Glycogen Synthase Kinase 3Я Interaction Protein Functions as an A-kinase Anchoring Protein*DS

106 Pages·2010·6.06 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.285,NO.8,pp.5507–5521,February19,2010
©2010byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
(cid:1)
Glycogen Synthase Kinase 3 Interaction Protein Functions
as an A-kinase Anchoring Protein*□S
Receivedforpublication,July23,2009,andinrevisedform,November17,2009 Published,JBCPapersinPress,December11,2009,DOI10.1074/jbc.M109.047944
ChristianHundsrucker‡1,PhilippSkroblin‡1,FrankChristian‡,Hans-MichaelZenn§,ViolaPopara‡,MangeshJoshi‡,
JennyEichhorst‡,BurkhardWiesner‡,FriedrichW.Herberg§,BerndReif‡,WalterRosenthal¶(cid:1),
andEnnoKlussmann‡2
Fromthe‡LeibnizInstituteforMolecularPharmacology,Robert-Ro¨ssle-Strasse10,13125Berlin,the§InstituteofBiology,
UniversityofKassel,Heinrich-Plett-Strasse40,34134Kassel,the¶MaxDelbru¨ckCenterforMolecularMedicine,
Robert-Ro¨ssle-Strasse10,13125Berlin,andthe(cid:1)DepartmentofMolecularPharmacologyandCellBiology,Charite´-University
MedicineBerlin,14195Berlin,Germany
A-kinase anchoring proteins (AKAPs) include a family of ing molecules, including other protein kinases (e.g. protein
scaffolding proteins that target protein kinase A (PKA) and kinase C and protein kinase D), phosphodiesterases (e.g.
othersignalingproteinstocellularcompartmentsandthereby PDE4D), and protein phosphatases (e.g. PP1 and PP2B/cal-
confine the activities of the associated proteins to distinct cineurin).AfewAKAPspossesscatalyticactivity.Forexample,
regionswithincells.AKAPsbindPKAdirectly.Theinteraction AKAP-LbcisaRhoguaninenucleotideexchangefactor(1–3).
ismediatedbythedimerizationanddockingdomainofregula- Thus, AKAPs assemble multiprotein complexes and thereby
torysubunitsofPKAandthePKA-bindingdomainofAKAPs. coordinatecellularsignaling.AKAPsarerequiredformanycel-
Analysisoftheinteractionsbetweenthedimerizationanddock- lular processes, including vasopressin-mediated water reab-
ingdomainandvariousPKA-bindingdomainsyieldedagener- sorptioninrenalprincipalcellsand(cid:1)-adrenoreceptor-depen-
alizedmotifallowingtheidentificationofAKAPs.Ourbioinfor- dentincreasesofcardiacmyocytecontractility(2,4,5).
matics and peptide array screening approaches based on this ThePKAholoenzymeconsistsofadimerofregulatoryRIor
signaturemotifidentifiedGSKIP(glycogensynthasekinase3(cid:1) RIIsubunitsandtwocatalyticsubunits,eachboundtooneR
interactionprotein)asanAKAP.GSKIPdirectlyinteractswith subunit. Upon binding of two molecules of cAMP to each R
PKA and GSK3(cid:1)(glycogen synthase kinase 3(cid:1)). It is widely subunit, the catalytic subunits dissociate and phosphorylate
expressedandfacilitatesphosphorylationandthusinactivation theirsubstrates(6,7).TheinteractionofAKAPswithPKAis
ofGSK3(cid:1)byPKA.GSKIPcontainstheevolutionarilyconserved mediated by the PKA-anchoring domain of AKAPs and the
domain of unknown function 727. We show here that this dimerization and docking (DD) domain of R subunit dimers.
domain of GSKIP and its vertebrate orthologues binds both BecausemostAKAPspreferentiallyanchorRIIsubunits,PKA-
PKAandGSK3(cid:1)andtherebyprovidesamechanismfortheinte- anchoringdomainsaretermedRII-bindingdomains(RIIBD).
grationofPKAandGSK3(cid:1)signalingpathways. Thesedomainsarestructurallyconservedamphipathichelices,
14–18aminoacidresiduesinlength(8,9).Basedonrecently
described determinants of the RIIBD/DD domain interaction
A-kinase anchoring proteins (AKAPs)3 are a family of (8–10), we developed a bioinformatics and peptide array
scaffoldingproteinscharacterizedbytheabilitytobindcAMP- screeningapproachtoidentifynewAKAPs.Foroneofthedis-
dependent protein kinase (protein kinase A (PKA)). They coveredproteins,GSKIP(GSK3(cid:1)interactionprotein),weshow
tetherPKAinthevicinityofitssubstrates,therebyfacilitating thatitfunctionsasanAKAP.
theirphosphorylation.Inaddition,AKAPsbindfurthersignal- Mammaliancellsexpresstwoisoformsofglycogensynthase
kinase-3(GSK3),GSK3(cid:2)andGSK3(cid:1),whichareconstitutively
activeandphosphorylateabroadrangeofsubstrates,thereby
*ThisworkwassupportedbyDeutscheForschungsgemeinschaftForscher-
gruppe806,ProjectKL1415/4-1,theEuropeanUnionProjectTheracAMP, participatingintheregulationofvariousprocesses,including
Proposal037189,andtheGoBioProgramoftheGermanMinistryofEdu- energymetabolism,proteinsynthesis,sortinganddegradation,
cationandScienceGrantsFKZ0315097and0315516.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains and transcription (11, 12). The activity of GSK3 isoforms is
supplementalFig.1andTable1. decreased through phosphorylation by other protein kinases
1Bothauthorscontributedequallytothismanuscript. such as Akt/protein kinase B, p70/p85-ribosomal S6 kinase,
2Towhomcorrespondenceshouldbeaddressed:Leibniz-Institutfu¨rMole-
p90-ribosomalS6kinase,andPKA(13–15).Phosphorylationof
kularePharmakologie,CampusBerlin-Buch,Robert-Ro¨ssle-Str.10,13125
Berlin, Germany. Tel.: [phone]; Fax: [phone]; E-mail: Ser-21ofGSK3(cid:2)andSer-9ofGSK3(cid:1)byanyoftheabovemen-
[email protected]. tioned kinases leads to inactivation. The different kinases
3Theabbreviationsusedare:AKAP,A-kinaseanchoringprotein;DUF,domain appeartoregulatedifferentpoolsofGSK3(cid:1).Forinstance,stim-
of unknown function; GST, glutathione S-transferase; GSK3(cid:1), glycogen
synthasekinase3(cid:1);GSKIP,GSK3(cid:1)interactionprotein;HRP,horseradish ulationofadrenoceptorsonskeletalmusclecellsleadstoPKA-
peroxidase; RII, type II regulatory subunit of PKA; RIIBD, RII-binding mediated phosphorylation of GSK3(cid:1), whereas stimulation of
domain;8-AHA-cAMP,8-aminohexylaminoadenosine3(cid:1)-5(cid:1)-cyclicmono- theinsulinreceptoronthesecellsleadstoproteinkinaseB-me-
phosphate; CFP, cyan fluorescent protein; PKA, protein kinase A; DD, diatedphosphorylationofGSK3(cid:1)(16).Theformationofsuch
dimerizationanddocking;GAPDH,glyceraldehyde-3-phosphatedehydro-
genase;FSK,forskolin;MOPS,4-morpholinepropanesulfonicacid. pools is likely to require scaffolding proteins. The AKAPs
FEBRUARY19,2010•VOLUME285•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5507
This is an Open Access article under the CC BY license.
GSKIPIsanAKAP
AKAP220andMAP2(microtubule-associatedprotein2)each YFP-RII(cid:2)usedinthisworkwasdescribedpreviously(24).To
recruitPKAandGSK3(cid:1)andfacilitatePKAphosphorylationof generateCFP-AKAP18(cid:2),humancDNA(seeabove)wasampli-
GSK3(cid:1) (17, 18). The phosphorylation by PKA appears to fied by PCR (forward primer 5(cid:1)-CCCCGAATTCCATGG-
require binding of both PKA and GSK3(cid:1)to the respective GCC-3(cid:1)andreverseprimer5(cid:1)-CGCGGTCGACCATTTCCT-
AKAP(13,19). GTTCTCATTGTCA-3(cid:1)) and inserted into pECFP-N1 via
Here,wedemonstratethattheevolutionarilyconservedand EcoRIandSalIrestrictionsites.Allconstructsweresequenced
widely expressed protein GSKIP (20) functions as an AKAP, priortoexpression.
recruitsPKAandGSK3(cid:1),andfacilitatesPKAphosphorylation Generation of Recombinant GSKIP—To obtain His-tagged,
of GSK3(cid:1). GSKIP and its vertebrate and invertebrate ortho- full-length GSKIP, a cDNA fragment encoding full-length
loguesfromfungitoHomosapienscontaintheevolutionarily GSKIP(139aminoacidresidues)wasgeneratedbyPCRusing
conserveddomainofunknownfunction727(DUF727),which the vector CFP-GSKIP (see above) as template. The forward
we show here to interact with RII subunits in the vertebrate (5(cid:1)-CAGCCATATGGAAACAGACT-3(cid:1))andreverseprimers
orthologues, thus conferring an AKAP function. RII binding (5(cid:1)-TGTGCTCGAGTCAGGACT-3(cid:1)) contained NdeI and
wasnotobservedforinvertebrateorthologues.Theinteraction XhoIrestrictionsites,respectively.ThecDNAwasclonedinto
withGSK3(cid:1),however,appearstobeconservedinbothinver- thevectorpET28((cid:2))(Merck)viatheserestrictionsitestoyield
tebratesandvertebrates. the vector His-GSKIP. His-tagged proteins were expressed in
EscherichiacolistrainBL21-DE3andaffinity-purified.TheHis
EXPERIMENTALPROCEDURES
tag was removed by thrombin protease cleavage as recom-
Peptide Arrays, RII Overlay, and Protein Overlay—Peptide mendedbythesupplierofthevectorpET28((cid:2)).
spots were generated by automatic SPOT synthesis as Preparation of Affinity-purified Anti-GSKIP Antibodies—A
describedpreviously(10,21,22).Peptidescontaining“difficult rabbit polyclonal antiserum was raised against recombinant
sequences”(23)wereexcludedfromtheanalysis.Forexample, full-lengthGSKIP(139aminoacidresidues;Biogenes,Berlin,
cysteines may oxidize to sulfoxides or undergo dimerization. Germany).Specificantibodieswereisolatedbyaffinitychroma-
(cid:1)-Sheet-formingsequencescausecollapseofthepeptide-resin tographyoftheantiserausingGSKIPimmobilizedonthiopro-
matrix. Under such conditions, diffusion of reagents into the pyl-Sepharose6B(GEHealthcare)(24).
matrixislimited,andcouplinganddeprotectionreactionsmay CellCultureandTransfection—HEK293cells(ATCC,Man-
be incomplete (21, 23). The interaction of spot-synthesized assas, VA) were grown to 40–60% confluency in Dulbecco’s
peptideswithRIIsubunitsofPKAwasinvestigatedbyRIIover- modified Eagle’s medium supplemented with 10% fetal calf
layassayusingradioactivelylabeledRIIsubunits(10,24,25).To serum.Cellsweretransfected48hafterseedingwithplasmid
determine the binding of GSK3(cid:1), peptide spot membranes DNA(1(cid:3)g/60-mmdish)encodingCFP-GSKIPorCFP-GSKIP-
were blocked for 1 h with blocking buffer (3% bovine serum V41P/L45PusingTransfectin(Bio-Rad)andanalyzed24–48h
albumin in Tris-buffered saline (cid:2) 0.05% Tween 20 (TBS-T)) post-transfection. SH-SY5Y cells were grown in Dulbecco’s
before incubation with 1 (cid:3)g/ml glutathione S-transferase modified Eagle’s medium/F-12 supplemented with 10% fetal
(GST) or 1 (cid:3)g/ml GST-GSK3(cid:1)(Cell Signaling Technology, calfserumand1%nonessentialaminoacids.
Danvers, MA), at 4°C overnight. Membranes were washed Detection of GSKIP in Cells by Laser Scanning Microscopy—
threetimeswithTBS-Tandincubatedwithananti-GSTanti- Glasscoverslips(30mm)werecoatedwith100(cid:3)g/mlpoly-L-
body (Millipore, Billerica, MA) in blocking buffer for 1 h at lysinein35-mmcellculturedishes.24hpriortotransfection,
roomtemperature.Thereafter,horseradishperoxidase(HRP)- 200,000 HEK293 cells were seeded in Dulbecco’s modified
coupled anti-rabbit antibody (Dianova, Hamburg, Germany) Eagle’smedium/fetalcalfserum.Cellsweretransfectedwith0.5
was added (1 h, room temperature), and after washing three (cid:3)g of YFP-RII(cid:2)and 1 (cid:3)g of CFP-GSKIP, CFP-GSKIP-V41P/
timeswithTBS-T,anECLreactionwascarriedoutusingLumi- L45PorCFP-AKAP18(cid:2)usingLipofectamineTM2000(Invitro-
Light substrate (Roche Diagnostics). Signals were visualized gen).24haftertransfection,cellwerecoveredwithphosphate-
withtheLumi-ImagerF1(RocheDiagnostics). buffered saline and analyzed by confocal laser scanning
Cloning—Total RNA was isolated from human neuroblas- microscopy, using a Zeiss LSM 710 (Carl Zeiss GmbH, Jena,
toma SH-SY5Y cells using TRIzol and reverse-transcribed Germany).
using SuperScript one-step reverse transcription-PCR with Immunoprecipitation and cAMP-Agarose Pulldown, Prepa-
Platinum Taq according to the manufacturer’s instructions rationofTissueLysates,andWesternBlotting—Untransfected
(Invitrogen).TheresultingcDNAservedasatemplatetoobtain HEK293 cells or HEK293 cells transiently expressing CFP-
full-lengthGSKIP(420bp)byPCR(forwardprimer5(cid:1)-ATGC- GSKIPorCFP-GSKIP-V41P/L45Pandtissuesfromwildtype
GCTAGCTATGGAAACAGACTGTAATCCCATG-3(cid:1) and Wistarratswerelysedinlysisbuffer(10mMK HPO ,150mM
2 4
reverseprimer5(cid:1)-GCATACCGGTCCTGACTGTCCATCTC- NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, pH 7.4)
TTTTCAAAG-3(cid:1)).PCRproductswereligatedintothevector supplementedwithproteaseinhibitors(1mMbenzamidine,0.5
pECFP-N1(BDBiosciences)viatherestrictionsitesNheIand mMphenylmethanesulfonylfluoride,3.2(cid:3)g/mltrypsininhibi-
AgeI to yield the vector CFP-GSKIP. Valine 41 and leucine torI-S,1.4(cid:3)g/mlaprotinin)andproteinphosphataseinhibitors
45weresubstitutedbyprolineresidues(GSKIP-V41P/L45P) (50mMNaFand100(cid:3)MNa VO )(24).Thelysateswerecleared
3 4
by site-directed mutagenesis (QuikChange site-directed by centrifugation (20,000 (cid:3) g, 4°C, 10 min). Endogenous
mutagenesiskit,Stratagene,LaJolla,CA)todisruptthehelical GSKIP,CFP-GSKIP,andCFP-GSKIP-V41P/L45Pwereimmu-
structureoftheRIIBD. noprecipitated with affinity-purified rabbit anti-GSKIP anti-
5508 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER8•FEBRUARY19,2010
GSKIPIsanAKAP
body (see above) and protein A-conjugated agarose (Sigma). dissociationphaseswererecordedfor300s.AftereachGSKIP
ForcAMP-agarosepulldown,thelysateswereincubatedwith injection,theentireproteincomplex(RsubunitsandGSKIP)
8-AHA-cAMP-agarose(4°C,3h;8-aminohexylaminoadeno- wasremovedbythreeshortinjections(30seach)of3Mguani-
sine3(cid:1)-5(cid:1)-cyclicmonophosphate;Biolog,Bremen,Germany)in diniumhydrochloride.Rateconstants(k andk )andequilib-
a d
theabsenceorpresenceofcAMP(50mM).Agarose-boundpro- riumbindingconstants(K )werecalculatedbasedonnonlin-
D
teinswerewashedfourtimeswithlysisbufferandelutedwith earregressionanalysisassuminga1:1Langmuirbindingmodel
Laemmlisamplebuffer(95°C).Thefollowingprimaryantibod- usingtheBIAEvaluationsoftware,version3.0(BiacoreAB,GE
ies were used for Western blot detection: rabbit anti-GSKIP Healthcare). For control, experiments were performed in the
(seeabove);rabbitglyceraldehyde-3-phosphatedehydrogenase presenceof5(cid:3)MPKAanchoringdisruptorpeptides(Ht31or
(GAPDH),phospho-GSK3(cid:2)/(cid:1)(p-GSK3(cid:2)/(cid:1)),andtotalGSK3(cid:1) AKAP18(cid:5)L314E (10)) using 500 nM GSKIP. As negative con-
antibodies (Cell Signaling Technology, Danvers, MA); mouse trols,doubleprolinesubstitutedpeptideswereusedunderthe
anti-(cid:1)/(cid:4)-tubulin (Calbiochem); goat anti-lamin A/C (Santa sameconditions.
Cruz Biotechnology, Santa Cruz, CA); mouse anti-RII(cid:2)(BD Enzyme-linked Immunosorbent Assay for Monitoring the
Biosciences).CFPwasdetectedwithmouseanti-greenfluores- GSKIP,PKA,andGSK3(cid:1)Interaction—Enzyme-linkedimmu-
centprotein(Clontech)(26).CorrespondingHRP-coupledsec- nosorbentassayswereconductedinCorning384-wellclearor
ondary antibodies were from Dianova, Hamburg, Germany. whiteflatbottompolystyrenehighbindmicroplates(Corning
SignalswerevisualizedwiththeLumi-ImagerF1(RocheDiag- B.V.,Schiphol-Rijk,theNetherlands).Theplateswerecoated
nostics).p-GSK3(cid:1)wasquantifiedbydensitometricanalysisof byadding20(cid:3)l/wellRII(cid:2)(50nM;preparedasdescribedinRef.
Westernblots(LumiAnalyst3.0software,RocheDiagnostics). 27) in coating buffer (phosphate-buffered saline containing 1
SubcellularFractionationofSH-SY5YCells—SH-SY5Ycells mMbenzamidine,0.5mMphenylmethanesulfonylfluoride,3.2
were scraped from 100-mm cell culture dishes in 500 (cid:3)l of (cid:3)g/mltrypsininhibitortypeI-S,1.4(cid:3)g/mlaprotinin,and1mM
fractionationbuffer(250mMsucrose,20mMHEPES,pH7.4,10 dithiothreitol) and incubation for 1 h atroom temperature.
mMKCl,1.5mMMgCl ,1mMEDTA,1mMEGTA,1mMdithi- Thereafter, the coating buffer was removed, and blocking
2
othreitol)supplementedwithproteaseandphosphataseinhib- buffer (coating buffer containing 0.3% skimmed milk powder
itors(seeabove).Cellswerehomogenizedusingaglass-Teflon and0.05%Tween20)wasadded(100(cid:3)l,1h,roomtempera-
homogenizer and centrifuged (10 min, 4°C, 700 (cid:3) g). The ture). After removal, monitoring of interactions of RII(cid:2)with
resultingnuclearpelletwaswashedwithfractionationbuffer, GSKIPand/orGSK3(cid:1)wascarriedoutinnewlyaddedblocking
centrifugedagain,andresuspendedinRIPAbuffer.Thesuper- buffer(20(cid:3)l/well,1h,roomtemperature).Proteinconcentra-
natantwascentrifugedat100,000(cid:3)gfor60min.Thepelletwas tionsareindicatedinthelegendtoFig.6A.Unboundprotein
considered the particulate and the supernatant the cytosolic wasremovedbywashingthewellsthreetimeswith100(cid:3)lof
fraction(24). washing buffer (phosphate-buffered saline containing 0.05%
Nuclear Magnetic Resonance Measurements—Isotopically Tween20).BoundHis-GSKIPwasdetectedwithmonoclonal
enrichedGSKIPwasexpressedinE.coliBL21-DE3strainusing anti-polyhistidineHRP-conjugatedantibody(Sigma;1:8000in
15N-NH ClasnitrogensourceinM9minimalmedium.1H,15N blocking buffer, 1 h, room temperature). Bound GSK3(cid:1)was
4
correlation experiments with 15N-labeled GSKIP and unlabeled detectedbyincubationwithrabbitanti-GSK3(cid:1)antibody(Cell
DDdomainwererecordedusingastandardheteronuclearsingle SignalingTechnology,Danvers,MA;1:1500inblockingbuffer)
quantumcoherencepulseprogramemployingWATERGATEfor and HRP-conjugated anti-rabbit IgG (1:3000, 1 h, room tem-
solvent suppression. All NMR experiments were recorded on a perature).Eachantibodyincubationstepwasfollowedbywash-
Bruker 600 MHz Avance spectrometer equipped with a triple ing.TheHRPreactionwasinitiatedbyadditionof3,3(cid:1),5,5(cid:1)-
channelcryoprobe.Thetemperaturewassetto30°Cinallexper- tetramethylbenzidine enzyme-linked immunosorbent assay
iments.Forinteractionstudies,thetypicalproteinconcentration substratesolution(Sigma)andterminatedafter30minbyadd-
was on the order of 0.1 mM. The acquisition time in the direct ing HCl (1 M; 20 (cid:3)l/well). The colored reaction product was
dimensionwasrestrictedto150ms.Atotalof256incrementswas quantified by measuring A in a GeniosPro plate reader
450nm
recordedintheindirectdimension. (Tecan,Durham,NC)with10flashes/well.Curveswerefitted
Surface Plasmon Resonance Measurements—Interaction basedonaone-site-bindingmodel,andK valueswerecalcu-
D
studieswereperformedemployingBiacore2000or3000instru- latedusingPrism4.0(GraphPadSoftware,SanDiego,CA).
ments(BiacoreAB,GEHealthcare)(27).Measurementswere Statistical Analysis—GraphPad Prism 5.01 for Windows
performedinrunningbuffercontaining20mMMOPS,150mM (GraphPad Software, San Diego) was used to perform two-
NaCl,0.005%Tween20,pH7.0,at25°Cinstrumenttempera- tailed t tests. One sample t tests were applied to determine
ture.Inbrief,8-AHA-cAMP(BioLog,Bremen,Germany)was significanceincomparisonwiththebasalvalue.
covalentlycoupledtoCM5sensorchips(researchgrade)using
RESULTS
NHS/EDC chemistry. RI or RII subunits, prepared according
Bertinettietal.(28)usingcAMPaffinitychromatography,were Bioinformatics-basedApproachtoIdentifyNewAKAPs—Hy-
injected in running buffer containing 1 mg/ml bovine serum drophobic amino acid residues in conserved positions of
albuminandreversiblycapturedonan8-AHA-cAMPsurface RIIBDsofAKAPsareimportantcontactsforRIIsubunitbind-
(surface concentration of 120–200 resonance units for each ing(Fig.1A)(8–10).Aconsensuspatternsummarizingthese
subunit). GSKIP was injected at different concentrations propertiesissuitablefortheidentificationofRIIBDswithina
(8–256 nM) at a flow rate of 30 (cid:3)l/min. Both association and givenAKAP(29).However,searchingaproteindatabasewith
FEBRUARY19,2010•VOLUME285•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5509
GSKIPIsanAKAP
FIGURE1.BioinformaticsapproachtoidentifynewAKAPs.A,multiplesequencealignmentofRIIBDsofseveralAKAPs.Aminoacidresiduesin
conservedpositionsareshadedingray.Theisoelectricpoints(pI)ofthepeptidesareindicated.Allsequencesexceptmurine(Mm)AKAP-Lbc,rat(Rn)
AKAP18(cid:5),andD.melanogaster(Dm)DAKAP550arederivedfromhumanAKAPs.AKAP isapeptidedesignedinsilico(10,52).B,flowchartforthe
IS
identificationofRII-bindingdomains.DepictedistheAKAPsearchpatternforscreeningoftheSwiss-ProtDatabaseforpotentialRII-bindingproteins.
Forthedatabasesearch,thepIvaluerangewaslimitedto3.0–6.4.Duplicatesandpeptidesnotaccessibletosynthesiswereremoved(Filter).The
remainingpeptides(2572)werespot-synthesizedandsubjectedtoRIIoverlayassay(supplementalFig.1).829peptidesboundRIIsubunits.C,829
peptidesweresynthesizedonthreemembranesandsubjectedtoRIIoverlay(Blocks1–3;forsequencessee supplementalTable1).Signalswere
detectedbyautoradiography.RowAoneachblockcontainstheindicatedcontrolpeptides,whichareestablishedRII-bindingpeptidesorinactive
versionsthereof.Dashedboxes,RIIBDsfrompreviouslyidentifiedAKAPs.Solidbox,peptidederivedfromtheproteinGSKIP.D,ofthe829RII-binding
peptides,27representingputativelynewAKAPs(andadditionally9derivedfrompreviouslydescribedAKAPs)werespot-synthesizedandsubjectedto
RIIoverlayintheabsence(control)orpresenceofthePKAanchoringdisruptorpeptideAKAP18(cid:5)-L314EortheinactivecontrolpeptideAKAP18(cid:5)-PP(10
(cid:3)Meach).RowAofeachmembranecontainstheindicatedcontrolpeptides;rowBcontainspeptidesderivedfrompreviouslyidentifiedAKAPs;androws
C–EcontaintheputativeRII-bindingdomainsoftheindicatedproteins(givenasabbreviations;forsequencesseeTable1).Box,RII-bindingdomainof
GSKIP.Displayedarerepresentativemembranesfromatleastthreeindependentexperiments.
5510 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER8•FEBRUARY19,2010
GSKIPIsanAKAP
suchageneralpatternfornewAKAPsidentifiedtoomanyfalse andinthepresenceofpeptideAKAP18(cid:5)-PP(Fig.1D;forpep-
positives (data not shown). Our recent observation that tidesequencesseeTable1).PreincubationofRIIsubunitswith
H-bondsandsaltbridgesinfluenceAKAP-RIIbinding(10)pro- peptideAKAP18(cid:5)-L314EreducedorabolishedRIIbindingto
vided the possibility to refine the search pattern. Charged the 27 RII-binding peptides. Thus, these peptides represent
amino acid residues, putative salt bridge, or H-bond partners putativeRIIBDsoftheircognateproteins(Fig.1D).Secondary
influencetheisoelectricpoint(pI)ofapeptide.Thus,although structure predictions (32) of the candidate peptides revealed
the number and positions of amino acids that function as highprobabilitiestoform(cid:2)-helices(datanotshown).
acceptors or donors of H-bonds or salt bridges vary among GSKIP Binds PKA with High Affinity in Vitro—Among the
AKAPs, the calculated pI for RIIBDs should be limited to a cognateproteinscontainingaputativeRIIBD,GSKIPwaschar-
certain range. Indeed, the calculated pI for peptides derived acterizedwithregardtoitsAKAPfunction.Thesequenceofthe
fromseveralRIIBDsofAKAPsrangefrom3.43to6.23(Fig.1A). putative RIIBD (TDMKDMRLEAEAVVNDVLFAVNNMF) is
TakingintoaccounttherangeofpIvalues,theconservedposi- located between amino acid residues 28–52 of full-length
tioningofhydrophobicaminoacidresidues,andthattheDD- GSKIP (Fig. 1D and Fig. 2A; Table 1). GSKIP is also named
domain of the RII subunit dimer forms a nearly symmetric C14orf129afteritschromosomallocalization,chromosome14,
groove(30),andthusassuminga2-foldmirrorsymmetryfor openreadingframe129(Swiss-ProtID,GSKIP_human).
the RIIBDs led to the following search pattern: (AVLISE)XX- Initially,weaimedtovalidatethelocalizationoftheRIIBD
(AVLIF)(AVLI)XX(AVLI)(AVLIF)XX(AVLISE), where amino withinGSKIP.Forthispurpose,anarrayofoverlappingpep-
acid residues in parentheses denote alternatives of the amino tidescoveringtheentiresequenceofGSKIP(139aminoacids)
acidresiduesinconservedpositionsofRIIBDs,andXstandsfor was spot-synthesized (25-mers, shift 1) and subjected to RII
any amino acid (Fig. 1, A and B). Utilizing this pattern and overlay assay. The cysteines in positions 5, 59, and 77 were
limitingtherangeofpIvaluesto3.0–6.4,theSwiss-ProtData- substitutedwithserinesascysteine-containingpeptidesarenot
basewassearchedforpotentialRII-bindingproteinswiththe accessible to spot-synthesis (see “Experimental Procedures”).
ScanSiteprogram.Thesearchretrieved(cid:4)11,700hitsfromDic- TheRIIsubunitsboundtoaseriesofoverlappingpeptides(Fig.
tyostelium discoideum, Saccharomyces cerevisiae, Drosophila 2A, C4–D6) such as is expected for a continuous binding
melanogaster, Danio rerio, Mus musculus, Rattus norvegicus, domain,i.e.decreasingaffinityuponincreasingdistancefrom
andHomosapiens.Thecognatesequencesofthehumanpro- the region of maximal binding. The RII subunits bound the
teinswereretrievedutilizingthebioperlpackageandthecor- previously identified peptide TDMKDMRLEAEAVVNDVL-
respondingidentificationnumbers(31).Aminoacidsequences FAVNNMFwiththehighestapparentbindingaffinity(Fig.2A,
were extracted from the cognate proteins and extended by 9 peptideC08).ThelowerpanelofFig.2Ashowsthesequences
aminoacidresiduesattheNterminusand4aminoacidresi- of the binding peptides with hydrophobic amino acid resi-
dues at the C terminus of the hit sequence to obtain 25-mer dues highlighted. They are in conserved positions of the
amino acid sequences. In total, 4519 peptides were derived amphipathic helix binding the DD domain (for comparison
from 2352 human proteins. These peptides were further fil- seeFig.1,AandB).TheRIIsubunitsboundadditionalpeptides
teredtoexcludeduplicatesandso-calleddifficultsequencesnot (F1–5andJ5–L5)withlowapparentbindingaffinitiessuggest-
accessibletopeptidespotsynthesis(see“ExperimentalProce- ing that GSKIP contains a second RII-binding site. However,
dures”) (21, 23). For example, the RIIBD of mAKAP (also furtherexperimentsindicatedthatsitesotherthantheRIIBDin
termedAKAP6)wasexcludedasitcontainsacysteineresidue. positions 28–52 of GSKIP are not involved in RII binding in
The filtering reduced the number of peptides to 2572. They cells(datanotshown).
werespot-synthesizedandsubjectedtoRIIoverlayassaytotest The influence of individual amino acid residues within the
theirabilitytobindRIIsubunits(supplementalFig.1andsup- RIIBDofGSKIPontheinteractionwithRIIsubunitswasdeter-
plemental Table 1). Peptide spots were overlaid with 32P-la- mined by probing a peptide substitution array of the GSKIP
beledRIIsubunitsofPKA,andbindingwasdetectedbyauto- RIIBDforRIIbinding(Fig.2B).Inthisarrayeveryaminoacid
radiography.ThisapproachissummarizedinFig.1B. residue of the RIIBD (vertical sequence) was substituted with
Approximatelyone-third(829)ofthe2572peptidesbound the amino acid residues indicated above the array (Fig. 2B).
RIIsubunits(Fig.1C,Blocks1–3).Thepeptidesequencesare Whenhydrophobicaminoacidresiduesinconservedpositions
indicatedinsupplementalTable1.Toselectpeptidesbinding oftheRIIBDweresubstitutedbypolarorchargedaminoacid
toRIIsubunitsinatypicalAKAPmannerattheDDdomain,all residues,RIIboundwithapparentlyloweraffinity,orbinding
RII-bindingpeptideswerespot-synthesizedagainandprobed was abolished. The nature (e.g. the size) of the hydrophobic
forRIIbindingwithhumanRIIsubunitspreincubatedwiththe residuesatparticularpositionsappearstobeimportantforthe
peptide AKAP18(cid:5)-L314E. The peptide was derived from the interactionwithRIIsubunitsbecausesubstitutionofahydro-
RIIBDofAKAP18(cid:5).ItbindsRIIsubunitswithsubnanomolar phobicaminoacidresiduewithanotherhydrophobicresidue
affinity (K (cid:5) 0.7 (cid:6) 0.5 nM for binding to RII(cid:2)) at the DD frequentlydecreasedtheinteractionwithRII.Forexample,the
D
domainandtherebypreventstheinteractionofRIIwithAKAPs Val-Valsequenceatpositions40and41withinGSKIPshows
(10).Asacontrol,RIIsubunitswereincubatedwiththepeptide variabletoleranceforisoleucine,leucine,tyrosine,andtrypto-
AKAP18(cid:5)-PP,whichcontainstwoprolinesanddoesnotbind phan(Fig.2B,solidboxes).AsexpectedfromatypicalAKAP,
RIIsubunits(10).Amongthe829peptides,27(andadditionally introductionofprolineintotheRIIBDreducesorabrogatesthe
9 derived from previously described AKAPs) reproducibly interactionwithRIIsubunitsasthe(cid:2)-helicalstructureofthe
interactedwithRIIsubunitsintheabsenceofpeptide(control) coreRIIBDislost(Fig.2B,dashedbox).
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TABLE1
SequencesofpeptidesfromFig.1D
SpotnumberingcorrespondstopositionsofpeptidespotsonthemembraneinFig.1D.PartA,controlpeptides.PartB,peptidesderivedfromAKAPcandidates.Protein
identification(ID)andprimaryaccessionnumbers(AC)relatetoentriesintheSwiss-ProtDatabase.AbbreviationsforproteinsasshowninFig.1D,andthecorresponding
fullnamesofthecognateproteinsareindicated.
Next, it was investigated whether full-length GSKIP func- His-taggedGSKIPwasinjectedasananalyteatdifferentcon-
tionsasanAKAP.cDNAencodingfull-lengthGSKIP(amino centrations(8–256nM).Associationanddissociationratecon-
acidresidues1–139)wasobtainedfromhumanneuroblastoma stantsweredetermined,andequilibriumbindingconstantsof5
cells(SH-SY5Y)andutilizedforthegenerationofrecombinant and43nMwerecalculatedforRII(cid:2)andRII(cid:1),respectively(Fig.
GSKIPfusedwithaHistag(His-GSKIP).Thetagwasremoved 3BandTable2).Theinteractionswerereducedby80%ifthe
by thrombin protease cleavage, and the interaction of GSKIP experiments were carried out in the presence of the peptide
withtheDDdomainofRII(cid:2)wasanalyzedbyNMRexperiments AKAP18(cid:5)-L314E(datanotshown).GSKIPdidnotbindtoRI
(Fig. 3A). The spectra confirmed that the above described subunits(datanotshown).
RIIBDislocatedatpositions28–52oftheproteinandbindsthe In addition, we tested whether immunoprecipitated full-
DDdomain.ResonanceattenuationinthepresenceoftheDD length GSKIP binds RII subunits. Human full-length GSKIP
domaincouldbeassignedtoresidueswithintheRIIBD(Leu-35, wastransientlyexpressedinHEK293cellsasafusionwithcyan
Val-41,Phe-46,andAla-47)butalsowithinthe(cid:1)-sheetregion fluorescent protein (Fig. 3C, CFP-GSKIP). A fusion protein
ofGSKIPandintheC-terminalhelixinproximitytotheRIIBD containingtwoprolinesintheRIIBDofGSKIP(CFP-GSKIP-
(Fig.3A,D112,L114,F122,andG123). V41P/L45P)wasgeneratedasanegativecontrol.Thefusions
To quantify the interaction of GSKIP with recombinant were immunoprecipitated with rabbit anti-GSKIP-antibody
human RII(cid:2)- and RII(cid:1)-subunits, Biacore technology was and tested for RII binding in an RII overlay (Fig. 3C, upper
employed.Rsubunitswerecapturedon8-AHA-cAMPsensor panel). RII subunits bound to immunoprecipitated proteins
surfacestoyieldanaveragesurfaceconcentrationof120–200 correspondinginsizetoCFP-GSKIP(48kDa).Nosignalwas
resonance units (see “Experimental Procedures” for details). detectedwhentheprecipitationwascarriedoutwiththecor-
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FIGURE2.RII-bindingdomainofGSKIP.A,mappingtheRII-bindingdomainofGSKIP.TheentiresequenceofGSKIP(139aminoacidresidues)wasspot-
synthesizedasoverlappingpeptides(25-mers,24aminoacidresidueoverlap)andprobedforRIIbindingbyRIIoverlayassay.Signalsweredetectedby
autoradiography.PeptidesequencesbindingRII(positionC06–D06)aredisplayedinthelowerpanel.Hydrophobicaminoacidresiduesinconservedpositions
ofRII-bindingdomainsareshadedingray(compareFig.1A).Ascontrols,PKAanchoringdisruptorpeptides(rowM)weresynthesized.B,influencesofsingle
aminoacidresidueswithintheGSKIPRII-bindingdomainontheinteractionwithRIIsubunits.Thescheme(left)depictsthelocationoftheRII-bindingdomain
(RII-BD)withinGSKIP.PeptidesderivedfromtheRII-bindingdomain(positions28–52withinGSKIP;vertical)andtheindicatedcontrolpeptidesinrowZwere
spot-synthesized.TheaminoacidresiduesoftheRII-bindingdomainweresubstitutedwiththeresiduesindicatedatthetopofthemembrane.Interactionof
thepeptideswithRIIsubunitswasrevealedbyRIIoverlayassayanddetectionbyautoradiography.Solidboxes,peptidessubstitutedataminoacidresiduesin
conservedpositionsoftheRII-bindingdomain.Dashedbox,introductionofprolinesintotheRII-bindingdomainreducesorabolishesRIIbinding.Aminoacid
residuesareindicatedbysinglelettercode.AandB,displayedarerepresentativemembranesfromatleastthreeindependentexperiments.
responding preimmune serum. In addition, the precipitated GSKIP Is Widely Expressed and Functions as an AKAP in
proteinsboundRIIsubunitsonlyintheabsenceofthepeptide Vivo—Homogenatesfromvariousratorgansweresubjectedto
AKAP18(cid:5)-L314E(seeabove).Asexpected,theintroductionof SDS-PAGEandWesternblottingwithourrabbitanti-GSKIP
twoprolinesintopositions41and45abolishedbindingtoRII antibody(Fig.4A).GSKIPwasdetectedinalltestedorgans.The
subunits.Takentogether,thedatashowthatfull-lengthGSKIP highestGSKIPproteinlevelswerefoundintestisandbrain(Fig.
bindsPKAatitsRIIBDbetweenaminoacidresidues28and52 4A).TheseresultsareinaccordancewiththoseofChouetal.
andthusfunctionsasanAKAP.Similaramountsofthefusions (20)whodemonstratedubiquitousexpressionofGSKIPmRNA
ofCFPwithwildtypeGSKIPandCFPwithGSKIP-V41P/L45P and with data base entries showing organism-wide GSKIP
were precipitated (Fig. 3C, lower panel). This was revealed expression, e.g. Serial Analysis of Gene Expression (SAGE)
whentheamountsofprecipitatedproteinswerecomparedby studies as summarized under the gene card homepage of
Westernblottingwithanti-greenfluorescentproteinantibody. the Weizmann Institute. To determine whether endogenous
The experiment also confirmed the specificity of the anti- GSKIPbindsregulatorysubunitsofPKAandthusfunctionsas
GSKIP antibody. This antibody precipitated the fusions con- anAKAPinvivo,cAMP-agarosepulldownassaysandWestern
tainingtheGSKIPversionsandCFPbutnotCFPalone,orother blottingwerecarriedoutusinglysatesderivedfromvariousrat
proteinscorrespondinginsizetoCFP(28kDa)werespecifically tissues (Fig. 4B). Cyclic AMP-agarose precipitates regulatory
detected(Fig.3C,lowerpanel). subunitsofPKA,includingtheirinteractingpartners.Co-pre-
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TABLE2 moiety,whichisknowntotargetvariousproteinstothenucleus
ApparentrateandequilibriumdissociationconstantsofGSKIP (36).SimilarlytotheGSKIPversions,YFP-RII(cid:2)wasdistributed
bindingtoRII(cid:2)andRII(cid:1)
throughout the cytoplasm but was not found in the nucleus,
Surface plasmon resonance measurements using Biacore technology were per-
formedtodeterminerateconstants(k andk)andequilibriumbindingconstants probablyduetothehighermolecularweightofthisfusionpro-
(KD)fortheinteractionofHis-taggedGaSKIPwdithhumanRsubunits.RII(cid:2)andRII(cid:1) teinincomparisonwithCFP-GSKIP.Togetherwithourmolec-
werereversiblycapturedvia8-AHA-cAMP,whichwascovalentlycoupledona
CM5sensorchip(notshownintheplot).His-GSKIPwasinjectedoverRII(cid:2)and ularinteractionandpulldownstudies(Figs.3and4),ourdata
RII(cid:1)surfaces(Fig.3B).Associationanddissociationphasesweremonitoredfor5 indicatethatGSKIPandRII(cid:2)co-localizeandformcomplexes
min,respectively.NobindingwasdetectedwhenRI(cid:2)andRI(cid:1)werecaptured(data
inthecytosolofcells.WecomparedthelocalizationofGSKIP
notshown).
k k K toanotherAKAP,AKAP18(cid:2),whichislipid-anchoredandthus
a d D
M(cid:7)1s(cid:7)1 s(cid:7)1 nM locatedintheplasmamembrane.
RII(cid:2) 1.3(cid:3)105 6.6(cid:3)10(cid:7)4 5 RII-bindingDomainofGSKIPIsLocatedwithintheDomain
RII(cid:1) 2.8(cid:3)104 1.2(cid:3)10(cid:7)3 43 of Unknown Function 727—Screening the InterPro data base
(37)revealedthatGSKIPorthologuescontainthe“domainof
unknownfunction727”(DUF727;InterProdatabaseaccession
cipitatedGSKIPwasdetectedbyimmunoblottingwithrabbit
numberIPR007967)basedontheirproteinsequencesimilarity.
anti-GSKIPantibody.Immunoblottingrevealedabandofthe
expected molecular weight (Fig. 4B, 17 kDa, (cid:7)cAMP), which DUF727spansaminoacidresidues32–139ofhumanGSKIP.
Thisdomain,coveringalmostthefull-lengthofGSKIP,includ-
wasnotdetectedifthecAMP-agarosepulldownwascarriedout
inthepresenceofexcesscAMP((cid:2)cAMP).Asacontrol,RII(cid:2) ingtheRIIBD,isconservedwithinmetazoans.Forexample,the
domainshowsa68%aminoacididentitybetweenzebrafishand
subunits were detected by Western blot. In addition to full-
lengthRII(cid:2),severalotherbandsweredetected.Thesemaybe humanproteins.TotestwhetherthemembersoftheDUF727
familybindRIIsubunits,peptideshomologoustotheRIIBDof
degradation products mainly observed in kidney and lung,
GSKIP were identified in various vertebrate and invertebrate
whichareprotease-richtissues(33).ThedatashowthatGSKIP
familymembers,spot-synthesized,andsubjectedtoRIIoverlay
functionsasanAKAPinvivo(Fig.4B).
To investigate the cellular localization of GSKIP, we pre- assaywithhumanRII(cid:2)subunits(Fig.5A).Allpeptidesderived
paredsubcellularfractionsofSH-SY5YcellsexpressingGSKIP fromtheindicatedvertebrateproteinsbindtohumanRII(cid:2)sub-
endogenously.Theenrichmentofnucleiandcytosolwasmon- units.BindingwasabolishediftheRIIsubunitswerepreincu-
itoredbydetectinglaminA/C(nucleus)andGAPDHandtubu- bated with the peptide AKAP18(cid:5)-L314E, suggesting that the
lin(bothcytosol).Apparently,GSKIPislocalizedexclusivelyin cognateproteinsfunctionasAKAPs.Thehighdegreeofcon-
thecytosol(Fig.4C).Thisresultisinlinewithapredictionusing servation implies that the AKAP function of GSKIP and its
the subcellular localization prediction program pTarget (34), orthologuesisinvolvedinthecontrolofmechanismscommon
which calculated a cytoplasmic localization of GSKIP with a toallvertebrates.ThesequenceidentityoftheputativeRIIBDs
high confidence of 87.6%. The soluble fraction also contains ofthevertebrateandinvertebratespeciesislow, e.g.between
high amounts of GSK3(cid:1)and PKA RII(cid:2)subunits. These two the human GSKIP (Q6POR6) and the C. elegans orthologue
proteinswerealsopresentinthenuclearandmembranefrac- (Q9XWX6)(cid:8)40%(Fig.5A).Inaddition,therearefewerhydro-
tions. This has been shown previously for both proteins (35). phobicaminoacidresiduesintheinvertebratesequencescom-
Laserscanningmicroscopicanalysiswascarriedouttoexamine pared with those in conserved positions of the vertebrate
whether GSKIP and RII(cid:2)co-localize in the cytosol. For this sequences.Thesedifferencesmostlikelyaccountfortheobser-
purpose, the two proteins were expressed in HEK293 cells as vation that binding of human RII(cid:2)subunits to the peptides
fusionswithCFPandyellowfluorescentprotein,respectively. derived from the indicated invertebrate proteins was appar-
CFP-GSKIP is distributed throughout the cells, including the entlyweakorundetectable(Fig.5A).Thus,itappearsthatthe
nucleus (Fig. 4D). The RII-binding deficient mutant CFP- invertebrateproteinsmayeitherbelowaffinityAKAPsordo
GSKIP-V41P/L45Pdisplaysasimilardistribution.Thenuclear not possess an AKAP function. We have confirmed that our
localizationoftheGSKIPversionsismostlikelyduetotheCFP approachcanidentifybothvertebrateandinvertebrateAKAPs.
FIGURE3.GSKIPfunctionsasanAKAPinvitro.A,GSKIPandtheDDdomainofhumanregulatoryRII(cid:2)subunitsofPKAweregeneratedandNMRmeasure-
mentsperformed.1H-15NheteronuclearsinglequantumcoherencespectraoftheGSKIPdomainintheabsence(red)orpresenceofDD(black)at1:0.5ratioare
shown.DisappearanceoftheGSKIPresonancesiscausedbecauseofchemicalexchangebroadeningand/orincreasedtransverserelaxationrateduetohigh
molecularweightofthecomplex.Lowerpanel,ProteinDataBankstructureofGSKIP(ProteinDataBankcode1sgo)TheRII-bindingdomain(black)encompasses
theamphipathic(cid:2)1-helixofGSKIP.TheconservedhydrophobicaminoacidresiduesAla-37,Val-40,Val-41,Val-44,Leu-45,andVal-48(seealsoFig.1A)are
depictedasball-and-stickmodels.ThecontactinterfaceforRIIbindingishiddenbya(cid:1)-sheet.B,surfaceplasmonresonancemeasurementstodeterminethe
associationanddissociationrateconstants(seeTable2)forthebindingofGSKIPtoPKAregulatoryRIIsubunits.HumanRII(cid:2)andRII(cid:1)subunits(upperandlower
panel,respectively)werecapturedon8-AHA-cAMPsensorchips,andHis-GSKIPwasinjectedintotheBiacoreinstrumentintheindicatedconcentrations.The
plotsshowrepresentativeexperiments.Eachexperimentwasrepeatedatleastthreetimesusingdifferentproteinpreparationsanddifferentimmobilization
ratesoftheregulatorysubunits.RU,resonanceunits.C,immunoprecipitated(IP)GSKIPbindsRIIsubunits.Cyanfluorescentprotein(CFP;lane1)andthe
indicatedfusionsofhumanfull-lengthGSKIPwithCFP(lane2)andGSKIP-V41P/L45PwithCFP(lane4)weretransientlyexpressedinHEK293cells.CFP-GSKIP-
V41P/L45PcontainsprolinesintheRII-bindingdomaindisruptingthe(cid:2)-helicalstructureandthuspreventingtheinteractionwithRII.Thecellswerelysed,and
GSKIPwasimmunoprecipitatedwithanti-GSKIPantibodies,ortheprecipitationwascarriedoutwiththecorrespondingpreimmuneserum.Precipitated
proteinswereprobedforRIIbindingbyRIIoverlayassayintheabsence(control)orinthepresenceofthePKAanchoringdisruptorpeptide,AKAP18(cid:5)-L314E(10
(cid:3)M).Signalsweredetectedbyautoradiography.Lane3,molecularweightstandard.Lowerpanel,Westernblot(WB)withanti-greenfluorescentprotein
antibody(alsodetectsCFP(26))forthedetectionofCFP,CFP-GSKIP,orCFP-GSKIP-V41P/L45PinHEK293cellstransientlyexpressingtheproteins.Proteinswere
detectedinprecipitatesobtainedwithrabbitanti-GSKIPantibody(IP,leftpanel)orobtainedwithpreimmuneserum(middlepanel)orincelllysates.
FEBRUARY19,2010•VOLUME285•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5515
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leucine (indicated in Fig. 5B by an
asterisk)wassubstitutedbyproline
inthepeptidesderivedfromverte-
bratesequences.40outof41puta-
tive DUF727 GSK3(cid:1) interaction
peptides bound GST-GSK3(cid:1) (Fig.
5B). No binding of GST to any of
the peptides or of GST-GSK3(cid:1)to
Leu 3 Pro mutant peptides was
observed. In summary, the data
indicatethatGSK3(cid:1)bindingiscon-
served in almost all GSKIP ortho-
logues/DUF727proteinsfromfungi
tomammalsandthatallvertebrate
DUF727proteinspossessanAKAP
function.
GSKIPFormsaTernaryComplex
with PKA and GSK3(cid:1)and Facili-
tates PKA Phosphorylation of
GSK3(cid:1)—Weaimedtoelucidatethe
functionoftheGSKIP/PKAinterac-
tion. GSKIP interacts with GSK3(cid:1)
through amino acids 115–139 of
GSKIP(20).Wethereforehypothe-
sized that GSKIP facilitates PKA
FIGURE4.GSKIPiswidelyexpressedandfunctionsasanAKAPinvivo.A,lysatesfromtheindicatedrat
organsweresubjectedtoWesternblot(WB)analysiswithananti-GSKIPantibodyand,asaloadingcontrol, phosphorylation of GSK3(cid:1). Ini-
anti-GAPDHantibody(167(cid:3)goftotalproteinperlane).B,cAMP-agarosepulldownswereobtainedfromthe tially, we investigated whether
indicatedrattissuelysates(3mgoftotalproteinineachsample)intheabsence((cid:7)cAMP)orpresence((cid:2)cAMP;
50(cid:3)M)ofcAMP.RII(cid:2)subunitsofPKAandGSKIPweredetectedwithspecificantibodiesbyWesternblotting. GSKIP forms a ternary complex
skel.,skeletal.C,indicatedsubcellularfractionswereobtainedfromSH-SY5Ycells.10(cid:3)goftotalproteinfrom withGSK3(cid:1)andPKAinanenzyme-
eachfractionwasanalyzedforthepresenceoftheindicatedproteinsbyWesternblotting.D,GSKIPandRII(cid:2)
linkedimmunosorbentassay-based
co-localizeinHEK293cells.HEK293cellsweretransientlytransfectedtoexpressYFP-RII(cid:2)andfusionsofCFP
withwildtypeGSKIP(CFP-GSKIPWT),GSKIP-V41P/L45P,orasaplasmamembranemarkerAKAP18(cid:2). approach(Fig.6A).Ifwellsofmicro-
titerplateswerecoatedwithGSKIP,
D.melanogasterDAKAP550(alsotermedneurobeachin(38)or GSK3(cid:1)bound as detected with anti-GSK3(cid:1)and secondary
rugose) contains two RII-binding sites (39). Both sites were HRP-coupled antibodies. If GSKIP was omitted, i.e. wells were
identifiedbyourAKAPcandidatesearch,andbindingwascon- blocked with blocking solution without GSKIP, signals were
firmedbyRIIoverlaywithhumanRII(cid:2)(datanotshown). hardlydetectable(Fig.6A,leftpanel).Ifincreasingconcentrations
AninteractionofGSKIPwithGSK3(cid:1)hasbeendemonstrated ofGSK3(cid:1)incombinationwithconstantamountsofGSKIPwere
forthehumanproteins(20,40)butalsofortheC.elegansortho- addedtoRII(cid:2)-coatedwells,acomplexconsistingofthethreepro-
loguesofGSKIP(Q22757)andGSK3(cid:1)(41).Axininteractswith teinsformed.Complexesaccumulatedwithincreasingconcen-
GSK3(cid:1). The GSK3(cid:1)-binding sites of GSKIP and of axin are trationsofGSK3(cid:1),asdetectedwithanti-GSK3(cid:1)andsecondary
homologous (20). The axin/GSK3(cid:1) interaction is brought HRP-coupled antibodies. As negative controls, wells were
aboutbyfourhydrophobicaminoacidsofanaxin(cid:2)-helixbind- blockedandincubatedwithGSKIPandGSK3(cid:1)orcoatedwith
ingtoahydrophobicgrooveinGSK3(cid:1)(42).Sequencecompar- RII(cid:2)and incubated with GSK3(cid:1)in the absence of GSKIP. In
ison revealed that three amino acids of axin, essential for the neither negative control was significant binding of GSK3(cid:1)
bindingtoGSK3(cid:1),areconservedin41of51membersofthe observed.ThisindicatesthatneitherGSKIPnorGSK3(cid:1)bound
DUF727family,i.e.theGSKIPorthologues(Phe-122,Leu-126, toblockedwellsandthatGSK3(cid:1)interactsindirectlywithRII(cid:2)
andLeu-130inhumanGSKIP,seeFig.5B).These41proteins via GSKIP. Thus, GSKIP is a scaffolding protein recruiting
alsohaveahydrophobicaminoacidinthepositioncorrespond- GSK3(cid:1)andRIIsubunitsandpositionsthetwoproteinsinclose
ing to Leu-133 in human GSKIP. In this position, various proximity.
hydrophobicaminoacidsarepermissiveforGSK3(cid:1)bindingas Next,weexaminedwhetherGSKIPfacilitatesPKAphosphory-
seen in axin-1 and axin-2, which contain valine and leucine lation of GSK3(cid:1). HEK293 cells expressing GSK3(cid:1) endog-
residues. To test whether the putative GSK3(cid:1) interaction enously(Fig.6B)weretransientlytransfectedtoexpressCFPor
domainsoftheDUF727proteinsbindGSK3(cid:1),peptidespotsof afusionofCFPwithGSKIPandleftuntreatedorweretreated
the corresponding sequences were incubated with recombi- withforskolin(20(cid:3)M,30min).Abasallevelofphosphorylated
nant GST-GSK3(cid:1) (serine was used for peptide synthesis GSK3(cid:1) (p-GSK3(cid:1)) was detectable in both CFP and CFP-
instead of cysteine, see “Experimental Procedures”). Binding GSKIP-expressingcells.Thelevel,however,washigherincells
wasdetectedwithananti-GSTantibody.Asnegativecontrols, expressingCFP-GSKIP.Forskolinisadirectactivatorofaden-
thepeptidespotswereincubatedwithGST,andinaddition,the ylyl cyclases and induces a rise in cAMP activating PKA. In
5516 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER8•FEBRUARY19,2010