Vol. 185, No. 3, 1992 June

30,

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

1992

THE PHOSPHATE

GROUPS OF THE HIGH MOBILITY Pl STRENGTHENS

Anne Carine Ostvold”‘“, aNeurochemical

Laboratory,

1091-l

097

GROUP LIKE PROTEIN

ITS AFFINITY FOR DNA

lngunn Hullsteinb

and S&fen G. Lalandb

Institute of Basic Medical Sciences, University of Oslo, Oslo,Norway

bDepartment

of Biochemistry,

University of Oslo, Oslo, Norway

Received May 11, 1992

SUMMARY: PCA soluble proteins isolated from rat liver and proliferating HeLa interphase cells were subjected to chromatography on columns containing immobilized s.s and d.s. DNA. Pi from rat liver was eluted from S.S. and d.s. DNA between 0,20 and 0,45 M NaCI, while dephosphorylated Pl was not retained by S.S. and d.s. DNA columns at 0,25 M, suggesting that phosphate groups enhance the affinity of Pl for DNA. Pl from proliferating HeLa interphase cells exhbit increased affinty for d.s. as well as S.S. DNA when compared to rat liver Pl . The higher extent of phosphorylation in proliferating cells supports the finding that phosphate enhances rather than redu0 1992 Academic PESS, 1°C. ces the affinity of Pl for DNA.

It is now established

that higher eukaryotic cells contain a set of HMG like proteins ( 1,2,3 )

closely related to those described by Johns et.al. ( 4,5 ). The highly phosphorylated Pl has an amino acid composition Nucleolin is a phosphoprotein phosphorylated

nucleolin

which resembles that of HMG 1,2 as well as nucleolin

in vivo, and active rRNA transcription

horylated by CK-2 in situ. As judged by phosphopeptide additional Pl

phosphorylation

is phosphorylated

is correlated

( 8,9 ). In growing cells both Pl ( 10 ) and nucleolin

seems to be mainly phosphorylated

nuclear protein ( 2,6,7 ).

to highly ( 8,ll

) are phosp-

maps, Pl from in vivo labelled hepatocytes

by CK-2 while Pl from proliferating

HeLa cells has

SeVeral

sites in vivo ( 12 )

by protein kinase C, CaM kinase II and protein kinase A in vitro ( 13 ). Both

nucleolin and Pl are further modlfied in mitosis, and both proteins are substrates for p34 cdmkinase in vitro and in vivo ( 12,14 ). HMG 1,2 exhibit preferential

binding to s.s DNA

( 15 ) while nucleolin binds exclusively to

s.s.DNA in vitro ( 16 ). The present study has examined whether Pl binds to s.s or d.s DNA and to *To whom correspondence should be addressed at University of Oslo, Neurochemical Laboratory, P.O. Box 1115, Blindern 0316, Oslo 3, Norway. Abbreviations used: PCA, perchloric acid, TCA, trichloroacetic acid, S.S. DNA , single stranded DNA, d.s. DNA , double stranded DNA , HMG, high mobility group, CaM kinase II, calcium / calmodulin kinase II CK-2, casein kinase 2. 0006-291X/92

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Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

185, No. 3, 1992

what extent phosphorylation

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AND BIOPHYSICAL

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affects this binding. In addition to DNA binding domains, HMG 1,2 and

nucleolin also consist of highly acidic stretches which can be considered for histones or other basic non histone chromatin

proteins, modulating

as potential binding domains their physiological

function. In

order to investigate whether Hl or other PCA soluble proteins influence the affinity of Pl for DNA, both purified Pi and PCA extracted proteins were subjected to chromatography

MATERIALS

on immobilized

DNA.

AND METHODS

Materials : Single stranded DNA Sepharose was purchased from Bethesda Research Laboratory while double stranded DNA cellulose was obtained from Pharmasia. 32P ( orthophosphate ) was from the Institute of Energy Technology ( Kjeller, Norway ). E.Coli phosphatase ( type Ill R ), protamine hydrochloride and phenylmethylsulphonylfluoride were obtained from Sigma Chemical Co. Single stranded spesific S, nuclease was purchased from Amersham. Cell culture and radiolabellina: HeLa cells were grown and labelled as described previously Preparation of perchforic acid extracts: Extraction of tissues and cells were carried out as in ( 2 ). Phosphatase treatment of perchloric acid extracted proteins: Phosphatase ( Ptase ) treatment of extracted proteins was carried out as in ( 2 ). Purification of Pl: PCA extracted proteins were dissolved in water ( 10 mg / ml ) and protamine hydrochloride dissolved in water ( 1 rng I ml ) was added to a final concentration of 0,2 mg / ml. The pH was adjusted to 8,0 by addition of 05 M Tris HCI pH 8,0. The precipitate formed was washed with acetone I 0,l N HCI ( 1:8 ) and finally acetone. Pi was separated from protamine by preparative acetic acid-urea gel electrophoresis as described in ( 2 ). Western immunoblottina: This was carried out as in ( 21 ). Affinity purified anti rat Pl raised in rabbis was used Chrornatoaraphv on S.S. DNA aqarose: DNA agarose containing 05 - 1 ,O mg calf thymus single Stranded DNA per ml agarose was packed in a column ( diameter 0.6 cm, bight 22 cm ) and washed with 1 ,O mM Tris HCI pH 75. The column was equilibrated with 1 ,O mM Tris HCI / 0,05 M NaCI, and the applied proteins were dissolved in the same buffer. The column was then washed with 15 ml 1,O mM Tris HCI I 0.2 M NaCl before elution of the proteins with 50 ml of a linear gradient from 0,2 - 0,6 M NaCl in 1 ,O mM Tris HCI pH 7,5 at a flow rate of 0,4 ml per min. and a fraction volume of I ml. The column was subsequently washed with 15 ml 1 ,O mM Tris HCI / 2M NaCI. The conductivity in each fraction was measured with a conductivity meter, Radiometer/Copenhagen. Proteins in 5 ml pooled fractions were precipitated with TCA (25 % final concentration) and analysed by SDS I 15% polyacrylamide gel electrophoresis according to Laemmli ( 17 ). ChromatOoraDhv on d. s. DNA cellulose: DNA cellulose containing 1 ,S mg native calf thymus DNA per ml cellulose was treated with single-stranded DNA spesific S, nuclease according to lsackson et al. ( 15 ). The column had a diameter of 0,6 cm and the hight was 8 cm. The column was washed with 1 ,O mM Tris HCI pH 7.5 and the applied proteins was dissolved in the same buffer. The column was washed with 1Oml 1 ,0 mM Tris HCI / 0,05M NaCI, and the proteins eluted with 50 ml of a linear gradient from 0,05 - 0,6M NaCl in 1OmM Tris HCI pH 7.5 at a flow rate of 5 ml per hour and a fraction volume of 1 ,O ml. The column was subsequently washed with 15 ml ml 1 ,O mM Tris HCI / 2M NaCl The fractions was analysed as described for s.s DNA chromatography.

RESULTS Purified Pl from rat liver was subjected to colums containing

immobilized s.s and d.s DNA respectively.

The eluted fractions were analyzed by Western immunoblotting phoresis. As seen in Fig.1 purified Pl was eluted from

gel electro-

s.s DNA colums between 0,20 and 0,45 M

NaCI. This was also the case with d.s. DNA ( not shown ). 1092

after SDS polyacrylamide

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No. 3, 1992

185,

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a

b

AND BIOPHYSICAL

d

c

Fig. Western lmmunobiots of purified Pl column with a linear salt gradient from 0,20 to were separated by SDS gel electrophoresis and 2pg purified Pi (a) material not attached at 0,05 0,35 M (e), 0,40 M (f), 0,45 M (g).

PCA extracts from mammalian

cells contain in addition to Hl and Pl the high mobility

( HMG ) proteins and some minor protein components.

and 04

S.S. DNA.

efg

from rat liver eiuted from a s.s DNA agarose 0,60 M NaCi . The proteins in 5ml pooled tractions probed with anti PI antibodies. M NaCl (b), Pl eluted at 0,20 M (c), 0,25 M (d),

DNA binding capacity of Pl , PCA soluble proteins containing

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To investigate a possible effect of Hl on the

from rat liver were fractionated

As seen in Fig. 2 , PCA solubilized

group

on a colum

Pl from rat liver was eluted between

0,20

M as was the case with purified Pi. Hence the presence of Hl or the other PCA soluble

proteins did not affect the affinity of Pl for S.S. DNA.

a

bcdef

9

h

0.7 I 0.6 *

0,5

z a

0.4, 0.3

02

: t

t

0.1 t 5 1015202530254D455D556065 Fraction

number

Fia. Eiution profile of PCA soluble proteins from rat liver eiutsd from a S.S. DNA agarose column with a linear salt gradient from 0,20 to 0,60 M . Western lmmunobiots of the eiuted proteinsseparated by SDS gel eiectrophoresis and probed with anti Pl antibodies are shown at the top. PCA extracted proteins from rat liver (a), proteins eluted at 0,20 M (b), 0.35 M (c), 646 M (d), 045 M (e), 0,50 M (f), 0,60 M (g), 2pg Pl standard (h).

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185, No. 3, 1882

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AND BIOPHYSICAL

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E.Coli Ptase dP1 Hl

g h i i kl abc d ef w. A Coomassie stained SDS polyacrylamlde gel of protein fraCtiOnS obtained when fractlonatlng dephosphotylated PCA soluble proteins on s.s DNA agarose as in Fig. 1. Dephosphorylated PCA extract from rat liver (a): proteins not attached at 0,05 M NaCl (b), proteins eluted at 0,20 M (c), 0,24 M (d), 0,28 M (e), 0,31 M (f), 0,39 M (g), 0,43 M (h), 0,47 M (i), 0,51 M (j), 055 M (k), 0,60 M (I).

It has been shown that Pl probably consists of a population different degrees ( 2 ). Dephoshorylation

by alkaline phosphatase

of molecules phosphorylated

to

removes all the phosphate groups

which have been shown to be linked to serine and threonine. To examine the effect of the phosphate groups on the affinity of Pl for DNA, a PCA extract from rat liver was treated with alkaline phosphatase prior to to chromatography

on S.S. and d.s. DNA columns. It is seen from Fig. 3 that dephosphorylated

Pl was not retained by the s.s column at 0,25 M NaCI, indicating that the prescence of phosphate groups enhances the affinity of Pl for S.S. DNA. A similar result was found for d.s. DNA ( results not shown ) Since Pl in proliferating cells is phosphorylated

to a greater extent than Pl from non proliferating

Cells as judged by the number of phosphopeptides rat hepatocytes and proliferating from proliferating

in thermolytic peptide

maps from in vivo labelled

HeLa cells ( 12 ) , it seemed of interest to compare the affinty of Pl

cells and rat liver for DNA.

labelled in vivo with with =P-phosphate

PCA soluble proteins from proliferating

were fractionated

on the S.S. DNA column.

HeLa

cells

As seen in Fig.4

Pl from HeLa cells was eluted from the S.S. DNA column between 0,22 and 0,60 M NaCI. In contrast to Pl from rat liver, a subfraction

remained bound

at 0,60 M and was subsequently

NaCI. When PCA soluble proteins from HeLa interphase

a d.s. DNA column, and the collected fractions probed with anti Pl antibodies, eluted within the same range of salt concentration

eluted at 2,0 M

cells were subjected to chromatography

as for the S.S. DNA column

on

Pl was found to be ( Fig. 5 )

DISCUSSION Protein Pl is a nuclear protein present in all mammalian a HMG like protein and has also many properties

species and cell types examined so far. It is

in common with nucleolin. 1094

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185, No. 3, 1992

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

M.W

Pl Hl

a

h

bcdefg

i

jklmn

m Autoradiogram of a SDS polyacrylamide gel of protein fractions obtaineo when fractbnatlng PCA soluble proteins from HeLa cells labelled In vlvo wlth 32P and on a S.S. DNA agams Proteins

COlUlllll.

The proteins

were

eluted

as in Fig.1,

Material

not attached

at 0,05 M NaCl (a),

eluted at 0120 M (b), 0,21 M (c), 0,25 M (d), 0,28 M (e), 0,32 M (1) 0,37 M (g), 0,41 M (h), 0,45 M 0) 050 M (j), 0,56 M (k), 0,60 M (I) 0,60 M (m), 2,0 M NaCl (n).

Thermolytic

peptide maps of Pl from , rat liver, calf liver, pig liver and HeLa cells imply a high

degree of homology in Pl from different species ( 21 ). Pl is a phosphoprotein of phosphotylation erating

and the pattern of thermolytic phosphopeptides

Cells, cells in log phase growth

phosphorylation

and metaphase

varies when comparing

arrested

cells ( 12 ).

pattern in Pl may reflect different functions in proliferating

and may also explain the differences

in vivo but the degree non prolif-

The different

and non proliferating

cells,

in the affinity of Pl for both S.S. and d.s. DNA when comparing

Pl in PCA extracts from rat liver and HeLa cells. It might also explain the broad elution profile of Pl when compared to other proteins present in the PCA extract. Since dephosphorylation in a crude PCA extract dramatically decreases

the affinity of Pl for DNA while the binding of Hl ( Fig.2

and 3 ) is not affected, the DNA binding can not be solely due to electrostatic basic regions in Pl and the anionic phosphate

backbone

In contrast to HMG I ( 22 ) and probably poly dA - poly dT

( unpublished

of the proteins

nucleolin

interactions

between

of DNA. ( 23 ). Pl does not bind preferentially

results ). Purified Pl from rat liver does not introduce chromatin

*

Pl abcdef

ghijk

I

m western lmmunobbts of a SDS polyacrylamide gel of protein fractions obtained when fractionating PCA soluble proteins from prollferatlng HeLa cells on d.s. DNA cellulose. The proteins were eluted with a salt gradient from 0,05 - 0,60 M NaCl and oroteins in 5ml pooled fractions Separated

to

by SDS gel electrophoresis.

Proteins

not attached

at 0,05 M NaCl

(a),

proteins

eluted

0.07 M (b), 0,12 M(c), 0,17 M (d), 0,23 M (e), 0.28 M (f), 0,34 M (g), 0,38 M (h), 044 M (i), 0,53 M (j), 0,60 M (k), 2,0 M NaCl (I).

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at

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185, No. 3, 1992

condensation nucleosome

when microinjected assembley

AND BIOPHYSICAL

into starfish oocytes ( unpublished

( D.Tremethick,

personal

communication

proteins have been shown to be highly phosphorylated probably

regulated

RESEARCH COMMUNICATIONS

data ) and Pl has no effect on ). Several transcriptional

( 24,25 ) . The bioactivity of these factors iS

by multiple kinases in vivo ( 26 ). Sequence

specific DNA binding by a bovine

testes ATF / CRE binding factor is dependent

on the level of phosphorylation.

when the natural protein is dephosphorylated

or when it is hyperphosphorylated

in vitro ( 27 ). The effect of Pl on transcription The broad elution heterogenous sequences

in DNA.

and replication

The binding is reduced

population

by protein kinase A

has however not yet been tested

profile of Pl from both S.S. and d.s. DNA columns

phosphorylated

activator

might be due to a

of Pl molecules or that Pl has different affinity

Pl might also be part of a heterodimer

for

different

or multimer complex in vivo and hence

difficult to test by employing in vitro systems. The findings that Pl is a substrate for several kinases in vivo and in vitro, and that it remains bound to DNA at physiological highly regulated

ionic strength, indicate however a

nuclear function in vivo.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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