ZAP-70: An Essential Kinase in T-cell Signaling

Haopeng Wang1, Theresa A. Kadlecek1, Byron B. Au-Yeung1, Hanna E. Sjölin Goodfellow1,

Lih-Yun Hsu1, Tanya S. Freedman1, and Arthur Weiss1
1
Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis,
Department of Medicine, University of California, San Francisco, San Francisco, California 94143
Correspondence: [email protected]

ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical role in the events involved
in initiating T-cell responses by the antigen receptor. Here we review the structure of ZAP-70,
its regulation, its role in development and in disease. We also describe a model experimental
system in which ZAP-70 function can be interrupted by a small chemical inhibitor.

pproximately 18 years ago, a T-cell receptor and activation, much effort has been focused on
A (TCR)–z chain-associated 70 kDa tyrosine
phosphoprotein (therefore termed ZAP-70) was
studying the regulation, structure, and function
of ZAP-70.
identified in TCR-stimulated Jurkat cells (Chan How does ZAP-70 mediate TCR signaling?
et al. 1991). The importance of ZAP-70, a cyto- Our lab proposed a model by which the TCR
plasmic tyrosine kinase mainly expressed in T initiates signaling via sequential interactions
cells, was rapidly revealed by the following ob- with cytoplasmic tyrosine kinases (Weiss 1993).
servations: ZAP-70-deficient patients have no When the TCR interacts with peptide antigen
functional T cells in their peripheral blood and bound to a MHC complex molecule on antigen
suffer from severe combined immunodeficiency presenting cells, coreceptor-associated Lck is
(SCID) (Arpaia et al. 1994; Chan et al. 1994; brought into proximity of the CD3 complex and
Elder et al. 1994); T-cell development in ZAP-70 phosphorylates tyrosines in the immunoreceptor
deficient mice is arrested at the transition from tyrosine-based activation motifs (ITAMs). When
the CD4 þ CD8 þ double positive (DP) stage, doubly phosphorylated, ITAMs recruit ZAP-
where positive selection occurs (Negishi et al. 70 via a relatively high affinity interaction by
1995b; Kadlecek et al. 1998); and a Jurkat-derived binding to the tandem SH2 domains of ZAP-
cell line (p116) deficient in ZAP-70 expression 70. Recent studies suggest that this binding event
fails to activate downstream signaling events after leads to the release of ZAP-70 from its autoin-
TCR stimulation (Williams et al. 1998). Because hibited conformation, which exposes the regu-
ZAP-70 plays a critical role in T-cell development latory phosphorylation sites for Lck-mediated

Editors: Lawrence E. Samelson and Andrey Shaw

Additional Perspectives on Immunoreceptor Signaling available at www.cshperspectives.org
Copyright # 2010 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a002279
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

1
H. Wang et al.

phosphorylation. In addition, tyrosines in the STRUCTURE OF ZAP-70

activation loop of the ZAP-70 kinase domain
are then phosphorylated by Lck or by ZAP-70 ZAP-70 is composed of two SH2 domains and
itself in trans to further promote its catalytic a carboxy-terminal kinase domain (Fig. 2A).
activity. A number of signaling proteins, includ- Unlike Src-family kinases with an amino-
ing the linker for the activation of T cells (LAT) terminal SH3-domain and a SH2-domain, the
and the SH2-domain-containing leukocyte amino terminus of the spleen tyrosine kinase
protein of 76 kDa (SLP-76), are subsequently (Syk) family of kinases, of which ZAP-70 is a
phosphorylated by active ZAP-70. The phos- member, consists of two tandem SH2 domains
phorylated LAT and SLP-76 proteins function that are separated by a linker region, termed
as scaffolds to recruit many other signaling mol- interdomain A. In ZAP-70, these SH2 domains
ecules (Fig. 1). The consequences of these early bind to doubly phosphorylated ITAMs of TCR z
signaling events eventually lead to T-cell activa- chain, and are responsible for bridging ZAP-70
tion, proliferation, and differentiation. to the activated TCR. Another linker region,
Here we discuss the structure and regulation interdomain B, connects the SH2 domains to
of ZAP-70, as well as the role of ZAP-70 in T-cell the kinase domain (Chan et al. 1992).
development and disease. We also highlight The crystal structure of a truncated version
a recently developed chemical-genetic approach of ZAP-70 containing the tandem SH2 domains
of inhibiting the ZAP-70 kinase activity and its complexed with a doubly tyrosine phosphory-
potential usage in studying the role of ZAP-70 lated ITAM peptide reveals interesting features
in T-cell responses, both in vitro and in vivo, about its structure and clues to its regulation.
which will facilitate and justify further effort The most unusual feature of the ZAP-70 SH2
in developing an inhibitor of wild-type ZAP- ITAM complex is that the phosphotyrosine
70 protein. binding (PTB) pocket of the amino-terminal

MHC
MHC MHC

CD4 CD4 CD4

TCR TCR TCR
α β α β α β
ε ε ε ε ε ε
δ γ δ γ δ γ
CD3

Lck Lck Lck

ZAP-70
ζζ ζ ζ ζζ

ZAP-70 ITAM motif ZAP-70 Phosphorylation

[(D/E)xxYxxI/Lx(6-8)YxxI/L]
of LAT & SLP76

Figure 1. A sequential model for T-cell activation. Following TCR engagement, CD4-associated Lck is brought
into proximity of the CD3 complex and phosphorylates the ITAMs ( phoshorylation depicted as red dots).
Doubly phosphorylated ITAMs then interact with the tandem SH2 domains of ZAP-70. After ITAM binding,
ZAP-70 can be phosphorylated by Lck, which results in activation of ZAP-70 catalytic activity and its
autophosphorylation. Active ZAP-70 subsequently phosphorylates LAT and SLP-76, which function as
scaffolds to recruit many other signaling molecules and lead to T-cell activation, proliferation, and
differentiation (not shown).

2 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

 ZAP-70

A Vav-1
c-Cbl CrkII Lck
292 315 319 492 493

Y Y Y Y Y
N-SH2 I-A C-SH2 I-B Kinase domain

Inactive ZAP-70

N-lobe
C-SH2
Y315F
Y319F

W131 P396

activation loop

C-lobe
N-SH2

B e
ob Tyr315
N-l Tyr492
Tyr493
Tyr319
N-lobe
be
C-lo
C-S C-
H2 SH
2
N-

ITAM binding
SH

N- C-lobe
SH
2

CD3 ITAM

Figure 2. Structural organization of autoinhibited ZAP-70, and model of autoinhibition. (A) Schematic of
ZAP-70 domains and phosphorylated tyrosine residues with binding molecules (top) and a crystal structure
of autoinhibited ZAP-70 (PDB code 2OZO, bottom). The amino-terminal SH2 domain, interdomain A,
carboxy-terminal SH2 domain, interdomain B, and the kinase domain are shown in cyan, yellow, blue, red,
and orange, respectively. The side chains of residues involved in the aromatic –aromatic interactions between
interdomain A, interdomain B, and the kinase domain are labeled and colored in dark blue. Dotted lines
represent disordered regions. (B) Model for the activation of ZAP-70 following ITAM binding. The SH2
domains, interdomain A, interdomain B, and kinase domains are colored as in (A). The doubly
phosphorylated ITAM is depicted in pink. When ZAP-70 shows an autoinhibited conformation it is
incompatible with binding to phosphorylated ITAMs. Following ITAM binding, conformational changes in
ZAP-70 promote disassembly of the interface mediating the autoinhibited conformation, and exposure of
tyrosines in interdomain B, leading to their phosphorylation that further destabilizes the interface.

Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279 3

H. Wang et al.

SH2 domain is formed by residues contributed fact that Y492 and Y493 are unphosphorylated.
from both the amino- and carboxy-terminal Its bilobed structure is well conserved compared
SH2 domains that are brought into juxtaposi- to the catalytic domains of many protein kin-
tion by ITAM binding. As in most SH2 domains, ases. Common to these kinases, ZAP-70 is com-
the carboxy-terminal SH2 PTB pocket is posed of an amino-terminal lobe containing a
completely composed of residues within the five-stranded antiparallel b-sheet and a single
carboxy-terminal SH2 domain. In addition, helix, and a carboxy-terminal lobe that is pre-
interdomain A appears to form a coiled-coil dominantly helical with short strands of b-
domain that facilitates extensive contact be- sheets. An extended linker connects these lobes,
tween the two SH2 domains and stabilizes their and the ATP-binding site is located at the junc-
interaction (Hatada et al. 1995). This structure tion between the two lobes (Jin et al. 2004).
suggests that the SH2 domains do not function The ability of the catalytic domain to adopt an
independently, and that interactions between active conformation without activation loop
the domains are important for specific recogni- phosphorylation suggests that additional mech-
tion of the phospho-ITAMs of the activated anisms are used in the full-length protein to reg-
TCR. Mutational studies addressing minimal ulate its activation.
spatial requirements between tyrosines in ZAP-70 contains several tyrosine residues
ITAM sequences for signaling also support this that are phosphorylated following TCR stimu-
model (Ottinger et al. 1998). lation and are known to be important in its
Comparison of the crystal structures and regulation, activity, and association with other
NMR analysis of the bound and unbound signaling molecules. Y292, Y315, and Y319 are
SH2 domains reveals two very different confor- located within interdomain B and when phos-
mations. Binding to phospho-ITAM peptide phorylated are thought to serve as docking sites
induces large movements between the SH2 for downstream signaling molecules (Fig. 2A).
domains, and the formation of the complete Regulation of ZAP-70 kinase activity is also
amino-terminal PTB pocket. Compared to a dependent on tyrosine residues in interdomain
more rigid structure in the bound form, inter- B. Mutation of both Y315 and Y319 to phenyl-
domain A shows a flexible structure in the ananine renders the kinase inactive. In contrast,
unbound form. This more rigid structure is mutation of these same residues to alanine re-
likely to contribute to the specificity of bind- sults in increased basal kinase activity (Brdicka
ing to the appropriately spaced and phosphory- et al. 2005). Additionally, some function is
lated ITAM (Folmer et al. 2002). Additionally, maintained in a mutant lacking most of inter-
studies using antibodies against regions within domain B, suggesting that it may be involved
interdomain A show binding to ZAP-70 only in an autoinhibitory mechanism (Zhao and
when it is not associated with the z chain, fur- Weiss 1996). It was hypothesized that mutation
ther enforcing the idea of a conformational of Y315 and Y319 to phenylalanine stabilizes an
change upon docking to the ITAM (Grazioli autoinhibited conformation and tyrosine phos-
et al. 1998). phorylation may impede the autoinhibited
Following TCR engagement, two tyrosine conformation (Brdicka et al. 2005).
residues located in the activation loop of the Indeed, the crystal structure of autoinhib-
kinase domain (Y492 and Y493) are phos- ited ZAP-70 has also been solved at 2.6 Ang-
phorylated. It is thought that this phosphory- strom resolution using a recombinant protein
lation displaces the activation loop from the with Y315 and Y319 in interdomain B muta-
catalytic site of the kinase, leading to complete ted to phenylalanine (Fig. 2A). In the crystal
activation of the kinase (Watts et al. 1994). The structure the catalytic domain in autoinhibited
crystal structure of a protein consisting of the ZAP-70 adopts a conformation that is charac-
kinase domain (aa327-606) in complex with the terized by many of the features of the inactive
ATP-competitive inhibitor Staurosporin, shows conformation of Src. Helix aC in the N-lobe
an active-like kinase conformation, despite the of the kinase domain is rotated away from the

4 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

 ZAP-70

active site, the salt bridge between Glu386 and mutants when the interface is disrupted (Deindl
Lys369 that is required for catalytic activity is et al. 2009).
disrupted, and a short 310-helix at the base of A multistep model for activation of ZAP-70
the activation loop packs against the displaced has been proposed that involves spatial and
helix aC. Although this structure contains temporal coordination of several key events
many common features of autoinhibited Src (Fig. 2B). In resting cells ZAP-70 is in a dynamic
family kinases, the mechanism for its autoin- equilibrium, and transitions in and out of the
hibitory regulation is novel. A hydrogen bond- autoinhibited conformation. Following TCR
ing network involving interdomains A and B stimulation and Lck-mediated phosphorylation
and the back of the kinase domain serves to sta- of tyrosine residues of the ITAM, the tandem
bilize the hinge region of the catalytic domain, SH2 domains of ZAP-70 can then bind the dou-
thereby stabilizing the inactive conformation. bly phosphorylated ITAM. The binding of the
This notion is supported by comparison of two SH2 domains requires their realignment
energy displacement data from the active and from the autoinhibited conformation. Thus,
inactive kinase domains. Two interactions serve a conformation of ZAP-70 that disrupts the
to stabilize the autoinhibited conformation. interactions stabilizing interdomain B interac-
First, two a-helices from interdomain A (viewed tion with interdomain A as well as with the cat-
as a single helix in the tandem SH2-ITAM alytic domain is favored, thereby destabilizing
bound structure) are docked onto the carboxy- the interactions key to the maintenance of the
terminal lobe of the kinase domain. A proline autoinhibited state of ZAP-70. This allows for
residue at the junction of these two is wedged increased accessibility and Lck-mediated phos-
between two tyrosines on the back of the phorylation of Y315 and Y319 in interdomain
C-lobe of the catalytic domain. Interdomain B B, phosphorylation of Y493 of the activation
is positioned between the kinase domain and loop of the kinase, and subsequent full acti-
interdomain A. Additionally, critical residues vation of the kinase.
stabilizing this conformation include a set of
perpendicular aromatic-aromatic interactions
between W131 and P296 with F315 and F319
that create a rather stable hydrophobic environ- REGULATION OF ZAP-70
ment (Deindl et al. 2007). The hydroxyls of
Recruitment of ZAP-70 to the TCR
Y315 and Y319 in the wild-type kinase would
presumably decrease the stability of the hydro- ZAP-70 activation can be regulated both by
phobic interactions in this region and allow binding to phosphorylated ITAMs of the TCR
for more conformational flexibility in wild-type and by phosphorylation of multiple tyrosine
ZAP-70. Of considerable interest, the alignment residues on ZAP-70 (Fig. 2A). Imaging studies
of the tandem SH2 domains in the auto- have shown that in resting T cells, ZAP-70
inhibited structure is incompatible with bind- is distributed throughout the cytoplasm, but
ing to phosphorylated ITAMs, because the is rapidly recruited to the plasma membrane
PTB pocket of the amino-terminal SH2 domain following TCR stimulation and ITAM phos-
is not appropriately apposed to the carboxy-ter- phorylation (Sloan-Lancaster et al. 1997; Sloan-
minal SH2 domain to complete the PTB site. Lancaster et al. 1998). ZAP-70 mobility in the
Studies of mutant kinases, predicted to interfere cytoplasm is rapid, and decreases as ZAP-70
with the interface interactions, that were overex- associates with the plasma membrane (Sloan-
pressed in 293Tepithelial cells and in the Jurkat Lancaster et al. 1998). Whereas the recruitment
cell line deficient in ZAP-70 validate this to the plasma membrane is enhanced by Lck
structure. In vitro kinase activity, TCR-induced activity and phosphorylation of the ITAMs,
tyrosine phosphorylation, intracellular Ca2þ involvement of other proteins such as the acti-
response, as well as ITAM peptide binding vated forms of ezrin and RhoH, a member of
were all increased compared to WT ZAP-70 in the Rho GTPase family, has also been reported

Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279 5

H. Wang et al.

(Sloan-Lancaster et al. 1997; Gu et al. 2006; Ilani phosphorylated. In addition to stabilization of

et al. 2007). the active conformation, these are docking sites
Following TCR ligation, TCR microclusters for other molecules controlling the function of
are formed in which signaling is initiated and ZAP-70 and downstream signaling. Phosphory-
sustained. The microclusters continue to form lated Y319 is a positive regulator of ZAP-70
in the periphery of the immune synapse (IS) function and is well documented to bind Lck.
and later translocate to the center of the synapse. Overexpression of Y319F ZAP-70 mutant in
ZAP-70 accumulates at these microclusters. Jurkat cells resulted in a dominant-negative
Although the initial clustering of TCR is inde- effect, with reduced TCR-triggered activation
pendent of tyrosine phosphorylation events, of NFAT and IL-2 induction. Further, it con-
the activity of Lck and ITAM phosphorylation ferred impaired up-regulation of ZAP-70 cata-
are required for ZAP-70 accumulation (Sloan- lytic activity and reduced phosphorylation
Lancaster et al. 1998; Campi et al. 2005; of its substrates LAT and SLP-76 (Di Bartolo
Yokosuka et al. 2005; Varma et al. 2006). et al. 1999). Additionally, Y319F ZAP-70 ex-
pressed in ZAP-70 deficient Jurkat cells failed
to bind Lck and to reconstitute TCR triggered
Positive Regulation of ZAP-70 Function
signaling (Williams et al. 1999). Further, muta-
Although critical, binding to TCR-z and pertur- tions of the Lck SH2 domain abrogated binding
bation of the autoinhibitory conformation of Lck to ZAP-70 and T-cell activation (Pelosi
alone is insufficient for complete ZAP-70 acti- et al. 1999; Williams et al. 1999). The binding
vation (van Oers et al. 1994; Chan et al. 1995; of Lck to phosphorylated Y319 may thus help
Zhao et al. 1999; Jin et al. 2004). Phosphory- prevent reversion to the autoinhibitory con-
lation of tyrosine residues, by the action of Src formation, promote Lck mediated phosphory-
family kinases such as Lck, and/or by ZAP-70 lation of Y493, and facilitate activation of
itself, is also important. Requirements for full downstream signaling. The importance of
catalytic activity include phosphorylation of Y319 phosphorylation in positive regulation
tyrosine residue Y493 in the activation loop of ZAP-70 was confirmed in a mouse model
of the kinase domain of ZAP-70. Y493 is a expressing Y319F ZAP-70, which resulted in
preferred phosphorylation site for Lck but can severe defects in calcium mobilization and
also be a substrate for ZAP-70 autophosphory- thymocyte selection (Gong et al. 2001).
lation (Watts et al. 1994; Chan et al. 1995; Kong Vav-1 has been shown to interact with ZAP-
et al. 1996). If phosphorylation is prevented 70 via its SH2 domain, and mutation of Y315
by mutation of this residue to phenylalanine, to phenylalanine abrogates binding of Vav to
Lck is unable to activate ZAP-70 (Chan et al. ZAP-70 in DT40 cells and causes a decrease in
1995; Wange et al. 1995). Reconstitution studies antigen receptor mediated tyrosine phosphory-
of the Syk deficient DT40 B-cell line showed lation of Vav, SLP-76, Shc and ZAP-70 itself
that Lck mediates phosphorylation of Y493 (Katzav et al. 1994; Wu et al. 1997). Further,
and that catalytic activity of ZAP-70 is required although not having large effects on intrinsic
for downstream signaling (Chan et al. 1995; kinase activity, the Y315F mutation was re-
Kong et al. 1996). When overexpressed in the ported to alter ZAP-70 binding affinity for phos-
Jurkat T-cell line, ZAP-70 mutated both on phorylated ITAMs, reducing its capacity to
Y492 and Y493 to phenylalanines (Y492F, trigger Erk activation in murine T cells (Di
Y493F) impaired TCR-mediated signaling. How- Bartolo et al. 1999). This suggested that the
ever, the mutant was able to bind Lck, suggest- Vav/ZAP-70 interaction via phospho-Y315
ing that Lck association with ZAP-70 is not promotes ZAP-70 function. However, because
dependent on activation loop phosphorylation Vav phosphorylation was not affected by the
(Mege et al. 1996). ZAP-70 mutation in vivo, the importance of
Following TCR engagement, the tyrosine Vav SH2 binding to ZAP-70 at Y315 for its phos-
residues Y315 and Y319 in interdomain B are phorylation is not clear (Magnan et al. 2001).

6 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

 ZAP-70

Actin polymerization may be influenced by involved in ZAP-70 association with the Crk
ZAP-70 interaction with members of the CT10 family member, CrkII, which has been impli-
regulator of kinase (Crk) family. Crk adaptor cated in negative regulation of TCR signaling.
protein can bind to phospho-Y315 on ZAP- Lck-dependent CrkII interaction with ZAP-70
70. A model has been proposed in which phos- was detected after pervanadate treatment of
phorylated ZAP-70 associates with Crk-like Jurkat cells, and mutation of Y315 to phenyl-
(CrkL)-WIP-WASP complexes allowing redis- alanine eliminated this association (Gelkop and
tribution of the complex to lipid rafts and Isakov 1999; Gelkop et al. 2005). One possibility
the IS following TCR triggering. PKCu, whose is that negative regulation by CrkII is exerted by
activity is indirectly dependent on Lck and complex formation between Crk, p85 PI3K, and
ZAP-70, can phosphorylate WIP thereby re- the E3 ubiquitin ligase Cbl (Gelkop et al. 2001).
leasing WASP from WIP-mediated inhibition. Several studies have also described a nega-
WASP can then be activated by membrane tive regulatory role for Y292 in interdomain
bound Cdc42, leading to actin polymerization, B. Mutation of Y292 to phenylalanine enhanced
whereas WIP binds newly formed actin and NFAT induction in DT40 cells in response to
thus helps to stabilize the IS (Sasahara et al. BCR stimulation, without directly affecting ki-
2002). Studies of mouse models have further nase activity or TCR-z binding. Phosphorylation
confirmed a detectable, though modest, posi- of Y292 may therefore promote association
tive regulatory role of Y315 in T-cell activation of ZAP-70 with a negative regulatory protein
and thymocyte selection (Gong et al. 2001; (Zhao and Weiss 1996). One strong candidate
Magnan et al. 2001). Whether this is mediated is c-Cbl, known to diminish TCR-mediated acti-
by specific interactions of ZAP-70 with Vav, vation (Murphy et al. 1998; Naramura et al.
CrkL or other molecules, in addition to stabi- 1998). ZAP-70 has been reported to associate
lization of an active conformation, has yet to with Cbl following TCR stimulation and phos-
be clarified. phorylation of Y292 was required for this inter-
action, as well as for the negative effect of Cbl on
signaling (Lupher et al. 1996; Lupher et al. 1997;
Negative Regulation of ZAP-70 Function
Rao et al. 2000; Magnan et al. 2001). Cbl may
Phosphorylation of specific tyrosine residues promote ubiquitination and degradation of
has also been implicated in negative regulation TCR-z using ZAP-70 as an adaptor protein,
of ZAP-70 function. Y492 is phosphorylated possibly also affecting ZAP-70 function (Wang
following TCR triggering. Mutation of this tyro- et al. 2001; Naramura et al. 2002; Davanture
sine to phenylalanine elevates ZAP-70 kinase et al. 2005). However, because of discrepancies
activity, showing a negative regulatory role for in phenotypes of c-Cbl knockout mice, mice ex-
this site. Furthermore, Y492F mutant ZAP-70 pressing the ZAP70 Y292F mutation, and mice
was able to restore antigen receptor signaling expressing a mutation in the tyrosine kinase
in Syk deficient DT40 B cell line, indicating binding domain of c-Cbl, the mechanism for
that phosphorylation of this tyrosine residue is the regulatory effect of Y292 remains undefined
not required for ZAP-70 function (Watts et al. (Murphy et al. 1998; Naramura et al. 1998;
1994; Chan et al. 1995; Wange et al. 1995). Magnan et al. 2001; Thien et al. 2003).
Y492, like Y493, is positioned in the activation
loop of the kinase domain, and could thus
SYK FAMILY KINASES AND THEIR ROLE IN
potentially influence accessibility of the cata-
T-CELL DEVELOPMENT
lytic site. However, the exact mechanism for
Y492 influence on kinase activity has yet to be ZAP-70 and Syk, the only two members of the
revealed (Chan et al. 1995; Wange et al. 1995; Syk kinase family, provide both overlapping
Jin et al. 2004). and distinct functions in immunoreceptor
Although able to recruit positive effectors signaling. Both ZAP-70 and Syk consist of
of TCR signaling, Y315 was also shown to be two tandem N-terminal SH2 domains and a

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H. Wang et al.

carboxy-terminal kinase domain. Although peptide in the amino-terminal PTB pocket is

the tandem SH2 domains of the two kinases closely associated with the side chain of Y238
share 57% sequence identity, crystal structures and K242 from the C-terminal SH2 domain
of the tandem SH2 domains complexed with a (Fig. 3A), revealing that the amino-terminal
phosphorylated ITAM peptide reveal important SH2 domain requires residues of the carboxy-
differences between ZAP-70 and Syk. As dis- terminal SH2 domain to complete this PTB
cussed earlier, in the ITAM-bound state, both pocket. These features of the ZAP-70 structure
SH2 domains of ZAP-70 are tightly apposed, confer high selectivity for interaction with the
making extensive contacts with each other and activated TCR:CD3 complex (Hatada et al.
the phosphorylated ITAM. The amino-terminal 1995). Surprisingly, the PTB site of Syk is
SH2 domain of ZAP-70, unlike its carboxy- self-contained within the amino-terminal SH2
terminal SH2 domain, has an incomplete PTB domain. The interaction between phosphotyro-
pocket. The phosphotyrosine of the ITAM sine in the amino-terminal PTB site and

A N-terminal phospho-tyrosine binding pocket

ZAP-70 Syk

B β selection Positive selection

DN1 DN2 DN3 DN4 ISP DP SP

CD44+ CD44+ CD44– CD44– CD8+ CD4+ CD4 + CD8–/

CD25– CD25+ CD25+ CD25– TCRβ– CD8+ CD4 – CD8+

Expression:
Syk
ZAP-70

Function:
Syk
ZAP-70
Figure 3. Comparison of ZAP-70 with Syk. (A) Cartoon structures of the amino-terminal phosphotyrosine
binding pocket in ZAP-70 (PDB code 2OQ1, left) and in Syk (PDB code 1A81, right). The amino-terminal
SH2 domain, carboxy-terminal SH2 domain, and phosphorylated ITAM peptide are shown in cyan, blue,
and pink, respectively. There are six different conformations in the crystal asymmetric unit of Syk. The Syk
structure shown here is representative of three of six conformations. In the other three conformations, only
one residue (Lysine) in the carboxy-terminal SH2 domain seems to interact with the phosphotyrosine of the
ITAM. However, the electron density for the side-chain of this Lysine is absent in all three conformations
(not shown), suggesting that this interaction may not contribute significantly to the binding. (B) Expression
and function of Syk family kinases throughout T-cell development.

8 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

 ZAP-70

residues of carboxy-terminal SH2 domain competitive repopulation assays using ZAP-70-

is significantly reduced compared to ZAP-70 deficient or Syk-deficient versus WT hemato-
(Fig. 3A), suggesting that the amino- and poietic stem cells were performed in irradiated
carboxy-terminal SH2 domains of Syk can host mice. The Syk-deficient, but not ZAP-70-
function independently (Futterer et al. 1998; deficient, thymocytes are specifically impaired
Kumaran et al. 2003). In addition, the asym- in initial pre-TCR signaling and cell-cycle
metric unit of Syk crystals contains six different entry at the DN3 stage. Despite overlapping
conformations, reflecting a remarkable flexibil- expression of both kinases, only ZAP-70 is
ity in the relative orientation of the two tandem responsible for sustained pre-TCR/TCR sig-
SH2 domains. These features may provide the naling and cellular proliferation during the
molecular basis for Syk interaction with a DN4, ISP and DP stages (Palacios and Weiss
greater variety of ITAMs and other binding part- 2007). These data suggest a model where Syk-
ners that vary considerably in the length of the dependent pre-TCR signaling is replaced with
spacer region between the two phosphotyro- ZAP-70-dependent pre-TCR signaling in early
sines (Futterer et al. 1998; Kumaran et al. 2003). T-cell development (Fig. 3B). To further test
Although both Syk and ZAP-70 are ex- this model, the generation of a knockin mouse
pressed during T-cell development, they have in which Syk is targeted into the ZAP-70 locus
distinct expression patterns and play different could provide further insights. ZAP-70-like
roles. The early thymic progenitors transit expression of Syk within DN4, ISPand DP stages
through four stages as CD4/CD8 double nega- would allow testing of whether ZAP-70 has a
tive (DN) cells (DN1 – 4) before up-regulating unique function in generating DP thymocytes
CD8 and CD4 to become double positive before positive selection.
(DP) thymocytes, and finally become single
positive (SP) thymocytes. Thymocyte matura-
tion requires two critical checkpoints: pre-TCR ZAP-70 AND DISEASE
mediated b-selection at the DN3 stage and
Chronic Lymphocytic Leukemia (CLL)
TCR-mediated positive/negative selection at
the DP stage. Syk and ZAP-70 proteins are B-chronic leukemia (B-CLL) is a B-cell malig-
inversely expressed across T-cell development. nancy caused by accumulation of monoclonal
Whereas Syk is highly expressed in early devel- CD5þ B lymphocytes in the blood, bone mar-
opment (DN1-DN3), ZAP-70 is highly ex- row, lymph nodes, and spleen (Chiorazzi et al.
pressed in later development (after DN4). 2005). Early studies showed that the mutational
However, there is overlapping expression of status of the immunoglobulin heavy chains of
both kinases during some transitory stages, B-CLL was associated with a distinctly different
including the DN3, DN4, and “immature prognosis, with 50 percent of cases in each
single-positive” (ISP) stages (Palacios and Weiss group. Subsequent gene expression profiling
2007). The positive/negative selection at the DP showed that ZAP-70 expression was the most
stage is completely arrested in ZAP-70 knockout discriminating feature between the two sub-
mice but is not affected by Syk deficiency types (Rosenwald et al. 2001). In fact, ZAP-70
(Negishi et al. 1995b). Neither ZAP-70- nor protein expression in B-CLL patients appears
Syk-deficient thymocytes have any obvious to have more predictive value than IgVh muta-
defect at the b-selection stage; however, the tions, and its expression level appears to be
double knockout of both kinases results in an constant during the course of disease (Rassenti
absolute block at this stage, indicating that Syk et al. 2004).
and ZAP-70 kinases can be functionally redun- Despite the prognostic value of ZAP-70 ex-
dant during pre-TCR signaling at the DN3 stage pression in B-CLL, the presence of what might
(Cheng et al. 1997). be considered a T-lineage specific gene in B-
To determine whether Syk or ZAP-70 is pref- cell lineage derived leukemic cells is unexpected.
erentially used to mediate pre-TCR signaling, Whether ZAP-70 expression plays a functional

Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279 9

H. Wang et al.

role in B-CLL progression is unclear. ZAP-70 facilitating BCR recruitment to Lyn. In support
expression in B-CLL is associated with enhan- of this notion, ZAP-70 expression in B-CLL is
ced BCR signaling (Chen et al. 2002). ZAP-70 associated with efficient translocation of BCR
can couple to the BCR and up-regulate Syk to lipid rafts in response to BCR engagement.
phosphorylation and activation as well as down- As a result, phosphorylation of the BCR by
stream signaling events. Transduction of ZAP- lipid-raft-associated Lyn kinase is significantly
70-deficient B-CLL cells with adenoviral vectors stronger in ZAP-70þ CLL cells (Allsup et al.
expressing ZAP-70 increased BCR-induced 2005). Signaling enhancement of Syk by ZAP-
tyrosine phosphorylation of several BCR signal- 70 in B-CLL may represent an amplification
ing molecules as well as mobilization of intra- mechanism that compensates for the low
cellular calcium (Chen et al. 2005). Together, expression of IgM and CD79 in B-CLL.
these results suggest that the function of ZAP- In summary, ZAP-70 can enhance the intra-
70 in CLL cells may be to enhance BCR cellular signaling capacity of the Ig expressed in
signaling. CLL. Whether such increased intracellular sig-
The finding that ZAP-70 can enhance BCR naling influences the survival or proliferation
signaling in CLL B cells seems counterintuitive, of CLL cells, leading to disease progression
given that there are similar levels of Syk ex- remains unknown. However, ZAP-70 is an
pression in both ZAP-70þ or ZAP-702 CLL attractive candidate for treatment, owing to its
B cells and Syk has approximately 100-fold grea- participation in BCR signaling in CLL.
ter in vitro kinase activity than does ZAP-
70 (Latour et al. 1996). A recent study showed
SCID
that ZAP-70 promotes BCR signaling inde-
pendently of its kinase activity, as both wild- Loss of function or expression of ZAP-70 in
type ZAP-70 and a catalytically inactive humans leads to SCID. This form of SCID is
ZAP-70 mutant induced similar increases in characterized by normal numbers of nonfunc-
calcium and protein tyrosine phosphoryla- tional CD4þ T cells and an absence of CD8þ
tion (Chen et al. 2008). Consistent with these T cells in the peripheral blood of these patients
observations, the interaction between ZAP-70 (Elder et al. 1994; Hivroz and Fischer 1994).
and downstream effector molecules appeared As shown in Figure 4, the ZAP-70 mutations
unchanged in the CLL B cells (Gobessi et al. occurring in human SCID, which abolish
2007). Together, these results suggest that ZAP-70 expression, are mostly located in the
ZAP-70 may function as an adaptor protein to kinase domain (Arpaia et al. 1994; Chan et al.
facilitate BCR signaling or compete for a nega- 1994; Elder et al. 1994; Gelfand et al. 1995;
tive regulator of Syk. Matsuda et al. 1999; Noraz et al. 2000; Toyabe
How might kinase-inactive ZAP-70 enhance et al. 2001). Mutations causing transcriptional
Syk activation? Previous studies of DP thymo- loss or destabilized protein have also been
cytes showed that ZAP-70, rather than Syk, reported (Fig. 4) (Gelfand et al. 1995; Matsuda
promotes more efficient TCR-z chain phospho- et al. 1999; Noraz et al. 2000). A patient with a
rylation by recruiting Lck into the TCR-z com- missense mutation within the highly conserved
plex (Ashe et al. 1999). The role of ZAP-70 DLAARN motif of kinase domain has been
in augmenting TCR ITAM phosphorylation reported, resulting in a moderate decrease in
occurs when expression of Lck and the TCR ZAP-70 stability and complete absence of kinase
are limiting in DP thymocytes, and is independ- activity (Elder et al. 2001). Interestingly, an
ent of its kinase activity. Similarly, B-CLL cells identical spontaneous mutation in DLAARN
express lower levels of IgM and CD79b. There- motif has also been reported in mice (Wiest
fore, it is conceivable that, when limiting num- et al. 1997), but still leads to a discordant
bers of CD79b ITAMs are available in B-CLL phenotype of the peripheral T-cell compart-
cells, ZAP-70 positively affects Syk activity by ment of humans and mice.

10 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

 ZAP-70

Human muations 1 2 3/4 567 8 9

N-SH2 I-A C-SH2 I-B Kinase domain
Mouse muations a b c d
Human SCID mutations
Mutant allele DNA mutation Consequence to protein
1 transcript defect No protein
2 C to A transition at nucl. 448 Missense (P to Q at residue 80, temperature-sensitive allele)
3 C to T transition at nucl. 1602 Missense (R to C at residue 465)
4 G to A transition at nucl. 1603 Missense (R to H at residue 465)
5 13 bp deletion (nucl. 1719-1731) Deletion (premature termination at residue 538)
6 C to T transition at nucl. 1729 Missense (A to V at residue 507)
7 C to A transition at nucl. 1763 Missense (S to R at residue 518)
8 G to A transition at nucl. 1833 Splicing error (9 bp in-frame addition; L-E-Q 3 addition at residue 541)
9 A to T transition at nucl. 1923 Missense (M to L at residue 572, temperature-sensitive allele)

Hypomorphic ZAP-70 mutants

Mutant mouse Mutant Name Mutation Autoimmune phenotype

a SKG W to C at residue 163 autoimmune arthritis, hyperglobulinemia, RF autoantibodies

b YYAA Y to A at residue 315 and 319 no arthritis, RF autoantibodies
mrd/mrd
c ZAP-70 I to F at residue 367 no anti-DNA autoantibody, no hyper-IgE
mrt/mrt
d ZAP-70 W to R at residue 504 no anti-DNA autoantibody, no hyper-IgE
mrd/mrt
c&d ZAP-70 both I367F and W504R anti-DNA autoantibody, hyper-IgE

Figure 4. Mutations of ZAP-70 in Human SCID and hypomorphic mutant mice.

Regardless of the mutation, the lack of CD4þ thymocytes from the patients are able
ZAP-70 or complete loss of its function affects to transduce signals via their TCRs as measured
T-cell maturation differently in humans and by calcium flux and tyrosine phosphorylation
in mice. In ZAP-70-deficient mice, T-cell devel- (Gelfand et al. 1995). The differential signaling
opment is blocked at the DP stage of thymocyte capacity between CD4þ thymocytes and pe-
differentiation, resulting in no mature T cells in ripheral T cells may be explained by the obser-
the periphery (Negishi et al. 1995a; Kadlecek vations that Syk is present at higher levels
et al. 1998). The selective deficiency of pe- and later in development in human thymocytes
ripheral CD8þ cells in humans is caused by a compared to mouse thymocytes (Chu et al.
defect in thymic T-cell development. In a thy- 1998; Chu et al. 1999). These findings suggest
mic section from a ZAP-70 deficient SCID that higher levels of Syk in human thymocytes
patient, DP T cells are present in the thymic cor- may be able to partially compensate for the
tex, but only CD8þSP cells are absent from the loss of ZAP-70 in SCID patients’ CD4þ thymo-
medulla (Arpaia et al. 1994). This observation cytes but not in peripheral T cells. Further
suggests that positive selection of CD8þ cells down-regulation of Syk expression in the
is specifically blocked. It is possible that human peripheral T cells and/or the absolute
ZAP-70 plays different roles in selection of requirement for ZAP-70 may explain the signal-
human CD4þ and CD8þ T cells and is indis- ing defect seen in peripheral CD4þ T cells lack-
pensable for maturation into CD8þ T cells. ing ZAP-70. The mechanism for the preferential
Although positive selection of CD4 SP cells selection of CD4þ cells remains unclear. It
occurs in these patients, the peripheral CD4þ T is possible that greater association of Lck
cells do not respond normally to mitogens or to with CD4 rather than CD8 may allow more
stimulation by anti-CD3 antibodies, as shown efficient activation of ZAP-70, sufficient to
by a failure to proliferate and produce IL-2. overcome a threshold leading to CD4 but not
Interestingly, unlike peripheral CD4þ T cells, CD8 lineage selection (Wiest et al. 1993). Taken

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H. Wang et al.

together, studies on human ZAP-70 immuno- B of ZAP-70. YYAA mice show defects similar
deficiency reveal the critical role of ZAP-70 sig- to SKG mice. For instance, YYAA mice show
naling both in mature T cells and in thymic diminished TCR signaling, impaired T-cell
development. development, and defective positive and nega-
tive selection. However, in contrast to the
SKG mice, which develop both arthritis and
Hypomorphic ZAP-70 Mutant Mice rheumatoid factor autoantibodies on the
Balb/c background, YYAA mice only develop
Although ZAP-70 deficiency typically leads to rheumatoid factor autoantibody. Quite sur-
the complete absence of T cells in the periphery prisingly, in addition to impaired negative
of mice, several ZAP-70 mutant mice with par- selection seen in the YYAA mice, the suppressive
tial defects in TCR signaling have been reported. activity of Tregulatory cells in these mice is also
The SKG mouse has a spontaneous missense profoundly defective. This raises the question of
mutation (W163C) in the C-terminal SH2 why very limited autoimmunity is observed in
domain of ZAP-70 and develops autoimmune the YYAA mice. Based on the extent of deletion
arthritis in response to innate immune activa- of Vb TCR genes to endogenous superantigens,
tion (Sakaguchi et al. 2003). This mutation our findings suggest that TCR-mediated nega-
greatly impairs the binding of ZAP-70 to the tive selection is more impaired in the SKG
TCR-z chain, thereby altering TCR signaling than in the YYAA mice. Thus, it appears that
and affecting thymic development, particularly different TCR repertoires result from different
positive and negative selection, and the dif- sensitivity to negative selection in these mice.
ferentiation of T cells. As a consequence of The difference in TCR repertoires may be
impaired negative selection, it is thought that responsible for their different susceptibility to
the thymocytes showing the strongest affinity arthritis.
for self-peptide/MHC ligands are positively Recently, a hypomorphic allelic series of
selected instead of being deleted. This presum- ZAP-70 have been generated through N-ethyl-
ably leads to a shift of T-cell repertoire that N-nitrosourea (ENU)-induced mutations, and
would otherwise have been eliminated during these mutations are located within the kinase
negative selection in WT mice. Adoptive trans- domain of ZAP-70 (Siggs et al. 2007). A graded
fer studies have suggested that expansion of change in TCR signaling potential created by
these autoreactive CD4þ T cells that escaped combining two ZAP-70 hypomorphic muta-
negative selection is a key cause of autoimmune tions (mrd and mrt) has revealed that the bal-
arthritis in SKG mice. SKG arthritis resembles ance between thymic selection and function of
human rheumatoid arthritis (RA) in many effector T cells is differentially regulated. In
ways, such as presentation of autoimmune arth- these ZAP-70 allelic mutants, this results in
ritis, hyper-g-globulinemia, vasculitis, nodule the coincident occurrence of both autoimmun-
formation, and rheumatoid factor production ity (spontaneous production of anti-DNA
(Sakaguchi et al. 2006). Therefore, SKG mice autoantibodies) and immune dysregulation
may be a suitable model for understanding the (hyper-IgE syndrome). Interestingly, although
cellular mechanism for autoimmune arthritis both SKG mice and ZAPmrt/mrd mice show
and might also provide useful insights into the defective negative selection, they show different
development of autoimmunity in general. autoimmune syndromes. SKG mice develop
Another hypomorphic ZAP-70 mutant arthritis but not anti-DNA autoantibody,
mouse has recently been reported by our lab whereas ZAPmrt/mrd mice develop anti-DNA
and has revealed mechanistic insights into antibody but not arthritis. There are two possi-
how signaling defects may result in autoimmu- bilities that might explain the difference in
nity or autoimmune disease (Hsu et al. 2009). autoimmune syndromes in SKG, YYAA and
This ZAP-70 YYAA strain has knockin alanine ZAPmrt/mrd mice. First, the genetic background
mutations of Y315 and Y319 in the interdomain could play a role in the resultant phenotypes.

12 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

 ZAP-70

Alternatively, quantitative and qualitative dif- mutant kinase to accommodate a larger analog
ferences in TCR signaling resulting from ZAP- of PP1, one that would be too large to inhibit
70 mutations may skew the TCR repertoire in the WT kinase (Bishop et al. 1998). This tech-
ways that manifest as different autoimmune nique has allowed our lab to study the require-
syndromes in these mice. Collectively, ZAP-70 ments for ZAP-70 kinase activity by T cells for
hypomorphic alleles extend our understanding productive TCR signaling leading to T-cell
of how a quantitative difference in T-cell sig- activation.
naling may influence the outcome of immune An analog-sensitive ZAP-70 (ZAP-70AS)
responses as well as identify a threshold bet- mutant was generated by mutating the gate-
ween susceptibility toward autoimmunity ver- keeper residue M414, to alanine, along with a
sus autoimmune disease. second mutation, C405V, to stabilize the ATP-
binding domain (Levin et al. 2008) (Fig. 5).
Expression of ZAP-70AS in ZAP-70 deficient
ZAP-70 AS A THERAPEUTIC TARGET
P116 Jurkat T cells showed that, indeed, expres-
Because of its critical role in TCR signaling, sion of ZAP-70AS could reconstitute TCR sig-
ZAP-70 is an attractive target for therapy. A spe- naling. Additionally, stimulation of ZAP-70AS-
cific inhibitor for ZAP-70 could potentially expressing Jurkat cells in the presence of the
block T-cell activation at a level of signal trans- inhibitor 3-MB-PP1 resulted in a rapid, dose-
duction very proximal to the TCR. Additionally, dependent inhibition of downstream signals,
ZAP-70 expression is relatively restricted to T including phosphorylation of LAT and SLP-76,
cells, NK cells, and basophils (Au-Yeung et al. as well as impaired calcium mobilization and
2009). Thus, effects of specific ZAP-70 inhi- ERK phosphorylation. Furthermore, the
bition would be limited to these cell types. ZAP-70AS system showed the dynamic role of
However, NK cells and basophils also express ZAP-70 catalytic activity in the regulation of
Syk, which may be able to compensate for the the calcium flux, as inhibitor addition at the
lack of ZAP-70. This appears to be the case plateau of the calcium response resulted in a
for ZAP-70 deficient NK cells, which are still rapid decrease in intracellular free Ca2þ down
able to exert cytolytic activity (Negishi et al. to basal levels within seconds.
1995b). This suggests that inhibition of ZAP- These studies further highlight the critical
70 activity may specifically target T-cell res- importance of ZAP-70 kinase activity in the
ponses, while leaving innate immunity intact. transduction of signals from the TCR. In
Such an inhibitor could be particularly useful in the future, it will be of great interest to test the
settings of allograft rejection or T-cell-mediated ZAP-70AS system in mature, primary T cells.
autoimmunity. Such experiments will enable investigation of
To date, the development of a highly specific, whether ZAP-70 activity is required for naı̈ve,
cell permeable small molecule inhibitor of ZAP- effector, and memory T-cell responses. Addi-
70 has been difficult. Although some potential tionally, using the ZAP-70AS system may
ZAP-70 inhibitors have been described (Hira- eventually allow us to model the effects of a
bayashi et al. 2009), their specificity and potency ZAP-70 inhibitor in pathological T-cell re-
in T cells or in vivo have not been shown. sponses in vivo. Until then, however, the analog-
In the absence of an available inhibitor, sensitive approach could be applied to the study
our lab has engineered a ZAP-70 mutant that of other signaling kinases to analyze the tempo-
retains catalytic activity, yet can be inhibited ral requirements for kinase activity in TCR
by an analog of the kinase inhibitor PP1. This signal transduction and T-cell activation.
chemical-genetic approach takes advantage of
the presence of a conserved, bulky “gatekeeper”
ACKNOWLEDGMENTS
residue within the ATP-binding region of
tyrosine kinases. Mutation of the bulky gate- H.W., T.A.K., B.B.A-Y., H.E.S.G., and L-Y.H.
keeper residue to glycine or alanine allows the contributed equally to this work.

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H. Wang et al.

A ZAP-70 kinase domain B

C-lobe N-lobe

3-MB-PP1

ATP-binding site

C
‘gatekeeper’ residue steric clash-
WT ZAP-70 WT ZAP-70 bulky inhibitor
cannot fit

3-MB-PP1

ZAP-70 M414A ZAP-70 M414A

(analog-sensitive) (analog-sensitive)

3-MB-PP1

Figure 5. Chemical-genetic strategy to generate an inhibitor-sensitive ZAP-70. (A) Structure of the ZAP-70
kinase domain (PDB code 1U59). C- and N-lobes are indicated. The ATP-binding site is boxed. (B)
Chemical structure of the inhibitor 3-MB-PP1. (C) Schematic of the analog-sensitive strategy. Left, Diagrams
show the WT (top) and analog-sensitive mutant (bottom) ZAP-70 ATP-binding regions, modeled using
PyMol. Circles identify the region of the gatekeeper residue. Right, Diagrams show the model of 3-MB-PP1
binding within the WT (top) and analog-sensitive mutant (bottom) ZAP-70. The presence of the
“gatekeeper” residue in the ATP-binding site of WT ZAP-70 prevents the binding of the bulky 3-MB-PP1
inhibitor. In the analog-sensitive ZAP-70 mutant (bottom), the enlarged ATP-binding pocket allows binding
of 3-MB-PP1. The 3-MB-PP1 model is derived from the crystal structure of the kinase domain of
cryptosporidium parvum calcium dependent protein kinase in complex with 3-MB-PP1 (PDB code 2WEI).
Top, white arrow points to the steric clash between 3-MB-PP1 and the gatekeeper residue, indicating that
3-MB-PP1 is not compatible with binding to WT ZAP-70.

14 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002279

 ZAP-70

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