Phosphorylation of cardiac sarcoplasmic reticulum by a calcium-activated, phospholipid-dependent protein kinase

Vol.

96,

No.

October

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

pages

16, 1980

1378-1383

PHOSPHORYLATION OF CARDIAC SARCOPLASMIC FETICITLHM BY A CALCIUM-ACTIVATED, PHOSPHOLIPID-DEPENDENT Constantinos Cardiovascular

Division, School

Received

September

J. Limas

Department

of Medicine,

PROTEIN KINASE.

of Medicine,

Minneapolis,

University

Minnesota

of Minnesota

55455

?,lS?O

SUMMARY: Cardiac sarcoplasmic reticulum is phosphorylated by a cytosolic Ca2*-activated, phospholipid-dependent protein kinase. This phosphorylation is independent of cyclic nucleotides and enhanced by unsaturated diacylglycerols; saturated diacylglycerols, mono- and trilycerides are ineffective. k Diacylglycerol stimulation is due to increased Ca sensitivity of the kinase reaction. Protein kinase catalyzed phosphorylation results in enhanced Ca2+transport ATPase activity and may be an important determinant of cardiac sarcoplasmic reticulum function. INTRODUCTION: be modulated studied

Calcium

through

example

is

the cyclic

ponent

(phospholamban)

kinase

also

site

other

by calmodulin distinct

(3,ft).

It

from is

Ca2+ ions, calcium

likely

recently

described

than

which

transport

protein

(2).

promotes

complex

by this (5-7)

interactions sites

organelle.

cardiac

the

of the calcium

transport

b of is

of-

of phospholamban

AMP-dependent between

reticulum

com-

control

protein

different

study,

kinase kinases,

regulate

we report

as substrate

at a

protein

reticulum

phospholipid-dependent

sarcoplasmic

stimulates

phosphorylation

regulatory

In the present

The best

Phosphorylase

on the sarcoplasmic

Ca2+ -activated,

utilize

(1).

the phosphorylation the cyclic

which

can

a 22,000-dalton

through

Additional

reticulum

kinases.

kinase

reticulum probably

thatinfluencedby that

protein

by phosphorylating

phospholamban

ase can also activity

activity

calcium

sarcoplasmic

by endogenous

AMP-dependent

and phosphorylation

transport

by cardiac

of the sarcoplasmic

enhances

components

transport

phosphorylation

and Ca 2+ -ATPase

Ca2+ uptake

fered

ion

protein

that

a kin-

and modify

ATPase.

MATERIALS AND METHODS: Experiments were carried out on adult male Sprague-Dawley rats. Isolation of sarcoplasmic reticulum: The animals were killed by cervical dislocation and their hearts were rapidly excised and chilled in crushed 0006-291X/80/191378-06$01.00/0 Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

1378

Vol.

96, No.

3, 1980

BIOCHEMICAL

AND

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RESEARCH

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ice. The myocardial tissue was washed twice with a solution containing 10 mM NaHC03-5 mM NaN3 and was then homogenized in the same solution with a Polytron PT-20 homogenizer three times at a rheostat setting of 3 for 5 set with 15 set rest intervals. The homogenates were then processed for sarcoplasmic reticulum isolation as described by Sumida et al. (8). Ca'+-activated ATPase' . ATPase activity was determined at 25'C in a medium containing 40 mM histidine-HCl buffer (pH 6.8), 5 mM MgCl 5 mM ATP, 120 mM KCl, 5 mM NaN3, calcium-EGTA buffer containg 1 UM free Ca 24 and 50-80 ug microsomal protein in a total volume of 0.5 ml. Reaction mixtures were pre-incubated at 25OC for 5 min. after which the reaction was started by the addition of microsomal protein. To determine "basal" (Mg2+-ATPase) activity, reactions were carried out in the presence of EGTA (0.2 mM) instead of the calcium-EGTA buffer. At various intervals after the start of the reaction, 100 ul aliquots were added to tubes containing 25 ml of 25% (w/v) trichloroacetic acid and 125 pM inorganic phosphate. After centrifugation at 3,000 g for 10 min. inorganic phosphate was determined (9) in the supernatant. Protein kinase isolation: Phospholipid-dependent protein kinase (kinase C) was isolated essentially by the method of Takai et al. (5). Rat hearts were homogenized in four volumes of 20 mM tris-HCl (pH 7.5)-50 mM 2-mercaptoethanol2 mM EDTA (buffer A) using a Polytron PT-20 at a setting of 4 for 30 sec. The homogenate was centrifuged at 20,000 g for 40 min. and the resulting supernatant was filtered through glass wool. The extract was applied to a DEAE-cellulose column previously equilibrated with buffer B (buffer A containing 70 mM NaCl). After the column was washed with 100 ml of buffer B, the first elution was carried out with 250 ml buffer B containg 10V5M cyclic AMP. The second elution was carried out with a linear gradient (70-300 mM) NaCl in buffer A containing 10m5M cyclic AMP. Fractions of 10 ml were collected and assayed for protein kinase activity (Figure 1). Protein kinase assay: The assay medium for protein kinase contained, in a total volume of 0.25 ml, 5 umol tris-HCl (pH 7.5), 1.25 umol MgC12, 25 nm01 ]u-~~P]ATP (Amersham/Searle, Arlington Heights, Ill., sp. act. 6.1 Ci/mmol), 10 ug protein kinase C, 50 ug sarcoplasmic reticulum or histone HI (Sigma Chemical Co.) and various concentrations of CaC12 or lipids. Incubation was carried out at 37'C for variable lengths of time and the reactions were stopped by the addition of 2 ml of 25% trichloroacetic acid; precipitates were collected on Millipore filters (0.45 urn pore size) and processed for radioactivity counting.

Figure

1:

Isolation extract

of protein was

passed

kinase through

C from rat heart a DEAE-cellulose

cytosol. cclumn

Cardiac previously

equilibrated with 20 m?Jtris-HCl (pH 7.5)-50 mM 2-mercaptoethanol-2 mM EDTA-70 ml4 NaCl (buffer B). After the column was washed with buffer buffer B containing gradient of N&l were assayed for text.

B, the first lo-%l cyclic (---) in buffer. protein kinase

1379

elution was carried out with AM’P and then with a linear Aliquots from the eluates (0 0) as described in the

Vol.

96, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

Time

Figure

isolate

protein

kinase

C from rat

cardiac

protein

kinase

has properties

the enzyme from

cardiac

rat

(data

not

sarcoplasmic

sarcoplasmic

are

when either

histone

is

is

seen.

carried

similar

kinase

examined

shown

in Table

out with

Phosphatidylserine

as substrate

is

the most

pholipids

fatty

on the protein

diacylglycerols, SR is

acids

mono-

significantly

The nature

less

of the protein

kinase,

or tri-acylglycerols. stimulated

endogenous

lipid

followed

of phos-

by saturated

1380

of cardiac

of exogenous

by diolein

by

containing

the effects

by the addition

kinase

requirement

phospholipid

not shared

phos-

ineffective

Phosphorylation

C enhancement

this

When protein

potentiate a property

kinase

protein

Diacylglycerols

significantly

kinase

are

a definite potent

by cyclic

for

is

for

by micromolar

that

enhances

used as substrate.

phosphatidylinositolandphosphatidylcholine. unsaturated

for

nucleotides

as substrate,

HI

described

activated

The requirements

1: cyclic

HI or SR is

previously is

to

Hl as substrate,

the possibility

significantly

phosphorylation.

histone

to those it

(5) was utilized

and is unaffected

can serve

2, this

With

Specifically,

We then

et al.

cytosol.

and phospholipids

reticulum

reticulum

of Takai

cardiac

(5-7).

shown).

in Figure

phorylation

assay

cerebrum

of calcium

C. As shown

(mid

The procedure

RESULTS AND DISCUSSION:

nucleotides

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Effect of protein kinase C on SR phosphorylation. The assay mixture contained ZOtitris-HCl (PH 7.5), 5 mM MgC12, 50 ug cardiac SR, 25 nmol [Y-~~P]ATP, 10s5M CaC12, and 50 pg protein kinasa C. Reactions were carried out at 37OC for various lengths of time as described in the text. Control mixtures (0) were identical except for omission of prctein kinase C. Results represent meanf SE for six comparisons.

2:

concentrations

RESEARCH

lipids.

revealed

by

Vol.

96, No.

BIOCHEMICAL

3, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Lipid dependency of protein kinase C. Reactions were carried out at 37'C for 10 min. with 50 g protein kinase C, lo-514 CaC12, and the indicated additions. Phospholipids (16 pg) and diacylor triacylglycerols (6 pg) were first solubilized by sonication in the incubation buffer. Results are expressed as percentage of protein kinase activity in controls, in the absence of added lipids (4.8 nmol/mgflO with histone and 1.6 nmol/mg/lO min. with cardiac SR as substrate).

With

ADDITIONS

PROTEIN

KINASE

Histone

Hl(50

ACTIVITY ng)

With

SR (50

100

100

Phosphatidylinositol

346

127

Phosphatidylserine

359

136

Phosphatidylcholine

117

108

Phosphatidylethanolamine

108

104

+ diolein

489

146

None

(Control)

Phosphatidylserine II

+ dilinolein

426

138

I,

+ diarachidonin

501

151

11

+ dipalmitin

352

130

11

+ distearin

361

126

11

+ triolein

348

128

a study

of the

Ca2+ dependency

diolein

significantly

so that

lower

increases

concentrations

of the reaction the

sensitivity

are needed

(Figure of

to maximally

the

min

3). kinase

activate

Addition to

the

pg)

of calcium

enzyme.

10%

03

Time (mtn)

Figure

3:

Calcium dependency of protein kinase C activity. Reactions were carried out at 37°C for 10 min. with 50 ug histone HI, 50 ug protein kinase C, various concentrations of CaC12. and 6 pg diolein (l-l), 10 ug phosphatidylserine (AI, or 6 ug diolein plus 10 ug phosphatidylserine (a). Results represent mean f SE for six experiments.

Figure

4:

Effect of protein kinase C on the calcium transport ATPase of cardiac sarcoplasmic reticulum. Reactions were carried out at 25°C in the presence of 50 ug SR alone (c), or 50 JIM protein kinase C plus 10 up phosphatidylserine and 6 ug diolein (a,). Results represent mean + SE for six experiments.

1381

Vol.

96, No.

3, 1980

BIOCHEMICAL

Experiments reticulum

on the Ca2+ transport

strongly

functional

suggest

that

implications. of both

we have also

a stimulatory

found

sarcoplasmic

These

results

represent

reticulum.

actions

other

our

with

laboratory.

dependent though

that

it

protein

ation in view sensor

which

kinase calcium enzyme. is

levels

to the formation

since

it

The enhancement

especially

effects

hormonal,

intriguing

of protein

kinase

would

to the

depends

it

by the latter

C catalyzed

phosphoryl-

features.

First,

can be envisaged

as a

stimuli

on a shift

in the

sarcoplasmic

1382

cell

pump phos-

to glycerolipid

across

major

provides

is possible

by membrane

require

because

Since

stimulation

fluxes

sensitivity

al-

to the calcium

of diacylglycerol

C on the

C (6),

to phosphatidylinositol

of calcium

AMP-

and a link

transport

or neural

it

cyclic

(4).

transport

enzyme it

its

relevant

in

enzyme which,

explored. kinase

calcium

not

inter-

has two attractive

of the

calcium

and the possible

related

of calcium

Secondly,

with

of cardiac

investigation

kinase

may

the function

the Ca"-ATPase

by protein

relates

C. Such activation

under

not

concentration

may be particularly

leads

studies,

phosphorylation

regulating

stimulate

reticulum

reticulum.

to metabolic,

breakdown

clearly

stimulated

calcium

has been associated

response

for

from calmodulin-dependent

and diglycerides This

(Figure

C on Ca2+ uptake

C catalyzed

are currently

dependency

of cytoplasmic

olism.

C has

activity

kinase

of phosphorylation

C is

sarcoplasmic

of the calcium

pholipids

mechanism

does not is

kinase

in the enhancement

of the sarcoplasmic

kinase

In preliminary

of protein

possibility needs to be further of mechanisms involved, protein

of cardiac

COMMUNICATIONS

sarcoplasmic

ATPase

and diolein.

protein

kinases

and differs

participates

stimulates

effect

kinase

b kinase

enzyme; this Regardless

by protein

The site(s)

calcuim-dependent,

phosphorylase

phosphorylation

phospholipid

that

Protein

kinase

of the cardiac

kinase

and important

sarcoplasmic

RESEARCH

reticulum.

indicate

a novel

BIOPHYSICAL

ATPase

Protein

4) in the presence

by cardiac

AND

metabturnover

membranes

in

(10).

Phosphatidylinositol

which

activates

increases

in intracellular

Ca2+ sensitivity

by unsaturated a mechanism reticulum.

protein

for

of the diglycerides

amplifying This

mechanism

the would

Vol.

96, No.

3, 1980

be primarily

BIOCHEMICAL

important in which

failure, decreased

capacity

lower

in disease

states,

intracellular

mediated

stimulation

circumstances,

an important

BIOPHYSICAL

levels

reticulum of protein

adaptation

RESEARCH

such as cardiac

calcium

of the sarcoplasmic

Diacylglycerol

reticulum

AND

for

hypertrophy

are associated

to accumulate kinase

maintaining

COMMUNICATIONS

C may be, cardiac

and with

calcium. under

these

sarcoplasmic

function.

ACKNOWLEDGMENT: This study was supported the National Heart, Lung, and Blood Institute,

by a grant [HL 218501 from NIH, Bethesda, Md.

REFERENCES (1) (2) (3) (4) (5)

Katz AM (1979), Adv Cyclic Nucleotide Res 11, 303-345. Schwartz A, Entman ML, Kaniike K, Lane LK, Van Winkle IvB, Bornet EP (1976), Biochim Biophys Acta 426, 57-72. Katz S, Remtulla MA (1978), Biochem Biophys Res Commun 83, 1373-1379. LePeuch CJ, Haiech J, Demaille JG (1979). Biochemistry 18, 5150-5157. Takai Y, Kishimoto A, Inouc M, Nishizuka T (1977), J Biol Chem 252, 7603-7609.

(6)

(7) (8) (9) (10)

Takai Y, Kishimoto A, Twasa Y, Kawahara Y, Mori T, Nishizuka J Biol Chem 254, 3692-3695. Kishimoto A, Takai Y, Mori T, Kikkawa D, Nishizuka Y (l?SO). Chem 255, 2273-2276. Sumida M, Wang T, Mandel F, Froehlich JP, Schwartz A (1978), Chem 253, 8772-8777. Martin JB, Doty DM (194?), Anal Chem 21, 965-967. Michell RH (1979), Trends Biochem Sci 4, 128-131.

1383

Y (1979), J Biol J Biol