Postsynaptic densities contain a subtype of protein kinase C

Vol.

140,

No. 2, 1986

October

30,

BIOCHEMICAL

EIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

POSTSYNAPTIC

DENSITIES

Marlene

Wolp,

September

18,

CONTAIN

A SUBTYPE

Susan Burgess’,

Department of Molecular 3030 Cornwallis Road,

Received

AND

1986

Biology, Research

OF PROTEIN

U. K Misra

KINASE

691-698

C

and Naji Sahyoun*

The Wellcome Triangle Park,

Research Laboratories, North Carolina 27709

1986

SUMMARY. Protein kinase C or an isoenzyme thereof appears to be a significant component of postsynaptic densities (PSDs) from rat brain. This cytoskeletal organelle binds 4B-phorbol 12,lIdibutyrate (PDBu) with a B,*, of about 20 pmollmg protein and an apparent K, of 3.3 nM. Ca2 * and phosphatidyl serine (PS) stimulated the endogenous phosphorylation of a subset of PSD polypeptides with M, values between 16,000 and 22,000. Finally, a monospecific protein kinase C antibody reacted with a Mr 70,000 PSD polypeptide which migrated on SDS-PAGE slightly ahead of the M, 77,000 purified enzyme. These data suggest that protein kinase C or a similar enzyme can be integrated into a cytoskeletal system and may play an important role in postsynaptic function. 10 1986

Academic

Press,

Protein

kinase

cultures and

of fetal

neuronal

contrast

mature

(13-15);

indicate

differentiation,

brain

after

highly

may

much

treatment

with

in

a MrB7,000

regulation

the enzyme

enzyme

which levels

increase

Ca”

protein

kinaseC

brain

during in neurons

kinase

remains

(1.6).

where

(8).

Postulated

release

(9-12)

act on neurotransmitter

in brain

dramatically

chelators

polypeptide

abundant

concentrations

of the

of neurotransmitter also

is especially

higher

subpopulation

enriched

B-SO (7) and include

that

a major

even

are

enzyme

attaining

tissues,

fraction

substrates: function

C is a ubiquitous

neurons

to other

particulate

Inc.

receptors

(l-4).

brain

development

than

in glia

associated

Moreover,

and

for

the

of ion

(5). with

synaptosomes

it phosphorylates roles

Primary

two kinase

channel

and/or

transmembrane

region

lying

In the

from notable

in neuronal conductance signalling

systems The postsynaptic the postsynaptic and long-term calmodulin-dependent

density

membrane. neuronal

(KD)

represents

This cytoskeletal communication

(16).

protein

(17-19)

kinasell

an electron-dense organelle

is thought

The major

constituent

which

* Present address: Theodor Kocher Institut, Universitaet ’ Present address: Glaxo Inc., Research Triangle Park, *To whom correspondence should be addressed. Abbreviations:

PDBu, 4B-phorbol density; SDS-PAGE,

12,13-di butyrate; sodium dodecyl

appears

in tight

to feature

prominently

of cortical to play

Bern, Freiestrasse NC 27709

K, phosphatidyl sulfate-polyacrylamide

dual

structural

to

in short-

and midbrain

1, 3000

serine;

apposition

PSDs is as well

Bern 9, Switzerland

PSD, postsynaptic gel electrophoresis

as

Vol.

140.

No. 2, 1986

catalytic

roles (20).

associated

BIOCHEMICAL

Neurotransmitter

with

the PSD.

PSDs, particularly

because

and other

kinases

protein

out utilizing

phorbol

receptors

Thus,

it was

this enzyme (24).

ester

AND

and

cogent

ion

and (@‘P)ATP were supplied

(21-23)

whether

can act on cytoskeletal

COMMUNICATIONS

also appear

protein

polypeptides,

is present

receptors,

of the kinase

phosphorylation

and immunochemical

polypeptide

to be tightly

kinaseC

and characterization

MATERIALSAND 3H-PDB~ and calmodulin

RESEARCH

channels

to determine

The detection

binding,

BIOPHYSICAL

in

ion channels,

in PSDs were

carried

reactivity.

METHODS

were obtained from New England by Sigma. Nitrocellulose membranes

Nuclear. were

Unlabeled PDBu, ATP, from Millipore Corp.

PS

Synaptosomes were derived from cortices and midbrains of 150-200 g Sprague-Dawley rats; nuclei were removed from the brain homogenates and synaptosomes were separated from myelin and mitochondria on a 0.8/1.0/l .2 M sucrose-density gradient (25). Synaptosomes were treated with 2mM EGTA and 1 mM EDTA to release bound calmodulin and protein kinasec, and they were extracted with 1% Triton X-100 to prepare the cytoskeletal fraction. PSDs were purified from this fraction by centrifugation over a 1.011.512.0 M sucrose-density gradient (25). Protein kinase C was purified from rat brains by affinity recycling on human erythrocyte inside-out vesicles followed by phenyl-Sepharose chromatography (26). Protein kinase C antiserum was raised in a rabbit by intradermal injection of 60-80 ug of antigen mixed with complete Freund’s adjuvant followed by booster injections at 3- to 5-week intervals. Monospecific antibodies were subsequently affinity purified, utilizing electrophoretically pure kinase immobilized on Gene-Screen (New England Nuclear) membranes (27.28). Recognition of PSD polypeptides by this antibody preparation was examined by immunoblotting: polypeptides were resolved on 7.5% acrylamide Laemmli gels (29), electrophoretically transferred to nitrocellulose sheets (30). blocked with a 3% albumin solution and incubated with a 1:250 antibody dilution (31). Goat anti-rabbit IgG coupled to alkaline phosphatase (Cappel) was then used to visualize the immunoreactive polypeptides (32). 3H-PDB~ binding to PSDs, synaptosomes and purified protein kinase C was performed in triplicate at 24’C as described earlier (26), and the bound ligand was retrieved by filtration over Whatman GFK glass filters. Nonspecific binding was measured in the presence of 1 PM PDBu and was lessthan Zoom of total binding. Phosphorylation of PSDs was carried out in a reaction mixture of 50 PI of 50 mM Tris/HCI pH 7.6, 12.5 PM ATP, 1 uCi (y-“P)ATP, 1 mM dithiothreitol, 10 mM MgCI>, 1 mg/ml polyethyleneglycol 20,000 and CaCI,, calmodulin or PS where indicated. The reaction was carried out for 1Omin at 3O”C, stopped with 1% SDS-solution and resolved on a 7.5-15% gradient SDS polyacrylamide gel. Proteins were measured by the method of Bradford (33).

RESULTS Phorbol binding (Fig.

ester

was lA,B).

a linear The

remained

receotor(s)

at 100 nM ‘H-PDBu, PSD phorbol

function

binding

unaltered

ester

in PSDs. Specific

of PSD concentration,

of 30 nfv:

with

and a similar receptor

half

K, value. from

In five

linearity

reduction

was then

was 6.5 nM for the pure

deviation

3H-PDB~

protein

separate

at higher

equilibrium

the binding

protein

the PSD contained the

concentrations

Scatchard

of ligand.

692

with

The

0.5 uM

at 24°C PDBu,

1 mM

EGTA for

CaCI,.

kinase

(Fig. 2).

for

number

1Omin

The

70%,

a high plot

evident.

by about

by substituting

to the purified

experiments,

after

by 80%

PS reduced

was obtained

whereas

to PSDs was clearly

be displaced

omitting

compared

of ‘H-PDBu reaching

could

0.5 UM 4a-phorbol;

3H-PDB~ this

binding

affinity

The

site with

PSDs (Fig. of high-affinity

but even The K, for about

2) showed binding

a

Vol.

140,

No. 2, 1986

BIOCHEMICAL

ca,

AND

250

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

.

5 E

200

4 3

150

2 2

100

g

50

,I

J

:/10

20

30 TIME

40

50

60

(mm)

PSD (pg protein) fig.

1.

Deoendence of ‘Ii-PD8u bindina on time of incubation of ‘H-PD8u (15.8Cilmm) was incubated with 15 pg of intervals (upper panel), or with different amounts of panel). 0.1 mM CaCll and 20 pg/ml PS were present in all

3l-GPDBu Fig. 2.

and PSD concentration. PSD protein for different PSD protein for 40min assays.

3~~

(nM)

‘Ii-PD8u bindina to PSDs and to purified ‘H-PD8u were incubated with 40 ng pure (closed circles) for 45 min in the presence binding is shown (A) and a Scatchard plot regression method (8).

693

BOUND

30 nM time (lower

@nob)

orotein kinase C. Varying concentrations of enzyme (open circles) or 10 Pg PSD protein of 0.1 mM CaCI, and 20 ug/ml PS. Specific of the data was obtained by the least square

Vol.

140,

No. 2, 1986

BIOCHEMICAL

AND

1

Fig. 3.

sites

was

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

23456

Stimulation of endoaenous ahosbhorvlation of P5Ds bv Ca2’/s. 10 ug of PSD was phosphorylated as described in “Materials and Methods’ with the specified additions. Lane 1,200 UM CaCI, and 1 ug/ml calmodulin; lane 2, 1 mM EGTA; lane 3,200 PM CaCI,; lane 4, 200 PM CaCI,, 1 119 P5, 100 nM PDBu; lane 5, as in lane 4 with 20 ng purified protein kinase C; lane 6, autophosphorylation of 20 ng protein kinase C in the presence of 200 uM CaCII, 1 Pg P5, 100 nM PDBu. M, x lo” values for standard proteins are shown to the right.

1Opmollmg

protein,

and

about

twice

that

amount

when

the

low-affinity

sites

were

included. Q*+/PS-dependent also

assessed

phosphorvlation

by Ca’+/phospholipid-dependent

substrates.

No Ca’+/PS-dependent

substrate.

However,

lated

(Fig. 3).

200 PM

CaCI,

between

16,000

augment

the

from

PSD-associated

Four

relatively

and

20 pg/ml

to

phosphorylation

and 22,000

1 min.

also

two

Mr polypeptides

lation

resulted

characteristically lmmunochemical

solubilize

in non-denaturing

weight

3, lane 4).

of these

same

not shown).

calmodulin

molecular

polypeptides

PS (Fig. 3, arrows); (Fig.

due

Interestingly, in increased

these

Addition

several

agents.

other

of protein Therefore,

when

histone

evident

of the However,

was

the

assay

presence

MI

of

values

C did not further time

reduced,

was

while

presence

C substrates,

reduced increased

of exogenous

in the

kinase

phosphory-

apparent

kinase

the

phosphorylation protein

111s was used as

32P in the presence had

when

in the

exogenous

to be selectively

protein

kinase

in PSDs was

and type

incorporated

However,

endogenous labeling

endogenous

of exogenous

became

kinaseC

of

phosphoproteins

to endogenous

(Fig. 3, lane 1).

detection

of protein

proved

polypeptides

polypeptides.

polypeptides

involved

presence

was detected

endogenous

phosphorylation

of the

C (data

lower

low

The

phosphorylation

phosphorylation

phosphorylation

10min

kinase

in PSDs.

protein of Ca2* and

especially

Ca’+Icalmodulin-dependent

of the

phosphory-

PSD components. kinaseC we

in PSDs. elected

PSDs are

to analyze

notoriously for

their

difficult putative

protein

to

Vol.

140,

BIOCHEMICAL

No. 2. 1986

Fig. 4.

by immunoblotting

purified

soluble

enzyme

estimated

with

pure

soluble

counterpart

component because

by direct

binding

(lanes

which

1 and

(Fig. 4, lane 2). However,

in the same PSDs derive

region

from

PSDs was markedly

as the

attenuated

was

protein synaptosomes

Further,

relative

to the

the

0.9 or

A less intensely C appeared

doublet;

this

immunoreactivity

1.8 pmol

staining

a minor

of antigen

of pure PSD

band

to co-migrate

is not

of

(Fig. 4, lane 3) in also

with

its

immunoreactive

a surprising

of both

amount

reactivity

of presumed

at M, 70,000

also contained

presumed

the

1.6 pmol

visualized

kinase

PSD immunoreactive

synaptosomes.

employing

4, respectively).

at M,77,000.

synaptosomal

Fig. 4 confirms

antibody

band

migrates

In contrast,

purification.

rabbit

an immunoreactive

antigen

at Mr 66,000.

than

its monospecific

by 3H-PDB~ examined;

to the

appeared

rather

enzyme

was also

contrast

RESEARCH COMMUNICATIONS

Western blot analvsis of ourified orotein kinasec. svnantosomes and PS&. Purified protein kinaseC, synaptosomes and PSDs were electrophoresed on a 7.5% SDSpolyacrylamide gel, transferred to nitrocellulose and incubated with a rabbit monospecific protein kinaseC antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit antibody. Lane 1, purified protein kinase C, 0.9 pmoles ‘H-PDBu receptors; lane 2, synaptosomes, 3.2 pmoles ‘H-PDBu receptors; lane 3, PSD, 1.6 pmoles 3H-PD8u-receptors; lane 4, purified protein kinase C, 1.8 pmoles ‘H-PDEu receptors. Molecular weight standards were used as in Fig. 3 (not shown) to estimate the M, values of the immunoreactive polypeptide bands (lines to the right). The same immunoreactive bands were observed in five independent experiments.

kinaseC

kinase

AND BIOPHYSICAL

finding

synaptosomes

estimated

and

by ‘H-PDBu

binding, DISCUSSION Postsynaptic underwent

densities

Ca’+lPS-sensitive

protein

kinaseC

protein

kinase

antibodies.

kinase

in several

cytoskeletal aspects,

rat

brain

endogenous These

C in this cytoskeletal

This putative brain

from

contained

high-affinity

phorbol

ester

and

cross-reacted

with

phosphorylation,

observations

constitute

strong

evidence

for

differs

from

binding

sites,

monospecific the

presence

of

organelle. subpopulation including

of enzyme,

the following: 695

however,

1) the PSD enzyme

is tightly

purified associated

soluble with

Vol.

140,

the

No. 2, 1986

cytoskeleton,

display

resisting

a twofold

apparent

displays

phorboi

antibody

ester

protein

kinaseC

Thus,

weak

)H-PDBu data

suggests

integrated

that

into

post-translational coding

genes

and mRNA

ester

receptor

unlikely

cannot

species

ester

binding

polypeptides

and reacts

with

chelators;

contain

2) PSDs

one

histones

in

brain

or more

a Ca”/PS-

protein

kinase

that

has a lower

a doublet

mRNA

The presence

in a preliminary

report

which

are

C

as evidence

for

of the

between

interpretation

a modified

protein

PSD enzyme

may

stem

C isoenzymes

a gene

to the existence

(35-38).

The altered

affinity

of several

of the PSD phorbol or may

of Mr 110,000/115,000 may

from

has, in fact,

be ascribed

PSD (31)

is

covalent

distinct

and may

in the

C which

from

protein

PSD kinase,

of the

kinase

of brain

discovery

authentic

a dissociation

plausible

kinase

‘H-PDBu

observations).

from

recent

protein

with

or may derive

(34)

enriched

and

unpublished

that

from

synaptosomal

activity

of the corresponding

Our highly

different

solubilized

A more

possibility

splicing,

for such isoenzymes property

the

(U. K. Misra,

represents

M, value

enzyme.

environment.

PSDs raised

completely

that

columns

receptor

alternative

coding

and

was

be construed

The lower

be an intrinsic

in its molecular

soluble

phosphorylation.

PSD phorbol

described

features

Ca”

basic

against

by the finding

and ion-exchange

cytoskeleton.

may

COMMUNICATIONS

and

histone-phosphorylating

Ca’+/PS-dependent

for the soluble

recently

phosphorylate

fractions

Ca’+/PS-dependent

modification,

been

and/or

3H-PDB~,

synaptosomes

subcellular

was rendered

the

the

for

antibody

toward

cross-reactivity

and

enzyme not

RESEARCH

detergents

for PSD components

in these

on gel filtration

binding

soluble

monospecific

cross-reactivity

with

antibody

non-ionic

3) PSDs do

affinity

receptor

co-purified

the

BIOPHYSICAL

kinase.

C. This possibility

receptors

that

lower

the soluble

The weak

kinase

4) the

AND

with

than

as well;

and

an apparently than

affinity

sites

reaction;

Mr value

solubilization

higher

low-affinity

dependent

the

BIOCHEMICAL

reflect

protein

pertain

to this

special kinase

C-

molecular

environment. The presence enzyme

plays

synaptic

of protein

a role

in presynaptic

compartments

permitting

dual

terminals.

The integration

from limited

kinase

a soluble subset

signalling

compartment, of substrates

role of the enzyme

C or a protein

kinase

C isoenzyme(s)

(28) and

postsynaptic

parallels

that

of

calmodulin-dependent

through

both

enzymatic

of protein

kinase

possibly in its proximity.

in PSD function.

systems

C into

leading

signalling.

at the

level

to faster

responses of these

696

and/or

substrates,

it was of interest

kinase

of the the

to note

that

Its occurence

protein

the PSD precludes

The nature

Accordingly,

neuronal

in the PSD implies

need

pre-

II

in both (39),

thus

and postsynaptic

for

its translocation

to its action naturally, that

this

four

on a more

determines low

the

molecular

Vol.

140,

No. 2, 1986

weight

PSD polypeptides

these

polypeptides

Protein

kinaseC

equally

well

values

were

were and

suggests

the that

and the number

designated both

L,-L,

kinases

(42,43),

phosphorylated reaction

may

the PSD, and

far

possibly

C of other

greater

protein

Because

it would

by Ca’+/PSresult

for

the

endogenous

abundance

C phosphorylates

polypeptides

(four) light

to speculate

and calmodulin-dependent

representing

in actin-myosin an aspect PSD components

at least

these

two

of

protein

kinase

II.

shared

substrates

calmodulin-dependent these

substrates

approximate

chains

COMMUNICATIONS

reaction;

phosphorylate

of the

kinase

myosin

RESEARCH

calmodulin-dependent

kinase

be interesting

in alterations

less abundant

BIOPHYSICAL

in a Ca’+/PS-dependent

calmodulin-dependent

of these

(40,41).

AND

phosphorylated

substrates

the

despite

observation

kinase

BIOCHEMICAL

from

those other

kinases interactions

very

of brain

tissues

on whether

of synaptic

plasticity.

cannot,

of course,

PSD.

myosin

light

The light

M.

chains,

substrates chains

for

are really

Such a phosphorylation

thus

altering

The

phosphorylation

be ruled

This

efficiently.

are suitable

myosin

in the (44).

enzyme.

the topography by protein

out.

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698