Phosphorylation of microtubule-associated protein 2 by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Vol. 109, No. 3, 1982 December 15, 1982

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS Pages

975-981

PHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN 2 BY CALMODULIN-DEPENDENT PROTEIN KINASE (KINASE

II)

WHICH OCCURS ONLY IN THE BRAIN TISSUES Takashi Department

Received

November

Yamauchi

and Hitoshi

Fujisawa

of Biochemistry, Asahikawa Medical Asahikawa 078-11, Japan

College,

3, 1982

Microtubule-associated protein 2 (MAP 2) from the rat brain was phosphorylated by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues. The apparent Km for MAP 2 of Kinase II was 0.2 uM. The maximum incorporation of phosphate into MAP 2 by the action of Kinase II was about 5 mol of phosphate per mol of MAP 2, while that by the action of CAMPdependent protein kinase was about 3 mol of phosphate per mol of MAP 2. When microtubule-associated proteins were incubated with both Kinase II and CAMP-dependent protein kinase together, about 7 mol of phosphate were incorporated into 1 mol of MAP 2.

Calcium

ions

are known to play

the physiological little

is

Ca2+.

known about

Recent

level the

functions

dependent

studies

that

three

different

substrate

cytosol

(6).

in the

brain

through (3-5).

that

the

specificities

(7)

protein

Kinase

II,

and was found

of the

proteins

in

of calmodulindemonstrated kinases

in rat

was found

in

but

2+ Ca regulates

activation

are present

roles

the action

We have recently

calmodulin-dependent

One of them, tissues

(1,2),

of a number of endogenous

kinases

distinct

system

mechanisms of

have demonstrated

system especially protein

nervous

the molecular

of phosphorylation nervous

of the

a number of important

brain

to occurs

to be involved

with

only

in the

Abbreviations: MAP 2, microtubule-associated protein 2; Hepes, (2-hydroxyethyl)-l-piperazineethane sulfonic acid; Mes, 2-(N-morphorino)ethanesulfonic acid; EGTA, ethylene glycol bis(B-aminoethyl ether)-N,N,N', N'-tetraacetic acid; SDS, sodium dodecyl sulfate.

4-

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Vol. 109, No. 3, 1982

activation

of tryptophan

monooxygenase Kinase

BIOCHEMICAL

II

(8),

cerebellum

may play sis

but

in the

also

found

as well

in the a role

nervous

not only

other

To understand

the it

nervous

system,

protein

substrates. was identified

system.

physiological

function

Kinase cortex

to endogenous This

suggests

regulation

and

in the

analyze

communication,

as an endogenous

broad

protein that

of Kinase

to

II

Kinase

II

of monoamine biosynthe-

functions function

of

of catechol-

system.

cerebral

3-

and to have relatively

be important In this

and tyrosine

biosynthesis

in the

respect

in the

(6,8)

nervous

brainstem

physiological

will

of the

central

with

RESEARCH COMMUNICATIONS

one physiological

to occur

as the

specificities

also

brain

that

may be the regulation

was, however,

substrates

5-monooxygenase

indicating

amines and serotonin

substrate

AND BIOPHYSICAL

protein

its

nervous II

system.

in the endogenous

MAP 2 from the substrate

rat

of Kinase

EXPERIMENTAL PROCEDURES [v-~~PIATP (3000 Ci/mmol) was purchased from Radiochemical Centre, Amersham. Kinase II was purified about 720-fold with a 36% yield and to near homogeneity from the extract of rat cerebral cortex. The purification procedures will be published elsewhere. CAMP-dependent protein kinase from bovine heart was prepared by treatment of alumina Q-gel. the method of Rubin et al. (9) without Phosphorylase kinase from rabbit muscle was prepared by the Myosin light chain kinase from chicken method of Cohen (10). gizzard was prepared by the method of Hartshorne et al. (11). Rat brain calmodulin was prepared by the method of Wang and Desai Myosin from rabbit skeletal muscle was prepared according (121. to the procedure of Perry (13). Rat brain microtubule protein was purified by two cycles of assembly-disassembly according to a modification of the method of Shelanski et al. (14) as described by Karr et aZ. (15). Microtubule-associated proteins were purified by phosphocellulose column chromatography according to the procedure of Weingarten et aZ. (16). The content of MAP 2 of this preparation was determined to be about 60% of total protein by disc gel electrophoresis in the presence of 0.1% SDS. Phosphorylation of MAP 2 by Kinase II or CAMP-dependent The standard protein kinase was carried out at 30 OC for 3 min. incubation mixture for phosphorylation )ZJ&J Kinase II cogtained 50 mM Hepes buffer, pH 7.0, 0.05 mM [yP]ATP (1 X 10 cpm/nmol), 5.@ W(CH8COO18, 0.12 mg Ca"la,c,":; ;i Ey;ipOi; W,c;;;;~~l;;, mlcrotubul -ass ciated protein The phosphorylation of MAP 2 by in a total volume of 0.07 ml. CAMP-dependent protein kis$se was carried out under the same and Kinase II were replaced conditions except that Ca ,calmodulin 976

II

Vol. 109, No. 3, 1982

6IOCHEMICAl

AND BIOPHYSICAL

RESEARCH COMMUNKATIONS

The incorpoby 0.01 rn! @MP and CAMP-dependent protein kinase. ration of [ PIphosphate into MAP 2 was analyzed by SDS-polyacrylAfter the reaction, 0.03 ml of amide disc gel electrophoresis. an SDS-stop solution containing 0.1 M boric acid-sodium acetate buffer, pH 8.5, 25% glycerol, 5% SDS-and 5% mercaptoethanol was added to the reaction mixture. The mixture was subjected to SDSpolyacrylamide disc gel electrophoresis after heating at 100 “C for 3 min. A$$er electrophoresis, gels were sliced into 2-mm sections and [ Plradioactivity of the section which corresponded in mobility to MAP 2 was measured in 10 ml of water by Cerenkov counting. When the phosphorylated proteins were analyzed by SDS-polyacrylamide slab gel electrophoresis, the phosphorylation reaction was terminated by the addition of 0.01 ml of an SDS-stop solution containing 8% SDS, 10% mercaptoethanol and 30% glycerol in 0.18 M Tris-HCl buffer, pH 6.7. The mixture was then boiled for 3 min, and an aliquot was subjected to SDS-polyacrylamide slab gel electrophoresis. autoradiography was carried After electrophoresis, out as described previously (5) SDS-polyacrylamide disc gel electrophoresis was carried out with 8% polyacrylamide gels in the presence of 0.1% SDS by the method of Davies and Stark (17). SDS-polyacrylamide slab gel electrophoresis was carried out with 7.5% polyacrylamide gels in the presence of 0.1% SDS according to the procedure of Maize1 (18). Protein was determined by the method of Lowry et aZ. (19) with bovine serum albumin as the standard.

RESULTS AND DISCUSSION Microtubule-associated in the

presence

phorylated

electrophoresis

only

followed

of

radioactivity

as described radioactivity

[

32

both

followed under

protein

was found

protein

kinase

to that

into

of MAP 2 by Kinase protein

with

kinase.

gel 1,

When the

MAP 2.

the

appeared

extent

of each gel about

gel

section

80% of the

CAMP-dependent l),

in accord

with

of the phospho-

to be higher

Phosphorylase 977

disc

microtubule-associated

MAP 2 (Fig.

However, II

of I!AP 2.

counting

total

phosphorylated (20).

II

As shown in Fig.

Procedures,"

to co-migrate

observations

slab

by SDS-polyacrylamide

by Cerenkov

"Experimental

also

Kinase

into protein was observed Plphosphate Ca2+ and calmodulin and the position

corresponded

incorporated

CAMP-dependent

by SDS-polyacrylamide

sample was analyzed

electrophoresis

rylation

of

with

or Ca2+ , and then phos-

by autoradiography.

incorporation

phosphorylated

ealier

were analyzed

in the presence

of the

were incubated

and absence of calmodulin

proteins

a marked

proteins

kinase

than

that

by

from rabbit

Vol. 109, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A, Autoradiosraph Kinase

II

B, Protein A-Kinase

12345678

Staining 9

I

-Mt-,

K

-220

-94 -60

-40

MAP

-

+

+

+

+

+

+

-

-

Kinase Ca2+ CaM CAMP

+ t t -

t t t -

t t -

t t -

t t -

t t

t

t -

t t

Autoradiograph showing the phosphorylation of MAP 2 .-Fig. 1. Microtubuleby Kinase II or CAMP-dependent protein kinase. associated proteins (4.1 ug of MAP 2) were incubated with 0.35 ng of Kinase II or 2 ug of CAMP-dependent protein kinase as described under2$he "Experimental Procedures" in the presence and absence After the incubation of Ca calmodulin or CAMP as indicated. at 30 "6 for 3 min, SDS-polyacrylamide slab gel electrophoresis and autoradiography were carried out as described under the A, autoradiograph; B, protein staining "Experimental Procedures." of line 2 with Coomassie blue. The standard proteins and their assumed molecular weights were: myosin heavy chain, 220 K; phosThe phorylase b, 94 K; catalase, 60 K and aldolase! 40 K. abbreviations used are: MAP, microtubule-associated proteins; A-Kinase, CAMP-dependent protein kinase; CaM, calmodulin.

muscle

and

significantly Table

light

myosin

phosphorylate I summarizes

of MAP 2 by Kinase which

tion

II.

was one order

dependent

protein

kinase

from chicken

the

catalytic

The Km for

of magnitude

properties

did

not

MAP 2 were

25

lower

nM and

978

of phosphorylation

MAP 2 of Kinase than

reported previously 2+ producing a half and Ca

of

gizzard

MAP 2.

kinase

of calmodulin

phosphorylation

chain

0.8

that

II

was 0.2 ug,

of the

(21).

CAMP-

The concentra-

maximum activation r&l,

respectively.

of

Vol. 109, No. 3. 1982

BIOCHEMICAL

Table I.

Properties

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of MAP 2 phosphorylation

Parameter

by Kinase II

Value

Vmax

nmol of phosphate/min/ mg of Kinase II

194

Km, MAP 2

0.2 1.IM

Ka, Calmodulin

25

Ka, Ca2+

nM -

0.8 I.IM -

The incorporation of [ 32PIphosphate into MAP 2 was analyzed by SDS-polyacrylamide disc gel electrophoresis as described under the "Experimental Procedures.*' The Vmax and Km values were obtained from double reciprocal plots of data in the presence of 0.09 ug of Kinase II. The concentration of MAP 2 was determined as 60% of microtubule-associated proteins. The value of molecular weight for MAP 2 was 270,000 (22). The Ka for calmodulin was desgrmined in the presence of 1.3 pg of Kinase II/ml. The Ka for Ca was determined in the presence of 1.3 pg of Kinase II/ml, 1 mM - EGTA 200 nM calmodulin.

Fig.

2 shows the

relationship

of MAP 2 by Kinase

II

The maximum extent

of phosphorylation

approximately to about weight

and that

between

of MAP 2 to be 270,000

maximum extent

of phosphorylation

protein

was about

kinase

was somewhat higher was incubated kinase

by CAMP-dependent

with

together,

than both

about

protein

kinase.

of MAP 2 by Kinase

18 nmol of phosphate/mg

5 mol of phosphate/m01

the phosphorylation

of MAP 2, which

of MAP 2, taking (22).

II

was

corresponded

the molecular

On the other

hand,

the

of MAP 2 by CAMP-dependent

3 mol of phosphatefmol

of MAP 2, which

that

(20).

of earlier

Kinase

II

report

and CAMP-dependent

7 mol of phosphate

When MAP 2 protein

were incorporated

into

1 mol of MAP 2.

(23)

Calcium

ions

and the

Ca2+ effects

demonstrated hand, kinase

were reported

on microtubule

to be mediated

MAP 2 was found (20,21,27,28)

MAP 2 was demonstrated

to inhibit

microtubule

assembly-disassembly

by calmodulin

to be a substrate

(24-26). for

and the CAMP-stimulated to be inhibit 979

assembly

the rate

were

On the other

CAMP-dependent

protein

phosphorylation

of

and extent

of

Vol. 109, No. 3. 1982

BIOCHEMICAL

AND EIOPHYSICAL

RESEARCH COMMUNICATIONS

J Time (min)

Fig. 2. Time course of phosphorylation of MAP 2 by Kinase II and CAMP-dependent protein kinase. Microtubule-associated proteins (4.1 pg of MAP 2) were incubated with 0.35 pg of Kinase II (x-x), 2 pg of CAMP-dependent protein kinase (04). or 0.35 pg of Kinase II and 2 pg of CAMP-dependent protein kinase 2+ (w), or without protein kinase (o--- a) in the presence of Ca calmodulin and CAMP. After the incubation at 30 'C for the times' indicated, 0.03 ml of SDS-stop solution was added to the reaction mixture. The solution was subjected to SDS-polyacrylamide disc gel electrophoresi32after heating at 100 OC for 3 min. Incorporation of [ Plphosphate into MAP 2 was determined as described under the "Experimental Procedures."

microtubule-associated (29,30),

indicating

in the

regulation

together

with

the

protein-stimulated the

of microtubule

the earlier

phosphorylation

role

in the

central

assembly of MAP 2

of phosphorylation function.

reports

described

of MAP 2 by Kinase

regulation

nervous

importance

microtubule

II

of microtubule-mediated

The present above, may play

studies,

suggest

that

an important

functions

in the

system.

ACKNOWLEDGEMENTS The authors would like to thank Mr. Andrew Grenville who kindly read the first draft and offered suggestions for correcting the English phrasing. This work has been supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

REFERENCES 1. 2.

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Rev.

57,

Vol. 109, No. 3. 1982

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BIOCHEMICAL

AND BIOPHYSICAL

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