Phosphorylation by protein kinase C of a synthetic heptapeptide bearing a lysine residue on the C terminal side of serine

Vol. 142, No. 1, 1987 January 15, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 155-161

PHOSPHORYLATION BY PROTEIN KINASE C OF A SYNTHETIC HEPTAPEPTIDE BEARING A LYSINE RESIDUE ON THE C TERMINAL SIDE OF SERINE Hiroki Kondo I, Y shimi Babal, Katsunori Ta ski', Kiyoshi Kondo s , and Hiroyuki Kagamiyama 3 1

Laboratory of Bioorganic Industrial Chemistry, Nagasaki 2Department of Medical College, Takatsuki,

Chemistry, Department of Nagasaki University, 852, Japan Chemistry, Osaka Medical Osaka 569, Japan

Received November 4, 1986 A peptide, Ala-Ser-Gly-Ser-Phe-Lys-Leu, which corresponds to Ala103 'L Leul09 of Hl histone, was synthesized and tested as substrate for protein kinase C. The serine residue at position 4 was phosphorylated specifically. Another peptide lacking the lysine at position 6 was not phosphorylated by the same enzyme, indicating the importance of that basic residue as the recognition site for protein kinase C. 0 1987 Academic Press, Inc.

It

has become increasingly

lipid-dependent

protein

role

in the

4).

Many proteins

enzyme, basic

protein

the

at

present

vicinity

residue. terminal

However,

least

it

determinant

Abbreviations: phatidylserine; thiohydantoin;

of

one basic

remains

substrate

kinase

factor

amino acid

receptor

and myelin

residue This

is

sites

derived

therefrom

residue

is always

serine

or threonine

whether the

(I-

by this

whose phosphorylation

to be clarified

activity.

a vital

and stimuli

be phosphorylated

phosphorylated

basic

C) plays

signals

The information

of the

or the C-terminal

to

growth

examples

(5-7).

has shown that

Ca2+- and phospho-

extracellular

been found

sole

that

(protein

the epidermal

are

the

of

have

have been determined

in

kinase

transduction

of which

clear

the

N-

primary

question

may be best

kemptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly; PS, phosAEAP, 2-aminoethyl-3-aminopropyl; PTH, phenylHPLC, high-performance liquid chromatography.

155

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Copyrkht 0 I987 rights of reproduction

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Vol. 142, No. 1, 1987

BIOCHEMICAL

explored

using

synthetic

indicated

that

peptides

acids

on the

substrates that

the

protein

enzyme

the

C-terminal

the

substrate

peptides.

of

C (11-13).

protein

kinase

C as well

multiple

but

calf

of

insight

the

molecule

into

the

factors

and proteins

residues

103-109

Phe-Lys-Leu

in

residue

the

Phe-Leu

(peptide-2).

rylates

peptide-l

Since

the

it

the

prepared

not

for

was found

for by

kinase been

Cis

substrate

histone: that

another

comparison: protein

suggested

from

in

Dr. Y.

based

and further

at

located

(numbering

was felt

that

debatable.

protein

kinase

on

that

phosphorylated

a heptapeptide of

claimed

as a substrate

has also

Ser-106

in peptide-1,

It but

It

this

sequence

be important was also

still

is

as

residues

C is

communication

dictating

(peptide-1).

might

basic

kinase

by protein

we prepared

have

evident

protein

To confirm

can serve

thus,

employed

is

amino

is,

sites.

site

histone).

having

(personal

basic

others

as by CAMP-dependent

modified

have

residue

while

It

often

distinct

sites

peptides

this

protein

One possible

thymus

lysine

of

This

mutually

one of the

Nishizuka).

(10).

reports

serine

peptides

serine

RESEARCH COMMUNICATIONS

carrying

C (8,9),

is a protein

kinase

middle

of the

specificity

protein

the

side

phosphorylates

Hlhistone

that

as kemptide

kinase

side

Earlier

such

N-terminal for

AND BIOPHYSICAL

on the

to gain activity

of

representing Ala-Ser-Gly-Serthe peptide

penultimate lacking

Ala-Ser-Gly-Serkinase

C phospho-

peptide-2.

MATERIALS

AND METHODS

Materials. The following materials were obtained from DEAE cellulose and P81 paper, Whatman. the specified sources. SP-Sephadex, Sephadex G-10, Phenyl Sepharose, and AH Sepharose, Pharmacia. Biogel P-150, BioRad. PS, dj-olginp,q;~r~;;;SIIIS histone (Hl rich), Sigma. Carrier-free [Yphosphate, Japan Atomic Energy Research Institute. [Y- 'PIATP was either prepared by the method of Glynn and Chappell (14) or purchased from ICN. Homogeneous catalytic subunit of bovine heart CAMP-dependent protein kinase was prepared by J. Kinoshita in our laboratory. solid tially

Peptide Synthesis. phase method (15). the same as those

Peptide-l and -2 were prepared by the Experimental procedures were essendescribed previously (16). Crude 156

Vol. 142, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

peptides were purified by SP-Sephadex and Sephadex G-10 chromatography. The purity and identity of the peptides were assessed by high voltage paper electrophoresis at pH 3.6 and amino acid analysis. Enzyme Purification and Assay. Protein kinase C was purified from porcine spleenaccording essentially to the method of Humble -et al. (17) and from rat brain by the method of Kitano Most of the experiments were carried out with et al. (18,191. -the former preparation, but the results presented in Table 1 were obtained with the latter preparation. The assay mixture (200 ~1) consisted of the following components: 20mM Tris-HCl, pH 7.5, ImM 2-mercaptoethanol, 5mM MgC12, 0.5mM CaCl 10 w3lyp PS, 1 pg/ml diolein, 200 ug/ml type 111s histone, 3'0 I-IM [YP]ATP (100-300 cpm/pmol) and enzyme. Assay was usually carried out at 3O'C for 5 min. Phosphorylated histone was collected on ~81 paper. Peptide Phosphorylation. The composition of the assay mixture was identical to that described above except for substitution of peptide for histone. The phosphorylated peptide was separated from other assay components by highvoltage paper electrophoresis in pyridine-acetic acid-water (1:10:89) buffer, pH 3.6, on Toyo Roshi filter paper No. 5lA. The dried paper was exposed to Fuji X-ray film for 2-3 days to locate the radioactive spots, the areas representing the phosphorylated peptide were cut out and subjected to scintillation counting. Peptide Sequencing. The phosphorylated peptide was sequenced by automated Edman degradation on an LKB 4030 solid phase sequencer equipped with an autoconverter (LKB 4025). The sample was coupled to 2-aminoethyl-3-aminopropyl glass through the amino groups (20). The PTH derivatives of amino acids were identified by HPLC on an LS 410 C18 column (Toyo Soda) by isocratic elution according to the method of Zimmerman -et al. The PTH derivatives were also monitored for radio(21). activity. Miscellaneous. Protein was quantitated by the method of Lowry et al. (22) with bovine serum albumin as the standard. Radioac!v%y associated with filter paper was determined with 5 ml of toluene-based scintillation cocktail on an Aloka liquid scintillation counter LSC-703. Amino acid analysis was carried out on a Jeol JLC-6AH amino acid analyzer. High-voltage paper electrophoresis was run using a Toyo HPE-406 apparatus.

RESULTS AND DISCUSSION Phosphorylation was carried Methods. voltage peptide-l product

out

of

under

The progress paper with with

synthetic

the

the

conditions

of the

electrophoresis

peptides

reaction at

described

pH 3.6 (23).

of

0.47 toward 157

cathode

C and

by high-

Incubation

generated the

kinase

in Materials

was followed

enzyme and [y- 32P]ATP

a mobility

by protein

of

a radioactive relative

to

Vol. 142, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 1. Effects of Ca2+ and lipids phosphorylation of peptide-l by protein Conditiona

32P Incorporatedb,

Complete -Ca2+ -PSI diolein -Ca2+, PS, d iole in a

of

peptide-1.

diminished

This

272 254 57

phosphorylation

markedly

by the

In

peptide

(Table

1).

system

was 9 times

Ca2+,

fact,

than

Rates

complete

system

determined

The data

were

analyzed

were:

wand

V,,,

of

phosphorylated

up to peptide

as described

water,

subjected

and then

of Ser

were

and Lys6 AEAP-glass,

(0.34

identified

were not

at

identified

and the

seventh

initial

serine

in

rate

this

mol of

Edman

degradation

because residue 158

3

Vmax

phosphosubstrate

it

was necessary

Thus

extracted

2,

the

was also

(1.09 and

they

5,

peptide The

paper

from

the

(Fig.

nmol),

the

peptide.

by high-voltage

Gly

the

conditions.

u.M, while

for

above,

nmol),

of

equation.

residues,

per

mol

Steps

absence

phosphorylation

was phosphorylated.

to

complete

mg.

l

was separated

electrophoresis

phospholipids

in the

parameters

two

SO.1

was

the

111s histone

3 nmol/min

them

in

to be 100

type

possesses

was phosphorylated

nmol)

23

Henri-Michaelis-Menten

The kinetic

which

derivatives

79

obtained

under the

Sigma

mg.

l

As peptide-l to determine

that

was determined

analogously. Km 1.0

22

of Ca2+ and/or

of peptide

using

peptide-l

was 19 nmol/min rylated

28

peptide-l

phosphorylation

greater

were

of

omission

PS and diolein.

The Km for

+ 2 * +

518

cpm

The complete system contained 0.10 mM CaC12, 10 ug/ml PS and 1 ug/ml diolein as activators. Mean f SD of duplicate runs. The background radioactivity (19 cpm) was subtracted.

b

that

on the kinase C

paper

The PTH

1).

and Phe

(1.02

Ala1

respectively.

were

was unable

attached to remain

with

to

the

on the

Vol. 142, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Cycle

Fig. 1. Determination of the phosphorylation site by Edman degradation. Peptide-l was phosphorylated under the conditions specified in Materials and Methods. Approximately 5 nmol of phosphorylated peptide-l was subjected to the analysis. About 1.5 nmolof the sample was estimated to be linked to the AEAP glass. glass

after

activity

Step

6.

was released

presumably

to partial

degradation,

Fig.

position lacks

the

extent

amino acid

step,

demonstrates

recognition

site

nor peptidekinase,

C.

potential

identical

that

awaits

histone

molecule.

kinase

C is

capable

of

lysine

residue

on the the

course that

of Edman the

words, for

at

which

to any

indicating

this

the

serine

Peptide-2,

peptide

that

to be phosphorylated lysine

kinase.

serves

Neither

by CAMP-dependent

SerlO6

of

by protein

on the

with

1 and 6, due

as a peptide-l

protein

(24).

suggest

possibility

consistent

the

was phosphorylated

to be modified

this

Steps

in the

conditions,

for

In other

as expected

of radio-

was not phosphorylated

in peptide-l

These data

including

unambiguously

lysine,

under

amount

phosphorylated.

is essential

kinase

site

every

dephosphorylation

penultimate

by protein

of

1

at

a considerable

4 was preferentially

detectable this

Although

kinase

identification It

also

phosphorylating C-terminal

results

Hl histone

side

reported 159

has the

C, though of

a peptide serine.

recently

proof

the phosphorylation

became clear

of

final

that

bearing This by Ferrari

protein a basic finding et --

is al.

Vol. 142, No. 1, 1987

BIOCHEMICAL

Meanwhile,

(IO).

kemptide

it

carrying

can serve

basic

as substrates

results

we conclude

phosphorylate

or the

protein

kinase

arginine

or Three

lysine

residue,

This

on the

N-terminal

for

kinase

(8,9).

this

that basic

more

residues

on the

points

should

when placed

at

a peptide

to

activity

of

peptides

and proteins.

residues

in

peptide-1,

words, the

the

serine

a good rylated useful activity

enzyme

appears

to be modified.

substrate

for

by CAMP-dependent as a substrate in

a complex

the

kinase

the

mixture

Firstly,

selective of

for

protein

a single is

protein the

of the

strict

recognition

assay

that

In

other of

peptide-l

it

is

this of

serine

is

C.

makes

kinase

substrate two

lysine

C and that kinase

C

(24).

kinase

fact

C-

having

side

for

to the

Finally,

protein for

serines

Secondly,

show rather

protein

kinase

position,

requirement

by protein to

protein

a substrate

one closer

phosphorylated

the

be discussed.

minimal

the

for

an appropriate

the

to

CAMP-dependent

N-terminal

become

C is able

on either

sense

preference

of serine

these

kinase

than

as

side

acids

this

versatile

is probably

preferentially

In

such

From

protein amino

side.

exhibits

lysine

for

residues

as being

further

sufficient

peptides

bearing

which

RESEARCH COMMUNlCATlONS

that

N-terminal

may be regarded

known

tentatively

a site

terminal

c.

has been

AND BlOPHYSICAL

not

is phospho-

compound

protein

kinase

kinases.

ACKNOWLEDGEMENTS The authors are grateful to Dr. Y. Nishizuka and his collaborators for kindly informing of their unpublished results as well as for helpful discussions. They also thank Ms. J. Kinoshita for her expert technical assistance. REFERENCES Nishizuka, Y. (1984) Nature 308, 693-698. 2. Takai, T., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. 3. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 13341-13348. 4. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U ., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851 1.

160

T.,

C

Vol.

142,

5.

No.

1, 1987

Hunter,

T.,

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BIOPHYSICAL

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Merrifield, R.B. (1963) J. Am. Chem. Sot. 85, 2149-2154. Kondo, H., Uno, S., Komizo, Y., and Sunamoto, J. (1984) Int. J. Peptide Protein Res. 23, 559-564. Humble, E., Heldin, P., Forsberg, P., and Engstrom, L. (1984) J. Biochem. 95, 1435-1443. Kitano, T., Go, M., Kikkawa, U., and Nishizuka, Y. (1986) Methods Enzymol. in press. Kondo, H., Kinoshita, J., Matsuba, T., and Sunamoto, J. (1986) FEBS Lett. 201, 94-96. Enzymol. 47, 277-288. Laursen, R.A. (1977) Methods Zimmerman, C.L., Apella, E., and Pisano, J.J. (1979) Anal. Biochem. 77, 567-573. Lowry, O.H., Resebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Zetterqvist, O., Ragnarsson, U., Humble, E., Berglund, L., and Engstrom, L. (1976) Biochem. Biophys. Res. Commun. 70, 696-703. Glass, D.B. and Krebs, E.G. (1980) Annu. Rev. Pharmacol. Toxicol. 20, 363-388.

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