Purification of a protein kinase from human Namalwa cells that phosphorylates topoisomerase I

Vol. 109, No. 4, 1982 December 31, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1222-1227

PURIFICATION OF A PROTEIN KINASE FROM HUMAN NAMALWA CELLS THAT PHOSPHORYLATES TOPOISOMERASE I John

S. Mills,

Harris

Busch and Egon Durban

Department of Pharmacology Baylor College of Medicine Houston, Texas 77030 Received

November

17, 1982

A nuclear protein kinase which phosphorylates phosphoprotein 110/8.4, recently identified as topoisomerase I, has been purified approximately 330 fold from a 10 mM Tris extract of human Namalwa cells. The kinase was chromatographed on DEAE-Sephacel and further purified by affinity chromatography on phosvitin-Sepharose. The protein kinase exhibited a high affinity (Km = 0.3 PM) for topoisomerase I; its affinity for phosvitin was approximately 100 fold lower (Km = 25 uM). Topoisomerase nuclear

extracts

permits tion ization

nuclear

in phosphorylated

enzymes

analysis (3,4).

I was recently

II or poly(A)

RNA polymerase These

type

Km was 0.3 uM which

describes

kinase

which

shows a high

from Novikoff

(1,2). are

can be phosphorylated

report

of a protein

form

polymerase

of enzyme activity

This

purified

Other also

the

phosphorylates for

by protein

such as

--in vivo

(3,4).

kinases

which

of phosphoryla-

and preliminary topoisomerase

topoisomerase

cell

enzymes

to the degree

purification

affinity

nuclear

phosphorylated

--in vitro

in relation

ascites

character-

I --in vitro;

its

I.

MATERIALS AND METHODS [Y-~'P]ATP and Aquasol 2 were obtained from New England Nuclear; BoltonHunter reagent from Amersham; dithiothreitol, casein, phosvitin from Sigma Chemical Corp.; leupeptin from Peptide Institute, Inc., Japan; protein markers and protein determination solution from Bio-Rad Laboratories; CNBr-Sepharose and DEAE-Sephacel from Pharmacia. The Namalwa cells (a Burkitt lymphoma cell line used for synthesis of interferon) were the generous gift of Mr. Fred K'lein, Dr. John Douros, and Dr. Vincent DeVita of the Frederick Cancer Research Laboratories, Frederick, MD, and the USPHS, Bethesda, MD. 10 mM Tris extracts of Namalwa nuclei were prepared according to the method of Chan et al. (5). The kinase assay was essentially that of Dahmus (6). The assay sxture contained 150 ug phosvitin/40 mM 4-morpholine propane sulfonic acid (MOPS), H 6.6/6 mM magnesium acetate/O.2 mM EGTA/O.lO mM ATP, and 2 x.106 dpm[--32P P ATP, and enzyme in a final volume of 0.1 ml. The samples were incubated at 37' for 20 min., and the reaction was stopped by the addition of 1 ml 10% trichloroacetic acid plus 1.5% (w/v) pyrophosphate. The precipitates were collected on nitrocellulose filters and washed twice with 2 ml portions of trichloroacetic acid-pyrophosphate. The filters were then counted with Aquasol scintillation fluid. Protein 110/8.4 (topoisomerase I) was purified from Novikoff 0006-291X/82/241222-06$01.00/0 Copyright 0 1982 by Academic Press, Inc. AN righrs of reproducfion in any form reserved.

1222

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

hepatoma ascites cells and Namalwa cells as described (1). Protein concentration was determined by the method of Bradford (7). Phosvitin-Sepharose was prepared according to Thornburg et al. (8) except that CNBr-Sepharose from Pharmacia was used. For proteiniodination, 100 uCi Bolton-Hunter reagent was placed in a 12 x 75 mm test tube and dried with a stream of air. The reagent was resuspended in a 300 ul aliquot from DEAE-Sephacel and the pH was raised to 9.5 using 1 M potassium borate buffer (pH 10.4). The sample was incubated for 20 min. at 4'C and the reaction was stopped with 10 r.1 of ethanolamine. The iodinated sample was added to the remainder of the DEAE-Sephacel pooled sample. The specific activity was determined by precipitation with 10% trichloroacetic acid and was 1800 cpm/ug protein. The starting material for purification was a 10 mM Tris-HCl (pH 8.0) nuclear extract; extracts of higher ionic strength were not chosen for further purification since they contained other proteins which bound more tightly to the phosvitin-Sepharose column. Novikoff hepatoma DNA was prepared as described (1). RESULTS Purification

of Protein

The 10 mM Tris-HCl of the g for

total

(pH 8.0)

nuclear

kinase

activity

protein

16 hrs.

Kinase

The supernatant

extracts

was then

had been equilibrated

column

was washed

with

sulfonyl

fluoride

(Fig.

M NaCl containing 1).

0.4 M NaCl.

Hunter

reagent

These

fractions

as described

f F

activity

with

eluted pooled

in Materials

column After

gradient

uM leupeptin/l 15-18

and iodinated

with

from mM PMSF

at approxiBolton-

and Methods.

1.6 1.2

0.16 0.12

2 -2

0.8

0.08

% -

0.4

0.04

.E G E

4

8

12

16

FRACTION

20

24

NUMBER

Separation of protein kinase by DEAE-Sephacel chromatography. Namalwa Tris extract supernatant was applied to a DEAE-Sephacel column and eluted with a 100 ml gradient from 0.1 M to 0.6 M NaCl as described in the text. Fractions of 3.5 ml were collected. Aliquots were assayed for protein kinase Protein concentration (0) was determined by the method of BradPeak activity fractions were pooled as indicated by the bracket. 1223

x

the

mM phenylmethyl-

a linear

in fractions

30%

at 100,000

(pH 8.0).

(pH 7.0)/l

(pH 7.0)/100

were

centrifuged

10 mM Tris-HCl

Nuclei.

approximately

to a DEAE-Sephacel

mM MOPS-HCl

10 mM MOPS-HCl

of Namalwa

contained

were

the enzyme was eluted

The peak of kinase

mately

which

applied in

0.1 M NaCl/lO

(PMSF),

Extracts

in nuclei,

(1 x 10 cm) which

to 0.6

from Tris

0.1

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

loo0 500 J c

1.5

100 80

8 ;

1.0

z 0 z 2 5

0.5

f

60

8

40

5 -

20 4

8

12

16

20

24

28

32

FRACTION NUMBER Fig. 2. Phosvitin-Sepharose chromatography of protein kinase. Pooled DEAESephacel fractions were iodinated as described under "Materials and Methods". The specific activity was 1800 cpm/pg protein. The pooled fractions were applied directly to the column and eluted with a 100 ml gradient from 0.2 M NaCl to 1.2 M NaC1/40% (v/v) ethylene glycol. Aliquots of 10 pl were assayed for protein kinase activity (o and 100 ~1 were precipitated with 10% trichloroh . acetic acid to determine I 5 I incorporated into protein (0). Fraction size was 3 ml. The gradient began at fraction 4. Peak of activity was eluted at approximately 0.7 M NaC1/20% ethylene glycol.

The (1

x 25

enzyme-rich cm)

Beginning NaCl

and at

to

1.2

Protein

with

10 ml

washed

4,

M NaC1/40%

kinase

was

well

was

step. and its

Total Proteina (mg) Tris

Extract

15

(pH

bulk

a gradient

of

the

from

protein;

0.2

for

stored

of the enzyme was 333 fold.

I

SCHEME OF NAMALWA

PROTEIN

KINASE

Total Activity (pmoles/min)

Specific Activity (umoles/min/mg)

Yield

Fold Purification

0.042

0.003

100%

1.3

7.2

0.028

0.004

67%

1.3

DEAE-Sephacel

1.2

0.016

0.014

382

4.7

PhosvitinSepharose

0.016

0.016

1.0

38%

fic

activity

1800

was determined where protein cpm/ug

protein).

1224

by the method was estimated

333

of Bradford (7) by 1251-counts

20%

1 year.

Tris Extract Supernatant

a Protein concentration for phosvitin-Sepharose

fold in

over

M

(Fig.

a 70

was

was stable

purification

mM PMSF.

mM PMSF

enzyme

activity

column

8.0)/l

mM leupeptin/l

The purified

TABLE PURIFICATION

with

0.1

the

a phosvitin-Sepharose mM Tris

eluted in

from

overall

on

M NaCl/lO

glycol

at -2O'C the

placed

0.2

column

in this

I,

then

separated

glycol

in Table

the

ethylene

occurred

ethylene

As shown

was

fraction

purification (v/v)

sample

except (speci-

2).

Vol. 109,

No.

8lOCHEMlCAL

4, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. 3. 32PO~ incorporation into topoisomerase I. Topoisomerase I was dialyzed against 10 mM Tris (pH 7.4)/0.25 M KC1/0.5 mM OTT/O.5 mM PMSF/50% (v/v) ethylene glycol for 1 hr. Topoisomerase I (3 ug) was incubated with[yJ2F$ATP (10 JJM, 20 Ci/mMole) and 10 mM Mg++ in the absence or presence of 15 ng of purified protein kinase for 15 min. at 37'C and electrophoresed on SDS-7.5% polyacrylamide gels according to the method of Laemmli (9). Lane 1, molecular weight markers: phosphorylase B (94,000), bovine serum albumin (68,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (21,000), lysozyme (14,000); lane 2, topoisomerase I alone; lane 3, topoisomerase I plus purified protein kinase. A: Coomassie blue stained gel; B: Autoradiogram of the gel.

Phosphorylation bation

with

the

topoisomerase In

the

Fig.

3B).

was

0.3

the

which

I.

somerase

of

the

When

compared I was uM

(36

may

that

80-90%

presence

of no of

the

of

the

(800

32P01,

was

protein

or

and higher

pg/ml),

was

1225

kinase

100

that uM

3,

detected

(2.5

was

found

product

of

the

affinity

for

phosvitin. mg/ml)

Fig.

3B).

(lane

towards

casein,

incuinto

(lane

degradation

than and

incorporated

32PO~

protein

phosvitin

Following

kinase

labeling

purified

rds

Kinase.

incorporated

loo-fold 25 PM

the

significant

a contaminant

towa

of

Protein

purified

be

approximately pg/ml),

I by

mixture,

lo-20%

activity with

Topoisomerase

enzyme,

Approximately

Kd polypeptide

I was

kinase I in

absence

merase

of

2,

in

a 40

topoiso-

topoisomerase for

topoiThe

for

Km

topoisomerase

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A

6

I

I

I

I

0.2

0.4

0.6

0.8

L

1.0

DNA (pglml)

Fig. 4. Analysis of kinetic parameters of protein kinase with substrates topoisomerase I, phosvitin and casein. Purified topoisomerase I was dialyzed for 2 hr. against 10 mM potassium phosphate (pH 7,4)/l mM PMSF, lyaphilized and resuspended in kinase assay buffer. A buffer blank was prepared by adding pOtaSslum phosphate buffer (pH 7.4) to klnase assay buffer so that blank and sample had the same conductance. Final phosphate concentration in the assay was 20 mM. A. Phosvitin (a) and casein (h) were assayed in the range of 0.25 to 2 mg/ml. 6. Topoisomerase I (0) was assayed In the range of 0.005 to 0.04 mg/ml. The Km values determined by linear regression analysis were: 0.036 mg/ml (0.3 vM) for topoisomerase I, 0.8 mg/ml (25 uM) for phosvitin and 2.5 mg/ml (100 pM) for casein. Fig. 5. Effects of Novikoff hepatoma DNA on protein kinase activity. Protein kinase was assayed with 1.6 ug of either Novikoff hepatoma topoisomerase (o), Namalwa topoisomerase (A) or phosvitin (a) as substrate and the indicated final concentration of Novikoff hepatoma DNA in the assay.

I,

phosvitin,

GTP as The

and

casein

substrate;

Vmax

for

the

(Fig. Km for

phosvitin

topoisomerase

and

4). ATP

The and

casein

enzyme

GTP was

was

1.4

was

equally

16 uM and

umol/min/mg

active 20 uM,

and

0.7

with

ATP

or

respectively. umol/min/mg

for

I.

Phosphorylation was

enhanced

by

tin

phosphorylation

concentrations

of low

topoisomerase

concentrations was

not

decreased

I from of

Novikoff

enhanced

this

both

by

low

Novikoff

hepatoma

and DNA

concentrations

Namalwa

(Fig. of

DNA.

cells

5).

PhosviHigher

DNA

activity. DISCUSSION

This nuclear

report

extracts

describes of

Namalwa

the cells.

330-fold

purification

The 1226

kinase

of has

a loo-fold

a protein higher

kinase affinity

from

BIOCHEMICAL

Vol. 109, No. 4, 1982

AND BIOPHYSICAL

...k 2

L

6

6

10

ELUTION

Figure

1.

Effect Cells B(a)P ilution

dosage

of x-rays

comparison

and the elution

providing

Initial and cells

treated

similarly-treated

alkaline from

elution cells

determine

the

treatment

B(a)P-induced elute

at

was sensitive kinetics

were

exposed

had

been

interstrand

protein

of

of the

cell

after

that

to proteinase a series of

450

x-rays

rad

profiles the

prior

crosslinks

to

this

proteinase both

irradiation

was

cells.

presence

of DNA markedly

Such a phenome of the between

enzyme.

As

the

K to

shown

in

effect

and control

DNA

these

of proteinase

K removed

B(a)P-treated

to

the elution

to distinguish

in the

when

prior

or crosslinking

In order

cells

obtained

immediately

to

of

cells.

of control

profiles

irradiated

crosslinking

with

and treated

can be seen that

B(a)P

allowing

rate

determined

the

lysates

crosslinking,

with

performed

M to

used as a point

control elution

in control

molecules.

sensitivity

therefore

8 hours

It

with

either

were

production

1A).

observed

experiments

in nature, of

treated

10

as described in Materials and Methods. with proteinase K (100 us/ml) in the prior to alkaline elution.

were

to

(Figure

6

l ,O)

between for

to that

from

a similar

Having protein

10m6

cells

to adjacent

possibilities lB,

with

6

elution of DNA from HTE-B cell:B with DMSO (control or 10 cells were x-irradiated prior

the alkaline

M B(a)P

compared

non may result strands

compared

analysis

that

decreased

period

maximum resolution

experiments

L

TIME (HOURS)

of B(a)P on the alkaline were incubated for B hours in DMSO (&O). As required, (0,O). Alkaline elution performed Cell lysates were treated lysing and EDTA solutions

B:

2-

RESEARCH COMMUNICATIONS

Figure

of

cells

to

x-irradiation.

the

observed

K digestion

crosslinking and hence

of experiments these

lesions

effect

may be assumed

was performed and

1293

whether

of B(a)P

or

to be DNA-

to investigate not

they

treatment

were

the removed