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Archives of Biochemistry and BiophysicsVol. 368, No. 2, August 15, pp. 401–412, 1999Article ID abbi.1999.1316, available online at http://www.idealibrary.com on

hosphorylation-Dependent Association of the Ras-RelatedTP-Binding Protein Rem with 14-3-3 Proteins1

. S. Finlin and D. A. Andres2

epartment of Biochemistry, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0084

eceived April 12, 1999, and in revised form May 21, 1999

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Rem belongs to a subfamily of Ras-related GTPaseshat includes Rad, Gem, and Kir. These proteins arenique among the Ras superfamily since their expres-ion is under transcriptional regulation and they con-ain distinct amino and carboxyl termini. To gain in-ight into the cellular function of Rem, we have under-aken an expression screen using a mouse embryoDNA library to identify Rem-interacting proteins andnd that Rem interacts with a series of 14-3-3 isoforms

e, h, u, and z). Immunoprecipitation studies demon-trate an interaction that is independent of the nucle-tide state of Rem. Rem is phosphorylated in vivo, andinding of Rem to 14-3-3z is abolished by pretreatingem with protein phosphatase 1. Thus, the associationf Rem and 14-3-3z is phosphorylation-dependent. Ex-mination of the interaction between 14-3-3z and var-ous Rem deletion mutants mapped a critical bindingite to the C-terminus of Rem. Finally, we demonstratehe interaction of Rad but not the newly identifiedem2 protein with 14-3-3 proteins. These results sug-est that 14-3-3 may allow the recruitment of distinctroteins that participate in Rem-mediated signalransduction pathways. © 1999 Academic Press

Rem is the newest member of a growing subfamily ofas-like GTPases3 that includes Rad, Gem, and Kir

The nucleotide sequences for rat Rem2 have been submitted to theenebank Data Bank with Accession No. AF084464.1 This work was supported by NIH Grant EY11231 and a grant

rom the American Heart Association, Kentucky Affiliate.2 To whom correspondence should be addressed. Fax: 606-323-

037.3 Abbreviations used: GST, glutathione S-transferase; HMK,

AMP-dependent protein kinase from heart muscle; SH2, Src homol-gy 2 domain; GTPase, guanosine triphosphatase; GTPgS, guanosine9-3-O-(thio)triphosphate; PAGE, polyacrylamide gel electrophore-
is; DTT, dithiothreitol; HA, influenza hemagglutinin epitope; PCR,olymerase chain reaction; BSA, bovine serum albumin; MAP, mi-
bp

003-9861/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

1–4). These GTPases have several unique structuraleatures which are distinct from those of other Ras-likeTPases. These include several nonconservativemino acid substitutions within the regions known toe involved in guanine nucleotide binding and hydro-ysis, unique effector domains, extended N- and C-ermini, and a conserved C-terminal sequence thoughto mediate membrane association but lacking the clas-ical CAAX motif necessary to direct protein isopreny-ation (5, 6). The members of this Ras subfamily arelso subject to transcriptional regulation. In mice, Rems most highly expressed in cardiac muscle and at mod-st levels in the lung, kidney, and skeletal muscle. Thedministration of lipopolysaccharide results in a gen-ral repression of Rem mRNA levels, making Rem therst Ras-like GTPase to be shown to be regulated byepression (1). Rad expression has been shown to bencreased in the skeletal muscle of some type II dia-etic patients (2), and Gem expression is induced in the1 phase in mitogen-activated T lymphocytes (3),hereas Kir expression is induced in pre-B cell linesxpressing BCR/ABL or v-abl (4). Although the physi-logical function of these proteins remains to be deter-ined, Rad has been implicated as an inhibitor of

lucose uptake in several cultured cell lines (7).Members of the Ras GTPase superfamily share the

bility to function as nucleotide-dependent molecularwitches. In the GDP-bound form these proteins arenactive, but respond to extracellular stimuli by ex-hanging GTP for GDP, thereby triggering an activat-ng conformational change. Once activated, Ras-re-

ogen-activated protein; MEK, MAP kinase kinase/ERK kinase;AK, p21-activated kinase; RGK, Rad, Gem, and Kir Ras subfamily;b, kilobase pair(s); KSR, kinase suppressor of Ras; MEKK, mitogen-ctivated protein kinase/extracellular signal-regulated kinase ki-ase; NP-40, Nonidet P-40; IPTG, isopropyl b-D-thiogalactoside;
ME, b-mercaptoethanol; PP1, protein phosphatase 1; PBS, phos-hate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.
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402 FINLIN AND ANDRES

ated GTPases are able to interact with and activatearious effector proteins, which in turn stimulate sig-aling cascades that induce a variety of cellular re-ponses (8–10). In this way, Ras-related GTPases reg-late a wide range of physiologically important cellularignaling pathways by controlling the assembly of pro-ein signaling complexes at specific cellular locations.o date, six subfamilies of the Ras superfamily haveeen identified: Ras, Rho, Rab, Ran, ARF, and theem/Rad/Gem proteins (11). These broad subfamiliesre related by primary sequence relationships but alsoy regulation of common cellular activities, includingell growth (Ras), cytoskeletal organization (Rho), nu-leocytoplasmic transport (Ran), or vesicular transportRab and ARF).

The likelihood that Rem may control signaling cas-ades through its specific association with a variety ofellular proteins, stimulated our interest in searchingor proteins that interact with Rem. As described in theollowing study, four members of the 14-3-3 family ofroteins were identified as Rem-binding proteins. 14--3 proteins constitute a family of abundant acidicroteins that have been highly conserved throughoutvolution (12), reflecting the fundamental importancef these proteins in cellular physiology. 14-3-3 proteinsave been shown to interact with a number of differentignaling proteins including, Raf (13–15); Bcr and Bcr-bl (16); polyoma middle tumor antigen (17); kinaseuppressor of Ras (KSR) (18); the Bcl family memberAD (19); phosphotidylinositol 3-kinase (20); Cdc25

21); the platelet adhesion receptor (22); insulin recep-or substrate-1 (23); protein–tyrosine phosphatase H124); mitogen-activated protein kinase/extracellularignal-regulated kinase kinase-1 (MEKK-1), -2, and -325); Cbl (26); and protein kinase C (27). Crystallo-raphic studies indicate that each 14-3-3 molecule con-ains a large cleft that can bind to a-helices from ap-ropriate substrates in a phosphoserine dependentanner (28). This, together with their ability to formomotypic or heterotypic dimers (29), suggests that4-3-3s might serve as molecular adapters, bringingifferent proteins into close proximity.Since the original cloning of Rem, we have under-

aken a series of studies designed to determine theunction of Rem in regulating cellular physiology. Thedentification and characterization of cellular Rem-in-eracting proteins is one approach that has been pur-ued. In this study we describe the generation of aecombinant Rem molecule that is readily phosphory-ated by the catalytic subunit of heart cAMP-depen-ent protein kinase, enabling us to use it as a molecu-ar probe to screen cDNA expression libraries for po-ential Rem-associated proteins, similar to thepproach used to identify retinoblastoma-binding pro-eins (30). Using this strategy, we have identified four
4-3-3 proteins (e, h, u, and z) from a 14-day whole
ouse embryo cDNA library capable of interactingith Rem. Stable complexes between Rem and 14-3-3zere reconstituted in HEK 293 cells. These complexesere shown to contain at least two additional proteins,hich might be endogenous components of a Rem sig-aling complex. Although both Raf-1 and Rem formomplexes with 14-3-3z, they appear to do so indepen-ently. We have demonstrated that Rem is normallyhosphorylated in vivo and that Rem binds 14-3-3z initro in a phosphorylation-dependent manner. Deletionapping was used to locate a critical 14-3-3 binding

omain to residues 265–282 at the C-terminus of Rem.n addition, we have demonstrated the in vitro associ-tion of the Rem-related GTPase Rad but not Rem2ith 14-3-3 proteins. Thus, Rem and other members of

he RGK GTPase family are the first Ras-related GT-ases to be shown to interact with 14-3-3 family pro-eins. These results raise the possibility that 14-3-3roteins might function as scaffold-like molecules inhe regulation of Rem activity by directing protein–rotein interactions that control the composition orellular location of Rem-mediated signal transductionascades.

XPERIMENTAL PROCEDURES

Plasmids. Standard molecular biology techniques were used (31).ll site-directed mutations and PCR reaction products were verifiedy DNA sequence analysis. PCR was performed using Pfu polymer-se (Stratagene, La Jolla, CA) according to the manufacturer’s in-tructions. To construct pGexKG HMK, a plasmid in which Remontains a heart muscle kinase (HMK) site at its N-terminus, theamHI/PstI fragment of pGexKG (32) was ligated into the BamHI/stI site of pGex2TK (Pharmacia) to expand the available restrictionites within the polylinker. HMK Rem pGexKG was created byubcloning the BamHI/XhoI fragment of pRem Gex (1) into theamHI/XhoI site of pGexKG HMK. pRem TrcHisA was created byubcloning the BamHI/XhoI fragment of pRem express (1) into theamHI/XhoI site of pTrcHisA (Invitrogen). Eukaryotic expressionectors allowing expression of recombinant proteins bearing threeopies of the influenza hemagglutinin (HA) epitope tag at their-terminus were created as follows. HA Rem pKH3 was generatedy subcloning the BamHI/EcoRI fragment of pRem pTrcHisA intohe BamHI/EcoRI site of pKH3 (33). To construct HA RemCDNA3.1, the HindIII/EcoRI fragment of HA Rem pKH3 was li-ated to HindIII/EcoRI digested pcDNA3.1(1) (Invitrogen). HA RadKH3 was created by generating a PCR product with a 59 BamHI sitesing the mouse expressed sequence tag AA444424 (which containedhe full-length mouse Rad cDNA; the full-length cDNA sequence cane retrieved using GenBank Accession No. AF084466) (Genome Sys-ems Inc.). The Rad PCR product was subcloned into the EcoRV sitef pZero (Invitrogen), the BamHI/EcoRI fragment from this plasmidsolated and subcloned to the same sites in pKH3 to create mRadKH3. A manuscript describing the full cDNA cloning of rat Rem2,he fourth and newest member of the RGK family, is in preparation.4

owever, the Rem2 cDNA sequence can be retrieved from Genbanksing Accession No. AF084464. rRem2 pKH3 was created by gener-ting a PCR product with a 59 BamHI site, subcloning the PCRroduct into the EcoRV site of pZero to create rRem2 pZero, andubsequently ligating the BamHI/EcoRI fragment of rRem2 pZero

4 B. S. Finlin and D. A. Andres, unpublished data.

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403CHARACTERIZATION OF Rem AND Rad AS 14-3-3 BINDING PROTEINS

nto pKH3. The C-terminal deletion constructs HA Rem1–282 pKH3,A Rem1–265 pCDNA3.1, and HA Rem1–244 pCDNA3.1 were created by

enerating a PCR product with a 59 BamHI site and an in-frameermination codon and subcloning the blunt products into the pZerocoRV site. The Rem1–282 BamHI/EcoRI fragment was subsequently

igated into the BamHI/EcoRI site of PKH3 to make HA Rem1–282

KH3. The Rem1–265 and HA Rem1–244 BamHI/XhoI fragments wereubcloned into the BamHI/XhoI site of HA Rem pCDNA3.1 to makeA Rem1–265 pcDNA3.1 and HA Rem1–244 pcDNA3.1. HA Rem18–297

as created by generating a PCR product with a 59 BamHI sitemmediately upstream of codon 18. The BamHI/NheI fragment ofhis PCR product was then subcloned to the BamHI/NheI site of HAem pCDNA3.1. Rem18–77Gex was generated by PCR and the productubcloned to the BamHI/SmaI site of pGexKG. 59 BamHI sites werentroduced to 14-3-3 z and 14-3-3 u by PCR, the resulting productsere subcloned into pZero, and the BamHI/EcoRI fragment was

eleased and ligated into the same sites of pTrcHisA.Generation of 32P-labeled Rem. The plasmid HMK Rem pGexKGas transformed into BL21(DE3) bacteria (Novagen). RecombinantST-HMK-Rem was expressed and purified by glutathione–agaroseffinity chromatography, and the glutathione S-transferase (GST)as cleaved with thrombin as described previously (1), yieldingMK-Rem. HMK-Rem (10 mg) was incubated with 2.0 mCi [32P]ATP

6000 Ci/mmol, NEN) and 100 U HMK (Sigma) in 100 ml of 20 mMris pH 7.5, 100 mM NaCl, 12 mM MgCl2 for 30 minutes on ice. Theeaction was stopped by addition of 400 ml of stop buffer (10 mMhosphate, 10 mM sodium pyrophosphate, 1 mg/ml bovine serumlbumin). The probe was dialyzed against four changes (50 ml each)f dialysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12 mM MgCl2,0 mM GDP) to remove unincorporated label and stored in multipleliquots at 220°C.Rem interaction cDNA library screen. Because Rem is expressed

n a variety of tissues, a 14-day mouse embryo lEXlox cDNA libraryNovagen) was selected for screening and plated at 40,000 plaques/late (150 mm) in BL21(DE3) bacteria. Once plaques reached 0.5–1m in size, plates were overlaid with nitrocellulose filters and incu-

ated overnight at 4°C. The plates were then placed at 37°C andncubated for another 4 h, and the primary filters were removedmmediately to Hyb75 (20 mM Hepes, pH 7.6, 75 mM KCl, 0.1 mMDTA, 2.5 mM MgCl2, 1 mM DTT, 0.05% NP-40) (30). Secondarylters were generated by overlaying the plates with a second set ofitrocellulose filters and incubating for 4 h at 37°C. These filtersere immediately combined with the primary membranes in Hyb75.ilters were blocked in Hyb75 with 1% nonfat milk (Carnation) forh at 4°C. Bacterial extract containing recombinant HMK-GST wasrepared from BL21DE3 cells transformed with pGexKG HMK. Theacteria were grown at 37°C in LB medium to an A600 5 0.6. Proteinroduction was induced with 0.5 mM IPTG for 4 h. The bacteria wereelleted, resuspended in Hyb75, and broken using a French pressureell, and the 100,000g cleared supernatant was used as a supplemen-al blocking agent. Library filters were incubated with Hyb75 con-aining 250 mM KCl, 1% nonfat milk, 400 mg/ml HMK-GST bacterialxtract, 10 mM GDP, and 200,000 cpm/ml [32P]HMK-Rem probe for6 h at 4°C with shaking. Filters were washed with four 100-mlashes of Hyb75 supplemented with 10 mM GDP for 30 min, dried,nd exposed to Kodak X-OMAT film for 4 h at room temperature.ore than 100 positive plaques were detected, of which 66 were

elected at random for further characterization. Seven of these po-ential positives were selected for secondary and tertiary screening.ositive plaques were isolated, and plasmids containing libraryDNAs were rescued by in vivo CRE-mediated excision according tohe manufacturer’s protocol. DNA sequence analysis of these plas-ids revealed that each contained a full-length cDNA encoding

ither the z, e, h, or u isoforms of the 14-3-3 family of proteins. Theemainder of the primary positive plaques were rescued by CRE-
xcision and the plasmid pools were analyzed by PCR using 14-3-3ene specific primers. Of the 66 original positives, 58 were found to
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ontain one of the four initially identified 14-3-3 genes, while theemaining 8 plaques were shown to be false-positives. Thus, all of theositive plaques were shown to encode 14-3-3 family proteins.In vitro analysis of 14-3-3 binding. The ability of recombinantem and HMK phosphorylated recombinant Rem (see below) to bind4-3-3 proteins was analyzed using an in vitro Ni21-NTA Sepharoseead pull-down assay. Recombinant fusion proteins containing sixistidine residues (His6-tagged), mouse 14-3-3z and 14-3-3u, wererepared as follows. 14-3-3z pTrcHisA and 14-3-3u pTrcHisA wereransformed into the bacterial strain DH5a. The cells were grown,ysed, and resuspended in column buffer (20 mM Tris, pH 7.9, 500

M NaCl, 10% glycerol, 1 mM bME, and 10 mM imidazole), and theleared supernatant was subjected to Ni21-Sepharose affinity chro-atography using a 10–750 mM imidazole gradient under the con-

itions recommended by the manufacturer (Pharmacia). The histi-ine-tagged 14-3-3 proteins (.90% pure as judged by SDS–PAGE)ere dialyzed against two changes of buffer containing 20 mM Tris,H 8.0, 150 mM NaCl, 5% glycerol. After dialysis, the proteins weretored in multiple aliquots at 270°C. The identity of the recombinantroteins was confirmed by immunoblotting with anti-14-3-3 antibodyS.C. 629, Santa Cruz Biotech.). When necessary, the histidine tagas removed by enzymatic cleavage of the protein bound to Ni21-epharose using an EKMAX kit (Invitrogen) according to the man-facturer’s instructions. Recombinant Rem was produced as de-cribed previously (1). To detect His6-14-3-3 binding to Rem, 1 mgem and 2 mg His6-14-3-3z were incubated in a 100-ml reactionontaining standard Ni21 binding buffer (20 mM Tris pH 7.5, 250 mMaCl, 1 mM Mg21, 10 mM GDP, 0.1% Triton X-100, 50 mM imida-

ole). After incubation for 1 h on ice, 20 ml of a 50% slurry ofi21-Sepharose was added to each reaction and incubated for 30 mint 4°C with end-over-end rotation. The Ni21-Sepharose beads werehen pelleted in a microfuge for 30 s at 14,000 rpm, and the super-atant was collected. The pelleted Ni21-Sepharose beads wereashed on ice three times with ice-cold assay buffer (1 ml), afterhich the pellet was resuspended in 30 ml of assay buffer and 20 mlDS sample buffer, which disrupted the Ni21-Sepharose interactionnd solubilized the Ni21–His6–14-3-3 complex. The supernatant (50l) and pellet fractions were then analyzed for the presence of Remy immunoblotting with rabbit anti-Rem (5723) antibody at a 1:1000ilution using goat anti-rabbit secondary antibody (Zymed) at a:10,000 dilution.Phosphorylation of recombinant Rem. Phosphorylation of recom-

inant Rem by cAMP-dependent HMK was assessed by incubation ofmg of wild-type Rem with or without 1 U/ml HMK for 1 h on ice in

0 mL of HMK reaction buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12M MgCl2, 1 mM ATP, and 1 mCi [32P]ATP). Following incubation,

he reaction was run on a 10% SDS–PAGE gel, stained with Coo-assie blue, dried, and exposed to Kodak X-OMAT film for 10 min at

oom temperature. Phosphorylated recombinant Rem for bindingtudies was generated by incubating 10 mg recombinant Rem (lack-ng the N-terminal HMK consensus site) on ice with 100 U HMK forh in 100 ml HMK reaction buffer. Incorporation of radioactivity waserified by SDS–PAGE and autoradiography before use in bindingssays.Analysis of RGK family proteins/14-3-3 interactions. HEK 293

ells were maintained in Dulbecco’s modified Eagle’s medium con-aining 5% (v/v) fetal calf serum and 55 mg/ml gentamicin and plated4 h prior to transfection at 70% confluence. Monolayers of HEK 293ells were transiently transfected with 20 mg of mammalian expres-ion plasmid encoding hemagglutinin epitope tagged Rem, Rad,em2, or Rem variant DNA/100-mm dish using the calcium phos-hate technique as described previously (34). The cells were har-ested at 48 h, washed with 2 3 10 ml PBS, and transferred in 1 mlce-cold PBS to a 1.5-ml tube. The cell pellets were then lysed with aontes probe sonicator (six bursts/10 s duration) on ice and centri-

uged at 4°C for 10 min at 100,000g, and the supernatant wasransferred to a fresh tube. The protein concentration of the 105g

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upernatant (S100) was determined using the Bradford assay. 14-3-3inding was assessed using 40 mg of the S100 fractions and histidine-agged 14-3-3 proteins as described above. Inclusion of the expressedA-tagged proteins in the Ni21-Sepharose pellet was analyzed by

mmunoblotting with the anti-HA mAb 12CA5. All immunoblotsere subsequently reprobed with anti-Raf-1 antibody (1 mg/ml) as aositive control for 14-3-3 activity. For competition experiments 1 to0 mg of recombinant 14-3-3z, from which the histidine tag had beennzymatically removed, was preincubated with the S100 fraction forh on ice prior to addition of His6-14-3-3z. For experiments in which

he GDP/GTP state of Rem was evaluated for its effect on 14-3-3inding, 20 mg of the S100 fraction was brought to 1 mM EDTA andupplemented with either 10 mM GDP or GTPgS for 5 min at roomemperature. The Mg21 concentration was then adjusted to 1 mMnd incubation continued for 10 min at room temperature. Theseonditions have been shown to allow for efficient nucleotide exchangen recombinant Rem (unpublished observation). 14-3-3 binding wasssayed as described above, except that 10 mM GDP or GTPgS wasncluded in all assay buffers.

Dephosphorylation of HEK 293 extracts. An S100 fraction wasrepared from transiently transfected HEK293 cells expressing wild-ype Rem (see above) and dialyzed against 20 mM Tris, pH 7.4, 50M NaCl, and 1 mM MgCl2. The S100 fraction (100 mg) was then

reated either with protein phosphatase 1 (PP1) 10 U (New EnglandioLabs), 10 U PP1 and 2 mM okadaic acid (CalBiochem), or withoutP1 in a 100-ml reaction for 60 min at 30°C in PP1 buffer (50 mMris, pH 7.0, 0.01 mM EDTA, 1 mM MnCl2, 1 mM MgCl2, 5 mM DTT,nd 0.01% Brij 35). Twenty microliters (20 mg S100 protein) was thennalyzed for His6-14-3-3 interaction (see above).Antibodies and immunoblotting. Immunoblotting was carried

ut as described previously (34) using enhanced chemiluminescenceetection according to the manufacturer’s protocol (SuperSignal,ierce). A polyclonal anti-Rem antibody (5723) was created by pro-ucing antibody against amino acids 18–77 of Rem as follows. Theem18–77 Gex plasmid was transformed into BL21(DE3) bacteria.ecombinant Rem18–77 fusion protein was expressed and purified byST affinity chromatography as described previously (1). The fusionrotein (250 mg) was denatured by incubation at 65°C for 10 min andsed to immunize rabbits as described previously (34). The produc-ion of anti-Rem antibody was evaluated by immunoblotting againstuthentic Rem. Rabbit anti-Raf-1 (S.C.-133) and panreactive rabbitnti-14-3-3 (S.C.-629) antibodies were from Santa Cruz. Secondaryntibodies were from Zymed. For coimmunoprecipitation studies 5g of 12CA5 anti-HA antibody was crosslinked to 1 ml protein G

epharose Fast Flow resin (Pharmacia) using dimethylpimelimidatessentially as described (35).Metabolic labeling and immunoprecipitation. HEK 293 cellsere transiently transfected with 20 mg of pKH3 or Rem pKH3 asescribed (see above). Dishes were refed 43 h after transfection withysteine- and methionine-free medium containing 5% dialyzed fetalalf serum. After a 1-h preincubation, 600 mCi [35S]methionine.1000 Ci/mmol) was added to each plate. After an additional 4-hncubation at 37°C, the radiolabeled cells were rinsed two times withBS, scraped from the dish, and pelleted by centrifugation. Radio-

abeled cell pellets were lysed by sonication in PBS (see above). An100 supernatant was generated by adding 100 mg of radiolabeledell lysate (50 ml) to 50 ml IPA buffer (20 mM Tris, pH 7.5, 150 mMaCl, 1% NP-40, 1 mM PMSF) and incubating the mixture for 10in on ice, followed by centrifugation at 100,000g for 10 min. The

upernatant (S100) was removed and incubated with 12.5 mg ofither 12CA5 anti-HA antibody or rabbit preimmune sera for 2 h at°C on a rocking platform. Twenty-five microliters of a 50% slurry ofrotein A-Sepharose (Pharmacia) (which had previously been prein-ubated with 100 mg of unlabeled HEK 293 cell extract and washedith IPA) was added and incubated for 1 h at 4°C with rotation. The

mmune complexes were then washed three times with 1 ml IPB (20M Tris, pH 7.5, 150 mM NaCl, 0.2% NP-40), 1 3 1 ml IPC (20 mM t

ris, pH 7.5, 500 mM NaCl, 0.2% NP-40), and once with 1 ml IPD (20M Tris, pH 7.5, 150 mM NaCl). Twenty microliters of 23 SDS–AGE sample buffer was added, and the samples were heated at5°C for 5 min. The samples were resolved on 10% SDS–polyacryl-mide gels, transferred to nitrocellulose membranes, treated withmplify (Amersham) for 15 min, dried at 45°C for 15 min, andxposed to Kodak X-OMAT AR film for 16 h at 270°C with anntensifying screen.

Coimmunoprecipitation analysis. HEK 293 monolayers wereransiently transfected with HA Rem pCDNA3.1 (20 mg DNA/dish)nd after 48 h of recovery placed under drug selection (250 mg/mleocin) (Invitrogen) to generate stable cell lines expressing recombi-ant HA-Rem. After 14 days of drug selection, individual coloniesere isolated, expanded, and tested for Rem expression by immuno-lotting with 12CA5 anti-HA antibody. One of these cell lines, HAem pcDNA3.1-1 was used for the coimmunoprecipitation studies. Aingle confluent plate of HEK 293 cells or HA Rem pcDNA3.1-1 cellsas harvested, resuspended in IPA, and lysed by sonication, and a00,000g supernatant (S100) was prepared. For analysis of 14-3-3oimmunoprecipitation, 1 mg of each S100 lysate was incubated with0 ml of a 50% slurry of protein G covalently crosslinked to 12CA5 (asescribed above) in a 300-ml reaction for 2 h at 4°C with gentleotation. Immune complexes were pelleted, washed, and resolved byDS–PAGE as described (see above). 14-3-3 proteins were detectedy immunoblotting using a 1:500 dilution of S.C. 629. For analysis ofaf coimmunoprecipitation, 10 mg of 12CA5 was incubated with 1 mgf each S100 lysate and 20 ml protein G-Sepharose in a 300-mleaction for 2 h at 4°C. The immune complexes were processed asescribed above, and Raf immunoblotting was carried out using S.C.33 (1:100 dilution).

ESULTS

Rem interacts with members of the 14-3-3 gene fam-ly. To generate purified, radiolabeled Rem proteinor use as a molecular probe, Rem was expressed as ausion protein in which the peptide recognition se-uence (RRASV) for the catalytic subunit of cAMP-ependent HMK (36) was placed at its N-terminus.his allowed the in vitro phosphorylation of recombi-ant Rem by incubation with commercial protein ki-ase and [g-32P]ATP. To isolate Rem-interacting genes,pproximately 400,000 cDNA clones from a 14-dayouse embryo cDNA library were screened in dupli-

ate using 32P-radiolabeled Rem. The Rem probe re-ealed more than 100 potential candidate clones, fromhich 66 were subjected to secondary and tertiary

creening as described under Experimental Proce-ures. After this analysis, 58 of the initial clones con-inued to interact with Rem. Sequence analysis re-ealed that all of the clones contained full-lengthDNAs which encoded either the e, h, u, or z 14-3-3enes. Thus, using Far Western interaction cloningnd a mouse embryo library, Rem was found to interacttrongly with a series of 14-3-3 proteins.Phosphorylation is required for the association of

4-3-3 proteins and Rem. We next conducted experi-ents to determine whether 14-3-3z binds to Rem in

itro. For this purpose, recombinant wild-type Remas purified from bacteria. Recombinant 14-3-3z pro-

ein modified to contain six histidine residues at the

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-terminus and therefore able to bind Ni21-Sepharoseesin was also produced. To detect the binding of wild-ype Rem to histidine-tagged 14-3-3z, we incubated theroteins together under various conditions, isolatedhe complex by absorption to Ni21-Sepharose beads,nd visualized the bound Rem protein by SDS gellectrophoresis and immunoblotting (Fig. 1A). Whenis6-14-3-3z was incubated with recombinant Rem, the

mmunoblotable Rem protein remained in the super-atant (Fig. 1A, lane 4). Because previous studies have

IG. 1. In vitro association of recombinant Rem and 14-3-3z ishosphorylation dependent but guanine nucleotide independent. (A)ach assay contained (in a final volume of 100 ml) standard Ni21-inding buffer plus either 1 mg of recombinant Rem (Rem) or heartuscle kinase (HMK) phosphorylated recombinant Rem (P-Rem)hich had been preloaded with either GDP or GTPgS as describednder Experimental Procedures. Recombinant His6-14-3-3z fusionrotein (2 mg) was then added as indicated by the plus sign. Afterncubation for 1 h on ice, 20 ml of a 1/1 (v/v) suspension of Ni21-epharose was added to each tube. The incubation was continued for0 min at 4°C with rotation, and the samples were pelleted andashed. Aliquots (50 ml) of the supernatant (S) and pellet fractions

P) were subjected to SDS–PAGE on a 10% polyacrylamide gel,ransferred to nitrocellulose filters, and subjected to immunoblotnalysis with anti-Rem polyclonal antibody as described under Ex-erimental Procedures. (B) Phosphorylation of recombinant Remas assessed by incubation of 1 mg of Rem with (1) or without (2) 1/ml HMK and [g-32P]ATP (1 mCi) for 1 h on ice. The reactions were

esolved by 10% SDS–PAGE, and the migration of recombinant Remas detected with Coomassie blue staining. The dried gel was ex-osed to Kodak XAR-5 film for 10 min at room temperature. The datare representative of two to five separate experiments.

hown that 14-3-3 proteins bind to specific phospho- p

erine-containing motifs (37), we reasoned that HMKreatment of Rem was necessary for 14-3-3 binding. Asxpected, when the wild-type Rem protein was firstubjected to phosphorylation with HMK, a fraction ofhe modified Rem protein (P-Rem) was now detected inhe Ni21-Sepharose pellet (Fig. 1A, lane 4). Inclusion inhe pellet fraction was His6-14-3-3z dependent, sincehosphorylated Rem was not pelleted by Ni21-Sepha-ose beads alone (Fig. 1A, compare lanes 2 and 4). Toonfirm that wild-type Rem was a substrate for HMK,em was subjected to in vitro phosphorylation withAMP-dependent protein kinase from heart muscle. Ashown in Fig. 1B, Rem was phosphorylated in theresence, but not the absence, of HMK. Binding of Remo His6-14-3-3z was independent of the guanine nucle-tide state of Rem. Thus, phosphorylated Rem wasound in the Ni21-Sepharose pellet when bound to ei-her GTPgS or GDP (Fig. 1A, lane 4).

To examine the interaction between Rem and 14--3z in a cellular context, the cDNA encoding full-ength Rem modified to express a N-terminal HApitope tag was introduced into HEK 293 cells byransfection. Cell lysate prepared from these cells washen incubated with recombinant His6-14-3-3z, theomplex isolated with Ni21-Sepharose beads, and ana-yzed by immunoblotting with anti-HA antibody. HA-agged Rem was found in the Ni21-Sepharose pellet in

His6-14-3-3z-dependent, but guanine nucleotide-in-ependent manner (Fig. 2A, compare lanes 2 and 4).he specificity of the binding to Ni21-Sepharose wasonfirmed by the finding that Rem failed to bind toi21-Sepharose after incubation with either wild-type4-3-3z (not shown) or in the absence of His6-14-3-3zFig. 2A). When the blots were reprobed with an anti-ody specific for Raf kinase, a known 14-3-3z bindingrotein (13–15), endogenous Raf was found to associateith the Ni21-Sepharose pellet in a His6-14-3-3z-depen-ent manner (Fig. 2A, bottom). Although not shown,his control was used in all remaining His6-14-3-3zssociation experiments to confirm His6-14-3-3z activ-ty. To confirm that 14-3-3 proteins and Rem associaten vivo, anti-HA immune complexes from untrans-ected HEK 293 cells and HEK 293 cells transfectedith the cDNA encoding HA epitope tagged Rem werenalyzed. As shown in Fig. 2B, endogenous 14-3-3 pro-eins were present in the anti-HA immune complexesrom transfected HEK 293 cells.

These findings suggest that posttranslational modi-cations, particularly phosphorylation, may play an

mportant role in regulating the 14-3-3 binding inter-ction. Indeed, recent reports have shown that mem-ers of the 14-3-3 protein family bind to specific phos-hoserine-containing motifs (37, 38). As seen in Fig.C, the specific interaction of Rem and His6-14-3-3zas totally abolished by the pretreatment of Rem ex-
ressing HEK cell lysates with the phosphoserine/thre-

onosdoinprtRwrbwTpt

pptRadtfotpariapTkmdttpgrT3

FHtwrboawPttattlSSlpmwithout (none, top) or with 10 U of protein phosphatase 1 (PP1,bottom) in a 100-ml reaction at 30°C for 1 h. 20 ml of the reactions (20

mrwmSl1r


nine directed phosphatase PP1. This disruption wasot due to proteolytic degradation of Rem, since theverall level and integrity of the protein remained con-tant throughout the PP1 treatment (Fig. 2C). In ad-ition, pretreatment of the HEK lysate with 2 mMkadaic acid, a potent serine/threonine phosphatasenhibitor, completely reversed the PP1 inhibition (dataot shown). These results suggest that the in vivohosphorylation of Rem, on either serine or threonineesidues, is required for 14-3-3 association. To confirmhat Rem is phosphorylated in vivo, lysates from HA-em transfected cells metabolically labeled with 32Piere immunoprecipitated with anti-HA antibody. This

esulted in the isolation of a single radiolabeled 46-kDaand, which comigrated on SDS–polyacrylamide gelsith authentic HA-tagged Rem (data not shown).hese results confirm that the phosphorylation of Remlays a critical role in its association with 14-3-3 pro-eins.

Mapping of the 14-3-3 binding site in Rem. Usinghosphoserine peptide libraries it was recently re-orted that 14-3-3 protein binding is directed towardwo predominant phosphorylated consensus motifsSXpSXP and RXY/FXpSXP (where X is any aminocid and pS indicates the phosphorylated serine resi-ue) present in nearly all known 14-3-3 binding pro-eins (37, 38). Although the phosphorylation sites ofull-length Rem have not been characterized, a seriesf in vitro serine phosphorylation sites have been iden-ified within the C-terminus of the closely related Radrotein (39). In addition, while Rem does not containn exact 14-3-3 consensus motif, it does contain threeelated amino acid sequences. These are the sequencesncluding Ser-18 (RRA[S]TP), Ser-262 (RRA[S]LG),nd Ser-290 (RSK[S]CH) (where [S] is the putativehosphoserine residue in the 14-3-3 consensus site).he first two sequence motifs also contain perfect HMKinase sites ([RRXS/T] or [RR/KXS/T]) in which theodified serine residue would correspond to the pre-

icted phosphoserine in the 14-3-3 binding motif. Inhe initial binding assays, we were only able to detecthe binding between Rem and 14-3-3z using HMKhosphorylated wild-type Rem (Fig. 1A), strongly sug-esting that the phosphorylation of serine/threonineesidues within Rem by HMK was critical for binding.hese findings suggested that one of the potential 14--3 binding sites containing HMK consensus motifs

g lysate) was then incubated with 2 mg of His6-14-3-3z, Ni21-Sepha-ose was added to each tube, and the samples were pelleted andashed as described under Experimental Procedures. Aliquots (50l) of the supernatant (S) and pellet fractions (P) were subjected toDS–PAGE on a 10% polyacrylamide gel, transferred to nitrocellu-

ose filters, and subjected to immunoblot analysis with anti-HA

IG. 2. In vitro and in vivo association of Rem with 14-3-3. (A)EK 293 cells were transfected with a vector expressing HA-epitope-

agged Rem protein. Each assay contained 40 mg of cytosolic extract,hich had been preloaded with either GDP or GTPgS. 2 mg of

ecombinant His6-14-3-3z fusion protein was then added as indicatedy the plus sign. All assay tubes were incubated on ice for 1 h, 20 mlf a 1/1 (v/v) suspension of Ni21-Sepharose was added to each tube,nd the incubation was continued for 30 min at 4°C. The samplesere then pelleted and washed as described under Experimentalrocedures. Aliquots (50 ml) of the supernatant (S) and pellet frac-

ions (P) were subjected to SDS–PAGE on a 10% polyacrylamide gel,ransferred to nitrocellulose filters, and subjected to immunoblotnalysis with anti-HA monoclonal antibody 12CA5. The blots werehen reprobed with an anti-Raf antibody (bottom). (B) Epitope-agged Rem protein was immunoprecipitated from 1 mg of total cellysate (S100) with anti-HA monoclonal antibody 12CA5. 20 mg of the100 lysate and washed immune complexes were then subjected toDS–PAGE on a 10% polyacrylamide gel, transferred to nitrocellu-

ose filters, and subjected to immunoblot analysis with anti-14-3-3olyclonal antibody. (C) HEK293 cells were transfected with a plas-id expressing HA-Rem and cell lysates (100 mg) were incubated

2CA5 antibody. The data are representative of a typical experimentepeated three times.

mTgs3RflfRastaHp

a31pm

srrcrbtit

Fcd(HPt ysio


ight represent the 14-3-3 interaction domain in Rem.o begin to identify the 14-3-3 binding domain weenerated HA-tagged Rem protein mutants containinguccessive truncations at their N- and C-terminus (Fig.A). HEK 293 cells were transiently transfected withem expression plasmids and the binding of the trans-

ected gene products to His6-14-3-3z in vitro was ana-yzed with an anti-HA antibody. His6-14-3-3z bound toull-length Rem protein and two deletion mutants,em1–282 and Rem18–297, from which 15 and 17 aminocids were removed from the N- and C-terminus, re-pectively (Fig. 3B). Additional C-terminal deletionshat removed 32 or 53 C-terminal residues (Rem1–265

nd Rem1–244, respectively) eliminated the binding tois -14-3-3z. As a control, the membranes were re-

IG. 3. Mapping of the 14-3-3 binding region of Rem. (A) Schemonsensus sequences for GTP binding and hydrolysis (G1–G5) and tomains. The position of five deletion mutations (below) and four serin40 mg) from HEK 293 cells transiently transfected with and expris6-14-3-3z, Ni21-Sepharose was added to each tube, and the sarocedures. Aliquots (50 ml) of the supernatant (S) and pellet fracransferred to nitrocellulose filters, and subjected to immunoblot analf three to five separate experiments.

6

robed with an anti-Raf antibody. Comparable e

mounts of Raf could be detected in all of the His6-14--3z pellets (data not shown). Thus, residues critical for4-3-3 binding are contained within the region encom-assing residues 265–297 within the extended C-ter-inus of Rem.Although Rem does not contain a perfect 14-3-3 con-

ensus, and residues 265–282 contain only one serineesidue (Ser-278) which would not be predicted to di-ect 14-3-3 association, a plausible 14-3-3 binding siteentered at Ser-262 lies just above this deletion. Weeasoned that the Rem1–265 deletion protein might note recognized by the kinase needed to phosphorylatehe Ser-262 recognition motif, resulting in a disruptionn 14-3-3 binding. To determine the importance ofhese putative phosphoserine residues, we replaced

c diagram of Rem, with dark bars designating the position of thestripped bars indicating the position of putative 14-3-3 interactiono alanine point mutations (above) are denoted by arrows. (B) Lysatesng various HA-tagged forms of Rem were incubated with 2 mg ofles were pelleted and washed as described under Experimentals (P) were subjected to SDS–PAGE on a 10% polyacrylamide gel,s with anti-HA 12CA5 antibody. Examples shown are representative

atihee t

essimp

tion

ach of these serines with alanine by site-directed mu-

tRpaatealHmryCatebpw

dfRetprhaf1w4Rt1HbstRfftbep1cHlqa

ad

S

FpwRadsPfins


agenesis to generate the constructs RemS18A, RemS262A,emS278A, and RemS290A (Fig. 3A). These mutants werelaced in HA-tagged mammalian expression vectorsnd transfected into HEK 293 cells to determine theirbility to interact with His6-14-3-3z in vitro. The pro-ein products of these three expression vectors wereach expressed as revealed by immunoblotting with annti-HA antibody (data not shown). However, whenysates from the transfected cells were incubated withis6-14-3-3z, it was found that each of the alanineutants bound His6-14-3-3z (data not shown). These

esults are therefore inconclusive. While deletion anal-sis clearly indicates that residues 265–282 within the-terminus of Rem are critical for 14-3-3 association,nd additional studies suggest that Rem phosphoryla-ion is necessary, we have not been able to define thexact serine-containing sequence(s) essential for 14-3-3inding. Additional studies, including in vivo phos-hoamino acid analysis and additional mutagenesis,ill be needed to address this issue.A subset of the RGK family interacts with 14-3-3. To

etermine if 14-3-3 proteins associated with other RGKamily members, full-length Rem, Rad, Gem/Kir, andem2 with the HA epitope tag at their N-termini werexpressed in HEK 293 cells. Rem2 is a new member ofhe RGK gene family, and the characterization of thisrotein will be described in another manuscript.4 De-egulated expression of Gem has been reported to in-ibit cell growth in a number of cell lines (3). Repeatedttempts to express HA-tagged Gem/Kir in HEK cellsailed, and Gem/Kir was therefore not characterized for4-3-3 binding. Immunoblotting with the HA antibodyas used to confirm the expression of each protein (Fig.A). Incubation of the HEK 293 lysates expressingem, Rad, and Rem2 with His6-14-3-3z demonstrated

hat both Rem and Rad, but not Rem2, associated with4-3-3z (Fig. 4A). This experiment was repeated usingis6-14-3-3u. No binding of Rem2 to His6-14-3-3u coulde detected, although Rem was bound (data nothown). To extend these observations, we examinedhe interaction of endogenous 14-3-3 proteins withem, Rem2, and Rad. Anti-HA immune complexes

rom untransfected HEK 293 and HEK 293 cells trans-ected with the cDNA encoding either HA epitope-agged Rem, Rem2, or Rad were analyzed. Immuno-lotting with the HA antibody was used to confirm thexpression and efficient immunoprecipitation of eachrotein (Fig. 4C). As shown in Fig. 4B, endogenous4-3-3 proteins were present in the anti-HA immuneomplexes from transfected HEK 293 cells expressingA-Rem and HA-Rad but not HA-Rem2 (compare

anes 2–4). The inability of 14-3-3 to bind to Rem2 isuite surprising because of the high degree of amino
cid conservation within the C-termini of Rem, Rad, m
nd Rem2, including in the putative 14-3-3 bindingomains (Fig. 5).Rem is present in a cellular complex with 14-3-3.

ince both Rem and Raf can interact with 14-3-3z, they

IG. 4. Selective interaction of RGK family members with 14-3-3rotein. Lysates (20 mg) from HEK 293 cells transiently transfectedith and expressing full-length HA-tagged forms of Rem, Rad, andem2 were incubated with 2 mg of His6-14-3-3z, Ni21-Sepharose wasdded to each tube, and the samples were pelleted and washed asescribed under Experimental Procedures. Aliquots (50 ml) of theupernatant (S) and pellet fractions (P) were subjected to SDS–AGE on a 10% polyacrylamide gel, transferred to nitrocelluloselters, and subjected to immunoblot analysis with anti-hemaggluti-in antibody 12CA5. Examples shown are representative of fiveeparate experiments.

ay both be recruited to the same protein signaling

cTfbFwaRaipdwprbTldpSbtaidAutpdpi

D

taRwapc

1rqctsd1mai3

ptnsscodno3mcbcapsswiytpist

p

Fwnocm . LU


omplexes and function in the same signaling cascades.o address this, immune complexes were analyzed

rom [35S]methionine-labeled HEK 293 cells that hadeen transfected with HA-tagged Rem. As shown inig. 6 (top), 35S-labeled endogenous 14-3-3 proteinsere found in anti-HA immune complexes. At least twodditional radiolabeled proteins were also identified inem immunoprecipitates, strongly suggesting that thenti-HA immune complexes include recombinant Remn association with additional cellular proteins. Theseroteins likely did not arise through the proteolyticegradation of Rem or 14-3-3, since probing the blotith anti-HA or anti-14-3-3 antibody detects intactroteins. Although unlikely, these bands might alsoesult from selective N-terminal proteolysis, becauseoth antibodies recognize amino terminal epitopes.he immunoprecipitates were contaminated by a low

evel of nonspecific cellular protein, which made theetection of endogenous Raf difficult. To overcome thisroblem, the anti-immune complexes were resolved onDS–PAGE and immunoblotted with anti-Raf anti-ody. Under conditions in which HA-Rem was quanti-atively immunodepleted from HEK 293 cell lysates bynti-HA antibody, Raf was not detected in anti-HAmmune complexes (Fig. 6, bottom). This situation isistinct from the association of the zinc finger protein20 with Raf, in which 14-3-3 proteins serve as molec-lar adapters to direct these protein–protein interac-ions (40). This suggests that while Rem and 14-3-3roteins may associate in HEK 293 cells, Rem and Rafo not appear simultaneously in the same 14-3-3 com-lexes and are therefore unlikely to directly collaboraten the regulation of signaling cascades.

ISCUSSION

In this study, we have demonstrated for the firstime an in vitro and in vivo association between Remnd a series of 14-3-3 proteins. Indeed, Rem is the firstas-related GTPase to be shown to interact directlyith members of the 14-3-3 gene family and must bedded to the growing number of signal transductionroteins that bind 14-3-3 molecules (27). Interaction

IG. 5. Comparison of the C-terminal amino acid sequences of moas performed using the CLUSTAL W1.6 program. Hyphens represumbers. Amino acid residues that are conserved in at least three of tverlines indicate two putative 14-3-3 binding sites, while asterisksonsensus motif. The rat Rem2 cDNA and predicted protein sequenceouse Rem (Accession No. U91601); hRad, human Rad (Accession No13052).

loning was used to initially identify the association of n

4-3-3 proteins with Rem. All of the clones selected forescreening after primary library analysis and subse-uently detected in secondary analysis were found toontain full-length 14-3-3 family members. The facthat all of the identified cDNA clones were full-lengthuggests that Rem might only interact with 14-3-3imers. This is consistent with the known structure of4-3-3, in which the C-terminus of each of the proteinonomers contains the binding groove which functions

s the protein binding region, while the N-terminusncludes the sequences needed for dimer formation (28,7).While, the biological consequence of binding 14-3-3

roteins is controversial, the importance of 14-3-3 pro-eins in controlling signal transduction pathways isow well established. 14-3-3 association has beenhown to decrease the enzymatic activity of proteinsuch as nitrate reductase (41), whereas binding in-reases the activity of tryptophan hydroxylase and ex-enzyme S (42, 43). However, 14-3-3 binding does notirectly influence the enzymatic activities of a largeumber of signaling proteins including MEK kinasesr protein-tyrosine phosphatase H1 (24, 25). Thus, 14--3 proteins may not always directly regulate enzy-atic function, but rather control the composition or

ellular location of signaling cascades. 14-3-3s haveeen proposed to play an important role in cell cycleontrol (44), mitogenic signaling pathways (27, 45),poptosis (19), and learning (46). Furthermore, 14-3-3rotein binding has been shown to protect phospho-erine residues from phosphatases (38, 42, 47). In Dro-ophila, D14-3-3 is an essential component in the path-ay regulating photoreceptor development and genet-

cally maps between Ras and Raf (48, 49). While ineast, 14-3-3 proteins are necessary for regulation ofhe Ras/mitogen-activated kinase pathway regulatingseudohyphal development (50). These studies clearlymplicate 14-3-3 proteins as key components in theignaling cascades regulating cellular mitogenesis andransformation.

Using in vitro binding assays, we demonstrate thathosphorylation of Rem, likely on serine residues, but

Rem, human Rad, human Gem/Kir, and rat Rem2. The alignmentgaps introduced for optimal alignment. Numbers indicate residue

four proteins in the alignment are placed in shaded boxes. The heavyote the position of the predicted phosphoserine residue within eachy be retrieved from GenBank using Accession No. AF084464; mRem,24564); hGem/Kir, human Gem and Kir (Accession Nos. U10550 and

useenthedenma

ot its guanine nucleotide status is a prerequisite for

bmp(t

ed11mrrtdsoR3dttweawTbtfvou1pmmtwtetca

twfhciltepwppi

3

FHtRm3

c2APs(Eetmpidctaba


inding (see Figs. 1 and 2). These findings are in agree-ent with previous findings of a requirement for phos-

horylation by other proteins that interact with 14-3-327, 37, 38). A series of Rem deletion mutants was used

IG. 6. Raf is not detected in the Rem/14-3-3 protein complex fromEK 293 cells. (Top) Monolayers of HEK 293 cells were transiently

ransfected with either pKH3 alone (lanes 1, 2, 6, and 8) or pKH3-em (lanes 3, 4, 5, 7, and 9). Cells were then radiolabeled for 4 h inethionine- and cysteine-free medium containing 100 mCi/ml Trans

5S-label. Rem was immunoprecipitated from detergent-solubilizedell extracts with either anti-HA monoclonal antibody 12CA5 (lanes, 4, 6, 7, 8, and 9), preimmune IgG (lanes 1 and 3), or with protein-Sepharose beads alone (lane 5) as described under Experimentalrocedures. The resulting immunoprecipitates were washed exten-ively and subjected to SDS–PAGE followed by autoradiographylanes 1–5) or immunoblot analysis (lanes 6–9) as described underxperimental Procedures. An arrow denotes the position of proteinsnriched within the Rem immunoprecipitate. The migration of HA-agged Rem, 14-3-3 proteins (Rem, 14-3-3), and molecular weightass standards are indicated. (Bottom) HEK 293 cell lysates pre-

ared from pKH3 alone (2) or pKH3-Rem (1) transfected cells weremmunoprecipitated with anti-HA monoclonal antibody 12CA5 asescribed under Experimental Procedures. The washed immunopre-ipitates were subjected to SDS–PAGE on a 10% polyacrylamide gel,ransferred to nitrocellulose filters, and subjected to immunoblotnalysis with either anti-HA 12CA5 antibody (HA) or anti-Raf anti-ody (Raf ). Examples shown are representative of three to five sep-rate experiments.

o locate the 14-3-3 binding domain in the C-terminal v

xtended region of Rem. Thus, while deletion of resi-ues 282–297 from full-length Rem did not disrupt4-3-3 binding, deletion of residues 265–297 abolished4-3-3 binding completely (Fig. 3B). Based on theseutants, the final 31 amino acids of Rem play a critical

ole in 14-3-3 dimer binding, and the 17 amino acidegion encompassing residues 265–282 might be par-icularly important in directing this interaction. In-eed, of the three potential serine phosphorylationites in Rem that share some features with the previ-usly characterized 14-3-3 consensus recognition siteSXSXP (underline indicates phosphoserine) (see Fig.A), two of these sites are disrupted in the Rem1–265

eletion mutant. However, the mutation of these puta-ive phosphoserine residues to alanine failed to disrupthe in vitro association of Rem with 14-3-3s. Therefore,hile the extended C-terminal domain is clearly nec-ssary for 14-3-3 binding, clarification of the exactmino acid(s) necessary for 14-3-3 dimer interactionith phosphorylated Rem will require further study.he ability of Rem to interact with at least four mem-ers of the 14-3-3 family in vitro raises the possibilityhat Rem might associate with a large number of dif-erent 14-3-3 homo- and heterodimers in response toarious cellular signals. It is possible that stimulationf distinct protein kinases might phosphorylate Rem innique ways to facilitate its interaction with various4-3-3 dimers. While we have not shown that Rem ishosphorylated within these putative 14-3-3 bindingotifs, if it is assumed that phosphorylation of theseotifs is important for the observed 14-3-3 association,

hen the amount of 14-3-3 protein associated with Remould be determined by the activity of the kinase(s)

hat phosphorylate Rem, because 14-3-3 proteins arextremely abundant (12). Thus, the identification ofhe kinase involved in 14-3-3 binding is essential forlarifying the regulatory mechanism of 14-3-3 proteinssociation with Rem.A second intriguing finding of the current study was

he in vitro association of Rem and Rad, but not Rem2,ith 14-3-3s. Although members of the RGK Ras sub-

amily of GTPases share a high degree of sequenceomology, they have distinct tissue distributions andontain unique effector domains (1–4). This differences significant because the effector domains of the Ras-ike GTPases play a central role in defining their effec-or proteins interactions, and therefore greatly influ-nce their cellular function. The finding that 14-3-3roteins interact specifically with Rem and Rad but notith Rem2 in vitro suggests that 14-3-3 binding maylay a role in regulating the cellular activities of theseroteins, and might provide a mechanism for specify-ng their individual cellular roles.

Although we have not yet determined whether 14--3 binding directly alters the Rem GTPase cycle, pre-
ious studies have implicated 14-3-3 binding in regu-

lpiipltqswpmRad3

darepdfoacocmasse(tcstiSsmmMmRbrscvthi

labavawofwrt1upifiwTRti

R

1

1

1

1


ating the assembly of macromolecular signaling com-lexes. When epitope-tagged versions of Rem aremmunoprecipitated from labeled HEK 293 cell lysate,t is found in complex with a series of endogenousroteins in addition to 14-3-3 proteins (see Fig. 6, top,ane 4). It has been suggested that the recognition andethering of proteins using serine-phosphorylated se-uence motifs by 14-3-3 proteins is mechanisticallyimilar to that of the SH2-domain-containing proteins,hich utilize phosphorylated tyrosine residues to bringroteins into proximity (51). Thus, 14-3-3 proteinsight also play a structural or organizational role inem-controlled signal transduction pathways. Studiesre underway to identify these binding proteins and toetermine whether their interaction with Rem is 14--3 mediated.While the physiological role of Rem has yet to be

etermined, the finding of 14-3-3 protein interactionnd the results of expression studies using the Rem-elated GTPase, Kir, are quite intriguing. When over-xpressed in yeast, Kir leads to the induction ofseudohyphae, a development transition normally in-uced by nitrogen starvation (52). Genetic analysisrom this yeast system suggests that Kir acts upstreamf the p21-activated kinase (PAK) homologue STE20nd results in the specific activation of the MAP kinaseascade resulting in pseudohyphal development, with-ut activating the mating MAP kinase signaling cas-ade (50). In Saccharomyces cerevisiae, the 14-3-3 ho-ologs BMH1 and BMH2 have recently been shown to

ssociate with Ste20p. 14-3-3 binding to Ste20p is es-ential for the activation of the MAP kinase cascadeignaling pseudohyphal development, but it is not nec-ssary for viability or mating MAPK cascade signaling50). Therefore, 14-3-3 association might allow Ste20po bind additional cellular proteins that would serve asellular scaffolds or upstream activators. This is con-istent with the hypothesis that 14-3-3 proteins directhe organization and subcellular localization of signal-ng complexes (27). The yeast model in which PAK/te20p links the small Ras-like GTPase Cdc42 to tran-criptional and cytoskeletal events is paralleled inammalian cells, in which constitutively active PAKutants can activate the c-Jun N-terminal kinaseAP kinase cascade (53–55). These studies suggest aodel in which Kir, and perhaps other members of theGK family, might regulate cellular signaling cascadesy controlling the activity of STE20/PAK kinases. Theesults of the current study bolster this model anduggest that regulation may be achieved by the asso-iation of RGK family members and cellular kinasesia 14-3-3 dimers. Additional studies using Rem mu-ants which do not associate with 14-3-3 proteins mayelp to determine the presence of this putative signal-

ng pathway.1

Further study is necessary to elucidate the physio-ogical role of the 14-3-3 protein association with Remnd Rad in cellular signaling. In this regard it shoulde useful that Rem proteins which are unable to inter-ct with 14-3-3 proteins can be generated. This pro-ides an experimental basis for a detailed biochemicalnalysis of Rem function in vivo and to establishhether the 14-3-3 binding participates in Rem biol-gy. While we have not assigned a definite biologicalunction to Rem, here we report, an interaction of Remith 14-3-3 protein family members. This is the first

eport of the direct interaction between any member ofhe Ras and 14-3-3 gene families. Given that the Rem-4-3-3 complex preexists without obvious cellular stim-lation, we propose that 14-3-3 may serve as a scaffoldrotein for the docking of Rem with other members ofts signaling cascade. This idea is strengthened by thending that Rem apparently is found in associationith additional endogenous proteins in HEK293 cells.he recognition that Rem, and other members of theGK GTPase family, directly interact with 14-3-3 pro-

eins should facilitate further work on the role of Remn cellular signal transduction pathways.

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