Phosphorylation of translational initiation factor 3 (eIF-3) by cyclic AMP-regulated protein kinase

BlOCHEMlCAL

Vol. 83, No. 2, 1978 July 28,1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 379-384

Phosphorylation

of Translational

Initiation

Cyclic

AMP-regulated

Jolinda

A. Traugh

Protein

May

3 (eIF-3)

by

Kinase

and Tina

S. Lundak

Department

of Biochemistry

University

of California

Riverside,

Received

Factor

CA 92521

11,1978

SUMMARY initiation factor 3 (eIF-3) is phosphorylated by the Translational eIF-3 is a cyclic AMP-regulated protein kinases from rabbit reticulocytes. large molecular weight complex which facilitates binding of the ternary complex containing met tRNAfr GTP and initiation factor 2 to 40s ribosomal subunits. A single polypeptide with a molecular weight of 130,000 is modified. The phosphorylation is dependent upon the presence of cyclic AMP and is inhibited by the inhibitor protein diagnostic for cyclic Am-regulated protein kinase. Assuming a molecular weight of 700,000 for eIF-3, one mole of phosphate is incorporated per mole of eIF-3. Thus the phosphorylation of two interacting components of the protein synthesizing system, 40s ribosomal subunits and eIF-3, is controlled by cyclic AMP. INTRODUCTION Eucaryotic (21,

initiation

has been

a number complex

shown

of

sources

--et

subunits

to

al.

of

form

500,000-700,000,

subunits protein with protein

in

molecular have kinase

a molecular

have

80s and

been

This

met-tRNA of

(5)

3

the

f

40s

shown

consists weight

shown

weight

with factor

the

onto

factor has

to

to be phosphorylated (8). of

In 130,000

this

entry

405

(1,6).

subunits

from

the

ternary

In

molecular

of

the

weight

of

proteins

(2,3,7).

and

addition,

reassociation

by cyclic

modified

IF-M5

subunits

10 different 130,000

and

of

ribosomal

a reported

report is

(1)

ribosomal

prevents

approximately 35,000

40s

complex

eIF-3 of

IF-E3

facilitates

initiation

that

from

formerly

native

* GTP *eIF-2

ribosomes.

activities

(eIF-31,

be associated

(3-5).

formation

Thompson

range

to

consisting

stabilizes

factor

Four

which of

these

nucleotide-independent

we demonstrate by the

that

cyclic

the

subunit

AMP-regulated

kinases. 0006-291X/78/0832-0379$01.00/0 379

Copyright All rights

0 1978

by Academic Press, Inc. in any form reserved.

of reproduction

Vol. 83, No. 2, 1978

MATERIALS

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHODS

The type I and II cyclic AMP-regulated protein kinases were isolated by chromatography on DEAE-cellulose and phosphocellulose as previously described Free catalytic subunit was obtained from the type II enzyme by further (8). chromatography on phosphocellulose. A 10 ml column was equilibrated in Buffer A (25 mM potassium phoseate, pH 6.8; 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.02% sodium azide; and 1x10 M cyclic AMP). The sample (235 mg in Buffer A) was applied to the column, and the column was washed with two column volumes of Buffer A to remove contaminating protein and undissociated protein kinase. The free catalytic subunit was eluted with 0.4 M NaCl in Buffer A and stored at 4'C. The0 rleqac~~;~m_ij~tuu; MgCf2,

;:oo6p34~o mcL;;;;oali;;d;

~Oxm:;~i;HcZy~l[cH

i$;(tEe;f

.

l

indicated) and 24 enzyme units (EU) of type II cyclic AMP-regulated protein kinase or 35 EU of catalytic subunit; and eIF-3 (14-21 pg) or 40s ribosomal subunits (26 ug). An EU is that amount of enzyme which incorporates 1 pmol of phosphate into histone per min at 30°C. Purification of eIF-3 (2) and ribosomal subunits (9) have been described elsewhere. After incubation for 30 min at 30°C the reactions were terminated and analyzed by slab gel electrophoresis and autoradiography (8) or by precipitation with trichloroacetic acid (8). RESULTS AND DISCUSSION Phosphorylation protein

of

kinases

type

I

and

was

type

respectively; tography

subunits

electrophoresis identified

kinase

with

was examined

in

observed

addition

of

weight

of

either eIF-3

130,000

Essentially

In subunits radioactive

the

the

results same

was also

or

examined. into

obtained

with

phosphate As previously a single

protein

380

the

components

When the

cyclic with

protein bands

AMP.

Upon

a molecular

of

in the absence

cyclic

AMP.

of cyclic

kinase.

incorporation

into (9,11-131, 40s

were

1 show the results

presence

I protein

the

by chroma-

gel

band

was observed

IIIH

by polyacrylamide

of

a protein

in

and

no phosphorylated

absence

observed

II

The

the

(Y- 32 P)ATP.

in

AMP.

phosphorylation,

in Figure

the

type

as

modified

substrate,

phosphorylated of phosphate

were

the

and

mixture,

cyclic

reticulocytes

separated

expressed

of added

AMP-regulated

of

After

and

enzyme

presence

reaction

highly

rabbit

were

sulfate,

cyclic

identified

from

factor

II

absence

the

experiment,

phosphate

type

the

II

and absence

(previously

The data

no incorporation

Identical

AMP.

the

the

was

presence

type

phosphocellulose.

dodecyl

in to

I and

purified

and

sodium

experiment

the

partially

comprising

in

type

kinases

by autoradiography.

this

were

protein

were

by in

on DEAE-cellulose

individual

of

examined

II

10)

eIF-3

subunit

40s

ribosomal

incorporation was stimulated

of

Vol. 83, Nq. 2, ‘1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-69-

-359 -32.5-

+cA

+cA

No Addn. Figure

1.

Phosphorylation

AMP-regulated panel,

protein

addition

by cyclic

in

Right

1).

This

the

absence

by the cyclic

panel,

panel,

of

protein

addition

protein,

as S-13

multiple

+4os

and 40s ribosomal

Left

reticulocytes

protein-synthesizing lated

kinase.

AMP (Figure

and contains

observed

of eIF-3

of eIF-3.

and in rabbit 32,500

+elF-3

(91,

complex, AMP-regulated

cyclic eIF-3

kinase

as S-6

liver

weight

sites.

and 405 ribosomal

381

Middle

in rat

has a molecular

kinases.

alone.

subunits.

nucleotide.

protein

by cyclic

of 40s ribosomal

identified

phosphorylation the

subunits

(12),

of approximately

No phosphorylation Thus

two components

subunits,

were

was in

the

phosphory-

Vol. 83, No. 2, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I too

50

Enzyme

Figure

2.

Quantitation

concentrations to

of

reaction

the

reaction

eIF-3

using

of

This

phosphorylation

protein

Attempts phorylation

the been

40s

(12,141

reticulocytes by

correlation

cyclic

of the

to

cyclic

AMP

(121,

and

(ll),

pituitary

been

made

between

of

this

382

the

been

by phosphorylation

per

(Figure

observed

(Table AMP

and addition

in of

hamster

and

of

serum

and

specific

1). phos-

Although rat

liver

regeneration islet

2).

inhibitor

control.

conditions (151,

factor

cyclic

translational

were

incorporated

kinases of

tri-

kinase

heat-stable

protein

under

culture

protein

the

role

with

was

for

addition

has

slices tissue

added

phosphate

precipitation

phosphate

700,000

with

protein

EU were

enzyme,

of

the

subunits

in

II

by

of

correlate

this

and

type

AMP-regulated

of

AMP; has

1 mole

by

cyclic

ribosomal

the

directly

to

140

eIF-3.

weight

made

phosphorylation

glucagon

(16)

have of

increased

for

0 to

concentrations

up

inhibited

Increasing

from

from

increasing

a molecular

specific

of

measured

mixture,

eIF-3.

ranging

subunit was

was

into

subunit 14 ug

When

the

mole

catalytic

eIF-3

acid. to

Incorporation

catalytic

into

chloroacetic

“P

containing

free

incorporation

in

free

mixtures

Using

added

of

Units

with (12);

cell

tumor

(17,181; molecular

no

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 83, No. 2, 1978

Table

I.

Effects

of

heat-stable 32

Additions

tone

P Incorporated

In

lated

phosphorylation

synthesis

addition,

have

initiation

been

14.2

2.2

426.9

15.1

kinases,

these

No

enzymes

lysates

(20).

as

step

in

a

steps

in

of

the

other

initiation

subunit

on

(19).

Here

this

a

control

process. on

we

eIF-3

An

the

that

shown

have

been

been

to

of

protein

one

be

the

the of

of

synthesis

cell first

these

the

role

by

from

considered

the

of

modified

isolated

regulating of

protein

AMP-regulated

phosphorylation

examination

AMP-reguof

cyclic

been

mechanism

control

shown

the

has

cyclic

reactions

have

has

complex

of

partial

coordinate

be

reactions

the

role

by

and

sequence,

initiation

the

factor

and

potentially

phosphorylation

examining

subunits

(3-51,

18 EU of free inhibitor (I), 38 ug

phosphorylated

40s

initiation

the

40s

is

Since

could

studies

eIF-3,

complex

the

components

vitro

inconclusive

factors,

protein

--in

(pmol) +I

The reaction mixture contained catalytic subunit, 80 units of eIF-3 and 300 ng histone.

events.

protein

-1

eIF-3 His

inhibitor

two

subsequent

these

various

are

currently

for

the

in

progress. ACKNOWLEDGMENTS We wish

to

preparations

of

heat-stable from

the

thank

Drs.

eIF-3

C.

in

these

used

inhibitor United

William

protein.

States

Merrick studies

This

Public

and

and

research

Health

Brian

was

Safer

Dr.

Donal

supported

Walsh by

for

Grant

Schreier,

2.

Safer, Merrick,

3.

Sundkvist,

M. B.,

H.

Adams, W.

C. I.

and S. (1976)

C.

and

Staehelin,

Service.

L.,

T.

Kemper, Proc.

Staehelin,

(1973) W.

Natl. T.

383

Nature

N.

B.

242,

M.,

Berry,

K.

W.,

Lloyd,

Acad.

Sci.

USA

73,

2584-2588.

(1975)

3.

Mol.

Biol:99,

35-38. M.,

the

GM 21424

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