Direct phosphorylation of the IL-2 receptor Tac antigen epitope by protein kinase C

Vol. 135, No. 1, 1986

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages 239-247

February 26, 1986

DIRECT PHOSPHORYLATION OF THE IL-2 RECEPTOR TAC ANTIGEN EPITOPE BY PROTEIN KINASE C Masaaki Taguchi,l Wayne B. Anderson,2

1Laboratory of Molecular Immunoregulation, Response ModifiersProgram, Division of Cancer Treatment, Cancer Institute, NIH,Frederick Cancer Research Facility, Building 567, Room 211, Frederick,Maryland 21701 of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205

Biological National 2Laboratory

Received

Thomas P. Thomas,2 and William L. Farrarl,*

October

28,

1985

Summary: Phorbol esters induce a rapid phosphorylation of the antigenic epitope of the human IL-2 receptor identified by anti-Tat monoclonal antibody. The physiological activator of protein kinase C, diacylglycerol also stimulated the phosphorylation of the Tat epitope in intact activated human T lymphocytes. Stable derivatives of cyclic nucleotides had no effect on the stiImmunoprecipitated mulation of Tat phosphorylation with cultured lymphocytes. Tat derived from particulate membranes could serve as a direct substrate for purified protein kinase C --in vitro. The Ca2'/phospholipid dependency of the phosphorylation reaction substantiated that the phosphorylation of --in vitro Tat observed in intact cells stimulated by phorbol ester or diacylglycerol was the result of the physiological activation of protein kinase C. Q 1986 Academic

Press,

Inc.

Interleukin creted

by T lymphocytes

(l-3).

IL-2

lymphocytes

of IL-2 affinity

promotes as well

associated

IL-2

2 (IL-2)

with is

the growth as of large

believed

killer

to involve acquired

antibody

*To whom correspondence

by antigen,

granular cell

activity

the

interaction

should

anti-Tat

synthesized lectin,

and se-

or phorbol

esters

of antigen-sensitized

lymphocytes (4,

which

5).

have

with

activation. characterized

(6-8).

Although

been closely

The biological

of the peptide

or lectin

and biochemically

designated

peptide

and differentiation

by antigen

has been antigenically

monoclonal

a lymphocytotropic

upon stimulation

natural

receptor

is

activity a high-

The receptor with

for

a defined

the ligand-binding

be addressed.

Abbreviations: IL-2, interleukin-2; 2-acetylglycerol; DG, diacylglycerol; transferrin receptor; TAC, anti-IL-2 monoclonal antibody.

PK-C, protein kinase C; OAG, l-oleoylPHA, phytohemagglutinin; TRF, antireceptor antibody; RPC, control IgG

0006-291X/86 239

All

Copyright D 1986 rights of reproduction

$1.50

by Academic Press, Inc. in any form reserved.

Vol. 135, No. 1, 1986 characteristics defined 11)

of IL-2

(6,

little

BIOCHEMICAL

transduction.

studies

report

have

shown

of various

(15-16).

that

cells

infected have

also

(18).

demonstrate

that

vator,

diacylglycerol,

epitope

in intact

kinase

The study phorbol

lymphocyte

Materials

esters

normal

and the

kinase

C (12).

role

in signal

as phorbol

been shown

to stimulate

human IL-2

receptor,

state

data

protein

kinase

of the Tat

Tat

of

on surface

biochemical

Furthermore,

of

HTLV-infected

expressed

the phosphorylation

esters

effects

physiological

immunoprecipitated

the rapid

a constituative

provides

human T lymphocytes. --in vitro

protein

In contrast,

epitope

here

to stimulate

(10, signal

biological

of the

(17).

antigen

presented

stimulate

C phosphorylated

bilized

have recently

been shown to exhibit

of the Tat

of IL-2-receptor

as well

some of the

lymphocytes

accomplished

C has a crucial (13)

epitope

have been well

recently

system,

kinase

to mediate esters

Tat

was shown

hormones

of the antigenic

"autophosphorylation" membranes

protein

biological

Phorbol

on retrovirus

HUT102B2

IL-2

RESEARCH COMMUNICATIONS

receptor

mechanism

of a phosphotransferase

the phosphorylation Tat,

the biochemical

and has been suggested

oncogenes

Tat

of the cDNA for

In a previous

transduction (14)

cloning

about

membrane-association Recent

and the high-affinity

9) and the is known

AND BIOPHYSICAL

C acti-

antigen

purified derived

which

protein

from solu-

membranes.

and Methods

Preparation of Cultured Lymphocytes and Immunoprecipitation Gel Electrophoresis. Human peripheral blood T cells were separated using nylon wool columns and discontinuous Percoll density gradient sedimentation as described T cells were cultured for 2 days in RPMI-1640 supplemented with 5% FCS (19). and 2 ug/ml PHA (Wellcome) and were cultured for an additional 3 days in RPMI-1640 containing 5% FCS and purified IL-2 (100 units/ml, Cellular ProTen million cells were washed twice in a balanced salt solution, reducts). suspended in 2 ml of methionine-free (RPMI-1640 Selectamine medium) (Gibco) and preincubated at 37°C for 30 min. 0.5 mCi of NEN translation grade [35S]methionine was added and the culture was incubated at 37°C for 4 hr. Cells were washed once with cold RPMI-1640 and lysed with 10 mM Tris-HCl-NaCl pH After centrifugation at 100,000 g 7.5 buffer containing 1% Triton X-100. for 1 hr at 4'C, the supernatant was subjected to immunoprecipitation as described previously (8, 18). After extensive washing of the immunoabsorbents, bound antigens were eluted by boiling for 5 min in 2% SDS-sample buffer and resolved on SDS-polyacrylamide gel (7.5%). After staining with Coomasie brilliant blue R250, the gel was treated with ENHANCE (NEN) and dried. Fluorography was performed at -80°C. were cells

32P-0rthophosphate Labeling of Intact T Lymphocytes. Activated T cells prepared as described in figure 1. Five million million activated T were washed twice with Hepes-buffered saline and suspended in 1 ml of

240

Vol.

135,

No.

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

phosphate-free RPMI-1640 and preincubated at 37'C for 1 hr. Cells were labeled with 0.5 mCi of [32P]orthophosphate (Amersham) for 2 hr and then incubated with (+) or without (-> 100 rig/ml PMA for 10 min. The cells labeled with [32P]orthophosphate were also treated with 50 pg/ml OAG, 1 mM 8-bromo cyclic GMP, or 1 mM 7-chloro-CAMP Cells were washed once with for 10 min. cold RPMI-1640 and lysed with the buffer containing 1% Triton X-100 and 20 mM NaF. The same procedure for immunoprecipitation was done except use of the washing buffer containing 20 mM NaF (R = anti-RPC, T = antiTac). Bound antigen was eluted in 2% SDS-sample buffer by boiling for 5 min and resolved on Phosphoproteins were detected by SDS-polyacrylamide gradient gels (7.5-15%). autoradiography using intensifying screens (DuPont, Lightening Plus) and Kodak Exposure was done at -80°C. X-Gmat AR films. In Vitro Phosphorylation of Tat by Protein Kinase C. About 1 billion activated T cells were prepared as mentioned (fig. 1). Membranes were obtained by modification of a previously described procedure (12). Briefly, the cells were washed twice with cold phosphate-buffered saline and resuspended in the buffer; 20 mM Tris-HCl, pH 7.5, 5 mM EGTA, 2 mM EDTA, 0.33 M sucrose, Cells were disrupted in a Wheaton and 1 mM phenylmethylsulfonyl fluoride. Homogenates were layered on 41% sucrose in the same bufDounce homogenizer. fer and centrifuged at 100,000 g for 1 hr at 4°C. The membrane bands were removed from the interphase and then diluted lo-fold and collected by centrifugation at 100,000 g for 1 hr at 4°C. Membranes were solubilized with 1% Triton X-100. Immunoprecipitates were prepared as described. The phosphorylation reaction was carried out at 30°C for 15 min in an Eppendorf tube. The reaction mixture (total volume of 250 pl) contained 14 pg of purified rat brain protein kinase C (20), 20 mM Tris-HCl pH 7.5, 10 mM MgC12, 30 pl of immunoprecipitate, 20 mM NaF, 50 pM [Y-~~P] ATP (Amersham) with or without 1 mM CaC12, 5.0 pg of phosphatidylserine, and 1.0 pg of 1,2-dioleoylglycerol. The reaction was terminated by the addition of 50 pl of 100 mM ATP and 100 mM EDTA and the mixture was immediately centrifuged with a Beckman Microfuge for 15 sec. Results IL-2

receptors

identified

analysis,

reach

following

stimulation

T cells

were

to analysis

maximal

labelled

lysates

ceptor),

Tat

molecular

with

[35S]methionine

which (7).

1 shows

in HTLV-transformed

receptor), extracted of 60,000

The 40 Kd species

can be further

The 60 Kd Tat+ T-cells

of activated (PHA)

monoclonal

from daltons

(7)

but

normal

antibodies

this

T cells

had ap-

when separated

to be a precursor on the plasma

slightly

larger

than

molecular

weight

is

241

(IL-L-re-

immunoglobulin

daltons

and expressed

were

to Tat

activated

has been reported

was subjected

of labelled

control

and 40,000

Activated

electrophoresis

profile

or a nonspecific

glycosylated molecules

acrylamide

at d 5-7

(9).

lysate

the fluorographic

binding

T cells

and IL-2

and cellular

and SDS-poly

with

antigens

weights

by 7.5% SDS-PAGE.

brane

phytohemagglutinin

Figure

or by [3H]-leu-lys-IL-2

surface

with

TFR (transferrin

molecule

on the

immunoprecipitated

(RPC, IgGZa). parent

levels

by immunoprecipitation

(PAGE)-fluorography. cell

by anti-Tat

those consistent

mempresent with

Vol. 135, No. 1, 1986

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

927867TACw

~

30TAC TFR RPC of 35S-methionlne-labeled Tat. Activated T Fig. 1. Immunopreclpitation lymphocytes were labeled as described in Materials and Methods. Immunopreclpitates were applied to a 7.5% SDS-PAGE. Fluorography was performed at -80°C for 3 days.

that

described

on activated receptor

for

the Tat

T lymphocytes (TFR)

antigen

associated

(6-8).

The immunoprecipitation

at the apparent

molecular

with

weight

the IL-Z-receptor

found

of the

of 92 Kd agreed

transferrin

with

published

values. Phorbol

esters

have been shown to stimulate

of membrane-associated ma1 growth ascertain

factor whether

receptors (EGF),

activated

phosphate-free

RPMI-1640

myristate

terminated Tat

significantly

that

phosphate

in

in the

were

an additional

containing

lane

242

epider-

In order

to

similarly

were

stimulated

in with

The reaction

was

and immunoprecipitation Figure

1) of PMA, IL-2

or absence

for

[32P]orthophosphate

autoradiography.

The transferrin presence

were

10 min.

RPMI-1640,

by SDS-PAGE and ([+]

with

The cells

2 hr at 37°C.

22).

receptors

prelabeled

of a number

receptors

C (21,

or transferrin

(PMA) for

the presence

phosphorylated.

phosphorylated

for

high-affinity

and somatomedin

T cells

acetate

and TRF was analyzed

strates

not

with

insulin,

IL-2-receptors

phosphorylated,

phorbol

including

phosphorylation

receptor (2)

receptors (TRF)

of PMA.

of 2a demon(Tat)

complex

Limited

were was

phos-

BIOCHEMICAL

Vol. 135, No. 1, 1988

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

200120927887-

%--

78= 87-

-TAC 45-

4530301813-

1813-

+-+-+-

RTRTRT

_1-TAC

TFR

RPC

+=P

+cGMP

+yo’;;;E

Fig. 2. Immunprecipitation of phosphorylated Tat. S*P-orthophosphate labeled activated lymphocytes were stimulated with PMA (+) or control diluent (-) and cellular lysates precipitated with anti-Tat, anti-TRP, or control antibody anti-RPC. Panel B; labeled cells were stimulated with cyclic nucleotide derivatives or synthetic diacylglycerol, OAG. Immunoprecipitates were prepared with anti-Tat (T) control anti-RPC (R).

phorylation tion to

in

the

vate

protein

by

protein

of

have

been C,

intact

phorylation alternatively

control

kinase

with

or

non-Tat-associated

binding

esters

Tat

a few

unrelated

nonspecific

phorbol

of

of

of

Tat.

peptides

the

to to

Whether

of

C cannot

be

is

PK-C

which

a protein

A sepharose

be

from

intact

cell

attributed

with

Since and

in

the

protein

activity

phoskinase

is

phorbol

regulated

in

part

also by

have

intracellular

considerable

protein

levels

of

243

cyclic

kinase

ester

AMP

A and and

cyclic

C

regulated treat-

merit. T lymphocytes

acti-

phosphorylation

of

whose

stimula-

matrix.

involved

substrate kinase

PMA

was

PMA-stimulated may

a direct

inferred

after

physiochemically of

that

Tat

protein

associate

suggests

seen

(RPC)

the

demonstration

a substrate kinase

was

immunoprecipitate

shown

cells

peptides

G activities GMP,

Vol. 135, No. 1, 1986

respectively

(23).

stimulated

with

glycerol

with

performed. which

is tion

exogenously

Cyclic

In order

protocol

as described

for

direct

protein

fig.

(20)

and [u-32P]-ATP

Fig.

3 shows

that

the

lysates

cells

were

were

immunopreci-

diacylglycerol

substitutes

induced

concerning

Accordingly,

of Tat

the

in the presence

or absence

immunoprecipitate

with

issue

2 R +

‘3 T + +

(Fig.

of whether

an in.vitro

protein

of phospholipids

phospholipid; Lane PK-3, phospholipid

3, PK-C plus plus control

kinase and Ca2+.

phosphorylated

4 R + PSlDi +CK

244

Tat ppt. RPC ppt.

and

phospholipid

Tat

prepared

ImmunoprecipiFig. 3. of Tat by purified PK-C. --In vitro phosphorylation tates of Tat were prepared from isolated particulate membranes and admixed with purified PK-C plus Tat without phospholipid; Lane 2, PK-C with anti-RPC without Lane 4,

2b,

reconstituwere

purified

was directly

DG

on Tat phosphorylation.

immunoprecipitates

in vitro

(DG) OAG,

endogenous

phosphorylation

C enzyme activity,

1 T +

for

had no effect

evidence

kinase

synthetic

1 and incubated

the Tat

[32P]orthophosphate GMP, or 1-oleoyl-2-acetyl-

Cell

derivatives

was designed. in

only

C (24),

nucleotide

with

(T or R) and SDS-PAGE autoradiography

to intact

kinase

to obtain

a substrate

diacylglycerol.

or anti-RX

protein

labeled

AMP, 8Br-cyclic

2b shows that

when added

6).

7-Cl-cyclic

anti-Tat

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T cells

a synthetic

Figure

and activates lane

Activated

either

(OAG),

pitated

BIOCHEMICAL

cofactors;

in

C

BIOCHEMICAL

Vol. 135, No. 1, 1986 vitro

in the

presence

phatidylserine

and diolien

phosphorylation the

phorylation

of the

range

antigen

electrophoresis

the major

phosphorylated

was observed

phosphorylation direct

protein

C for

(lanes

phospholipid

(data

not

were

kinase

(intact

shown).

cell)

C in the

not

Taken

occur

immunoconjugate

together,

and in vitro

layer

revealed

No phosphoty-

stimulation

of Tat

(OAG) as well

by purified

evidence

of IL-2

in the

and thin

and serine.

Tat

Phos-

in the molecular

diacylglycerol

phosphorylation

(23).

hydrolysis

Tat

threonine

of immunoprecipitated

situ

substantiated

C and phospholipids Acid

phos-

of detectable

co-factors

(RPC) did

2 and 4).

residues

C activators

of phospholipids

by PMA and the synthetic

in

kinase

of the phosphorylated

phosphorylation

C provided

addition

kinase

RESEARCH COMMUNICATIONS

The absence

immunoprecipitate

of protein

cellulose

rosine

the

kinase

control

of Tat

protein

(diacylglycerol).

of protein

or absence

weight

phospholipid

of Tat without

dependence

presence

of the

AND BIOPHYSICAL

as the

protein

for

kinase

a direct

Tat-associated

role

of

receptors.

Discussion The data identified

in

by protein

Towbridge

(17)

of Tat

kinase

did

not

Tat

residues.

phosphorylated observed

between

these

presented

here,

however,

of the Tat

epitope

The data

previously Although protein

confirm

implied

kinase

to be modified

provide

may serve the

HUT102B2

by protein

does not

supposition

for

of phorbol consequences

other

kinase

cellular

C (21-22).

245

Shackelford

HUT102B2.

Unstimulated

reconcile

In conconstituatively

the

differences

T lymphocytes.

the

that

role

The data

the protein

of protein of protein to cultured

of Tat

phosphorylation

receptors

have been kinase

moiety

kinase

esters

Protein

and

phosphorylation

express

substrate

cells

the

evidence

as a direct

receptor

phosphorylation.

HTLV-infected

conclusive

the physiological unknown,

cells

IL-2

in intact

induced

shown that

by the addition

C are

esters

Tat

with

the

Recently,

demonstrable

Our data

studies

2,3).

infected

have

that

phosphorylated

phorbol

possess (18)

is

C (figs.

that

et al.

demonstrate

moiety

on HTLV-retrovirus

cells Depper

study

Tat

demonstrated

expressed

HUT102B2

vity.

this

by the antigenic

and --in vitro

trast,

presented

C acti-

kinase cells. by reported

C has been

C

Vol. 135, No. 1, 1986 demonstrated

to phosphorylate

which

resulted

kinase

(25).

tion

BIOCHEMICAL

of receptors

phorbol

ligands

for

insulin

(21,

22).

C phosphorylation

finity

of the

associated membrane tain

IL-2

of IL-2

receptor

which

circumstances. receptors signaling

studies

it

have

is

event

shared

Frank

Ruscetti,

receptor

affinity

for

a role

of protein

interesting

to speculate

may mediate under

a variety the

factors,

The

respeckinase

transduction. that

alterations

protein of af-

of physiological

phosphorylation

C may suggest

by growth

the

(22). their

and signal

occur

tyrosine phosphoryla-

interactions

receptors

kinase

to stimulate

suggested

Alternatively,

by protein

(EGF)

C on B lymphocytes

receptor

of receptor-ligand

kinase

pathological

had reduced

findings,

factor

been shown

and somatomedin

These

of the present

growth

RESEARCH COMMUNICATIONS

of the EGF receptor-associated

have also

cells

C in the regulation In view

activity

esters

ester-treated

tive

the epidermal

in diminished Phorbol

AND BIOPHYSICAL

of membrane-

a common crucial tumor

or

promoters,

transand cer-

oncogenes.

Acknowledgements We thank Ronald

Drs.

Herberman

for

the helpful

Joost

Oppenheim,

suggestions

Suzanne

in the preparation

Beckner,

and

of the manu-

script. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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