Characterization of immune type interferon (IFNγ)-induced cytoplasmic protein kinase activity in mouse L cells

Vol. 104, No. 2, 1982 January 29, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 422-429

CHARACTERIZATION OF IMMUNE TYPE INTERFERON (IFNy)-INDUCED CYTOPLASMIC PROTEIN KINASE ACTIVITY IN MOUSE L CELLS Kenzo

Ohtsuki,

Department

Received

November

Barbara

A. Torres,

of Microbiology, Galveston,

and Howard

M. Johnson

University of Texas Texas 77550 U.S.A.

Medical

Branch

30, 1981

SUMMARY: Mouse immune interferon (IFNV) induced double-stranded RNA-independent protein kinase activity in the cytoplasmic fraction of mouse L cells as measured against a histone substrate. Chromatographic purification separated the activity into three distinct kinases of imolecular weights of approximately 1OOK (kinase I), 70K (kinase II), and 70K (kinase III). Partially purified IFNY was as effective as crude in inducing protein kinase activity. Induction was blocked by treatment of IFNY with specific anti-IFNr antibody or by treatment of the L cells with actinomycin D. Kinase II activity was different $$orn that of kinases I and III in that it showed Ca-dependence in the absence of My , was inhibited in activity by the SH binding agent N-ethylmaleimide, and could use the cellular enzymes RNase A The described protein kinases could play an and hexokinase as substrates. important role in mediation of IFNY effects, particularly where phosphorylated enzyme substrates were shown to have altered activity.

It

has been demonstrated

interferon protein

(IFN)

results

in increased

phosphorylation

have partially

of cellular

purified

protein

kinase,

resulting

in the

inhibition

Recently,

we reported

for

IF/$

biological

from

(9).

study

properties

of mouse L cells. cytoplasmic

protein

kinase

total

and ribosomal

phosphorylates

also

kinases

induces

that

(37K)

synthesis protein

dsRNA-

of eIF-2, (4,7,8). kinase

of If?&-induced

virus-induced

ribosomal

IFNs as was also

shown

and characterize

the

(10-12).

kinase

against

422

determine

activity

IFNy induces

0006-291X/82/020422-08$01.00/0 Cop.yright 0 1982 by Academic Press, Inc. AN rights of reproduction m any form reserved.

We and others IFN-induced

subunit

protein

of induction

to further

as measured

small

with

and increased

(l-7).

dsRNA-dependent

for

A synthetase

of IFNy-induced We found

the

of cells

activity

proteins

of viral

observed

was undertaken

treatment

a ribosomal-associated

The kinetics

that

of 2',5'-oligo

The present

that

of initiation

that

was different

induction

distinct

which

in mouse L cells

kinase

laboratories

and characterized

dependent

activity

by several

in the

an increase a histone

cytoplasmic

in at least substrate.

fraction three A possible

Vol. 104, No. 2. 1982 biological

role

BIOCHEMICAL

of the

IFNY-induced

AND BIOPHYSICAL kinases

in the

RESEARCH COMMUNICATIONS

IFN-induced

biochelnical

events

is discussed.

MATERIALS

AND METHODS

Chemicals - [Y-32P]ATP (19.0 Ci/mol) was obtained from Amersharn Corp. Casein, phosvitin, pancreatic ribonuclease A (RNase A), dithiothreitol (DTT), calf thymus histone (type II-a) and CAMP were obtained from Sigma Chemicals Corp. DEAEConcanavalin A-Sepharose and Sephacryl cellulose (DE-52) was obtained from Whatman. S-200 were obtained from Pharmacia. - IFNywas produced in C57Blj6 mouse spleen cell Mouse IFNy preparation cultures using staphylococcal enterotoxin A as an inducer as previously described (13). The produced IFNywas partially purified by adsorption to controlled Poreglass beads and elution with 20% ethylene glycol and 1 M NaCl in phosphate buffered Following dialysis against PBS, the IFNYfraction was applied on saline (PBS) (14). a concanavalin A-Sepharose column and eluted with 0.25 M a-methyl-Dgmannoside in PBS. The final product had a specific activity of approximately 10 units/mg protein. Treatment of thq cells with IFNs - Confluent monolayers of mouse L cells (about lo8 cells) in 150 cmL flasks were treated with 300 reference units/ml of IFN for 12 hrs at 37'C. The cells were harvested by scraping with a rubber policeman, centrifuged, washed twice with PBS, and cytoplasmic fractions were prepared as described below for testing protein kinase activity. Cytoplasmic fraction Icrude kinase fraction1 1 The cytoplasmic fractions fgom untreated and IFN -treated cells were prepared as follows. The cells (about 10 cells) were gently homogenated with a glass homogenizer in 3 ml of 10 IBM Tris-HCl 2 mM dithiothreitol (DTT) and 10 mM KCl, and then (pH 7.5) containing 2 mM CaCl The supernatant was recentrifuged at high centrifuged for 20 min at 12,8;0 rpm. speed (105,000 Xg for 45 min) to remove ribosomes from the fraction. The obtained supernatant was concentrated to 1.0 ml using polyethylene glycol 20000 powder. The concentrated solutions were dialyzed overnight at 4°C against 10 mM Tris-HCl (pH 7.5) containing 0.5 M KCl, 2 mM DTT and 10%glycerol (Suffer A). The dialyzed fractions were used as crude kinase preparations (cytoplasmic fractions) for the purification and kinase assay in this study. Assay of protein kinase activity - The complete reaction mixture (0.1 ml) contained 39 mM Tris-HCl (pH 7.5), 4 mM DTT, 10 ug of bovine serum albumin (BSA), 100 uE1 [yP]ATP (2,000 cpm/pmol), the indicated ayyunts o$+substrate (histone or or Ca and the indicated other proteins), the indicated amounts of either Mg amounts of protein kinase purified or unpurified from the cytoplasmic fraction of After incubation for the periods indicated at 37Y, the enzyme mouse L cells. reaction was terminated by the addition of 0.5 ml of 0.2 M sodium pyrophosphate containing BSA (1 mg/ml) and 0.5 ml of 20% trichloroacetic acid. The precipitate WJ~ collected by filtration through a Whatman glass filter (type GF/F) and the [ Plradioactivity of the filter was determined by a liquid scintillation spectrometer after drying. Protein measurement - Protein concentration of the enzyme preparations was determined by the method of Lowry et al. (15). RESULTS IFNY-induced

cytoplasmic

When mouse L cells reference (histone

units/ml)

for

phosphorylation

as compared

with

that

protein

kinase

(about

lo8

12 hrs

at 37'C,

with

cells)

activity were the

cells

(Table

423

exposed

activity

CY-~~ P]ATF)frorn

of untreated

in mouse L cells to crude of cytoplasmic

such cells 1).

mouse IFNv (300

This

protein

was significantly increased

protein

kinase increased kinase

BIOCHEMICAL

Vol. 104, No. 2. 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 1. Characterization of interferons as stimulators cytoplasmic protein kinase activity in mouse L cells.

of

ProteQ kinase activity (pm01 cv- P]ATP/pg histone)

Treatments None Mouse IFNy Mouse IFNy + actinomycin Mouse IFNo/@ Human IFNa Mouse IFNY + anti-mouse

18.5 63.8 22.8 40.2 19.1 23.0

D IFNv antiserum

Crude kinase fractions (cytoplasmic fractions) were prepared from untreated mouse L cells or cells treated with 300 units/ml of mouse IFNy (crude), or5cells treated with 300 units/ml of human IFNa (specific activity 10 units/mg) for 6 hrs at 37*C. Actinomycin D (1 vg/ml) was Protein kinase added to the cell cultures at the same time as IFNy. activity of the fractigns was assayed using either histone (type 34-A) in the presence of Mg (3 mM) or phosvitin in the presence of Ca (3 mM) as descri!>ed in Materials and3Methods. Anti-mouse IFWy antiserum was preincubated with mouse IFNY (10 units/ml) for 30 min at 37OC before exposure to the cells, at a concentration that completely neutralized the antiviral activity.

activity

was also

However,

no increase

human leukocyte

pg/ml),

in the

interleukins strongly kinase

inhibits

antiserum

activity

suggest

Tris-HCl

(pH 7.5)

by the

antiserum

fractions S-200

and P-II) The kinase

molecular were activity

of

is

antiviral

state

with

factor

(unpublished the

treated

by IFNs.

the

in D (1

Also,

kinase lymphokines,

data).

increase

with

resistance

protein

against

for

was used.

actinomycin

induced

IFNv to induce

responsible

These

of total

data

protein

of mouse L cells,

kinases

from

untreated

(1.5

0.5

appreciable

not reactive

fraction

column

containing

by their

addition

cytoplasmic

in cells

mouse IFNv together

stimulating IFNv is

units/my)

was observed

of antiviral

of IFNy-induced

a Sephacryl

acceptor.

mouse

in the

through

(P-I

with

induce

the ability

This

that

The cytoplasmic

activities

not

the development

cells.

IFNv (lo5

activity

does

treated

neutralized

purification

kinases

which

cells

purified

kinase

1 and 2 or colony

activity

Partial

of protein

or from

which

anti-IFNv

when partially

IFN (IFNu)

L ce?ls,

mouse

obtained

X 50 cm) previously

M KCl,

size

and IFNv-treated

of the

of 10 PM CAMP, whereas

histone

first

peak

the activity 424

(or (P-I)

casein)

with

10 mM kinase

1, two distinct

as a phosphate

was significantly

of the

passed

to separate

As shown in Fig.

using

were

equilibrated

2 mM DTT and 10% glycerol

differences.

detected

cells

second

peak

stimulated (P-II)

was

Vol. 104, No. 2. 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

3s 5 8 22

0

1

0

0 1

30

40

50

Fraction

Fig.

slightly and

1.

Separation filtration. untreated units/ml) Aliquots described (Xl.

inhibited P-II)

by

from

cells

to

IFN.

37°C)

of

the

treatment

protein kinase activities by Sephacryl S-ZOO gel The cytoplasmic fractions were prepared from cells (0) and cells treated with mouse IFNY (300 for 2 hrs (A) and 6 hrs (O), respectively, at 37'C. (5 ~1) were assayed against a histone substrate as in the Materials and Methods. Absorbance at 280 nm

CAMP.

Total cells

The

activity

of

the

specific

order

to

peak cells

number

of

IFNY-treated

second

60

(P-II with

protein

kinase

activities

increased

with

increased

(histone

kinase) IFNv

time

both

increased

units/ml)

fractions

of

phosphorylation/pg

was

(300

of

exposure

of

protein/min

approximately

as

(P-I

compared

at

3-fold with

that

the

6 hr of

after

untreated

cells. In IFNu-treated column. buffer tne

The

other

kinase

2).

The

between The

active

respectively, molecular

the

was

0.2

were

eluted

as

active P-II

was

between

fractions

0.05

0.3 in

M KC1 the

immediately kinases

two

on

between

0.15

M and

and

designated

separated

into

two

kinases:

(Fig.

0.1 3).

Only

and

as 425

the

kinase

column by

III

M KC1

0.2 as the

other II

from

M KC1

the

I.

first

On

peak III)

was RNase

were 20000.

a Sephacryl

in

kinase

phosphorylated

jlycol on

P-II)

(kinase

chromatography

polyethylene deteri,rined

and

a DEAE-cellulose

pooled

M and

and

(P-I

were

DEAE-cellulose

II

kinases applied

peak

concentrated I,

distinct

separately

a single

fractions kinase

M and

of

the

fractions

eluted

and weight

characterize

the

P-I

hand, II)

eluted A.

cells,

(Fig.

(kinase

further

pooled, The

S-200

column

Vol. 104, No. 2. 1982

BIOCHEMICAL

AND BIOPHYSICAL

r

Kinase

RESEARCH COMMUNICATIONS

I

0.6

Fraction

Fig.

were

DEAE-cellulose column chromatography of P-I fraction from the cytoplasmic fraction of IFNy-treated cells. The P-I kinase fraction was applied to a column (1.5 X 8 cm) of DEAEcellulose previously equilibrated with 10 mM Tris-HCl (pH 7.5) containing 2 mM OTT and 10% glycerol. The column was washed with 10 ml of the same buffer and fractions (0.5 ml) were collected. Elutions were carried out with a linear gradient (0 M to 0.5 M KCl) in the buffer. Aliquots (5 ~1) were assayed as described in the Materials and Methods. Histone phosphorylating activity (0) and absorbance at 280 nm (X).

2.

lOOK,

approximately

marker

proteins.

318-fold

with

Requirements

activity for

histone

70K and 70K,

Kinases

I,

II

yields

respectively, III

and

kinase

conditions or casein

for were

were

as measured

purified

about

by comparison

250-fold,

350-fold

activity

of the

suitable

kinases

phosphate

Kinase

are

summarized

acceptors.

in Table

The optima1

II

I

0 50

30 Fraction

3.

and

activities

I

Fig.

with

of 45%, 25% and 38%, respectively.

IW-induced

The optima1 Either

number

70

number

DEAE-cellulose column chromatography of P-II fraction from the cytoplasmic fraction of IFNY-treated cells. The OEAEcellulose column chromatography of P-II kinase was performed as described in Fig. $+ Histone phosphorylating activity was in the presence of Mg (5 mM) (0).2+RNase phosphorylating activity was in the presence of Ca (3 mM) (0). Absorbance at 280 nm (X).

426

2.

Vol. 104, No. 2. 1982

BIOCHEMICAL

Table

Reaction

mixtures

Complete Complete Complete Complete Complete Complete Complete

+ +

2.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Requirements for activity of from IFNY-treated cells

(pm01 Kinase

Protejs &I

kinases

kinase activity P]ATP/Pq histone) Kinase II

36.6 0.2 0.3 0.1 32.9 58.5 32.6

Mg $1 Mg + Ca2+ Histone DTT + NEM CAMP dsRNA

the

Kinase

4.9 0.1 6.4 0.1 0.1 2.8 5.0

III

15.4 0.1 0.1 1Z 14:3 14.6

The complete reaction mixture (0.1 ml) contained 20 mM Tris-HCl (pH 7.5), 3 mM MgCl or 3 rnlJ2CaC1 4 mM DTT, 10 Pg of BSA, 20 Pg of histone (type II-A), 106 PM [YPlATsland either kinase I, kinase II or kinase III at 0.5 ug each. The reaction mixture was incubated for 10 min at 37OC. The concentration of CAMP, N-ethylmaleimide (NEM) and dsRNA (poly rI:poly rC) was 3 PM, 1 mM and 100 rig/ml, respectively.

concentration absolute

of requirement

contrast

to

this,

ethylmaleimide

dsRNA

(poly

rig/ml

to

II

(3 uM) an

for

was

was

on

on

by

the

2+

I

other

three

I and

activated

absence

activity stronyly

of

iiig

2+

activities

.

an In

protein

2.6-fold.

Nthe

stirlruldt.inJ at

Ca

had

2+

for

inhibited No

III

by

about

kinases.

kinase

Kinases

not

in

SH enzymes,

the

oM.

were

Ca

kinase

of

3-5

and

activated

inhibitor

rC)

was

activity,

enhanced

no effect

poly

1 mg/rnl)

The

substrate

3.

Kinases

casein

phosphate

is

CAMP

kinases

activity

effect

any

concentration

kinases

are

of (10

observed.

requirements

Table

and

II

had

r1:

Substrate

and

but

Mg

three

2+

kinase

(NEM),

kinase

the

for

phosphorylation.

of

for

My'+

in

requirements

in

a Ca-dependent

substrate

were

presence

of

the

the

presence

protein

requirements

of

partially

similar

Mg 2+

In contrast

acceptors.

hexokinase

III

I and

the

of

in

(5 to

kinase

(in

kinase

II

phosphorylation

r&l) , while this,

Ca 2+

of

purified

the are

hexokinase

II

kinase (3

of

rtil).

These

absence

of

different

histone,

and

from

show The

those

A were both

results

of

in

lphosvitin

RNase

phosphorylated

MJ*+).

summdrized

RNase

that

data kinases

poor

kinase show

ttldt

I and

A II the

III.

DISCUSSION We have dependent

previously

protein

kinase

shown activity

that

mouse in

IFNy

mouse

can

L cells 427

induce that

ribosome-associated phosphorylates

dsRNAthe

a subunit

Vol. 104. No. 2. 1982 Table

BIOCHEMICAL 3.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Substrate requirement of the three distinct purified from the cytoplasmic fraction of IFNv-treated cells

kinases

Protein kinase activity (pnol [Y-~~P]ATP/@ histone) Kinase I Kinase II M9*+ Ca*+ M9*+ Ca*+

Substrate Histone Casein Mouse Igti RNase A

36.5 1.4 2.9 1.7

N.D. N.D. N.D. N.D.

4.4 2.8 3.1 3.7

Phosvitin Hexokinase BSA

:':' 0:l

N'D' ND N:O:

5'2 19 N:D.

Kinase M92+

6.3 2.2 4.8 5.7 2.8 9.1 N.D.

III Ca*+ N.D. N.D. N.D. N.D. ND N'D' N:D:

14.5 8.1 1.8 1.5 1:':' N:D.

The protein kinase activity of kinase I, kinase II and kinase III (all at 0.5 ug) was incubated, respectively, for 10 min at 37Y wish variojf substrates (20 pg each) in the presence of either Mg or Ca (3 mM). The activity of kinase I was assayed in the pr!:efz!z of 3 UM CAMP. N.D. = not detected.

of eukaryotic also

peptide

induce

cytoplasmic

measured

against

Sephacryl

S-200

separated

the

a histone gel

for

observation

that:

the

kinase

(b)

specific

(c)

actinomycin

activity anti-IFNv

kinases

kinases presence

(7-11).

They are,

CAMP-dependent required

Mg2+ for

of Ca2+ (absence

II),

three

(d)

however, activity,

of

and mouse IFNa/6, species

in that

they

are

functionally

activation,

but

of Mg2+).

Only

distinct

kinases

II

only

kinase

kinase 428

II

III).

of IFNYis

as evidenced

by the

protein)

with

crude

IFNvto but

induced

mouse IFNy,

induce

not

activity,

human IFNa,

in

induction.

dsRNA-independent,

known ribosome-associated,

while

weights

specificity all

as

using

units/mg

observed

IFNy can

chromatography,

kinases

the ability

demonstrating

the well

exchange

mouse IFNy (lo5

neutralized

similar

purification,

and 40K (kinase

as that

that

in L cells

of molecular

cytoplasmic

same level

induction,

ion

shown here

activity

Kinase

kinases

purified the

thus

them from

kinase

substrate.

distinct

of the

antiserum

are

We have

protein

70K (kinase

partially

D blocked

The three differentiates

I),

to about

activity,

exhibited

three

induction (a)

(9).

and DEAE-cellulose

into

the

eIF-2

casein)

filtration

kinase

kinase

(or

1OOK (kinase

responsible

factor

dsRNA-independent

activity

approximately

induced

initiation

which

dsRNA-dependent enzymes.

and III

did

Kinase not.

II was activated was sensitive

All

protein I three

in the

to NEM, an

BIOCHEMICAL

Vol. 104, No. 2, 1982 inhibitor

of SH enzymes.

kinase

II

while

the

from

kinases

others

phosphorylation

were

activities

provides

pleiotropic

effect determine

activities,

I and III,

requirement since

inactive.

it

that

possible

in regulation possible

and the

possible

data enhances

IFN y can induce

additional

also

against

suggest

that

function. substrates

of enzyme function

II

prote in kinase IFbcan

Studies for

kinase

activity.

cytoplasmic by which

differentiated

RNase and hexokinase

enzymatic

mechanisms

enzyme

alteration

RESEARCH COMMUNICATIONS

activity

several

of cellular

cellular

for

was active

Preliminary

of RNase A sign ificantly

The demonstration

further

Substrate

AND BIOPHYSICAL

are

exert in

the cytoplasmic as a result

a

progress

to

kinase of the

phosphorylation. ACKNOWLEDGMENTS ---This

study

was supported

by NIH Grant

AI 16248.

REFERENCES 1. 32: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Zilberstein, A., Federman, R., Shulman, L., and Revel, M. (1976) FEDS Letters 67, 119-124. Lebleu, F., and Lengyel, P. (1976) Proc. Natl. Acad. Sci. USA 73, 3107-3111. Samuel, C.E., Farris, D.A., and Eppstein, D.A. (1977) Virology 83, 56-71. Samuel, C.E. (1979) Proc. Natl. Acad. Sci. USA 76, 600-604. Gupta, S.L. (1979) J. Virol. 29, 301-310. Gupta, S.L., Rubin, B.Y., and Holmes, S.L. (1979) Proc. Natl. Acad. Sci. USA 76, 4817-4821. Ohtsuki, K., and Baron, S. (1979) J. Biochem. 85, 1495-1502. Ohtsuki, K., Nakamura, M., Koike, T., Ishida, N., and Baron, S. (1980) Nature 287, 65-67. Robbins, C.H., Kramer, G., Saneto, R., Hardesty, B., and Johnson, H.M. (1981) Biochem. Biophys. Res. Commun. (in press). Hovanessian, A.G., Riviere, Y., Robert, N., Svab, J., Chamaret, S., Gullion, H.C., and Montagnier, L. (1981) Ann. Virol. (Inst. Pasteur) 132E, 157-188. Baglioni, C., and Maroney, P.A. (1980) J. Biol. Chem. 255, 8390-8393. Breeze, R.J., Dougherty, J.P., Pichon, J., Jayaram, B.M., and Lengyel, P. (1981) J. Interferon Res. 1, 191-201. and Johnson, H.M. (1979) Infect. Immun. 23, 80 Osborne, L.C., Georgiades, J.A., 86. Georgiades, J.A., Langford, M.P., Stanton, G.J., and Johnson, H.M. (1979) IRCS Med. Sci. 7, 559. Lowry, O.H., Rosebrough, M.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.

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