Protein kinase in human plasma analogous to that present in control and interferon-treated HeLa cells

Vol. 103, No. 4,198l December

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31, 1981

PROTEIN PRESENT

KINASE IN

IN

HUMAN PLASMA

CONTROL

Department *

Received

TO THAT

AND INTERFERON-TREATED

Ara G. Hovanessian, Pierre

ANALOGOUS

Pouillart*,

Pierre Pierre

Rollin, Sureau

HELA Yves Rivisre,

and Luc Montagnier

of Virology, Institut Pasteur, 25 rue 75724 Paris CBdex 15, France

Institut

Curie,

25 rue

November

4, 1981

d'Ulm

- 75232

CELLS

Paris

du Dr Roux,

Cddex 05, France

SUMMARY - A protein kinase activity analogous to that found in interferontreated HeLa cells is detectable in human plasma. This kinase activity is manifested by the phosphorylation of an endogenous 72,000 molecular weight protein which is stimulated markedly by Mn2+. The protein kinase activity in human plasma can phosphorylate'calf thymus histones and is independent of cyclic AMP. The phosphate is linked to the phosphorylated 72,000 molecular weight protein by its serine and threonine residues. The level of protein kinase activity in 50 different normal human plasma that we analysed varied from one individual to the other. Treatment of two patients with fibroblast (8) interferon resulted in an enhanced level of the protein kinase activity in the plasma. These results suggest that such protein kinase activity in human plasma may reflect the presence and action of circulating interferon. INTRODUCTION Interferon-treated protein

kinase

67,000

activity

(p67K kinase)

respectively

(l-3).

the ~67~ kinase level with

is

activity

is

enhanced

inducers

mouse and human cells manifested

by the phosphorylation

and 72,000 Besides

its

also

detectable

several

fold

of interferon

can be assayed

(p72K kinase) presence

purification

on poly(I).poly(C)-Sepharose

of a protein

kinase

kinase

is

which

routinely

and dsRNAs

and efficiently (5).

in human plasma enhanced

weight

and plasma with

of a

of endogenous

molecular

in tissues

conveniently

levels

in interferon-treated

in mice treated

such as virus

activity

show enhanced

mouse cells, of mice and its

interferon (4). after

proteins,

or injected

Such kinase its

partial

Here we show the presence corresponding

in interferon-treated

to the p72K HeLa cells

(3).

Abbreviations : p72K kinase, interferon-mediated protein kinase in human cells ; p67K kinase, interferon-mediated protein kinase in mouse cells ; &RNA, double-stranded RNA ; poly(1) .poly(C), polyinosinicpolycytidilic acid ; poly(A) .poly(U), polyadenylic-polyuridylic acid.

0006-291X/81/241371-07$01.00/0 1371

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MATERIALS AND METHODS Blood was collected in polystyrene tubes containing heparin (100 u/ml) and aprotinin (100 u/ml ; Zymofren, Specia) and left 15-30 min at 4Oc. Plasma was collected after centrifugation (2000 x Q, 15 min) and stored at -8OOC. The p72K kinase wasassayedafter its partial purification On poly(I).poly(C)-Sepharose prepared in our laboratory (6). Plasma samples (100 Dl) were first diluted in an equal volume of NP40 buffer (10 mM Hepes pH 7.6, 10 mM KCl, 2 mM Mg(OAc)2, 7 mM 2-mercaptoethanol and 0.5% ~~40) before addition of poly(I).poly(C)-Sepharose (100 ill) equilibrated in Hepes buffer/glycerol (10 mM Hepes pH 7.6., 50 mM KCl, 2 mM Mg(OAC)2 7 mM 2-mercaptoethanol and 20% glycerol (v/v). Binding of the kinase to poly(I).poly(C)-Sepharose was carried out in polystyrene tubes at room The poly(1) .poly(C)temperature for 30 min with occasional gentle mixing. Sepharose bound kinase fractions were first washed (by centrifugation, 100 x g for 10 min) with Eiepes buffer/glycerol (3 x 5 ml) then followed with the same buffer (5 ml) but containing 5 mM M (OAC)~ and 5 mM MnC12. Phosphorylation (60 rain, 30°C) was with 10 rlM (y- 12 P)ATP (900 Ci/mmol ; All the samples Amersham, England) in Hepes buffer/glycerol as above. sample buffer and supernatants were heated (95', 5 min) in electrophoresis were analysed on polyacrylamide slab gels (10%) containing sodium dodecyl sulphate (SDS) as described previously (5, 6). For the analysis of the phosphoamino acids in the phosphorylated protein was recovered from the polyacrylamide 72~ protein, the 3ZP-labeled slab gel and was subjected to acid hydrolysis in 6 M HCl at 100°C for 4 hr. The HCl was removed by evaporation under vacuum and the hydrolysates were Phosphoserine, phosphothreonine (Sigma) dissolved in 50 ~1 of water. and phosphotyrosine (a generous gift of 0. Bensaude, Institut Pasteur An aliquot (10,000 cpm) Paris) were added to the radioactive sample. was analysed by high voltage electrophoresis on Whatman 3 MM paper at pH 3.5 (pyridine/acetic acid/H20, 1 : 10 : 289) at 2400 V for 1 hr. RESULTS AND DISCUSSION Figure kinase

1 shows the optimum

in human plasma.

conditions

The amount

for

the

assay

of phosphorylation

increased upon incubation at 30°C and was markedly enhanced ion concentrations (5 - 10 mM). Mg2+ at 2 to 10 mM slightly p72K kinase activity (Fig-l) but Ca 2+ had no apparent effect shown). Cyclic

The Protein AMP (Fig.1)

kinase

we assayed the protein 2+ 5 mM Mg , 10 mM Mn2+ during the kinase assay ATP dilutes the specific when cold

activity

or cyclic kinase for

was not modified

GMP (data activity

not

shown).

Mn 2+

at higher enhanced

the

(data not in the presence of this work BM ATP,

60 min at 30°C. markedly activity

ATP (1 mM) was added

Addition of cold ATP at 1 IIIM the incorporation of 32p since (y- 32p) ATP. On the other hand, 90 min after incubation with 10 no of reduced of the

shown),

rent

not

on the

72~ protein

Throughout with 0.01

by incubation

(Y- "P)ATP, no effect was observed (data not that the "P incorporated into the 72K protein the kinase reaction. Addition of UTP, CTP and W32 P)ATP in the kinase assay at the start of effect

of the p72K

of the

incorporation

of

32P (data

1372

is not

thus

suggesting

turned

over

during

GTP at 1 mM to 10 no the reaction shown).

had no appaATP,

therefore,

Vol. 103, No. 4,198l

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123

#As++

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Time

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RESEARCH

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Fig. 1 - Optimum conditions for the phosphorylation of 72K protein in partially purified fractions from human plasma: electrophoretic analysis on polyacrylamide (10%) slab gels containing SDS. An autoradiograph of a stained, dried gel is shown. The figure represents parts of the gels at the region of 72K protein. The poly(~).poly(C)-bound kinase fractions were incubated (30°C, 60 min ; unless otherwise indicated) in the presence of +9.01 uM (y-"'P)ATP (900 Ci/mmol) at different conditions, as indicated. 1 to 3+i incubation at Mg(OAc) concentrations of 2,s and 10 mM Mg .lanes lanes 1 to 4 incubatians at MnCl concentrations of respectively ; Mn all in the presence af 5 ti Mg(OAcf 0, 2, 5 and 10 mM respectively, time lanes 1 to 9 : incubations in the presence of 5 mM Mg(OAcj2 an ii IomM MnCl for 2, 5, 10, 15, 30, 45, 60, 90 and 120 min respectively ; ATP : incdations in the presence of 5 mM Mg(OAcj2 and 10 mM MnC12 at 0.01, 0.1 and 1 !&I ATP for 30, 60 and 90 min (lanes 1, 2 and 3 for each ATP concentration) ; Control : incubation at 5 mM Mg(OAc)2 and 10 mM MnCl2 ; CAMP lanes 1 to 5 : as the control but in the presence of 0.5, 1, 5. 10 and 50 DM CAMP (Sigma, Chemical Co.) ; 2,6 DAP lanes 1 to 5 : as control but in the presence of 0.5, 1, 2, 4 and 8 mM 2,6 DAP (2,6 diaminopurine, Nutritional Biochemical Corporation, Cleveland, USA). Each reaction was stopped by the addition of 100 1.11 of electrophoresis sample buffer and heating at 95'C for 5 min. Supernatants equivalent to 25 ~1 of plasma were analysed on polyacrylamide slab gels.

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03 Fig. 2 - Analysis An autoradiograph Methods) is shown. phosphoamino acid

of of

phosphoamino acids in the a part of the electrophoretic The positions of P51, origin markers were as indicated.

phasphorylated analysis and ninhydrin

72K protein. (Materials and stained

3 - ~72~ kinase activity in different normal human plasma. --Fig. Assay of p72K kinase was as described in Materials and Methods. An autcxaof stained, dried gels in the region of the 72~ protein is shown. diograph Samples 1 to 32 represent the p72K kinase activity in the plasma of 32 different normal individuals. Samples 33 to 37 and 38 to 40 represent the p72K kinase in the plasma of two-different individuals at 24 hr and 3 days interval, respectively. Each sample (l-40) represents kinase activity in 50 ul of plasma.

1374

Vol. 103, No. 4,198l

is the

only

BIOCHEMICAL

nucleotide

reaction.

triphosphate

The protein

2,6-diaminopurine the

kinase

(2,6

inhibitory

ation

cells

Figure

activity individual

the

activity

individual

33-37,

38-40).

3, samples

of p72K kinase

feron

produced

In a recent

review

conditions chial

systems

the

level

plasma

feron

(for

reflect protein.

of the

activity

is

viruses,

types

endotoxins, it

activity

is is

raised

worthidenti-

together, shown that

in the plasma

different with

to as "physiophysiological

of this,

same strain

enhanced

with

in health.

gut and the bron-

We have previously

of patients

in different

i.e.,

inducers

of mice of inter-

human &interferon activity levels of p67K

of mouse and human cells

see l-3).

remained

possible

observed

the residual For this

an exogenous

the

such as bacteria,

mice

of inter-

referred

In support

of time in the

level

different

in an enhanced level of p72K kinase phenomenon is in accord with enhanced

and p72K kinase

activity

is with

in an

period

differences the

under

of p72K

constant

conditions,

interferon

conditions.

treatment

that

of the ~67~ kinase

or inoculated

Similarly,

references

level

of different

interferon

(4).

It kinase

the

of the p67K kinase

seems to result (Fig. 4). This kinase

that

from one

a certain

reflect

associated

to agents

individuals.

varies

remained

may be produced tissue

physiological

with

this

phosphoryl-

was the level

for

natural

smokes and chemicals.

similar

treated

lymphoid

to note

in the

(7), it

in response

protein, here

under

since

by the

foreign while

by Bocci

interferon"

it

is possible

under

resi-

of p72K kinase

50 normal

Whatever

in the plasma

in an individual

acid

serine

this

activity

at least It

activity

by its but

more than

however,

conditions,

in the phosphoryl-

in the level

l-32).

normal

and

of serine.

of such kinase

(samples

with

activity

72K protein

involved mostly

that

we assayed level

in accord

kinase

of such phosphoamino

out variations

in an individual,

(Fig.

is

the

phosphorylated

than

under

level

logical

also

level

to the other

kinase

cal

were

to find

3 shows that

acids

was phosphorylated

in human plasma,

Figure

which

of

(6).

2 shows the results

residues

in the presence

on the protein

the amino

was at a much lower In an attempt

a result

COMMUNICATIONS

the phosphorylation

of the bond between

The 72K protein Threonine

ation

l),

in culture

we investigated

reaction.

for

RESEARCH

was inhibited

compound

the characterisation

analysis. dues.

activity

of this

from interferon-treated the phosphate

required

DAP ; Fig.

action

For

AND EIOPHYSICAL

(in reason

substrate,

that

the variations

in the

level

of ~72~

in an --in vitro assay system (Figs. 3 and 4), the plasma) phosphorylated state of the 72K therefore calf

thymus

we assayed histone,

1375

the phosphorylation as a test

for

of

an enhanced

BIOCHEMICAL

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B

Fig. 4 - Enhancement of the p72K kinase activity in the plasma of patients Patients (A and B) were treated with human fibrotreated with interferon. blast (8) interferon (8 x 10" units injected intravenously ; lo6 units/mg protein). The protein kinase activity was assayed before (lanes 1 in A An autoradiograph and B) or 24 hr (lanes 2 in A and B) after injection. of the stained, dried gel is shown. The numbers on the left give the molecular weights of protein markers in thousands : fi galactosidase, 130; phosphorylase B, 92; bovine plasma albumin, 69; ovalbumin, 46. Each sample represents protein kinase activity in 50 ~1 of plasma.

kinase

activity

(8).

of p72K kinase

The results

activity

phosphorylation

of exogenous

kinase

was as described

activity

of phosphorylation

of the

are due to different remain

may serve

as a marker

circulating cult

to test

for

with interferon we have recently enhanced

for

several

shown;

nated

against

rabies

under

these

conditions

in patients

life.

virus (9,

thus

and its

in

of interferon

the plasma,

of an organism

suggesting

lo).

1376

with

origin

activity,

Furthermore by methods

of interferon

treated

of the histone

activity.

(Fig. 4) or inducers of interferon. shown that the level of p72K kinase fold

assay

of

Variations in the level in human plasma, therefore

and action

the response

the level

the level

6).

of such kinase

half

in

with

in human plasma

of the p72K kinase

to test

not

kinase

the presence

low amounts

assay

differences well

observed

of protein

has a short

very

A simple

may be sufficient

(data

in reference

The detection

interferon

to date.

histone

of the p72K kinase

to be investigated.

that

correspond

72K protein

levels

The function

indicated

in the plasma

however, since

it is available

diffiup

therefore,

towards

treatment

In view of these, in the plasma is

poly(A).poly(U)

the production

or vacciof interferon

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ACKNOWLEDGMENTS Human B-interferon was kindly provided by Dr. A. Billiau, Rega Institute, Leuven, Belgium. This work was supported by grants from "Ligue Nationale Frangaise contre le Cancer" and "Fondation pour la Recherche Medicale". We thank Drs. B. Keil and T.T. Nguyen for advice in electrophoretic analysis of the phosphorylated amino acids. The authors acknowledge the excellent technical assistance of Mrs. N. Robert and S. Chamaret, A.G.H. thanks D.G.R.S.T. and "Fondation pour la Recherche Wdicale" for financial support. Members of Viral Oncology Unit at Institut Pasteur are also members of research team no. 147 of C.N.R.S.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Hovanessian, A.G. (1979) Differentiation 15, 139-151. Hovanessian, A.G., Meurs, E. and Montagnier, L. (1981) J. Interferon Res. 1, 179-190. Meurs, E., Hovanessian, A.G. and Montagnier, L. (1981) J. Interferon Res. 1, 219-231. Hovanessian, A.G., Riviere, Y., Robert, N., Svab, J., Chamaret, S., Guillon, J.C. and Montagnier, L. (1981) Ann. Virol. (Inst. Pasteur) 132 E, 175-188. Hovanessian, A.G., Brown, R.E. and Kerr, I.M. (1977) Nature 268, 537-540. 93, 515-526. Hovanessian, A.G. and Kerr, I.M. (1979) Eur. J. Biochem. Rev. 56, 49-85. Bocci, V. (1981) Biol. Roberts, W.K., Hovanessian, A.G., Brown, R-E., Clemens, M.J. and Kerr, I.M. (1977) Nature 264, 477-480. Hovanessian, A.G., Riviere, Y., Montagnier, L., Michelson, M., Lacour, J. and Lacour, F. Manuscript in preparation. Rollin, P. , Hovanessian, A.G., RiviSre, Y., Montagnier, L. and Sureau, P. Manuscript in preparation.

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