Protein kinase A and C site specific phosphorylations of LAP (NF IL6) modulate its binding affinity to DNA recognition elements

Protein kinase A and C site-specific

phosphorylations of LAP (NF-IL6) modulate its

binding affinity to DNA recognition elements.

C Trautwein, … , T Hunter, M Chojkier

J Clin Invest.

1994;

93(6)

:2554-2561.

https://doi.org/10.1172/JCI117266

.

LAP (NF-IL6 or C/EBP beta), is a liver transcriptional activator protein that confers

liver-specific gene expression. Because LAP has a characteristic phosphoacceptor sequence for

cAMP-dependent protein kinase A (PKA), we tested if in vitro phosphorylation of LAP by

PKA modulates its interaction with specific DNA sequences. The major PKA

phosphorylation site of LAP was identified as Ser105, which is a predicted PKA site. As

expected, this PKA phosphorylation site disappears after mutation of Ser105 to Ala. Kinetic

studies with LAP and LAP Asp105 (which mimics a phosphoserine residue) demonstrated

that phosphorylation of Ser105 itself has no effect on DNA binding. Phosphorylation of other

sites by PKA, identified in the region between Ser173 and Ser223 and at Ser240, by

analysis of truncated and mutated LAP peptides, resulted in an inhibition of DNA binding.

LAP was also phosphorylated by purified protein kinase C in vitro, and the major

phosphoacceptor was shown to be Ser240 within the DNA-binding domain of LAP.

Phosphorylation of LAP at this residue or introduction of a Ser240 to Asp mutation resulted

in marked decrease in its binding to DNA. These results suggest that site-specific

phosphorylations of LAP modulate transactivation of its target genes.

Research Article

Find the latest version:

Protein

Kinase A and C Site-specific

Phosphorylations of LAP (NF-1L6) Modulate

Its

Binding Affinity

to

DNA Recognition

Elements

Christian Trautwein,** PetervanderGeer, Michael Karin,t Tony Hunter,' and MarioChojkier*

*DepartmentsofMedicine(VeteransAffairsMedicalCenter)and*Pharmacology, and Centerfor MolecularGenetics, Universityof

California, San Diego, California92161;and

ISalk

Institutefor Biological Studies, LaJolla, California92037

Abstract

LAP(NF-1L6or

_C/EBPj#),

is a livertranscriptionalactivator

protein that confers liver-specific gene expression. Because

LAP has a characteristic phosphoacceptor sequence for

cAMP-dependentproteinkinaseA(PKA), we tested if in vitro

phosphorylationofLAP by PKA modulates its interaction with

specific DNAsequences. The major PKA phosphorylation site

ofLAP wasidentifiedasSer

15,

whichis a predicted PKA site. As expected,this PKAphosphorylationsitedisappears after

mutation of Ser"_{to Ala.Kinetic studies with LAP and LAP}

Asp1"

(whichmimicsaphosphoserine residue) demonstrated

thatphosphorylation of Ser"_{itselfhasnoeffectonDNA}

bind-ing. Phosphorylation ofother sites by PKA, identified in the

region between

Ser'"

and

Ser223

and at

Serm',

by analysis of

truncated and mutated LAP peptides, resulted inaninhibition of DNA binding. LAP was also phosphorylated by purified proteinkinase Cin vitro,and the major phosphoacceptor was

showntobe

Ser2'

within the DNA-binding domain ofLAP.

Phosphorylation ofLAP atthis residue or introductionof a

Ser2'

toAsp mutation resulted in marked decreaseinits bind-ingtoDNA.These resultssuggest thatsite-specific

phosphor-ylationsofLAPmodulatetransactivationof itstarget genes. (J.

Clin. Invest. 1994.

93:2554-2561.) Key

words:

liver-specific

gene

expression

* albumin gene

transcription

*

acute-phase

genes*

C/EBP

*

IL6-DBP

Introduction

During

the development ofhigher eukaryotes, certaingenes

become

activated

ina

cell-type-specific

manner. This

tissue-specific

geneactivation is controlled by transcription factors thatare,

themselves,

enriched in particular celltypes such as

the

hepatocyte.

For

example,

thealbumin geneis

specifically

expressed in the

liver

after birth,

and this

expression

is

regu-latedpredominantlyatthelevelof transcription( 1,2). We havereported the

isolation of

acDNAclone

encoding

a

Portions of this workwerepresentedatthemeetingonRegulationof Liver GeneExpression in Health and Disease,Cold Spring Harbor Laboratory, ColdSpring Harbor,NY, 5-9May 1993.

AddresscorrespondencetoMarioChojkier,Department of Medi-cine and Center for MolecularGenetics(91

l-D),

Universityof Cali-fornia, SanDiego,SanDiego,CA 92161. Dr.Trautwein'scurrent ad-dress is Department ofMedicine,Medizinische HochschuleHannover,

Hannover,Germany.

Receivedfor publicationI November 1993 andinrevisedform11

February1994.

liver-activator protein, LAP, a major albumin promoter D-site

binding proteinthat confersliver-specificgeneexpression(3).

LAP, a member ofthe C/EBP family (4), has also been named

NF-IL6 (5), IL6-DBP (6), AGP/EBP (7), C/EBP,3 (8),and CRP2(9).LAPstimulates the transcription of chimeric genes containing albumin promoter D-elements both in vivo and in

vitro (3, 6).LAPsharesextensivesequencehomology(71%) initsDNA-bindingandleucinezipperdomainswith C/EBPa, and these proteins show indistinguishableDNA-binding speci-ficity andreadily heterodimerize (3, 9). Anotherproductofthe LAP gene has been identified, LIP (liver-inhibitory protein),

whichconsists of the 152 amino acids of the COOHterminus ofLAP. LIP can be translated from LAP mRNA and it behaves as anantagonistofLAP-induced transcription from the albu-min promoter (10).

In response toenvironmental signals, modulation oftarget gene expression can be achieved through posttranslational

modificationsoftranscriptional activators (11). For example, phosphorylation and dephosphorylation of nuclear proteins have beenshown to be crucial to theability ofthese proteinsto bind to cognate DNA sequences, activate genetranscription,

and/or affectprogressionthroughthe cellcycle (12-14).

Like-wise,activation of signal transduction pathwaysmayresultin the phosphorylation of LAP andregulation of itsactivity as

suggestedpreviously (3).Inthisconnection,it has been shown that activation of the cAMP-dependent protein kinase A

(PKA)'in ratpheochromocytoma PC12cellscausesincreased

phosphorylation

and nuclear translocation ofLAP and con-comitant induction ofc-fostranscription (15). In pituitary

G/C cells,calcium-regulated phosphorylationofSer276 within the leucine zipper domain of LAP stimulates transcription

from aCa2+-calmodulin-dependent proteinkinase Il-respon-sive element(13).We haverecentlyshown that

phosphoryla-tion of LAPatSer'05 isstimulated upon activation ofprotein kinase C (PKC) by treatment ofHepG2 cells with phorbol

esters, and that this

phosphorylation

enhances LAP

transacti-vationactivity ( 16).

Prompted by thesereportsofLAP

phosphorylation

in in-tact cells, we have studied the ability of PKA and PKC to

phosphorylateLAP invitro and have tested the effect of phos-phorylation byPKAand PKConthe

ability

of LAPto

recog-nize itscognate DNAelements. Weshow thatSer05 isa

major

phosphoacceptorfor PKA invitro, but that this posttransla-tional modificationof LAP doesnotaffect its

binding affinity

for its cognate response element in target genes.

PKA-me-diated

phosphorylation

of other

sites,

identified in theregion

betweenSer"73andSer223 and of

Ser21

,resulted inan inhibi-tion ofDNA

binding.

We identified the main in vitro

phos-The Journal of ClinicalInvestigation,Inc. Volume93, June 1994, 2554-2561

phorylation site for PKC as

Ser2"

within the DNA-binding

domain of LAP. Phosphorylation atthis site markedly

im-paired the binding of LAPtoits DNA recognition element.

Methods

Plasmidconstruction and site-directed mutagenesis. Thefull-length

cDNAofLAPsubcloned inpBS (Stratagene Inc.,LaJolla, CA)wascut with SphI/EcoRIandreligated toobtain

_pBS-LAP,121.

LAP<, corre-spondstothemajorform of LAPexpressedin the liver(3),because of thepreferentialtranslation from the second AUG ofLAP mRNA(10).

Deletionof the first 21 amino acids hasnoeffectonthefunction of

LAPas a trans-acting factororon itsbinding toDNA. Inthis

manu-script,

LAP,,21

is referredto asLAP,asreportedpreviously(3). For site-directedmutagenesis,the cDNAof pBS

LAP,,21

wasusedto

pre-paresingle-strandedDNAin theEscherichia colistrain JM 109using

thehelper phage M l 3 KO7,accordingtoSambrooketal.(17).

Site-directedmutagenesiswasperformed by usingthemutagenesiskit avail-ablefrom AmershamCorp.(ArlingtonHeights, IL)accordingtothe

supplier's instructions. The following oligonucleotides served as primers: Alal05 mutation, 5'-TAACC GTA GTC GGC CGG CTT

CTTGCTC-3';Asp'05mutation,5'-TA ACC GTA GTC ATC CGG

CTTCTTGCTC-3'; Ala240mutation,5'-CCG CTT GTC GCG AGC

CTTGCG CACCGC-3';Asp240mutation,5'-CCGCTT-GTC GCG GTC CTT GCG CAC CGC-3'.

Theresulting mutants were sequenced, and fragments containing thecorrect mutations were introduced into the

_LAPa1

cDNA. For

expression inmammalian cells the wild type (wt) and mutant LAP cDNAswere subclonedinto cytomegalovirus-driven expression vec-tors.Thesesameconstructs werealsoinserted into bacterialexpression

vectors,transformed into the E.colistrain BL 21 /DE-3/pLys S ( 18), and purifiedas describedpreviously (3, 19). A smaller segmentof LAP wasremovedfrom the LAP clone encoding amino acids 187 to 297 and insertedinto the vectors described above, for the expression of

_LAPsmA

.

Phosphorylation ofLAPin vitro byPKA. For PKA

phosphoryla-tionreactions,3pgof either recombinant purifiedLAPormyosin light chain(SigmaChemicalCo.,St.Louis, MO)wasassayedin the pres-enceofthefollowing (mM): 10MgCl2,20

2-(N-morpholino)ethane-sulfonic acid(MES),2EGTA,1EDTA,and

_100MgM

ATP,containing

20ACiof[y32P]ATP(3,000Ci/mmol;AmershamCorp.)(finalspact 5,000dpm/pmol).Reactionswereinitiatedbytheaddition of 2,ulof

purifiedPKAcatalyticsubunit(5mg/ml;21U/mg)(kindly provided

by Dr. SusanTaylor,UniversityofCalifornia,SanDiego),andcarried

outfor 20 minat30°C.Proteinswereprecipitatedonice with 2.5 volof

100% ethanol aftertheaddition of 5 Mgserumalbumin asacarrier.

Phosphorylationwasassessedbyautoradiographyafter SDS-gel

electro-phoresison a10%polyacrylamidegel(20).

Phosphorylation ofLAPin vitrobyPKC.For PKCreactions, 3

4g

of

recombinantpurifiedLAP(20 ng/36Ml)wasassayed at30°C in the presence of 200 Mg/mlfreshly sonicatedphosphatidylserine (Sigma Chemical Co.), 70mM Tris-HCl (pH 7.5),0.1 mMCaCl2, 7 mM

MgCl2, 5Mugserumalbumin, and 1Mlofpurifiedbovine brain PKC (100 ng/ml)containingamixture of the a,fi, and yisoforms (21) (kindly provided by Dr. Gordon Gill, University of California, San

Diego). The reaction was initiated by the addition of 100 AM

[ L32p]ATP (5,000 dpm/pmol).Forphosphatasetreatment, 2 U of potato acidphosphatase (Boehringer Mannheim Corp., Indianapolis, IN) was added to the kinase reaction at 30°C for 15 min. The phosphatasereaction was stopped by placing the samples at4°C,and the reactions were terminated by adding an equal volume of4x

Laemmli sample buffer. Proteins were resolvedona 10%

SDS-poly-acrylamidegel.

Tryptic phosphopeptide mapping, phosphoamino acid analysis, and

manual Edman degradation. For tryptic mapping, proteins were blot-tedonto a_{nitrocellulose membrane}

_(Schleicher

_and

_Schuell,

_Keene,

NH)

in 1%

SDS,

20% methanol,400mM

_glycine,

50mM

_Tris-HCI,

pH8.3, for 2 h at 200 mA. Proteins were visualizedby autoradiography

of the wet filter. The LAP band was excised and then washed twice with freshly made 0.05 M NH4HCO3. The immobilized LAP protein was

digestedwith 1OMg TPCK-trypsin(WorthingtonBiochemicalCorp.,

Freehold, NJ) by incubationfor4hat370C in 150 mlNH4HCO3. 0O Mg of fresh enzymewasthenadded forafurther 3-h incubation.

After the digestion samples werecentrifuged for 10 minat14,000g, the supernatantsweretransferredtofresh tubes andlyophilized. The pelletwasoxidized in 50 Ml performic acid for 60 minonice. Immedi-ately after the incubation 400 Ml ofwaterwasadded, the samplewas frozenondry ice and lyophilized. The samplewasdissolved inpH 1.9 buffer andspottedon a I00-Amcellulose TLC plate. Phosphopeptides were separated in thefirstdimensionbyelectrophoresisatpH 1.9at1 kVfor 30 min (22).Separationin the second dimensionwasdoneby chromatographyinphosphochromatographybuffer (22).

Forphosphoaminoacidanalysisthetryptic digestwasdissolved in 50 Ml 6 NHCl and incubated for 1 hat 110C,lyophilized,and dis-solved in 5 ml ofpH 1.9 buffer. Thesamplesweremixed with 1Mgof the unlabeledphosphoaminoacidsphosphoserine, phosphothreonine,

andphosphotyrosine, and spottedon a _lO-Amcellulose TLC plate.

Sampleswereseparated byelectrophoresis for 20 minat2 kV in the firstdimensionatpH 1.9 and for 20 minat2 kVatpH3.5 buffer(5% glacialaceticacid,0.5% pyridineinwater)in the second dimension. Theunlabeledphosphoaminoacids markerswerevisualizedby ninhy-drinstaining,and32P-labeledphosphoamino acidsweredetectedby

autoradiography.

Edmandegradation wasperformed exactlyasdescribed by Boyleet al. (22). The samples from eachcyclewerespotted 1 cmaparton a 100-Mm cellulose TLC plate andelectrophoresedinpH 1.9 buffer for 25 minat1kV.

Gel retardation assays.For thegelretardationassays1ngof recom-binantbacterially expressed LAPwasused. An oligonucleotide (5'-TGGTATGATTTTGTAATGGGG-3') spanningthe D-site of the al-bumin promoter(1ng)wasusedasaprobe.Free DNAand

DNA-pro-tein complexeswereresolvedon a6%polyacrylamide gelasdescribed previously(3).

Results

In vitro

phosphorylation of

LAPatSer'05 by PKA. As we re-ported previously (3), analysis of the amino acidsequenceof

LAP revealed possible phosphoacceptor sites for

PKA-me-diated phosphorylation intheNH2 terminus of LAP.

There-fore weinvestigated whetherLAPcould beasubstrateof phos-phorylation by PKA in vitro. Purified bacterially expressed LAP wasphosphorylatedby PKA and the products were

sepa-rated by SDS-PAGE. Fig. 1 Ashowsthatphosphorylationof

LAPoccurredinthepresence(lane 2)but notintheabsence (lane1) ofPKAcatalyticsubunit. Myosin light chainwasused

as a

positive

controlforPKAphosphorylation (lane 3). Minor

phosphorylation

products (lanes2and3)could be due to

con-taminating

proteins in the PKA preparation orin the LAP

preparation.

Next,we setout toidentify the site(s)phosphorylated in

LAPbyPKA.Phosphoamino acidanalysis showed that LAP wasphosphorylatedmainlyon serine by PKA although a low levelof threonine phosphorylation was also detected (Fig. 1

B). As a nextstep,phosphopeptides resulting from atryptic

digest

of phosphorylatedLAP wereresolvedbyelectrophoresis

and chromatography in two dimensions on cellulose TLC

plates. Severalpeptideswerefoundto bephosphorylated(Fig.

2A).The arrowheadindicatesthemainPKAphosphopeptide (spot

I).

53%ofthe

_[32PI

phosphate

incorporated

intoLAPby

A

B

PKA - + + ATP + +

97 69

-46 - - _

Uo

- LAP

30 -21- _

14

-1 2 3

Figure1.Phosphorylation of LAP by PKA in vitro. (A) Recombinant LAP was phosphorylated by PKA in vitro as described in Methods. Thereactionwasperformedinthe absence (lane 1) or in the presence (lane 2) of PKA and

[y-32P]ATP.

Thearrow indicates LAP with an apparent molecular massof36kD as reportedpreviously(3, 10). As acontrolmyosinlight chainwasphosphorylatedbyPKA (lane 3). (B)Phosphoaminoacidanalysis of LAP afterphosphorylationby PKA invitro. Phosphorylated LAP was hydrolyzed in 6 NHCI,and phosphoamino acids were separated bytwo-dimensional

electropho-resisonTLC plates. The arrowhead indicates the migration of the

phosphoserinestandard. A trace of phosphothreonine is evident just totherightofthe phosphoserine. The origin is in the right lowercorner.

A

*AI

_.4 4i

B

,A

I

.1

radioactivity

being

contained in several other phosphopep-tides.A

computer-assisted prediction

of the mobilities of LAP

tryptic

peptides

during separation

onthin

layer

cellulose

plates

indicated that the

peptide containing Ser'05

is themostlikely candidate for spot I(KPS

°5DYGYVSLGR).

In this tryptic

peptide

Ser105 is threeamino acids away from the

NH2

ter-minus. This

prediction

was confirmed

using

manual Edman

degradation during

which free

[32P]

phosphate

was released

after the third

cycle.

As a moredefinitive

proof

of theidentification ofthis

phos-phorylation site,

theserine codonat

position

105wasmutated toanalanine

codon,

thus

introducing

aresidue thatcannotbe

phosphorylated. Fig.

2 Bshows that

PKA-phosphorylated

LAP

Ala'05

lacksspotI,as

expected

ifthis

peptide

containsP.Ser

O5.

Similar results were obtained when

Ser'05

was mutated to

Asp'05

(notshown). These results confirm that

Ser'05

is the main site ofLAP

phosphorylated

by

PKAin vitro.

Phosphorylation of

LAP

by

PKA

changes

the

binding

tothe

albumin promoter

D-site. Because Ser'05 is the main PKA

phosphorylation site of

LAP invitro and becauseour

previous

analysis

ofLAP

phosphorylated

in vivo also showed strong

phosphorylationat

Ser'05

(16),we

investigated

whether

phos-phorylation

ofLAPbyPKAhas anyeffectonthe

binding

of

LAP to theD-site of the albuminpromoter. This

analysis

is

potentially

complicated

by the fact that in additionto

Ser'05,

otherserines inLAP are

phosphorylated by

PKAin

vitro,

and

these

phosphorylations might

alsoaffect

binding

totheD-site.

To analyzetheeffect ofin vitro phosphorylation ofthese

differentsiteson LAPDNA

binding,

we

phosphorylated

both

wt LAPandatruncatedform ofLAPlacking

Ser'05

_(LAPsMA;

amino acids 187-297) withPKAand

performed

gel shift

stud-ies.Asmalldecreaseof - 30% inthe

DNA-binding affinity

of

wt LAP wasobserved

after phosphorylation (Fig.

3A),

initially

suggesting

that

phosphorylation

of

Ser'05

inhibitedDNA

bind-Figure 2. The main PKAphosphorylationsite of LAP isSer'05.(A)

Trypticmapof 32P-labeledwtLAP. LAP wasphosphorylatedin the presence of PKA and

[y-32PJATP

and separated by SDS-PAGE as showninFig. 1A.After transfer ontonitrocellulose,tryptic peptide

mappingwasperformedasdescribed in Methods. The resultant

pep-tideswere separated on cellulose TLC plates byhighvoltage electro-phoresisatpH 1.9(horizontal dimension)followed by ascending

chromatography(vertical dimension).Theoriginis in the left lower

cornerand the cathode was on theright.Themainpeptide,spotI, is indicated withanarrowhead. (B)Trypticmapof32P-labeled mu-tantLAPAla'05.LAPAla1o0 wasphosphorylatedand processed as

describedunder A.

ing. However, sincephosphorylationofthe truncated

LAPsmA,

which lacks

Ser'05,

decreasedDNAbinding by 65% (Fig. 3

B,

lanes I and2),itappearedthattheinhibitory effectof PKA

phosphorylation on wt LAPDNAbindingwasdueto phos-phorylationof sites otherthan Ser105. From other experiments

with various truncatedformsof LAPdescribed

by

Wegneret al. (13), we localized the additional PKA phosphorylation

sites to a region just upstream of the

DNA-binding

domain between Ser173_{andSer223 (notshown),and to}

_Ser2"

_withinthe

DNA-bindingdomain. To provethatphosphorylationatsites

otherthan Ser'05 inhibited DNAbinding,weinvestigatedthe effect of PKAphosphorylation oftheLAPAla'05mutant. A

dramatic 80%decrease in

binding

totheD-site

oligonucleo-tide resultedfrom thephosphorylation ofmutant LAP Ala'05 byPKA(Fig. 3 C). Similar resultswereobtainedafter

phos-phorylationof mutant LAP

Asp"'0

by PKA (notshown).We conclude that the inhibitory effects ofPKAphosphorylation

onDNA

binding

aredueto phosphorylation of sites located

closeto orinthe

DNA-binding

domainthat are common to wt

+ + - - PKA

+ - + - ATP

-~I

B

+ + PKA + - ATP

C

+ -+

b

If

1 2

1 2 3 4 1 2 3

the absenceof the main Ser05phosphorylationsite in allthree

mutantLAPproteinsleadstostrongerphosphorylationofthe

sites closetoorintheDNA-bindingdomain.Inaddition, phos-phorylation ofwtLAPonSer105mayinduceaconformational

change in LAP that masks the phosphorylation sites in the

vicinity of the DNA-bindingdomain. As will be discussed

be-low, phosphorylationof

Ser2"

byPKAis mostlikely responsi-ble for the observeddecrease in LAPDNA-binding activity.

Asanotherwayofshowing that phosphorylation of Ser'05

itself hadnoeffectonDNAbinding,amutantLAPproteinwas

expressedinwhich the serineatposition 105 isreplaced byan

aspartate,which shouldmimic the effect of Ser'05

phosphoryla-tion by introducing a negative charge. DNA-binding curves wereobtained forwtLAPand the LAP Ala'05 and LAPAsp'05

mutantproteinstomonitorpossible changesinbinding dueto

thephosphorylation of Ser

105.

Fig.4shows that the DNA-bind-ing affinities ofthe three different LAP formstotheoligo D-site

are very similar. These results confirm that the decrease in

DNAbinding ofthe LAP protein afterPKAphosphorylation is relatedtothephosphorylation site(s) closetothe DNA-bind-ing domain and that phosphorylation of Ser'05 itself has no

effectonDNAbinding.

In vitro

phosphorylation of

LAP

by

PKC.Inarecentstudy,

Li et al. (23) showed that PKC phosphorylates a threonine residue in the DNA-binding domain of myogenin, which in

turnblocksDNAbinding. Several likely phosphoacceptor sites for PKCphosphorylation arepresent intheDNA-binding do-main of LAP. Therefore, weinvestigated whether LAP could

bephosphorylated initsDNA-binding domain by PKC. The trypticmapof LAPphosphorylated by PKC (Fig. 5 A) showsa

patternrelatedtothat found for LAPphosphorylated by PKA (comparedtoFig.2A). However, the intensity of three basic phosphopeptides (spots II) is muchgreaterrelative to spotI than with PKA.To obtainmoreinformation aboutthese three

spots, weperformed Edman degradationonthese three

phos-phopeptides. The threespotsshowed release of free phosphate either afterthe firstorthe secondcycle of Edman degradation.

This analysis identified Ser223 and

Ser2'

as good candidate

acceptorsitesforphosphorylation by PKC. The relative mobili-ties of thethreespotssuggested that they might be derived by incomplete trypsin digestion ofasingle phosphorylation site.

Specifically,

Ser21

was alikely candidate because the Arg and

Lys around

Ser21

occurinpairs (see Fig. 5), which is knownto

result inpartial tryptic digests (22).

Figure 3. PKA phosphorylation of LAP modulates its PKA affinitytothe albumin D-site. (A)wtLAPwasincubated ATP with

(+)

and without

(-)

PKAat30'Cinthepresence

(+)

orabsence

(-)

of ATPasindicated.I ngof each

re-b actionwasincubated witha_32P-labeledsite D

oligonu-cleotide andprocessed asindicated in Methods. The

DNA-protein complexeswereresolvedon a6%

nondena-turingpolyacrylamide gel.Thepositionsof free(f)and bound(b)DNAareindicated.(B) LAPSMA (aminoacids

189-297)wasincubated withPKAat30'Cinthe

pres-enceorabsence ofATPasindicated in Methods.1 ngof

LAPsmAwasincubated witha32P-labeledsite D oligonu-cleotide andprocessedasindicated in Methods.(C)

Mu-tantLAP

_Ala'05

wasincubated with

(+)

and without f (-) PKAorATPat30'C. 1ngof proteinwasincubated

with the 32P-labeled site D oligonucleotide andprocessed

asindicatedinMethods.

We noted thatoneof the predicted phosphopeptides

gener-atedby trypsin digestion of thesequencearound

Ser2"

in LAP would be the same asthat generated by digestion of the se-quencecontaining the main PKC phosphorylation site in the

cytoplasmic tail of theasubunit oftheIL2-receptor (Fig. 5 B).

Therefore, a synthetic IL2 receptor peptide described by Woodgett et al. (24) was phosphorylated by PKC, digested

with trypsin and the peptideswere separated intwo dimen-sions. Thetrypticmap(Fig. 5C)showed themorebasic of the

IL2 receptor-derived phosphopeptides comigrated with the middlepeptide of thespotsIIcluster (Fig. 5 A). On the basis of this information and their relative mobilities, wetentatively

identify themostacidicspotinthis clusteras

P.Ser240-Arg-Asp-Lys, the centralspotasP.Ser24-Arg, and themostbasicspotas

Lys-P.Ser240-Arg-Asp-Lys.

From these experiments we concluded that Ser21 is the

main PKCphosphorylation site. Therefore, the serine codonat

position 240wasmutatedtoanalaninecodon, and

recombi-nant-LAP

Ala21

was phosphorylated by PKC in vitro. The

tryptic mapofLAP

Ala21

showed thedisappearance of the three mainPKCphosphopeptidespots(Fig. 5 D), indicating that all three are related to each other and confirming that

Ser21

is the mainPKCphosphorylation site in LAP.

Phosphorylation of

LAPat

Ser2"0

blocks the bindingtothe

albuminpromoterD-site.Themain PKCphosphorylation site

ofLAPis

Ser21

.This site islocated in thebasic region of the

DNA-binding domain of LAP andalikely candidate for the

decrease in DNAbinding seenwiththe

LAPsm,

protein and

theLAPmutants atposition 105 after PKA phosphorylation. As - 90% of thephosphate_wasincorporated_atSer24' due_to

PKC phosphorylation, we determined the percentage of

P.Ser240

afterphosphorylation of LAP by PKC. Stoichiometric analysis showed that> 50% of LAPmoleculeswere

phosphor-ylatedat

Ser21

(datanotshown).

Totestwhether PKC phosphorylationaffected the binding ofLAPtoDNA,weperformed gel shift experiments with

PKC-phosphorylated LAP and a32P-labeled oligonucleotide

span-ning the albuminpromoterD-site. The binding ofLAPtothe

cognate DNAwasnearly completely abolished by PKC phos-phorylation (Fig. 6 A, lane 1), when compared with control incubations withATPorPKC alone(Fig. 6 A, lanes 3 and 4).

Thedecreaseinbindingwaspartially reversed bypotatoacid phosphatasetreatment after the phosphorylation ofLAP by PKC(Fig. 6 A, lane 2). The failuretoobtain complete reversal

0 2 4 6 8 10 12 14

wasprobably dueto thecontinued presencein theincubation

of

ATP,

which acts as acompetitive inhibitorofthe

phospha-tase.Treatmentwithphosphatase had noeffectonthe phos-phorylationof theoligonucleotide (Fig. 6).

To test whether the inhibition of DNA bindingdetected afterphosphorylationof LAP at

Ser2"

can bemimicked bythe introduction of an aspartate atthisposition,wemade and

ex-pressedan LAP

Asp2"

mutant.Thebinding of LAP

Ala240

to theD-site wascomparableto that ofwt LAP, whereas LAP

Asp21

did notbindtotheD-site, (Fig. 6

B,

lane3). This shows

thatthepresenceofaspartateatposition 240 mimics theeffect ofphosphorylation of

Ser21

and establishes that phosphoryla-tion of Ser2'"

by

PKC issufficienttoblock DNA

binding.

Discussion

B

0

D 0 .0

C..

0

CC

0

Figure4. DNAbi

Asp105 32P-labele4

amountofrecoml (C).TheDNA-p turing polyacrylar boundwasquanti

imaging and plott protein.Theperco

LAP (ng)

LAP canmodulate its affinity for the D-site of the albumin

promoter. We started by investigating whether PKA could phosphorylate LAP in vitro, because ofapriorreportthat LAP isphosphorylatedinPCl2 cells afterinductionofcAMP

path-ways. Inthat study itwasnotdeterminedwhether PKA

phos-phorylatesLAPdirectly,and the LAPphosphoacceptor site(s) were notidentified (15). Here we demonstratethat purified

recombinant LAPcanbephosphorylatedin vitro by the

cata-lytic subunit of PKA. Usingacombination of phosphoamino

acid analysis, phosphopeptide mapping, and Edman degrada-tion, we identified the major PKA phosphorylation site as

Ser'05 (Fig.7), whichis partofacharacteristic

phosphoaccep-torsequence(SKKPS'05) for PKA (consensus R/K R/K X

S/T)

(3). We confirmed ouridentification by showing that

0 2 4 6 8 10 12 14 this PKA phosphorylation site disappears after itsmutation

(Ser105toAla,orSer 105toAsp). Wegneretal. (13)wereableto

LAPAla1'

(ng) phosphorylate recombinant mouse LAP in vitro with

Ca2+-calmodulin-dependent kinase II at Ser276 (Fig. 7), but they reported only weakphosphorylation with the PKA catalytic subunit. Theapparentdiscrepancy between these resultscanbe

reconciled, since the recombinant LAP protein used by Wegner and co-workers (13)wasderived froma mouseLAP

cDNAclone that lacks the corresponding seine, having ala-nine instead in the_{corresponding position (8),}whichcannot

bephosphorylated. BothratandhumanLAPscontain

Serl05

(3, 6, 9).The_significanceofthis difference between the mouse

LAPsequenceand the other vertebrateLAPsequencesis

un-clear.

Ourresults indicate thatphosphorylationofSer105doesnot

alter the bindingof LAP to the cognate albumin promoter

D-site.Inparticular,wefound that recombinant LAPAsp'05

mu-_____._,_,_._,_,_,_ . tantproteinin which theAspmimicsP.Ser'05, displayed

un-0 2 4 6 8 10 12 14 changed DNA-binding affinity. Moreover, although

phosphor-ylation ofwtLAPbyPKAdecreased DNA-binding activityof

LAPAsp1

l(ng)

LAP,the same was true for the Ala'05 mutant LAP protein.

This indicates that PKA phosphorylation of sites otherthan

indingaffinity ofwtLAP,LAPAlat05,andLAP

Ser'l5

in LAP results in decreasedbinding affinityfor the

albu-doligo-D ( 15pg.)was incubated with an increasing m. st.

binantwtLAP(A),LAPAla'05 (B), andAsp'05

men

promoter

D-site.These

additional

PKA

phosphorylation

* ' '

~~~~~~~~~~~sites

were

mapped

tothe

region

between Ser175_{and Ser}223_in rotein complexeswereresolvedon a6%

nondena-mide gel. The fraction of theprobespecifically close proximity to the DNA-binding domain and to _Ser24.

ified (percentspecificshifted probe) by phospho- Based on results obtained with PKC, it seems likely that PKA-Ledas afunction oftheamountof recombinant dependent phosphorylationof

Ser21

isresponsiblefor the de-entageofspecificoccupancyisindicated. creasedDNAbinding.

A

0

0-D

Q

50

40

30

20

10

B

IL-2receptor peptide Y-Q-R1F

LAP(aa 233 -244) N-N- I-)

I

v

Hw4

El

D

The major phosphoacceptor for PKC in vitrowasmapped

to

Ser2"

within the DNA-bindingdomain of LAP (Fig. 7). Phosphorylation ofSer2" orintroduction ofanAsp240

muta-tion, which mimicsP.Ser240,resulted in marked reduction in

A

+ + - ₊ PKC

+ + + - ATP

_ + - - PPase

w~9rnP ----b

ss.'&.f

1 2 3 4

B

_O St _b

M-110

f

1 2 3

Figure 6. PKC phosphorylation ofLAPinhibits its bindingtothe al-buminD-site.(A)wtLAPwasincubated with (+)andwithout(-) PKC inthepresence(+)orabsence(-) ofATPat30°C.AfterPKC phosphorylation, the sampleinlane 2wasadditionally incubated

withphosphatase for 15minat30°C. AfterPKC phosphorylation andphosphatasetreatment, 1 ngofeach samplewasincubated with a32P-labeledsite-D oligonucleotide. The DNA-proteincomplexes

wereresolvedon a6%nondenaturing polyacrylamidegel. The posi-tions of free(f)andbound (b)DNAareindicated.(B) 1ngofwt

LAP(lane 1),mutantLAPAla240(lane 2),orLAPAsp240(lane3)

wasincubated witha32P-labeledsite-D oligonucleotide.The DNA-proteincomplexeswereresolvedasindicatedunderA.

RIQ-R!K!S-RR1T-I Figure 5. The main PKC phosphorylation site ofLAP is Ser2"'. (A) Recombinant LAP wasphosphorylated byPKC in vitro

A-V-RfKfS-RfD-K-A as described in Methods. Phosphorylation

productswereseparatedon a10%SDS-gel

andblottedontoanitrocellulose mem-brane. Tryptic mappingwasperformedas described in Methods. The main phospho-peptidesareindicatedasspots II.(B) Se-quencecomparisonbetween thesynthetic

IL-2 receptor peptide and LAPatamino acids232-247. Thepossible trypsin cleav-age sitesareindicated.(C)Thesynthetic

IL-2 receptorpeptidewasphosphorylated by PKC,andtryptic mappingwas per-formedasdescribed in Methods. (D) Mu-tantLAPAla240wasphosphorylated by PKC in vitro. Identification and tryptic

mappingofphosphorylatedLAPwas per-formedasdescribed in Methods.

the binding of LAPto its DNA recognition element. LIP, a

liver-inhibitoryprotein(10), lacks the NH2 terminal domain ofLAP, includingthephosphoacceptoratSer'05,but it is

sus-ceptibletothe other phosphorylations(Fig. 7). This differen-tial phosphorylation may providea mechanism for selective modulation of LAP activity, when compared with LIP activity. Our previous study of LAP phosphorylation in HepG2

hep-atomacells identified Ser05as aphysiologicalphosphorylation

site and showed that its phosphorylationwasstimulatedupon

activation of PKC with concomitant enhancement of

tran-scriptional activation (16). Since we have shown here that PKC doesnotphosphorylate Ser05in vitro, this effect of PKC

islikelyto beindirect. In addition, aconstitutively activated

derivative of PKC that ispredominantlynuclear (23) didnot

stimulate LAP activity orphosphorylation (our unpublished

observations). Most probably, activation of cytoplasmic PKC results in activation of downstream protein kinases including

onedirectlyresponsible forphosphorylationof LAPonSer

105.

It alsoseemsunlikely that PKA is responsible for phosphoryla-tion of Ser05inHepG2 cells, since in preliminary experiments

wehavefound thattreatmentwithforskolintoelevatethe in-tracellular level of cAMP in HepG2 cells didnotaffect either the extent ofLAP phosphorylation at Ser'05 or its nuclear

translocation (our unpublished observations). Likewise, other PKA activatorsorinhibitors didnot leadto eitherhigher or

lower levels of LAP phosphorylation, respectively. In some

waysthis is surprising since activation of PKA leadstonuclear translocation of the C subunit. In contrast, in PC 12 cells cAMP-induced phosphorylation of LAP is associated with its nuclear translocation ( 15). These results indicate that different mechanisms may be responsible for the phosphorylation of

LAPinhepatoma and PC 12 cells (15).

Inourprevious study, phosphorylation ofSer21 wasnot

readily detected in intact HepG2 cells ( 16), but in subsequent experiments we have found that there isalowbasal level of

LAP phosphorylation at Ser240in hepatoma cell lines.

How-ever,phosphorylation ofSer240wasnotincreased by activation ofPKCorPKAin intact cells, eventhough both protein

ki-nases canphosphorylate Ser240in vitro( 16, andour

unpub-A

TranscrptionalActivation

, s

SKKPS'°S

152 DNA Binding Dimerization 297

VRKS24OR SRELS276

152 LIP ,..

187 LAPsmJA L.

Figure 7. Phosphoacceptor domains of LAP.Theinvitro

297 _{phosphorylation sites of LAP}

_ (Ser'°5, Ser24, and Ser276)

areshown. LIPstarts at resi-due 152 and endswith resi-297 due297,whileLAPsm

in-cludes residues 187-297.

lished observations). Taken together these observations sug-gestthatprotein kinases other thanPKAand PKCcan phos-phorylate LAPat Ser'-4 and such protein kinases might be regulated by other signaling pathways whose activation would result in decreased activation of LAPtargetgenes.

What is the potential biological relevance of LAP phos-phorylation in vivo? Therearepotential parallels between the

regulation of LAP by phosphorylation, and the regulation of other leucinezipper transcription factors by phosphorylation. c-Jun,amember ofthe leucine zipper family, has five major

phosphorylation sites, whicharelocatednearthe

DNA-bind-ing domain and in the transactivation domain within the NH2 terminus(25-27). The affinity of c-Jun for AP-1 sites is de-creased when it isphosphorylated in the region adjacenttothe DNA-binding domain (25, 28). In contrast, c-Jun showsan

increase in transcriptional activity after phosphorylation oftwo

serines withinits transactivation domain (26, 27). Phosphory-lation also affects the activity of CREB. PKA phosphorylates CREBat

Ser'"I

invitro and in vivo(29, 30). Phosphorylation of CREBat.Ser'33, which lies in the major transactivation do-main, leadstoaseveralfold increase in transactivation froma

chimericreportergenewith aCREB-binding site without af-fecting DNA binding (29). Interestingly,thisPKA-mediated phosphorylation of CREB plays a role in liver-specific gene

regulation (31 ). The tyrosine aminotransferase (TAT)geneis

regulated inahepatocyte specificmannerby CREB.

PKA-me-diated pathways stimulate transcription of the TATgeneby

CREB inarathepatoma cell line FTO-2B (31 ).

Ourexperiments with themutant LAPAsp 105, which

ex-hibits increased transactivation fromanLAP-responsive DNA

element,supportthe conclusion thatphosphorylation of LAP inits transactivation domain stimulates transcription of

spe-cifictargetgenesin vivo( 16).Thispositive regulation is

analo-goustothestimulatory effect of phosphorylating the transacti-vation domain of other members of the leucine zipper family (26, 29). Conversely, LAP displaysadecreasedaffinity for its

cognateDNA-binding sites when phosphorylated in vitroin

theDNA-binding domainorinitsproximity by PKCorPKA,

respectively. This resembles the inhibitory effect ofphosphory-lation of c-Jun in thevicinity of the basic domain, which is known to be physiologically relevant (25, 28). LAP is also potentially positively regulated by phosphorylation of Ser276in

the leucinezipper by calmodulin-dependent protein kinaseII,

which would be activated upon elevation of intracellular Ca2+ (13).

Inthisreportwehave identified and characterized the

site-specific phosphorylations of LAP critical for its interaction withcognatecis-elements intargetgenes.Now it will be

possi-bletostudy the LAP phosphorylation site(s) in the normalrat

liveraswellasunder physiologicalconditions thatareknown

toactivatevarioussignal transductionpathways. Experiments intransgenic animals, includinganLAP"knock-out" with and without overexpression of various deleted/mutated LAP

trans-genes, shouldclarify the role of thissite-specific

phosphoryla-tion ofLAPonliver-specificgeneexpression during

develop-mentand underphysiological stimulation.

Acknowledgments

WearegratefultoSusan Taylor and GordonGill (University ofCalifor-nia, San Diego) for providing preparations of PKAand PKC,

respec-tively.We thank Sam Maddox forpreparing the manuscript. C. Trautweinwassupported byapostdoctoral fellowship fromthe

DeutscheForschungsgemeinschaft(Germany). Research inM. Choj-kier'slaboratorywassupported by NationalInstitutes ofHealthgrants

DK-38652,DK-46971,and GM-47165, and bygrantsfromthe Depart-mentof Veterans Affairs.Research inM.Karin'slaboratorywas

sup-ported by National Institutes of Health grantsCA-50528 and HL-35018. Research in T. Hunter'slaboratorywassupported by National Institutes of HealthgrantCA-39780.

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