Peptides from myelin basic protein as substrates for adenosine 3′, 5′-cyclic monophosphate-dependent protein kinases

Vol. 61, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATlOh,~

PEPTIDES FROM MYELIN BASIC PROTEIN AS SUBSTRATESFOR ADENOSINE 3’,

5 '-CYCLIC

MONOPHOSPHATE-DEPENDENTPROTEIN KINASES

Peter Russell

Received

Daile and Patrick R. Carnegie Grimwade School of Biochemistry University of Melbourne Parkville, 3052, Australia

October

15,1974

Summary - A simple electrophoretic assay demonstrated that peptides from enzymic digests of the basic protein of human myelin were effective substrates for adenosine 3', 5'-cyclic monophosphate-dependent protein kinases from bovine cardiac muscle and brain. From a peptic digest a peptide of 17 amino acid residues was isolated and when used as a substrate a Km of 1.9 x 10m4M was found for the cardiac kinase. 4 major dependent

problem

protein

or threonine suggested whereas

tion

to

(2J)

of the basic

system.

This

protein

kinases

established

within

pro+

protein

that

could

(1)

be of importance

the enzyme may recogrise of the substrate.


for

ami.,o acid

a number c.f species

serine

Langan

In

the phosphcryla-

from the central

and its

of CAMP-

specific

we are studying

.:. of myelin

role

proteins.

structure

problem

(4,5,6,',

for

substrate

considered

this

recognise

structure

of the primary resolve

the biological

is how they

the tertiary

others

some feature

understanding

kinases

residues that

attempts

in

nervous

CAMP-deponder: seyuence

is weli

(*,9,10).

MATERIALS Human myelin digested protein purified

basic

as described kinase

on DEAE-cellulose

against

was prepared

previously

was prepared

160 and 180mM potass,um dialysis

protein

(8,9,10).

from bovine (11).

Cyclic cardiac

The fraction

phosphate

5mM potassium

and enzymically

buffer,

phosphate

muscle

eluting

pH 7.0, buffer,

AMP-deperden' and between

was used afte2mM Na2EDTA,

Vol. 61,No.

3, 1974

pH 7.0.

It

BIOCHEMICAL

contained

(Sigma Type IIA) ferred lY,

0.33

or myelin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mg protein/ml

and with

basic

as substrates

protein

4.2 and 3.4 nmoles of Pi/mg

when incubated

specified Glynn

in Table

and Chappell

mmole.

It

substrate

for

5 min at 30°C with

1.

[v-~~P]ATP

(12)

was diluted

it

protein

trans-

respective-

the solutions

was prepared

and had a specific with

histone

by the method of

activity

ATP as described

of

in the

2-2.5

Ci/

text.

METHODS Electrophoretic ~1) of

assay for

the incubation

mixture

1 cm bands on a sheet line

with

blotting

of Whatman

out

a liquid (Du Pont,

plate

petroleum

diffusion

of the

All

film.

in a Packard

for

Results

cocktail. of

total

100:3:

for

radioactivity

a Triton

there

TF

a heavy cooled was more

(25OC).

The

out

and counted

X-lOO-toluene

each spot were expressed

recovered

cases as pmoles Pi incorporated

"Freon"

Kodak RP/S X-Omat

were cut

with

was

converted

in a tank

temperature

spots

counter

of

but

18-48 hr with

and in selected

per mole of peptide.

RESULTS Electrophoretic

assay for

at pH 6.5 provided

a simple

protein

kinase.

method for

853

Paper electrophoresis

following

a

removed with

and applying

effective

radioactive

scintillation

scintillation as percent

the

along

was carefully

(Frankfurt)

plate

at the higher

paper was autoradiographed

throughly

25

onto

acid:water,

Electrophoresis

was equally

spots

(57 x 34 cm)

a 1 cm depth

to the cooling

spirit

dried

The paper

apparatus

the paper.

(e.g.

(18OOV, 60-9OmA at l°C)

system by adding

onto

1).

moisture

in a "Pherograph"

Delaware)

1) was rapidly

3MM paper

Electrophoresis

cooled

A sample

(pyridine:acetic

and the excess

paper.

carried

X-ray

(Table

pH 6.5 buffer

897, by vol.),

with

substrates.

37 cm from the anode end (Fig.

saturated

glass

peptide

the phosphory-

to

Vol. 61, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1234K567

Figure

Electrophoretic assay 1. Autoradiography of electrophoretogram of phosphorylation of increasing amounts of peptic peptide Pl from myelin basic protein as described in Fig. 2B. 1, 0.5 nmoles Pl; 2, 1.0 nmoles; 3, 1.5 nmoles; 4, 2.5 nmoles; 5, 6.25 nmoles; 6, 12.5 nmoles; 7, 25 nmoles; and K, no peptide present. Spots in ascending order represent Pi, ATP, protein kinase or a protein in the kinase preparation and peptide Q, the phosphorylated form of peptide Pl.

lation

of protein

gave similar protein

and peptide

results

substrates

to the usual

could

detected.

enzymic

digests

reproducible

1, Table

precipitation

be monitored

(Fig.

ATPase or phosphoprotein

readily

(Fig.

assays

and had the added advantage

ATP concentration contaminating

substrates

With

a series

of the myelin

to within

Peptides as substrates and peptic

digests

substrates

for

incubation

conditions

any

activity samples of

results

were

f 2.3%. for

protein

of myelin

protein

protein,

for

2A) and thus

duplicate

kinase for

basic

kinase. protein

(Table

1).

phosphorylation

a54

It

changes in

phosphatase

of six

basic

that

1).

Tryptic, functioned

thermolytic as

Under identical the peptic

and

Vol. 61, No. 3, 1974

BIOCHEMICAL

AND BiOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 Phosphorylation bY

of myelin

CAMP-dependent

basic

protein

protein

kinase

and enzymic

from cardiac

digests

muscle.

Incubation

Substrate

1

Endogenous substrate Precipitation assay Electrophoretic assay

7.0 5.4

109 84

Human myelin basic protein Precipitation assay Electrophoretic assay

82.5 82.4

1279 1277

0.1 0.1

Tryptic digest basic protein

40.3

625

0.05

2

Percent 32~ incorporated

pmoles Pi incorporated

moles Pi/mole

of

Thermolytic digest of basic protein

74.8

1159

0.09

Peptic digest basic protein

72.5

1124

0.09

33.1

513

0.04

of

Main 32P-peptide, in peptic digest

Q,

To incubations 2, 3, 4 and 5 were added 50~1 of basic protein of enzymic digests, each 5 mg/ml in 1OOmMsodium acetate 50~1 of buffer was added to incubation 1. buffer, pH 6.5; Incubations also contained 50~1 of each of solutions (b), (c) and (d) as specified in Fig. 2A and were incubated for 1 hr at assay 25~1 of the incubation 3oOc. For the precipitation For mixture was treated as described by Li and Felmly (13). the electrophoretic assay 40~1 of incubation mixture was applied to the electrophoresis paper.

thermolytic protein. paper

digests

were almost

The phosphorylated electrophoresis

pyridine:acetic distribution

as good substrates

peptic

digest

was examined

at pH 6.5 and chromatography

acid:water (30:20:6:24, by vol.). of 32 P in the peptides was determined.

855

as the intact by

in butan-l-01: From this

the

One peptide,

Vol. 61, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 0

20

40

60

TIME

I20

240

0

5

(min)

IO PEPTIOE

15

20

25

(n moles)

Fi .gure 2. Phosphorylation of peptide Pl A) Time: Separate tubes wereincubated for each time interval. The incubation mixture contained in each tube 10~1 of each of the following solutions: (a) peptide Pl in H20 (25 nmoles/lOOul); (b) 14mM magnesium acetate, 1.2mM EGTA (ethylene glycol bis (f3-aminoethyl ether)-N,N' tetraacetic acid) and 40)~M CAMP; (c) 31).1M [y-32PlATP (1.5 x lo5 cpm per 10~1) in 1OOmM sodium acetate buffer, pH 6.5; (d) cardiac protein kinase (0.33 mg/ml). At appropriate times tubes were removed from the constant temperature bath (30°C) and 25~1 applied to the electrophoresis paper. B) Concentration: The incubation mixture in each con=ined varying amounts of peptide Pl (in 10~1) [Y-~~PIATP solution added at 111uM (1.4 x 105 cpm Incubation time was 5 min. Other conditions were 2A.

Q, contained the

33% of the

total

radioactivity

tube and the per 10~1). as for

incorporated

into

digest. Peptic

peptides

protein

(8,lO)

kinase.

Five

were isolated

and tested basic

for

substrate

peptides

phosphorylated

tion,

Q.

chromatographic

This

properties,

corresponded,

peptide

with

to those

of peptide

protein

The amino acid

the protein

activity

after

and

composition

Pl from myelin

basic

sequence of peptide

Pl is

856

and

phosphoryla-

had electrophoretic

and amino acid

were identical (10).

activity

showed some substrate

the most readily to peptide

from the unphosphorylated

which

Acetyl-

Vol. 61, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Ala-Ser-Gln-Lys-Arg-Pro-Ser-Gln-Arg-His-Gly-Ser-Lys-Tyr-~u-Ala1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Thr. 17 Phosphorylation detailed

of peptide

Pl.

Peptide

In Fig.

2A the

transfer

study.

demonstrated conditions

as a function used the

of

time.

concentration

Pl,

enzyme was shown to exhibit

a Km of

1.9 x 10-4 M for

2 x 10-5 M for

Km of phoretic using

assay,

and also

assay.

2B a maximum of 0.09

eluted

phosphate

buffer,

peptides,

including

by Miyamoto

peptide

a

by the electro-

and Kakiuchi

conditions

(6)

used in Fig.

per mole of for

1% hr with

14

kinase

between also

from bovine

which

180 and 205mM potassium

phosphorylated

several

in the presence

Pl,

brain,

peptic

of CAMP but

not

absence. Mild

acid

hydrolysis

in peptide

major

site

(4) :

whether

this

pH 7.0,

kinetics

mole Pi per mole was incorporated.

of protein

from DEAE cellulose

the

of

compared with

determined

Under the

under

became limiting.

This

mole Pi was incorporated

of 0.35

A preparation

serine

Pl.

Pl is

the phosphorylation

When 12.5 nmole Pl was incubated

nmole ATP a total

in its

peptide

reported

that

Michaelis-Menten

the whole protein

a precipitation

peptide.

is obvious

for

for

P to peptide

of ATP rapidly

2B where more ATP was available

with

32

of

It

In Fig. the

Pl was selected

site

Q.

With

of phosphorylation the cardiac

remains

(4)

showed the presence

the intact

protein

by rabbit and brain

of phospho-

serine-12

was a

muscle protein

kinases

also

kinase

phosphorylated

to be determined. DISCUSSION'

The use of the electrophoretic clearly

demonstrated

phosphorylate

quite

that small

assay

CAMP-dependent peptides.

857

It

for protein

is

protein

kinase

kinases

noteworthy

that

could no

Vol. 61, No. 3, 1974

isolated

acidic

although

they

The ability

BIOCHEMICAL

or neutral

several

of peptides

to act

recognises

proteins

around

specific

conformation

phosphorylation

peptides

contained

kinase

some feature the serine of

may also

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were phospnorylated

serine

and threonine

as substrates

indicated

of the primary

structure

to be phosphorylated. the peptide

chain

residues.

close

that of

However, to the

the

site

some of

be necessary.

We thank Mr. G. Luxford for myelin basic Acknowledgements: protein, Ms. S. Murray for amino acid analysis and Dr. P. R. The work was supported by the National Dunkley for comments. Health and Medical Research Council of Australia and in part by Multiple Sclerosis Society. Grant No. 835-A-6 from the National REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10

11. 12. 13.

Advances in Cyclic Nucleotide Lanqan, T. A. (1973) Research 3, 99-153. Huanq, T. S., Bylund, D. B., Stull, J. T. and Krebs, E. G. FEBS Letters 42, 249-252. (1974) Advances in Protein Chemistry 28, Taborsky, G. (1974) l-210. Carnegie, P. R., Kemp, B. E., Dunkley, P. R. and Murray, A. W. (1973) Biochem. J. 135, 569-572. Carnegie, P. R., Dunkley, P. R., Kemp, B. E. and Murray, A. W. (1974) Nature 249, 147-150. J. Biol. Chem. 249, Miyamoto, E. and Kakiuchi, S. (1974) 2760-2777. Steck, A. J. and Appel, S. H. (1974) J. Biol. Chem., in press. Carnegie, P. R. (1971) Biochem. J. 123, 57-67. Dunklev, P. R. and Carneqie, P. R. (1973) Research Methods in Neurochemistry 2, 219-245. Dunkley, P. R. and Carnegie, P. R. (1974) Biochem. J. 141, 243-255. KUO, J. F. (1972) Methods in Cyclic Nucleotide Research 3, 199-220. Glynn, I. M. and Chappell, J. B. (1964) Biochem. J. 90, 147-149. D. A. (1973) Anal. Biochem. 52, Li, H. C. and Felmly, 300-304.