Vol.

184,

No.

April

30,

1992

BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

COMMUNICATIONS

RESEARCH

Pages

893-899

CYCLIC AMP-DEPENDENT PHOSPHORYLATION OF THE INOSITOL-1,4,5-TRISPHOSPHATE RECEPTOR INHIBITS Ca2+ RELEASE FROM PLATELET MEMBRANES Todd

M. Quinton

University

Received

March

12,

and William

of Louisville, Louisville,

L.

School KY 40292

Dean

of Medicine

1992

SUMMARY: Purified internal platelet membranes were treated with the catalytic of protein kinase A to determine its effect on inositol-1,4,5subunit Release kinetics were monitored using trisphosphate (IP3)-mediated Ca2+ release. rhod-2, a Ca2+-specific fluorophore. Protein kinase A maximally inhibit-d the rate of IP,-mediated Cazf release by approximately 30% at saturating IP, (10 PM). This inhibition was eliminated by pretreatment with a specific kinase inhibitor peptide. Partial purification of the platelet IP3 receptor showed that both Since endogenous kinases and added Akinase directly phosphorylate the receptor. the IP3 receptor is phosphorylated in the absence of added kinase, the observed inhibition (30%) by protein kinase A does not represent the maximal effect of phosphorylation. 4' 1992 Academic Press, Inc.

Ca2+ is an important many

metabolic

activation and

system low

(2).

located

Ca2+ and

due to reduced

phosphorylation

the

dense

dense

CaZt is sequestered process

both

Tohmatsu

addition,

tubular

permeabilized

platelets

IPs-sensitive

stores,

al. caused

although

of CAMP-dependentphosphorylation available platelet

Ca2+ both dense

Abbreviations: effect; IP3,

by stimulating

tubular

inositol

membranes reported

that et al.

of

dense

tubular

in maintaining activation

shown

that

CAMP-dependent

increases

the

and in intact

in the amount failed

effect at

which

platelets

(8),

by CAMP.

CAMP to this

CAMP may Ca2+ release

In

saponin-

of Ca2+ released to observe

Thus,

and inhibiting

rate

function of

this

is

of

in isolatedmembranes. Ca2+ uptake

component

part

addition (10)

the

involved

of platelet

a 50% decrease O'Rourke

(6,7)

of

least

has been

membranes

and

regulation

Ca2+-ATPases

Platelet

and at

to inhibition (9)

is

activation. It

system

in isolated

et

membrane

CAMP (3),

Ca2+ (4,5).

integral

is regulatedby

system

Ca2' for

contribute

may

plasma

tubular

increase

cytoplasmic df

the

in the

an

Ca2+ concentration in

that

involved

Ca2+ is

platelets,

in providing

by agonists

messenger

both

Consequently,

resting

and this

In

The intracellular

channels

inhibited is

processes.

(1).

Ca2'

intracellular

from effect

decrease in the

system.

EC,, > concentration 1,4,5-trisphosphate.

required

to

elicit

50% of

the

maximum

Vol.

184,

No.

2,

1992

The effect appears

to

increase

in the

be

phosphorylation

This system

has to IP,)

and liver

for

in brain the

opposite

as that

may be a consequence

paper

examines

membranes

the

on the

of

effect rate

IP,

effect

observed of

et

microsomes,

EC,, for

MATERIALS

COMMUNICATIONS

These forms release

of

in

of of

IP, platelet

a lo-fold

CAMP-dependent

Thus,

differences the

Ca2+ release

reported

in liver,

(12).

phosphorylation

of IP,-mediated

(11)

Ca2+ release

in brain. multiple

al. while

5-fold on

RESEARCH

on IPa-mediated

Supattapone

IP,

the

BIOPHYSICAL

phosphorylation

tissue-specific. EC,,

AND

CAMP-dependent

decreased

phosphorylation sensitivity

of

BIOCHEMICAL

CAMP-dependent liver

(increased between

receptor dense

brain

(13-15). tubular

Ca2+.

AND METHODS

Materials. Outdated human platelets were obtained from the Louisville chapter of the American Red Cross. Rhod-2 was purchased from Molecular Probes, Inc. The catalytic subunit of CAMP-dependent protein kinase (protein kinase A) heparinagarose and protein kinase inhibitor peptide of Cheng et a1.(16) were purchased from Sigma Chemical Co. Thapsigargin and inositol 1,4,5-trisphosphate were Wheat germ agglutinin-agarose was a product of purchased from LC Services, Inc. Pharmacia. [T-~~P]ATP was purchased from Amersham and [3H]IP3 was from New England Nuclear. Platelet internal membranes were purified according to Dean (17). Ca2+ Methods. release rates were determined from the rate of change in fluorescence of rhod-2. Fluorescence was monitored with an SIM-Aminco SPF-500C spectrofluorometer using an excitation wavelength of 553 nm and an emission wavelength of 576 nm. A typical assay mixture consisted of 2.5 pM Ca2+, 10 mM Tes buffer at pH 7.5, 100 5 PM rhod-2, and 0.2 mg platelet internal mM KCl, 50 mM K,HPO,, 10% glycerol, titration of rhod-2 was carried membrane protein in 2 mL at 37O C. A complete out under the above assay conditions (with 5 mM MgATP added) to determine the linear range of Ca2+ response and the relationship between Ca2+ concentration and emission magnitude. MgATP was added to a concentration of 5 mM to initiate uptake of Ca2+ into membrane vesicles. The amount of Ca z+ loaded into the vesicles was normalized for each assay by allowing a decrease of 2.0 fluorescence units (corresponding to about 10 nmol Ca2+) during uptake. Thapsigargin (200 nM) (23) was added to inhibit uptake. IPs was then added to a concentration of 10 PM to initiate Ca2+ release from vesicles (19). CAMP-dependent protein kinase was incubated at 30' C for 10 minutes in the absence or presence of protein kinase inhibitor, and was added after the addition of ATP and membranes. Rates of Ca2+ release were determined from first order plots of the fluorescence data (20) using the expression:

ln[ l- (Fobs - F,dFmax - F,d 1 where Fobs is the observed fluorescence at a time after IP3 addition, Fmin is the fluorescence at the point of IP, addition, and F,,, is the maximum level of fluorescence after IP3 addition. Zero time was at the point of IP, addition. Rates were obtained by multiplying the rate constant derived from the first order Since protein kinase A did not plot by the total amount of Ca2+ released by IP,. affect the extent of release, the same value of total Ca2+ was used for completed on the same day with the same calculating rates for experiments membrane preparation. The platelet IP3 receptor was partially purified after solubilizationwith Triton X-100 as described by Mourney et al. (21). Purified platelet membranes (5 mg/mL) were solubilized with 1.5% (w/v) Triton X-100 at pH 7.7 in 50 mM Tris containing 0.15 M NaCl, 1 mM EDTA and with PMSF (40 PM), leupeptin (10 pg/mL), pepstatin A (10 pg/mL) and aprotinin (20 pg/mL) added to inhibit proteolysis. After centrifugation at 100,000 x G, the supernatant was applied to a heparinagarose column equilibrated with solubilizationbuffer containing 0.1% TritonX100 and 0.25 M NaCl. The IP3 receptor was eluted with a 0.5 - 1.0 M NaCl gradient and was then applied to a wheat germ agglutinin-agarose column (15) 894

Vol.

184, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

equilibrated with solubilization buffer containing 0.1% Triton X-100 and 0.6 M NaCl. The IP3 receptor was eluted with 0.2 M N-acetylglucosamine. IP3 binding measurements with 5 nM [3H]IP3 (21) indicated approximately loo-fold purification of the receptor to a level of 15 pmol IP3 bound/mg protein. SDS polyacrylamide gel electrophoresis was carried out according to Laemmli (22) on 5% acrylamide gels. Phosphorylation of platelet membranes was accomplished as described by Adunyah and Dean (7). Platelet membranes (75 pg) were treated with 2.5 pg of catalytic subunit of CAMP-dependent protein kinase in the presence of 10 mM Tes buffer, 0.1 M KCl, 0.05 M NaPO,, 1 mM MgATP and 20 $i of [f-32P]ATP. After incubation for 5 min at 30" C, the reaction was stopped by chilling to 0" C and membranes were removed by centrifugation at 100,000 x G. In control experiments protein kinase A was omitted, Phosphorylated membranes were solubilized with Laemmli sample dilution buffer (22), electrophoresed on 5% polyacrylamide gels, and phosphorylation was visualized by autoradiography.

RESULTS In

order

simultaneous addition to

to re-uptake

of

inhibit

of

Ca2+,

has been

all

forms

of

endoplasmic

membrane

Ca2+-pumps

transport

assays

thapsigargin.

in The

that

our

the total

shown

presence

that

membrane rates

were

release

activity

rates was

reticulum-type (Fig.

1) of

and

absence

Ca2+ uptake

preparation determined

is from

by devoid semilog

1.

Effects

by

prior

to

is

sufficient while

2 show (Fig. 200 of

not

typical 2)

of

Ca2+ 200

100

200

300

400

of thapsigargin.

500

600

700

plasma

plots

800

(see

membrane inset

900

(WC)

IP,, and catalytic

subunit of protein kinase A on Ca2+ uptake and release. Ca'+ uptake was measured using rhod-2 as described in "Materials and Methods". Thapsigargin (tg, 200 nM), IP, (10 PM) and protein kinase A (PKA, 100 units) were added at the times indicated by the arrows. The lower trace was offset by two fluorescence units to enable comparison of the two traces. Both traces are from the same membrane preparation at the identical protein concentration (0.2 mg/mL). The inset shows semilog plots of fluorescence data after the addition of IP, used to determine first order rate constants.

895

I-M

nM thapsigargin

2.6 nmollmin

Time

Figure

(23)

1 and

damped

inhibited

Ca'+-ATPases,

Figures

I 0

not

200 nM thapsigargin

(18).

inhibition

purified

Ca2+ release

Ca2+

Ca'+-ATPase

It

plasma

Ca'+-ATPase.

IP,-mediated

IP,.

affecting

indicates

obtain

in

Vol.

184,

No.

2,

1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

500

700

COMMUNICATIONS

6-

0

200

100

300

400 Time

600

800

900

(set)

Figure 2. Effect of protein kinase A on IP,-mediated CaZt release and r-e-uptake. Experimental detail is the same as in Fig. 1 except thapsigargin was not added to inhibit Ca'+ uptake and the data were obtained from a different membrane preparation. Arrows indicate the extent and duration of the IP3-induced CaZ+ release from the time of addition of IP,. The Ca2+ uptake rates obtained from the indicated lines are 0.5 nmol/min in the absence of PKA and 0.6 nmol/min after addition of IP3.

Fig.

1) as described

IPs-mediated kinase

Ca2+ release

addition

(Fig.

inset),

was not

extent

of

kinase (vertical return

arrow) to the

was reduced

the

experiment

was inhibited Table

was inhibited membrane

when

peptide

inhibited

arrows.

The

at I shows

that

(16)

prior

was to

the

first

I at In order

loo-fold

(see

was not

10 PM IP, to identify Methods)

significantly

(data

not

time

rate

of

mixture rate

to

arrow) for This

of

four effect

kinase

(Table of

the

Ca"

required

a protein

100 units from

by the

inhibition

of

different

of

in one membrane

assay the

and

(horizontal

with

of

rate

A

I).

To

kinase

was

Ca2+ release

results

at

presented

shown).

the platelet

and the

and the addition

Ca'+-

extent

release

incubated

ensure that 10 PM IPs was saturating, determined at 20 and 50 PM IP,. The inhibition concentrations

maximum

was 26 + 10%.

the

in Table

the

of re-uptake

The average

effect

efflux

in

of kinase to

the

both

both

Ca'+ release

16%.

addition

the

of IP,

by 100 units

kinase

2,

addition

the point

by a maximum of the

Fig.

with

When the

reduction

is due to stimulation in

1 and 2,

IP,-mediated

of Ca2+ released.

addition

the

in Figs.

that

was inhibited

of

by

50% by kinase

preparations

rate

kinase

shown

Ca2+ concentration

eliminated

inhibitor

2),

demonstrated

As shown the

amount

(20)

Ca'+-ATPase

A reduced

total

indicated

et al.

process. When the

of thapsigargin

by 40%.

preparation different was

absence In

the

(Fig. as

Meyer

order

kinase

affecting

inhibited

(6,7).

a first

protein

CaZ+ efflux

in the

section.

Ca2+ release.

l),

without

ATPase release

is

inhibited

thapsigargin (see

in the Methods

IP3 receptor,

partially

purified 896

the receptor receptor

is

shown

was purified in Fig.

3.

Vol.

184,

No.

2,

1992

BIOCHEMICAL

Table protein

and

kinase (units)

Effect kinase

I.

added

lane

high

Ca2+ release" (nmol/min) 3.1 +/0.1 2.6 +/0.1 2.6 +/0.1 3.1 +/0.1 3.1 +/0.2 __ - _ _ _ _ _ _ inhibitor.

receptor l),

but

by

added

is

close

a minor

protein

protein

is of

in our kinase

reported

membrane (Student's

the

purified

weight

membrane

by both

IP3

preparations. t-test).

of

by Mourney

et

4).

250 kDa (Fig.

al.

This

result

is

3,

The IP3

(Fig.

kinases

3,

(lane

suggests

on Ca2+ release

receptor

(21).

preparation

endogenous

phosphorylation

because

kinase Ca2+ release

(n-3).

4 different at P