Adenovirus type 2 coded single-stranded DNA binding protein: in vivo phosphorylation and modification.

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JOURNALOF VIROLOGY, May 1977, p. 402-411

Vol.22,No. 2

Printed in U.S.A.

Adenovirus Type

2 Coded Single-Stranded DNA Binding

Protein:

In

Vivo

Phosphorylation and Modification

YUN-HUA JENG,* WILLIAM S. M. WOLD, KENJI SUGAWARA,' ZVEE GILEAD, AND

MAURICE GREEN

InstituteforMolecular Virology, Saint Louis UniversitySchoolofMedicine, St. Louis, Missouri 63110

Received for publication 30 November 1976

The adenovirustype2-coded single-strandedDNAbinding protein (DBP) was shownto be aphosphoprotein and to exist in at least two forms that differ in mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After a 30-min pulse with [35S]methionine or 32PO4, 35S- or 32P-labeled DBP had a nominal molecularweightof 74,000, whereas aftera30-minlabel followed bya 20-h chase, 35S- and 32P-labeled DBP hadanominalmolecular weight of77,000. Both large and small forms of 35S-and32P-labeledDBPboundtosingle-stranded

DNA-cellulose columns andwereeluted by0.4 to 0.6 MNaCl; both forms also wereimmunoprecipitated byantiserumagainst adenovirustype 1-simian virus 40-inducedtumorcells(thisantiserumcontainsantibodiesagainstDBP) andby

monospecific antiserum against95 to 99% purified DBP. Withhighlypurified

32P-DBPlabeled7 to 10 hpostinfection, it wasshownthat the32p radioactivity was firmly associated with protein material (i.e., not contaminating nucleic acids or phospholipids) and had properties expected of a phosphoester of an aminoacid;paperelectrophoresisof acidhydrolysatesof thispreparation identi-fied phosphoserine but notphosphothreonine. Phosphoserine but not phospho-threonine was also identified in acid hydrolysates of another preparation of 32P-DBP labeled for30 min, chased for20 h, and then immunoprecipitated by adenovirus type 1-simianvirus 40 antiserum.

Human adenovirus 2 (Ad2) is an oncogenic DNA virus that replicates in the nucleus of permissive human cells (reviewedinreference 18 and in W. S. M. Wold, M. Green, and W. Buttner,In D. P.Nayak, ed.,Molecular Biology

ofAnimal Viruses, in press). The productive infection proceedsin an"early"stage,when23 to 32%of the asymmetricgenome isexpressed asmRNA, andin a"late"stage,which follows the initiation of viral DNA replicationat 6 to 7 h postinfection (p.i.). Virus-coded or -induced proteinssynthesized earlyafter infection

prob-ably are involved in viral DNA replication, regulation of viral gene expression, and trans-formation ofnonpermissive cells. A protein of 75,000 (75K) daltons, which binds to single-stranded DNAbutnotto native DNA, is pro-ducedinlarge quantities in early stages of Ad2 infection(24, 27). Ananalogous Ad5DNA bind-ing protein (DBP) of 72K was isolated from abortively infectedmonkey cells (28). Recently anAdl2DBPof 58K to 60K has beendescribed (29). Ad2 and Ad5 DBPs of 41K to 45K are also

IPresentaddress: Department ofMicrobiology,Faculty ofMedicine, Kyoto University,Konoe-cho,Yoshida, Sakyo-hu,Kyoto, Japan606.

synthesized, butpeptide analyses (20), genetic

studies (29), and immunoprecipitation studies (27a) indicate that these are derived from the larger DBP (possibly degradation products). Ad2 DBP has been showntobe viral codedby cell-free translation of hybridization-purified Ad2 earlymRNA into a 72Kpolypeptidewith a peptide map similar to that of DBP isolated from infectedcells, and theDBPgenehasbeen

mapped in Ad2 EcoRI-B fragment (Ad2 map position, 0.59 to 0.71) (16). Recently, Ad2 DBP has been purified to 95 to 99% homogeneity; sucrosegradientandgel filtration studies indi-cate that it does not have a typical globular protein structure (27).

DBPisprobably involved in viral DNA repli-cation, since itis found in both nuclear mem-brane and soluble Ad2 DNA replication com-plexes (24, 33, 34; M. Arens, T. Yamashita, R. Padmanabhan, T. Tsuruo, and M. Green, man-uscript in preparation). Genetic studies indi-cate that DBP functions in the initiation of viral DNAreplication (30). DBP issynthesized inlargequantities, which suggests that its role is stoichiometric rather thancatalytic (28), as inthecase of T4gene 32protein (1). Many Ad2-402

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PHOSPHORYLATED Ad2 DNA BINDING PROTEIN 403

transformed cells neither containtheDBPgene (23) nor synthesize DBP (15, 27a; P. H. Galli-more, personal communication), indicating that DBP does not function to maintain cell transformation.

Many cellular processes are controlled by modifications, such as adenylation (10), meth-ylation (4), and phosphorylation (22), of key regulatory proteins. We, therefore, investi-gatedwhether Ad2 DBPundergoes posttransla-tional modification. Inthiscommunication, we demonstrate that Ad2 DBP is phosphorylated atserineresidues, and that at least two forms of DBPexist that differ inmobility by sodium dodecyl sulfate (SDS)-polyacrylamide gel elec-trophoresis.

MATERIALS AND METHODS

Chemicals. [35S]methionine (Met) (400Ci/mmol),

[3H]leucine (40Ci/mmol), and inorganic32P04 (car-rier-free) were obtained from New England Nuclear Corp.;

1-,f-D-arabinofuranosylcytosine

(ara-C),

phos-phoserine, phosphothreonine, pancreatic DNase

(EC 3.1.4.5), and Pronase were secured from Sigma Chemical Co.; acrylamide came from

East-man Kodak Co.; N,N-methylenebisacrylamide,

N,N,N',N'-tetramethyladenediamine (TEMED),

and ammoniumpersulfate were procured from

Bio-Rad Laboratories; SDS was bought from BDH

Chemical, Ltd.; pancreatic RNase A (EC 2.7.7.16)

and Escherichia coli alkaline phosphatase (EC

3.1.3.1) wereboth obtained from Worthington

Bio-chemical Corp.; and pancreatic RNase T, was

ac-quiredfromCalbiochem.

Preparation of labeled protein extracts. Human

KBcells (clonal line) were grown at370CinEagle

minimal essential medium (MEM) containing 5%

horse serum. Cells were infected at 6 x 106cells/ml

with Ad2 (strain 38-2) at 100 PFU/cell in MEM

without horse serum. The purification of Ad2 has

beendescribed (9). After1hofadsorption,cells were

suspendedat 3.5 x 105cells/mlinMet-free or phos-phate-free MEM with 5% horse serum. At 4 h p.i.,

ara-Cwasadded to aconcentrationof 20

gg/ml.

At

0.5 h priorto labeling, cells werecentrifuged and

resuspended at 6.5 x 105cells/ml in warm Met-free

orphosphate-freeMEMwith5%horseserumand20

,ugof ara-Cper ml. Cellswerepulse-labeledfrom 7

to 7.5 h p.i. with 25 ,uCi of [35S]Metor0.5 mCi of

32PO4 per ml. Chased samples were prepared by

washing cells twice in warm, complete MEM

con-taining 5% horse serumand20,.gof ara-C perml,at

the endof thelabelingperiod, andthenincubating

cellsinfresh washmedium for20h.Harvested cell

pellets were washed twice with cold

phosphate-buffered saline lackingCa2+ and Mg2+. Cellswere

thensuspendedat 107cells/mlinreticulocyte

stan-dardbuffer (10 mM Tris-hydrochloride[pH 7.4], 10

mMNaCl, 1.5mMMgC12), placedinanicebath for

15min, andsonicallydisrupted. Cellextracts were

digestedwith 25 Mugof DNaseperml at37°Cfor1h

in 5 mM MgCl2. EDTA wasadded to 10mM, and

extractswere furtherdigested with 20 ,ug each of

RNase A andT1 permlat370C for 30 min.Extracts

were thendialyzed for 16 h at40C against 10 mM Tris-hydrochloride (pH 7.5) and 1 mM 2-mercapto-ethanol, and the samples wereeither analyzed di-rectlyorstoredat -70°C. Mock-infected protein

ex-tracts were similarly prepared, except that virus

wasnotadded.

Analytical and preparative SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel experi-ments were prepared as described (11, 14, 26).

Gra-dientgels of8to 21%polyacrylamide with 0.1%SDS

were formed as slabs between glass plates; slabs

were 1.5 mm thick and 100 or 270 mm long. A

stacking gel of 5% polyacrylamide and 0.13% bis-acrylamide (analytical gels contained up to 20 sam-ple wells) was cast above the separating gel.

Elec-trophoresis was performed at 7°C with a constant

currentof20mA. After electrophoresis, analytical

gelsweredried under vacuum and autoradiographed

with Kodak single-coated panoramic dental X-ray

film (DF-85). Apparent molecular weights of

pro-teinswereestimatedby comparison of their

migra-tion rate in SDS-polyacrylamide gels with known

molecular weight standards. Cylindrical gels (0.6 by

10cm) containing 6%polyacrylamide were

electro-phoresed at 2 mA/gel for 3 h.Gels were fractionated

into 2-mm slices with aGilson gelfractionator and

counted in 5 ml ofAquasol in a liquid scintillation

counter.

To localize protein bands in preparative gels, a

lightly loaded analytical gel with labeled protein

markers was run in parallel with each batch of

preparative gels. The gels were kept wet during

autoradiography, and thelocalized phosphoprotein

bands were cut out and elutedfrom gels with

agita-tion at 37°C in 10volumes of 5mMNaHCO3

contain-ing 0.05% SDS. After 16 h, acrylamide fragments

wereremoved by centrifugation, and the

superna-tant was dried. A small portion ofprotein from

preparative gels was checked forpurity by

analyti-calSDS-polyacrylamide gelelectrophoresis.

Immunoprecipitation of DBP from crude

ex-tracts and DNA-cellulose purified materials.

Indi-rect radioimmunoprecipitation was carried out as

described (7, 8) with minormodifications.

Immuno-globulin G (IgG)wasisolated fromanantiserum (a

gift from R. Gilden) against extracts of Adl-simian

virus 40(SV40)-induced hamstertumorcells(6); this

antiserum contains antibodiesto DBP(7, 8).

Sam-ples from infected and mock-infected extractswere

mixed with 20 Mg of Adl-SV40 IgGornonimmune

IgG in 240-,ul reaction mixtures and incubated for 18

hat4°C.Then45 ul of goat serumanti-hamster IgG

wereadded and incubation continued for2hat37°C.

Immunoprecipitates were collected by

centrifuga-tion for 1.5 min at 10,000 x g in a Beckman 152

microfuge, washed threetimeswith150M1 of

phos-phate-buffered saline containing0.5 M urea, 0.5%

Nonidet P-40, and1%deoxycholate, suspendedinan

equal volume of gel electrophoresis sample buffer

(11), and electrophoresed on SDS-polyacrylamide

gels.

Someimmunoprecipitations wereconductedwith

guineapigIgG against95to 99%purifiedDBP

(anti-DBP) (27a); these utilized a modified

radioimmuno-precipitation buffer (10mM sodiumphosphate [pH

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404 JENG ET AL.

7.2], 150 mM NaCl, 1% Triton X-100, 1% sodium

deoxycholate, and 0.1% SDS) for both incubation

and washing (5). Portions of DBP-containing

elu-atesfrom DNA-cellulosewereadjustedto1x

radio-immunoprecipitationbuffer and mixed with 60 Ag

ofanti-DBP guineapig IgGornonimmune guinea

pigIgG; the reactionswere thenincubated for 18 h

at4VC. Then 60 pul of goat serum anti-guinea pig

IgG were added to form theimmunoprecipitates.

Purification of32P-labeled DBP. Adetailed

de-scription of the DBP purification method is

pre-sented elsewhere (27). Briefly, infected cells were

simultaneously labeled with 2 jtCi of [3H]leucine

and 0.5mCi Of32PO4per mlfrom7to10hp.i.inthe

presence ofara-C. Cytoplasmic extracts were

pre-pared and chromatographed on a single-stranded

DNA-cellulose column (24). Bound proteins were releasedfrom the columnby stepwise elution with

buffer A (20 mMTris-hydrochloride[pH 8.1],50mM

NaCl, 5 mMEDTA, 1 mM2-mercaptoethanol, and 10%glycerol)containing0.05M, 0.2 M, 0.4 M, 0.6 M,

and 2.0 MNaCl,respectively.Fractionseluted with

0.6 M NaCl were pooled, treated with 1% sodium

deoxycholate for 1 h at 40C, and dialyzed against

buffer B (10 mMTris-hydrochloride[pH7.5], 5mM

NaCl, 1 mM 2-mercaptoethanol, 0.02% Nonidet

P-40, 0.4 M urea, and 10% glycerol). The dialyzed

material wasthenappliedto aDEAE-Sephadex

col-umnequilibrated withbufferB, andproteins were

eluted withalinear gradientofbufferBand 0.3 M

NaCl inbufferB.The major elutedradioactivepeak

containing DBP was pooledand analyzed by SDS-polyacrylamidegelelectrophoresis.

Identification of phosphorylated amino acids. 32P-labeled DBP purified by DNA-cellulose and DEAE-Sephadexchromatographywasfurther puri-fied by electrophoresis in preparative SDS-poly-acrylamidegels asdescribedabove. The32P-labeled

DBPband was eluted from the gel and dried, and

thedriedresidue washydrolyzedin 1mlof 6 N HCl at 1100C for2 h. A 2-h period was selected as the hydrolysistime tomaximize hydrolysis ofthe

pep-tide bondwhilelimitinghydrolysisof the

phospho-rylated amino acid. Under theconditionsofpartial

acid hydrolysis used, about 20% of the liberated phosphoserine would be hydrolyzed (12). The hy-drolysatewasdriedanddissolved in 16pjlofwater containing 40 ,ug ofunlabeled phosphoserine and phosphothreonineeach andsubjectedtopaper elec-trophoresis (2). Electrophoresis wascarried out on

Whatman no. 1filterpaper with a formicacid-acetic

acid-water (30:90:880)mixture (pH 1.9) at 600 V for

16 h at 40C. The paper was dried, and unlabeled

phosphoamino acidmarkers developed with

ninhy-drinreagent (Sigma). Theposition ofradioactivity

wasdeterminedwith aPackardradiochromatogram

scanner.Rechromatographywasperformedby

elut-ing theradioactive spots from the paper

chromato-gram withwater andsubjectingthe driedmaterial

topaperelectrophoresisasdescribed above.

RESULTS

Pulse-chase experiments showing that

35S-and 32P-labeled DBP decrease in mobility in

SDS-polyacrylamide

gel

electrophoresis

dur-ing

chase

periods.

Ad2-infected and mock-in-fected KB cellswere labeled with [35S]Met or

32PO4

for 30

min,

orfor 30 minfollowed

by

a

20-h chase. To prevent the transition to late

stages

of

infection,

ara-C was added 4 h

p.i.,

and cellswerelabeled in thepresenceof ara-C. Whole cell extracts were

prepared,

and

poly-peptides

wereresolved

by

SDS-polyacrylamide

gradient

slab

gel

electrophoresis. Figure

1 shows an

autoradiogram

ofone such

experi-ment. After a 30-min

pulse,

35S-labeled DBP

migrated

withan apparent molecular

weight

of 74K (lane

A),

but after a 20-h chase the

apparent

molecular

weight

of DBP had in-creasedto 77K (lane C). The

35S-labeled

DBP band was not

present

in lanes

representing

pulse

(lane B)- or

pulse-chase

(lane

D)-la-beled,

mock-infectedcellextracts.

Analyses

of

proteins

labeled with

32P04

indicated that

DBP,

and no other Ad2-induced

polypeptide,

was associated with the

phosphate

label (in

Fig.

1 cf. lane E with

F,

and lane G withH).

The 32P-labeled DBP behaved like35S-labeled

DBP in

pulse-chase

experiments:

32P-labeled

DBP hada nominal molecular

weight

of 74K

aftera30-min

pulse

(lane E) and 77K aftera

20-h chase (lane G). In

Fig.

1,

there are no

apparent

virus-specific

32P-labeled

polypep-tides of 41Kto45K

daltons, i.e.,

representing

the DBP

subspecies

oftheseapparent molecu-lar

weights

(20).

Apparently

the 32P-labeled

DBP

subspecies

were present in insufficient amounts tobeobserved in the

autoradiogram,

because in other experiments these

polypep-tides were shown to be

phosphorylated

(data

not

shown).

Immunoprecipitation by

Adl-SV40

IgG

of

pulse-labeled

and

pulse-chase-labeled

35S-and

32P-DBP.

Antisera

against

cell extracts of

Adl-SV40-andAd2-SV40-induced hamster tu-morcells containantibodiestoAd2 DBP(7,8).

Asshownin

Fig.

2,

IgG

fromAdl-SV40

antise-rum

precipitated

35S-labeled DBP from cells labeled for 30 min

(lane

F),

or for 30 min followed

by

a20-h chase (lane J). The

immu-noprecipitated DBPs showed the same appar-ent molecular

weight change

as untreated

samples

(lanes A and

C,

pulse-labeled and

pulse-chase-labeled

infected cells); i.e., the chased DBP hada

slightly

reducedmobilityin the

gels.

In control experiments, IgG from nonimmunehamstersdidnot

immunoprecipi-tate 35S-DBP from pulse-labeled (lane E) or

pulse-chase-labeled

(lane I) infected cell

ex-tracts;

also,

no 35S-labeled polypeptides were

immunoprecipitated

from mock-infected cell extracts

by

Adl-SV40

IgG

(pulse-lane

H,

chase-lane L) or normal hamster

IgG

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http://jvi.asm.org/

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ABC

D E F

G

H

4

I.

4

77K>

s4

74KI

*Pd

Hi9*

1"n s * P;4

_¢ ¢ 9* <>

n"= z

i__

_V

b__* ..*

u * * is ^

u

"g2.:

_ b ^

.,-_

u___*w _.w.

_

h __

-=-S0|i -

wt-~ ~ V

IMI

mlI

M I M

C

35 32

S -met PG 4

FIG. 1.Autoradiogram of SDS-polyacrylamide

gel electropherogram showing two electrophoretic forms(anapparentmolecularweight of74K,aftera

30-mmn pulse,and77K,aftera30-mmn pulse followed

bya20-h chase) of35S- and 32P-labeled DBP. Ad2-infected (I) and mock-infected (M) KB cells were

pulse-labeled with [358]Metor32P04, 7 to 7.5 hp~i. (p),or7to 7.5hp.i.followed bya20-hchase in thepresenceofara-C. Total cellproteinextractswere

prepared andanalyzed by SDS-polyacrylamide gel

electrophoresis followed by autoradiography. (AB)

us;(C,D) pulse-chase; (EF)3) pulse;

(0 pulse-chase.

(pulse-lane G, chase-lane K). Adl-SV40 IgG also precipitated32P-DBP from both pulse (lane R)- and pulse-chase-labeled (lane V) in-fected cell extracts. Again, the chased 32P-la-beledDBPhadaslightlyreducedmobility. As with 35S-labeled DBPs, the 32P-labeled DBPs immunoprecipitated from pulsed and pulse-chased infected cellextractshad the same ap-parentmolecularweight as 32P-labeledDBPin samplesnotimmunoprecipitated(lanesMand 0). Controlimmunoprecipitationexperiments performedwith32P-labeledinfected and mock-infected cell extracts revealed no detectable 32P-labeledDBP (lanesQ, S,T, U, W, andX). Thus DBP-specific antisera immunoprecipi-tated74Kand 77K formsof both 35S- and32p_ labeled DBP. High-titer monospecific guinea pig antiserum against 95 to 99% purified DBP also precipitated 35S- and 32P-labeled DBP (data notshown).

Pulse-labeled and pulse-chase-labeled 32S-and 32P-DBPs that bind to single-stranded DNA-cellulose. Ad2 DBP binds to single-stranded DNA and canbe eluted from single-stranded DNA-cellulose with0.6 M NaCl (24). Totestwhether both74K and 77K forms of 35S-and32P-labeledDBPhavea similaraffinityfor DNA, infectedand mock-infected KB cellswere labeled with V35S]Metor32PO4for 30min(or for 30minfollowedby a 20-h chase), and cytoplas-micextracts were chromatographed on single-stranded DNA-cellulose columns. About 90 to 95% of the labeledproteins passed throughwith

the void volume in 50 mM NaCl. The column was washed with buffers of increasing ionic

strength(seeabove). Proteins eluted with0.6 M NaCl were electrophoresed on SDS-polyacryl-amide gels. As shown in Fig. 3, pulse-labeled 35S-DBP(lane A) and 32P-DBP (laneE), aswell as pulse-chase-labeled iS-DBP (lane C) and 32P-DBP (lane G), boundto DNA-celluloseand were eluted by0.6 MNaCl. Both pulse-chased 35S- and32P-labeled DBPs had lower mobilityon

the gel. Thus both 74K and77K forms of ",S-and 32P-labeled DBPs bind to single-stranded DNA and are eluted from the column at the sameNaCl concentration.

Purification of 32P-labeled DBP by DNA-cellulose and DEAE-Sephadex chromatogra-phy and preparative SDS-polyacrylamide gel electrophoresis. The experiments described above demonstrate that 32p labelisapparently firmly associated with DBP (i.e., notremoved by SDS and by chromatographyon DNA-cellu-lose). For further studiesonthephosphorylated component, 32P-labeled DBP was purified by sequential DNA-cellulose and DEAE-Sephadex

chromatography (which yieldsDBP of95to99% purity) (27), followed by preparative SDS-gel

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[image:4.504.50.242.57.550.2]

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3EKFIH

K

LMNO,

1>

~~~~~~~~~A.a

ii~.~ ~ ~ ~ ~ ~ ~ * 4

FIG. 2. Autoradiogram ofSDS-polyacrylamide gel electropherogram showingthatAdl-SV4OIgG

immu-noprecipitatesboth 74K(seen after30-mmnpulse)and 77K(seenafter30-mmnpulse followedby20-h chase) forms of35S- and32P-labeledDBP.Ad2-infectedandmock-infectedcellswerelabeled with[35S]Met or32O (from7to7.5hp.i.,or7to 7.5hfollowedbya20-hchase),totalcellextractswereprepared,and immuno-precipitationexperimentswereperformedwithAdl-SV4Oand nonimmunehamsterIgGs.AthroughDand M throughP,respectively,show 35S- and32P-labeledextractselectrophoresed before immunoprecipitation:

pulse-labeledextractsare inA(Ad2 infected),B(mockinfected), M(Ad2infected),and N(mock infected);

pulse-chase-labeledextractsareinC(Ad2infected),D(mockinfected),0(Ad2infected),and P(mockinfected).E

throughL(35S-labeledextracts) andQthroughX(32P-labeledextracts)show the resultsofthe

immunoprecipi-tationexperiments. (EandQ)Infected pulse-labeledcellsversusnonimmunehamsterIgG;(FandR)infected

pulse-labeledcells versus Adl-SV4OIgG; (GandS) mock-infected, pulse-labeled cells versus nonimmune IgG; (Hand T) mock-infected,pulse-labeledcells versusAdl-SV4O IgG;UIand U) infectedchase-labeled

cellsversusnonimmuneIgG; (J andV)infectedchase-labeled cellsversusAdl-SV4OIgG;(KandW)

mock-infected,chase-labeled cellsversus nonimmuneIgG;and(LandX)mock-infected,chase-labeled cellsversus Adl-SV4OIgG.

electrophoresis. Infected cell proteins were

la-beled lightly with [3H]leucine and copurified with 32P-labeledproteins. Each stage of purifi-cation was monitoredby gel

electrophoresis.

A labeling time of7 to 10 h p.i. was chosen to obtainmaximal synthesis of DBP,asindicated by preliminary experiments. The cytoplasmic extract was adsorbed to a single-stranded

DNA-cellulose column, and bound proteins were eluted with buffers of increasing ionic strength. 32P-labeled polypeptides eluted with each NaCl concentrationwereanalyzedbygel

electrophoresis,

and the results are shown in Fig. 4. Lanes A and B, respectively, show marker 35S-labeled polypeptides from infected and mock-infected cell extracts; these extracts werepreparedby the"cycloheximide

enhance-ment" procedure to increase the levels of la-beledAd2-inducedearly polypeptides(11). Ad2-induced

polypeptides

of 74K (pulse form of

DBP), 21K, 19K,

17.5K (faint), 15K,

11.5K,

11K, and 8K are apparent in this autoradi-ogram.

Figure

4shows the32P-labeled

polypep-tides presentin totalcell(laneC)and

cytoplas-mic (lane D)extracts, and thefollowing NaCl

eluates from DNA-cellulose: 0.05 M (lane

E),

0.2 M(laneF), 0.4M (lane G), 0.6M (lane

H),

and 2.0 M NaCl

(lane

I). 32P-labeledDBPwas elutedmainlywith 0.6 MNaCl (lane H)andto alesserextentwith0.4MNaCl (laneG). Thus

32P-labeled DBP is eluted from DNA-cellulose

atthesameNaClconcentrationsas3H-labeled DBP reported previously (24, 28). 32P-labeled

DBP is notapparent inlanes C and D,

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PHOSPHORYLATED Ad2 DNA BINDING PROTEIN

A

BCD E F

G

H

0

--

#1

.4

DEAE-Sephadex. 32P-labeled proteins co-chro-matographed with the 3H-labeled proteinsthat wereused to monitor the purification. The peak fractions were pooled and analyzed by electro-phoresis on cylindrical gels. The 3H- and 32P_ labeled DBP comigrated in the cylindrical gel

(datanotshown), suggesting the covalent asso-ciation of 32p label with purified DBP. The re-maining material was electrophoresed on a pre-parative slab gel; the band correspondingto32p_ labeled DBP was cut from the gel, and the 32p_ labeled DBP was eluted and chemically ana-lyzed as described below.

DBPcontains phosphoserine but not phos-phothreonine. The phosphate linkage proper-ties of the 32P-labeled DBP band isolated by preparative gel electrophoresis, as described

M

I

Ml

Mi

I

M

p c p IC

[image:6.504.50.241.61.383.2]

35S

-met

32P04

FIG. 3.Autoradiogram of SDS-polyacrylamide

gel electropherogram showingthatboth74K(pulse)

and 77K(chase) forms of 35S-and 32P-labeledDBP

bindtosingle-stranded DNA-cellulose columnsand

are elated by 0.6 MNaCl. Ad2-infected (I)

mock-infected (M) cellswerepulse-labeled with [35S]Metor

32PO47to 7.5 hp.i. (p),orthesameperiod followed

bya20-h chase(c). Cytoplasmic extracts were

pre-pared and chromatographed on single-stranded

DNA-cellulose columns. Bound proteinswereelated

with 0.05M,0.4M,and 0.6 MNaCl. Theproteins

elated by 0.6 M NaCl were analyzed by

SDS-poly-acrylamide gel electrophoresisandautoradiography.

(A,B) 35S pulse; (C,D), 35Spulse-chase; (E,F) 32p

pulse; (G,H) 32ppulse-chase.

senting total cell and cytoplasmic extracts (which contain DBP),becauseequal

radioactiv-ity was applied to each lane, and 32P-labeled

DBPwas enriched in the0.4 and 0.6 M NaCl

eluates from DNA-cellulose. The 74K 32P-la-beled polypeptide eluted from DNA-cellulose

was showntobe DBPby immunoprecipitation of both the 0.4 and 0.6 M NaCl eluates with monospecific IgG against purified DBP (data not shown).

The 0.6 M NaCl eluate from DNA-cellulose was further purified by chromatography on

74K

21 K

19K

17. 5K

. t

15K

11.5K

K

1K

-I

8K-A B

C

D

E

F G

H

I

ii

* * 0

_T.

__., I

S

[image:6.504.255.449.256.485.2]

I M

FIG. 4. Autoradiogram of SDS-polyacrylamide

gel electropherogram showing the 32P-labeled

poly-peptides elated from a single-stranded

DNA-cellu-lose column by various NaCl concentrations.

Ad2-infectedcellswerelabeled with32PO47 to 10 hp.i.,

and thecytoplasmicextractwaschromatographedon

asingle-strandedDNA-cellulose column. The bound

proteinswereelated withincreasingNaCl

concentra-tions.(A)Ad2early infectedKB cells extracts labeled

with[35S]Met 7to8 hp.i. in thepresenceofara-C

after cycloheximidepretreatment(i.e.,Ad2early

pro-teinmarkers); (B) mock-infected,35S-labeled cell

ex-tractstreated thesame asinfected cells. (C through I)

show 32P-labeled polypeptides as follows: (C) total

cell extracts; (D) cytoplasmic extract before

DNA-cellulose; (E)DNA-celluloseflow-through and0.05

MNaCl wash; (F) 0.2 M NaCl eluate; (G) 0.4 M

NaCI eluate;(H) 0.6MNaCleluate;and (I)2.0M

NaCleluate.

77K

74K

lt

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408 JENG ET AL.

above, were analyzed. As shown in Table 1,

treatment with hot trichloroacetic acid or

or-ganicsolvents didnotsolubilize significant 32p counts,suggesting that the 32p labelwas

associ-ated with protein andnotwith RNAorDNAor

phospholipidscontaminatingtheDBP

prepara-tion. Pronasetreatmentcompletely solubilized the32P counts,which establishesthatthe radio-activity was associated with DBP. The

phos-phate linkage was at most 20% resistant to treatmentwith alkali, anditwas90%resistant

toacid,asexpected foraphosphoester of serine orthreonine; this excludedphosphohistidineas a major component of DBP, because

phos-phoimide linkages are stable to alkalai and sensitive toacid (10, 19). Consistentwiththis,

alkaline phosphatase partially solubilized 32P

counts, indicating a phosphomonoester bond

(17).

To identifythe phosphoamino acid, thesame

preparation of 32P-labeled DBP was partially

hydrolyzed bytreatmentwith6NHClat110°C for 2 h, mixed with 40 ,g each of "carrier" phosphoserine andphosphothreonine, and elec-trophoresedonpaperatpH 1.9. Phosphoserine andphosphothreonine markers,notmixed with sample,werealso electrophoresed. The

electro-pherogramwas developed with ninhydrin and

then autoradiographed. The carrier phospho-serineandphosphothreonine inthesample mi-grated slightly behind the marker phospho-aminoacids, possibly becausetrace amountsof materials (e.g., oligoacrylamide) had retarded the mobility of phosphoamino acidsinthe

sam-ple. Tworadioactivespotsappearedonthe

au-toradiogram of thiselectropherogram:one

coin-cided with inorganic phosphate and the other with the carrier phosphoserine ninhydrin spot inthe sample; thissuggeststhat serine butnot threonineresidues ofDBParephosphorylated.

To verify that the radioactivity was in

phos-phoserine, the carrier phosphoserine spot was

eluted, evaporated repeatedly, and reelectro-phoresed on paper. As shown in Fig. 5, the

radioactivity again coincided with phosphoser-ine andinorganic phosphate. In the reelectro-phoresisexperiment,themobility of the carrier phosphoserine inthe samplecoincided exactly with the markerphosphoserine. These results indicate thatserine butnotthreonine residues of DBP arephosphorylated. The 32p04 spot on

thepaperelectropherogramspresumablyarose

by hydrolysisof32P-labeledphosphoserine resi-dues of DBP, because the first electrophoresis step should have eliminated inorganic phos-phate from thecarrierphosphoserine.

In the above experiment, DBP was labeled

from7 to10hp.i.,sothatitisnotclear whether

TABLE 1. Propertiesof the phosphatelinkage

associated withhighly purified 32P-labeled DBPa

Trichloroacetic Treatment acid-insoluble 32p

counts(%)

Control 100

10%Trichloroacetic acid, 20 min at 100

900C

10%Trichloroacetic acid, 20 min at 100

90'C plus ethanol-ether (3:1)

ex-traction, 30 min at 60'C

0.1 N HCl, 15 min at 1000C 90

0.1 N KOH, 15 minat1000C 25

Pronase, 1 h at370C 0

Alkaline phosphatase, 1hat370C 30

a Portions corresponding to about 500 cpm were treated with trichloroacetic acid, trichloroacetic acid

plus organic solvents, HCl, KOH, Pronase, or

alka-line phosphatase (19). One hundred ,ug of bovine

serum albumin wasadded at the end of the

incuba-tion, and the proteins were precipitated with cold

10% trichloroacetic acid. The acid-insoluble

materi-als were collected on glass-fiber filters, washed,

dried,and counted forradioactivity.

both the pulse and pulse-chase forms of DBP contain phosphoserine. To demonstrate that the chased form contains phosphoserine, Ad2-infected cells were labeled with32P04,from7to 7.5 hp.i., thenchasedwith normal MEM for20 h. Total cellular proteins were extracted and immunoprecipitated by Adl-SV40 IgG; then the immunoprecipitates were electrophoresed on SDS gels. As described above, the 32P-labeled DBPband was cutfrom the gel, the DBPwas eluted andwasacid hydrolyzed, andthe hydrol-ysatewassubjectedtopaperelectrophoresis.As shown inFig. 6, the 32P countscoincided with phosphoserine and inorganic phosphate. Thus

the chased formof DBP containsphosphoserine

but notphosphothreonine.

DISCUSSION

Wehave shown that Ad2DBPis

phosphoryl-ated as indicated by (i) identification of 32p_

labeled DBPininfected cell extracts, (ii) bind-ingand elution of32P-labeledDBPfrom

single-strandedDNA-cellulose, (iii) immunoprecipita-tion of32P-labeled DBP by Adl-SV40IgG and monospecific anti-DBPIgG, and (iv) identifica-tionof32P-labeledserine inpurifiedand immu-noprecipitated DBP. Pulse-chase experiments also indicated that DBP exists in atleasttwo forms that differinmobility by

SDS-polyacryl-amide gel electrophoresis. After a short (30 min) pulse, DBP had an apparent molecular

weightof 74K, but, afterapulse followedby a 20-h chase, the apparent molecular weight of DBPincreased to77K, and the74Kformwas no

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MI g 9 P-serine P-ttreonire

FIG 2

FIG. 5. Identification of32P-labeledphosphoserine inhighlypurified32P-labeledDBP.Ad2-infectedcells

werelabeled with 32PO4from 7 to 10 h p.i., and32P-labeled DBP was purified from cytoplasmic extracts by

sequential chromatography on DNA-cellulose and DEAE-Sephadex (27), followed by preparative

SDS-polyacrylamide gel electrophoresis. The32P-labeledDBPband was elated from the gel, partially hydrolyzed in

HCl,mixed withunlabeled "carrier" phosphoserine and phosphothreonine, and electrophoresed on paper as

described inthetext.Phosphoserine andphosphothreonine"markers" (i.e.,notmixed with the 32P-labeled

DBPsample) were also electrophoresed. The "carrier"phosphoserine and phosphothreonine inthe sample

migrated slightly behind the marker phosphoserine and phosphothreonine. The radioactivity coincided with phosphoserineinthe sample and with inorganic phosphate. The phosphoserine radioactive spot was elated

fromthe paper chromatogram and electrophoresed a second time. The results of the second electrophoresis are

shown in this figure. Ninhydrin spray was used to locate the position of unlabeled amino acids, and a Packard radiochromatogram scanner was used to determine the position of radioactivity. The origin is located to the

extremeright of the paper scan.

+ pPi P-serine P-threonine

4 e | *4-is

I t

---

-

.3

_---_-#S

_

L

---

'3

-

-FIG. 6. Identification of 32P-phosphoserine in pulse-chase labeled 32P-DBP after immunoprecipitation by

Adl-SV4OIgG and preparativeSDS-polyacrylamide gel electrophoresis. Ad2-infected cells were labeled with 32PO4,from 7 to 7.5 h p.i., then chased in normal MEM for 20 h. 32P-labeled DBP was immunoprecipitated

from cytoplasmic extracts byAdl-SV4O IgO, then electrophoresed onSDS-polyaciylamidegels. 32P-labeled

DBP waselatedfrom the gel, acid hydrolyzed, mixed with carrier phosphoserine and phosphothreonine, and

subjected to paperelectrophoresis. The figure shows the correspondence between sample radioactivity and

carrier phosphoserine present in the sample. "Marker"phosphothreonine and phosphoserine (i.e., not mixed

with the sample) had the same electrophoretic mobilities as"carrier"phosphoserine and phosphothreonine in

the sample.

longerobserved.Both74Kand 77Kforms

of35S-and 32P-labeled DBPboundto single-stranded

DNA and were immunoprecipitated by Adl-SV40IgG.

Other workers have commented on altera-tions ofAd2 DBP mobility on

SDS-polyacryl-amide gels. Anderson et al. (3) reportedthat late after infection Ad2 DBP appeared as a diffuseband, and occasionallyas adoublet. In studies of viral-specific proteins during late stages ofinfection, by Walter andMartin (31) withAd2-SV40hybrids,and Weber(32)withan

Ad2latepolypeptide-processingmutant, a sim-ilar reduction inthe mobility of this polypep-tide (presumably) was observed. Lewis et al. (16) found that DBP synthesized by cell-free translation of early Ad2 mRNA occasionally appearedas adoublet. Both formswere identi-calby peptide analysis andwereverysimilarto DBPisolated from infected cells. The results of our pulse-chase experiments, together with data of other workers, may indicate that 74K DBP isthe primary translationproduct thatis then phosphorylated and further modified in VOL. 22, 1977

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410 JENG ET AL.

some unknown manner that affects the DBP mobility in gel electrophoresis. Both 74K and 77Kforms of35S-and 32P-DBPcouldbe detected when cellswerepulse-orpulse-chase-labeledin the presence of ara-C, which indicates that DBP can be phosphorylated and converted to the apparentlargerform in the absence of viral DNA replication. Highly purified DBP has a frictional ratio of 1.86 and tends to aggregate unless treated with detergent, which suggest that DBP does nothavetypicalglobularprotein structure and has hydrophobicsurfaceregions (27). Possiblyphosphorylation and other post-translational modifications contribute tothese physical properties of DBP.

The roles ofphosphorylation and other possi-ble posttranslational modifications in DBP function areunknown. Both 74K and77Kforms of 35S- and 32P-labeled DBP bind to single-stranded DNA, but we have no accurate esti-mateoftheaffinityofeach formtoDNA.Also, it is not known whether nonphosphorylated

DBP will bind to DNA. Protein phosphoryla-tion isbelievedtoplayakeyregulatoryrolein

eukaryotic cell metabolism. The role of phos-phoproteins (modulated by adenosine

3',5'-cyclicmonophosphate)inintermediary metabo-lism iswell established, and protein phospho-rylation may beinvolvedinregulation of mem-brane function (22). Particularly germane to Ad2 DBP is that phosphorylation ofhistones

and especially acidic DNA binding proteins mayfunction in regulation of gene expression (13, 22,25). The SV40 T-antigen is phosphoryl-ated (R.Carroll, D. Livingstone, P. Tegtmeyer,

personal communication),andthis protein may

havemanybiologicalfunctions,including initi-ationof viral DNA replication and expression of late genes, as well as maintenance of the trans-formed phenotype of SV40-transformed cells. Ad2 DBP seems to be an excellent model sys-temtostudy the general rolethat phosphoryla-tion plays in the biochemistry of eukaryotic DNA binding proteins.

ACKNOWLEDGMENTS

Wethank H. Thornton for cell culture assistance and R. V.Gilden for a gift of the Adl-SV40 antiserum. This work

was supported by Public Health Service grant AI-01725 fromthe National Institute of Allergy and Infectious

Dis-eases andbyresearch contractNO1 CP 43359 within the Virus Cancer Program ofthe National Cancer Institute. W.S.M.W. is a research fellow of the Canadian Medical Research Council. M.G. is a Research Career Awardee of the National Institute of Health (Public Health Service grant 5K6-A14739 from the National Institute of Allergy andInfectious Diseases).

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