Loss of endogenous nuclear protein A24 lyase activity during chicken erythropoiesis

Vol. 100, No. 4,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

June 30, 1981

COMMUNICATIONS Pages 1464-1470

LOSS OF ENDOGENOUS NUCLEAR PROTEIN A24 LYASE ACTIVITY DURING CHICKEN ERYTHROPOIESIS Ira

L.

Goldknopf,

Department

Received

Sheila Cheng, Michael and Harris Busch

of Pharmacology, Houston,

W. Andersen

Baylor College Texas 77030

of Medicine

May 8, 1981

SUMMARY To study the endogenous protein A24 lyase activity of erythroid nuclei during chicken erythropoiesis, 3H-protein A24 was incubated with nuclei of mature erythrocytes and premature rythroid cells, Lyase activity was m3asured as conversion of 5 H-protein A24 into 3H-histone 2A and H-ubiquitin (Andersen, N. R., Goldknopf, I. L., and Busch, H. (1981) M.W., Ballal, Biochemistry 20, 1100-1104). The transcriptionally active nuclei of immature erythroid cells from anemic blood had substantial endogenous lyase activity. The activity was 1.34 fold higher in the anemic bone marrow nuclei from more immature cells. On the other hand, transcriptionally inactive nuclei of mature erythrocytes from normal chicken blood had negligible lyase activity. This effect parallels the loss of the ubiquitin portion of protein A24 in mature chicken erythrocytes (Goldknopf, I. L., Wilson, G., Ballal, N. R., and Busch, H. (1980) J. Biol. Chem. 255, 10555-10558). INTRODUCTION Chromatin

protein

and ubiquitin the

carboxyl

linkage This

linkage

(8).

portion

down during

is

group

in

of

converted

protein

of protein

is

into

study

119 of

histone

which

cleaves

of the

1464

in which

by an isopeptide

component

0006-291X/81/121464-07$01.00/0 Copyrighr 8 1981 bv Academic Press, Inc. All rights of reproducfion in any form reserved.

attached

showed that

2A (1)

structure

its

A24 accompanied

maturation

of histone

a branched

lysine

A24 lyase

A previous

final

a conjugate

of ubiquitin

e-NH2

conjugate enzyme

arranged

terminal

to the

by the

tin

(2-4)

A24 is

2A (5-7).

polypeptides the

loss

isopeptide

of the

transcriptional chicken

erythrocyte

ubiquishut(9).

Vol. 100, No. 4,198l

The present activity

BIOCHEMICAL

report

AND

shows that

is also

lost

in

BIOPHYSICAL

endogenous

the

mature

MATERIALS

RESEARCH

protein

chicken

COMMUNICATIONS

A24 lyase

erythrocytes.

AND METHCDS

Blood from normal and phenylhydrazine treated (9,lO) adult leghorn hens was collected in 1% heparin to a final concentration of 0.05%. Bone marrow cells obtained from the tibia and femur of phenylhydrazine treated chickens were placed in 5 volumes of 0.05% heparin, phosphate buffered saline (PBS-PMSF; 10 mM sodium phosphate, pH 7.4, 150 mM sodium chloride, 1 mM phenylmethylsulfonylfluoride). After centrifugation the cells were washed twice in at 1,000 x g for 10 minutes, Nuclei were obtained (11) by homogenizing the cells PBS-PMSF. in RSB-NP40-PMSF (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgC12, 0.5% Nonident P40, 1 mM PMSF) and centrifugation at 3,000 g Nuclei were washed twice in RSB-PMSF. Purifor 10 minutes. fied protein A24 was obtained as described previously (12). Protein A24 was labeled with 3H in vitro by reduction methylation (8,13A. To determine endogenous protein A24 lyase activity, H-labeled protein A24 (1 pg N 100,000 cpm) was incubated with nuclei (40 Ilg DNA) (14) in a final volume of 40 1.11 containing 50 mM Tris, pH 7.5, 5 mM MgC12, 0.1 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 1 mM leupeptin, for 90 minutes at 370, a modification of conditions established previously for isolated nucleoli (8). 40 ~11 of 0.1 M sodium phosphate, pH 7.1, 6 M urea, 1% sodium dodecylsulfate, 5% 2-mercaptoethanol, 10% glycerol was added to the reaction mixtures followed by heating to 100° for 2 minutes and one-dimensional electrophoresis in 12% polyacrylamide, 0.1% sodium dodecylsulfate, 0.1 M sodium phosphate, pH 7.1, 6 M urea was performed as described (8). were stained in Coomassie Brilliant Blue R, treated The gel with E$ HANCE (New England Nuclear), dried and fluorographed. Fluorographs were scanned with white light in Transidyne 2955 Scanning Microdensitometer and the scans analyzed by planimetry. Unincubated nuclei (40 Llg DNA) were also subjected to electrophoresis and the gels stained in Coomassie brilliant blue R to analyze nuclear proteins.

RESULTS To measure equivalent of the

lyase

amounts proteins

showed patterns series

(15).

clear

content

a compensatory

of

activity

during

of nuclei these

nuclei

includes

of erythrocyte decrease

in

erythropoiesis,

(40 ug DNA) were used. on SDS gels

characteristic This

chicken

of the

lysine

1465

rich

la-c)

differentiating

a progressive specific

(Figure

Analysis

erythroid

increase lysine histone

rich

in

the

histone

1 (16-21)

nu5 and and

Vol. 100, No. 4,198l

Figure

1

nonhistone

BIOCHEMICAL

proteins

earlier

stage

the

progressively (la)

tein found

(h)

in mature

as reported

previously

If-i).

stages

~24 into

anemic

H-histone

erythrocytes

blood

erythrocytes

with

decreased la)

pro-

was also

from

(g).

1466

A24 followed

to detect early

converted 3 2A and H-ubiquitin; not

to

and normal

(Figure

3H-protein

and fluorography

did

(lb)

lc)

(9).

incubated

The nuclei

(Figure

a markedly

of erythropoiesis

3

COMMUNICATIONS

when comparing

bone marrow

In addition,

A24 content

(Fig.

mature

mature

(10).

were

is evident

of anemic

more

by electrophoresis

This

(14,22).

cells

cells

The nuclei

ty

RESEARCH

Separation of Nuclear Proteins and The Products of Protein A24 Lyase on Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis. SDS-urea-phosphate polyacrylamide gel electrophoresis was performed as described in Materials and Methods. (a-e) Coomassie blue stained gels of (a) untreated chicken erythrocyte nuclei (40 ug DNA); nuclei (40 Dg DNA ) fran (b) anemic blood and (c) anemic bone marrow of phenylhydrazine treated chickens; purified protein A24 (d and Dbi(f-i) fluorographs of: (f) bH-protein quitin (e). A24 incubated alone (1 ug, 100,000 cpm); and with nuclei (40 Mg DNA) of (g) normal chicken erythrocytes; (h) anemic blood; and (i) anemic bone marrow of phenylhydrazine treated chickens. Anemic blood and bone marrow were taken simultaneously from the same 6 phenylhydrazine treated chickens. Purified ubiquitin was the gift of G. Goldstein of Ortho Pharmaceuticals.

the

blood

AND BIOPHYSICAL

In

(i)

the

lyase

activi-

and intermediate

a portion but,

addition

of 'H-protein the nuclei

the

lyase

from

BIOCHEMICAL

Vol. 100, No. 4,1981

Quantitative into

BIOPHYSICAL

2A and 3H-ubiquitin

~24 lyase

in nuclei

Untreated chicken erythrocytes Protein

A24

RESEARCH

Tabie 1 of percent of 3H-protein

analysis

3H-histone

AND

COMMUNICATIONS

A24 converted

by endogenous protein

(40 ng DNA) from:

Erythroid cells from phenylhydrazinc treated chicken* blood bone marrow 71.3

61.7

Histone 2A

10.1

14.5

Ubiquitin

19.0

23.9

Relative

100

Activity

*Circulating

-

1.0

blood and bone marrow were obtained

**

groups of 6 phenylhydrazine

Average of the relative

in 3H-histone

activity than

treated

chickens.

3H-protein

decrease in

two

~24 and increases

2A and 3H-ubiquitin.

was in the

simultaneously

Average of data obtained with

from the same chickens. separate

1.34**

1.34

fold

blood

higher

in the nuclei

of anemic chickens

(Table

from bone marrow 1).

DISCUSSION Several

lines

of evidence

protein

A24 IS a function

portion

of

the molecule

The rates

24).

A24 are parallel

mitosis

(23)

tin

portion

portion lost

turns

over

that

throughout

synthesis

and drop

In quiescent

unstimulated

(24).

chicken

down and chromatin

A24 turns Protein

over

is

highly

(23, into

during chromo-

the ubiqui-

the histone ubiquitin

2A are

(9) when transcription

condensed

1467

levels

lymphocytes

A24 and free

erythropoiesis

interphase

from metaphase

while

of

The ubiquitin

and conjugation

to minimal

A24 is absent

of protein

metabolism

activity.

when protein

does not during

of nuclear

of ubiquitin

protein

somes (25).

indicate

(15).

On the

shuts other

Vol. 100, No. 4,19Bl

hand,

BIOCHEMICAL

protein

nucleoli

A24 content

(26-28)

where

free

free

(35-39)

are

apparently It

A24 might

effect

why cleavage

accompanies

activation

lysates

whether

presence

of the

ubiquitin

erythroid

cells

shutdown

reflects

jugation

in

the

and loss

of

a control cell

of protein

mechanism

(40,41)

perhaps This 2A bond

and repression also

in rabbit

A24 lyase activity

(32-34).

conjugation

are

it

histones

- histone

of proteins studies

where

conjugation

(9,41).

chromatin

this

(29)

nucleosomes

Ubiquitin

of protein

other

(40,41,25),

state

Further

hypertrophic

octamers

presence

adjacent

active

degradation (42,43).

the

that

chromatin

chromatin.

proteolytic

protein

by ubiquitin

of nucleolar

nucleolar

in

fractions

2A and the

of

COMMUNICATIONS

of nucleosomes

core

speculated

of

may explain

decreased

disrupted

flexibility

a potentially

cyte

the

histone

has been

stabilizing

cedes

a subset

not

condensation

of extra

in

between

(33,34,38).

inhibiting

(29-31).

2A in

RESEARCH

chromatin

found

found

histone

The interactions

some active

is

A24 is

BIOPHYSICAL

is markedly

and in

ubiquitin

Protein replaces

AND

needed in

involving

reticuloto test

immature

during

pre-

chicken

transcriptional ubiquitin

con-

nucleus.

ACKNOWLEDGEMENTS ject

These Grant

studies were supported by the Research Program CA 10893 Pll of the National Cancer Institute.

Pro-

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