Partial purification and characterization of a novel bovine adrenocortical protein kinase that phosphorylates endogenous 120,000 dalton peptide

Vol.

96,

No.

September

2, 1980 30,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 601-610

1980

PARTIAL PURIFICATIONANDCHARACTERIZATION OF A NOVELBOVINE ADRENGCGRTICAL PROTEINKINASETHAT PHOSPHORYLATES BNDOGENOUS 120,000 DALTONPEPTIDE YOSHIKAZUKURODAand RAMESHWAR K. SHARMA Department of Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 Received

August

4,198O SUMMARY

We describe partial purification of a novel protein kinase (designated PR 380) from bovine adrenal cortex. The enzyme binds neither cyclic AMP or cyclic GMP, nor phosphorylates the known exogenous substrates such as histones, phosphorylase b, casein, phosvitin, or protamine, in the presence or absence of the cyclic nucleotides. The protein kinase activity of the enzyme was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. Addition of exogenous calcium, calmodulin, or EGTA did not affect the process of phosphorylation. It catalyzes, however, the phosphorylation of an endogenous 120,000 dalton polypeptide. We, therefore, conclude that PK 380 is a cyclic nucleotide- and calciumor calcium-calmodulin independent protein kinase, that phosphorylates the endogenous substrate, a 120,000 dalton polypeptide. Recently, we have found that PK 380 specifically phosphorylates eukaryotic initiation factor 2 a. This enzyme therefore, might play an important role in the translation control of eukaryotic cell. INTRODUCTION Investigations lation

is

metabolic

exist

that protein phosphory-

process that mediates several intracellular

activities

(l-3).

of eukaryotes

nucleotide-dependent

To date cyclic

calcium-

AMP-binding autophosphorylating in nature (for a review see ref.

independent protein

The phosphorylation

or cyclic

AMP-dependent, cyclic

calcium-calmodulin-dependent, cyclic

have indicated

an important physiological

catalyzed by cyclic kinases.

by varius laboratories

nucleotide-independent

are

protein

GMP-dependent, calcium-dependent,

and cyclic protein 4).

reactions

nucleotide-independent,

and

kinases have been reported

to

The cyclic

kinases show a broad specificity

nucleotide-dependent for

their

and

substrates and

The abbreviation used are: SDS, sodium dodecyl sulfate; EDNA ethylenediaminetetraacetate; EGTA, ethylene glycol bis (8-aminoethyl ether) N,N,N*,N'-tetraacetic acid.

0006-291X/80/180601-10$01.00/~ 601

Copyright 0 1980 by Academic Press, Inc. All righrs of reproduction in any form reserved.

Vol.

96, No.

defining

2, 1980

their

challenging

late

specific

problems

This novel

BIOCHEMICAL

report

biological

deals

with

role

in metabolic

the partial

purification

protein

the commonly used

kinase,

This

dependent on either

designated

substrates

exogenous

or phosphorylase

120,000 dalton.

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

reactions

is one of the most

in biology.

adrenocortical

phosvitin,

AND

b,

such as histone,

protamine,

an endogenous

nucleotides-independent

or calmodulin

for

of a

PK 380, that does not phosphory-

phosphorylates

but

enzyme is cyclic

calcium

and characterization

its

catalytic

casein,

polypeptide and also

of

is not

activity.

MATERIALS AND METHODS Histone fate

(type

(grade

I),

II-A),

cyclic

b, cyclic

phosphorylase

human y-globulin,

beef liver

other

cellulose

reagents

[3Hlcyclic

Protein ation

kinase

gel was prepared

was a gift

from

as described

[3Hlcyclic

W.Y.

activity

(6, 7) with

250 ul incubation chloride,

Cheung.

mixture

Calcium

When cyclic

mixture

was assayed

by the incorporas previously

as follows.

contained

2On@lTris-HCl

was

AMP-dependent protein

contained

1 x 1O"M

in addition,

(200 cpm/pmol)

All

commercially.

(pH 7.5),

10 mM magnesium

1 x 1O-7M [v-32 PIATP (8 to 20 x lo3 cpm/pmol) and an appropriate

of PK 380. reaction

minor modifications

[ y-

GMP (40

by Koike and Hamada (5).

grade and were obtained kinase

Dr.

inhibitor,

from Bio-Rad.

and

sul-

were obtained

of 32P from [v-32 PIATP into exogenous or endogenous substrates

described

tion

AMP (40 Ci/mmol),

Kinase Assay - Protein

protamine

protein

A-1.5m were purchased

Calmodulin

were analytical

phosvitin,

and horse spleen apoferritin

and Boi-Gel

Ci/mmol) were from Amersham.

casein,

AMP-dependent

catalase,

from Sigma. DEAE-cellulose 32PIATP (3,000 Ci/mmol),

phosphate

nucleotides,

kinase

1 uM cyclic in

the

activity

aliguot

was determined

the

AMP, and [y- 32P]ATP concentra-

presence

or

absence

GMP-dependent protein

kinase

of

exogenous

substrates.

For the determination

of cyclic

activity

the

composition

of the reaction

1 uM cyclic

GMP and 1 x 10-5M [y- 32PIATP (200 cpm/pmol) were used. Incubation was

mixture

was

the same except 100 n+l magnesium chloride,

602

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96, No.

carried acetic

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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1 min at 37OC and the amount of 32 P incorporated

out for

acid precipitable

material

an exogenous substrate,

was measured (6, 7).

into

trichloro-

When protamine

ice cold 25% trichloroacetic

was used as

acid was used to precipitate

the protein. Cyclic

Nucleotide

determined aliquot

by

Binding

modification

a

Assays

- Cyclic

(6) of the Gilman procedure

of PK 380 was incubated

AMP (1 x 10m5 cpn/pmol)

lo5 cpm/pmol).

The reaction

terminated

2 ml of ice cold 70% saturated

precipitate

was

ammonium sulfate,

cocktail

Polyacrylamide

(9, 10) with amide.

toluene

Denaturing prepared

kinase

identical conditions distilled

prior

disc

by SDS gel

radioactivity

was in

gels

with

25% saturated in 5

in a nondenaturing

consisted

as described

of 5% polyacrylat 2 mA/gel at 4OC.

200~1 20mM Tris-HCl

(0.5 x 12

by native

(pH 7.5)

containing

cm)

gel electrophoresis

Blue,

gel scanner for

and

[y-

mixture

The protein

by liquid

scintillation peroxide

Scintiverse. 603

was analyzed.

at 55Onm. In a separate 10 min with

0.1% SDS were

by Engbaek --et al.

destained

the sample buffer

electrophoresis.

30% hydrogen

diffusion

The incubated

to pH 6.8 with

determined

0.5 ml of

20 min the

radioactivity

and Laemmli (11) as modified

Brilliant

a Gilford

adjusted

with

(0.5 x 6 cm) were prepared

gels

to electrophoresis.

water,

thrice

and counted for

Separating

enzyme sample was incubated

fractionated

dissolved

Coomassie

with

After

activity.

10 ug of enzyme isolated

acid and traced

GMP (1 x

g/l).

extracted

polyacrylamide

with

(pH 4.0),

of the enzyme and was

to pH 6.8 and electrophoresed

by the method of Favre

were stained

was

An appropriate

or [3H]cyclic

- For electrophoresis

(6).

1.5mm slices,

for protein

washed

disc gels

some modification

Samples were adjusted

assayed

(4

Gel Electrophoresis

Gels were cut into

(12).

in

activity

(8).

ammonium sulfate.

were dried,

polyacrylamide

system,

by the addition

on GF/C filters,

and the filters

ml of Omnifluor

stacking

was initiated

collected

binding

at O°C for 60 min in 50mM sodium acetate

2mMEDTA and 3 x 10m8Mof [3H]cyclic

with

nucleotide

with

7.5% acetic

experiment,

an

32 P]ATP under standard was dialyzed

against

of Engbaek (12), band associated

counting

at 6S°C,

Gels

of gel

and mixed

with

and with

slices, 7 ml

Vol.

96, No.

BIOCHEMICAL

2, 1980

Protein

Determination

using the Bio-lad

AND

- Proteins

reagent

with

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

were measured by the method of Bradford

bovine

(13)

serum albumin as a standard.

RESULTS AND DISCUSSION Purification

of PK 380 - All

Step I:

procedures

ammonium sulfate

Hcmcgenixation,

cellulose

gel

dissected

out of the adrenal

potassium

phosphate

(buffer

chromatography

buffer

A, pH 7.0)

(750 ml) solid

precipitate

was dissolved

- Bovine glands

The dialyzed

same buffer,

gradient, 7.0).

20-400 Ml,

buffer.

with

containing

kinase

peak

complete

separation

chromatography

precipitate

dissolved

column

NaCl gradient

NaCl and was clearly proteins

(Fig.

in

1).

The the same

cellulose

A, washed with

linear

phosphate)

potassium

2 1 of

phosphate

and 2 mM EDTA (pH 120 mM potassium AMP-dependent

was obtained

but

- To the pooled PK 380 fractions, concentration

20 mM Tris-HCl

the same buffer.

the

buffer (buffer

The dialyzed

500 ml of buffer

(0 - 4OOmM) in buffer resolved

from cyclic

B.

solid

of 60% saturation. containing

The 6 mu 2-

B, pH 7.5) and dialyzed fraction

(2.8 x 16 cm) preequilibrated

The column was washed with linear

buffer

phosphate

at approximately

2 mM EDTA and 10% glycerol

against

DEAB-cellulose

to a calcium

against

of PK 380 peak from cyclic

was added to a final

overnight

and 2 Ml EDTA

overnight

a 3 1 of

ammonium sulfate

mercaptoethanol,

800 ml of 20 I@!

of these enzymes was not achieved.

DEAE - cellulose

was

(400 g) was

was added to 60% saturation.

with

(200 mM potassium

phosphate

To the postmitochondrial

6 Ml 2-mercaptoethanol

Some resolution

protein

2 min.

A and dialyzed

The PK 380 peak was obtained

phosphate

Step II:

for

equilibrated

and eluted

tissue

6 mM 2-mercaptoethanol

was applied

column (5 x 20 cm) previously the

adrenocortical

ammonium sulfate

fraction

and calcium

and homogenized with

containing

in buffer

out at 0-4OC.

precipitation

in a Waring blender

supernatant

buffer.

were carried

with

B and eluted

was applied

to a

the same buffer. with

1 1 of the

PK 380 peak emerged at 0.17 M

AMP-binding

and cyclic

GMP-binding

Vol.

96, No.

2, 1980

BIOCHEMCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

\’ 1

FKm

2

01

FRACTION

NUMBER

F 30

02 ’ X3

3

30 40 50 60

RACnON

NWlBER

Fig.

1 DEAF,-cellulose column chromatography of PK 380 from bovine adrenal Approximately 450 mg protein obtained by elution from calcium cortex. phosphate cellulose gel was applied to a column of DEAE-cellulose (2.8 x 16 cm) equilibrated with 20 mM Tris HCl, 6 n@I 2-mercaptoethanol, 2 mM EDTA and 10% glycerol, pH 7.5. The column was washed with 5 column volume (500 ml) of the equilibration buffer and eluted with 11 of the linear gradient of NaCl (0 - 0.4M) in the same buffer. Fractions (6ml) were analyzed for protein kinase (c), cyclic AMP binding (0) and cyclic GMP binding activity (A) as described under "Materials and Methods".

Fig.

2 (A) Bio-Gel A-1.5m column chromatography of PK 380 from bovine adrenal cortex. About 29mg protein obtained by elution from DEAE-cellulose was applied to Bio-Gel A-1.5m (2.5 x 60cm) equilibrated with 20 mM Tris-HC1, 2mercaptoethanol, 2mM EDTA and 10% glycerol, pH 7.5. Elution was performed with the same buffer at flow rate of 25 ml/h. Fractions (5 ml) were analyzed for protein kinase (o), cyclic AMP binding (0) and cyclic GMP binding activity (A) as described under "Materials and Methods". (B) Estimation of molecular ,:_ight of PK 380 by gel filtration. 1. horse spleen apoferritin (Mr = 480,000), 2. beef liver catalase (Mr = 248,000) and 3. human y-globulin (Mr = 160,000) were used as standard proteins.

Step III:

Gel-filtration

chromatography

-

The

tions were combined and solid ammoniumsulfate The precipitate same buffer. preparative

The dialyzed

preparation

was applied

column (2.5 x 60 cm) previously

AMP- nor cyclic

equilibrated

against the

to a Bio-gel

A-1.5m

with buffer 8, and

The PK 380 peak obtained corresponded to a

weight of 380,000 (Fig.

neither cyclic

was added to 60% saturation.

was dissolved in buffer B and dialyzed overnight

eluted with the same buffer. molecular

PK 380 peak frac-

DEAE-cellulose

2).

The enzyme thus isolated

GMP-binding activity

605

nor cyclic

showed

AMP- or cyclic

Vol.

96, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

Table Purification

of

Purification of protein assay procedure used is

kinase described

Fraction ~_______-----_-~--------~-----

was carried out under "Materials

9,690

2.9

described and Methods".

in

the

The 1 fold purification activity mentioned under

b)

GMP-dependent

kinase

conducted

this

on

was

Units

fold

1030

1

2.3

1030

23

18

522

164

80

230

727

arbitarily

calcium

taken

phosphate

activities.

All

fraction.

The

Purification

One unit was defined as that amount of enzyme which Btalyzed ation of one pmole of inorganic phosphate from [ yP]ATP substrate per min at 37OC.

a)

text.

0.11

449

29

as

Cortex

Total Unit a) --------------

Units/mg

A-1.5m

Adrenal

Bovine

v

DEAE cellulose Bio-Gel

from

Specific Activity

phosphate gel

COMMUNICATIONS

1

Total Protein

60% Ammonium sulfate of postmitochondrial supernatant b) Calcium cellulose

PK 380

RESEARCH

Table

kinase I

as

1

cellulose

incorporendogenous

based

on

1030

units

gel purification

properties

shows

the into

the

of

summary

step.

the of

enzyme were purification

procedures.

Table histone,

II

shows that

protamine,

distinguished at

observed

that

for

is yet

this

phosvitin,

PK

utilize

least

In order AMP-, cyclic

PK 380.

Table

380

from

one

of

other

b.

substrates This

nucleotide-independent

mentioned

such as

characteristic kinases

Curiously,

substrates.

the phosphorylation

of PK 380.

which it

was

The reason

undetermined. the phosphorylation

GMP-, calcium,

Further

above

exogenous

or phosphorylase

cyclic

b inhibited

to differentiate

III

not phosphorylate

casein

the

phosphorylase

enzyme was incubated EGTA.

PK 380 did

with

and calmodulin-dependent the cyclic

shows that evidence

of PK 380 from

nucleotides

none of that

these

PK 380 is

606

protein , calcium

agents

chloride,

affected

different

kinase,

from

that

of cyclic

the isolated calmodulin,

the phosphorylation cyclic

AMP-dependent

or of

Vol.

96, No.

BIOCHEMICAL

2, 1980

AND

Table Substrate

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

II

Specificity

of PK 380

The enzyme (26ug) was assayed under scydard3$onditions as described under "Materials and Methods" except that 1 x 10 M [y- PlATP (5,000 cpm/pmol) was used and 1OOug each of various protein substrates was added as indicated. ---__----__-_____ Acid Precipitable Substrate radioactivity ---~------------------~---(cm)

PK 380

13,600

+Histone

12,500

+Protamine

11,200

+Phosvitin

13,000

+Casein

14,000

+Phosphorylase b ~-------.--_-----------_______

1,600

Table Effect

_-----

III

of cyclic nucleotides, calcium and calmodulin on the phosphorylation of PK 380

Protein kinase from Bio-Gel A-1.5m was dialyzed against 20 mM Tris AC1 (pH 7.5) buffer containing 10% glycerol. The enzyme (15ug) was assayed under standard conditions as described under "Materials and Methods" except that each compound described below was added as indicated. Acid precipitable radioactivity

Addition -_l_l_----------

--

(Cpn)

PK 380

10,400

+cyclic

AMP (1uM)

10,500

+cyclic

GMP (UM)

9,800

+EGTA (0.8aW)

10,500

+Ca2+ (0.4 mM1

10,100

+Calmodulin (80ug/ml)

9,900

+Ca2+ (0.4 mM) and Calmodulin (80Wml)

10,200

607

Vol.

96, No.

BIOCHEMICAL

2, 1980

AND

Myosin (Mr = 200,000), 6-galactosidase bovine serum albumin (Mr 94,000), used as standard proteins.

protein

kinase cyclic cyclic

regulatory

dependent

Fig.

of of

It

when

PK 380 is

of

120,000

DNAse

,

kinase

and

protein

kinases

by cyclic

not

the

shown). by

32 PIATP

[y-

was

and it

further

120,000

phospholipase

In cyclic

analyzed

phosphory-

that

or

eliminated

the

substantiated

dalton,

C,

digestion

indicating

nor

was pronase the

phosphate

treated before

phosphory-

was

originally

polypeptide.

therefore

protein

band

(data

of

.

substrate

pronase

inhibitor

conditions,

substrate,

KNAse,

Only

dalton the

dalton

subunit

inhibited

with

polypeptide.

endogenous

electrophoresis.

into

AMP or

the

the

be readily

denaturing

incubated

regulatory

PK 380

of

phosphorylated

dalton

the

Neither

could

under

120,000

or

activity

holoenxyme

phosphorylated

120,000

is

kinase

PK 380 was

experiment.

kinase.

inhibitor.

nature

gel

incorporated

cyclic

the

chloroform-methanol,

lation

COMMUNICATIONS

= 116,500), phosphorylase b (Mr = and ovalbumin (Mr = 43,000) were

inhibitor

protein

single

The

analysis

kinase

electrophoresis

endogenous peptide

(Mr = 68,000)

following

kinase that gel

follows.

with

protein

protein

polyacrylamide an

the

AMP-responsive

3 shows

The as

by

inhibited

a cyclic

lates

provided

AMP-dependent

subunit

contrast,

by

was

AMP-dependent

adrenal

AMP

RESEARCH

gel electrophoresis of PK 380. PK 380 (1Oug) 3. SDS disc polyacrylamide from native gel (5%) was subjected to SDS disc polyacrylamide gel electroThe gel was stained, destained and scanned at 559~ (0). In a phoresis. parallel experiment, PK 380 (1Oug) was preincubated with [y- PIATP prior to The protein band associated with radioactivity (-0) was electrophoresis. determined by counting individual gel slices dissolved in hydrogen peroxide.

Fig.

with

BIOPHYSICAL

obvious can the GMP

be

that

380

differentiated

following nor

PK

is from

First,

criteria. do

these

nucleotides

608

a

cyclic

the

nucleotide-independent

cyclic it

activate

has

nucleotide-dependent no the

binding

affinity

phosphorylation

for of

Vol.

96, No.

exogenous

lase

2, 1980

BIOCHEMICAL

substrates

such as histone,

b.

regulatory

Second,

the

subunit

of

unable to inhibit PK 380 is far dependent either

Furthermore,

it

not phosphorylate

casein

casein

kinase

kinase

protein

Third,

molecular

the

kinases

might

designed

dalton

polypeptide

with

the --in vitro

of this

enzyme.

of GMPby This

and calmodulinPK 380 does from the

(19-21).

factor-2a.

have some role

studies

(14).

is different

present, we cannot assign any physiological role . Recently', we have found that PK 380 &I &, specifically initiation

weight

In addition,

At

eukaryotic

are

is not affected

(15-17)

it

that

the

kinase

of calmodulin

(18).

indicating

or

AMP-dependent or cyclic

in the presence

or phosvitin

inhibitor

the enzyme activity

protein

or phosphory-

AMP-dependent

from the calmodulin-dependent

but calcium-dependent

COMMUNICATIONS

phosvitin,

protein

cyclic

alone or by calcium

differentiates

reported

AMP-dependent

than any of the known cyclic

kinases.

RESEARCH

casein,

the endogenous phosphorylation.

protein

independent

BIOPHYSICAL

protamine,

adrenocortical

greater

calcium

property

cyclic

AND

It is therefore

in the translational

to correlate

and the phosphorylation protein

translation

But, before such studies

the enzyme is purified

to homogeneity

that

of eukaryotic

free

are conducted, and further

this

define

of 120,000 factor-2a such a role

it is a prerequisite

characterized

enzyme Future

initiation will

system

enzyme.

cell.

of the phosphorylation of eukaryotic

in cell

this

phosphorylates

a possibility

control

the relationships

to

that

kinetically.

ACKNOWLEDGEMENTS We thank Dr. W.Y. Cheung for the gift by grants Institute

from National

Science

of calmodulin.

Foundation

This work was supported

(PCM 7800860)

and National

(CA-16091). REFERENCES

1.

Greengard,

P. (1978) Science 199, 146-152.

2.

Rubin, C.S. and Rosen, O.M. (1975) Ann. Rev.

1.

Kuroda,

Y. and Sharma, R.K.

Manuscript

Biochem.

in preparation.

44,

831-887.

Cancer

BIOCHEMKAL

96, No.

3.

Krebs,

4.

Sharma, R.K., Biol.

5.

Koike,

6.

Shanker, G., Ahrens, 76, 66-70.

7.

Sharma, R.K., 577-586.

McLaughlin,

C.A. and Pitot,

8.

Gilman,

(1970) Proc.

Natl.

9.

Orstein,

10.

Davis,

2, 1980

AND

Vol.

E.G. and Beavo, J.A. Ahrens,

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

(1979) Ann. Rev. Biochem. 48, 923-959.

H., Shanker,

G. and Moore, R.E.

(In Press)

Cell.

Mol.

M. and Hamada, M. (1971) Methods Enzymol. 22, 339-342.

A.G.

Ii. and Sharma, R.K.

(1979) Proc. H.C.

Natl.

Acad. Sci.

(1976) Eur. J. Biochem.

USA 65,

Acad. Sci. USA 67, 305-321.

L. (1964) Ann. N.Y. Acad. Sci. 121, 321-349. B.J.

(1964) Ann. N.Y. Acad. Sci. 121, 404-427.

11.

Favre,

M. and Laemmli, U. (1973) J. Mol. Biol.

12.

Engbaek, F., Gross, C. and Burgess, R.R. (1976) Mol. 295.

13.

Bradford,

14.

Cheung, W.Y. (1970) Biochem. Biophys. Res. Commun.8,

15.

Dabrowska, R., Sherry, J.M.F., Biochemistry 17, 253-258.

16.

Yagi, K., Yazawa, M., Kaikiuchi, Biol. Chem.253, 1338-1340.

17.

Waisman, D.M., Singh, T.J. and Wang, J.H. 3390.

18.

Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695.

19.

Yamamoto,M., Criss, W-E., Takai, Y., Yamamura,H. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 5049-5052.

20.

Dahmus,M.E. and Natzle,

21.

Hathaway, G-M. and Traugh, J.A.

M.M.

(1976) Anal. Biochem. 2,

J.

so, 575-599. Gen. Genet. 143, 291-

248-254. 533-538.

Aromatorio, D.K. and Hartshorne, D.J. (1978) S., Ohshima, M. and Uenishi, K. (1978) J. (1978) J. Biol.

Chem. 253, 3387-

(1977) Biochemistry 16, 1901-1908. (1979) J. Biol.

610

Chem. 254, 762-768.