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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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