflect

enzyme activity

concentration

is

casein

linear

48h and 96h cultures.

that

(8;

into

and almost

incorporation

dilution high

Fig.

10 min.

does indeed

the

the 20-fold

first

both

and also

affecting

32 P incorporation

that

between

Since

we are

kineses

interested

There

on the in the

in the muscle-specific

24h and 96h of culture,

at 48h and 72h.

385

protein

is,

at both

measurements times,

an

Vol.

97, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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------%

32P cp

205

01 O

30

0

IO

20

Extracf

.Time lmin)

I/JI.,

Time-course cultures was Each point

L--&--J

02

0

0

0.1 0.2

Added

05 I.0 2.0

Ca2’

%iKF-

of used is

Figure 1B. Effect of from 48h (O.lm~ Ca*+; cultures were used in subtracted. Each point incubations.

dilution of the extract on open circles) and 96h (0.5mM the experiments illustrated. is the average of triplicate

subtracted.

the protein kinase assay. and zero-time background a single determination.

of 32

L?--T

03

lmMl

An extract 300cpm

Added

from been

has

. P Incorporation. Ca*‘; filled Zero-time (48h) or

Ca2’

(mM)

96h,

Extracts circles) backgrounds duplicate

were (96h)

2+ Ca I” the culture medium on the levels of 48h (o----o) and 96h of culture, and with 10e6M or absent (o o) in the assay of 96h extracts. duplicate or triplicate incubations. With 48h significant effect of cyclic AMP (not shown). cpm incorporated per pg of protein in the extract.

at

Figure 2. Effect of external cytoplasmic protein kinase cyclic AMP present (a l) Each point is the average of extracts, there was also no Results are expressed as 32P

2+

Effect Figure 3. rotein kinase in 1-’ - S.E.M. of triplicate of nuclear protein.

protein

increased

gradual

AMP

effect

up

in

the at

experiments,

minor

kinase

decrease

cyclic

of

of external Ca in the culture medium on the levels of nuclei isolated at 72h of culture. Results are given assays, and expressed as 32 P cpm incorporated per pg

to

assay various

cyclic

AMP

component

of

was

the

for

the

nuclear

isolated

from

72h

cultures

the

therefore

assays not

Fig. differentiation

were

directly

4 shows in

2mM Ca of

in

response

.

There

of

the

2+

any

culture

described

though

level

was 72h

times

and

a stimulation

Ca

no

Ca

2+

5%,

The

(9).

under

1OOuM

and

effect

suggesting

similar

a subsequent of

a longer

concentrations,

protein

rather

to

Over

is

a basically

performed

up

significant

activity

enzyme

2+

extracts.

of

phosphorylating

shows

to

series

the that,

at

most,

AMP-dependent,

kinase

activity

different

conditions

nuclei 3),

and

comparable. the culture.

increase

in The

cyclic pattern

AMP and

386

levels absolute

which levels

occurs are

only as

in (Fig.

of

average

cyclic

response

10 -6M

during almost

even are

a

Vol.

97, No.

8lOCHEMlCAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

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Cyclic AMP Ipmoles/ mg.profein) 2 i;-

al---.-l 40

0

4

60

Figure 4. differentiation. (range of

Cyclic

AMP Each

duplicates

05

80

Time

Ih)

levels (p p$int is < - 5%).

moles /mg the average

of of

total protein) duplicate

Ca2+

during determinations

ImM1

myoblast

2+ 5. Effect of external Ca in the culture medium on intracellular AMP levels. Results are expressed as % of the level in p moles /mg in cultures with Ca2* -depleted medium alone (zero added-Ca2+). Each represents a different time of harvesting cultures between 48h and POh open circles; 55h: crosses; 66h: filled squares; 72h: open squares; filled circles).

Figure cyclic protein curve (48h: POh:

by Moriyama and Murayama

identical

to those

different

assay methods.

The absence

elsewhere

(11)

be due to insufficient

appropriate

described

may simply

culture

period.

The effects

here

are

stimulation

of cyclic

of the sharp

The pattern

Ca2+ (Fig. 5) is somewhat

external

Added

rather

variable

AMP levels

using

time-points

to that

in magnitude

quite

peak of cyclic

of response

similar

(lo),

during

of cyclic of protein

between

AMP described the

AMP levels kinase

levels.

experiments

but a

Ca 2+ concentrations

by intermediate

to

was always

observed.

(l), protein

it

Comparing

these

would

seem that

kinase

results

with

effects

MM-CKP accumulation

are

elevated.

In an earlier

study

Ca 2+ on MM-CPK levels

of external is

reduced

AMP and

when cyclic

DISCUSSION

CPK levels

cyclic

rule

out

involvement

that

one hypothetical

2+

cyclic

AMP be highest

at very

involved

that

cl),

precise

Ca -and

explanation,

in some form has not

AMP-dependent low Ca2+ . though

for

protein

kinases

The present

the possibility

been eliminated.

387

explanation

Ca 2+ effects and required

experiments of cyclic

clearly nucleotide

on

Vol.

97, No.

2, 1980

Elevated opposed

8lOCHEMlCAL

cyclic

to actively-dividing,

indeed

increase

into that

the

0.05-0.2mM

extracellular

highest

Berridge

(2)

Ca

2+

the observed would

for

suggest

that

Correlations

given

cell

they

its

proteins

do not

are mediated

enzyme that

the changes (Figs.

the

therefore

yet

justify

the conclusion

AMP effects

protein

kinase

myogenesis described

here,

and both

of proteins

(17),

2 and 3).

activity

casein

redistribution

on cell

it since

Leakage

does not nuclear

the

intra-

levels

(Fig.2)

a role. protein

synthesis

extent

that in a 2+ of Ca on

The effects

seem sufficient

that

is

Ca 2+ effects,

external

to account

not all

muscle-specific 2+ as CPK, a notable Ca

receptor Ca

2+

(15).

The data,

effects

on CPK levels

division. may be,

in part

activities between appear

at least,

are cyclic nucleus

the same AMP-independent

and cytoplasm

to be responsible

and cytoplasmic

of enzyme from

388

but

by modulating

play

to external

acetylcholine

actually

Ca 2+ itself

kinase

still

However,

of response

cell-surface

phosphorylates

Although during

same pattern

by cyclic

The nuclear

might

is

division,

if

even to the (14).

on CPK levels.

show the

cell

protein

exclusive

media.

In some circumstances,

and muscle-specific

been described,

to decline

incorporation

AMP acting

might

division

effects

therefore,

changes

cell

(13)

being

occur

between

incorporation

exception

(16).

phosphorylation

response

expect,

and Ca2+-depleted

explained

AMP-independent

[3H]-thymidine

a premature

Ca 2+ does mediate

protein

have

normal

AMP does

One might

in

AMP

as

cyclic

(12).

increased

cyclic

quiescent,

when

about

(13).

can be best

may be mutually

opposite

bring

with

division,

in cyclic

4),

C3H]-thymidine

intracellular

populations

C3H]-thymidine for

cell

Even if

.

these

(Fig.

to both

COMMUNICATIONS

cultures,

in cyclic

5) would

since

with

declining

increase

relative

that

changes

qyoblast

are

AMP can be associated

signal

associated

Ca 2+ concentrations

suggests

more direct cellular

similar

RESEARCH

in myoblast

division

however,

at intermediate cyclic

usually and,

Ca 2i- (Fig.

the case,

elevated

in

rather

incorporation

is not

BIOPHYSICAL

48h and 96h of culture

DNA and cell

therefore,

C3H]-thymidine

are cells

between

incorporation

This

AMP levels

AND

enzymes

the nucleus

during

for

may any of

show similar preparation

in

Vol.

97, No.

of cytoplasmic would

BIOCHEMICAL

2, 1980

have

extracts little

effect

by cytoplasmic

the

that

capable

intermediate

cyclic

these

changes

muscle-specific

protein

likely

Ca

2+

protein

but

this

of nuclei

It are

in response

would

both

through

independent for

cyclic seem,

potentially

cells

activity

responsible

accumulation

certain

(8).

in muscle

of cyclic-AMP

4 and 5) are

to affect

concentrations

kinase

subsequently

(Figs.

cells

phosphorylation

the levels are

experiments, Contamination

here

in intact

external

AMP-dependent

and by increasing

Whether

described

(O.l-l.OpM)

protein

in these

COMMUNICATIONS

(17).

AMP levels range

out

RESEARCH

conclusions.

phosphorylations

of increasing

increasing levels

protein

overall

negligable

in cyclic

concentration

AMP-dependent therefore,

is

BIOPHYSICAL

been ruled

on the

proteins

The changes within

has not

AND

by

cyclic

AMP

enzymes.

observed

changes

to Ca 2+ , however,

in

has not

been established. ACKNOWLEDGEMENTS Support

We thank the Muscular Dystrophy Group of Great and Prof. R. J. Cole for providing laboratory

Britain for facilities.

financial

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Reimann, E.M., 1986-1995.

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