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

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<ul><li><p>Phosp f tGTP-B ei</p><p>B. S. FinDepartmen e, 8</p><p>Received Ap</p><p>Rem bethat incluunique amsion is untain distinsight intotaken ancDNA librfind that R(e, h, u, astrate anotide statbinding oRem withof Rem anaminationious Remsite to thethe interaRem2 progest thatproteinstransduct</p><p>Rem isRas-like G</p><p>hs ws.</p><p>acilveni, aatA</p><p>5,jehilsstrres-ionedtie</p><p>ases Kin</p><p>l fuRup</p><p>beto</p><p>switches. In the GDP-bound form these proteins areinactive, but respond to extracellular stimuli by ex-</p><p>The nucleGenebank D</p><p>1 This worfrom the Am</p><p>2 To whom1037.</p><p>3 AbbreviacAMP-depenogy 2 domain59-3-O-(thio)sis; DTT, ditpolymerase</p><p>0003-9861/99Copyright 1All rights of r</p><p>Archives of Biochemistry and BiophysicsVol. 368, No. 2, August 15, pp. 401412, 1999Article ID abbi.1999.1316, available online at http://www.idealibrary.com onchanging GTP for GDP, thereby triggering an activat-ing conformational change. Once activated, Ras-re-</p><p>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.</p><p>correspondence should be addressed. Fax: 606-323-</p><p>tions used: GST, glutathione S-transferase; HMK,dent protein kinase from heart muscle; SH2, Src homol-; GTPase, guanosine triphosphatase; GTPgS, guanosine</p><p>triphosphate; PAGE, polyacrylamide gel electrophore-</p><p>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</p><p>lin and D. A. Andres2</p><p>t of Biochemistry, University of Kentucky College of Medicin</p><p>ril 12, 1999, and in revised form May 21, 1999</p><p>longs to a subfamily of Ras-related GTPasesdes Rad, Gem, and Kir. These proteins areong the Ras superfamily since their expres-</p><p>der transcriptional regulation and they con-ct amino and carboxyl termini. To gain in-</p><p>the cellular function of Rem, we have under-expression screen using a mouse embryo</p><p>ary to identify Rem-interacting proteins andem interacts with a series of 14-3-3 isoforms</p><p>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-</p><p>deletion mutants mapped a critical bindingC-terminus of Rem. Finally, we demonstratection of Rad but not the newly identified</p><p>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</p><p>the newest member of a growing subfamily ofTPases3 that includes Rad, Gem, and Kir</p><p>(14). TfeatureGTPaseaminobe involysis, uterminito medisical CAlation (also subis mostest leveadminieral repfirst Rarepressincreasbetic paG1 phwhereaexpressologicamined,glucose</p><p>Memabilityhiothreitol; HA, influenza hemagglutinin epitope; PCR,chain reaction; BSA, bovine serum albumin; MAP, mi-</p><p>bME, b-merphate-buffer</p><p>$30.00999 by Academic Presseproduction in any form reserved.he Ras-Relatedns1</p><p>00 Rose Street, Lexington, Kentucky 40536-0084</p><p>ese GTPases have several unique structuralhich are distinct from those of other Ras-likeThese include several nonconservative</p><p>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</p><p>(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</p><p>in mitogen-activated T lymphocytes (3),ir expression is induced in pre-B cell lines</p><p>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).</p><p>rs of the Ras GTPase superfamily share thefunction as nucleotide-dependent molecularcaptoethanol; PP1, protein phosphatase 1; PBS, phos-ed saline; PMSF, phenylmethylsulfonyl fluoride.</p><p>401</p></li><li><p>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</p><p>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</p><p>Since thtaken a sfunction oidentificatteractingsued. Inrecombinalated bydent protelar probetential Rapproachteins (30)14-3-3 pr</p><p>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,</p><p>igcoxesW</p><p>orya pgto</p><p>tiof t-3Ko bhe</p><p>s mulinlo</p><p>s.</p><p>ME</p><p>ds.ireseqtags. Ta hstI</p><p>of phinghog thollowth</p><p>usninHI</p><p>.1,Hins crmo</p><p>enged</p><p>.). T(Invandma</p><p>h an, thcessPCRintontl</p><p>Fin</p><p>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</p><p>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-</p><p>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</p><p>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</p><p>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-</p><p>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</p><p>which mnalingcompledently.phosphvitro inmappindomainIn addiation owith 14the RGPases tteins. Tproteinthe regproteincellularcascade</p><p>EXPERI</p><p>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</p><p>4 B. S.ht be endogenous components of a Rem sig-mplex. Although both Raf-1 and Rem form</p><p>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</p><p>was used to locate a critical 14-3-3 bindingresidues 265282 at the C-terminus of Rem.</p><p>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</p><p>ation of Rem activity by directing proteinteractions that control the composition orcation of Rem-mediated signal transduction</p><p>NTAL PROCEDURES</p><p>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</p><p>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</p><p>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</p><p>nuscript describing the full cDNA cloning of rat Rem2,d newest member of the RGK family, is in preparation.4</p><p>e Rem2 cDNA sequence can be retrieved from Genbankion No. AF084464. rRem2 pKH3 was created by gener-</p><p>product with a 59 BamHI site, subcloning the PCRthe EcoRV site of pZero to create rRem2 pZero, and</p><p>y ligating the BamHI/EcoRI fragment of rRem2 pZero</p><p>lin and D. A. Andres, unpublished data.</p></li><li><p>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</p><p>was createdimmediatelythis PCR prRem pCDNAsubcloned tointroduced twere subcloreleased and</p><p>Generatiowas transfoGST-HMK-Raffinity chrowas cleavedHMK-Rem.(6000 Ci/mmTris pH 7.5,reaction waphosphate,albumin). Thof dialysis b10 mM GDPaliquots at 2</p><p>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</p><p>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.</p><p>In vitro analysis of 14-3-3 binding. The ability of recombinantRem and HMK phosphorylated recombinant Rem (see below) to bind</p><p>otel-doreased</p><p>d rl, 1upehy</p><p>ecoed</p><p>lyze50muwas, Sovee usrev</p><p>2g s</p><p>mMterharitheteasn</p><p>e pple</p><p>biliellenobusidiluory</p><p>emild</p><p>HMl2,ionlue,</p><p>peas-temly S</p><p>is oe m% (r toe trmiRe</p><p>chn48BSrob4</p><p>ed</p><p>403CHARACTERIZATION OF Rem AND Rad AS 14-3-3 BINDING PROTEINSthe BamHI/EcoRI site of PKH3 to make HA Rem1282</p><p>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</p><p>by generating a PCR product with a 59 BamHI siteupstream of codon 18. The BamHI/NheI fragment of</p><p>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</p><p>o 14-3-3 z and 14-3-3 u by PCR, the resulting productsned into pZero, and the BamHI/EcoRI fragment was</p><p>ligated into the same sites of pTrcHisA.n of 32P-labeled Rem. The plasmid HMK Rem pGexKGrmed into BL21(DE3) bacteria (Novagen). Recombinant</p><p>em was expressed and purified by glutathioneagarosematography, and the glutathione S-transferase (GST)with thrombin as described previously (1), yielding</p><p>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</p><p>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)</p><p>uffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12 mM MgCl2,) to remove unincorporated label and stored in multiple</p><p>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</p><p>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</p><p>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-</p><p>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</p><p>yb75 supplemented with 10 mM GDP for 30 min, dried,to Kodak X-OMAT film for 4 h at room...</p></li></ul>

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