RACK1 is involved in endothelial barrier regulation via its two novel interacting partners

RESEARCH Open Access RACK1 is in ol ed in endo helial ba ie egula ion ia i s wo no el in e ac ing pa ne s Ani a Bo a kó, Pál Ge gely and Csilla Cso os * Abs ac Backg ound: RACK1, ecep o o ac i a ed p o ein kinase C, se es as an ancho in mul iple signaling pa hways. TIMAP, TGF-βinhibi ed memb ane-associa ed p o ein, is mos abundan in endo helial cells wi h a egula o y e ec on he endo helial ba ie unc ion. The in e ac ion o TIMAP wi h p o ein phospha ase 1 (PP1cδ) was cha ac e ized, ye li le is known abou i s u he pa ne s. Resul s: We iden i ied wo no el in e ac ing pa ne s o RACK1, namely, TGF-βinhibi ed memb ane-associa ed p o ein, TIMAP, and a nesyl ans e ase. TIMAP is mos abundan in endo helial cells whe e i is in ol ed in he egula ion o he ba ie unc ion. WD1-4 epea s o RACK1 we e iden i ied as c i ical egions o he in e ac ion bo h wi h TIMAP and a nesyl ans e ase. Phospho yla ion o TIMAP by ac i a ion o he cAMP/PKA pa hway educed he amoun o TIMAP-RACK1 complex and enhanced ansloca ion o TIMAP o he cell memb ane in ascula endo helial cells. Howe e , bo h memb ane localiza ion o TIMAP and ansendo helial esis ance we e a enua ed a e RACK1 deple ion. Fa nesyl ans e ase, he enzyme esponsible o p enyla ion and consequen memb ane localiza ion o TIMAP, is p esen in he RACK1-TIMAP complex in con ol cells, bu i does no co-immunop ecipi a e wi h TIMAP a e RACK1 deple ion. Conclusions: T ansien pa allel linkage o TIMAP and a nesyl ans e ase o RACK1 could ensu e p enyla ion and anspo o TIMAP o he plasma memb ane whe e i may a end in main aining he endo helial ba ie as a phospha ase egula o . Keywo ds: Endo helial cell, P enyla ion, RACK1, TIMAP Backg ound The ascula endo helium unc ions as a semi-pe meable ba ie be ween blood and he in e s i ium. Endo helial cell (EC) ba ie egula ion is unde in ense in es iga ion, since EC ba ie dys unc ion is a well-known ea u e o acu e lung inju y (ALI) and i s mo e se e e o m, acu e espi a o y dis ess synd ome (ARDS) [1]. Ba ie enhan- cing o p o ec ing p ocesses a e no explo ed in de ails ye , howe e , se e al s udies indica e he signi icance o dephospho yla ion o key cy oskele al/memb ane asso- cia ed a ge s by speci ic p o ein phospha ases [2]. TIMAP (TGF-βinhibi ed memb ane-associa ed p o ein) p o ein has been conside ed as a membe o he MYPT (myosin phospha ase a ge ing) amily o he egula o y subuni s o p o ein phospha ase 1 (PP1) based on i s s uc u al ea u es [3]. TIMAP and MYPT3 a e he mos closely ela ed membe s wi hin he amily. Bo h p o eins con ain anky in epea s, a PP1 ca aly ic subuni (PP1c) binding mo i and a C- e minal p enyla ion mo i ; he la e one media es hei associa ion wi h he plasma memb ane [3]. A bipa i e nuclea localiza ion signal (NLS) is also p esen in TIMAP; acco dingly, i was de ec ed in he nucleus o ascula endo helial cells [4], bu i s signi icance is no known ye . In iew o he ac ha TIMAP mRNA syn hesis is down- egula ed by TGF-β1 [3], he ansc ip ional ep ession o TIMAP could be an impo an componen o he TGF-β1 pa h- way, as well as apop osis and endo helial ba ie in eg i y. TIMAP is mos abundan in endo helial cells, ye li le is known abou i s exac unc ion and i s in e ac ing pa - ne s. We ha e demons a ed speci ic p o ein-p o ein in e ac ion be ween TIMAP and PP1cδ(a.k.a. PP1cβ); and he e ec o TIMAP phospho yla ion on his in e - ac ion was also cha ac e ized [4,5]. S udies made on TIMAP-deple ed human pulmona y a e y endo helial * Co espondence: [email p o ec ed] Depa men o Medical Chemis y, Uni e si y o Deb ecen Medical and Heal h Science Cen e , Egye em é 1, Deb ecen, H 4032, Hunga y © 2013 Bo a kó e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 h p://www.biosignaling.com/con en /11/1/2 cell (HPAEC) monolaye s indica ed ha TIMAP has a posi i e egula o y e ec on he endo helial ba ie unc- ion. In he absence o TIMAP he e ec s o ba ie unc- ion comp omising agen s ( h ombin and nocodazole) we e enhanced and he e ec s o ba ie unc ion p o- ec ing agen s (sphingosine-1-phospha e and ATP) we e a enua ed [4]. Ou wo k e ealed ha he ERM (ez in- adixin-moesin) p o eins, which connec ac in ilamen s o he plasma memb ane, a e in e ac ing p o ein pa ne s o TIMAP and hey we e iden i ied as TIMAP-PP1c subs a es [4,5]. We also showed he in ol emen o TIMAP in PKA-media ed ERM dephospho yla ion as pa o endo helial cell ba ie p o ec ion by TIMAP [4,5]. The non-in eg in laminin ecep o 1 (LAMR1), which is in ol ed in he egula ion o cell mo ili y, was also desc ibed o in e ac wi h TIMAP. They co-localize a he plasma memb ane o endo helial cells and TIMAP egula es he dephospho yla ion o LAMR1 by PP1c [6]. Se e al po en ial TIMAP-binding p o eins we e iden i- ied by bac e ial wo-hyb id sc eening. Among hem a e he cys eine and glycine ich p o ein 1 and he euka yo ic ansla ion elonga ion ac o 2, which a e bo h in ol ed in he o ganiza ion o he ac in cy oskele on. Howe e , hese in e ac ions in mammalian cells ha e no been e i- ied ye [7]. In a hun o new TIMAP-binding pa ne s, we ound ha RACK1 ( ecep o o ac i a ed kinase 1, a.k.a. guan- ine nucleo ide-binding p o ein subuni be a-2-like 1, GNB2L1, o human lung cance oncogene 7 p o ein) in e ac s wi h TIMAP in pulmona y a e y endo helial cells. RACK1 is a sca olding/ancho ing p o ein ha con- ains se en T p-Asp (WD) epea s p edic ed o o m a se en-bladed p opelle s uc u e. The e o e RACK1 is capable o in e ac simul aneously wi h se e al signaling molecules [8], sugges ing he possibili y o mul iple p o- ein in e ac ion su aces. I has been named by i s bind- ing o ac i a ed PKC [9], bu se e al o he in e ac ing pa ne s we e also iden i ied [10-14]. Upon ac i a ion o PKC wi h PMA, RACK1 co-localizes wi h he S c y o- sine kinase a he plasma memb ane [15]; and RACK1 unc ions as a subs a e, as well as a binding pa ne and inhibi o o he kinase [15,16]. Recen ly, i was also demons a ed ha RACK1 is a co e componen o he euka yo ic 40S ibosomal subuni [17-19], localized on he head egion close o he mRNA exi channel, sugges - ing a physical link be ween he euka yo ic ibosome and cell signaling pa hways in i o [20]. In acco d wi h i s s uc u al ea u es, all hese ecen indings imply he in- ol emen o RACK1 in nume ous signaling pa hways. He e, we epo and cha ac e ize he in e ac ion o RACK1 wi h TIMAP, a p o ein in ol ed in he egula ion o endo helial ba ie unc ion, and wi h a nesyl ans- e ase, he enzyme ca alyzing p enyla ion o TIMAP in endo helial cells. We show he signi icance o hese in e - ac ions in he p enyla ion o TIMAP, consequen ly in i s a ic o he plasma memb ane. Resul s RACK1 binds TIMAP-PP1c complex in endo helial cells To iden i y new in e ac ing pa ne s o TIMAP in endo he- lial cells, GST pull-down assay was pe o med. BPAEC lys- a e was incuba ed wi h GST- agged ecombinan ull leng h TIMAP p o ein immobilized on glu a hione Sepha ose. GST p o ein incuba ed wi h he cell lysa e and GST-TIMAP incuba ed wi h he lysis bu e we e used as nega i e con- ols. A e he necessa y washing s eps he elu ed p o eins we e sepa a ed on SDS-PAGE s ained wi h BlueSil e solu- ion and he pa e n o he p o ein bands in he h ee sam- ples we e compa ed. An ex a band a abou 34 kDa was iden i ied in he sample o GST-TIMAP incuba ed wi h BPAEC lysa e (Addi ional ile 1: Figu e S1). I was cu om he gel and was u he analyzed by LC-MS/MS (D . Janáky, Uni e si y o Szeged). Bo ine RACK1 (Gene: GNB2L1, Ac- cession numbe : P63243) was iden i ied using Swissp o and Unip o TREMBL da abase. This esul was con i med by Wes e n blo analysis o he pull-down samples using an i- RACK1 an ibody (Figu e 1A). Speci ic band o RACK1 can be seen in he o al cell lysa e (posi i e con ol) and in he GST-TIMAP sample which was incuba ed wi h he cell lys- a e, howe e , no RACK1 signal was de ec able in he wo nega i e con ol samples sugges ing a speci ic in e ac ion be ween TIMAP and RACK1. Immunop ecipi a ion expe imen s we e u ilized o e i y he in e ac ion o he endogenous p o eins in endo helial cells. RACK1 was shown o be p esen in he immunop e- cipi a e (IP) o TIMAP and ice e sa, TIMAP co- immunop ecipi a ed wi h RACK1 (Figu e 1B). Sinceweha eshownea lie ha TIMAPhasas ong in e ac ion wi h PP1cδ[4,5], he p esence o PP1cδin he TIMAP-RACK1 complexes we e also examined. As i was expec ed, he phospha ase was p esen in bo h he RACK1 and he TIMAP IP complexes (Figu e 1C). To es whe he RACK1 binds PP1c di ec ly wi hou he a endance o TIMAP, immunop ecipi a ion was made wi h RACK1 an i- body om HeLa cells ha do no exp ess endogenous TIMAP [3]. No in e ac ion was de ec ed be ween RACK1 and PP1cδin HeLa cells (Figu e 1C), he e o e one may conclude ha RACK1 in e ac s wi h PP1cδ ia TIMAP. To u he e i y his esul , RACK1 was immunop ecipi a ed om con ol, non-siRNA and TIMAP speci ic siRNA ans ec ed EC. PP1cδwas no de ec ed in he RACK1 IP om TIMAP deple ed cells (Figu e 1D). In addi ion, mam- malian cons uc s we e c ea ed o exp ess ecombinan wild ype and unca ed TIMAP. The unca ed o m does no con ain he PP1c binding mo i , as he i s 68 amino acids a e dele ed; consequen ly i is expec ed no o bind he phospha ase. HeLa we e ans ec ed wi h emp y Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 2 o 14 h p://www.biosignaling.com/con en /11/1/2 pEGFP, wild ype TIMAP/pEGFP (GFP-TIMAP w ) o unca ed TIMAP/pEGFP (GFP-TIMAP ΔPP1c) plasmids and GFP o he endogenous RACK1 we e immunop ecipi- a ed. The IP complexes we e p obed o RACK1, GFP and PP1cδ(Figu e 1E). Al hough HeLa con ains PP1cδ, he phospha ase was no p esen in he RACK1 IP excep in wild ype TIMAP o e -exp essing HeLa. Mo eo e , PP1cδ was no de ec able wi h he unca ed o m o TIMAP in he RACK1-GFP-TIMAP ΔPP1c complex, only in he RACK1-GFP-TIMAP w complex. These esul s imply ha PP1cδis p esen in he la e complex due o i s in e ac ion wi h TIMAP, bu he e is no di ec binding be ween PP1cδ and RACK1; u he mo e, he p esence o his phospha ase is no a equi emen o TIMAP-RACK1 in e ac ion. Mapping he TIMAP-RACK1 in e ac ion domains Se e al dele ion mu an s o TIMAP we e c ea ed o iden- i y i s domains conce ned in he RACK1 in e ac ion (Figu e 2A). The in e ace be ween bac e ially exp essed TIMAP and endogenous RACK1 was mapped by GST pull-down assay. Su p isingly, RACK1 was able o bind bo h o he N- e minal egion (TIMAP 1–290) o TIMAP con aining he nuclea localiza ion signal (NLS), he PP1c- binding mo i and he i e ANK epea s, as well as o he C- e minal egion (TIMAP 291–567) wi h he ea lie iden i ied PKA and GSK3βphospho yla ion si es [21] and he C- e minal CAAX p enyla ion mo i . The la e was excluded as a signi ican egion o TIMAP in he in e - ac ion, since he C- e minal agmen missing he CAAX box (TIMAP 291–563) did no bind less RACK1 han TIMAP 291–567. To u he speci y he in e ac ing egion in he N- e minal sec ion o he p o ein, addi ional sho e ecombinan s we e es ed. When he N- e minal agmen was sho ened (TIMAP 1–165) we s ill could de ec bind- ing. The mu an s con aining only ANK4-5 and a egion wi h uniden i ied unc ion (TIMAP 165–290) o ANK1-3 (TIMAP 67–165) did no bind o RACK1. The e o e i was concluded ha none o he ANK epea s a e conce ned. The e y N- e minal egion o TIMAP (spanning amino acids 1–34) do no a ec he binding ei he . The sho e- gion o he po en ial NLS, howe e , appea ed o be essen ial by he compa ison o he binding abili y o TIMAP 35–165 and TIMAP 52–165 o endogenous RACK1 as he only di - e ence be ween hese wo agmen s is he p esence o ab- sence o he NLS mo i , espec i ely. The β-p opelle s uc u e o RACK1 due o i s se en WD epea s o e s mul iple docking si es o se e al in e - ac ions. The associa ion o na i e TIMAP o bac e ially exp essed ull leng h GST-RACK1, N- e minal (1–180) o C- e minal (137–317) GST-RACK1 unca ed o ms we e s udied in GST pull-down assays (Figu e 2B). Ou esul s clea ly indica e ha only he N- e minal hal o RACK1 is in ol ed in he RACK1-TIMAP in e ac ion. Fo skolin ea men a enua es he RACK1- TIMAP in e ac ion RACK1 ( e iewed in [22]) and TIMAP [5,21] a e ecog- nized o be ela ed o se e al kinases, and upon he Figu e 1 RACK1 in e ac s wi h PP1cδ ia TIMAP. (A): Bac e ially exp essed glu a hione S- ans e ase (GST) and GST- agged wild- ype TIMAP we e loaded on o glu a hione-Sepha ose as desc ibed in Ma e ials and Me hods. A e a washing s ep he esin samples we e incuba ed wi h BPAEC lysa e (CL) o cell lysis bu e (LB). Non-binding p o eins we e washed ou and he bound p o eins we e elu ed wi h 10 mM glu a hion. Wes e n blo p obed wi h RACK1 speci ic an ibody (A) o he endo helial cell lysa e (CL) and he elu ed ac ions a e he pull-down a e shown. (B,C): RACK1 o TIMAP was immunop ecipi a ed om lysa es o BPAEC (B) and BPAEC o HeLa (C) cells as desc ibed in Ma e ials and Me hods. IP complexes we e p obed o TIMAP and RACK1 (B) o PP1cδ(C). CL: cell lysa e, Ø AB: con ol o IP om BPAEC wi hou he addi ion o an ibody. (D,E): RACK1 o GFP was immunop ecipi a ed om non ans ec ed (CL), non-siRNA, TIMAP speci ic siRNA (si TIMAP), pEGFP-C1 (GFP), pEGFP-C1 TIMAP WT (GFP-Tw ) o pEGFP-C1 TIMAPΔpp1c (GFP-TΔ) ans ec ed HeLa cell lysa es. IP complexes we e p obed o GFP, RACK1 o PP1cδ. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 3 o 14 h p://www.biosignaling.com/con en /11/1/2 ac i a ion o ce ain kinases changes in hei p o ein- p o ein in e ac ions we e desc ibed. Namely, PKCs and RACK1 mu ually in luence each o he , bu RACK1 may pa icipa e in he cAMP/PKA pa hway as well [10,12,23]. Recen esul s indica e ha TIMAP is a a ge o PKA-p imed GSK-3βmedia ed phospho yla ion on si es Se 337 and Se 333, espec i ely [5,21]. Thus we nex es ed he e ec o he ac i a ion o PKC and PKA on he TIMAP-RACK1 in e ac ion challenging EC wi h PMA and o skolin, espec i ely. The a enua i e o es o a i e consequences o PMA o o skolin ea men on he in e ac ion we e es ablished by GST pull-down assays i s . Equal amoun s o bac e - ially exp essed GST-TIMAP o GST-RACK1 we e loaded on o glu a ion Sepha ose 4B as desc ibed in Ma e ials and Me hods and un ea ed, o skolin o PMA chal- lenged endo helial cell lysa es we e added o he esin. Bound p o eins in he elua es we e analyzed by Wes e n blo (Figu e 3A). The amoun o RACK1-TIMAP com- plex was conside ably lowe a e he ac i a ion o he cAMP/PKA pa hway ( o skolin), on he o he hand, PMA ea men o EC had no signi ican e ec . These indings we e u he s eng hened wi h endogenous p o- eins only, when IP complexes o RACK1 and TIMAP d awn om EC a e he same ea men s we e analyzed by Wes e n blo (Figu e 3B). T unca ed wild ype, S333A/S337A phospho yla ion de icien , and S333D/ S337D phosphomimic mu an s o a TIMAP agmen spanning amino acids 331–567 we e o e exp essed in E. coli and we e u ilized in pull-down expe imen s. Consis - en wi h he abo e desc ibed indings, he amoun o RACK1 bound o he phospomimic TIMAP agmen was dec eased compa ed o he amoun o RACK1 bound o wild ype TIMAP o he phospho yla ion de i- cien agmen (Addi ional ile 2: Figu e S2). These da a sugges ha he phospho yla ion s a e o TIMAP may be an impo an ac o in i s in e ac ion wi h RACK1. Ac i a ion o he cAMP/PKA pa hway a ec s localiza ion o TIMAP TIMAP localizes o he cell memb ane and i is also p esen in he nucleus and in he cy oplasm su ounding he nucleus in HPAEC monolaye [4]. We in es iga ed whe he he RACK1-TIMAP complex o ma ion has any e ec on he subcellula localiza ion o TIMAP. To modu- la e he in e ac ion, HPAEC monolaye s we e subjec ed o agen s a ec ing he phospho yla ion le el o TIMAP and he subcellula localiza ion was de ec ed by immuno luo - escence s udies o he monolaye s o by Wes e n blo o subcellula ac ions (Figu e 4A,B). Con ocal images on Figu e 4A show ha he applied e ec o s did no change he cy oplasmic localiza ion o RACK1 (Figu e 4A b,e,h,k). On he o he hand, upon o skolin ea men , he amoun o nuclea TIMAP dec eased pa allel wi h i s mo e p o- nounced appea ance in he cell memb ane (Figu e 4A d) compa ed o he un ea ed sample (Figu e 4A a). When cells we e p e ea ed wi h a PKA inhibi o , H89, no ansloca ion o TIMAP o he cell memb ane was obse ed upon o skolin challenge, p o ing he in ol e- men o PKA ac i i y (Addi ional ile 3: Figu e S3). Since PKA phospho yla ion o TIMAP on Se 337 p imes i s GSK3βphospho yla ion on Se 333 [5,21], AR-A014418, a selec i e GSK-3βinhibi o [24], was employed alone o as Figu e 2 Domain mapping o TIMAP-RACK1 in e ac ion. (A): GST-TIMAP pull-down o endogenous RACK1. GST, ecombinan GST- TIMAP WT o addi ional GST-TIMAP agmen s (depic ed in he uppe pa o panel A) we e loaded on o glu a hione-Sepha ose as desc ibed in Ma e ials and Me hods. The immobilized p o ein samples we e incuba ed wi h BPAEC lysa e. Wes e n blo o he pull- down elua es p obed wi h an i-RACK1 an ibody is shown. CL: o al cell lysa e. (B): GST-RACK1 pull-down o endogenous TIMAP. GST, ecombinan RACK1 WT o GST-RACK1 agmen s (depic ed in he uppe pa o panel B) we e es ed in pull-down assay. The immobilized samples we e incuba ed wi h BPAEC lysa e. Wes e n blo o he pull-down elua es was p obed wi h an i-TIMAP an ibody. Rep esen a i e da a o a leas 3 independen expe imen s a e shown. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 4 o 14 h p://www.biosignaling.com/con en /11/1/2 p e ea men be o e addi ion o o skolin o p e en PKA p imed phospho yla ion o TIMAP by GSK-3β. Wi hou o skolin, no TIMAP was de ec ed in he plasma mem- b ane when GSK-3βwas inhibi ed (Figu e 4A g); also, he e ec o o skolin was s ongly a enua ed in he p esence o AR-A014418 (Figu e 4A j). Me ged images indica e co- localiza ion o RACK1 and TIMAP in he egion o cy o- plasm ha is a he close o he nucleus in con ol and GSK-3βinhibi ed cells cells (Figu e 4A c,i,l), bu co- localiza ion was no de ec able in he cells ea ed exclu- si ely wi h o skolin (Figu e 4A ). Memb ane and nuclea ac ions o HPAEC we e iso- la ed by cell ac iona ion as desc ibed in Ma e ials and Me hods and he amoun o TIMAP in he ac ions was de ec ed by Wes e n blo (Figu e 4B). Pa allel wi h he esul s o he immuno luo escen s aining, he amoun o TIMAP inc eased in he memb ane ac ion a e o sko- lin, bu i was signi ican ly lowe ed in he p esence o GSK-3βinhibi o compa ed o he con ol. Fo skolin chal- lenge in GSK-3βinhibi ed cells caused signi ican inc ease in he TIMAP le el in he memb ane ac ion compa ed o he ex emely ain signal ound in he same ac ion o cells ea ed only wi h he kinase inhibi o . Fu he mo e, Wes e n blo analysis o he nuclea ac ions, as expec ed, demons a ed opposi e ends o he changes. The leas amoun o TIMAP was de ec ed in he nuclea ac ion o he o skolin ea ed cells, while inhibi ion o GSK-3β caused a la ge inc ease, howe e , o skolin signi ican ly mode a ed his e ec o he inhibi o . The e was no signi ican change in he amoun o RACK1 in TIMAP IP o GSK-3βinhibi ed cells compa ed o con ol. As expec ed, less RACK1 was associa ed o TIMAP a e o skolin challenge, bu no e ec o o skolin was de ec able a e AR-A014418 p e ea men o EC (Figu e 4C). These esul s sugges ha in e ac ion o TIMAP wi h RACK1 may exis in he cy oplasm o EC and i is a ec ed by double phospho yla ion (PKA and GSK-3β) o TIMAP. E ec o RACK1 deple ion on TIMAP RACK1 was deple ed in HPAEC cells using silencing RNA duplexes speci ic o RACK1 (GNB2L1). The e iciency o silencing was con i med bo h a mRNA and p o ein le el o RACK1 by RT-PCR and Wes e n blo (Figu e 5A,B). We de ec ed abou 70-80% and 50% dec ease in he mRNA and p o ein le el o RACK1, espec i ely, in he deple ed cells compa ed o he con ol o non silencing RNA ans ec ed cells. P ime s o B55, egula o y subuni o p o ein phospha ase 2A, we e used as endogenous con- ol o RT-PCR. In he same se o expe imen s he mRNA and p o ein le el o TIMAP we e e alua ed as well; in e - es ingly, bo h inc eased in he RACK1 deple ed HPAEC (Figu e 5A,B). This obse a ion may sugges ha RACK1 could be in ol ed in he egula ion o TIMAP ansc ip- ion, bu elabo a ion o his assump ion would equi e u - he examina ion. Immuno luo escen s aining and Wes e n blo analysis o memb ane ac ion o RACK1 siRNA ans ec ed HPAEC e ealed loss o TIMAP in he plasma memb ane (Figu e 5C,D). This o e s ano he plausible in e p e a ion o he inc eased amoun o TIMAP, namely, RACK1 silenced cells simply y o compensa e o he lowe ed memb ane localized TIMAP. Figu e 3 TIMAP-RACK1 in e ac ion is a enua ed by he cAMP/PKA pa hway. (A) GST, ull-leng h GST-TIMAP (uppe pa ) o GST-RACK1 (lowe pa ) we e immobilized on glu a hione-Sepha ose and incuba ed wi h cell lysa es o non ea ed (c ), o skolin (50 μM o 30 min) (FRSK) o PMA (1 μM o 30 min) ea ed BPAEC. The elu ed p o eins we e es ed by Wes e n blo using an i-RACK1 and an i-TIMAP an ibodies. (B) Endogenous TIMAP o RACK1 was immunop ecipi a ed om BPAEC lysa es a e he same ea men s desc ibed o panel A. IP complexes we e p obed o TIMAP and RACK1. Shown a e ep esen a i e da a o means ± SE om a leas 3 independen expe imen s. P o ein le els we e quan i ied by densi ome ic analysis. Elu ed p o eins we e no malized agains o al p o ein le els. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 5 o 14 h p://www.biosignaling.com/con en /11/1/2 Nex i was es ed whe he he ba ie unc ion o EC monolaye s could be a ec ed by RACK1 deple ion as a consequence o loss o TIMAP in he plasma memb ane. Endo helial ba ie o ma ion (a achmen and sp eading o EC) o con ol and RACK1 deple ed HPAEC we e ol- lowed by ECIS measu emen (Figu e 6A). The numbe o con ol and siRNA ans ec ed cells inocula ed in ECIS wells was iden ical; also he e was no no able di e ence in he cell densi y o he samples and no dead cells we e obse ed in he wells a e he ECIS measu emen s. The impedance alues measu ed a 1 h a e he s a o he measu emen we e signi ican ly lowe o he RACK1 silenced sample. Du ing he 20 hou s o he expe imen , his di e ence became mo e p onounced, implying ha he o ma ion o endo helial ba ie was damaged in he ab- sence o RACK1. In addi ion, he e ec o o skolin and sphingosin 1-phospha e, wo ba ie enhancing ago- nis s, was s ongly a enua ed in RACK1 deple ed EC (Figu e 6B,C). RACK1 aids a nesyla ion/memb ane anspo o TIMAP Since co-localiza ion o TIMAP and RACK1 was no de ec ed in ou p e ious expe imen s in he cell memb ane, we could exclude ha RACK1 would be di ec ly in ol ed in he anspo o TIMAP. Ye , we hypo hesized ha he p enyla ion o TIMAP leading o i s mo emen o he plasma memb ane may equi e he ancho ing p ope y o RACK1. Indeed, we de ec ed in e ac ion o a nesyl ans- e ase wi h RACK1 in pull-down assay, he binding egion in RACK1 is in he N- e minal WD1-4 egion (Figu e 7A). Mos impo an ly, TIMAP and a nesyl ans e ase co- immunop ecipi a ed om HPAEC wi h no mal RACK1 Figu e 4 GSK3βinhibi o esul s in loss o memb ane localized TIMAP. (A) Immuno luo escence s aining o con luen HPAEC wi hou (a-c) (CTR), o wi h a ious ea men s as ollows: 50 μM o skolin (FRSK) o 30 min (d- ); 20 μM GSK3βinhibi o , AR-A014418, (GSK3 inh) o 4 h s (g-i); o 20 μM GSK3βinhibi o o 4 h s ollowed by 50 μM o skolin o 30 min (j-l) using an i-TIMAP (a,d,g,j: g een) and an i-RACK1 (b,e,h,k: ed) p ima y an ibodies was pe o med. Rep esen a i e da a o a leas h ee independen expe imen s a e shown. Scale ba s: 100 μm. (B) Subcellula ac iona ions o HPAEC cells a e he same se o ea men s as lis ed in panel (A) we e made as desc ibed in Ma e ials and Me hods. The ac ions we e analyzed wi h an i-TIMAP, an i-CD31 as memb ane, an i-lamin A/C as nuclea and an i-β- ubulin (no shown) as cy oplasmic ma ke an ibody. Shown a e ep esen a i e da a o a leas 3 independen expe imen s. Quan i a i e analysis o TIMAP signals is also shown. CD31 o lamin A/C bands we e used o p o ein le el no maliza ion. The esul s a e p esen ed as means ± SE om 3 independen expe imen s. S a is ical analysis was done wi h S uden ’s - es . Signi ican changes a e indica ed by as e isks; * (P < 0.05), ** (P < 0.01), o *** (P < 0.001). (C) TIMAP was immunop ecipi a ed om HPAEC a e he same se o ea men s as lis ed in panel (A). To al cell lysa es and he IP complexes we e p obed o TIMAP and RACK1. Addi ional bands in IP samples co espond o IgG. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 6 o 14 h p://www.biosignaling.com/con en /11/1/2 le el only, bu no om RACK1 deple ed cells (Figu e 7B), sugges ing a pi o al ole o RACK1 in TIMAP p enyla ion. Discussion Vascula EC ba ie in eg i y is c i ical o issue and o gan unc ion [1,25]. The uniquely high exp ession o TIMAP p o ein in endo helial cells [3] implies i s signi i- cance in undamen al ac i i ies o his cell ype. In ac , ou p e ious indings indica ed i s in ol emen in he egula ion o endo helial cell ba ie unc ion [4]. S ill, only a ew o i s p o ein in e ac ions we e iden i ied [5-7]. In a sea ch o u he pa ne s o TIMAP we ecognized and p o ed by di e en me hods ha TIMAP binds he adap o p o ein, RACK1. This wo k was ocused on he cha ac e iza ion o his no el in e ac ion. RACK1 is known as a sca olding p o ein which belongs o he WD- epea con aining p o eins. I seems ha Figu e 5 RACK1 deple ion modula es mRNA and p o ein le els o TIMAP and a ec s he memb ane localiza ion o TIMAP. (A) HPAEC g own on 6-well pla e was ans ec ed wi h small in e e ing RNA (siRNA) as desc ibed in Ma e ials and Me hods. To al RNA was isola ed om non ans ec ed (c ), non silencing RNA, o RACK1 speci ic siRNA (siGBN2L1) ans ec ed cells and analyzed by RT-PCR using speci ic p ime pai s o RACK1, PP2A B (i ele an con ol) o TIMAP. (B) Wes e n blo analysis o non ans ec ed (c ), non silencing RNA, o siGBN2L1 ans ec ed cells using RACK1, TIMAP and ac in speci ic an ibodies. Ac in was es ed as loading con ol. The amoun o RACK1 o TIMAP signal was exp essed as a io o RACK1/TIMAP:β-ac in signal densi y. The e o ba s co espond o SE om 3 independen ans ec ions. S a is ical analysis was done wi h S uden ’s - es . Signi ican changes a e indica ed by as e isks; ** (P < 0.01), o *** (P < 0.001). (C) Immuno luo escence s aining o con luen non ans ec ed (c ) (a-c), non silencing RNA (d- ) o siGBN2L1 ea ed (g-i) HPAEC using an i-TIMAP (a,d,g: g een) and an i-RACK1 (b,e,h: ed) p ima y an ibodies is p esen ed. Scale ba s: 100 μm. (D) Memb ane ac ions o non ans ec ed (c ), non silencing RNA o siGBN2L1 ans ec ed HPAEC we e isola ed as desc ibed in Ma e ials and Me hods. To al cell lysa e (CL) was also loaded as con ol. The ac ions we e analyzed wi h an i-TIMAP and an i-CD31 an ibodies. Shown a e ep esen a i e da a o a leas 3 independen expe imen s. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 7 o 14 h p://www.biosignaling.com/con en /11/1/2 RACK1 has no p e e ence o a common s uc u al ea- u e in i s binding pa ne s. Among he RACK1 in e ac - ing p o eins some con ain S c homology (SH2) domains [14], plecks in homology (PH) domains like dynamin [23,26], C2 and V5 domains in PKCs [27,28] o PDZ domains [29]; bu he e is also example o a whole spe- ci ic s uc u al con o ma ion equi emen on he pa ne ’s side [30]. Ou esul s indica e ha RACK1 binds o he NLS egion a he N- e minal o TIMAP, bu he e is/a e u he associa ion si e(s) wi hin he C- e minal hal e- gion o TIMAP sugges ing a mo e complex su ace o he in e ac ion. I should be no ed ha he PKA/GSK-3β phospho yla ion si es, Se 337/333 o TIMAP a e p esen in his egion. Mo eo e , we showed ha he si e/ egion in RACK1 esponsible o TIMAP binding is wi hin he N- e minal hal (WD 1–4) o he p o ein. RACK1 o ms homodime s ia he ou h WD epea [31], he e o e bo h he N- and C- e minal mu an s es ed we e designed o con ain WD 4 as desc ibed by o he s [32]. Thus he bind- ing egion can be u he na owed o WD 1–3, bu eluci- da ion o he exac binding si es in TIMAP and RACK1 equi es addi ional esea ch. Ano he PP1 ela ed p o ein, CPI17 (PKC po en ia ed PP1 inhibi o ), was ecognized as binding pa ne o RACK1 by yeas wo-hyb id sc eening [33]. In e es ingly, binding o he dime o m (ca aly ic and sca olding subuni ) o PP2A, ano he majo Se /Th p o ein phospha ase, was shown o a C- e minal WD e- pea in RACK1 [32,34]. The mu ual o exclusi e binding o he wo subuni s o PP2A was no esol ed. We ound ha PP1cδis p esen in he RACK1-TIMAP complex as TIMAP is i s egula o y/ a ge ing subuni , bu does no bind di ec ly o RACK1. Al hough RACK1 and PKC a e in ima ely ela ed o each o he ( o e iew see [22]), ha seems i ele an in he TIMAP-RACK1 ela ion, as PKC ac i a ion o EC did no change hei binding. On he o he hand, ac i a- ion o he cAMP/PKA pa hway had signi ican e ec no only on he in e ac ion, bu also on he localiza ion o TIMAP. The second messenge cAMP is known as endo- helial ba ie s abilize [35,36]. Upon cAMP/PKA Figu e 6 RACK1 deple ion a enua es EC ba ie unc ion. Non ans ec ed (c ), non silencing o RACK1 speci ic silencing RNA ans ec ed HPAEC we e pla ed (5 x 10 5 cells/well) on o wo 8W10E a ays 48 h pos - ans ec ion. (A): The ini ial esis ance alues a he beginning o he measu emen we e abou 300 Ωand he impedance was measu ed o 20 h s a e he seeding. (B,C): A e o e nigh incuba ion and basal TER moni o ing (900–1200 Ω), HPAEC monolaye s we e ea ed wi h 50 μM o skolin (B)o 1μM sphingosine 1-phospha e (S1P) (C). Rela i e esis ances ha we e de ec ed a he ime o maximal TER inc ease o o skolin/S1P ea ed cells a e shown o each sample. The esul s a e p esen ed as means ± SEM a leas o ou chambe s o each ea men . Figu e 7 TIMAP in e ac s wi h a nesyl ans e ase ia RACK1. (A) GST-RACK1 pull-down o endogenous a nesyl ans e ase (FNTA). GST, ecombinan RACK1 WT o GST-RACK1 agmen s we e es ed in pull-down assay. The immobilized samples we e incuba ed wi h BPAEC lysa e. Wes e n blo o he pull-down elua es was p obed wi h an i-FNTA an ibody. (B) TIMAP o FNTA was immunop ecipi a ed om non ans ec ed (c ), non silencing RNA o siGBN2L1 ans ec ed HPAEC. To al cell lysa es and he IP complexes we e p obed o TIMAP and FNTA. Addi ional bands in IP samples co espond o IgG. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 8 o 14 h p://www.biosignaling.com/con en /11/1/2 ac i a ion o EC, we de ec ed en ichmen o TIMAP in he plasma memb ane and i s ansloca ion om he nu- cleus. Since pa allel ansloca ion o RACK1 did no hap- pen om he cy oplasm o EC ei he o he memb ane o o he nucleus, his sugges s ha sepa a e signaling pa h- ways egula ing nuclea expo and memb ane a ic o TIMAP could be ini ia ed simul aneously. RACK1 is connec ed o he cAMP/PKA signaling pa h- way h ough i s in e ac ion wi h cAMP phosphodies e ases [12,37]. Simila o ou esul s, in hippocampal neu ons dis- socia ion o RACK1 om i s binding pa ne , Fyn kinase, occu s upon ac i a ion o he PKA pa hway [38]. RACK1 ansloca ed o he nucleus in glioma and neu oblas oma cell lines upon PKA ac i a ion by o skolin [39] o media e he exp ession o a b ain-de i ed neu o ophic ac o . In con as , no ansloca ion o RACK1 upon o skolin ea - men o EC was obse ed in ou expe imen s. Howe e , i was e ealed ecen ly ha phospho yla ion by PKA o se- quen ial phospho yla ion by PKA and GSK-3βonly sligh ly modula ed hebindingo TIMAP oPP1cδ[5]. The dis- socia ion cons an o he complex was abou he same, only he a e o dissocia ion dec eased o a small ex en . In i o phospha ase assays indica ed ha double phospho yla ed o m o TIMAP allowed PP1c ac i i y owa d phospho-moesin subs a e, bu mono- o non- phospho yla ed o m o TIMAP inhibi ed he phospha ase. PKA/GSK-3βphospho yla ion si es, Se 337/333 o TIMAP a e p esen in he C- e minal egion which was shown o bind RACK1, hus he phospho yla ion o hese side chains may a ec no only he egula o y e ec o TIMAP on PP1cδ, bu he binding o TIMAP o RACK1 as well. The phospho yla ion may di ec ly impai he connec ion by in- ducing con o ma ion change o TIMAP, o may ini ia e in e ac ions wi h o he binding pa ne s leading o he loss o RACK1-TIMAP complex. Ou esul s clea ly demon- s a e ha signi ican loss in TIMAP-RACK1 complex ol- lows PKA p imed GSK-3βphospho yla ion o TIMAP. On he con a y, when he TIMAP-RACK1 in e ac ion was diminished by deple ion o RACK1, TIMAP was no ound in he plasma memb ane o he silenced cells sug- ges ing a pi o al ole o RACK1 in p enyla ion/memb ane localiza ion o TIMAP. Signi icance o RACK1 in mem- b ane localiza ion o a Vang p o ein was also ecognized by RACK1 knockdown ecen ly [40]. P enyla ion o TIMAP a he C- e minal CAAX box [3] by a nesyl ans e ase [41] is equi ed o i s memb ane localiza ion. E en ually, de iciency o memb ane ancho ed TIMAP may be he esul o he lack o i s p enyla ion. Ou esul s indica ed ha bo h TIMAP and a nesyl ans e ase bind o he N- e minal hal o RACK1 and he in e ac ion be- ween TIMAP and a nesyl ans e ase was diminished in RACK1 deple ed cells. These con i m he assump ion o RACK1 being he ancho ing su ace o p enyla ion o TIMAP. Since TIMAP is in ol ed in he egula ion o EC ba ie unc ion [4], RACK1 should also be ega ded as a pa icipan in main aining ba ie in eg i y, h ough he egula ion o TIMAP p enyla ion. The pulmona y ascula endo helium unc ions as a semi-selec i e ba ie be ween blood and su ounding issues and con ols biological p o- cesses such as p o ein and luid anspo o in lamma ion. Endo helial ba ie dys unc ion is he p ima y cause o ascula leak and pulmona y edema in sepsis and is an es- sen ial componen o angiogenesis, umo me as asis, and a he oscle osis. The e o e, he main enance o ascula EC ba ie in eg i y may ha e p o ound clinical impo ance. In ag eemen wi h he conclusion ha RACK1 is in ol ed in he main enance o ba ie in eg i y, we ound decele a ed ba ie o ma ion in RACK1 deple ed EC. Consis en wi h his, i was shown by o he s ha RACK1 egula es cell ad- hesion [42], mo eo e , silencing o RACK1 inhibi ed cell p oli e a ion and dec eased mig a ion and adhesion cap- abili y o ca cinoma cells [43]. A ecen pape [44] desc ibed he in ol emen o RACK1 in Gβγ-media ed adhe ens junc ion assembly in EC. They s udied he unc- ion o Gβγ in e-annealing o adhe ens junc ions a e h ombin challenge/PAR ac i a ion. RACK1, no mally bond o Gβγ, is eleased a e h ombin and ha igge s Fyn ac i a ion ia he ocal adhesion kinase. In RACK1 deple ed cells, hey de ec ed slowe eco e y o TER a e h ombin. Ou TER measu emen wi h h ombin ea ed RACK1 deple ed cells showed he same esul (no shown). S ill, he posi i e e ec o cAMP/PKA ac i a ion ( o skolin ea men ) on TER was signi ican ly a enua ed in RACK1 silenced EC. Mo eo e , sphingosin-1-phospha e, a well- known ascula s abilize [45,46], had also ailed o in- c ease TER in RACK1 deple ed cells. I seems, ha RACK1 may be in ol ed in mul iple signaling pa hways conce ned in EC ba ie egula ion. Conclusions The ancho ing p o ein RACK1 was ecognized as a new TIMAP binding pa ne in EC and he egions o he in e ac ing su aces we e iden i ied. The in e ac ion is ansien , ou esul s indica ed ha cAMP/PKA ac i a ion a ec ed hei binding and e oked a change in localiza ion o TIMAP om he nucleus o he cell memb ane. We p opose ha he phospho yla ion o TIMAP pool bound o RACK1 in he cy osol may ini ia e a con o ma ion change o TIMAP which acili a es i s p enyla ion and ansloca ion o he cell memb ane. The cy osolic pool o TIMAP is “ e- illed” om he nucleus, ge s p enyla ed as well and mo es o he plasma memb ane (Figu e 8). RACK1 suppo s his p ocess by p o iding a simul aneous ancho ing su ace o TIMAP and a nesyl ans e ase. This ensu es p enyla ion and subsequen ly memb ane anspo o TIMAP, whe e i may ul ill i s ba ie main- aining ole as a PP1 egula o y p o ein. Bo a kó e al. Cell Communica ion and Signaling 2013, 11:2 Page 9 o 14 h p://www.biosignaling.com/con en /11/1/2