Journal of Cellular Biochemistry 90:1224–1241 (2003)

RIFLE: A Novel Ring Zinc Finger-Leucine-Rich RepeatContaining Protein, Regulates Select Cell AdhesionMolecules in PC12 Cells

Baolin Li,1 Yuan Su,1 John Ryder,1 Lei Yan,1 Songqing Na,1 and Binhui Ni1,2,3*1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 462852Department of Psychiatry, Indiana University Medical School, Indianapolis, Indiana 462023Department of Anatomy and Cell Biology, Indiana University Medical School, Indianapolis, Indiana 46202

Abstract Cell adhesion molecules play a critical role in cell contacts, whether cell–cell or cell–matrix, and areregulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containingprotein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation ofglycogen synthase kinase-3a/b (GSK-3a/b) resulting in the inhibition of GSK-3 kinase activity and increase of b-cateninlevels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulationof these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell–cell and cell–matrix adhesion. Moreover, increase in cell–cell adhesion but not cell–matrix adhesion by RIFLE can be mimicked byselective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates com-ponents of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells. J. Cell. Biochem. 90: 1224–1241,2003. � 2003 Wiley-Liss, Inc.

Key words: ring zinc finger; leucine-rich repeats; cell adhesion; GSK-3b; b-catenin; kinase activity

The family of leucine-rich repeat (LRR) pro-teins constitutes a large number of proteins thathas been recently divided into six subfamiliestypified by distinct lengths (20–29 residues)and consensus sequences [Kajava, 1998]. Mem-bers of this family are known to participate in avariety of biological processes. For instance, theLRR is a structural module involved in mole-cular recognition processes such as cell adhe-sion, signal transduction,DNArepair, andRNAprocessing [Iozzo, 1999]. Like the LRR, the ringzinc finger (RZF) motif has also been found ina variety of eukaryotic proteins of diverse evolu-tionary origin that are involved in various

cellular processes such as oncogenesis, develop-ment, signal transduction, and apoptosis [Iuchi,2001; Kroncke, 2001; Laity et al., 2001; Paboet al., 2001]. RZF is known to play a role inmediating protein–DNA binding and protein–protein interaction, both of which are charac-teristic features of a transcriptional factor.

Cell adhesion to neighboring cells or to thesurrounding extracellular matrix (ECM) is avital function necessary for cell survival, migra-tion, proliferation, and differentiation duringCNS development [Adams and Watt, 1993;Sastry andHorwitz, 1996; Hynes, 1999; Bensonet al., 2000]. Gene regulation in response tosignals from the environment is usually requir-ed for cell adhesion interactions. This processinvolves signal-dependent-transcriptional re-gulation and subsequent modulation of adhe-sion-related molecules. The cadherin–catenincomplex is well known for mediating homotypiccalcium-dependent cell adhesion and recogni-tion in diverse tissue types and is regulated bycomponents of theWnt/wingless signalingpath-way, specifically glycogen synthase kinase-3(GSK-3)/b-catenin [Papkoff and Aikawa, 1998;

� 2003 Wiley-Liss, Inc.

*Correspondence to: Binhui Ni, PhD, or Baolin Li, PhD,Lilly Research Laboratories, Eli Lilly and Company, LillyCorporate Center, Indianapolis, IN 46285.E-mail: Binhuini@yahoo.com, or Li_Baolin@Lilly.com

Received 6 May 2003; Accepted 23 July 2003

DOI 10.1002/jcb.10674

Dierick and Bejsovec, 1999; Novak andDedhar,1999; Waltzer and Bienz, 1999]. GSK-3a and bare closely related serine/threonine kinasesthat act as inhibitory components of Wnt/wingless signaling during embryonic develop-ment. Inhibition of GSK-3 kinase activity bydominant negative GSK-3 mutants leads toactivation of the Wnt signaling pathway inDictyostelium, Drosophila, Xenopus, and mam-malian cells [Cadigan and Nusse, 1997; Dale,1998; Wodarz and Nusse, 1998]. In PC12 cells,both lithium and dominant negative GSK-3mutants can mimic Wnt signaling [Stambolicet al., 1996; Bournat et al., 2000]. Recentstudies, along with the elucidation of the GSK-3b three-dimensional structure, have signifi-cantly enhanced our understanding of themole-cular basis for b-catenin regulation by GSK-3.Activation ofWnt signaling results in inhibitionof GSK-3 kinase activity by disrupting a multi-protein complex comprising GSK-3 and its sub-strates such as b-catenin. GSK-3 is then unableto tag b-catenin by phosphorylation for degra-dation which in turn leads to the stability ofb-catenin [Cohen and Frame, 2001; Woodgett,2001]. Moreover, b-catenin is able to associatewith the C-terminal cytoplasmic domain of cad-herins to link extracellular adhesion with actincytoskeleton [Nagafuchi and Takeichi, 1988;Ozawa et al., 1990; Knudsen et al., 1995; Rimmet al., 1995] and/or is translocated to thenucleuswhere it forms a complex with Tcf/Lef-1 tran-scription factors [Kikuchi, 2000].In this study, we identify a novel ring zinc

finger-leucine-rich repeat containing protein,RIFLE, by using Incyte database screening to aconserved RZF region and reveal that RIFLEprotein contains three other putative functionalmotifs in addition to the RZF domain: LRRs, aleucine zipper domain and a nuclear localiza-tion signal (NLS) domain. Although largelydivergent in sequence, RIFLE shares similarstructural motifs with other RZF–leucine zip-per containing protein such as c-RZF. C-RZFis a putative transcriptional factor that wasidentified by subtractive hybridization fromgene expression that occurs when the substrateadhesion molecules cytotactin/tenascin bindto neurons [Tranque et al., 1996]. This pro-mpted us to investigate whether RIFLE mayplay a role in regulating cell adhesionmoleculesand cell adhesion in the PC12 pheochromocy-toma cells, a neural crest-derived tumor cell linewith both neuronal and epithelial characteris-

tics [Greene and Tischler, 1976; Franke et al.,1986].

MATERIALS AND METHODS

Cloning of RIFLE cDNA and Constructionof RIFLE Expression Vector

A partial RIFLE coding cDNA fragment wasidentified by searching the Incyte cDNA data-base using a conservedRZF region of a neuronalapoptosis inhibitory protein (NAIP) gene asbait. A 3.14 kbRIFLE cDNA containing an openreading frame was identified by screeningthe human brain cDNA library with standardmethods. The 3.14 kb cDNA was cut out ofpBluescript vector with Spe I, and then ligatedinapBI–EGFP expressionvector usinganEcoRV site to form the pBI–EGFP–RIFLE expres-sion vector. The pBI–EGFPexpression vector isa bi-directional doxycycline regulated plasmidthat expresses EGFP simultaneously withRIFLE. In addition, a DNA fragment codingthe hemagglutinin (HA) epitope was cloned atthe 30-end of the RIFLE cDNA and placed intoa pcDNA3.1(þ) vector to form the pcDNA3.1/RIFLE–HA expression vector.

Cell Culture

All cells were cultured at 378C and 5% CO2.Naive PC12 cells were cultured in DMEM(GIBCO, Gaitherburg, MD) supplementedwith 10% horse serum (GIBCO), 5% fetalbovine serum (Clontech, Palo Alto, CA) and 1%Pen/Strep. pcDNA3.1/RIFLE–HA tranfectedcells and tet-off system parental PC12 cells(Clontech) were maintained in naive PC12medium containing Geneticin (GIBCO, 200 mg/ml). pBI–EGFP–RIFLE tet-off transfectedclones were cultured in parental cell mediumcontaininghygromycin (Clontech,200mg/ml)butwithoutDoxycycline for continual onexpression.

Transfections

Twenty-four hours prior to transfection,1� 106 parental cells were seeded on individualcollagen IV 60-mm plates (Becton Dickinson,FranklinLakes,NJ). Cellswere cultured to 80%confluence. The transfection of the pBI–EGFP–RIFLE construct into parental PC12 tet-off cellswas performed by using lipofectamine-plus fol-lowing the manufacture’s instruction (Gibco/BRL). After selecting with hygromycin (200 mg/ml) for 2 weeks, 20 individual clones werepicked, expanded, and frozen down for further

RIFLE Regulates Cell Adhesion 1225

analysis. For maintenance of the RIFLE trans-fected cells, the concentration of G418 andhygromycin were kept the same as in theselection (200 mg/ml) process. Due to leakinessof the vector, pBI–EGFP–RIFLE transfectedcells were maintained under continual expres-sion conditions, that is without the presence ofDoxycycline.

pcDNA3.1/RIFLE–HA was transfected intonaive PC12 cells using the Lipofectaminemethod as described above. Following a 2-weekselection with G418 (500 mg/ml), pcDNA3.1/RIFLE–HA PC12 colonies were pooled andmaintained in standard PC12 culture mediumcontaining 200mg/mlG418.ApBI–EGFP vectorand GSK-3b KK (kinase dead) construct wasintroduced into PC12 tet-off cells (Clontech)using electroporation as previously described[Melemed et al., 1997]. Cells were selected inmedia supplemented with 200 mg/ml G418 and100 mg/ml hygromycin. Individual clones con-taining the GSK-3b KK construct were picked,characterized, and maintained under selectionmedia conditions and, as with pBI–EGFP–RIFLE clones, without Doxycycline for contin-ual on expression.

Northern Blot Analysis

Total RNA was isolated from RIFLE andvector control cells using Trizol (GIBCO) follow-ing manufacture’s instruction and subjected toNorthern blot analysis as described previously[Li et al., 1994]. Full length RIFLE cDNA wasused as a Northern probe. The Clontech multi-ple tissue northern (MTN) blot was also used inthis analysis.

Cell–Cell Adhesion Assays

PC12 cells stably transfected with pcDNA3.1/RIFLE–HA or GSK-3b KK (kinase dead) con-structs were analyzed for cell–cell adhesion byexamining defined cell clusters following themethod of Rothlein’s [Rothlein and Springer,1986] with modification. Briefly, PC12 cellswere cultured in complete medium for at least24 h. The cells were washed twice with 1� PBSand re-suspended in tissue culture mediumwithout serum to a concentration of 2� 106 cells/ml. Five microliters of calcein AM stock solu-tion (Molecular Probes, Pittsburgh, PA) wasadded into 1 ml of cell suspension to achieve afinal concentration of 5 mM. The mixture wasincubated at 378C for 30min. Cells werewashedtwice with pre-warmed (378C) culture medium

and re-suspended in culture medium at 2�106 cells/ml. One hundred microliters of thecalcein-labeled cell suspension (2� 106 cells/ml)was combined with either 1 mM Caþþ or 1 mMEGTAandwas transferred to a glass slide. Cellswere incubated on glass slides at 378C. Theformation of aggregated cell clusters (n� 5cells) was examined after 1–2 h using phasecontrast microscopy. For quantitative aggrega-tion assay, cells were allowed to settle sponta-neously and the degree of aggregation wasscored at indicated time. The number of freecells and aggregated cells were quantified.Percent aggregation was determined by the fol-lowing equation: percent aggregation¼ 100�(number of aggregated cells forming clusters/total number of cells). Total number of cellsequals the number aggregated cells formingclusters plus the number of free cells. Cell–celladhesion antibody neutralization studies weredone using the same procedure adding either E-cadherin antibody (DECMA-1, Sigma) [Zanteket al., 1999] or control antibody IgG (Rabbit IgG,KPL, Gaithersburg, MD) together with calcein.The final concentration for both antibodies was44 mg/ml.

Cell–Matrix Adhesion Assays

Cell–matrix adhesion assays were performedon CytoMatrix cell adhesion strips (Chemicon,Temecula, CA) pre-coated with human collagenIV or human fibronectin by the manufacturerwith one row of each strip pre-coated with 10%bovine serum albumin (BSA) as a negative con-trol. PC12 cells were sub-cultured 1 day prior toadhesion assay to reach 90–100% confluence.The cells were detached from the flasks using acell scraper and a single cell suspension of5� 106 cells per ml was prepared by pipettingthe cells up anddownabout 20 times followed bycounting using a Coulter particle counter(Coulter Cooperation, Miami, FL). During thepreparation of the cell suspension, the CytoMa-trix cell adhesion strip wells were re-hydratedwith 1� PBS for 15 min at room temperature.One hundred microliters of the cell suspensioncontaining 5� 105 cells was plated in each wellof the CytoMatrix cell adhesion strips and wereallowed to attach for 1 h at 378C in a humidified5% CO2 atmosphere. The non-adherent cellswere removed by gently washing the cells withPBS containing Ca2þ and Mg2þ and then theadherent fraction was quantified using crystalviolet staining. Briefly, after washing the wells,

1226 Li et al.

100 ml of 0.2 % crystal violet in 10% ethanolwas added to each well and the plates wereincubated at room temperature for 5 min. Thestaining solution was then removed and thewellsweregentlywashed3–5 timeswithPBS toremove the excess stain. One hundred micro-liters of solubilization buffer (a 50/50mixture of0.1 m NaH2PO4, pH 4.5 and 50% ethanol) wasthen added to each well. Following solubiliza-tion, the absorbance of each well was measuredin a microplate reader (Labsystems, Franklin,MA) at 540 nm with a reference wavelengthof 690 nm. The OD540 values of each RIFLEclone and vector control were then subject tostatistical analysis using the Student’s t-test.

Western Blot Analysis

At time of study, for analyzing cell mem-brane proteins, cells were harvested via cellscraper (Fisher, Pittsburgh, PA), washed withPBS and then dounced homogenized (Kontesdounce) in lysis buffer (10 mM K2HPO4 pH 7.2,1 mM EDTA, 5 mM EGTA, 10 mM MgCl2,50 mM b-glycerophosphate, 1 mM Na3VO4,2 mMDTT, 1% Triton X-100, 1 mMMicrocystin,COMPLETE protease inhibitor tablet (Roche,Indianapolis, IN)) and incubated on ice for30 min. The total lysate was clarified bymicrocentrifuging at 14,000 rpm for 30 min at48C. Total protein concentration of the super-natants was determined using the BCA ProteinAssay (Pierce, Rockford, IL). For analyzingintracellular proteins, cells were detached fromflask or platewith 0.25% trypsin–EDTA, rinsedonce with 1� PBS, then flash frozen in liquidnitrogen, and stored at �808C for futureanalysis. Whole cell lysate was prepared byre-suspending cell pellets in lysis buffer (seeabove), then following same procedure as scrap-ing harvested cells. Thirty micrograms of totalprotein from each samplewas then fractionatedon a 10%NuPAGE Bis-Tris gel and transferredonto a 0.2 mm nitrocellulose membrane (Novex,San Diego, CA) using a Hoefer Transblotter(Semiphor, San Francisco, CA). The membranewaswashed twice for 5min each in TST (10mMTris-HCl, 150 mM NaCl, 0.1% Tween-20,pH 7.5), blocked with 5% BSA–TST for 1 hat room temperature on a shaker platform(Lab-Line) and then incubated with respectiveprimary antibody (b-catenin, E-cadherin, a-actinin, and GSK-3 protein antibodies werepurchased from Upstate Biotechnology (LakePlacid, NY); phospho-GSK-3 and phosphor-

PKC antibodies were purchased from CellSignaling Technology (Boston, MA); intercell-ular cell adhesionmolecule (ICAM), cadherin-5,and vascular cell adhesion molecule (VCAM)antibodies were purchased from Santa Cruz(Santa Cruz, CA); HA antibody was orderedfromRoche (Indianapolis, IN)) overnight at 48C.The following day, the membrane was washedthree times for 5 min each with TST. An appro-priate HRP conjugated secondary antibody wasdiluted 1:2,000 in 5% milk–TST and incubatedwith the membrane for 1 h shaking at roomtemperature. Finally, themembranewaswash-ed as above followed by one wash with PBS for5 min. Respective protein on the membranewas then visualized by Chemiluminescence(Amersham Bioscience, Piscataway, NJ).

GSK-3b Kinase Activity Assay

The GSK-3 kinase activity was measuredusing a CREB substrate peptide as described[Wang et al., 1994]. Briefly, the kinase reactionoccurred in a 50 ml total volume containing20 mM MOPS pH 7.4, 25 mM b-glycerolphosphate, 5 mM EGTA, 1 mM NA3VO4, 1 mMDTT, 15 mM MgCl2, 100 mM cold ATP, 200 mMCREB peptide (KRREILSRRPpSYR, AnaSpec,San Jose, CA), 10 ml whole cell lysate, and 5 mCig-33P-ATP. The reactions were incubated for30 min at 308C in a Costar round-96 polypropy-lene plate. Reactions were then stopped withthe addition of 10% H3PO4 and transferred to aMillipore MAPH-NOB 96-well phosphocellu-lose plate. Next, the reactions were incubatedat room temperature for 1.5 h, filtered andwashed oncewith 320 ml 0.75%H3PO4, and thenfiltered and washed twice with 160 ml H3PO4 atthe same concentration using a vacuum mani-fold (Millipore, Bedford, MA). The filter platewas then placed in a carrier plate and 100 mlof Microscint 20 (Packard, Meriden, CT) wasadded to each well. The plate was sealed withsealing tape and incubated overnight at roomtemperature. The following day, the filter platewas read for 33P on Top Count (Packard,Albertville, MN). Finally, CPMwas normalizedto CPM per mg of total protein.

Statistical Analysis

All statistical analysis for Western blot andcell adhesion studies was completed using theStudent’s t-test with a standard P value thresh-old of �0.05.

RIFLE Regulates Cell Adhesion 1227

RESULTS

Cloning and Tissue Distribution of RIFLE

We used a conserved RZF region of a NAIPgene as bait to search the Incyte database andidentified a 1.2 kb cDNA fragment. This 1.2 kbcDNA fragment was subsequently used toscreen a human brain cDNA library. Twoindividual cDNA clones were identified andsubjected to further sequence analysis. BothcDNAs with different lengths contained anidentical open reading frame that encoded aprotein of 702 amino acids with a calculatedmolecular mass of 80 kDa. The first start codon(methionine) of the longest open reading framewas preceded by two consecutive in-frame stopcodons at �15 and �54 bp. The longer cDNA(3,144 bp), comprised of an open codingsequence, a long 50-flanking region and 30-untranslated region (Fig. 1A), was namedRIFLE and was used in the studies describedbelow.Database searching revealed thatRIFLEis a novel protein with no significant homologywith any known cDNAs or proteins contained inthe database. Protein sequence analysis revealsthat RIFLE has a LRR motif (amino acid 59–150), a NLS (amino acids 234–242), a leucinezipper domain (amino acids 528–556), and aRZF motif (amino acids 654–702) (Fig. 1B).

RIFLE is composed of four LRRs, each ofwhich consists of 24 residues, a general charac-teristic of the LRR family. The consensus se-quence of LRR in RIFLE (Fig. 1C,D), however,differs from that of all known six subfamilies.There are two and four amino acid residuesfollowing the second and the third leucine, re-spectively in the LRR consensus sequencesof RIFLE instead of the usual one and twoamino acids at the same positions in the LRRconsensus sequences of the known six sub-families (Fig. 1D), suggesting that RIFLEmight belong to a novel LRR subfamily [Kajava,1998].

The tissue distribution of RIFLE expressionwas examined in multiple human tissues anddifferent human brain regions by using Clon-tech RNA blots. The full length RIFLE cDNAwas used as the Northern blot probe. RIFLEwas mainly expressed in human brain, heart,skeletal muscle, kidney, and liver (Fig. 1E).Further, RIFLE was expressed in all brainregions examined (Fig. 1F). The molecular sizeof the RIFLE mRNA as measured by theNorthern blots is consistent with the calculated

length of the longer clone 3.14 kb, confirmingthat the longer clone is essentially a full lengthhuman RIFLE cDNA.

The non-tagged RIFLE cDNAwas cloned intothe pBI–EGFP expression vector (pBI–EGFP–RIFLE). RIFLE expression at the mRNA levelfrom three independent colonies stably trans-fected with pBI–EGFP–RIFLE was confirmedby Northern blot analysis (Fig. 2A). ControlPC12 cells have no detectable endogenousRIFLE and the three colonies (#10, #14, and#23) express RIFLE mRNAs at similar levels.The expression of RIFLE at the protein levelfrom the pooled population of the PC12 cellstransfected with pcDNA3.1/RIFLE–HA wasmeasured by Western blot using an anti-HAaffinity antibody. Sub-cellular distribution ofRIFLE was analyzed in the RIFLE–HA/PC12cells as previously described [Ahmed et al.,1993] (Fig. 2C). RIFLE is expressed in thecytosol and nuclear fractions, consistent withthe presence of NLS and RZF motifs. Boththe pCDNA3.1/RIFLE–HA and the pBI–EGFP–RIFLE cells expressed RIFLE withoutcausing changes in cell viability as assessed byMTT analysis and cell proliferation (data notshown).

Expression of RIFLE Facilitates b-CateninProtein Accumulation

We further investigated the PC12 cells ex-pressingRIFLE to assessRIFLE’s potential rolein the regulation of cell adhesion related mole-cules. We surveyed the expression of severaladhesion proteins in response to RIFLE expres-sion in PC12 cells. We found that b-cateninprotein, a mammalian homolog of Armadillo, issignificantly increased in independent linesof pBI–EGFP–RIFLE expressing PC12 cells(Fig. 3A). RIFLE, however, does not change theexpression of other adhesion related moleculessuch as cadherin-5, ICAM-2 (Fig. 3B,C), ICAM-1, VCAM-1, P-cadherin, and E-selectin (datanot shown) under the same conditions. The b-catenin protein levels were quantified by densi-tometry analysis and shown to be statisticallyelevated in the pBI–EGFP–RIFLE cells ascompared to control PC12 cells transfected withvector alone (Fig. 3D). These data suggest thatRIFLE selectively regulates b-catenin proteinlevels. b-Catenin is a key player in the WNTsignaling pathway and therefore this data sug-gest that RIFLE may mimic or augment WNTsignaling.

1228 Li et al.

Fig. 1. Panel A: Nucleotide and deduced amino acid sequenceof RIFLE. The deduced amino acid sequence beginning with theinitiating methionine is given. Numbering of nucleotide andamino acids is shown at the top. The leucine-rich repeat (LRR)sequences are underlined; the sequences of the nuclearlocalization signal (NLS), the leucine zipper and ring zinc finger(RZF) are framed. Panel B: Schematic structure of RIFLE proteinshowing the localizations of LRR, NLS, leucine zipper, and RZFmotifs. Panel C: Alignment of leucine repeat sequences showingthe localizations, length, and consensus sequences. Panel D:Comparison of the LRR consensus sequences between RIFLE andother LRR containing proteins. Unlike the one derived from theknown six subfamilies of LRR proteins, there are two and fouramino acid residues following the second and the third leucine,

respectively in the LRR consensus of RIFLE, suggesting that RIFLEmight belong to a novel LRR subfamily. Panel E: Northern blotanalysis of human tissue distribution of RIFLE. Lane 1: RNAmolecular marker; lanes 2–13, RNA from whole brain, heart,skeletal muscle, colon, thymus, spleen, kidney, liver, smallintestine, placenta, lung, and peripheral blood leukocyte,respectively. Panel F: Regional distribution of RIFLE expressionin human brain. Shown is RNA blot analysis (1mg) of mRNA usingClontech prepared human mRNA blot. Lane 1: RNA molecularmarker; lanes 2–9 are loaded RNA from cerebellum, cerebralcortex, medulla, spinal cord, occipital pole, frontal lobe,temporal lobe, and putamen, respectively. Both panel E and Fare probed using RIFLE full length cDNA.

Fig. 1. (Continued)

1230 Li et al.

RIFLE Regulates E-Cadherin Expression

b-Catenin is a well-known cell adhesion as-sociated molecule that can physically associatewith the C-terminal cytoplasmic domain ofcadherins [Huber et al., 1997]. In light of theevidence that b-catenin and cadherin areusually co-regulated, particularly byWnt/wing-less signaling [Porfiri et al., 1997; Stewart et al.,2000], we sought to investigate whether in-creased levels of b-catenin by RIFLEmodulateseither E-cadherin or N-cadherin in the RIFLE/PC12 cells by examining cadherin proteinlevels. While N-cadherin levels did not differsignificantly betweenRIFLEand control cells, astriking change was observed, by Western blot-ting analysis, in E-cadherin expression in theRIFLE/PC12 cells. RIFLE, as with b-catenin(Fig. 4A,B), elevated the E-cadherin expressionlevels (Fig. 4C, compare lanes 1 and 2) but didnot affect the N-cadherin (Fig. 4D) or a-actininexpression levels (Fig. 4E), suggesting thatE-cadherin is selectively co-regulated with b-catenin by RIFLE. The protein levels of RIFLE,b-catenin, E-cadherin, and N-cadherin werequantified bydensitometry analysis normalizedwith a-actinin (Fig. 4F–I) showing that b-catenin (Fig. 4G) and E-cadherin (Fig. 4H) pro-

tein levels are significantly elevated in theRIFLE/PC12 cells, respectively as compared tocontrol PC12 cells transfectedwith vector alone.Our data is consistent with the findings thatexpression of Wnt-1 in PC12 cells results inincreased E-cadherin levels [Bradley et al.,1993; Hollmann et al., 2001].

Expression of RIFLE Regulates Componentsof WNT/Wingless Signaling

Wnt/wingless signaling is one of the best-knownpathways involved in the regulation of b-catenin protein levels. Knowing that b-catenincomplexeswithand that it’s level is regulated byGSK-3, we examined the Wnt/wingless signal-ing associated with GSK-3 in the RIFLE–HA/PC12 cells. The Wnt/wingless gene family con-stitutes a large number of developmentally re-gulated genes involved in cell–cell signaling ina wide range of animal phyla. GSK-3a or b iso-form, a major player inWnt/wingless signaling,is a serine/threonine kinase known to directlyregulate b-catenin levels by phosphorylating b-catenin on specific serine/threonine residuesmarking b-catenin for ubiquination. Cells ex-pressing RIFLE consistently accumulated b-catenin while stimulating the phosphorylationof both GSK-3a (Ser21) and GSK-3b (Ser9)

Fig. 1. (Continued)

RIFLE Regulates Cell Adhesion 1231

Fig.

1.

(Continued

)

1232 Li et al.

with predominant phosphorylation occurringon GSK-3a (Fig. 5A). This phosphorylationoccurred without change in GSK-3a or b proteinlevels (Fig. 5B) or phosphorylation of sev-eral other kinases such as PKCb (Fig. 5C),PKCa, PKCe, Erk1, or Erk2 (data not shown).Phosphorylation (Ser21/9) of GSK-3a and bwere quantified by densitometry analysis inFigure 5D,E, respectively. RIFLE significantlystimulated an increase in GSK-3a/b phosphor-ylation (Ser21/9) of, most notably, GSK-3a.Since increased Ser21/9 phosphorylation of

GSK-3 is known to inhibit its kinase activity,this data suggested that RIFLE conceivably re-gulates b-catenin levels via inhibition of GSK-3kinase activity, resulting in increased b-cate-nin. To determine if RIFLE inhibits GSK-3kinaseactivity in theRIFLE–HA/PC12cells,wenext carried out experiments to directly mea-sure the GSK-3 kinase activity. GSK-3 activitywas measured in RIFLE and control PC12cells using a specific GSK-3 CREB peptidesubstrate and our data, derived from fiveindependent experiments, showed that GSK-3kinase activity was significantly suppressed byRIFLE expression in the PC12 cells (Fig. 5F).

This is in full agreement with the increase inSer21/9-phosphorylation of GSK-3 by RIFLEand correlates with the observed b-cateninstability.

Elevation of b-Catenin/E-Cadherin Levels byRIFLE Is Accompanied by Increases in

Cell Adhesion

Based on the fact that RIFLE up-regulatessome adhesion-related molecules, we investi-gated RIFLE’s effect on cell–cell and cell–matrix adhesions.We found thatRIFLEexpres-sing PC12 cells show increased cell–cell adhe-sion forming clusters in the presence of Caþþ

(Fig. 6A)while control cells grew inadissociatedpattern and remained in a single cell pattern(Fig. 6B) in the cell–cell adhesion assay. Ourdata demonstrates that the expression ofRIFLE significantly increases the Caþþ-medi-ated cell–cell adhesion as compared to controlPC12 cells or to the RIFLE PC12 cells in theabsence of Caþþ (plus EGTA) (Fig. 6C), suggest-ing that RIFLE mediates a Caþþ dependentcell–cell adhesion event. In order to directly ad-dress whether the observed cell–cell adhesionin RIFLE–HA/PC12 cells results from RIFLE

Fig. 2. Panel A: Expression of RIFLE in PC12 cells. RNA blotanalysis of 25 mg of total RNA isolated from RIFLE transfectedPC12 cell colony #10, #14, and #23, respectively. Lane 1: PC12cell stably transfected with pBI–EGFP vector as a control. Lanes2–4 represent three independent clones #10, #14, and #23 ofPC12 cells stably transfected with pBI–RIFLE. Panel B is anethidium bromide stained RNA gel showing the equivalentloading of the RNA in the corresponding RNA blot. Panel C: Sub-

cellular distribution of RIFLE in PC12 cells stably transfected withpcDNA3.1–RIFLE–HA. Shown is a Western blot probed withHA-tag antibody. Samples were analyzed in duplicate. Lanes 1 &2, 5& 6, 9& 10 (labeled with R) are PC12 cells expressing RIFLE.Lanes 3 & 4, 7& 8, 11& 12 (labeled with C) are PC12 cells stablytransfected with pcDNA3.1 vector as controls. RIFLE is pre-dominantly expressed in the cytosol and nuclear fractions.

RIFLE Regulates Cell Adhesion 1233

elevated E-cadherin levels, we carried outE-cadherin neutralization experiments usingan anti-E-cadherin antibody. Indeed, E-cad-herin antibody blocked the formation of cell–cell adhesion clusters in RIFLE–HA/PC12 cellsin the presence of Caþþ (Fig. 6E) while antibodycontrol IgG had no effect under the sameconditions (Fig. 6D), suggesting that elevatedcell–cell adhesion in RIFLE–HA/PC12 cells isE-cadherin mediated. Quantitative and statis-tical comparison of the antibody neutralizationexperiment is shown in Figure 6F. We also as-sessed whether RIFLE enhances cell–matrixadhesion in PC12 cells. Our data revealed thatRIFLE enhanced the cell attachment to theECM molecules collagen IV (Fig. 6G) and fibro-nectin (Fig. 6H).

Finally, we used a GSK-3b kinase deficientmutant to mimic RIFLE’s effect on b-catenin/E-cadherin and cell adhesion. We made severalkinase dead (KK) and vector stable PC12 celllines for use in studying GSK-3b kinase defi-cient effects in PC12 cells. We found that theGSK-3 kinase dead mutants increased b-cate-nin protein (Fig. 7A) and E-cadherin protein(Fig. 7B) as compared to the vector control,mimicking RIFLE’s effects in augmenting b-catenin stability and E-cadherin protein levels.Endogenous cdk5 protein serves as a loadingcontrol in Figure 7A,B. The quantitative eleva-tion of b-catenin, GSK-3b, and E-cadherin are,after being normalized with cdk5 protein levels,shown in Figure 7C–E, respectively. Similarly,we also used lithium, a GSK-3 inhibitor. Wedetermined the optimal dose of lithium forPC12cells without affecting cell survival to be 25 mM(data not shown). Naive PC12 cells were treatedwith lithiumat25mMforup to 24hand the cellswere then collected and prepared for b-catenin/E-cadherin expression analysis. b-Catenin andE-cadherin protein levels were increased 8 hafter lithium treatment and remained at thesame level up to 24 h (Fig. 7F,G). a-Actininlevels served as a loading control (Fig. 7H).Elevated protein levels of b-catenin and E-cadherin were quantified by densitometry ana-lysis, normalized with a-actinin, and shown inFigure 7I,J, respectively.

GSK-3 kinase dead stable lines were subject-ed to both cell–cell and cell–matrix adhesionanalysis. The stable PC12 cells expressing theGSK-3 kinase deficient mutant significantlyenhanced the cell–cell adhesion in the presenceof Caþþ as compared to their corresponding con-trols (without Caþþ and plus EGTA) (Fig. 8A).Interestingly, theGSK-3 kinase dead stable celllines, unlike RIFLE expressing cells, failed toenhance cell–matrix adhesion on either col-lagen IV (Fig. 8B) or fibronectin (Fig. 8C). Thisdata indicates that inhibition of GSK-3 kinaseactivity alone is sufficient to regulate b-cateninand E-cadherin protein levels and related cell–cell adhesion but not cell–matrix adhesion.Therefore, our data suggests that inhibition ofGSK-3 kinase activity and associated elevationofb-catenin/E-cadherinprotein levels byRIFLEcontribute to RIFLE-mediated cell–cell adhe-sion as with Wnt/wingless signaling. RIFLEmediated cell–matrix adhesion may depend ona yet unexamined signaling pathway(s) moreclosely related to cell–matrix adhesion.

Fig. 3. Overexpression of RIFLE results in elevated proteinlevels of b-catenin in PC12 cells. Panels A–D are Western blotsprobed with antibodies against b-catenin, cadherin-5, ICAM-2,and a-actinin, respectively. a-Actinin blot was served as loadingcontrol. Lane 1 is PC12/pBI–EGFP control; lanes 2 & 3 are clone#10 and #23 of PC12/pBI–RIFLE.Panel E is a statistical analysis ofb-catenin expression levels following overexpression of RIFLE.Densitometry of corresponding bands was measured; data fromthree independent cell lines of three experiments were pooled forstatistical analysis (P< 0.05).

1234 Li et al.

DISCUSSION

In this article, we identify and characterize anovel ring zinc finger-leucine-rich repeat con-taining protein, designated as RIFLE, and pri-marily focus on its biological functions in theregulation of cell adhesion molecules such as b-catenin and E-cadherin as well as cell–cell andcell–matrix adhesion in PC12 cells. Expressionof RIFLE in the PC12 cells results in accumula-tion of b-catenin, a key mediator of the Wnt/wingless signaling pathway, presumably bynegatively regulating GSK-3, enhancing Ser21and Ser9 phosphorylation of GSK-3a and b, re-spectively, and eliciting an inhibitory effect onGSK-3 kinase activity. RIFLE also co-regulatesb-catenin and E-cadherin expression withoutsignificant change in N-cadherin or cadherin-5.Consistent with RIFLE’s enhancement ofthese cell adhesion molecules, RIFLE/PC12cells augment cell adhesion without causing

significant change in cell proliferation or cellsurvival. Moreover, the effect on b-catenin sta-bilization, elevated E-cadherin expression, andcell–cell adhesion by RIFLE can be mimickedby lithium, a well-known GSK-3 inhibitor, orkinase deficient GSK-3 mutants (KK) in PC12cells. Our data suggest that elevation of b-catenin protein with co-regulation of E-cad-herin via inhibition of GSK-3 kinase activity issufficient for the observed cell–cell adhesionbut not for the cell–matrix adhesion mediatedby RIFLE. Taken together, we conclude thatRIFLE is a novel ring zinc finger-leucine-richrepeat containing protein that mediates compo-nents of Wnt/wingless signaling and cell–celladhesion in PC12 cells. A proposed model forhow RIFLE may regulate cell–cell or cell–matrix adhesion was summarized in Figure 9.

There is a similarity between Wnt-1 andRIFLE signaling in PC12 cells. First, bothlead to suppression of GSK-3 kinase activity

Fig. 4. RIFLE expression in PC12 cells enhances b-catenin andE-cadherin protein levels. Panels A–E are parallel-loaded blotssubject to Western analysis with antibodies against HA (forRIFLE), b-catenin, E-cadherin, N-cadherin, and a-actinin,respectively, as indicated. Lane 1 is RIFLE–HA PC12 cells andlane 2 is vector/PC12 cells. E-cadherin expression but not N-cadherin is co-regulated with the b-catenin in response to RIFLEexpression in the PC12 cells (n¼3). a-Actinin blot serves as a

loading control. Non-specific binding is indicated by ns. PanelsF–I are quantitative comparisons pooled from triplicate experi-ments for RIFLE (F), b-catenin (G), E-cadherin (H), and N-cadherin (I), respectively, where R and C represent RIFLE andcontrol, respectively. All quantitative data were normalizedbased on a-actinin loading control. *Indicates the significantdifferences of R vs. C (*P<0.05 in F–H).

RIFLE Regulates Cell Adhesion 1235

increasing b-catenin protein levels, a key med-iator of the Wnt/wingless signaling pathway,and both elevate E-cadherin protein [Bradleyet al., 1993; Hinck et al., 1994; Hollmann et al.,2001]. The selective regulation of RIFLE onGSK-3 kinase activity and cell adhesion mole-cules is evident and robust. We examined over10 other closely related cell adhesion-relatedmolecules such as P-cadherin, N-cadherin, andcadherin-5 and a variety of different kinasessuch as Erk1, Erk2, PKCa, PKCb, and PKCeand found that theyarenot regulatedbyRIFLE.Interestingly, RIFLE appears to have someselectivity for GSK-3a over the b isoform,evidenced by the enhanced phosphorylation ofthe GSK-3a isoform. There is considerableevidence that Wnt/wingless signaling is aconserved regulatory pathway for GSK-3. Forexample, the mammalian homolog of SggZw3,GSK-3, has been shown to be inhibited by

Drosophila Wg protein in fibroblasts [Cooket al., 1996] and embryos [Ruel et al., 1999].Further, expression of Wnt-1 in the PC12 cellsleads to increases in b-catenin, E-cadherin, andconsequent cell adhesion [Bradley et al., 1993;Hollmann et al., 2001]. Although the precisemechanism underlying the co-regulation of b-catenin and E-cadherin is not clear, a variety ofstudies suggest that there is a cross talkmechanism by which expression of these twomolecules are co-regulated. Indeed, overex-pression of the E-cadherin, for example, resultsin the up-regulation of three major cateninsincluding b-catenin in the E-cadherin-negativeDunning clones [Luo et al., 1999]. Interestingly,suppression of basal extracellular signal-regu-lated kinase (Erk) activity in PC12 cells resultsin the up-regulation of both cadherin andb-catenin, enhancing cell adhesion [Lu et al.,1998]. The fact that either lithium or the kinase

Fig. 5. Overexpression of RIFLE in PC12 cells increases thephosphorylation at Ser21/9 of GSK-3a/b and results in lowerGSK-3 kinase activity. Panels A–C are parallel loaded gelssubjected to Western blot analysis with antibodies againstphospho-GSK-3a/b, GSK-3a/b protein, and phospho-PKCb,respectively as indicated. Lane 1 is RIFLE–HA/PC12 cells andlane 2 is vector/PC12 cells. Panels D and E are quantitativecomparisons pooled from triplicate experiments for phospho-GSK-3a (D) and phospho-GSK-3b (E), respectively, where R and

C represent RIFLE and control, respectively. The quantitative datafor phospho-GSK-3a/b were normalized with GSK-3a/b proteinlevels. *Indicates the significant differences of R vs. C (*P<0.01in panel D; *P<0.05 in panel E). Panel F shows significantinhibition of GSK-3 kinase activity in RIFLE–HA/PC12 cells(black bar) compared to vector transfected PC12 control cells(white bar). Data represents the average from five independentexperiments (*P<0.05).

1236 Li et al.

Fig.

6.

Ove

rexp

ress

ion

ofR

IFLE

inPC

12

cell

sin

crea

ses

both

cell

–ce

llan

dce

ll–

mat

rix

adhes

ion.P

anelA

show

sre

pre

senta

tive

cell

–ce

llcl

ust

ers

form

edin

RIF

LE–

HA

/PC

12

cell

sin

the

pre

sence

of

Caþ

þ.Pan

elB

show

sth

esi

ngl

eor

two

cell

pat

tern

sse

enin

vect

or/

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sin

the

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sence

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þ.Pan

elC

:The

quan

tita

tive

com

par

ison

of

cell

–ce

llad

hes

ion

inPC

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tran

sfec

ted

wit

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wit

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ora

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nce

(plu

sEG

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nar

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imen

ts(*P<

0.0

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elsD

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neu

tral

ized

the

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hes

ion

(E)i

nR

IFLE

–H

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sw

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anti

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olI

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isons

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ell–

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repre

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(*P<

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elsG

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the

quan

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ysis

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–m

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(coll

agen

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repre

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nd

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espec

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ly(*P<

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1).

RIFLE Regulates Cell Adhesion 1237

Fig.

7.

Ove

rexp

ress

ion

of

kinas

e-defi

cien

tG

SK-3b

muta

nt

(KK

)co

nst

ruct

resu

lts

inac

cum

ula

tion

ofb-

cate

nin

and

E-ca

dher

inin

nai

vePC

12

cells.

Pan

elsA

andB

:PC

12

cell

sst

ably

tran

sfec

ted

wit

hve

ctor

const

ruct

(lan

e1)a

nd

the

kinas

e-defi

cien

tGSK

-3b

muta

nt(

KK

)const

ruct

(lan

e2)w

ere

subje

cted

toW

este

rnblo

tan

alys

isw

ith

anti

bodie

sag

ainstb-

cate

nin

/GSK

-3b/

CD

K5

(A)

and

E-ca

dher

in/C

DK

5(B

),re

spec

tive

ly.

CD

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pro

tein

serv

esas

alo

adin

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els

Aan

dB

.Lan

es1

and2

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anel

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are

PC

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sst

ably

tran

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ted

wit

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ctor

(clo

ne

#18)

and

kinas

e-defi

cien

tG

SK-3b

muta

nt

(KK

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one

#36),

resp

ecti

vely

.Pro

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-cat

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and

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inpan

elA

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den

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elsC–E.

Pan

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:Par

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ple

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subje

cted

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ith

anti

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ainst

b-ca

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nin

asin

dic

ated

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elH

(a-a

ctin

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serv

esas

load

ing

contr

ol.

Lanes

1–3

are

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vest

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and

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hfo

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ing

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ium

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t.The

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ange

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orm

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ifica

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5).

1238 Li et al.

deficient GSK-3 mutant can independentlymimic the effect of RIFLE or Wnt-1 on GSK-3b-catenin/E-cadherin pathway suggests thatinhibition ofGSK-3 alone is sufficient in leading

to the subsequent elevation of b-catenin andE-cadherin.

Secondly, both Wnt/wingless and RIFLEsignals enhance cell adhesion. Overexpres-sion of Wnt-1 results in an apparent increasein cell–cell adhesion via increase in b-catenin/E-cadherin [Bradley et al., 1993; Bournat et al.,2000]. Overexpression of RIFLE substantiallyincreases Caþþ dependent cell adhesion, pre-sumably via a similar mechanism through b-catenin/E-cadherin. The classic cadherins witha single transmembrane domain are known tobe involved in Ca2þ-dependent cell–cell adhe-sion. Interestingly, RIFLE enhances not onlycell–cell adhesion but also cell–ECM adhesionto collegen IV and fibronectin. It is unclear ifWnt-1 can enhance cell–matrix adhesion, butWnt-5a protein was shown to participate inregulation of cell to collagen adhesion via di-scoidin domain receptor 1 (DDR1) [Jonssonand Andersson, 2001]. It is conceivable thatRIFLE regulates cell–cell and cell–matrixthrough different signaling pathways. Oneclearly being the GSK-3/b-catenin/E-cadherinsignaling pathway associated with cell–celladhesion, a notion that is supported by in-creases in Caþþ dependent cell–cell adhesionfollowing inhibition of GSK-3 kinase activity byRIFLE or by lithium or kinase deficient GSK-3mutants. The other probably by a GSK-3 un-related signalingmechanism(s) since inhibitionof GSK-3 alone at least by the GSK-3 (KK)mutant is unable to enhance the cell–matrixadhesion like that observed in the RFILE/PC12cells. We, however, cannot exclude the possibi-lity that the regulation of b-catenin/E-cadherinby RIFLE may facilitate the cell–matrix adhe-sion process. Indeed, it has been reported thatthe E-cadherin/b-catenin complex is able toregulate both cell–cell and cell–matrix adhe-sion with Ezrin [Hiscox and Jiang, 1999].

Despite the similarity between RIFLE andWnt/wingless signaling, signaling mediated byRIFLE is clearly different. For example, RIFLEdoes not significantly influence PC12 cell differ-entiation and neurite outgrowth in response toNGF stimulation whereas expression of theWnt-1 oncogene in PC12 cells does inducemorphological and biochemical changes, includ-ing lack of differentiation in response to growthfactors [Bournat et al., 2000]. Although theprecise mechanism by which RIFLE inhibitsGSK-3 activity is unclear, RIFLE appears not toimpact on PKCa, b, and e activity while Wnt/

Fig. 8. Overexpression of kinase-deficient GSK-3b (KK) innaı̈ve PC12 cells mimics the RIFLE mediated effects in cell–celladhesion but not cell–matrix adhesion. Panel A: Cell–celladhesion of the GSK-3b kinase dead (KK)/PC12 cells in thepresence or absence (plus EGTA) of Caþþ. The stable PC12 cellsexpressing GSK-3b (KK) significantly enhanced the cell–celladhesion as compared to the multiple controls indicated. PanelsB (collagen IV) and C (fibronectin) show no effect of expressedGSK-3b dead kinase on PC12 cell–matrix adhesion (n¼ 3,*P< 0.05).

RIFLE Regulates Cell Adhesion 1239

wingless signaling abrogates GSK-3 kinaseactivity via an intracellular pathway involvingPKC [Cook et al., 1996].

In conclusion, RFILE represents as a novelprotein that regulates cell adhesion molecules,such as b-catenin and E-cadherin, and celladhesion in PC12 cells. GSK-3 is a criticalcomponent in the RIFLE signaling cascade,particularly in the RIFLE-mediated cell–celladhesion. Our study suggests a new mechan-ism/pathway by which a novel protein, RIFLE,signals involving crosstalk between compo-nents of Wnt/wingless and cell adhesion.

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