Vol.

183,

March

No.

16,

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1992

725-732

CALMODULIN-BINDING PROTEINS OF CALCIUM-INDEPENDENT TYPE IN RAT BRAIN SYNAPTOSOMAL MEMBRANES: THEIR LOCALIZATION AND PROPERTIES Naoki National

1* , Akiko

Natsukari

Institute for Transport,

IDivision

Miwa,

Physiological Myodaiji,

Sciences, Okazaki 444,

of Neuropsychopharmacology, Pennsylvania, Philadelphia,

'Department of Biochemistry, Medicine, 3600 Handa-cho,

Received

and Michiya

January

31,

2

Fujita Division Japan

of Active

The Medical PA 19129

College

Hamamatsu University Hamamatsu 431-31,

School Japan

of of

1992

Some properties of calmodulin(CaM)-binding proteins (CaMBPs) of the Ca2+-independent type were investigated in the synaptosomal membrane (SM) from rat brain using the [1251]CaM gel overlay method. When SM was prepared in the presence of Ca", Ca"-independent CaM binding was decreased, whereas the Cazt-dependent type was not altered. All Cazt-independent-type CaMBPs were membrane-bound and scarcely present in the soluble fractions. When SM was heat-denatured, the 24/22.5-kDa CaMBPs could no longer be detected by [l"]CaM binding and a new component with higher molecular mass (>ZOOkDa) was shown to bind CaM in a Cazt -independent manner. A possible effect of CAMP- and Ca2+/CaM-dependent phosphorylation on CaM binding was also examined.

Calmodulin(CaM) closely Although of the

the

with

intracellular

the

located

will

regulatory

(see

ref.

of CaM has been is

help

the

protein a review).

studied

not well-known.

us understand

localizations

1 for

(2).

Knowing

that where

relationship(s)

of CaM and CaMBPs and their

functions. showed

[ lz51]CaM

synaptosomal

CaMBPs of the

should

CaM,calmodulin; membrane.

gel

membranes

CaPt-dependent

*To whom correspondence Abbreviations: SM,synaptosomal

calcium-binding activities

proteins(CaMBPs)

We previously of EGTA-washed

neuronal

intracellular

physiological

the

a ubiquitous distribution

CaM-binding

CaMBPs are

between

is

associated

type

profiles

overlay

(SM)

from

were

identified

rat

of the brain

Some of

by immunological

be addressed. CaMBP,CaM-binding

(3).

CaMBPs

protein;

Vol.

183, No. 2, 1992 and enzymic comparing found

properties effects

analyses, them with

In this are

BIOCHEMICAL

report, only

in

AND BIOPHYSICAL

and others those

from

we show that the membrane

different of phosphorylation

from

those

were

tentatively

other

identified

by

tissues.

CaMBPs of the fraction

Ca 2t -independent

and have

of the

on their

RESEARCH COMMUNICATIONS

physico-chemical

Ca2'-dependent

CaM-binding

type

type.

properties

Possible were

also

examined.

MATERIALS

AND METHODS

Bovine brain CaM was purchased from Sigma Chemical Co. [ 125 IJCaM (92.5 uCi/ug, bovine brain) was purchased from New England Nuclear. All other chemicals were of reagent-grade. Male Sprague-Dawly rats (125-150 g) were obtained from Shizuoka Laboratory Animal Center (Hamamatsu). The EGTA-washed lysed SM was prepared from rat brain cortex and cerebellum by a modified method of Malnoe et al. (4) as described previously (5). Briefly, the brain was homogenized in medium A (2 mM EGTA,SO ug/ml PMSF, 0.2 ug/ml leupeptin, 0.5 mMDTT.5 mMTris-Xl, pH 7.5) containing 0.32 M sucrose and centrifuged at 1,OOOxg for 20 min. The supernatant was further centrifuged at 10,OOOxg for 15 min and the The pellet was suspended in medium A supernatant (S2) was recovered. containing 0.32M sucrose, diluted with medium B (medium A at pH 8.1) and centrifuged at 27,OOOxg for 30 min. The pellet was resuspended and centrifuged at 27,OOOxg for 30 min. The final pellet was suspended in medium C (60 mM Tris-HCl, pH 7.5, 5.0 mM MgS04, 0.5 mM DTT) (P,), and stored at -80°C. In addition to EGTA-washed SM "untreated" and "Ca"-treated" SMs were prepared according to the same procedures as EGTA-washed SM except that EGTA was excluded from or 100 uM CaC12 was added to the medium, respectively. [1251]CaM gel overlay was performed by a modified procedure of Hertzberg et a1.(6) as described previously (3). The membrane preparations (2-3 mg protein/ml) were mixed with l/3 vol. of the sample buffer consisting of 125 mM Tris-HCl, pH 6.6, 3X(w/v) SDS, lOX(w/v) glycerol and S%(v/v) 2-mercaptoethanol. A sample (15-25 ug protein) of each membrane preparation was immediately subjected to SDS-PAGE(12X gel) in the Laemmli system (7) without heat-denaturation unless otherwise stated. The final gels after pretreatment were incubated in the medium (50 mM Tris-HCl. pH 7.5, 0.2 M NaCl) containing 0.02% NaN3, 10 n&l [1251]CaM (1.8~10~ cpm/ml) and either 100 uM CaC12 or 1.0 rnkfEGTA. After the gels were exhaustively washed, they were stained, destained, dried and then exposed to X-ray films for 24-80 h with an enhancing screen. Phosphorylation was performed according to the conventional procedures. The reaction mixture contained 60 mM Tris-HCl,pH 7.5, 5.0 ug protein of SMs, 1.0 mM mu MgS04, 0.5 mM DTT, 100 uM ATP, 110-125 EGTA and 0.943 mM CaC12 (pCa 6.0) and either 1 uM CaM or 40 uM CAMP in a total volume of 100 ul. The reaction was initiated by adding 30 ul of SM and allowed to proceed for 1 min at 3O'C. The reaction mixture then received 25 ul of the sample buffer, followed by heating 726

Vol.

183,

No.

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

for 2 min at 100 'C, and a 20-ul aliquot of the mixture was subjected to SDS-PAGE (12X gel), followed by [ 1251]CaM gel overlay as described above. Protein content was determined by the method of Lowry et a1.(8).

RESULTS CaMBPs of cerebral

cortical

classified

by their

those

mass higher

with

with

mass lower The bigger

1351127 1, panel

k,

of proteins

l-4)

1, panel

(S2),

A, lanes

cerebellar

their

with

1).

molecular

variously

relative

enriched based

cerebral

In the

k = 58 k > soluble

was 58 k = 49 k >> 235K = 1351127

A kDa

12345676

235135/127g=

B kDa

Fig.1. [ 1251]CaM and Ca2+-treated

rzel overlay SMs (lanes

for CaMBPs. 2 and 4) and

EGTA-washed corresponding

(lanes 1 and soluble

3)

fractions (lanes 5-8) were obtained from cerebral cortex (lanes 1.2,s and 6) and cerebellum (lanes 3.4.7 and 8) as described in the Method. T:z5same amount of protein (18 ug) was subjected to SDS-PAGE (12%) and I]CaM gel overlay was performed [ CaC12 (A) or 1 mM EGTA (B), followed a representative of six experiments

in

the presence by autoradiography.

with

three

of

different

in

5-8).

abundance

> 235 kDa for 3 and 4).

masses

(lanes

1 and 2) and 1351127

SMs (lanes

abundance

and those

fractions

their

was 49 k > 58 k > 135/127k

SMs (Fig.

two groups: (Fig.

They were

and soluble

of Ca2+,

tentatively

components")

components")

58 k, and 49 kDa. presence

235 k = 49 kDa for fractions

mass on SDS-PAGE into

consisted

A, lanes

SMs were

49 kDa ("bigger

34 kDa ("smaller

in the

on CaM binding cortical

than

components

of 235 k,

When assayed

molecular

than

SMs (Fig.

and cerebellar

either The

100 )H data is

preparations.

kDa

Vol.

183,

No.

2, 1992

for

the

cerebral

= 49 kDa for or Cazt

cortex

the

in the

dependent "untreated not k, while the

22.5

24-kDa

Soluble

were

assayed

lanes

l-8).

devoid There

panel

A),

the

enhanced with

presence

binding

the

Ca2+-

final

components

to that

k

EGTA

of an

of EGTA-washed

SM

A, lanes

in

bigger

Heating definite component

and 22.5-kDa heating

Ca2+ -independent Fig.

was prepared

cortex

and

(Fig.

[ 1251]CaM

1, panels

binding

and CaPt-treated 1, panel

of the

preparations A, four

31.5

A and

by the

k-kDa

pairs

of

and 24-kDa

the Ca 2t-treated

cerebellar

CaM binding

SM (Fig.

of Ca 2t was characteristic

1,

of the

B).

from

both

no 24-kDa

the cerebral component

in the

a 22.5-kDa

presence

component

when

assayed

in

cortex

and

was detected

in

of Ca 2t (Fig.

1,

and ones with the

was

presence

the

lower of EGTA

1 and 2). on the

of the

components

(lanes

(filled

arrows). activity

electrophoresis

overlay

pattern

CaM binding

2, panels 24-kDa

staining not

was detected

B).

728

(Fig.

of the

(cerebellum;

(data

235 kDa component

had a

(Fig.

3-8;

panel

no longer could shown). at the 2, panel

2, panels

A

235-kDa

A and B, lanes

1 and 2) could Protein

aggregation

binding 1, panel

before

gel

and 3-8). (Fig.

those

to the

samples

[125 I]CaM l-2

was due to protein

corresponding

cerebral

in

in

assayed

detected

lanes

whereas

cerebellar

same membrane

components

absence

Although

was increased

arrows),

1 and

In fact, it was markedly 2t of EGTA. No Ca -independent CaM binding

of the membrane effect

of 34 k,

kDa

1. lanes

in EGTA-washed

of Ca *+(Fig.

the

masses

The 31.5

SM (Fig.

the

the

smaller

1, panel

1 and 2),

and B; compare

B).

to the Ca 2t-dependent

SM when

mass were B, lanes

both

components

B).

by molecular

4).

decreased

presence

(panel

when

CaM bindings

(Fig.

cortical

molecular

from

In contrast

the

cerebellum

compare

in

1, panel

EGTA-washed

markedly

by the

cerebral

this

on the

cortical

(lane

of the

the

components

(Fig.

was no difference

the

were

3 and 4).

Ca '+

between

in

(lane

after

influence

characterized

lower

absent

(S2)

However,

components

(panel

similar

was predominant

of added

5-8).

when

panel

of either

The corresponding

were

component

components

found

COMMUNICATIONS

Presence

components

in cerebral

fractions

cerebellum B. lanes

smaller

bigger A).

or

3) but was almost presence

bigger

kDa

was predominant

SM (lane in

had little

a CaM binding

components

24 k,

component Z),

RESEARCH

shown).

The smaller 31.5

of the

SM" showed

7 and 8).

medium

1, panel

BIOPHYSICAL

5 and 6) and 58 k >> 235 k = 135/127 (lanes

preparation (Fig.

AND

(lanes

cerebellum

CaM binding

preparations (data

BIOCHEMICAL

open

B, lane

2)

be observed not

determine

Unexpectedly.

a

position B, lanes

if

3-8;

Vol.

183,

No.

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A 12345676

kDa

31.524-

4

6 12345678

kDa

gel overlay for heat-denatured,& phosphorylated Fig.2. [125 I]CaM CaMBPs EGTA-washed SMs were prepared from --and then heat-denatured cerebral cortex (lanes l=and 7) or cerebellum (lanes 2,4,6, and 8) as described in the Method. Membrane proteins were subjected to SDSPAGE either after (lanes 3-8) or without (lanes 1 and 2) heatgel overlay was performed in the presence of denaturation. [ 1251]CaM either 100 p CaC12 (A) or 1 mM EGTA (B), followed by autoradiography. When indicated, proteins were phosphorylated in a CAMP- (lanes 5 and 6) and a Ca2+/CaM(lanes 7 and 8) dependent manners. To terminate the phosphorylation reaction, SMs were heat-denatured. The conditions of endogenous phosphorylation were described in the Method. On heating CaM binding of 235 kDa (open arrows) increased but that of 24/22.5 kDa bands (filled arrows) was lost. The data was a representative of two independent experiments

Membrane

proteins

dependent then

manner

tested

However, except

for

A,

lane

in

component

7).

were

their

CaM-binding

blur

between

58

also

slightly

noted

increased

that on

(Fig.

CaM

CaM

2,

binding

k-Da

in

or

a Ca 2+ICaM-

electrophoresed

and

49-kDa

this

and

lanes

profile

manner,

heterogeneity

was

a CAMP-

capacities in

a Ca2+/CaM-dependent

molecular It

in

heat-denatured,

alteration

a slight

in

They

remarkable

phosphorylated increase

phosphorylated

(3).

for no

were

5-8). was

which

bands

indicating mass

binding

range by

phosphorylation

the (lanes

detected

were

some (Fig. 235

2.

panel

kDa 5-8).

DISCUSSION Some

Ca2+ ions from

the

speculations

can

demonstrated membrane

in to

the

be made the soluble

about

present

the work.

compartment

729

of CaMJ3Ps to

susceptibility 1.

CaMBPs were

mobilized

as

reported

Saitoh

by

and

Vol.

183, No. 2, 1992

Schwartz

(9),

modified

the

induced

BIOCHEMICAL

2. EGTA stabilized activities

The abolition

the

possibility study.

could It

their

could

not also

CaM-binding

denaturation

smaller

corresponded,

70-,

235-kDa

calspectin 24/22.5-kDa

Synapsin

a Ca2+-dependent

which

tentatively There

three

Neuromodulin

(GAP43,

inner

surface

was found

that

CaM in the seemed a higher (15).

the

support affinity

It

B50, 24-kDa

previous

for

CaM in

was assumed

neuronal

cell

membrane

response

to an increase

phosphorylation rich

C kinase

independent

that in

by protein substrates type,

were

protein

4.1

The 58-

and 49-kDa

heterogeneity

subunits.

Mr=24,000) (11).

kinase

presence

C

sequester

more

This

in

with

work

SM bound

neuromodulin

of Ca2+ than could

associated present

of Ca2+.

that

absence

is

In the

cerebellar

observation

finding exhibited

its

presence

CaM at the

Ca 2t conditions and release 2t intracellular Ca and/or to

it

kinase

alanine-

low

(MARCKS)", found

of erythroid

on phosphorylation protein

of the

in the

were

and neurogranin.

Fl;

neuromodulin

under

84/82-kDa

from

membrane

the

protein,

distinct

pp46,

of Ca2+ than

aggregated

which

component

the

the

(14)

brain-specific

~57,

or not

such as

membrane

II

I,

a slightly

including

I and a related

plasma

synapsin

whether

components

These

The high

component

MARCKS protein,

of the

absence

to

was uncertain

for

which

to calspectin,

as CaM kinase

neuromodulin,

components

CaM binding.

well-known

(3).

on phosphorylation,

an increased

identified

are

substrates: the

showed

by heat-

substrates

respectively.

it

synaptosomal

membrane components

present

obtained

kinases

and 49-kDa

of multiple

and enzymically

showed

caused

contrary,

protein

showed,

However,

components. of bovine

this

in the

endogenous

mass basis,

immunologically

were

58-,

band

or a mixture

protein,

identified

was made up of a single

component

still

CaMBPs lost

change

on the

and beta-subunits,

CaM binding.

235-kDa

CaMBPs,

by

activity.

235-,

alpha-

weight

activated

astructural

larger

on the molecular II

molecluar

through

the

while

However,

staining

Ca*+ /CaM-dependent

included

CaM kinase

activity.

that

the

and

mass region

by protein

CaMBPs of SMs were

CAMP-dependentsubstrates

molecular

components and their

be supported

CaM-binding

Among the

24/22.5-kDa

be hypothesized

and that

and 3. Ca2+

such as Ca 2+-

an aggregation

CaM-binding

activity

Ca*+ -independent

of the

higher

Cazt -independent

of CaMBPs,

degradation.

indicate

to the

RESEARCH COMMUNICATIONS

a mechanism(s)

CaM binding

seemed to migration

retaining

structure

or proteolytic

of the

heat-denaturation

the

of CaMBPs by

phosphorylation

subsequent

AND BIOPHYSICAL

C (16).

The

a class

to release 730

"myristoylated

of CaMBPs of Ca'+-

CaM when

they

were

in

it

Vol.

183,

No.

2, 1992

BIOCHEMICAL

phosphorylated

by protein

AND

kinase

C (lO).It

(~17;

Mr=15,000-19,000)

also

(17).

These

neuromodulin

classified based

two CaMBPs, into

the

on both

binds

"smaller

BIOPHYSICAL

is

known

to CaM in the

that

defined

mass and the

COMMUNICATIONS

neurogranin

absence

and neurogranin,

components"

the molecular

RESEARCH

of Ca2+

can be in the

present

Ca2+-independent

work

manner

of

CaM binding. Another

CaMBP of synaptosomal

was reported (18).

to bind

However,

support

the

to calspectin

in the

present

assumption

corresponded

the

to cytosynalin.

More

was phosphorylated

CaM kinase

II.

Very

candidates

for

smaller

Identification

there

34-kDa

was found

cytosynalin

and interact

work

that

ACAMP-81(Mr=81,000), protein

membrane,

in the

by both

with

evidence in the

a new,

bovine

brain

histone type

is

in

(19).

protein

itself

is

work

CaMBP,

membrane

CaMBPs of Ca 2t-independent

of CaMBPs of this

to

present

acidic

CAMP-dependent

interestingly,

cytoskeleton

was little component

recently,

(Mr=35,000),

This

kinase

one of

the

(12,

13).

type

and

progress.

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Cheung,W.Y.

(1982)

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COMMUNICATIONS

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Arch.

Calmodulin-binding proteins of calcium-independent type in rat brain synaptosomal membranes: their localization and properties.

Some properties of calmodulin(CaM)-binding proteins (CaMBPs) of the Ca(2+)-independent type were investigated in the synaptosomal membrane (SM) from r...
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