FRAT1, a Substrate-specific Regulator of Glycogen SynthaseKinase-3 Activity, Is a Cellular Substrate of Protein Kinase A*

Received for publication, July 24, 2006, and in revised form, September 15, 2006 Published, JBC Papers in Press, September 18, 2006, DOI 10.1074/jbc.M607003200

Thilo Hagen‡§1, Darren A. E. Cross2, Ainsley A. Culbert¶, Andrew West�, Sheelagh Frame**3, Nick Morrice**,and Alastair D. Reith¶‡‡

From ‡Discovery Research Biology, ¶Neurology Centre of Excellence in Drug Discovery, �Computational, Analytical,and Structural Sciences, GlaxoSmithKline Pharmaceuticals, Harlow, Essex CM19 5AD, United Kingdom, ‡‡MedicinesResearch Centre, Stevenage, Herts SG1 2NY, United Kingdom, **Medical Research Council Protein Phosphorylation Unit,School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom, and §Wolfson Digestive DiseasesCentre, University of Nottingham, Nottingham NG7 2UH, United Kindom

FRAT1, like its Xenopus homolog glycogen synthase kinase-3(GSK-3)-binding protein, is known to inhibit GSK-3-mediatedphosphorylation of �-catenin. It is currently unknown howFRAT-GSK-3-binding protein activity toward GSK-3 is regu-lated. FRAT1has recently been shown tobe aphosphoprotein invivo; however, the responsible kinase(s) have not been deter-mined. In this study, we identified Ser188 as a phosphorylatedresidue in FRAT1. The identity of the kinase that catalyzesSer188 phosphorylation and the significance of this phosphoryl-ation to FRAT1 function were investigated. Protein kinase A(PKA) was found to phosphorylate Ser188 in vitro as well as inintact cells. Importantly, activation of endogenous cAMP-cou-pled �-adrenergic receptors with norepinephrine stimulatedthe phosphorylation of FRAT1 at Ser188. GSK-3 was also able tophosphorylate FRAT1 at Ser188 and other residues in vitro orwhen overexpressed in intact cells. In contrast, endogenousGSK-3 did not lead to significant FRAT1 phosphorylation incells, suggesting thatGSK-3 is not amajor FRAT1kinase in vivo.Phosphorylation of Ser188 by PKA inhibited the ability ofFRAT1 to activate �-catenin-dependent transcription. In con-clusion, PKA phosphorylates FRAT1 in vitro as well as in intactcells and may play a role in regulating the inhibitory activity ofFRAT1 toward GSK-3.

Glycogen synthase kinase-3 (GSK-3)4 is a serine/threoninekinase that phosphorylates multiple substrates in the cell and isinvolved in distinct cellular signaling pathways, including insu-lin/growth factor and Wnt signaling (1–3). GSK-3 is a consti-tutively active kinase. Upon stimulation of both insulin/growthfactor and Wnt-dependent signaling pathways, GSK-3 is inac-tivated. Insulin-dependent GSK-3 inhibition involves activa-

tion of phosphatidylinositol 3-kinase and Akt/protein kinase B,which then phosphorylates GSK-3 at an N-terminal serine res-idue (Ser21 in GSK-3� and Ser9 in GSK-3�) (4). The phospho-rylated N terminus is believed to act as a pseudosubstrate andcompete for substrate binding, thus leading to autoinhibition ofthe catalytic activity of GSK-3 (5, 6). Inactivation of GSK-3 inresponse to insulin stimulation leads to dephosphorylation andactivation of the GSK-3 substrates glycogen synthase andeukaryotic protein synthesis initiation factor 2B, ultimatelycontributing to the stimulation of glycogen and proteinsynthesis.In the Wnt signaling pathway, GSK-3 is known to phospho-

rylate �-catenin at defined residues at the N terminus, thustargeting the protein for ubiquitin/proteasome-mediated deg-radation (1, 7). �-Catenin phosphorylation occurs in a complexthat includes the tumor suppressor protein APC and the scaf-fold protein Axin. Activation of Wnt signaling through bind-ing of Wnt-secreted glycoproteins to their receptors leads toinhibition of the Axin-APC-GSK-3 complex. Consequently,�-catenin becomes stabilized and translocates into thenucleus, where it acts as a transcriptional coactivator of tran-scription factors of the TCF/LEF family, activating targetgenes such as c-myc and cyclin D1 (8–10). Themechanism ofWnt-dependent GSK-3 inactivation is distinct from theinsulin pathway and does not involve phosphorylation of theN-terminal serine (11, 12).The protein GSK-3-binding protein (GBP) was identified in

Xenopus (13). GBP inhibits GSK-3, leading to stabilization of�-catenin in Xenopus embryos and induction of a secondarybody axis (13). GBP is homologous to the mammalian T cellprotooncogene FRAT1 (frequently rearranged in advanced Tcell lymphomas 1) (14). Two homologs of FRAT1 have beencloned, FRAT2 (15) and Frat3 (16). However, no GBP/FRAThomologs appear to be present in the genomes of Drosophilaand Caenorhabditis elegans (2).All GBP/FRAT homologs have been shown to induce a sec-

ondary axis in Xenopus embryos, indicating that they all inhibitGSK-3 activity toward �-catenin (13, 15, 16). The mechanismby which GBP/FRAT inhibits GSK-3 activity toward �-cateninappears to involve preventing Axin from binding to GSK-3,probably by competition for a common (or closely overlapping)binding site onGSK-3.GBP, FRAT, and FRATtide, a 39-residuepeptide derived from FRAT1 that is sufficient to bind GSK-3,

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a Marie Curie Industry Host Fellowship (awarded to A. D. R.)throughout the duration of this work. To whom correspondence should beaddressed. Tel.: 44-115-8231079; E-mail: Thilo.Hagen@nottingham.ac.uk.

2 Present address: AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield,Cheshire SK10 4TG, United Kingdom.

3 Present address: Cyclacel Ltd., James Lindsay Pl., Dundee, DD1 5JJ UnitedKingdom.

4 The abbreviations used are: GSK-3, glycogen synthase kinase-3; GBP, GSK-3-binding protein; MS, mass spectrometry; PKA, protein kinase A; PKC, pro-tein kinase C.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 46, pp. 35021–35029, November 17, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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have all been shown to dissociate GSK-3 from the Axin com-plex (17–21).Interestingly, although FRATtide inhibits GSK-3-mediated

phosphorylation of �-catenin and Axin in vitro, it does notinhibit GSK-3 activity toward peptides derived from glycogensynthase or eukaryotic protein synthesis initiation factor 2B(19). In addition, using adenoviral expression of FRAT1 inPC12 cells, we have shown that FRAT1 overexpression resultsin �-catenin stabilization but does not alter glycogen synthaseactivity (22). These results indicate that FRAT1-mediatedGSK-3 inhibition is also selective in vivo.It is not known how FRAT activity toward GSK-3 is regu-

lated. A recent report demonstrated that murine Frat1 andFrat2 are phosphoproteins (23); however, the responsiblekinases have not been identified. We observed that FRAT1immunoprecipitated from cells is phosphorylated by one ormore endogenous kinases. We identified protein kinase A(PKA) as a kinase that phosphorylates FRAT1 in vitro as well asin cells. We also provide evidence that GSK-3 is not a majorFRAT1 kinase in vivo.

MATERIALS AND METHODS

Plasmids and Cell Culture—A full-length human FRAT1clone was tagged with the Myc epitope at the N terminus andinserted into the mammalian pcDNA3 expression vector togenerate Myc-FRAT1-pcDNA3. C-terminally 2�FLAG- orV5-tagged or untagged expression plasmids for GSK-3� orFRAT1 were generated by PCR amplification of the humanGSK-3� and FRAT1 open reading frame from HEK293 or fetalbrain cDNA and insertion into pcDNA3. pFC-PKA, encodingthe mouse PKA� catalytic subunit, was obtained from Strat-agene. Subconfluent HEK293T cells were transfected usingFugene reagent (Roche Applied Science) according to theman-ufacturer’s instructions. 48 h after transfection, the cells werewashed with ice-cold PBS, lysed in 2.5 ml of lysis buffer (25 mMTris/HCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mMsodium orthovanadate, 100 mMNaCl, 20 mM sodium �-glycer-ophosphate, 10 mM sodium pyrophosphate, 0.5% (v/v) TritonX-100, 0.1% (v/v) �-mercaptoethanol, Roche Applied Science“Complete” protease inhibitors, pH 7.5), and the lysates weresnap frozen in liquid nitrogen and then stored at �80 °C untilrequired. Lysates were precleared by centrifugation before use.Immunoprecipitation of Myc-FRAT1—10 �l of protein

G-Sepharosewas coupled to 5�g ofmonoclonal anti-Myc anti-body (clone 9E10; Autogen Bioclear), and the pellet was used toimmunoprecipitateMyc-FRAT1 from1ml of precleared lysate.The pellets were thenwashed three times in 1ml of Buffer A (50mM Tris/HCl, 0.1 mM EGTA, 0.1% (v/v) �-mercaptoethanol,pH 7.5) containing 0.5 M NaCl and then twice with 1 ml ofBuffer A. The washed immunoprecipitates were used for invitro kinase reactions or denatured in SDS-sample buffer andsubjected to SDS-PAGE.Immunoblotting—Cells were lysed as described above. Equal

amounts of protein lysate or immunoprecipitated protein weresubjected to SDS-PAGE, electrophoretically transferred tonitrocellulose membranes, and immunoblotted. The followingantibodies were used:monoclonal anti-Myc (clone 9E10; Auto-gen Bioclear), monoclonal anti-V5 (Serotec), monoclonal anti-

FLAG (M2; Sigma), monoclonal anti-GSK-3� (BD Bio-sciences), rabbit polyclonal anti-PKA� catalytical subunit (sc-903 (C-20); Santa Cruz Biotechnology), and polyclonal FRAT1phospho-Ser188-specific antibody, raised in rabbit and gener-ated against a peptide representing residues 182–194 in humanFRAT1 (LQQRRGpSQPETRT; where pS represents phospho-serine), which was conjugated to keyhole limpet hemocyanin.Blots were developed using the Amersham BiosciencesEnhanced Chemiluminesence kit. TheWestern blots shown todetect protein expression and phosphorylation in intact cellsand in vitro phosphorylation of FRAT1 are representative ofthree independent experiments.In Vitro Phosphorylation Assays—FRAT1 immunoprecipi-

tates were incubated on a shaking platform for 10 min with35 �l of Buffer A in the presence of the different proteinkinase inhibitors or recombinant kinases. The kinase reac-tion was then initiated by the addition of 10 �l of 50 mM

MgCl2, 0.5 mM ATP (standard ATP for mass spectrometryanalysis or [�-32P]ATP (200–1000 cpm/pmol) for autora-diography) and incubated on a shaking platform for 30 minat 30 °C. LiCl and the selective GSK-3 inhibitors SB216763and SB415286 were used as described previously (22,24–26). Following the reaction, the samples were denaturedin SDS-sample buffer and subjected to SDS-PAGE. The gelswere dried, and phosphorylation of Myc-FRAT1 was ana-lyzed using autoradiography, where the reactions contained[�-32P]ATP. Alternatively, the gels were stained with Coo-massie and Myc-FRAT1 bands excised for phosphorylationsite mapping using mass spectrometry.Mass Spectrometry Analysis—The Myc-FRAT1 bands were

excised from the SDS-polyacrylamide gel, and the protein wasthen digested in gel with trypsin (modified sequencing grade;Promega Corp.). Following digestion, the resulting peptideswere desalted using C18 ZipTips (Millipore Corp.) and ana-lyzed using electrospray ionization on an ion trap mass spec-trometer (ThermoFinnigan). Themass spectrometerwas set toacquire an ion map by fragmenting each ion within a givenrange. In this way, each peptide in the sample was fragmentedwithin the mass spectrometer to generate a tandemmass spec-trum (MS/MS). The ion map, a representation of the tandemmass spectra of all components in a given sample, was thenexamined to see if any peptides had potentially lost a phosphategroup. Identification of phosphorylated peptides was then con-firmed by interpretation of the corresponding MS/MS spectra.In Vivo Labeling—HEK293T cells were transiently trans-

fected withMyc-FRAT1-pcDNA3. 16 h after transfection, eachdish of cells was labeled with 5mCi of [32P]orthophosphate andtreated without or with the selective GSK-3 inhibitorsSB216763 (10�M) or SB415286 (40�M). After 4 h of compoundtreatment, the cells were washed with phosphate-bufferedsaline and then lysed in lysis buffer, as described above. Myc-FRATwas immunoprecipitated from lysates using monoclonal9E10 antibody, and immunoprecipitates were subjected toSDS-PAGE and transferred to nitrocellulose. Phosphate incor-poration into Myc-FRAT was determined by autoradiography.

�-Catenin-TCF/LEC-regulated Gene Reporter Assay—Lucif-erase activity in cells transiently transfected with a TCF/LEF-

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regulated luciferase gene reporter construct was determined asdescribed previously (24).

RESULTS

Ser188 in FRAT1 Is Phosphorylated in Vitro and in Vivo—Todetermine whether FRAT was modified by phosphorylation inintact cells, HEK293T cells transfected with Myc-FRAT1 were32P-labeled. Autoradiography showed that isolated Myc-FRAT1 was indeed phosphorylated, indicating that Myc-FRAT1 is phosphorylated in intact cells (Fig. 1a). Moreover,treatment of cells with selectiveGSK-3 inhibitors resulted in nosignificant reduction in FRAT phosphorylation in the case ofSB216763 and amoderate reductionwith SB415286, suggestingthe presence of compound-insensitive FRAT kinase activitiesin intact cells (Fig. 1a).When Myc-FRAT1 immunoprecipitated from transfected

HEK293T cell lysates was incubated with Mg2�/[32-P]ATP invitro, the protein became phosphorylated (Fig. 1b). SinceFRAT1 has no intrinsic kinase catalytic activity, it was con-cluded that the observed FRAT1 phosphorylation reflected thein vitro activity of one or more endogenous kinases that hadco-immunoprecipitated with Myc-FRAT1. In keeping withprevious reports of a FRAT-GSK3 complex, the presence ofthe GSK-3 inhibitors LiCl or SB415286 (Fig. 1b) suppressedthe phosphorylation of Myc-FRAT1 markedly. However,inhibition of GSK-3 activity did not completely abolish Myc-FRAT1 phosphorylation, raising the possibility of additionalco-immunoprecipitating kinase activities within the Myc-FRAT1 complex.Mass spectrometry (MS) was used to identify the sites in

Myc-FRAT1 that were phosphorylated. To this end, Myc-FRAT1 was immunoprecipitated from cells and subjected to invitro phosphorylation with standard ATP, followed by SDS-

PAGE and mass spectrometry analysis of the excised Myc-FRAT1 band. This approach is expected to detect residues thatare phosphorylated in intact cells or in vitro by endogenouscoimmunoprecipitating kinases. Data from a series of MS/MSion maps indicated that the tryptic peptide containing Ser188(residues 187–193) was phosphorylated. This peptide also con-tains a threonine residue (Thr192), which, unlike Ser188, is notconserved in FRAT2 (see Fig. 2a). It was, therefore, assumedthat the serine was the phosphorylated residue (see below).However, since not all tryptic peptides were analyzable bymassspectrometry, it is possible that there are additional phospho-rylation sites.Ser188 in FRAT1 Is Phosphorylated by PKA and PKC in Vitro—

Analysis of residues around Ser188 in FRAT1 suggested that thisresidue lay within a consensus sequence for phosphorylation byPKA (RRX(S/T)) (Fig. 2a). Ser188 also conforms to consensusmotifs for phosphorylation by protein kinase C (PKC), whichare (S/T)X(K/R), (K/R)XX(S/T), or (K/R)X(S/T) (27). To evalu-ate further the role of PKA and/or PKC in FRAT phosphoryla-tion, a FRAT1 mutant was generated in which Ser188 wasmutated to Ala, and in vitro phosphorylation of wild type andmutant Myc-FRAT1 by recombinant PKA and PKC was deter-mined in the presence of the GSK-3 inhibitor SB-415286. Asshown in Fig. 2, both PKA and PKC phosphorylated wild typeMyc-FRAT1. In contrast, no 32P incorporation into the S188A

FIGURE 1. Phosphorylation of Myc-FRAT1 in intact cells and in vitro byendogenous coimmunoprecipitating kinases. a, mock-transfected (�)and Myc-FRAT1 transfected (�) HEK293T cells were labeled with 32P in thepresence (�) or absence (�) of 5 �M SB216763 or 40 �M SB415286. Myc-FRAT1 protein was immunoprecipitated and subjected to autoradiographyas described under “Materials and Methods.” b, Myc-FRAT1 was expressed inHEK293T cells and immunoprecipitated. The Myc-FRAT1 kinase assay wasperformed as described under “Materials and Methods” in the presence (�) orabsence (�) of 50 mM lithium chloride or 50 �M SB415286.

FIGURE 2. In vitro phosphorylation of wild type and S188A mutant Myc-FRAT1 by PKA and PKC. a, alignment of human FRAT1 sequence surround-ing Ser188 with human and mouse orthologs and paralogs. Conserved resi-dues are shown in boldface type. b and c, immunoprecipitated wild type andS188A mutant Myc-FRAT1 were phosphorylated in vitro in the presence ofSB415286 and recombinant PKA catalytic subunit (Novagen) (b) or PKC(Sigma) (c), as described under “Materials and Methods.” The PKC kinase assaywas carried out in the presence of 100 �g/ml phosphatidylserine, 20 �g/mldiacylglycerol (1-stearoyl-2-arachidonoyl-sn-glycerol), and 0.5 mM CaCl2.Immunoprecipitates were then subjected to SDS-PAGE, and gels were driedfor autoradiography or transferred to nitrocellulose membrane for Westernblot analysis with monoclonal 9E10 antibody. 20 �M SB415286 was includedin all reactions in b and c.

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mutant could be observed with PKA, indicating that PKAexclusively phosphorylates FRAT1 at Ser188 in vitro (Fig. 2b). Incomparison, phosphorylation of Myc-FRAT1 by PKC was onlyslightly reduced in the S188Amutant compared with wild typeFRAT1 (Fig. 2c), indicating that PKCdoes not exclusively phos-phorylate Ser188 in vitro.

To characterize further the phosphorylation of Ser188 inFRAT1, a polyclonal phosphosite-specific pS188 antibody wasgenerated. Incubation of immunoprecipitated wild type Myc-FRAT1 with either recombinant PKA or PKC increased theimmunoreactivity of the phospho-Ser188-specific antibody, andthis was abolished in the S188A mutant (Fig. 3). This resultverified that the antibody only recognizes phosphorylation ofSer188, and confirmed that both PKA and PKC are able to phos-phorylate this site in FRAT1 in vitro.These findings indicated that recombinant PKA and PKC

could phosphorylate Ser188 in vitro. We next wished to deter-mine whether Ser188 is phosphorylated by a co-immunopre-cipitating protein kinase. Interestingly, the addition of 8-bro-mo-cAMP to the Myc-FRAT1 protein complex significantlyincreased the amount of phosphate incorporated into Ser188 invitro (Fig. 4a). Furthermore, consistent with this idea, the phos-phorylation of Ser188 resulting from treatment with 8-bromo-cAMP was strongly inhibited by PKA inhibitor, as determinedby autoradiography and Ser188 phosphospecific immunoblot-ting (Fig. 4, a and b). In contrast, 8-bromo-cAMP did notinduce phosphorylation of the S188AmutantMyc-FRAT1 (Fig.4b). In addition, PKA was observed by immunoblotting to co-immunoprecipitate with FRAT1 (Fig. 4b). We did not observestimulation of Ser188 phosphorylation when adding PKC acti-vators phosphatidylserine and diacylglycerol plus calcium toFRAT1 immunoprecipitates in the presence of MgATP (datanot shown). Together, these data indicated that PKA, but notPKC, is a co-immunoprecipitating kinase that mediates phos-phorylation of FRAT1 at Ser188.PKA Phosphorylates Ser188 in FRAT1 in Intact Cells—To

assess whether PKA and/or PKC can phosphorylate Ser188 inintact cells, HEK293T cells were transfected with Myc-FRAT1

and treated with activators of PKA (forskolin and 8-bromo-cAMP) or PKC (TPAandbryostatin-1) for 1 h. Phosphorylationof Ser188 was then measured in immunoprecipitates of Myc-FRAT1 using the phospho-Ser188-specific antibody. Fig. 5ashows that the PKA activators forskolin and 8-bromo-cAMP,but not the PKC activators TPA and bryostatin-1, were able toinduce phosphorylation of Ser188. To confirm phosphorylationof Ser188 by PKA in cells, PKAwas cotransfected with wild typeand S188A mutant Myc-FRAT1. As shown in Fig. 5b, cotrans-fection of PKA resulted in a strong signal in wild type but not inS188AmutantMyc-FRAT1. Taken together, these results indi-cated that PKA can phosphorylate Ser188 in intact cells as wellas in vitro and that PKA is more important to the regulation ofphosphorylation of Ser188 in a cellular context than PKC.Activation of Endogenous cAMP-coupled �-Adrenergic Re-

ceptors Induces the Phosphorylation of FRAT1 at Ser188—HEK293 cells express endogenous �2-adrenergic receptors(28). To determinewhether elevation of the cellular cAMPcon-centration through activation of an endogenous cAMP-cou-pled receptor induces Ser188 phosphorylation, HEK293 cellswere transfected with FRAT1-FLAG followed by treatmentwith norepinephirne for 2 h. As shown in Fig. 5c, norepineph-rine treatment led tomarked Ser188 phosphorylation of FRAT1.

FIGURE 3. Phospho-Ser188-specific immunoblot of wild type and S188Amutant Myc-FRAT1 after in vitro phosphorylation by recombinant PKAand PKC. Immunoprecipitated wild type and S188A mutant Myc-FRAT1 werephosphorylated in vitro using unlabeled ATP at a concentration of 0.5 mM inthe presence of recombinant PKA catalytic subunit or PKC plus PKC activatorsand 20 �M SB415286, as described in Fig. 2, followed by SDS-PAGE of immu-noprecipitates and Western blot analysis with phospho-Ser188-specific or9E10 Myc antibody.

FIGURE 4. Myc-FRAT1 phosphorylation at Ser188 is stimulated by 8-bro-mo-cAMP and inhibited by protein kinase A inhibitor. a, to measure Ser188

phosphorylation by a coimmunoprecipitating kinase, in vitro phosphoryla-tion of immunoprecipitated Myc-FRAT1 was carried out in the presence of 0.5mM unlabeled ATP, 20 �M SB415286, and 0.25 mM 8-bromo-cAMP or 1 �M

protein kinase A inhibitor 5-24 (PKI) (Calbiochem), as indicated. Immunopre-cipitates were then analyzed by SDS-PAGE and Western blotting with phos-pho-Ser188-specific or 9E10 Myc antibody. b, immunoprecipitated wild typeand S188A mutant Myc-FRAT1 were phosphorylated in vitro in the presenceof [�-32P]ATP, 20 �M SB415286, 0.25 mM 8-bromo-cAMP, and 1 �M proteinkinase A inhibitor 5-24 (PKI), as described under “Materials and Methods.”Immunoprecipitates were then separated by SDS-PAGE and analyzed byautoradiography or Western blotting with phospho-Ser188-specific, 9E10Myc, or PKA catalytic subunit antiserum.

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Phosphorylation of Ser188 by GSK-3 in Vitro and in IntactCells—Given that GSK-3 is known to interact with FRAT1 andthat we found that the kinase could induce marked phospho-rylation of Myc-FRAT1 in vitro, we determined whether thiskinase can also phosphorylate Ser188. As shown in Fig. 6a,Ser188 phosphorylation of immunoprecipitated FRAT1 in vitro,as measured using the phosphosite-specific antibody, wasinhibited in the presence of LiCl and SB415286, indicating thatco-immunoprecipitating GSK-3 can phosphorylate Ser188. Todetermine GSK-3-mediated phosphorylation of FRAT1 atSer188 in cells, cells transfected with FRAT1 were treated withGSK-3 inhibitors or cotransfected with an expression plasmidfor GSK-3�, followed by immunoblotting of cell lysates withSer188 phosphospecific antibody. Cotransfection of GSK-3�increased Ser188 phosphorylation, but to amuch smaller degreecompared with the effect observed when PKA was cotrans-fected (Fig. 6b). GSK-3 is a constitutively active kinase; thus, ifGSK-3 phosphorylates Ser188 in vivo, GSK-3 inhibitors wouldbe expected to reduce Ser188 phosphorylation. However, treat-ment of cells with SB415286 or LiCl had no effect on Ser188phosphorylation (Fig. 6b). When cells transfected with FRAT1

were treated with phosphatase inhibitor okadaic acid, amarkedincrease in Ser188 phosphorylation was detected (Fig. 6c).Again, no inhibition of okadaic acid-induced Ser188 phospho-rylation was observed in the presence of SB415286. Takentogether, these results indicate that whereas GSK-3 can phos-phorylate Ser188 in vitro or in cells when overexpressed, endog-enousGSK-3 does not contribute to FRAT1 phosphorylation atSer188 in intact cells.GSK-3-mediated Phosphorylation of FRAT1 at Ser188 Does

Not Require Priming—The majority of physiological GSK-3substrates require prior priming through phosphorylation at aSer or Thr residue at the n � 4 position (where n is the site ofGSK-3-mediated phosphorylation). Interestingly, the residuelocalized four amino acids downstream of Ser188 is a Thr. Inorder to gain further insight into the possible significance ofSer188 phosphorylation by GSK-3, we investigated whether apriming event is necessary.Mutation of Arg96 to Ala inGSK-3�prevents phosphorylation of primed substrates but does not

FIGURE 5. Phosphorylation of Myc-FRAT1 at Ser188 by PKA in intact cells.a, HEK293T cells were transfected with Myc-FRAT1. After 2 days, the cells weretreated for 1 h with 10 �M forskolin, 0.5 mM 8-bromo-cAMP, 0.1 nM tetradec-anoylphorbol 13-acetate (TPA), or 0.1 �M bryostatin-1 and then lysed forphospho-Ser188-specific immunoblotting. b, HEK293T cells transfected withwild type or S188A mutant Myc-FRAT1 were cotransfected with PKA� cata-lytic subunit as indicated. After 2 days, the cells were lysed for analysis ofSer188 phosphorylation. c, HEK293 cells were transfected with FRAT1-FLAG. 2days after transfection, cells were treated with 100 nM norepinephrine (NE) for2 h, as indicated, followed by immunoblotting with phospho-Ser188-specificand FLAG antiserum.

FIGURE 6. Phosphorylation of FRAT1-FLAG by GSK-3 in vitro and in intactcells. a, FRAT1-FLAG, immunoprecipitated from lysates of transfectedHEK293 cells, was incubated in the presence of 0.5 mM ATP and 20 �M

SB415286 or 30 mM LiCl as indicated, as described under “Materials and Meth-ods.” Immunoprecipitates were subjected to SDS-PAGE and immunoblottingwith Ser188 phosphosite-specific or FLAG antibody. b, cells were cotrans-fected with FRAT1-FLAG and GSK-3�-V5 (1.25 �g) or PKA� catalytic subunit(0.75 �g) as indicated. Treatment of cells with 20 �M SB415286 or 30 mM LiClwas for the last 10 h. Cell lysates were analyzed by SDS-PAGE and Westernblotting with the indicated antibodies. c, cells were transfected with FRAT1-FLAG and treated with 100 nM okadaic acid and 20 �M SB415286 for the last2 h as specified, and cell lysates were analyzed using the Ser188 phosphosite-specific antibody.

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affect phosphorylation of nonprimed substrates (6). BothV5-tagged (Fig. 6b) and untagged (Fig. 7a) R96A mutantGSK-3� retained the ability to phosphorylate FRAT1 at Ser188in cells. We then determined the effect of mutating the poten-tial priming site Thr192 in FRAT1. As shown in Fig. 7b, overex-pressed GSK-3� increased Ser188 phosphorylation of T192Amutant FRAT1, although slightly less compared with wild typeFRAT1. Furthermore, mutation of Thr192 had no effect onendogenous Ser188 phosphorylation (Fig. 7b). As expected,mutation of Thr192 did not affect phosphorylation of FRAT1 byPKA (Fig. 7b). These results strongly suggest that GSK-3-de-pendent phosphorylation of Ser188 does not require priming.Phosphorylation of FRAT1 by GSK-3 in Intact Cells—The

results presented in Fig. 1 suggest that although GSK-3 canphosphorylate FRAT1 in vitro, endogenous GSK-3 does notsignificanctly contribute to FRAT1 phosphorylation in intactcells. To further study the phosphorylation of FRAT1 in cells,HEK293 cells were co-transfected with FRAT1 andwild type orR96A mutant GSK-3�. Co-transfection with both GSK-3�plasmids resulted in a number of FRAT1 bands that migratedmore slowly in the SDS gel, which is probably due to phospho-rylation events (Fig. 8a). Interestingly, the R96A mutantinduced more phosphorylation of FRAT1 compared with wildtype GSK-3. This is also apparent in Figs. 6b and 7a, where theR96A mutant induced slower, Ser188-phosphorylated FRAT1bands compared with wild type GSK-3. This mobility shift wasnot due to Ser188 phosphorylation, since co-transfectedGSK-3� induced a similar mobility shift in both wild type andS188A mutant FRAT1 (Fig. 8b).Having observed a GSK-3-dependent slower migration of

FRAT1, we then determined whether phosphatase inhibitor

okadaic acid and GSK-3 inhibitor SB415286 affected FRAT1mobility. As shown in Fig. 8c, treatment with okadaic acid hadonly a marginal effect. No obvious difference in FRAT1 mobil-ity was observed in the absence or presence of SB415286 exceptupon longer exposure of the Western blot, when a faint slowband in the control (indicated by an arrow) disappeared whenSB415286 was added. These results, combined with the find-ings in Fig. 1, suggest that endogenous GSK-3 is not a majorFRAT1 kinase.Phosphorylation of Ser188 by PKA Inhibits the Ability of

FRAT1 to Activate �-Catenin-dependent Transcription—Thepresented data indicate that PKA, but not GSK-3, is a FRAT1kinase in vitro and in intact cells and that PKA-mediated phos-phorylation of FRAT1 occurs exclusively at Ser188. We there-fore wished to investigate the functional relevance of PKA-in-duced phosphorylation of Ser188 in FRAT1. It is well knownthat FRAT1 binds to GSK-3, resulting in increased stability ofcytoplasmic �-catenin and its translocation into the nucleus,where it activates transcription in conjunction with transcrip-tion factors of the TCF/LEF family. Thus, to measure FRAT1activity we utilized a TCF/LEF-dependent luciferase reporterassay (24). Transfection of either wild type or S188A mutantMyc-FRAT1 increased luciferase reporter activity to a similarextent (Fig. 9a). In order to examine the effect of Ser188 phos-phorylation, PKA was cotransfected with wild type or S188A

FIGURE 7. Ser188 phosphorylation by GSK-3� does not require priming.a, cells were cotransfected with FRAT1-FLAG and wild type (WT) or R96Amutant untagged GSK-3�, and cell lysates were analyzed using the indi-cated antibodies. b, cells were cotransfected with wild type or T192Amutant FRAT1-FLAG and wild type GSK-3�-V5 or PKA� catalytic subunit asindicated, and Ser188 phosphorylation was determined using phos-phosite-specific antiserum.

FIGURE 8. Phosphorylation of FRAT1 by GSK-3� in intact cells. a, cells werecotransfected with FRAT1-FLAG and wild type (WT) or R96A mutant untaggedGSK-3� and cell lysates were analyzed using FLAG antibody. b, cells werecotransfected with FRAT1-FLAG (wild type or S188A mutant) and wild typeGSK-3�-V5 followed by Western blotting with FLAG antibody. c, cells weretransfected with FRAT1-FLAG and treated with 100 nM okadaic acid and 20 �M

SB415286 for the last 2 h followed by immunoblotting of cell lysates withFLAG antibody.

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FRAT1. Co-expression of PKA resulted in a much lower stim-ulation of luciferase reporter activity by wild type FRAT1 com-pared with S188A FRAT1 (Fig. 9b). We noted that cotransfec-tion of PKA by itself led to a significant increase in luciferaseactivity. This is probably due to direct stimulation of geneexpression from transfected expression plasmids by PKA, ashas been previously been observed (29). We also noted signifi-cantly increased expression from other transfected plasmidswhen PKA was cotransfected (data not shown). Because of this

nonspecific effect of PKA, we couldnot directly compare the results inFig. 8, a and b. Nevertheless, whencomparing the effects of wild typeand S188A mutant FRAT1 in theabsence versus presence of PKA, theresults clearly indicate that Ser188-phosphorylated FRAT1 had amark-edly reduced ability to stimulateTCF/LEF-dependent reporter ac-tivity. These findings suggest thatPKA-mediated phosphorylation ofSer188 in FRAT1 inhibits its activitytoward GSK-3. This was not due toreduced binding of Ser188-phospho-rylated FRAT1 to GSK-3, sincewild type and S188A mutantFRAT1 from cells co-transfectedwith PKA immunoprecipitatedGSK-3� equally well (Fig. 9c).FRAT1 has been reported to medi-ate nuclear export of GSK-3 andrequire interaction with LRP5/6 toactivate �-catenin-dependent tran-scription (30, 31). However, PKA-dependent Ser188 phosphorylationdid not affect FRAT1 subcellularlocalization or its interaction withLRP6 (data not shown). On theother hand, we observed thatSer188 phosphorylation of wildtype FRAT1 by cotransfected PKAreduced its half-life comparedwith S188A FRAT1, as determinedby following FRAT1 degradationin the presence of cycloheximide(Fig. 9d). In contrast, in theabsence of PKA, no difference inthe half-life of wild type andS188A FRAT1 was observed (datanot shown). The shorter half-lifeof Ser188-phosphorylated FRAT1may contribute to its reduced abil-ity toactivate�-catenin-TCF/LEF-dependent transcription.

DISCUSSION

In this study, we investigated themechanism of FRAT1 phosphoryla-

tion.We identified two kinases, GSK-3 and PKA, that coimmu-noprecipitate with FRAT1 and phosphorylate FRAT1 in vitro.Furthermore, mass spectrometry studies identified Ser188 as aphosphorylated residue in FRAT1. Ser188 conforms to consen-sus motifs for phosphorylation by GSK-3, PKA, and PKC, andall of these kinases phosphorylated Ser188 in vitro.

Recently, both FRAT1 and FRAT2 were shown to be phos-phorylated in cells (23), andGSK-3 was found to phosphorylateFRAT2 in vitro (32). Our results indicate that GSK-3 phospho-

FIGURE 9. Ser188 phosphorylation of FRAT1 inhibits its ability to activate �-catenin/TCF/LEF-dependentluciferase reporter activity and reduces protein half-life. a and b, HEK293T cells were transfected with aTCF/LEF-regulated luciferase gene reporter construct and wild type (wt) or S188A Myc-FRAT1 (a) or wild typeand S188A Myc-FRAT1 plus PKA� catalytic subunit (b) for 2 days. �-Catenin-TCF/LEF-dependent luciferasereporter activity was expressed as Myc-FRAT1-induced increase over the mock-transfected control. The resultsrepresent the average of four (a) or three (b) independent experiments. c, lysates from cells transfected withPKA� catalytic subunit plus empty vector, wild type, or S188A mutant FRAT1-V5 were subjected to immuno-precipitation (IP) with a V5 antibody. Immunoprecipitates were then analyzed by SDS-PAGE and immunoblot-ting (WB) with a GSK-3� antibody. d, cells were cotransfected with PKA� catalytic subunit plus wild type orS188A mutant FRAT1-FLAG. 24 h after transfection, 40 �M cycloheximide was added, and cells were lysed at theindicated times, followed by Western blot analysis using FLAG antibody, as shown in the upper panel. The lowerpanel represents the average of the densitometry results of three independent experiments. Œ, wild typeFRAT1; f, S188A FRAT1.

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rylates Ser188 in FRAT1 in vitro as well as in cells when overex-pressed. However, the effect of transfected GSK-3� on FRAT1Ser188 phosphorylation was much smaller compared with thatof transfected PKA. Although most physiological GSK-3 sub-strates require priming throughphosphorylation of a Ser orThrat the �4-position, we have shown that phosphorylation ofThr192 is not necessary forGSK-3-mediated Ser188 phosphoryl-ation. Furthermore, inhibition of endogenous GSK-3 with spe-cific inhibitors had no effect on Ser188 phosphorylation. Giventhat GSK-3 is a constitutively active kinase, these results sug-gest that it does not contribute to Ser188 phosphorylation invivo. Transfection of GSK-3� also induced a slower migrationof FRAT1 that was not due to Ser188 phosphorylation, indicat-ing that the kinase can phosphorylate other residues in FRAT1when overexpressed. However, our in vivo labeling experi-ments (Fig. 1a) showed that the presence of GSK-3 inhibitorshad no effect or only a small effect on FRAT1 phosphorylation.This suggests that GSK-3 is not a major FRAT1 kinase in vivo.Given that phospho-Ser188 lies within a consensus sequence

for PKA and for PKC (see above), we studied their role inmedi-ating Ser188 phosphorylation in vitro and in cells. Althoughboth kinases phosphorylated Ser188 in vitro, activation ofendogenous PKA with forskolin and 8-bromo-cAMP, but notof endogenous PKC, resulted in FRAT1 phosphorylation atSer188 in intact cells. Furthermore, activation of endogenouscAMP-coupled �-adrenergic receptors with norepinephrinestimulated the phosphorylation of FRAT1 at Ser188. These find-ings clearly indicate that endogenous PKA, when activated, is aFRAT1 kinase that phosphorylates Ser188. We also noted thatalthough basal levels of phosphorylated Ser188 were very low toundetectable, the addition of phosphatase inhibitor okadaicacid increased Ser188 phosphorylationmarkedly. Okadaic acid-induced phosphorylation was not reduced by the GSK-3 inhib-itor SB415286 (Fig. 6c) or by inhibitors of PKA (H-89 and cell-permeable protein kinase A inhibitor 14-22) (data not shown).These results confirm that constitutively active GSK-3 is not aphysiological Ser188 kinase and that Ser188 phosphorylation byPKA, which is inactive under basal conditions, requires thecAMP-dependent dissociation of the catalytic from the inhibi-tory regulatory subunits. The okadaic acid-induced Ser188phosphorylation also indicates that in addition to PKA, a sec-ond unidentified kinase may mediate Ser188 phosphorylationunder basal conditions in vivo. Given that the basal phospho-rylation of FRAT1 at Ser188 was very low, the significance of thiskinase for the regulation of FRAT1 is not clear. However, it isalso possible that this unidentified kinase(s) becomes activatedunder certain conditions and then contributes significantly toSer188 phosphorylation of FRAT1.Functionally, phosphorylation of FRAT1 at Ser188 by PKA

inhibited the ability of FRAT1 to activate �-catenin-dependenttranscription, suggesting that PKA-mediated phosphorylationof FRAT1 reduces its inhibitory activity toward GSK-3. This isnot a result of reduced binding of Ser188-phosphorylatedFRAT1 to GSK-3 but may at least partially be due to decreasedFRAT1 protein stability.The FRAT homolog GBP is required for maternal Wnt sig-

naling in Xenopus (13). Mechanistically, FRAT/GBP preventsbinding of Axin to GSK-3 (17–21) and can also induce deple-

tion of GSK-3 on the dorsal side of the embryo (33). It has beenproposed that activation of the Wnt signaling cascade causesDvl to recruit FRAT/GBP into the �-catenin degradation com-plex, leading to dissociation of GSK-3 from Axin and conse-quently to stabilization of �-catenin (17, 34). More recently, itwas reported that the activation of Wnt signaling by FRAT1 ismediated through its interaction with the Wnt co-receptorLRP5 (31). However, a study by van Amerongen et al. (35),which used triple-knock-out mice lacking all three murine Frathomologs, demonstrated that Frat is dispensable for Wnt/�-catenin signaling in mammals. PKA has not been implicateddirectly in the transduction of the Wnt signal. We also couldnot detect any change in FRAT1 phosphorylation at Ser188 afterstimulation of the canonicalWnt pathway bymeans of cotrans-fection of Wnt3a or Dvl2 (data not shown). Thus, PKA-medi-ated FRAT phosphorylation is unlikely to play a role in trans-ducing the Wnt signal but may regulate GSK-3 activity toward�-catenin in a Wnt-independent manner. Alternatively,FRAT1 phosphorylation may also regulate GSK-3 activitytoward other cellular substrates.Ser188 as well as the PKA consensus at this site are conserved

in themammalian FRAT1 homologs, FRAT2 (Thr164 in humanFRAT2) and Frat3 (Ser181 in mouse Frat3), suggesting thatPKA-mediated phosphorylation of FRAT andmodulation of itsactivity is a general mechanism in mammals. However, ahomologous residue to Ser188 is absent in Xenopus andzebrafishGBP.Thus, regulation of FRATactivity by PKAwouldbe amechanism that is specific tomammals andmay reflect theevolution of additional levels of regulation of FRAT1 functionnecessary in mammalian cells.PKA is also known to phosphorylate GSK-3� at Ser9, result-

ing in inhibition of its catalytic activity (36, 37). Thus, PKAmayregulate GSK-3 activity at multiple levels. PKA-mediated phos-phorylation of GSK-3� at Ser9 would have the opposite conse-quence compared with PKA-dependent FRAT1 phosphoryla-tion. However, GSK-3 is known to exist in different pools in thecell. For instance, insulin, which inhibits GSK-3� via Ser9 phos-phorylation, leading to activation of glycogen synthase, doesnot stabilize �-catenin, whereasWnt-dependent GSK-3� inhi-bition is not mediated through Ser9 phosphorylation and doesnot activate glycogen synthase activity (11, 12). FRAT/GBP hasalso been shown to only inhibit the activity of GSK-3 towardspecific substrates (19, 20, 22), suggesting that the phosphoryl-ation of FRAT1 described in this study affects only specificGSK-3-directed phosphorylation events. A GSK-3-bindingprotein, p24, which is unrelated to FRAT/GBP but also inhibitsthe catalytic activity of GSK-3, has recently been identified (38).Interestingly, p24 is also a substrate for PKA, and similar towhat we have observed with FRAT1, phosphorylation of p24 byPKA reduces its inhibitory activity toward GSK-3 (38). In addi-tion, PKA-mediated phosphorylation may also regulate otherfunctions of FRAT, such as regulation of GSK-3 stability andnuclear export or its binding to kinesin light chains (30, 33, 39).In summary, we identified PKA as a FRAT1 kinase in vitro as

well as in intact cells. Phosphorylation of FRAT1 by PKA maybe a mechanism by which FRAT activity toward GSK-3 is reg-ulated in a Wnt-independent manner.

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Supplementary Data Fig.S1 Effect of PKC activators on Ser188 phosphorylation in FRAT1 immunoprecipitates

FRAT1 immonoprecipitates were incubated for 45 min at 30ºC in the presence of 0.5 mM MgATP, 20 µM SB415286, PKC activators (100 µg/ml phosphatidylserine, 20 µg/ml 1-stearoyl-2-arachidonoyl-sn-glycerol, 0.5 mM CaCl2), as indicated. After the kinase assay, Western blotting was performed to detect Ser188 phosphorylation and total FRAT1-FLAG amounts. As expected, no significant Ser188 phosphorylation was observed in the absence of ATP. Omission of the GSK-3 inhibitor SB415286 induced significant Ser188 phosphorylation, mediated by coimmunoprecipitating GSK-3, which served as a positive control. Addition of PKC inhibitors did not stimulate Ser188 phosphorylation.

Fig.S2 Half life of wild type and S188A mutant FRAT1 in the absence and presence of PKA

Cells were transfected with wild type or S188A mutant FRAT1-V5 (and cotransfected with PKAα catalytic subunit in the left panels). 36 hours after transfection, 40 µM cycloheximide was added and cells were lysed at the indicated times, followed by Western blot analysis using V5 and α-tubulin antibody, as shown in the upper panel. The densitometry results are shown in the bottom panels (▲ wild type FRAT1 + PKA; ■ S188A FRAT1 + PKA; ∆ wild type FRAT1, no PKA; □ S188A FRAT1, no PKA). As expected, the half life of wild type FRAT1 was shorter than that of S188A mutant FRAT1 when PKA was cotransfected. In contrast, no difference in half life was observed in the absence of PKA, confirming that the difference in half life is due to Ser188 phosphorylation. The observed difference in FRAT stability in the left versus the right panel (presence versus absence of PKA) may be due to the non-specific effect of PKA on the expression of transfected FRAT1 (see last paragraph of Results section).

Nick Morrice and Alastair D. ReithThilo Hagen, Darren A. E. Cross, Ainsley A. Culbert, Andrew West, Sheelagh Frame,

a Cellular Substrate of Protein Kinase AFRAT1, a Substrate-specific Regulator of Glycogen Synthase Kinase-3 Activity, Is

doi: 10.1074/jbc.M607003200 originally published online September 18, 20062006, 281:35021-35029.J. Biol. Chem. 

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