Endogenous activity of cyclic nucleotide-dependent protein kinase in plasma membranes isolated from Strongylocentrotus purpuratus sea urchin sperm

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 180, No. 3, 1991

Pages 1436-1445

November 14, 1991

ENDOGENOUS IN P L A S M A

Jesus

A C T I V I T Y OF C Y C L I C N U C L E O T I D E - D E P E N D E N T PROTEIN KINASE MEMBRANES I S O L A T E D F R O M Strongylocentrotus purpuratus SEA URCHIN SPERM

G a r c i a - S o t o a*, Luz Alberto Darszonb, c

M. A r a i z a a, M i r i a m B a r r i o s a, and Juan P. Luna-Arias a

aInstituto de Investigacion en Biologia Experimental, Facultad de Quimica~ Universidad de Guanajuato, A.P. 187, Guanajuato, GTO, Mexico bDepartamento de Bioquimica, Centro de Investigacion y de Estudios Avanzados and CDepartamento de Bioquimica, Centro de Investigacion sobre Ingenieria Gen~tica y Biotecnologla, Cuernavaca, Mexico Received September 27, 1991 SUMMARY: Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [T-32p]ATP showed in the presence of 1 ~M cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 ~M of cAMP. In contrast, higher concentrations (100 pM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a d e t e r g e n t - s o l u b i l i z e d fraction. Membranes pretreated with 200 p M cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit. ©1991AcademicP..... Inc.

In mediated

eukaryotic via

This

enzyme

(R)

subunits

the

form

results subunit

the

cells,

activation

is composed that

(I).

of

protein

*To whom correspondence

of

constitute cAMP

to

years, kinase

an

the

(C)

the

subcellular

has

been

1436

two

regulatory

activation, the

are

protein

and

inactive

should be addressed.

0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

cAMP

of a cAMP-sensitive

and concomitant

In recent

cAMP-dependent

effects

of two catalytic

together

R2C 2. Binding in release,

the

largely kinase.

regulatory

holoenzyme subunit

of

dimer

of the catalytic distribution subject

of

of

more

Vol. 180, No. 3, 1991

detailed cytosol

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

studies but

since

also

and microtubules Cell eventto

and

are essential these

The

the

i.e.,

in

has been already

membranes

reaction

exocytotic

that sperm must perform in

(5). In sperm from sea of

-an

processes

cyclic

are

urchins

order

and

many

accompanied

nucleotides

and

by

of

a

cytosolic

cAMP-dependent

shown in sea urchin sperm

soluble

protein

sperm,

protein

representing

(9,10).

On the

kinase as much

other

hand,

of plasma membranes

L. pictus and A. punctulata sea urchin

sperm

from

(speract and resact)

[y-32p]ATP

report,

the

invoked,

an

protein

early studies have shown that incubation peptides

the

(6,7,8).

presence

1.5% of the

structures,

acrosome

cellular

metabolism

phosphorylation

cellular

functions

the egg

species

increased

as

other

not only

(2-4).

motility

fertilize

other

in

it seems to be localized

results

into several membrane

involvement

of

with

GTP

plus

in the incorporation polypeptides

a cGMP-dependent

without testing the extractability

(11).

protein

from egg

of 32p In this

kinase

was

of the kinase actvity

from the sperm membranes. In this work, whether associate membranes

a

experiments

cyclic with

therefore

nucleotide-dependent

plasma

derived

were

membranes

from

of

flagella

protein

sea of

undertaken

urchin

the

sea

to study

kinase sperm. urchin

does Plasma sperm

Strongylocentrotus purpuratus were used in this study. METHODS M a t e r i a l s . Sea u r c h i n s (Strongylocentrotus purpuratus) w e r e obtained from Pacific Bio-Marine Labs (Venice, CA). Spawning was induced by intracoelomic injection of 0.5 M KCl. Chemicals were obtained from Sigma, except materials for electrophoresis (Bio-Rad Labs.) and [7-32p]ATP (Amersham). Isolation of sperm plasma membranes. Collected sperm (i ml) were washed twice by diluting them 15-fold in Ca 2+ and Mg2+-free artificial seawater and centrifuging at 3000 x g for 10 min. The flagella were then broken off from sperm by passing the cell suspension ten times through a 21 gauge hypodermic needle (12). This suspension was then centrifuged (5000 x g for 5 min) and the flagella remaining in the supernate used as the starting material to isolate plasma membranes as already described (13,14). Measurements of protein kinase activity. Sperm membranes were incubated in a reaction mixture (75 ~i) containing 20 mM Tris-HCl (pH 7.4), 5 mM magnesium acetate, 1 mM theophylline, 150 mM NaCI, 10 mM NaF, 25 M [7-32p]ATP (600,000-900,000 cpm/assay), 0.25 % Nonidet P-40 and the indicated concentration of cAMP or cGMP. The medium also contained 1 mM ouabain and 1 mM EGTA to preclude ATP h y d r o l y s i s by N a + , K +- and C a 2 + - d e p e n d e n t ATPase activities, respectively (15). The reaction was initiated with the addition 1437

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of membrane protein (20-30 ~ g) and allowed to incubate at 30 °C for 3 min; the reaction was terminated by adding 75 pl 20% trichloroacetic acid (TCA). Precipitates were collected into GF/B Whatmann filters, which were then washed with 5 ml TCA (10%) and 5 ml ice-cold ethanol, dried and counted for radioactivity. Basal i n c o r p o r a t i o n of 32p was d e t e r m i n e d by o m i t t i n g the c y c l i c n u c l e o t i d e s from the assay medium. V a l u e s of p r o t e i n k i n a s e activity represented the substraction of 32p incorporated in the p r e s e n c e and absence of cyclic nucleotides. For the cAMPdependent protein kinase activity, linearity with respect to incubation time and membrane protein concentration were observed up to 3 min and 0.6 mg/ml of membrane protein, respectively. The concentration of ATP to give maximal incorporation of radioactivity was between 25 and 50 ~M. For e l e c t r o p h o r e s i s and a u t o r a d i o g r a p h y , the r e a c t i o n m i x t u r e (50 ~i) was as above, except that 1 p C i / a s s a y of r a d i o l a b e l e d ATP was used. In this case, the r e a c t i o n was terminated with the addition of 50 ~i electrophoresis sample buffer (Tris-HCl, pH 6.8, 8% SDS, 32% glycerol, 4% 2mercaptoethanol and 0.008% bromophenol-blue). Samples were boiled for 3 min, and then s u b j e c t e d to S D S - p o l y a c r y l a m i d e gel electrophoresis according to Laemmli (16). Gels were stained with 0.025% Commassie Blue, destained, dried and exposed to Kodak XOMAT-AR5 films for visualization of phosphorylated proteins. Protein was determined by the method of Lowry et al. (17), with bovine serum albumin as standard. RESULTS Effect

of

cyclic

nucleotides

on

protein

phosphorylation.

Flagellar plasma membranes were incubated with radiolabeled ATP in the presence of various concentrations of cyclic nucleotides and then results

processed for electrophoresis and autoradiography. are

presented

nucleotides,

the

in

amount

Fig.

i.

In

the

absence

of r a d i o p h o s p h a t e

membrane polypeptides was very low

of

cyclic

incorporated

(lane A).

Addition

The into

of cAMP

stimulated in a dose-dependent manner phosphorylation of multiple polypeptides (lanes B-E). Polypeptides that become phosphorylated corresponded to

molecular weights of about 95.5,

60.2,

39.4 and 29 kDa. Although

50,

40.7,

not

88,

78.5,

resolved

66,

in the

gel of Fig. i, at least three other polypeptides of 110, 125 and 160 Kda also accumulated radiophosphate in the presence of cAMP. cGMP, on the other hand, also stimulated protein phosphorylation in membranes but higher concentrations (50-100 PM) were required for

maximal

effect

(lanes

F-I);

the

pattern

of

phosphorylated

polypeptides was essentially the same than that elicited by cAMP. Quantitation of protein phosphorylation was accomplished by incubating

the

concentrations were

processed

membranes

with

[ y -32p]ATP

of cyclic nucleotides. for determination

into acid-precipitable material

of

and

After incubation, radioactivity

various samples

incorporated

(see Methods). As illustrated in

1438

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

25

A

B

C

D

E

F

G

H

I

97 -

20

"o

66-

~

E

_

"~

cGMP

15

45-

~:~ z

10

29-

~ ~

5 O

Q

i

®

_,

i

i

_o

i

log [CYCLIC NUCLEOTIDEL M

Fig. 1. Effect of cyclic nucleotides on 32p incorporation into polypep£ides of sperm plasma membranes. Lanes are: A, without cyclic nucleotides; R, 0.5 ~M cAMP; C, i ~ M cAMP; D, 50 ~ M cAMP; E, i00 ~M cAMP; F, 0.5 ~ cGMP; G, i ~M cGMP; H, 50 ~ M cGMP and I, i00 ~M cGMP. Three other experiments made with different membrane preparations gave the same results. Fig. 2. Protein kinase activity in sperm plasma membranes as a function of the cyclic nucleotide concentration. Incorporation of radiolabeled phosphate into membrane protein was determined by measuring the radioactivity associated with an acid-precipitable membrane material, as outlined in Methods. Values are the mean of three determinations. The results shown are representative of four independent experiments.

Fig. ~M;

2,

cAMP

stimulated

half-maximal

100 ~ M

of

maximally

response

cGMP

were

was

protein

seen

necessary

to

phosphorylation

at about

0.6 ~ M .

observe

a

In

similar

at

1

contrast,

stimulatory

effect. The

results

presented

kinase

in

protein

preferentially

Extraction

cyclic

of

kinase

investigating protein

the

then

buffer

with

sperm

the

protein

mM and

sedimented

solution

mM dithiothreitol

membranes, We

EGTA the

protein

kinase

surface

of

could

thereby

tested of

of

that

a is

for last

(i00,000

200 m M

30

x g for 10 m M

1439

occur

during for

the

this

the

membrane-associated

at of

4

°C,

this

lh)

KCI,

sucrose).

sperm

cytosolic

accounting

plasma

min

minute

from

the

for

Accordingly,

(I0 m M NaCl, and

presence

membrane

kinase

extractability

activity. i0

plasma

membrane

activity.

first

the

to c A M P m o d u l a t i o n .

plasma

the

kinase

incubated

were

the

suggest

cAMP-dependent to

AMP-dependent

measured

during

the

Binding

preparation

the

sensitive

of

membranes.

above

and

10 m M This

possibility

by

membranes

were

sonicating

them

period.

Membranes

resuspended Tris,

treatment

pH

in

a

7.4,

0.i

allows

the

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1.

Extraction from membranes of the cAMP-dependent protein kinase

Pretreatment

Total enzyme activity

(9) None (control)

i00

i0 mM EGTA + sonication: Supernate Pellet

19.6 81.2

Supernate Pellet

105 9.4

Nonidet P-40:

500 pg of membrane protein were subjected to the indicated treatment, followed by ultracentrifugation (see the Text). Resuspended membranes and the respective supernate were assayed for cAMP-dependent protein kinase activity. Total activity (pmol/min) was calculated for each condition and expressed as the percentage with respect to that measured in nontreated membranes. Values represent the mean of two independent experiments, each made in triplicate.

removal

of

membranes. above

approximately

25%

of

proteins

associated

Treated membranes and the supernate

centrifugation

step

were

then

with

obtained

the

from the

incubated

under

p h o s p h o r y l a t i n g conditions, with and without 25 p M cAMP. As shown in Table I, 81.2% of the total protein kinase activity stimulated by

cAMP

remained

in

the

treated

membranes,

while

a

minor

percentage was recovered in the supernate.

with the

In an additional

experiment,

1% NP-40

°C)

activity

(i h, 4 of

membranes were

and then ultracentrifuged.

cAMP-dependent

protein

kinase

both the resuspended pellet and the supernate. i,

the

enzyme

activity

first

was

only

detected

was

incubated

Thereafter, measured

in

As shown in Table in

the

supernate

indicating the total solubilization of the protein kinase. Thus, resistant

to

the

detected

extraction

cAMP-dependent

by

treatments

protein

that

remove

kinase

is

proteins

loosely bound to the membrane surface.

The enzyme may be however

recovered

disrupting

in

a

soluble

form

after

the

membrane

structure with NP-40.

Removal

of

the

pretreated

with

manner

which

in

cAMP-dependent cAMP.

the

We

protein

addressed

inactive

in

kinase

a

holoenzyme ]440

from

membranes

preliminary

form

the

interacts

with

the

V o l . 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

Fig. 3. pretreated

Absence with an

B

C

of p r o t e i n phosphorylation in m e m b r a n e s excess of cAMP. Flagellar plasma membranes

were preincubated with cAMP and then ultracentrifuged and resuspended in buffer solution (see the Text). cAMP-pretreated membranes were incubated with [7 -32p] ATP alone (A), with 25 ~M cAMP (B) or 25 ~M cAMP plus 5 units/100 pl of bovine heart catalytic subunit.

membrane. the

This

presence

protein).

was

of

accomplished

cAMP

in

Sonication

by

excess

was

sonicating (200

carried

the

~moles

out

to

membranes

cAMP/ pg allow

in

membrane

the

cyclic

nucleotide to reach both sides of the membranes.

After i n c u b a t i o n

(30 min,

i00,000

4 °C),

membranes

r e s u s p e n d e d in buffer incubated

under

were

centrifuged

solution,

at

cAMP-treated membranes

phosphorylating

conditions

and,

p r o c e s s e d for electrophoresis and autoradiography. in Fig. the

3, protein phosphorylation

absence

or

presence

of

cAMP

was abolished in the

assay

This result may not be due to inactivation

x

g

were

and then

thereafter,

As i l l u s t r a t e d irrespective

(lanes

A

and

of the protein

of B).

kinase

since nontreated membranes were responsive to cAMP. Additionally, polypeptides

derived

from

cAMP-treated

susceptible to phosphorylation, of

a marked

catalytic

radiolabeling

membranes

remained

as demonstrated by the a p p e a r a n c e

upon

introduction

subunit from bovine heart

of

an

exogenous

(lane C).

DISCUSSION

The present

study shows that plasma membranes

flagella

of

the

sea

urchin

contain

an

endogenous

sperm

activity

1441

isolated

from

Strongylocentrotus purpuratus of

protein

kinase

that

is

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

preferentially

stimulated

by cAMP

rather

than

cGMP.

mentioned that in terms of purity these membranes free of material that

they

from intracellular

maintain

functionally

transport properties

(14,15,18,19).

Given

its

resistance

sonication,

but

that

detected

the

not

physiologically

that not

this

shown).

protein

was

is

of

10

their

ion

EGTA

and

mM

plausible

activity

the

might

membrane,

rather

kinase

be than

activity

is

form was also supported by the fact as

an

exogenous

a reliable

sea

in addition,

of

therefore

membrane-bound

ineffective

Protamine

kinase

and,

with is

kinase

with

from the cytosolic

protamine

it

be

are relatively

many

extraction

protein

That

active

detergent,

associated

artifactually. different

to

with

structures

It must

urchin

substrate

sperm

substrate for

(i0);

the

this

(data

soluble previous

observation was also shown in our assay system. It whole

has

been

sperm

respond

to

that

plasma

L.

urchins and

A

similar

membranes

pictus

GTP

phosphorylation

(ii).

independently These

sea

peptides

the

polypeptides nM.

the

egg

production,

protein

reported

of

by

level

effect

of

was

data

suggest

the

although

indication

participation

of

of

to

be

their caused

between a

cGMP

10-100

cGMP-dependent

intrinsic

association

of this kinase with the membranes was not documented.

We have not

found

the

same

phsphorylation discrepancies different amounts

sensitivity

in f l a g e l l a r may

be

sea urchin of

purpuratus

a

conditions,

to

plasma

explained species,

by

ii)

cGMP-dependent

sperm

and

including

cGMP-mediated membranes.

i) the

protein

iii) the

of an

via

several shown

from

punctulata

increasing,

by cAMP and cGMP at concentrations

kinase,

isolated

A.

and

the

kinase

variations

use

of

use

presence in

protein

These of of

apparent

cells

undetectable

activity the

distinct

from in

S.

experimental

plasma

membrane

preparations. The by

subcellular

cAMP

has

investigations example,

been

distribution recently

the

in several types

cAMP-sensitive

Golgi

apparatus,

poles

and

rough

microtubules

performed

in mammalian

cytosolic

enzyme,

of protein

protein

subject

of cells. kinase

endoplasmic (2-4). sperm

of

On

the

been

cells,

found

in

for the

spindle

hand,

studies

in addition kinase

appears

present in the outer plasma membrane and in the axoneme 1442

detailed

mitotic

other

that,

protein

stimulated

more

In somatic

has

reticulum,

indicate

a cAMP-dependent

kinases

to

the

to be

(20-22).

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The exact the

sperm

that

manner

membrane

in those

been

found

remains

cells in

subcellular

in which

absence

subunit.

Nevertheless, the

kinase

detected

to

experiments

isolated

from

preparation). nucleotides

membranes,

i.e.,

preference

for cAMP

the We

(data

was

not

plausible of sperm.

The

physiological

dependent,

protein

speculative. assume

could

be

bilayer

the

relevant

The

(ref.

with

of the

of the catalytic to c o n f i r m

membrane

~M for

to

via

its

importance

of

with the cytosolic

those

of

might

protein

cyclase

and

over

cGMP.

along

membrane-bound,

is

at

present it

is

and the

cAMPmerely

reasonable

in

the

membrane

substrates

placed

in

the

membrane

serve

as

surface; a

protein

this form of

reservoir

kinase

for

occurs

and probably

also

that the enzyme may be involved reaction,

likely

polypeptides for

the

here used,

acting

this

in

the

in the

in sperm

in concert

this

regard,

susceptible

possibly

the

the

it is expected

that become protein

the nature of the phosphorylated

membrane

flagellar

kinase

also

acrosome

substrates In

of

to

kinase.

not all the membrane

established.

response

of

this

Given the in vitro conditions physiological

details

distributes

a

activity

of the sperm flagellum,

and in the

found

in plasma

for

effect)

of compartmentalization,

suggests

also

the

that

half-maximal

that this kinase

occurrence for

we

15

that

similar

fact that the detected

head membrane,

hand,

to The

pretreated

protein kinase

head

or in the proximity of the membrane

plasma membrane motility

II serves

amounts of protein kinase activity

kinase

In terms

that

compartmentalization enzyme.

other

possibility.

shown)

anticipate

very

(0.4

It is therefore

sperm

can

identical

plasma membrane

to

the

has

the experiment

are necessary

binds

activity of cyclic nucleotide-dependent membranes cyclic

noteworthy kinase

of the dissociation

and the displacement

to

subunit.

In preliminary

membrane

same

binds

some

type

in membranes

other experiments

here

is

or

context,

this

as an indication

of the holoenzyme

regulatory

subunit

In this

to explore

subunits

It

protein

membranes

the regulatory

phosphorylation

cAMP was interpreted

holoenzyme

elucidated.

with

(2,4,22).

3 was made

of protein

whether

be

inactive

a cAMP-dependent

association

structures,

in Fig.

to

where

anchor the holoenzyme shown

the

only

phosphorylated

kinase.

1443

On

polypeptides known

of phosphorylation adenylate

that

cyclase

sea

are

the

other

remains

to be

urchin

sperm

is the guanylate (23,24).

Other

Vol. 180, No. 3, 1991

potential

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

substrates

function,

essential

might

under

be

phosphorylation receptor channels.

(26)

could

for

control

sperm by

include

Thus,

influence

the

transporters

and

acrosome

phosphorylation;

of the Ca 2+ channel may

ion

motility

cation

cAMP-dependent

(25)

and the

conductance protein

for

instance,

acetylcholine through

kinase

characterized in detail and its role in chemotaxis,

whose

reaction,

these

should

be

activation of

motility and acrosome reaction further explored.

This research was supported by The World Health O r g a n i z a t i o n , The T h i r d W o r l d A c a d e m y of S c i e n c e s , C O N A C Y T (Mexico) and by an International Research Scholar award from the Howard Huges Medical Institute to A.D. We thank Enrique Ramirez, Lucia Garcia and Irma Vargas for technical assistance, and Carmen Silva for help in preparing the manuscript.

ACKNOWLEDGMENTS:

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