Phosphorylation of phosphofructokinase by protein kinase C changes the allosteric properties of the enzyme

Vol.

129,

No. 3, 1985

June

28,

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1985

Pages

892-897

OF PHOSPHOFRUCTOKINASE BY PROTEIN KINASE PHOSPHORYLATION CHANGES THE ALLOSTERIC PROPERTIES OF THE ENZYME H. W. Hofer, Faculty D-7750

S.

and

Schlatter

of Biology, Konstanz,

University Fed. Republic

C

M. Graefe of Konstanz of Germany

Received May 20, 1985

The Ca'+and phospholipid - dependent protein kinase C from rat brain phosphorylates rabbit muscle phosphofructokinase at the same trypsin-labile site as cyclic AMP-dependent protein kinase. However, protein kinase C also effectively phosphoryIncubation of phosphofructolates one or more separate sites. Ca2+, kinase in the presence of protein kinase C, phospholipids, and ATP appears to affect the allosteric properties of phosphofructokinase by shifting the fructose 6-phosphate saturation curve to lower substrate concentrations in a time-dependent 0 1985 *ca&?!nic, manner and decreasing cooperativity of the enzyme. Press,

Inc.

The Ca2'-

and phospholipid

- dependent

protein

kinase

C

[l] has received attention because of its possible role in the regulation of cellular differentiation and tumor promotion (see Ref. [Z]). Although protein kinase C may be involved in signal transduction from al receptors [Z], little is known about the

nature function

of

its physiological in the regulation

substrates of energy

and about metabolism.

its

possible

Phosphorylation of muscle phosphofructokinase (EC 2.7.1.11) has been described in vivo [3, 41 and under the catalytic influence of cyclic AMP-dependent protein kinase (protein kinase

A)

in vitro [5 - 71. However, phosphorylation by protein kinase A does not cause a significant change in the catalytic or regulatory properties of phosphofructokinase. This report describes the in vitro phosphorylation of phosphofructokinase from rabbit skeletal muscle by a protein kinase C preparation from rat brain and the resultant changes in its regulatory properties. MATERIALS

AND

METHODS

Materials containing

Histone ca.

(Type III-S), phospholipids 80 8 phosphatidylserine)

0006-291X/85 $1.50 Cowright 0 1985 by Academic Press, Inc. AN rights of reproduclion in any form reserved.

892

(Brain and diolein

extract were

type from

II,

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3, 1985

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Sigma (St. Louis, MO.). Additional biochemicals were obtained from Boehringer Mannheim (Germayy) and other chemicals weryzfrom Merck (Darmstadt, Germany). [yP]ATP was prepared from [ Plorthophosphoric acid (The Radiochemical Centre, Amersham, U. K.) by a modification of the procedure described by Johnson and Walseth [8]. The preparation and assay of phosphofructokinase from rabbit skeletal muscle and of the catalytic subunit of protein kinase A from beef heart was performed as previously described [9].

Preparation of protein kinase C E'rotein kinase C was prepared from rat brain by a modification of the procedure described by Kikkawa et al. [IO]. The homogenate of 6 brains from freshly sacrificed rats was prepared in 6 volumes of Tris-HCl buffer (20 mM, pH 6.5), containing EDTA i2 mM) , EGTA (IO mM), sucrose (0.25 M), and aprotinin (20 units per ml) and centrifuged at 100,000 x g for 60 min. The supernatant was applied to a chromatography column (2.5 cm diameter, 10 cm length) containing DEAE cellulose (Whatman DE-52) which had been equilibrated with buffer A consisting of Tris-HCl (20 mM, EGTA (5 mM) , EDTA (2 mM) , 2-mercaptoethanol (50 mM) , and pH 7.5), aprotinin (20 units/ml). The column was washed with 120 ml of buffer A and 0.1 mM cyclic AMP to elute the catalytic subunit of protein kinase A, and another 200 ml of buffer A. The elution of protein kinase C was accomplished by a linear gradient from 0 to 300 mM NaCl dissolved in buffer A (total gradient volume 200 ml). The protein kinase C containing fractions were pooled, concentrated to about 5 ml by ultrafiltration, and applied to a column (1 cm diameter, 10 cm length) of Phenylsepharose 4 B (Pharmacia, Uppsala, Sweden) equilibrated with Tris-HCl buffer (20 mM, containing EDTA (0.5 mM) , EGTA (0.5 mM), 2-mercaptopH 7.5), ethanol (IO mM), aprotinin (20 units/ml), and NaCl (1.5 M). The column was washed with 30 ml of the same buffer and protein kinase C was eluted by linearly decreasing the salt concentration to zero (total volume of the gradient 40 ml). The protein kinase C containing fractions were concentrated by ultrafiltration to a final volume of 3 ml and frozen in liquid Nz after the addition of glycerol (final concentration 20 %). The preparation lost approximately 30 % of its initial activity during 1 week of storage at -65 OC. The enzymatic activity of the preparation was at least lo-fold stimulated by phospholipids. The preparation contained no phosphofructokinase activity.

Assay

of protein kinase C The assays of protein kinase C were performed as previously described for protein kinase A [b] and contained Tris-HCl buffer (0.1 M, pH 7.5), Mg-acetate (20 mM), CaClz (2 mM), phospholipids (200 pg/ml), diolein (3 kg/ml), histone (5 mg/ml), Control assays were made up and [y-3' PIATP i40 PM, 100 kBq/nmol). without phospholipids and diolein. Trypsin treatment of phosphofructokinase was performed as previously described [ 131. The procedure of CNBr and protease digestion and of peptide mapping was described in Ref. 151. Protein determinations were performed according to Bradford [Ill using bovine serum albumin as a standard, and electrophoreses were performed according to Laemmli [12] using 12.5 % polyacrylamide gels and 0.1 % sodium dodecylsulfate. 893

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No. 3. 1985

RESULTS

AND

AND

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DISCUSSION

Phosphorylation

of

phosphofructokinase

by protein

kinase

C

Phosphofructokinase has been shown to be phosphorylated by protein kinase A at a serine residue 6 amino acids from the Cterminus [14]. The phosphorylation site is labile to proteases under conditions enzyme intact

which

leave

the

catalytic

properties

of

the

[13].

During the phosphofructokinase

course of this from rabbit

by a preparation

of

protein

presence of phosphatidyl electrophoretic patterns phosphofructokinase in

study, muscle

kinase

it became apparent that was also phosphorylated

C from

rat

brain

in

the

serine, diolein, and Ca 2+ ions. The resulting from phosphorylation of the presence of either protein kinase A

or C, after digestion of phosphofructokinase with either trypsin, thermolysin, or CNBr, revealed partly identical phosphopeptides (data not shown). However, there were additional phosphopeptides with protein phosphorylates also

one or

kinase the more

C. These data same C-terminal

additional

suggest that protein kinase C site as protein kinase A and

sites.

Further evidence supporting protein kinase C phosphorylation moval of the C-terminal site of

the presence of site was obtained phosphofructokinase

an additional with the reby a brief

trypsin treatment. The incorporation of radioactive phosphate into the modified phosphofructokinase preparation, as catalyzed by protein kinase A and C, is shown in Fig. 1. An almost linear progress with time of [32 Plphosphate incorporation was observed in the presence of protein kinase C (Curve A), whereas protein kinase A had little effect on the trypsin-treated phosphofructokinase (Curve B). Fig. 2 shows an autoradiograph of an electrophoretic gel on which the phosphofructokinase samples were separated after trypsin treatment and subsequent phosphorylation in the presence of either protein kinase C or protein kinase A. Although protein kinase A phosphorylated the lower molecular weight proteolytic fragments, it did not phosphorylate the high molecular weight form of phosphofructokinase. On the other hand, protein kinase C had a preference for these high molecular weight forms. These data support the hypothesis that, in addition to the site on the C-terminal tryptic peptide, phosphofructokinase contains an extra phosphorylation site for protein kinase C. 894

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kDa ::yg:; 97.4 ’ 66.0 45.0

PFK-

A

01

C

D

02 Fig. 1 Incorporation of radioactive phosphate into phosphofructokinase (19.2 ug) after 20 set treatment with trypsin (I ug/mg phosphofructokinase). A: Incubation with protein kinase C in the presence of CaCIZ (2 mM), phospholipids (200 pg/ml), diolein (3 kg per ml). B: Incubation with protein kinase A (catalytic subunit). The incubations contained equal activities of the protein kinases, Mg-acetate (20 mM), and 40 ).LM ATP (specific radioactivity 75 kBq/nmol). Fig. 2 A and B: Autoradiographs of electrophoretic gels containing phosphofructokinase samples obtained by 5 min incubation as described in the Legend of Fig. 1. A: Sample incubated with protein kinase A. B: Sample incubated with protein kinase C. c: Staining pattern of the gel shown in Lane A with Coomassie Blue (identical phosphofructokinase samples were used in experiments A and B). D: Staining pattern of molecular weight markers. The position of the high molecular weight form of phosphofructokinase is indicated.

Influence

of

protein

kin&se

C on

the

properties

regulatory

of

phospho.Eructokinase

protein when

phate

Phosphofructokinase kinase C exhibited assayed

at

pH 7.0

in

and 2.0 mM ATP (c.

fructokinase subjected protein kinase C (Fig. (not

incubated in a time-dependent

shownj

exhibited

the

f.

presence

Fig.

the

presence increase

of

0.5

mM fructose

3, Curve

A).

However,

to the same incubation 3, Curve B) or protein a slight

decrease 895

of in

in

ATP and activity 6-phos-

phospho-

procedure without kinase A instead activity.

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No. 3, 1985

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AE=O Yiiz A

05.

.

/.A’

I// \

0

B

o------o

0

5

0 IO

ml”

3

Fig. 3 Activity of phosphofructokinase after incubation at pH 7.0 for different periods of time in the presence of ATP (2.0 mM), CaClz (4.0 mM), phospholipids (200 bg/ml), diolein (1 IN, MgClz and in the presence (A) or absence (B) of protein (3 us/ml), kinase C. The phosphofructokinase assays were performed in piperazine-N,N'bis(2-hydroxipropanesulfonate) buffer (0.1 M, pH 7.0) in the presence of MgC12 (4.0 mM), ATP (2.0 mM), fructose 6-phosphate (0.5 mM), NADH (0.2 mM), aldolase (10 fig/ml), triosephosphate isomerase (1 pg/ml), and glycerol l-phosphate dehydrogenase (3 ug per ml). Fig. 4 Substrate saturation curves of phosphofructokinase after 15 min incubation in the presence (A) or absence (B) of protein kinase C. The incubations and assays were performed as described in the Legend of Fig. 3, except that varying concentrations of fructose 6-phosphate were used in the assays. The curves represent graphs of optimum fits of the nonlinear form of the Hill equation v = (V,*S")/(K"+S") [15], where S is the substrate concentration, K a constant, and n the Hill coefficient. The parameters used for the graphs were for Curve A K = 1.18 mM, n = 1.18, and for Curve B V, = 0.144 vm = 0.215, K = 4.11 mM, n = 2.50.

Phosphofructokinase kinase of Fig. C led

incubated

C exhibited a typical sigmoidal 4), whereas incubation in the to

a distinct

reduction

of

the

in the absence of protein saturation

presence half-saturating

curve

(Curve

of protein

B

kinase

fructose

6-

a slight increase in V and reduced phosphate concentration, m' sigmoidicity of the saturation curve (Curve A, Fig. 4). Therefore, the action of protein kinase C may lead to a change in phosphofructokinase conformation which favours a high affinity for fructose 6-phosphate. Such a change in the regulatory properties of phosphofructokinase by protein kinase C could be of physiological importance. Further studies are needed to clarify 896

Vol.

129,

whether kinase tory

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

or not phosphofructokinase C in vivo and the physiological mechanism

suggested

on the

basis

is

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also a substrate significance of of

the

present

to protein a regula-

in

vitro

experiments.

ACKNOWLEDGMENTS We are indebted to Dr. R. Staron for advice during the preparation of the manuscript. The technical assistance of Mrs. S. Kirsch is gratefully acknowledged. The study was supported by the Deutsche Forschungsgemeinschaft (Grant Ho 650/5) and the Fonds der Chemischen Industrie, Frankfurt/M., Germany. REFERENCES

7.

Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. Nishizuka, Y. (1984) Trends Biochem. Sci. 9, 163-166. Hofer, H.W., and Furst, M. (1976) FEBS Lett. 62, 118-122. Uyeda, K., Miyatake, A., Luby, L.J., and Richards, E.G. (1978) J. 13iol. Chem. 253, 8319-8327. Riquelme, P.T., Hosey, M.M., Marcus, F., and Kemp, R.G. (1978) Biochem. Biophys. Res. Comm. 85, 1480-1487. S4rensen-Ziganke, B., and Hofer, H.W. (1979)Biochem. Biophys. Res. Comm. 90, 204-208. Riquelme, P.T., and Kemp, R.G. (1979) J. Biol. Chem. 255,

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Johnson,

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T.F.

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Hofer, H.W., and Sbrensen-Ziganke, B. (1979) Biochem. phys. Res. Comm. 90, 199-203. Kikkawa, U., Minakuchi, R., Takai, Y., and Nishizuka, (1983) Methods in Enzymology 99, 288-298. Bradford, M. (1976) Anal. Biochem. 72, 248-254. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Krystek, E., and Hofer, H.W. (1981) Biochem. Biophys. Comrn.

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