Calcineurin is a calmodulin-dependent protein phosphatase

vol. 106, No. 4, 1982 June 30, 1982

BIOCHEMICAL

CALCINEURIN

IS A CALMODULIN-DEPENDENT

Shiaw-Der

Yang,

RESEARCH COMMUNICATIONS Pages 1419-1425

AND BIOPHYSICAL

E. Ann Tallant

PROTEIN PHOSPHATASE

and Wai Yiu

Cheung

Departments of Biochemistry St. Jude Children's Research Hospital and The University of Tennessee Center for the Health Memphis, Tennessee 38101 Received

Sciences

May 19, 1982

SUMMARY: Calcineurin, one of the major calmodulin-binding proteins in the brain, dephosphorylates a phosphorylated protein termed inhibitor-l, a potent inhibitor of protein phosphatase 1. The phosphatase activity was Ca2+- and calmodulin-dependent and was reversed by ECTA or trifluoperazine, an antagonist of calmodulin. Using a radioimmunoassay of calcineurin and a phosphatase activity assay, we found that the two activities coincided in a sucrose density gradient and in a non-denaturing polyacrylamide gel. These results demonstrate that calcineurin is a calmodulin-dependent protein phosphatase. INTRODUCTION Calmodulin,

originally

of CaZ+-dependent dependent

of divergent

there

are numerous

ties

remain

regulated

a Mr of 80,000,

has been The large subunit ( lO-6 dent

(for

(for

inhibitor

four

protein

of this

apparently protein

60,000

is primarily

C&i-BPS0

calcineurin

sites

with

for

proteins,

(5)

in the

with

daltons, brain

calmodulin,

in

(5),

or calcineurin

--in vitro

adenylate

been

calmodulin-

dissociation

inhibits

by competition has not

with

activi-

and 16,500

found

a

In addition,

One of these

interacts

Ca 2+-binding

3).

whose biological

4).

(6),

Ca2+-

regulating

may be additional

of two subunits,

of phosphodiesterase, (4),

see ref.

see ref.

protein

as an activator

is a multifunctional

proteins

of calcineurin

Although

activities

review,

and they

review,

termed

2),

laboratory

in eukaryotes,

calmodulin-binding

This

M (7).

function

enzymes

ratios.

harbors

Ca2+-ATPase

ubiquitous

consists

subunit

(1,

protein

to be identified, enzymes

equimolar

in our

phosphodiesterase

regulatory

variety

discovered

and (7).

and the

small

constants calmodulin-depen-

cyclase calmodulin,

and erythrocyte the biological

clarified. 0006-291X/82/121419-07$01.00/0

1419

Cop.vrrghr 0 1982 .AN rights of reproductfon

by Acirdwnic Press, Inc. m an Y form reserved.

Vol. 106, No. 4, 1982 Recently, contains with

Stewart,

protein

--et al.

phosphatase

calcineurin.

calcineurin

BIOCHEMICAL

In this

is

indeed

(8)

AND BIOPHYSICAL

and suggested

communication,

a protein

that a calcineurin

reported

activity

RESEARCH COMMUNICATIONS

its

we present

phosphatase

whose

preparation

probable

evidence activity

identity to show that

is Ca 2+-

and

calmodulin-dependent. MATERIALS

AND METHODS

Chemicals and Reagents: lz51 (16-20 mCi/jlg) and [Y-~~P]ATP (2 mCi/mmol) were purchased from Amersham; [methyl-14C] methylated bovine serum albumin (0.016 mCi/mg) from New England Nuclear; Triton X-100, phenylmethylsulfonylfluoride, leupeptin, aprotinin, dithiothreitol (DTT), histone (Type II) and casein from Sigma; sodium dodecyl sulfate and Affi-gel blue from Bio-Rad; iodogen (1,3,4,6-tetrachloro-3a, 6a-diphenyl glycoluril) from Pierce Chemical Co. and Sephadex G-100 from Pharmacia. Goat anti-rabbit IgG serum was a gift from Dr. William Walker, St. Jude Children's Research Hospital. Bovine brain was generously supplied by Fineberg Packing Co., Memphis. Rabbit skeletal muscle troponin c was a gift from Dr. Thomas C. Vanaman, Duke University. Protein Preparations: Inhibitor-l was isolated from bovine brain according to a procedure developed for skeletal muscle (9). The catalytic subunit of CAMP-dependent protein kinase was isolated from bovine heart (10). Inhibitor-l, casein and histone were labeled with 32Pi by the cataCalmodulin was lytic subunit of CAMP-dependent protein kinase (11, 12). isolated from bovine brain (13). Calcineurin was also isolated from bovine brain (14), with minor modifications (15). Assay of Protein Phosphatase: Phosphatase activity was assayed by the release of 32Pi from [JLP]-labeled inhibitor-l. The assay mixture, in a final volume of 50 ~1 containing 20 mM Tris-HCl (pH 7.0), 0.5 mM DTT, 0.1 mM 50 ug bovine serum albumin and an appropriate CaC12, 2.5 x 10m7 M calmodulin, amount of calcineurin, was preincubated at 30°C for 5 min. The reaction was initiated with 10 ~1 of [32P]-labeled inhibitor-l (0.4 mg/ml). After 5 min, was determined (16). The amount the reaction was terminated; the 32Pi release of labeled phosphate released generally did not exceed 15% of the substrate originally added to the reaction mixture. Analytical Methods: Calcineurin was assayed by radioimmunoassay (5) Protein was either determined by the method with minor modifications (15). as a standard or estimated of Lowry, --et al. (17) with bovine serum albumin discontinuous polyacrylamide from the absorbance at 280 nm. Non-denaturing The gel was pre-run gel (6%) electrophoresis was performed as in (18). without sample to remove substances which might interfere with subsequent Sucrose density centrifugation determination of the phosphatase activity. using a linear gradient from 5% to 20% sucrose was run in a Beckman SW50.1 weight was determined rotor at 45,000 rpm for 16 h at 4°C; the molecular with [14C]-bovine serum albumin as an internal marker (19). Discontinuous polyacrylamide gel electrophoresis in the presence of SDS and 1 mM EGTA was performed at 22°C in a slab gel containing a linear gradient of 7.5-15% acrylamide (20). RESULTS AND DISCUSSION Calcineurin [3*P]-labeled (2.5

x

10T7

exhibited inhibitor-l

M),

the

some basal as substrate

activity

phosphatase (Table

was increased 1420

1).

activity

when assayed

In the presence

several-fold.

When Ca2'

with

of calmodulin was

BIOCHEMICAL

Vol. 106, No. 4, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 Effect

of Ca2+ and Calmodulin

on Protein

Phosphatase

Additions

Activity

' Phosphatase

of

cpm released/5 ?Jone

Calcineurin

Activity min

1223

EGTA

0

Calmodulin

(minus

Ca2+)

1081

Calmodulin

6400

Calmodulin

+ EGTA

43

Trifluoperazine

1152

Calmodulin Troponin

+ Trifluoperazine

1863

c

1208

Each reaction mixture in a final volume of 50 1~1 contained 4 ug inhibitor-l (25,000 cpm), 20 mM Tris-HCl (pH 7.0), 0.5 mM DTT, 50 ug bovine serum albumin, 1.3 x 10-7 11 calcineurin, 0.1 mI1 Ca2+ (unless indicated otherwise), and where present, 2.5 x 10-7 M calmodulin, 1 mF1EGTA, 2.5 x 10-7 M troponin c or 50 IIM trifluoperazine. The phosphatase was assayed as summarized under "Materials and Methods", and is expressed as cpm released per 5 min, the background having been subtracted.

omitted

from

the reaction

the phosphatase

activity

mixture, was slightly

when 1 mM EGTA was included abolished,

whether

troponin

c, a close

activity

at a concentration

troponin

c to 2.5 x 10-6

activity

slightly

that tissue

content

troponin

c free observed

a trace that

troponin

did

c does agent

not

M, lo-fold than

affect

higher

the basal

than level,

(data

not

shown).

substitute to block

the basal

activity

for

is

it

is

remains

in line

calmodulin

the biological but 1421

in an

lower

difficult that

than

of the high

in the physical

of troponin

This

of

resulted

Because

similarity

The possibility concentration

the concentration

calmodulin,

c,

muscle

phosphatase

but dramatically

and troponin

of calmodulin.

known

M. Raising

was

skeletal

stimulate

and

Moreover,

activity

Rabbit not

stimulatory

level.

phosphatase

did

and the close

a high

was not

the basal

was present.

of 2.5 x lo-.'

of calmodulin

not

all

of calmodulin,

of calmodulin. with

itself than

the assay,

of calmodulin

contaminant

anti-psychotic (21),

analogue

of calmodulin

properties

less

calmodulin

greater

chemical

lation

in

or not

in the presence

calmodulin

and

to prepare

the slight

stimu-

c may be contributed with

the general

(3).

Trifluoperazine,

activity

suppressed

the

by

finding

of calmodulin calmodulin-stimu-

an

Vol. 106, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

.c Z 6000 t 4 : 4000 ,o ,” 2Lf? 2000 a \ I

20% Bottom

1

IO

1

Fraction

aa!

1

I

I

20

‘0

1

5% TOP

Number

of calcineurin. A linear Figure 1. Sucrose density gradient centrifugation gradient of 5-20% sucrose in 20 mM Tris-HCl (pH 7.0), 1.0 ti DTT, loaded with 40 ug calcineurin was centrifuged at 45,000 rpm in the Beckman rotor SW50.1 for 16 h at 4oC. Fractions (about 200 ~1) were collected by puncturing the bottom of the tubes; phosphatase and radioimmunological activities were performed as outlined under "Materials and Methods".

lated

activity

to near

the phosphatase

activity

To demonstrate analytical native

its

the

assay

gel

symmetrical, cineurin tube

with also

10, although

calcineurin.

the peak

is not

as an internal

78,000,

a value

by gel

strikingly

filtration

demonstrate

is

of phosphatase corresponded

the

by polyacrylamide

conditions.

Two gels

phatase

that

to the

1 shows

and by phosphatase activity

peak

1251+alcineurin

This

is

using

for

at

probably

recognized

due to

by the

[14C]-bovine

antiserum

weight

calcineurin

is

of cal-

with

to a molecular 80,000

Figure

Radioimmunoassay

symmetrical.

close

was examined

other

10.

and

of obtained

(14).

To further

the

that

phosphatase,

performed.

competition

of calcineurin

marker

the notion

centrifugation

The phosphatase at tube

greatest

albumin

gradient were

gradient.

The peak activity

the

by radioimmunoassay

the

product

support

with

density

maximum activity

exhibits

some degradation

sucrose electrophoresis

density

results

of calcineurin

of calcineurin

in a sucrose

These

and calmodulin-dependent.

identity with

polyacrylamide profile

level.

is Ca2+-

experiments

the activity

basal

was sliced activity.

identity gel

of these

electrophoresis

used.

one was stained

to determine

radioimmunological

were

Again

the peak of phosphatase 1422

two proteins, under for

non-denaturing

protein activity

activity

calcineurin

pattern; and phos-

was symmetrical,

BIOCHEMICAL

Vol. 106, No. 4, 1982

AND BIOPHYSICAL

Sltce

Figure 2. calcineurin. 2 h. The

Non-denaturing The gel was reservoir buffer

RESEARCH COMMUNICATIONS

Number

discontinuous 6% polyacrylamide was replaced

polyacrylamide gel electrophoresis and pre-run without sample by a fresh buffer and 30 ~g of

cineurin was applied to the gel. At the end of electrophoresis, stained for protein with Coomassie brilliant blue and the other 2-mm slices. The protein, extracted from each slice by shaking 4oC

at

in

200

~1 of

20 ti

Tris-HCl

(pH

7.0),

1.0

phosphatase and for calcineurin as outlined anode is at the bottom of the gel.

although

that

seen in peaks

of the

the sucrose

were

mirror

density images

migrated

as a diffused

diffused

region

tase

activity;

presumably

radioimmunoassay

the main

however,

it

sodium

dodecyl

protein

bands

(Lane

of the A).

activity taminating

usually

sulfate

calcineurin

the

may be associated

with

(4-7). before

these calcineurin

preparation. with

Calcineurin

not

results

and not with

of protein 1423

electrophoresis. gradient

was examined 3 (Lane

pattern

that

the

some minor

in a

and 16,500, identical

sucrose

gradient

phosphatase proteins

phosphatase

co-migrates

in

B) shows two

appears

in the

that that

activity,

of 60,000

indicate

gel.

show any phospha-

density

centrifugation

The possibility a trace

2.

Figure

This

pattern

in Figure

during

weights

The

two activity

pattern

gel.

to molecular

of calcineurin

Collectively, resides

and the protein

corresponding

of the

polyacrylamide

sucrose

for

and Methods".

some immunological

from the

polyacrylamide

assayed

was

the

band did

showed

one gel cut into overnight

Further,

of calcineurin

fraction

1 was concentrated,

two subunits

1.

as shown

protein

due to some denaturation

Figure

to that

of Figure

of one another,

trailing

was

reminiscent

band in the non-denaturing

The peak activity

the

was not,

gradient

mM DTT,

in "Materials

of for cal-

con-

activity with

cal-

The

Vol. 106, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

A

B

RESEARCH COMMUNICATIONS

C

--

Electrophoresis of calcineurin on SDS-polyacrylamide gel. ElecFigure 3. trophoresis was performed on a 7.515% gradient polyacrylamide slab gel, in the presence of 1 mEI EGTA, as described in "Materials and Methods". Lane A, 10 ug of calcineurin before sucrose gradient centrifugation; Lane B, activity weight markers: peak from sucrose gradient of Figure 1; Lane C, molecular phosphorylase b (Mr = 94,000), bovine serum albumin (Mr = 67,000), ovalbumin (Mr = 43,000), carbonic anhydrase (Mr = 30,000), trypsin inhibitor (Mr = 20,100) and a-lactalbumin (Mr = 14,400). The anode is at the bottom of the gel.

cineurin but

under

it

our

appears

it

the

interest.

inhibits

(11).

In addition

to

Calcineurin

that

of inhibitor-l

has a rather

be totally

excluded,

broad

for

phosphatase

calcineurin

in this

by a CAMP-dependent 1 (22,

23),

which

has

study,

broad

that

of

many

other

inhibitor-l,

we have

catalyzed (data substrate

their not

used

casein

is not

specificity. 1424

thus

active.

regulatory

pro-

mechanism. and histone

dephosphorylation

shown),

kinase,

substrate

form of inhibitor-l like

is of

protein

by a phosphorylation-dephosphorylation

strates. to

used

of inhibitor-l,

is controlled

teins,

cannot

The dephosphorylated

the activity

Thus,

substrate

When phosphorylated

a protein

specificity

conditions

unlikely.

Inhibitor-l, special

experimental

suggesting

as sub-

at a rate that

comparable

the enzyme

BIOCHEMICAL

Vol. 106, No. 4, 1982 We have the that

caudate

shown that nucleus

calcineurin

and dendritic

is primarily microtubules

shown that

that

calcineurin

is

activities

are

whose with

a specific

nervous

calcineurin

and putamen

have

is (5).

rich

associated

increases

a protein regulated activity

in the brain,

with

with

phosphatase by calmodulin. will

RESEARCH COMMUNICATIONS

Immunohistochemical

in mouse basal

calcineurin

enzymic

AND BIOPHYSICAL

help

particularly studies

the postsynaptic

ganglia

(24).

synaptogenesis

More

More to define

densities recently,

we

The finding list

importantly, its

revealed

(25).

adds to the growing

in

function

of enzymes its

identity in the

system.

Acknowledgements: This work was supported by grants CA 21765, NS 08059 and GM 28178 from the National Institutes of Health and by ALSAC. We are grateful to Shawn Hawkins for excellent technical assistance and to Pat Nicholas for typing the manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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