Phosphorylation-Dependent Association of the Ras-Related GTP-Binding Protein Rem with 14-3-3 Proteins

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    Archives of Biochemistry and BiophysicsVol. 368, No. 2, August 15, pp. 401412, 1999Article ID abbi.1999.1316, available online at onchanging GTP for GDP, thereby triggering an activat-ing conformational change. Once activated, Ras-re-

    otide sequences for rat Rem2 have been submitted to theata Bank with Accession No. AF084464.k was supported by NIH Grant EY11231 and a granterican Heart Association, Kentucky Affiliate.

    correspondence should be addressed. Fax: 606-323-

    tions used: GST, glutathione S-transferase; HMK,dent protein kinase from heart muscle; SH2, Src homol-; GTPase, guanosine triphosphatase; GTPgS, guanosine

    triphosphate; PAGE, polyacrylamide gel electrophore-

    togen-activated protein; MEK, MAP kinase kinase/ERK kinase;PAK, p21-activated kinase; RGK, Rad, Gem, and Kir Ras subfamily;kb, kilobase pair(s); KSR, kinase suppressor of Ras; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase ki-nase; NP-40, Nonidet P-40; IPTG, isopropyl b-D-thiogalactoside;horylation-Dependent Association oinding Protein Rem with 14-3-3 Prot

    lin and D. A. Andres2

    t of Biochemistry, University of Kentucky College of Medicin

    ril 12, 1999, and in revised form May 21, 1999

    longs to a subfamily of Ras-related GTPasesdes Rad, Gem, and Kir. These proteins areong the Ras superfamily since their expres-

    der transcriptional regulation and they con-ct amino and carboxyl termini. To gain in-

    the cellular function of Rem, we have under-expression screen using a mouse embryo

    ary to identify Rem-interacting proteins andem interacts with a series of 14-3-3 isoforms

    nd z). Immunoprecipitation studies demon-interaction that is independent of the nucle-e of Rem. Rem is phosphorylated in vivo, andf Rem to 14-3-3z is abolished by pretreatingprotein phosphatase 1. Thus, the associationd 14-3-3z is phosphorylation-dependent. Ex-of the interaction between 14-3-3z and var-

    deletion mutants mapped a critical bindingC-terminus of Rem. Finally, we demonstratection of Rad but not the newly identified

    tein with 14-3-3 proteins. These results sug-14-3-3 may allow the recruitment of distinctthat participate in Rem-mediated signalion pathways. 1999 Academic Press

    the newest member of a growing subfamily ofTPases3 that includes Rad, Gem, and Kir

    (14). TfeatureGTPaseaminobe involysis, uterminito medisical CAlation (also subis mostest leveadminieral repfirst Rarepressincreasbetic paG1 phwhereaexpressologicamined,glucose

    Memabilityhiothreitol; HA, influenza hemagglutinin epitope; PCR,chain reaction; BSA, bovine serum albumin; MAP, mi-

    bME, b-merphate-buffer

    $30.00999 by Academic Presseproduction in any form reserved.he Ras-Relatedns1

    00 Rose Street, Lexington, Kentucky 40536-0084

    ese GTPases have several unique structuralhich are distinct from those of other Ras-likeThese include several nonconservative

    d substitutions within the regions known tod in guanine nucleotide binding and hydro-que effector domains, extended N- and C-nd a conserved C-terminal sequence thoughte membrane association but lacking the clas-X motif necessary to direct protein isopreny-6). The members of this Ras subfamily arect to transcriptional regulation. In mice, Remghly expressed in cardiac muscle and at mod-in the lung, kidney, and skeletal muscle. Theation of lipopolysaccharide results in a gen-ssion of Rem mRNA levels, making Rem thelike GTPase to be shown to be regulated by

    (1). Rad expression has been shown to bein the skeletal muscle of some type II dia-nts (2), and Gem expression is induced in the

    in mitogen-activated T lymphocytes (3),ir expression is induced in pre-B cell lines

    g BCR/ABL or v-abl (4). Although the physi-nction of these proteins remains to be deter-ad has been implicated as an inhibitor oftake in several cultured cell lines (7).

    rs of the Ras GTPase superfamily share thefunction as nucleotide-dependent molecularcaptoethanol; PP1, protein phosphatase 1; PBS, phos-ed saline; PMSF, phenylmethylsulfonyl fluoride.


  • lated GTPases are able to interact with and activatevarious effector proteins, which in turn stimulate sig-naling cascades that induce a variety of cellular re-sponses (810). In this way, Ras-related GTPases reg-ulate a wisignalingtein signaTo date, sbeen idenRem/Rad/are relateby regulacell growtcleocytopl(Rab and

    The likcades throcellular pfor proteinfollowingproteins w3-3 proteproteins tevolutionof these phave beensignalingAbl (16);suppressoBAD (19)(21); the ptor substr(24); mitsignal-reg(25); Cblgraphic sttains a lapropriatemanner (2homotypic14-3-3s mdifferent p

    Since thtaken a sfunction oidentificatteractingsued. Inrecombinalated bydent protelar probetential Rapproachteins (30)14-3-3 pr

    mouse embryo cDNA library capable of interactingwith Rem. Stable complexes between Rem and 14-3-3zwere reconstituted in HEK 293 cells. These complexeswere shown to contain at least two additional proteins,


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    402 FINLIN AND ANDRESde range of physiologically important cellularpathways by controlling the assembly of pro-ling complexes at specific cellular locations.ix subfamilies of the Ras superfamily havetified: Ras, Rho, Rab, Ran, ARF, and theGem proteins (11). These broad subfamiliesd by primary sequence relationships but alsotion of common cellular activities, includingh (Ras), cytoskeletal organization (Rho), nu-asmic transport (Ran), or vesicular transportARF).elihood that Rem may control signaling cas-ugh its specific association with a variety of

    roteins, stimulated our interest in searchings that interact with Rem. As described in thestudy, four members of the 14-3-3 family ofere identified as Rem-binding proteins. 14-

    ins constitute a family of abundant acidichat have been highly conserved throughout(12), reflecting the fundamental importanceroteins in cellular physiology. 14-3-3 proteinsshown to interact with a number of different

    proteins including, Raf (1315); Bcr and Bcr-polyoma middle tumor antigen (17); kinaser of Ras (KSR) (18); the Bcl family member; phosphotidylinositol 3-kinase (20); Cdc25latelet adhesion receptor (22); insulin recep-ate-1 (23); proteintyrosine phosphatase H1ogen-activated protein kinase/extracellularulated kinase kinase-1 (MEKK-1), -2, and -3(26); and protein kinase C (27). Crystallo-udies indicate that each 14-3-3 molecule con-rge cleft that can bind to a-helices from ap-substrates in a phosphoserine dependent8). This, together with their ability to formor heterotypic dimers (29), suggests that

    ight serve as molecular adapters, bringingroteins into close proximity.e original cloning of Rem, we have under-eries of studies designed to determine thef Rem in regulating cellular physiology. Theion and characterization of cellular Rem-in-proteins is one approach that has been pur-this study we describe the generation of ant Rem molecule that is readily phosphory-

    the catalytic subunit of heart cAMP-depen-in kinase, enabling us to use it as a molecu-to screen cDNA expression libraries for po-em-associated proteins, similar to theused to identify retinoblastoma-binding pro-. Using this strategy, we have identified fouroteins (e, h, u, and z) from a 14-day whole

    which mnalingcompledently.phosphvitro inmappindomainIn addiation owith 14the RGPases tteins. Tproteinthe regproteincellularcascade


    PlasmiAll site-dby DNAase (StrastructioncontainsBamHI/PPstI sitesites witsubcloninBamHI/XsubcloninBamHI/Xvectors acopies ofN-terminby subclothe BampCDNA3gated topKH3 wausing thethe full-lbe retrievtems Incof pZeroisolatedpKH3. Athe fourtHoweverusing Acating aproductsubseque

    4 B. be endogenous components of a Rem sig-mplex. Although both Raf-1 and Rem form

    with 14-3-3z, they appear to do so indepen-e have demonstrated that Rem is normallylated in vivo and that Rem binds 14-3-3z inhosphorylation-dependent manner. Deletion

    was used to locate a critical 14-3-3 bindingresidues 265282 at the C-terminus of Rem.

    n, we have demonstrated the in vitro associ-he Rem-related GTPase Rad but not Rem2-3 proteins. Thus, Rem and other members ofGTPase family are the first Ras-related GT-e shown to interact with 14-3-3 family pro-se results raise the possibility that 14-3-3ight function as scaffold-like molecules in

    ation of Rem activity by directing proteinteractions that control the composition orcation of Rem-mediated signal transduction


    Standard molecular biology techniques were used (31).cted mutations and PCR reaction products were verifieduence analysis. PCR was performed using Pfu polymer-ene, La Jolla, CA) according to the manufacturers in-o construct pGexKG HMK, a plasmid in which Remeart muscle kinase (HMK) site at its N-terminus, thefragment of pGexKG (32) was ligated into the BamHI/Gex2TK (Pharmacia) to expand the available restrictionthe polylinker. HMK Rem pGexKG was created by

    the BamHI/XhoI fragment of pRem Gex (1) into theI site of pGexKG HMK. pRem TrcHisA was created byhe BamHI/XhoI fragment of pRem express (1) into theI site of pTrcHisA (Invitrogen). Eukaryotic expression

    ing expression of recombinant proteins bearing threee influenza hemagglutinin (HA) epitope tag at theirwere created as follows. HA Rem pKH3 was generatedg the BamHI/EcoRI fragment of pRem pTrcHisA into/EcoRI site of pKH3 (33). To construct HA Remthe HindIII/EcoRI fragment of HA Rem pKH3 was li-dIII/EcoRI digested pcDNA3.1(1) (Invitrogen). HA Radeated by generating a PCR product with a 59 BamHI siteuse expressed sequence tag AA444424 (which containedth mouse Rad cDNA; the full-length cDNA sequence canusing GenBank Accession No. AF084466) (Genome Sys-he Rad PCR product was subcloned into the EcoRV siteitrogen), the BamHI/EcoRI fragment from this plasmidsubcloned to the same sites in pKH3 to create mRad

    nuscript describing the full cDNA cloning of rat Rem2,d newest member of the RGK family, is in preparation.4

    e Rem2 cDNA sequence can be retrieved from Genbankion No. AF084464. rRem2 pKH3 was created by gener-

    product with a 59 BamHI site, subcloning the PCRthe EcoRV site of pZero to create rRem2 pZero, and

    y ligating the BamHI/EcoRI fragment of rRem2 pZero

    lin and D. A. Andres, unpublished data.

  • into pKH3. The C-terminal deletion constructs HA Rem1282 pKH3,HA Rem1265 pCDNA3.1, and HA Rem1244 pCDNA3.1 were created bygenerating a PCR product with a 59 BamHI site and an in-frametermination codon and subcloning the blunt products into the pZeroEcoRV site. The Rem1282 BamHI/EcoRI fragment was subsequentlyligated intopKH3. Thesubcloned inHA Rem1265

    was createdimmediatelythis PCR prRem pCDNAsubcloned tointroduced twere subcloreleased and

    Generatiowas transfoGST-HMK-Raffinity chrowas cleavedHMK-Rem.(6000 Ci/mmTris pH 7.5,reaction waphosphate,albumin). Thof dialysis b10 mM GDPaliquots at 2

    Rem interin a variety(Novagen) wplate (150 mmm in size,bated overnincubated foimmediatelyEDTA, 2.5 mfilters werenitrocelluloswere immedFilters were4 h at 4C. Bprepared frobacteria werproduction wpelleted, rescell, and thetal blockingtaining 250extract, 10 m16 h at 4Cwashes of Hand exposedMore thanselected at rtential positPositive placDNAs werethe manufamids revealeither the z,remainder oexcision andgene specific

    contain one of the four initially identified 14-3-3 genes, while theremaining 8 plaques were shown to be false-positives. Thus, all of thepositive plaques were shown to encode 14-3-3 family proteins.

    In vitro analysis of 14-3-3 binding. The ability of recombinantRem and HMK phosphorylated recombinant Rem (see below) to bind


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    403CHARACTERIZATION OF Rem AND Rad AS 14-3-3 BINDING PROTEINSthe BamHI/EcoRI site of PKH3 to make HA Rem1282

    Rem1265 and HA Rem1244 BamHI/XhoI fragments wereto the BamHI/XhoI site of HA Rem pCDNA3.1 to makepcDNA3.1 and HA Rem1244 pcDNA3.1. HA Rem18297

    by generating a PCR product with a 59 BamHI siteupstream of codon 18. The BamHI/NheI fragment of

    oduct was then subcloned to the BamHI/NheI site of HA3.1. Rem1877Gex was generated by PCR and the productthe BamHI/SmaI site of pGexKG. 59 BamHI sites were

    o 14-3-3 z and 14-3-3 u by PCR, the resulting productsned into pZero, and the BamHI/EcoRI fragment was

    ligated into the same sites of pTrcHisA.n of 32P-labeled Rem. The plasmid HMK Rem pGexKGrmed into BL21(DE3) bacteria (Novagen). Recombinant

    em was expressed and purified by glutathioneagarosematography, and the glutathione S-transferase (GST)with thrombin as described previously (1), yielding

    HMK-Rem (10 mg) was incubated with 2.0 mCi [32P]ATPol, NEN) and 100 U HMK (Sigma) in 100 ml of 20 mM100 mM NaCl, 12 mM MgCl2 for 30 minutes on ice. The

    s stopped by addition of 400 ml of stop buffer (10 mM10 mM sodium pyrophosphate, 1 mg/ml bovine serume probe was dialyzed against four changes (50 ml each)

    uffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12 mM MgCl2,) to remove unincorporated label and stored in multiple

    20C.action cDNA library screen. Because Rem is expressedof tissues, a 14-day mouse embryo lEXlox cDNA libraryas selected for screening and plated at 40,000 plaques/m) in BL21(DE3) bacteria. Once plaques reached 0.51plates were overlaid with nitrocellulose filters and incu-ight at 4C. The plates were then placed at 37C andr another 4 h, and the primary filters were removedto Hyb75 (20 mM Hepes, pH 7.6, 75 mM KCl, 0.1 mMM MgCl2, 1 mM DTT, 0.05% NP-40) (30). Secondary

    generated by overlaying the plates with a second set ofe filters and incubating for 4 h at 37C. These filtersiately combined with the primary membranes in Hyb75.blocked in Hyb75 with 1% nonfat milk (Carnation) foracterial extract containing recombinant HMK-GST wasm BL21DE3 cells transformed with pGexKG HMK. Thee grown at 37C in LB medium to an A600 5 0.6. Proteinas induced with 0.5 mM IPTG for 4 h. The bacteria were

    uspended in Hyb75, and broken using a French pressure100,000g cleared supernatant was used as a supplemen-agent. Library filters were incubated with Hyb75 con-

    mM KCl, 1% nonfat milk, 400 mg/ml HMK-GST bacterialM GDP, and 200,000 cpm/ml [32P]HMK-Rem probe forwith shaking. Filters were washed with four 100-ml

    yb75 supplemented with 10 mM GDP for 30 min, dried,to Kodak X-OMAT film for 4 h at room...


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