Vol.

167,

March

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

16, 1990

SUBCELLULAR

LOCALIZATION

Nikodem

lDepartment

of Molecular

Grankowski’

Biology,

and

Institute

lInstitut

filr

Lublin,

Humangenetik,

8,

- Georg

KINASE

I

Issinger

Maria

Curie-Sklodowska

University,

Poland Universitlit

D-6650

January

Olaf

CASEIN

of Microbiology,

20-033

Received

OF

471-476

Homburg-3,

des Saarlandes FRG

1990

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the ,yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus. 01990 Academic Press, Inc.

Casein

kinases

cyclic the

type

nucleotides, protein

kinases

s,ubstrate

have

been

to mammalian other

cells.

from

contrast

CKI

and

to caseiu

kinase

cross-react

less

we know

some

paper

with

and

rDNA

transcription.

nucleolin

AND

CKI

found

the

daltons

antigens

of the

of

beginning the

from

cytoplasm

which

kinase

purification

little

was the

also

are

independent

chosen

These

eukaryotes

e.g.

in association

with

structure

with

of

according

enzyme.

simple

but

role

initiation

nonhistone

that

polyclonal

from

various

the

is known

physiological

translational

in

name the

kinases

has a monomeric

NII) the

and

of protein

to protein yeast various

a molecular

mass

(1).

(CKII,

show

is located

in

enzyme

about

(6,7) we

nuclear antibodies mammalian

nucleolus

which

about of their

factors

cell

for

is able

mRNP

(3),

spectriu

RNA CKI

types.

substrates CKI

(e.g.

against

endogenous

phosphorylatiou.

(2),

proteins

suggests

the

polymerases

isolated

We could

that

particles

it may

I and

from show

also

yeast that

CKI

be involved

II cells like in

METHODS

Biological materialHeRo-SV, is a human virus (10). Purification CKI from postribosomal

of

synthase

this

CKII

MATERIAL

been

II

for

organisms

The

to 45,000

much

In

has

The

used

in numerous

CKI

to a family

phospholipids. casein)

30,000

glycogen

@,9).

belong

components.

to phosphorylate (4,5),

NI)

(i.e.

found

intracellular

ranging

In

I (CKI, Ca +t or

Krebs II glioblastoma

mouse cell

ascites tumor cells were propagated in NMRI mice. line which was experimentally transformed by SV40

of CKZCKI from yeast cells was isolated and purified Krebs II mouse ascites tumor cells was isolated and supernatant from the cells was precipitated with 50%

as described before (11). purified as follows: the saturation of ammonium

0006-291X/90 471

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

167,

No.

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

sulphate. 2.3 g of protein in 110 ml were dialyzed overnight against 3 changes of 2 L starting buffer (20 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5 mM 2.mercaptoethanol, 1 mM EDTA, 1 mM EGTA, 0.5 mM pbenylmethylsulfonyl fluoride (PMSF) and then loaded on the DEAE (DE 52) cellulose column (40 x 2.5 cm). The column was equilibratd in the same buffer. The flow through material (1.2 g protein) containing the CKI activity was directly loaded onto a phosphocellulose column ( 1 x 12 cm) which bad been equilibrated with starting buffer. CKI activity was eluted with a linear gradient from 100-1000 mM NaCl (2 x 100 ml) in the same buffer. Fractions with casein kinase I activity were pooled, dialyzed against 2 L starting buffer and chromotographed on heparin-agarose (5 ml bed volume). The enzyme was eluted with a linear gradient from 100-400 mM NaCl (2 x 50 ml). The last step of the purification was achieved by gel filtration on a BioRad HPLC Model1 400 data station using a Biosil TSK column (300 x 7.5 mm) with starting buffer. An aliquot of 900 pl (740 pg protein) from the most active enzyme fractions after heparin-agarose chromatography was concentrated by ammonium sulphate precipitation (50%), centrifuged at 10,000 x g for 10 min and resuspended in 35 pl of starting buffer. 30 pl of this material were loaded onto the HPLC column. Casein

kinase

activity-

Casein

kinase

I activity

was determined

as described

earlier

(11).

Immunodetection of casein kinase I- When not otherwise specified 50 pg of protein were applied onto 12.5% PAGE. After electrophoresis proteins were blotted onto NITROSCREEN WEST (NEN, Dreieich, FRG) using a semidry blotting apparatus (BIOMETRA, Giittingen, FRG). Before the transfer the membrane and the blotting papers were soaked in blotting buffer (25 mM TRIS/HCL, 192 mM glycine,pH 8.3,). The transfer time was 60 min at 300 mA. Succesful transfer was verified by the use of rainbow marker (AMERSHAM, UK). The blot was blocked for 60 min in 1% non fat dry milk in PBS (GLUCKSKLEE, Mfnchen, FRG) followed by incubation with an anti-yeast CKI antibody raised in rabbits (12). The dilution of the first antibody was 500. to 1000.fold. When affinity-purified anti-CKI antibody was used the dilution was 25.50.fold. Incubation with the first antibody was 60 min at room temperature or at 4’C overnight. This step was followed by washing the membrane three times for 5 min in PBS/O.l% Tween 20. Incubation with the second antibody was for 60 min at room temperature. As a second antibody a phosphatase-conjugated goat anti-rabbit IgG preparation was used (MEDAC, Hamburg, FRG). The dilution of this antibody was 1000.fold in 1% milk/PBS. This step was followed by washing the membrane three times for 5 min in PBS/Tween 20 followed by an equilibration step in AP-buffer (100 mM TRIS/HCL, 100 mM NaCI, 5 mM MgC12, pH 9.5). Staining with NBT(p-nitro tetrazolium blue and BCIP (5.bromo-4-chloro-3-indoyl-phosphate) was as described earlier (13). Purification of affinity purified CKZ antibody10 pg of CKI were analyzed on a 12.5% PAGE and blotted onto nitrocellulose membrane (SCHLEICHER & SCHOLL, Dassel, FRG). After blotting the membrane was stained with Ponceau Red to visualize the transferred CKI (only for this purpose we used nitrocellulose, because the Nylon-based membrane does not allow easy staining of transferred proteins). The stained CKI band was excised from the membrane, destained in water and was then incubated in 100 pl anti-yeast CKI antiserum. Incubation was overnight at 4’C. The membrane was washed three times with PBS/Tween 20 and incubated for 1 min in 3 ml 100 mM glycine/HCI pH 2.2, 20 mM MgC12, 5 mM KCI. The membrane was transferred to a syringe and the glycine solution was forced through the paper twice. The eluted material was neutralized by adding l/10 the volume of 1 M TRIS base (0.3 ml). One ml of PBS was added and the solution was concentrated using a stirred Amicon ultrafiltration cell with a QM10 filter (14).

The

concentrated

material

(usually

1 ml)

was dialyzed

against

PBS.

Immunofluorescence - For immunofluorescense studies HeRo-SV cells were grown to subconfluency on cover glasses. Cells were fixed with methanol/acetone, washed with PBS and treated with trypsinlversene (0.05% trypsine, 0.05% EDTA, 2.5 mM NaOH in PBS) (15) for 20 see at room temperature. Incubation with the first antibody was for 45 min at 37’C in a humid chamber. Dilution of the first antibody was lo-to 50.fold in PBS. After this step the cover glasses were washed with PBS and then incubated in the presence of the second antibody (FITC-conjugated goat anti-rabbit serum, DIANOVA, Hamburg). Dilution of the second antibody was SO-fold in PBS. Incubation was 45 min at 37’C in a humid chamber. The cover glasses were washed in PBS and mounted to microscope slides and embedded in 90% glycerol/PBS. The presence of the FITC-labeled antibody was verified by immunofluorescense microscopy. 472

Vol.

167,

No.

RESULTS

AND

of

immunoreaction

CKI with

recognizes

only

one

CKI

whereas

predominant

yeast,

BIOPHYSICAL

in

the

with

antibody.

In

by anti-yeast

HeRo-SV

RESEARCH

COMMUNICATIONS

with

a molecular of

the

a molecular

addition

some

ascites

It

case

CKI antibodies.

and

anti-yeast-CKI-antibody.

polypeptide

polypeptide

CKI

cell extracts

from

the

the

yeast

mammalian

of

purified

yeast

AND

DISCUSSION

Characterization noblot

BIOCHEMICAL

2, 1990

mass

is

and

mass

of app.

minor

high

tumour

obvious

of app.

HeRo-SV

Fig.1

78 kDa

cell

that

45 kDa

ascites

is an immuextracts

the

yeast

(Fig.1)

crude

in

mass

antibody

the

extracts

case

also

is immunostained

molecular

after

by

the

polypeptides

of

another anti-

are

also

immunostained.

At the the

first

glance

antibody

precursor least

the

reacts molecules).

in Novik.off

The

molecular

crude

ascites

In

to

yeast

CKI

affinity major

ascites

the

Novikoff

order

There

same

reports

with

with or it

where

it was

2 polypeptides same

we

are

polypeptides

shown

as the dealing

that

situation

as described

earlier,

question

whether

additional

our

when

CKI

found

the

CKI

either

CKI

(e.g.

is associated,

37 and

II

that

of

activity

on the

Krebs

indicate

compounds

masses

ones in

may

related

of molecular

ballpark

that

other recognizes

85 kDa

(16,17).

immunoblot

mouse

activity

of

ascites was

at

the

crude

purified

from

cells.

the

antibody

CKI

antibody manner

in the

suggesting

alnswer

purified

are

cells are

extract

with

of the

an unspecific

masses

ascites

extracts

reaction

in

in

crude

antibody

polypeptide

extracts was

(Mr

from

prepared.

45,000)

the

HeRo-SV Only

was

the

isolated

polypeptides and

Krebs

antibody as

II mouse

that

described

recognized ascites

specifically in

the

by

our

tumor

cells

reacted

material

anti-

with

and

method’s

section.

ANTI-CKI

CKI F&J

HERO-SV

AFFI-ANTI-CKI

ASC

CKI

HERO-SV

ASC

Speci,ficity of polyclonal antibodies against yeast casein kinase I. CKI from yeast cells (0.2 pg) and app. 50 log crude extracts from HeRo-SV and Krebs II mouse ascites tumor cells were separated on 10% SDS-PAGE, blotted to NITROWEST and immunostained with either whole anti-CKI antiserum (dilution 500.fold) or with CKI antiserum (dilution 25.fold). The numbers on ‘!n.j right . immunoblots indicate the molecular masses in daltons I 10

473

affinity-purified hand side

of

aatithe

an the

Vol.

167,

Fig.1

No.

shows

the

the

whole

CKI

does

not

react

the

major

support

reaction

of the

antiserum

and

with

CKI the

in crude

the

mammalian

distance

protein

kinase

Isolation

of

CKZ from

from

yeast

and

cells

as described

in

were

similar

a gel

on

Fig.ZA

comparison intensity

the

purified of

correlates

the

quite

there

the with

material

BIOSIL the

peak CKI

and

same

have

is still

purified

in

recognizes

such

CKI

from

staining

both

cases.

some

described

that

despite

a well

yeast

that

the

been

surprising

ascites

TSK

were

HPLC.

to

yeast

was

that

migrates

activity.

cells.

the

for the

with

latter

one

intensity

of

These

results

unspecific

preserved

In

of 1D

In order

CKI

from

section.

proteins

an antibody large

to

evolutionary

cross-reaction

for

(11)

led

to

CKI

after

SDS

PAGE

to establish

identity

11 mouse

ascites

Krebs

Whereas

literature This

profile

applied

from

enzyme

in the

by

activity

tumor

we isolated method’s

described

polypeptide the

ZZ mouse sources

already

shows

from

Krebs

mammalian

to those

filtration

fractions

observations

it is still

mammals

the

antiserum

COMMUNICATIONS

It is obvious

although

virtually CKI

and

antiserum.

polypeptides,

is

Similar

extracts

CKI

anti-yeast

RESEARCH

1.

CKI

(Fig.2).

and

BIOPHYSICAL

cell

mass

45,000)

whole

However,

yeast

mammalian

molecular

extracts.

(14).

AND

affinity-purified

(Mr

the cell

between

casein

high

that

crude the

polypeptide

notion

microtubule

BIOCHEMICAL

2, 1990

the

the

inital

last

step

a virtually

and

by

is obvious

in

parallel.

It

same

position

as CKI

to

Coomassie

demonstrate

HPLC. by

at the

that

the

steps

introduced

stained

analyzed

order

purification

homogenous

analysis

included CKI

In

preparation

Fig.ZB

Coomassie

from

the

blue.

that

the

yeast

ascites

stained

of the tumour

on

main

For

staining the

polypeptide

gel is

Immunostained

HPLC

I

A’

I

I

C

B kDa

-92-69

-

-45-

f/

10

15

20

25

17

18

19

20

CKI

17

I8

19

20

FRACTIONS &&Analysis

of purified cosein kinase I. Chromatography of kinase on HPLC was as described under material and methods. The fractions, 0.25 ml, were collected and aiiquots of 20 ~1 were assayed for casein klnase activity (A). Aliquots from the peak fractions (200 ri) were diaiysed overnight against 20 mM NH4HC03, lyopbiiizcd and subjeeted to SDS PAGE (10%). Panel B shows the Coomnssie staining of the column fractions from panel A; panel C is an immunobiot from the samples shown in panel B. The numbers below the tracks refer to the fraction numbers of the column. The last track shows only CKI (2 pg) from yeast ceils wbicb served as a control.

474

CKI

Vol.

167,

indeed

No.

2, 1990

identical

with

an immunoblotting

CKI using

antibody

cross-reacts

Molecular

mass

mammalian

and

mammalian

was

contrast

mircroscopy

analyses.

As shown

the

cytoplasm

Fig.3D,F shown

show earlier

F&J

and

fractions from

which

and

RESEARCH

on

yeast

with

strongly the

antibody

cells.

The

behavior

gel

As one the

suggest

that

COMMUNICATIONS

a another

(Fig.ZC).

comigrates

data

related

BIOPHYSICAL

column

polypeptide

and can

purified

that

carried

CKI

CKI

from

from

from

yeast

can

of the

affinity-purified

out

see clearly

the

yeast.

yeast be

and

used

for

CKI.

further images in

Fig.3B

specifically

of

HeRo-SV

using

indirect

are

shown

cells in the

immunofluorescence to

the

immunological very

staining

examined

AND

anti-CKI-antibody

the

the are

Immunofluorescence body

we analyzed the

with

sources

detecting

BIOCHEMICAL

be

also

localized

immunofluorescence in

stained

Fig.3A,C,E. with

the

microscopy HeRo-SV

anti-yeast

CKI

(Fig.3B,D,F).

cells

antibody

were showed

used

anti.

Phase for

these

fluorescence

in

nucleoli.

microscopy

of CKII

in

nucleolus

the

and

nucleolin

(15,19).

both Here,

of which too,

we

have find

been some

staining of HeRo-SV cells with various antibodies. HeRo-SV cells were stained by indirect immunofluorescense using eitber (B) affinity-purified anti-CKI antiserum (30-fold), (D) anti-CKII antiserum (lo-fold), (F) anti-nucleolin antiserum (50fold). (A,C,E) show the corresponding phase contrast images.

Immmunofluorescence

475

Vol.

167,

No.

fluorescence has

signals

been

shown

Nucleolin

has

cytoplasm

(20).

mechanism

for

but

also

CKI

BIOCHEMICAL

2, 1990

in the previously

been

shown

The

transient

cytoplasmic and

AND

BIOPHYSICAL

cytoplasm.

This

observation

to

localized

in

be

recently exposure regulation

to

shuttle of shuttling

of nuclear

does

the

RESEARCH

not

come

cytoplasm

constantly proteins actvities

by

to a surprise

since

biochemical

means

between

the

to the

cytoplasm

which

may

COMMUNICATIONS

involve

nucleolus

and

may not

CKII (1). the

represent

only

a

nucleolin

CKII.

ACKNOWLEDGMENTS We thank Drs. B. Boldyreff and Tim Plant for critically Peter Striibele for carrying out some of the immuooblots. 246/B3 to O.-G. I. N. G. was supported by a SFB 246.

reading of This work

the manuscript. was supported

We thank by SFB

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Hathaway, G.M. and Traugb, J.A. (1982) Cm-r. Top. Cell. Regul. 21, 101.127. Hathaway, G.H., Lundak, T.S., Tabara, S.H., and Traugb, J.A. (1979) Methods Enzymol. 60, 495 - 5 11. Rittscbof, D. and Traugb, J.A. (1982) Eur. J. Biocbem. 123, 333-336. Hosey, M.M. and Tao, M. (1972) Biochemistry 16, 4578.4583 Simowski, K.W. and Tao, M. (1989) J. Biol. Cbem. 255, 6456.6461. DePaoli-Roach, A.A., Roach, P.J., and Learner, J. (1979) J. Biol. Cbem. 254, 12062.12068. Itarte, E. and Huang, K.-P. (1979) J. Biol. Cbem. 254, 4052.4057. Dabmus, M.E. (1981) J. Biol. Cbem. 256, 3332-3339. Duceman, B.W., Rose, K.M., and Jacob., S.T. (1981) J. Biol. Cbem. 256, 10755.10758. Fischer, H., Scbwecbbeimer, K., Heider, M., Bernbardt, S., and Zang, K.D. (1985) Cane. Genet. Cytogenet. 17, 257.268. Kudlicki, W., Szyska, R., Palen, E., and Gasior, E. (1980) Biocbim. Biopbys. Acta 633, 376. 385 Grankowski, N., Szyszka, R., and Pilecki, M. (1987) Acta Biocbim. Polon. 34, 45.49. Issinger, O.-G. Martin, T., Richter, W.W., Olson, M.O.J., and Fujiki, H. (1989) EMBO J. 1621.1626. Olmstedt, J.B. (1981) J. Biol. Cbem. 256, 11955.11957. Pfaff, M. and Anderer, F.A. (1988) Biocbim. Biopbys. Acta 969, 100.109. Dabmus, M. (1976) Biochemistry 15, 1821-1829. Dabmus, M. (1981) J. Biol. Cbem. 256, 3319.3325. Delpecb, M., Levy-Favatier, Moisand, F., and Krub, J. (1986) Eur. J. Biocbem. 160, 333-341. Liscbwe, M.L., Richards, R.L., Busch, R-K., and Busch, H. (1981) Exp.CeII Res. 136, 101. 109 Borer, R.A., Lebner, C.F., Eppenberger, H.M., and Nigg, E.A. (1989) Cell 56, 379.390.

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7,