Amino acid sequence at the phosphorylated site of rat liver pyruvate kinase

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AMINO ACID SEQUENCE AT THE PHOSPHORYLATED SITE OF RAT LIVER PYRUVATE KINASE Bror

Edlund,

iirjan

Jill

Andersson,

Zetterqvist

Institute

Vincent

and Lorentz

of Medical

Titanji,

Ulla

Dahlqvist,

Pia Ekman,

Engstrijm

and Physiological

University

of Uppsala,

S-751

Received

September

25,1975

Chemistry,

23 Uppsala,

Biomedical

Center,

Sweden

SUMMARY One dominating peptic phospQ?peptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-( P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3',5'-AMPstimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide prev' gqusly isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-( P)SerP-Leu. INTRODUCTION The main ferase:

isoenzyme

(L type)

EC 2.7.1.40)

phorylated

from

on serine

of liver

the

rat

residues

(1)

pyruvate

kinase

and pig

by cyclic

(2)

(ATP:phosphotrans-

has been

shown to be phos-

3 ',5'-AMP-stimulated

protein

kinase

(1,2). The activity decreased enzyme

enzyme at low phosphoenolpyruvate

by phosphorylation, has a regulatory

phopeptide pig

of the

liver

effect

was recently enzyme,

indicating

and its

-Ala-(32P)SerP-Leu

on its

isolated amino

experiments

phosphopeptide

from

rat

liver

chromatography

than

the

peptide

residue

in the of the

eicosanoic liver

activity

(1,2). digest

sequence

we found

that

amino

acid

two enzymes.

pyruvate

of the

One major (32P)-phos32 P-labelled

of the

was found

to be Leu-Arg-Arg-

kinase

is

the

kinase

corresponding

was eluted

earlier

the pig

liver

enzyme.

sequence

around

the

phosphorylated

In the

present

from

described.

paper

a peptic

the

digest

The amino

acid

( 32P)-

peptic

from

This

on gel suggested serine

isolation of a major of 32 P-labelled rat

sequence

of this

( 32P)-

was determined. MATERIALS

Rat liver

phosphorylation

a peptic

acid

c3* P)-phosphopeptide

pyruvate

phosphopeptide

the

is

(3).

In preliminary

difference

from

that

concentrations

pyruvate

kinase

AND METHODS

(EC 2.7.1.40)

was induced

in male

Sprague-

a

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Dawley rats byacarbohydrate-rich diet (4) and purified as recently described (4). 3000 units of protein kinase peak I purified from rat liver through hydroxyl apatite (4) and 35 mg of pyruvate kinase were mixed at O°C in 10 mM potassium phosphate buffer, pH 7.0, containing 16% glycerol, 30 mM KCl, 0.05 mM Fru-1,6-P2 and 0.05 mM dithiothreitol. The solution was tiJ$ated to P)ATP pH 8.0 by the addition of 0.55 ml of 0.5 M ammonium hydroxide. y-( (6 000 cpminmol) prepared as described previously (5,6) and cyclic 3',5'-AMP were added to a final concentration of 0.2 mM and 0.01 mM, respectively, and ;i;hincAuftz:i;; mini~u;:c~;~l~$j 1 was immediately transferred to a 30°C water P)ATP was removed by chromatography at 5OC on a 5 x 77 cm Sephadex G-50 column, equilibrated and eluted with 5 mM potassium phosphate buffer, pH 7.0. The incorporation of phosphate was calculated as mol of phosphate per mol of enzyme, assuming a tetrameric enzyme with a molecular weigh of 250 000 (1). 1 M HCL was added to the pooled fractions containing the 52 P-labelled enzyme to give a final concentration of O.C5 M, and 21 mg of pepsin (Boehringer-Mannheim, GmbH, type EPBK) were added. The digestion took place at 25'C for 2 hours. The digestion mixture was then chromatographed on a 7.2 x 52 cm Sephadex G-25 column, equilibrated and eluted with 50 mM pyridine-acetic acid buffer, pH 3.33* The radioactive peak appeared after 0.6 column volume. This radioactive ( P)-phosphopeptide material was further purified and digested with thermolysin (Sigma type P 1512) as described below. The total radioactivity of fractions was estimated by measuring the Cerenkov radiation (7). Amino acid sequence analysis was performed, using a dansyl-Edman method according to the description by Hartley (8). All preparations were carried out at room temperature (23O-25'C) unless otherwise stated. RESULTS Isolation

of the

incorporation phate

of phosphate

into

per mol of enzyme.

peptic

phosphopeptide

was applied in Fig. the

32 P-labelled

dominating

the

peptic

enzyme was found

30% of this

material

as described

1. The main part

of the

applied,

The molar

to be 3.1 mol of phos-

was obtained

as a 32P-labelled

The ( 32 PI-phosphopeptide

above.

to a 0.3 x 30 cm SP-Sephadex

radioactivity

phosphopeptide.

C-25 column

radioactive

peak,

material

and eluted

as described

corresponding

to 70% of

was pooled.

The ionic strength of the pooled material (62 ml) was reduced by chromatography on a 7.2 x 55 cm Sephadex G-25 column, equilibrated and eluted with 5 mM acetic acid. The ( 32 P)-phosphopeptide

material

(84 ml)

was then

19 ml of 0.25

M ammonium hydroxide

Sephadex

column,

pooled (v/v) amount

A-25

as shown

and lyophilized pyridine-water.

dissolved

The overall

yield

of 32P-labelled

thermolysin

performed

Further

digestion

of the

(87 nmol)

of the

were

dominating

of the 32 P-labelled

main

peptic ( 32

peptic

phosphopeptide

1517

on this

peak was ml) of 50%

obtained. in

the and

material.

( 32 PI-phosphopeptide

PI-phosphopeptide

of

on a 0.9 x 30 cm QAE-

was 60%, as calculated from acid sequence analysis

Amino

with

addition

1. The main radioactivity in a small volume (1.8

enzyme digested.

digestion

and isolation

to pH 8.5 by the

and chromatographed

in Fig.

and then

further

lysin

titrated

50% (v/v)

by thermoTo 600 91 pyridine-

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

l-

200

250

50

Effluent

100

(ml 1

32 1. Two successive chromatographies of a peptic ( PI-phosphopeptide from rat liver pyruvate kinase. A. Chromatography on SP-Sephadex C-25 column (0.9 x 30 cm) equilibrated with 0.05 M pyridine-acetic acid buffer, pH 3.2. Elution was performed with 100 ml of the same buffer, pH 3.2, and a linear gradient (total volume 400 ml) formed from 0.05 M and 0.3 M pyridine-acetic acid buffer, pH 3.2. The fraction volume was 2 ml. The elution volume indicated in the figure represents the effluent collected from the start of the gradient. B. Chromatography on a QAE-Sephadex A-25 column (0.9 x 30 cm) equilibrated and eluted with 0.05 M ammonium carbonate buffer, pH 8.5. 2.2 m frjstions were collected and the material was pooled as indicated. O--O = = P radioactivity. For details, see text. Figure

water

100 ~1 of 0.5 M ammonium carbonate

mixture

was then

equilibrated radioactive 2

ml

chromatographed

and eluted material

of 0.05

dissolved

0.6

column

ammonium carbonate Amino

acid

acid

residues

A-25

buffer,

sequence

peptic

(Fig.

The amino and that are

given

in

1518

pH 8.5.

The

dissolved

in

mg of thermolysin, After

on a 1.8

0.05

2 hours

x 40

cm

at

Sepha-

M ammonium carbonate appeared

was pooled

equilibrated

was Leu-Arg-Arg-Ala-SerP-Val-Ala-Glx-Leu. on the two phosphopeptides

with

material

phosphopeptide

-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu

0.4

This

G-25 column,

was then

was added.

radioactivity

column,

pH 8.5

analysis.

of the

pH 8.5,

added.

buffer,

by chromatography

This

volume.

QAE-Sephadex

It

pH 8.5.

and eluted

70% of the applied

pH 8.5. cm

buffer,

was interrupted

dex G-25 column,

0.9 x 30

buffer,

were

x 40 cm Sephadex

and lyophilized.

same

equilibrated

pH 8.5,

M ammonium carbonate

M ammonium carbonate

digestion

peak after

on a 1.8 0.05

was pooled

in 0.2 ml of the

25OC the buffer,

with

buffer,

as a well

and applied

and eluted

with

defined to a 0.05

M

2). acid

sequence was found

of the

first

12 amino

to be Asx-Thr-Lys-Gly-

of the thermolytic phosphopeptide The results of amino acid analysis Table

1.

BIOCHEMICAL

Vol. 67, No. 4, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Effluent

(ml1

material obtained after Figure 2. Chromatography of 53" c32 PI-phosphopeptide P-labelled peptic phosphopeptide with thermodigestion of the dominating equilibrated and eluted with lysin. A 0.9 x 30 cm QAE-Sephadex A-25 column, 0.05 M ammonium carbonate buffer, pH 8.5, was used. 2 ml fr2stions were P radioacticollected and the material was pooled as indicated, O--O = vity. For details, see text.

DISCUSSION From amino

acid

analysis

and the overlapping

-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu and Leu-Arg-Arg-Ala-(32 from the acid

of the

P)SerP-Val-Ala-Glx-Leu

thermolysin

sequence

amino

treated

acid sequence Asx-Thr( 32 PI-phosphopeptide

peptic

( 32 P)-phosphopeptide

of the

material it was concluded that the full 32 ( PI-phosphopeptide was Asx-Thr-Lys-Gly-Pro-

of the peptic

amino

-Glx-Ile-Glx-Thr-Gly-Val-Leu-Ar~-Ar~-Ala-~32P)SerP-Val-Ala-Glx-Leu. The amino site

of rat

acid liver

AMP-stimulated

obtained

pyruvate

kinase

protein

phosphorylation effects

sequence

of the

of the peptic

sequence

around

The amino similar

except

for

residue

acid the

following

pyruvate around strates

is

kinase

pig

exchange

of the

residue.

This

serine

protein

kinase

(2).

Furthermore,

a phosphorylated

for

this serine

AMP-dependent

with

is not

known

from rat this

protein

serine

leucine could

residue explain

might

suggest

have also kinase

1519

been (11-13).

residue is also 32 P)Ser?-Leu for

can phosphorylate (LO).

a different

a valine

why cyclic

liver

phosphorylation residue

3',5'is the

at present.

enzyme - Leu-Arg-Arg-Ala-(

the

it

leads

the phosphorylated liver

cyclic

that

to the demonstrated 37 P-labelled the remaining

acid

conservatory

of importance of cyclic

around

with

phosphopeptides

amino

phosphorylated

concluded that

Whether

contained

phasphorylated of the

phosphorylation found

(2,4,9).

sequence

the major

is therefore

residue

digest

to that

AMF-stimulated ture

the

after

It

serine

on enzyme activity

material

very

kinase.

constitutes

that Similar found

pig

3',5'liver

the primary

struc-

sequences in other

sub-

-

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

1

Amin32acid composition of the ( 32 P)-phosphopeptides obtained by digestion P)-labelled rat liver pyruvate kinase with pepsin and further d&esof ( tion of this phosphopeptide with thermolysin: 232nmol of the peptic ( P)phosphopeptide and 20 nmol of the thermolytic ( PI-phosphopeptide were hydrolyzed for 24 hours (both phosphopeptides) and 72 hours (the peptic phosphopeptide) at llO°C in sealed ampoules containing 6 M HCl. A one-column Durrum amino acid analyzer was used. Except for glutamic acid, numbers given within brackets represent the nearest integer of mol of amino acid per mol of phosphopeptide.

Amino

Phosphopeptide

acid

obtained

Pepsin Aspartic

acid

by digestion Thermolysin

1.0(l)

Threonine

1.ga(2)

Serine Glutamic

acid

0.8a(l)

0.8a(l)

1.8(3b)

1.0(l)

Proline

1.0(l)

Glycine

1.9(2)

Alanine

1.6(2)

2.0(2)

Valine

1.6'(Z)

1.0(l)

Isoleucine

l.OC(l)

Leucine

2.2c(2)

Lysine

1.0(l)

Arginine

2.4(2)

2.1(2)

Total

20

9

a corrected b

value

'the

to zero

time

1.7(2)

hydrolysis

estimated

from

amino

acid

same values

were

obtained

sequence from

analysis

analysis

times

of 24 and 72 hours

ACKNOWLEDGEMENTS This (Projects

work

was supported

13X-50

by the

Swedish

and 13X-4483).

1520

Medical

Research

Council

with

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES Ljungstrbm, O., Hjelmquist, G., and Engstram, L. (1974) Biochim. Biophys. Acta, 358, 289-298. Engstrijm, L., Berglund, L., Bergstrtim, G., Hjelmquist, G., and LjungstrGm, 0. (1974) in: Lipmann Symposium: Energy, Riosynthesis and Regulation in Molecular xology, pp. 192-204, Walter de Gruyter Inc, Berlin-New York. Hjelmquist, G., Andersson, J., Edlund, B., and Engstrbm, L. (1974) Biochem. Biophys. Res. Commun, 61, 559-563. Titanji, V., Zetterqvist, G., and Engstrtim, L. (1975) (submitted for publication). Engstrom, L. (1962) Arkiv Kemi, 19, 129-140. Msrdh, S. (1975) Biochim. Biophys. Acta, 391, 448-463. I. Msrdh, S. (1975) Anal. Biochem, 63, l-4. 8. Hartley, B.S. (1970) Biochem. J, 119, 805-822. 9. Ekman, P., Dahlqvist, U., Humble, E., and Engstrdm, L. (1975) (to be published). 10. Humble, E., Berglund, L., Titanji, V., Ljungstrom, O., Edlund, B., Zetterqvist, ij., and Engstrom, L. (1975) Biochem. Biophys. Res. Commun. (in press). 11. Larner, J., and Sanger, F. (1965) J. Mol. Biol, 11, 491-500. 12. Langan, T.A. (1969) Proc. Natl. Acad. Sci. U.S.A, 64, 1276-1283. 13. Cohen, P., Watson, C., and Dixon, G.H. (1975) Eur. J. Biochem, 51, 79-92.

1521