Molecula Cell, Vol. 10, 1269–1281, Decembe , 2002, Copy igh 2002 by Cell P ess
Mechanism o Rec ui men o WASP
o he Immunological Synapse and o
I s Ac i a ion Following TCR Liga ion
Ald ich synd ome (WAS), is exp essed only in hema o-
poie ic cells and is he i s iden i ied membe o an
expanding amily o p o eins in ol ed in signaling and
cy oskele al o ganiza ion ha includes N-WASP and
Sca /WAVE (De y e al., 1994; Machesky and Insall,
Yoji Sasaha a,
1,4
Rima Rachid,
1,4
Michael J. By ne,
1
Miguel A. de la Fuen e,
1
Robe T. Ab aham,
2
Na ayanaswamy Ramesh,
1,3
and Rai S. Geha
1,3
1
Di ision o Immunology
Child en’s Hospi al and
Depa men o Pedia ics 1998; Miki e al., 1996). WASP has an N- e minal Ena/
VASP homology domain 1 (EVH1) domain, a Cdc42/RacHa a d Medical School
Bos on, Massachuse s 02115 GTPase binding domain (GBD), a p oline- ich domain,
a G-ac in binding e p olin homology (VH) domain, a
2
Depa men o Pha macology
Duke Uni e si y Medical Cen e co ilin homology (CH) domain, and a C- e minal acidic
(A) segmen . WASP in e ac s wi h WIP ia i s N- e minalDu ham, No h Ca olina 27710
EVH1 domain (Ramesh e al., 1997), wi h Cdc42-GTP ia
i s GBD domain, wi h mul iple SH3 domain-con aining
p o eins ha include Nck ia i s p oline- ich egion, and
Summa y
wi h ac in and he A p2/3 complex ia i s VCA domain
(Abo, 1998). WASP exis s in cells in a closed inac i e
F-ac in polyme iza ion ollowing engagemen o he T
con o ma ion due o in amolecula in e ac ions ha in-
cell ecep o (TCR) is dependen on WASP and is c i i-
ol e he C- e minal acidic domain and a basic egion
cal o T cell ac i a ion. The link be ween TCR and
ha p ecedes he GBD domain. Binding o Cdc42-GTP
WASP is no ully unde s ood. In es ing cells, WASP
is hough o cause a con o ma ional change in WASP,
exis s in a complex wi h WIP, which inhibi s i s ac i a-
which allows he VCA domain o in e ac wi h and ac i-
ion by Cdc42. We show ha he adap o p o ein C kL
a e he A p2/3 complex (Higgs and Polla d, 2000; Kim
binds di ec ly o WIP. Fu he , TCR liga ion esul s in
e al., 2000; Roha gi e al., 2000). WASP plays a c i ical
he o ma ion o a ZAP-70-C kL-WIP-WASP complex,
ole in T cell ac i a ion and ac in eo ganiza ion. T cells
which is ec ui ed o lipid a s and he immunological
om WAS pa ien s and WASP⫺
/
⫺mice a e se e ely de i-
synapse. TCR engagemen also causes PKC␪-depen-
cien in hei abili y o inc ease hei F-ac in con en ,
den phospho yla ion o WIP, causing he disen-
sec e e IL-2, and p oli e a e ollowing TCR liga ion (Gal-
gagemen o WASP om he WIP-WASP complex,
lego e al., 1997; Snappe e al., 1998; Zhang e al., 1999).
he eby eleasing i om WIP inhibi ion. These esul s
WIP is a 503 aa long p oline- ich p o ein exp essed
sugges ha he ZAP-70-C kL-WIP pa hway and PKC␪
a high le els in lymphoid issues (Ramesh e al., 1997).
link TCR o WASP ac i a ion.
WIP binds ac in ia i s VH domain (aa 1–151) and WASP
ia i s ca boxy- e minal end (aa 416–488). A WIP-WASP/
In oduc ion
N-WASP complex is eadily de ec ed in es ing cells
(Ma inez-Quiles e al., 2001). In lymphocy es, ⬎95% o
In e ac ion be ween he T cell ecep o (TCR) and pep-
WASP is complexed wi h WIP (ou unpublished da a).
ide-loaded majo his ocompa ibili y complex (MHC)
WIP inhibi s Cdc42-media ed ac i a ion o N-WASP,
molecules on he su ace o an igen p esen ing cells
sugges ing ha one unc ion o WIP is o s abilize WASP/
(APCs) induces he o ma ion o molecula clus e s a
N-WASP in hei inac i e closed con o ma ion (Ma inez-
he con ac si e ha a e en iched in ilamen ous ac in
Quiles e al., 2001). Ano he unc ion o WIP is o s abilize
(F-ac in) (G akoui e al., 1999; Penninge and C ab ee,
ac in ilamen s (Ma inez-Quiles e al., 2001). WIP, like
1999). These sup amolecula ac i a ion clus e s (SMACs),
WASP, plays an impo an ole in T cell ac i a ion. T
also named immunological synapses (IS), con ain se -
cells om WIP⫺
/
⫺mice ail o p oli e a e, sec e e IL-2,
e al signaling componen s. They include s c and Syk
o inc ease hei F-ac in con en a e TCR liga ion. Fu he -
amily kinases, PKC␪and Cdc42-GTP (B omley e al.,
mo e, WIP⫺
/
⫺T cells a e de icien in conjuga e o ma ion
2001). The IS also con ains adap o p o eins such as
wi h supe an igen-p esen ing B cells and an i-CD3/ICAM-
SLP-76, Fyb, and Nck ha a e linked di ec ly o indi ec ly
1-con aining lipid bilaye s and ha e a diso ganized ac in
o p o eins such as WASP, Ena/VASP amily membe s
cy oskele on (An on e al., 2002).
ha a e in ol ed in ac in polyme iza ion (K ause e al.,
Recen da a shows ha WASP localizes wi h F-ac in
2000; Monks e al., 1998). The accumula ion o F-ac in
o he IS whe e i is hough o be ac i a ed by Cdc42-
a he T cell-APC in e ace is hough o s abilize a con-
GTP gene a ed ollowing ac i a ion o he exchange ac-
inuous con ac be ween T cells and APCs, which is
o Va and i s ec ui men o lipid memb anes (A ud-
equi ed o op imal T cell ac i a ion. Inhibi ion o ac in
chand an e al., 2000). The mechanism(s) by which
polyme iza ion by cy ochalasin blocks o ma ion o he
WASP is ec ui ed o he IS and is eleased om WIP
immunological synapse and T cell ac i a ion (Wul ing
inhibi ion o ini ia e ac in polyme iza ion is no well un-
and Da is, 1998).
de s ood. We show he e ha WIP binds o he adap o
WASP, he p oduc o he gene mu a ed in Wisko -
p o ein C kL and ha ollowing TCR liga ion, a C kL-
WIP-WASP complex is ec ui ed by ZAP-70 o lipid a s
3
Co espondence: na ayanaswamy. amesh@ ch.ha a d.edu (N.R.),
and he IS. TCR liga ion also causes PKC␪-dependen
[email p o ec ed] (R.S.G.)
4
These au ho s con ibu ed equally o his wo k.
phospho yla ion o WIP and disengagemen o WASP
Molecula Cell
1270
om he WIP-WASP complex, eleasing i om WIP inhi- ec ui he WIP-WASP complex o ZAP-70 ollowing TCR
liga ion. We i s p obed ZAP-70 immunop ecipi a es
bi ion.
om Ju ka cells be o e and 3 min a e an i-CD3 s imu-
la ion o C kL, WIP, and WASP. As expec ed, an i-CD3
Resul s
s imula ion esul ed in igo ous y osine phospho yla-
ion o ZAP-70 (Figu e 2A). C kL and WIP became eadily
The Adap o P o ein C kL Binds o WIP and
de ec able in ZAP-70 immunop ecipi a es a e an i-CD3
Associa es wi h ZAP-70 a e TCR Liga ion
s imula ion. The weak associa ion o hese p o eins wi h
In a sea ch o po en ial links be ween he ac i a ed
ZAP-70 in uns imula ed cells may be explained by he
TCR-ZAP-70 complex and he WIP-WASP complex, we
p esence o small amoun s o phospho yla ed ZAP-70
conside ed he possibili y ha an SH2-SH3 domain-con-
in hese cells. We we e unable o de ec WASP in ZAP-
aining adap o p o ein may link phospho yla ed ZAP-70
70 immunop ecipi a es.
o he p oline- ich WIP and WASP. Po en ial candida es
In a u he a emp o demons a e associa ion o
included he CT10 egula o o kinase (C k) amily o
ZAP-70 wi h WASP, we p obed WASP immunop ecipi-
adap o p o eins. The p o o ype, C kII, has one N- e -
a es o ZAP-70. Figu e 2B shows ha ZAP-70 cop e-
minal SH2 domain and wo SH3 domains. The second
cipi a ed weakly wi h WASP in uns imula ed cells. The
SH3 domain (SH3.2) is al e na i ely spliced ou o gi e
associa ion o WASP and ZAP-70 inc eased a e an i-
ise o C kI. A closely ela ed membe , C kL (C k like),
CD3 s imula ion, sugges ing ha WASP is ec ui ed o
esembles C kII in ha i also has wo SH3 domains and
ZAP-70 ollowing TCR liga ion. P obing o WASP immu-
is exp essed a high le els in hema opoie ic cells (Felle nop ecipi a es o C kL and WIP e ealed ha C kL, like
e al., 1998). GST-C kII and GST-C kL ha e been e- WIP, cop ecipi a ed wi h WASP in uns imula ed cells,
po ed o associa e ia hei SH2 domains wi h phos- sugges ing ha C kL, WIP, and WASP exis as p e-
pho yla ed ZAP-70 (Gelkop and Isako , 1999). WIP, bu o med complex, wi h WIP b idging WASP o C kL. C kI
no WASP, con ains wo copies o he consensus mo i and C kII we e no de ec ed in WASP immunop ecipi-
PxLPxK/R, which binds o he N- e minal SH3 domain a es (da a no shown). An i-CD3 s imula ion esul ed in
(SH3.1) o C k p o eins. dec eased associa ion o WASP wi h WIP and C kL,
To de e mine i WIP and WASP bind o C k p o eins, sugges ing ha TCR liga ion may pe u b he WIP-
we exp essed C kII and C kL as GST usion p o eins WASP complex. Simila esul s we e ob ained in no mal
and examined hei abili y o pull down Xp ess- agged T cell blas s (see Supplemen al Figu e S1 a h p://
ecombinan WIP and in i o ansc ibed and ansla ed www.molecule.o g/cgi/con en / ull/10/6/1269/DC1).
WASP. C kI was no examined because i is an al e na- Taken oge he , ou esul s sugges ha a C kL-WIP-
i e splicing unca ed p oduc o C kII. WIP bound o WASP complex is ec ui ed o phospho yla ed ZAP-70
C kL, bu no o C kII. (Figu e 1A). This indica es di ec ollowing TCR liga ion.
in e ac ion be ween WIP and C kL. WASP ailed o bind
o ei he C kL o C kII (Figu e 1A), e en in he p esence C kL, WIP, and WASP T ansloca e o GEMs
o he cons i u i ely ac i e Cdc42 mu an Cdc42L61 a e TCR Liga ion
(da a no shown). Using he yeas wo-hyb id sys em, Glycosphingolipids and choles e ol sel -associa e in
we ound ha he SH3.1 domain o C kL binds o a plasma memb ane mic odomains known as glycolipid
egion o WIP spanned by aa 321–415 (Figu e 1B). This en iched mic odomains (GEMs) o lipid a s. GEMs lo-
egion con ains he wo consensus binding mo i s o calize a he IS in an igen-s imula ed T cells (Bi e al.,
he C k SH3.1 domain (aa 332–337 and 399–404) and is 2001) and a e en iched in a numbe o molecules ele-
dis inc om he WASP binding si e (aa 416–488). an o ecep o signaling ha leads o F-ac in polyme -
We nex de e mined i C kL associa es wi h ZAP-70 iza ion and IL-2 gene exp ession. Signaling molecules
and WIP in T cells. Wes e n blo ing o C kL immunop e- ec ui ed o lipid a s a e TCR liga ion include phos-
cipi a es om Ju ka cells e ealed ha C kL associa ed pho yla ed CD3␨and i s associa ed ZAP-70. Since ZAP-
wi h ZAP-70 ollowing an i-CD3 s imula ion (Figu e 1C). 70 associa es wi h C kL, WIP, and WASP in ac i a ed
WIP cop ecipi a ed wi h C kL in uns imula ed Ju ka T T cells, we examined whe he hese p o eins ansloca e
cells, and his associa ion inc eased sligh ly a e TCR o GEMs ollowing TCR liga ion.
liga ion (Figu e 1C). Simila esul s we e ob ained in We used suc ose densi y g adien ac iona ion o T i-
PHA-de i ed T cell blas s de i ed om no mal subjec s on X-100 lysa es om Ju ka cells o analyze he ans-
(see Supplemen al Figu e S1 a h p://www.molecule. loca ion o C kL, WIP, and WASP o GEMs, which sedi-
o g/cgi/con en / ull/10/6/1269/DC1). WIP cop ecipi- men in ac ions 2–5 o he g adien , as e idenced by
a ed wi h C kL om pe iphe al blood T cell blas s o a he p esence o he glycosphingolipid GM1 in hese ac-
WAS pa ien wi h a poin mu a ion in he WASP gene ions. Small amoun s o 21 kDa phospho yla ed CD3␨,
(C73Y), who does no exp ess WASP p o ein (Figu e ZAP-70, C kL, WIP, and WASP we e p esen in GEMs
1D). These esul s sugges ha WIP associa es wi h be o e s imula ion. As expec ed, phospho yla ed CD3␨
ZAP-70 a e TCR liga ion and associa es cons i u i ely and ZAP-70 ansloca ed o GEMs a e an i-CD3 s imu-
wi h C kL independen ly o WASP. la ion. TCR liga ion caused ansloca ion o C kL, WIP,
and WASP o GEMs (Figu e 3C).
WIP and WASP A e Rec ui ed o ZAP-70
a e TCR Liga ion T ansloca ion o WIP and WASP o GEMs
Since WIP exis s as a p e o med complex wi h WASP Is Dependen on ZAP-70 and C kL
and since he WIP binding si es o C kL and WASP We used ZAP-70-de icien P116 Ju ka cells o examine
he ole o ZAP-70 in he ansloca ion o C kL, WIP, anda e dis inc , we examined he possibili y ha C kL may
Mechanism o WASP Ac i a ion Following TCR Liga ion
1271
Figu e 1. In e ac ion be ween C kL and WIP
(A) Pull-down assay using GST-C kL, GST-C kII usion p o eins and GST wi h pu i ied Xp ess- agged WIP o in i o ansla ed WASP. Bound
p o eins we e p obed wi h an i-Xp ess o an i-WASP mAbs. The le lane in each panel was loaded wi h WIP o WASP p o ein (“Inpu ”).
(B) Mapping by yeas wo-hyb id assay o he WIP binding si e in C kL (le panel) and o he C kL binding si e in WIP ( igh panel).
(C) Cop ecipi a ion o WIP and ZAP-70 wi h C kL in uns imula ed and an i-CD3-s imula ed Ju ka cells. C kL and con ol (“C l. Ig.”) immunop e-
cipi a es we e p obed o ZAP-70, WIP, and C kL. The igh lane in each panel was loaded wi h cell lysa es (“Lys.”).
(D) WIP associa ion wi h C kL is independen o WASP. Le : exp ession le els o WIP and WASP in lysa es om T cells o a WAS pa ien
(“p .”) wi h C73Y WASP mu a ion and a no mal con ol (“C l.”). Righ : C kL and con ol immunop ecipi a es om T cells o he pa ien and
no mal con ol we e p obed o WIP and C kL.
WASP o GEMs. The pa en P116 cell line was s ably he ans ec ed P116 clones was con i med by Wes e n
blo ing (Figu e 3A). In con as o wild- ype Ju ka cells, ans ec ed wi h ei he Myc- agged w (wild- ype) ZAP-
70 o Myc- agged kinase-dead (KD) ZAP-70. A leas he e was no ansloca ion o C kL, WIP, o WASP o
GEMs in ZAP-70-de icien P116 cells (Figu e 3B). T ans- wo clones ha showed equi alen su ace CD3 exp es-
sion o w Ju ka cells we e s udied o each cons uc , loca ion o all h ee p o eins o GEMs was es o ed in
P116 cells econs i u ed wi h w ZAP-70, bu no in P116wi h simila esul s. Exp ession o ZAP-70 p o eins in
Molecula Cell
1272
Figu e 2. ZAP-70, C kL, WIP, and WASP
Fo m a Complex ollowing TCR Liga ion and
A e Rec ui ed o Lipid Ra s
(A) ZAP-70 immunop ecipi a es om uns im-
ula ed and an i-CD3 s imula ed Ju ka T cells
and lysa es (“Lys.”) we e p obed wi h Abs o
phospho y osine (“PY”), C kL, WIP, and ZAP-
70 as a loading con ol.
(B) WASP immunop ecipi a es and lysa es
om he same cells we e p obed o ZAP-70,
C kL, WIP, and WASP as a loading con ol.
(C) Suc ose densi y g adien ac ions o T i-
on X-100 lysa es om uns imula ed and an i-
CD3 s imula ed Ju ka cells we e p obed o
CD3␨, ZAP-70, C kL, WIP, and WASP. The
isualized 21 kDa band co esponds o phos-
pho yla ed CD3␨. Chole a oxin B subuni was
used o p obe o GM1 ha cons i u i ely e-
sides in GEMs as a loading con ol.
cells econs i u ed wi h KD ZAP-70. These esul s sug- all ansloca ed o he T cell-APC in e ace (Figu e 4A
and Table 1). As expec ed, F-ac in also accumula ed a ges ha ZAP-70 and i s kinase ac i i y a e essen ial o
he ansloca ion o he C kL-WIP-WASP complex o he in e ace.
T ansloca ion o C kL, WIP and WASP o he T cell-lipid a s.
To examine he ole o C kL in he ansloca ion o APC in e ace was dependen on ZAP-70, because i
was ma kedly diminished in ZAP-70-de icien P116WIP and WASP o GEMs, Ju ka T cells we e s ably
ans ec ed wi h a C kL dele ion mu an ha lacked he cells. Bo h he pe cen ages o conjuga es ha con ained
hese p o eins a he in e ace and he amoun o accu-SH3.1 domain ha is essen ial o WIP binding
(C kL⌬SH3.1), o ec o alone. The mu an would com- mula ed p o eins we e dec eased (Figu e 4A and Table
1). Conjuga e o ma ion by hese cells was no mal (da ape e wi h na i e C kL o ZAP-70 binding. Two clones
ha showed equi alen su ace CD3 exp ession o w no shown). Consis en wi h p e ious da a (Mo gan e
al., 2001), he e was a much weake accumula ion o Ju ka cells we e s udied wi h simila esul s. Wes e n
blo ing e ealed ha he mu an was exp essed 2.5- F-ac in a he in e ace in P116 cells. T ansloca ion o
C kL, WIP, and WASP and F-ac in accumula ion a he old highe han endogeneous p o ein (Figu e 3C). Mu-
an C kL, like na i e C kL, ansloca ed o GEMs a e in e ace was comple ely co ec ed in P116 cells econ-
s i u ed wi h w ZAP-70, bu emained de icien in P116TCR liga ion, consis en wi h he no ion ha he mu an
can be ec ui ed by ZAP-70 ia i s SH2 domain. In con- cells econs i u ed wi h KD ZAP-70. T ansloca ion o
WIP and WASP o he T cell-APC in e ace was also as , WIP and WASP ansloca ed poo ly o GEMs in
C kL⌬SH3.1- ans ec ed cells (Figu e 3D). dependen on C kL, because i was ma kedly diminished
in C kL⌬SH3.1- ans ec ed Ju ka cells (Figu e 4A and
Table 1). Conjuga e o ma ion by hese cells was no mal
WIP and WASP T ansloca e o he T Cell-APC
(da a no shown).
In e ace in a ZAP-70- and C kL-Dependen Manne
Since C kL, WIP, and WASP ansloca e o GEMs, we
examined whe he hey also localize o he T cell-APC ZAP-70 and C kL Play an Impo an Role in Cellula
F-Ac in Inc ease and IL-2 Syn hesis ollowing TCRin e ace. Ju ka T cells we e incuba ed wi h Raji B cells
in he p esence o he supe an igen SEE, and T:B cell Liga ion by Supe an igen P esen ed by APCs
We nex in es iga ed he ole o ZAP-70 and C kL inconjuga es we e examined o localiza ion o C kL, WIP,
WASP, and F-ac in a e 10 min. C kL, WIP, and WASP he inc ease o o al cellula F-ac in con en and in IL-2
Mechanism o WASP Ac i a ion Following TCR Liga ion
1273
Figu e 3. Rec ui men o WIP-WASP Com-
plex o Lipid Ra s Is Dependen on ZAP-70
and C kL
(A) Exp ession o ZAP-70 p o ein in w Ju ka
cells and in P116 clones s ably ans ec ed
wi h ec o alone, wild- ype (“WT”) ZAP-70,
and a kinase-dead (“KD”) ZAP-70 mu an . Ly-
sa es we e p obed wi h an i-ZAP-70 mAb.
(B) Rec ui men o C kL, WIP, and WASP o
lipid a s in Ju ka cells, P116 cells, and P116
econs i u ed cells. F ac ions 2–5 o he su-
c ose g adien we e pooled and p obed o
ZAP-70, C kL, WIP, and WASP and o GM1
as con ol.
(C) Exp ession o C kL p o ein in Ju ka
clones s ably ans ec ed wi h ec o alone o
wi h a C kL mu an lacking he SH3.1 domain
(“⌬SH3.1”). Lysa es we e p obed wi h an i-
C kL mAb.
(D) Rec ui men o ZAP-70, C kL, WIP, and
WASP o lipid a s in C kL⌬SH3.1- ans-
ec ed Ju ka cells and con ols.
syn hesis ollowing TCR liga ion. P116 cells ailed o single consensus PKC phospho yla ion mo i (RxxS/
inc ease hei F-ac in con en a e an i-CD3 s imula ion TxR) ha su ounds S488. PKC␪, he majo PKC in T
and o sec e e IL-2 ollowing s imula ion wi h APCs and cells, is ac i a ed and ansloca es o GEMs and he IS
SEE. The la e esul is consis en wi h esul s ob ained ollowing TCR liga ion (Bi and Al man, 2001). This aised
wi h an i-CD3 s imula ion (Williams e al., 1998). These he possibili y ha PKC␪phospho yla ion o WIP may
de ici s we e comple ely co ec ed by econs i u ion pe u b he WIP-WASP complex. In addi ion o he C45
wi h w ZAP-70, bu no wi h KD ZAP-70 (Figu es 4B an ise um which ecognizes he ca boxy- e minal 45 aa
and 4C). C kL⌬SH3.1- ans ec ed Ju ka cells we e also o WIP (aa 459–503), we ha e aised ano he an ise um,
ma kedly impai ed in hei capaci y o inc ease hei C14, agains a 14 aa pep ide o WIP,
483
ESRSGSNRRER
F-ac in con en (Figu e 4B and Supplemen al Figu e S2 GGAP
496
, which con ains S488 (Figu e 5A). C14 Ab eac-
a h p://www.molecule.o g/cgi/con en / ull/10/6/1269/ i i y was ma kedly dec eased 5 min a e ea men o
DC1) and sec e e IL-2 ollowing TCR liga ion (Figu e 4C), Ju ka cells wi h an i-CD3 and was es o ed 30 min la e
bu had no mal calcium luxes (da a no shown). (Figu e 5B). In con as , C45 Ab eac i i y emained un-
changed. Reac i i y wi h C14 Ab was ully es o ed by
ea men o lysa es wi h alkaline phospha ase (Figu e
WIP Is Phospho yla ed by PKC␪
5C). Simila esul s we e ob ained in pe iphe al blood T
ollowing TCR Liga ion
cells (da a no shown). These indings indica e ha WIP
T ansloca ion o he WIP-WASP complex o he IS b ings
is phospho yla ed a e TCR liga ion and ha C14 Ab
i in o p oximi y wi h Cdc42-GTP. Howe e , since almos
ecognizes a nonphospho yla ed WIP epi ope, whe eas
all o he WASP (⬎95%) in es ing T cells is complexed
C45 Ab is insensi i e o phospho yla ion.
wi h WIP, which inhibi s i s ac i a ion by Cdc42, we
To explo e he iden i y o he enzyme ha esul s in
in es iga ed po en ial mechanisms o WASP ac i a ion
ollowing TCR liga ion. WIP, bu no WASP, con ains a WIP phospho yla ion, we i s examined he e ec o

Molecula Cell
1274
Figu e 4. Rec ui men o C kL, WIP, WASP, and F-Ac in a he T Cell-APC In e ace, F-Ac in Con en , and IL-2 Sec e ion A e Dependen on
ZAP-70 and C kL
(A) In acellula immuno luo escence s aining o C kL, WIP, WASP, and F-ac in in T cells s imula ed by SEE (5 ␮g/ml), and Raji B cells
p eloaded wi h CMAC (blue). Cells we e s ained simul aneously o C kL and WIP. In sepa a e expe imen s, cells we e s ained o WASP and
F-ac in. Magni ica ion is 600⫻.
(B) FACS analysis o in acellula F-ac in con en o cells ollowing CD3 c osslinking. Rep esen a i e esul s o h ee independen expe imen s
a e shown.
(C) IL-2 sec e ion by T cells s imula ed wi h SEE and Raji B cells. Supe na an s collec ed a 24 h we e assayed o IL-2 by ELISA. Means ⫾
SD o h ee independen expe imen s a e shown.
selec i e PKC inhibi o s. P e ea men o Ju ka cells To asce ain he ole o PKC␪in WIP phospho yla ion,
we examined splenic T cells om PKC␪⫺
/
⫺mice. An i-wi h o le in, an inhibi o o he nonclassical PKCs ␦and
␪(Villalba e al., 1999), inhibi ed WIP phospho yla ion CD3 s imula ion esul ed in loss o C14 Ab eac i i y in
T cells om w mice. In con as , he e was no loss o ollowing TCR liga ion, as e idenced by e en ion o C14
Ab eac i i y (Figu e 5D). In con as , G06976, an inhibi- C14 Ab eac i i y in T cells o PKC␪⫺
/
⫺mice (Figu e 5E).
ZAP-70, SLP-76, and Va -1 all play an impo an ole in o o he classical calcium-dependen PKCs ␣,␤, and
␥(Ma iny-Ba on e al., 1993), and he PKA inhibi o 14- he ac i a ion o PKC␪(He ndon e al., 2001; Villalba e
al., 2000). Following TCR liga ion, WIP phospho yla ion22 amide had no e ec (Figu e 5D and da a no shown).
Mechanism o WASP Ac i a ion Following TCR Liga ion
1275
Table 1. T ansloca ion o C kL, WIP, and WASP o he T-APC In e ace
Cells % o Conjuga es wi h Accumula ion a he T Cell-APC In e ace
C kL WIP WASP
SEE: ⫺⫹ ⫺ ⫹ ⫺⫹
Wild- ype Ju ka 8.5 ⫾5.7 26.5 ⫾5.0 15.0 ⫾1.2 36.0 ⫾2.3 8.0 ⫾2.2 34.0 ⫾2.4
P116/ ec o 7.5 ⫾3.4 10.5 ⫾5.7* 13.0 ⫾1.2 13.5 ⫾1.9* 4.0 ⫾1.5 6.0 ⫾2.3*
P116/ZAP-70 w 8.5 ⫾4.1 26.0 ⫾2.3 14.0 ⫾1.6 38.5 ⫾1.9 6.0 ⫾1.6 36.0 ⫾2.0
P116/ZAP-70 KD 9.0 ⫾6.0 13.0 ⫾6.0* 15.5 ⫾2.5 17.0 ⫾4.2* 5.0 ⫾2.4 9.0 ⫾3.6*
C kL/⌬SH3.1 9.5 ⫾5.2 34.0 ⫾5.0 12.0 ⫾2.0 26.0 ⫾2.5* 6.0 ⫾2.4 22.0 ⫾2.2*
Resul s ep esen he mean ⫾SD o ou expe imen s ( i y conjuga es we e examined in each).
*p ⬍0.01 compa ed o w Ju ka cells.
esul ing in loss o Ab C14 eac i i y was no de ec able in WASP immunop ecipi a es om an i-CD3-s imula ed
Ju ka cells (Figu e 2B). To con i m his esul , WIP im-in Ju ka T cells ha lacked ZAP-70 (P116 cells) o SLP-
76 (J14 cells) no in T cells om Va -1⫺
/
⫺mice. These munop ecipi a es we e p epa ed om an i-CD3-s imu-
la ed Ju ka cells using C45 Ab and we e p obed o esul s sugges ha PKC␪plays an impo an ole in he
phospho yla ion o WIP. WASP using a polyclonal an i-WASP an ibody ha ec-
ognizes he C- e minal 18 aa o WASP. An i-CD3 s imu-
la ion esul ed in a ma ked dec ease in he associa ionThe WASP-WIP Complex Is Dis up ed
ollowing TCR Liga ion o WASP wi h WIP (Figu e 6A). Simila esul s we e ob-
ained using a mAb ha ecognizes he WASP GBDThe WASP binding egion o WIP (aa 416–488) o e laps
wi h he egion ecognized by C14 Ab (aa 483–496). domain and wi h pe iphe al blood T cells (da a no
shown). I is unlikely ha he dec eased WASP signal isWe conside ed he possibili y ha WIP phospho yla ion
a e TCR liga ion may esul in he dissocia ion o WASP due o ailu e o he an ibodies used o ecognize WASP,
which may ha e been phospho yla ed ollowing TCR om WIP, which would allow i s ac i a ion by Cdc42.
In ac , we had obse ed dec eased amoun s o WIP liga ion, because ea men o WIP immunop ecipi a es
Figu e 5. WIP Is Phospho yla ed a e TCR
Liga ion by PKC␪
(A) Map showing WIP pep ides used o aise
C14 and C45 Abs.
(B) Reac i i y o lysa es om Ju ka cells be-
o e and a e an i-CD3 s imula ion (5 and 30
min) wi h C14 and C45 Abs.
(C) Re e sal o loss o C14 Ab eac i i y a e
an i-CD3 s imula ion (5 min) by ea men o
lysa es wi h alkaline phospha ase.
(D) E ec o he PKC inhibi o s on loss o
C14 Ab eac i i y a e an i-CD3 s imula ion
(5 min). Cells we e p e ea ed wi h 20 ␮M o -
le in o 62 nM G06976 o 30 min a 37⬚C.
(E) Role o PKC␪, Va -1, ZAP-70, and SLP-
76 in WIP phospho yla ion a e TCR liga ion.
Splenic T cells om PKC␪
⫺
/
⫺
and Va -1
⫺
/
⫺
mice and om w Ju ka T cells, P116 cells,
and J14 cells we e s imula ed wi h an i-CD3
o 5 min, lysed, and p obed wi h C14 and
C45 Abs.
Molecula Cell
1276
Figu e 6. WIP Dissocia ion om WASP ol-
lowing TCR Liga ion
(A) Dissocia ion o WASP om WIP ollowing
an i-CD3 s imula ion (5 and 30 min) o Ju ka
cells. WIP C45 Ab immunop ecipi a es we e
p obed o WASP and WIP as loading con-
ols.
(B) Re e sal o he dissocia ion o WIP and
WASP by ea men o cell lysa es wi h alka-
line phospha ase, 1 U/10 ␮la 25⬚C o 3 h .
(C) E ec o p e ea men wi h o le in (le
panel) and G06976 ( igh panel) on he disso-
cia ion o WIP and WASP a e TCR liga ion
(5 min). In all cases, WIP C45 Ab immunop e-
cipi a es om Ju ka cells we e p obed wi h
WASP polyclonal Ab and WIP C45 Ab as load-
ing con ol.
(D) Pull-down assay using w GST-WIP
401-503
usion p o ein and i s mu an s S405D, S486D,
and S488D wi h in i o ansc ibed and ans-
la ed WASP. Bound p o eins we e elu ed, un
on SDS-PAGE, and Wes e n blo ed o
WASP and GST as loading con ols.
wi h alkaline phospha ase did no inc ease he in ensi y binding si e (aa 416–488) as well as he poin mu an s
S488D, S405D, and S486D. A ini y p ecipi a ion usingo he WASP signal (da a no shown). I is also unlikely
ha he dec eased associa ion o WASP wi h WIP was ecombinan WASP showed ha w WIP
401-503
bound o
WASP. The S488D mu an , bu no he S405D and S486Ddue o deg ada ion o WASP, because he in ensi y o
he WASP band in cell lysa es emained unchanged a e mu an s, showed ma kedly dec eased WASP binding
(Figu e 6D). These esul s sugges ha phospho yla ionTCR liga ion, and no smalle molecula weigh bands
ha eac wi h an i-WASP we e de ec ed (da a no a S488 dis up s WIP binding o WASP.
shown).
We nex in es iga ed he ole o phospho yla ion in
he dissocia ion o he WIP-WASP complex ollowing Ac i a ion o PKC Is Essen ial o F-Ac in
Accumula ion and IL-2 Syn hesis, bu No o WASPTCR liga ion. T ea men o cell lysa es wi h alkaline
phospha ase p io o immunop ecipi a ion es o ed he T ansloca ion o he T Cell-APC In e ace
To es he hypo hesis ha WASP-media ed F-ac inassocia ion o WASP wi h WIP (Figu e 6B). Since WIP
is a a ge o PKC␪phospho yla ion, we examined he polyme iza ion may be dependen on PKC phospho yla-
ion o WIP and he subsequen elease o WASP ome ec o PKC inhibi o s on he dissocia ion o he WIP-
WASP complex. Figu e 6C shows ha p e ea men o WIP inhibi ion, we examined F-ac in accumula ion a
he T cell-APC in e ace in Ju ka cells p e ea ed wi hJu ka cells wi h o le in pa ially inhibi ed his dissocia-
ion, whe eas G06976 had no e ec . These esul s sug- PKC inhibi o s. F-ac in accumula ion a he in e ace
was diminished in cells p e ea ed wi h o le in, bu no ges ha WIP phospho yla ion by PKC␪ esul s in disso-
cia ion o he WIP-WASP complex. in cells p e ea ed wi h G06976 (Figu e 7A). In con as ,
accumula ion o C kL and WIP and WASP was no a -S488 is he only esidue in WIP ha alls wi hin a
consensus mo i o PKC phospho yla ion. We he e o e ec ed. Since SLP-76 is essen ial o PKC␪ac i a ion,
we also examined SLP-76-de icien J14 cells. C kL andexamined he e ec o an S488→D subs i u ion, which
mimics he nega i e cha ge o phosphose ine, on he WIP accumula ed no mally a he in e ace in hese cells.
Howe e , he amoun s o WASP and F-ac in ha accu-abili y o WIP o bind WASP. Since ull-leng h WIP is
poo ly exp essed in bac e ial sys ems, we cons uc ed mula ed a he in e ace we e diminished, al hough he
pe cen age o conjuga es wi h accumula ion o WASPa GST-WIP
401-503
usion p o ein ha con ains he WASP
Mechanism o WASP Ac i a ion Following TCR Liga ion
1277
Figu e 7. E ec s o PKC Inhibi o s and SLP-76 on he T Cell-APC in e ace, F-Ac in Con en , and IL-2 Sec e ion
(A) E ec o PKC inhibi o s and o SLP-76 de iciency (J14 cells) on he accumula ion o C kL, WIP, WASP, and F-ac in a he IS. Magni ica ion
is 600⫻.
(B) F-ac in con en ollowing TCR liga ion in w Ju ka cells un ea ed o p e ea ed wi h o le in o G06976, splenic T cells om w and
PKC␪
⫺
/
⫺
mice, and J14 cells we e analyzed by FACS. Rep esen a i e esul s o h ee independen expe imen s a e shown.
(C) E ec o PKC inhibi o s (uppe panel) and SLP-76 (lowe panel) on IL-2 sec e ion by Ju ka T cells o J14 cells s imula ed wi h SEE and
Raji B cells. Means ⫾SD o h ee independen expe imen s a e shown.
(D) Model o he ec ui men o WASP o GEMs and he IS and o i s ac i a ion ha esul s in ac in polyme iza ion ollowing TCR liga ion.