269

Adaptor proteins in lymphocyte activation

Erin Janssen
and Weiguo Zhangy

Adaptor proteins are unique, as they contain modular domains They differ from other signaling proteins as they lack
and lack intrinsic enzymatic activity. These proteins are scaffolds inherent catalytic activity. Instead, adaptors are primar-
for the organization of macromolecular complexes and they ily composed of modular domains that bind and recruit
recruit other proteins for correct localization during molecular other signaling proteins. These modules include Src
signal transduction. Numerous recent advances have been homology 2 (SH2) and PTB (phosphotyrosine-binding)
made through the elucidation of new adaptor proteins and the domains that recognize short phosphotyrosine (pY)
recognition of novel functions for previously identified molecules. motifs, SH3 and WW domains that bind proline-rich
In addition, the roles of adaptors in both the positive and negative (PR) regions, and pleckstrin homology (PH) domains
regulation of lymphocyte activation have been further clarified. that associate with phosphoinositides (reviewed in [1]).
Many adaptors contain multiple domains and act as
Addresses scaffolds to facilitate the formation of macromolecular
Department of Immunology, Duke University Medical Center, signaling complexes (Figure 1). Numerous comprehen-
Durham, North Carolina 27710, USA sive reviews on lymphocyte adaptor proteins have


e-mail: emj@duke.edu
y
e-mail: zhang033@mc.duke.edu
been published [2–7]. Here, we will focus on recent
advances in the field of adaptor proteins, highlighting
their part in the positive and negative regulation of
Current Opinion in Immunology 2003, 15:269–276 lymphocyte activation.
This review comes from a themed issue on
Lymphocyte activation Adaptors as positive regulators of
Edited by Andrey Shaw and André Veillette lymphocyte activation
LAT
0952-7915/03/$ – see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
Linker for activation of T cells (LAT) is an integral
membrane protein with a short extracellular domain
DOI 10.1016/S0952-7915(03)00044-X and a long cytoplasmic tail containing multiple tyrosine-
based motifs. LAT is localized to lipid rafts and is highly
Abbreviations phosphorylated by zeta-associated protein 70 (ZAP-70)
BCR B-cell receptor upon T-cell receptor (TCR) ligation. Its phosphorylated
BLNK B-cell linker tyrosines selectively recruit the SH2 domain-containing
Cbp Csk-binding protein adaptors Grb2 and Gads (for Grb2-related adaptor down-
CLNK cytokine-dependent hematopoietic cell linker
Dok downstream of tyrosine kinases
stream of Shc), as well as phospholipase C (PLC)-g1
Gads Grb2-related adaptor downstream of Shc (reviewed in [8]).
ITAM immunoreceptor tyrosine-based activating motif
LAB linker for activation of B cells From the examination of LAT-deficient mice it was
LAT linker for activation of T cells
discovered that LAT plays an essential role in T-cell
LAX linker for activation of X cells
MAPK mitogen-activated protein kinase development [9]. Recent studies demonstrate that the
NFAT nuclear factor of activated T cells four membrane distal tyrosines of LAT are crucial for its
NTAL non-T-cell activation linker function in pre-TCR signaling. In knock-in mice that
PH pleckstrin homology express LAT with mutations at the four distal tyrosines,
PLC phospholipase C
PR proline rich
thymocyte development is arrested at the CD4CD8
PTB phosphotyrosine-binding stage [10]. Furthermore, a LAT mutant containing only
RA rheumatoid arthritis these distal tyrosines is able to reconstitute both signaling
RasGAP RasGTPase-activation protein in LAT-deficient cells and thymocyte development in
SH2 Src homology 2
LAT-deficient mice [11].
SLAM signaling lymphocyte activation molecule
SLAP Src-like adaptor protein
SLP-76 SH2 domain-containing leukocyte protein of 76 kDa The importance of the LAT–PLC-g1 interaction was
TCR T-cell receptor examined using knock-in mice with a mutation at LAT
ZAP-70 zeta-associated protein 70 Tyr136, the residue critical in binding PLC-g1. Thymo-
cyte development is partially blocked in these mice
[12,13]. Mature T cells have defects in TCR-induced
Introduction PLC-g1 phosphorylation, calcium flux and NFAT (for
Adaptor proteins play important roles in the integration and nuclear factor of activated T cells) activation [12].
propagation of signals that lead to lymphocyte activation. Interestingly, an increase in the CD4þ:CD8þ T-cell ratio

www.current-opinion.com Current Opinion in Immunology 2003, 15:269–276

270 Lymphocyte activation

Figure 1

Y Y Y Y
LAB, LAT, LAX TM
Y Y Y Y Y
Y Y
SLP-76, BLNK PR SH2
Y
Y Y Y
CLNK PR PR SH2
Y

Nck SH3 SH3 SH3 SH2

Grb2, Grap SH3 SH2 SH3

Gads SH3 SH2 PR SH3

Y
Shc PTB PR SH2
Y
Y Y Y Y Y
Cbp TM PR PR
Y Y Y

SLAP SH3 SH2

Y Y
c-Cbl, Cbl-b SH2 RING PR
Y Y
Y Y Y
Dok PH PTB
Y Y Y Y

SAP SH2

Current Opinion in Immunology

A schematic of the adaptors mentioned in this review. The modular domains are illustrated. PH, pleckstrin homology domain; PR, proline-rich region;
PTB, phosphotyrosine-binding domain; RING, ring finger domain; SH2, src homology 2 domain; SH3, src homology 3 domain; TM, hydrophobic
transmembrane domain; Y, putative phosphorylation sites on tyrosine residues.

was observed. In the periphery of these knock-in animals,
CD4þ cells have an activated or memory phenotype with
only low levels of the TCR–CD3 complex on their sur-
face. With age these mice develop signs of an autoimmune
disorder, including splenic enlargement, eosinophilia,
increased numbers of CD4þ cells and inappropriate
secretion of type II cytokines (e.g. IL-4, IL-5, IL-10;
[12,13]). This highlights the effects that a single amino
acid substitution can have on significant processes such as
thymocyte selection and immune system homeostasis.
LAT localization was found to be exquisitely sensitive to
the redox state within the cell. In an oxidizing environ-
ment, such as that observed in the synovial T cells of
patients with rheumatoid arthritis (RA), LAT is no longer
found in the membrane [14,15]. It was proposed that, in
cases of chronic oxidative stress, disulfide bonds form
between LAT cysteine residues (Cys117 and either
Cys26 or Cys29) leading to a conformational change
and membrane displacement [15]. Given the importance
of LAT in T-cell signaling, these findings may help to

Current Opinion in Immunology 2003, 15:269–276 www.current-opinion.com


explain the hyporesponsive state of T lymphocytes in the
synovium of RA patients.
SLP-76
SLP-76 (for SH2 domain-containing leukocyte protein of
76 kDa) is a cytosolic adaptor that associates with LAT
via Gads or Grb 2. In contrast to LAT, SLP-76 has an
amino-terminal domain containing several tyrosines fol-
lowed by a PR region and an SH2 domain. The PR region
of SLP-76 constitutively associates with the carboxy-
terminal SH3 domain of Gads [16,17]. Reconstitution
studies with PR domain mutants show that deletion of
this region leads to a reduction in PLC-g1 phosphoryla-
tion, calcium flux and Erk activation after TCR ligation
[18,19]. Within this PR domain, a segment (P-1) was
identified that constitutively associates with the SH3
domain of PLC-g1. P-1 is necessary for maximal activa-
tion of PLC-g1 and Erk [20]. The PR region therefore
provides another pathway for PLC-g1 recruitment to the
LAT signaling complex in T cells. It is possible that
engagement of multiple PLC-g1 domains by LAT and
SLP-76 is required for full activation of PLC-g1 and its
stable incorporation into the complex.
Two recent papers examine the function of SLP-76
domains in T-cell development and activation [18,19].
Thymocytes from transgenic mice expressing a SLP-76
mutant with deletion of the amino terminus (D2–156) are
unable to develop past the CD4CD8 stage [19]. Mature
T cells do develop in mice expressing SLP-76 with muta-
tions in either the PR region or SH2 domain, but they fail
to upregulate CD69 [18] or proliferate appropriately in
response to anti-CD3 stimulation [18,19]. As discussed
above, the PR domain is vital in binding Gads; the
phenotype of the PR domain mutants is similar to that
observed in Gads-deficient mice [21]. The SH2 domain of
SLP-76 has been identified as the region involved in
binding the serine/threonine kinase HPK1, as mutation
of the SLP-76 SH2 domain leads to a significant reduction
in HPK1 activity. HPK1 may act as both a positive and a
negative regulator by promoting the Jnk mitogen activated
protein kinase (MAPK) pathway and inhibiting the path-
way leading to AP-1 activation [22]. Similar to PLC-g1, it
appears that HPK1 associates with the LAT complex in
two ways, through Gads [23] and SLP-76. Two SLP-76
homologs, B-cell linker (BLNK) and cytokine-dependent
hematopoietic cell linker (CLNK), also interact with
HPK1 in a similar manner [22,24]. Although CLNK
is believed to associate with a varied set of proteins to
SLP-76, it can rescue signaling in SLP-76-deficient Jurkat
T cells, possibly through its binding of HPK1 [24].
BLNK
Recent mapping of the tyrosine residues in BLNK
reveal three tyrosines that bind PLC-g2, and two tyr-
osines that bind the Tec family kinase Btk, the Vav
nucleotide exchange factor, and the adaptor Nck [25].
www.current-opinion.com
Lymphocyte adaptor proteins Janssen and Zhang 271
The phosphatase SHP-1 (for SH-containing tyrosine
phosphatase) was shown to disrupt the association of
Nck with BLNK by dephosphorylating these tyrosines.
The association with Nck may link BLNK with activa-
tion of the Jnk pathway [26]. Furthermore, mutation of
the five BLNK tyrosines evince that the complex of
BLNK with PLC-g2, Btk, Vav and Nck is vital for proper
B-cell receptor (BCR) signaling [25].
It is unclear how BLNK is recruited to the membrane.
One possibility is that BLNK directly associates with the
BCR complex. Indeed, the SH2 domain of BLNK can
bind non-immunoreceptor tyrosine-based activating motif
(non-ITAM) tyrosines on the Iga chain [27,28]. Muta-
tions in these two non-ITAM tyrosines (Tyr176,Tyr 204)
lead to an abrogation of calcium flux and MAPK activation.
Moreover, a chimeric protein composed of the platelet-
derived growth factor receptor (PDGFR), a mutated Iga
Tyr176/204Phe tail and BLNK, which is constitutively
targeted to the membrane, not only rescues but also
augments these signaling events [28]. This suggests that
one avenue for BLNK recruitment is through Iga.
LAB/NTAL
An alternative scenario for BLNK recruitment involves a
LAT-like molecule in B cells. This molecule, called
either linker for activation of B cells (LAB) or non-
T-cell activation linker (NTAL), was recently identified
[29,30]. LAB is expressed in B cells, natural killer
cells, monocytes and mast cells [30]. Similar to LAT,
LAB is localized to lipid rafts and contains multiple
conserved tyrosine motifs. When activated through the
BCR and other immune receptors, LAB is phosphory-
lated, probably by Syk, and interacts with Grb2. Unlike
LAT, however, LAB does not associate with PLC-g iso-
forms [29,30]. LAB is able to partially reconstitute
calcium flux and Erk activation in LAT-deficient Jurkat
T cells [30]. In addition, when LAB protein levels are
decreased in B cells using RNA interference, calcium flux
and Erk activation are diminished [29]. This demon-
strates that LAB operates in the calcium and Ras-MAPK
signaling pathways, similar to LAT. Furthermore, LAB
can effectively substitute for LAT in thymocyte devel-
opment. However, LAT and LAB do have some exclu-
sive functions, since when LAB is expressed in mature
T cells it causes aberrant activation [29]. We propose
that one function of LAB is to recruit BLNK to the
membrane in B cells through its association with Grb2,
with LAB–BLNK forming a functional equivalent of
LAT–SLP-76 (Figure 2).
Grb2, Gads and Shc
Grb2 is a small cytoplasmic adaptor containing a central
SH2 domain flanked by two SH3 domains. Grb2 plays a
significant role in activation of the Ras–MAPK cascades
by binding the Ras exchange factor, Sos, through its SH3
domains. Thymocytes from Grb2 heterozygous mice
Current Opinion in Immunology 2003, 15:269–276
272 Lymphocyte activation

Figure 2

(a) TCR (b) BCR

Cbp Cbp

LAT
SLAP LAB β α SLAP
Lyn PLC-γ2
Lck PLC-γ2
PLC-1γ Cbl
Cbl BLNK
SLP76 BLNK
Grb2/ Csk ? Csk ?
Grb2
Gads ZAP-70 Syk
Zap-70/ Syk/
HPK1 Src Src
HPK1
Src Src
kinase kinase

Current Opinion in Immunology

Proximal adaptors in lymphocyte signaling. (a) Following stimulation through the TCR, one early signaling event is the phosphorylation of tyrosines on
ITAMs present in the tails of CD3 components by the src kinase Lck. These phosphorylated residues provide docking sites for the SH2 domains of the
Syk family kinase ZAP-70, which is activated and goes on to phosphorylate the multiple tyrosines on LAT. This in turn leads to the recruitment of
LAT-associated proteins such as Grb2, Gads and PLC-g1. SLP-76 is also recruited to this complex via its constitutive association with Gads, and its basal
association with PLC-g1 may enhance the stabilization of this conglomerate. Furthermore, SLP-76 also binds the kinase HPK1. These proximal events lead
to the activation of the Ras-MAPK cascade and calcium flux, which culminate in gene transcription and initiation of effector functions. In addition, several
mechanisms exist to downregulate these actions. Cbp is phosphorylated in the absence of activation. In this phosphorylated state, Cbp recruits the
negative regulatory kinase, Csk. In addition, SLAP and SLAP-2 may bring c-Cbl in close proximity to Src and Syk family kinases, facilitating their
degradation. (b) Unlike the highly processed peptides in complex with MHC molecules recognized by the TCR, the BCR recognizes soluble antigen.
Despite these differences, there are striking similarities with regard to signaling through these immunoreceptors. Following BCR ligation, the src kinase Lyn
is activated and phosphorylates ITAMs present in the Iga and Igb chains. Just as in T cells, these phosphorylated motifs recruit a Syk family kinase.
One disparity, however, is that LAT is not expressed in B cells to recruit the SLP-76 family member BLNK to the membrane. Two current models for
this recruitment exist that are not mutually exclusive; BLNK may be recruited by non-ITAM tyrosines on the Iga chain, or the LAT-like adaptor LAB may
engage it. BLNK may then bring PLC-g2 into the complex, facilitating its subsequent activation and initiation of downstream signaling cascades.

show an attenuation of negative selection and stimula-
tion-induced apoptosis. In addition, a reduction of Grb2
expression in T cells leads to diminished activation of
Ras, Jnk and p38 MAPKs but normal Erk activation.
Gong et al. [31] also demonstrate that these MAPKs
require varied doses of phorbol 12-myristate 13-acetate
(PMA) for maximal activation. It is possible that the
observed effects are due to different activation thresholds
among the MAPKs, which subsequently affect T-cell
development and effector functions. In contrast to
Grb2, Grap (for Grb2-related adaptor protein) probably
functions in TCR signaling through downmodulation of
Erk activation. T lymphocytes from Grap-deficient mice
show increased proliferation and IL-2 production in
response to TCR stimulation [32].
Another Grb2 family member, Gads, plays a positive role
in lymphocyte activation. Similar to Grb2 and Grap, Gads
contains an SH2 domain and two SH3 domains with an
additional PR region (Figure 1). Its importance was
underscored recently in Gads-deficient mice, as thymo-
cyte proliferation and selection are severely impaired in
these mice. In addition, the association of SLP-76 with
a protein of approximately 40 kDa, probably LAT, is
significantly reduced [21].
The Grb2-related adaptor Shc contains an SH2 and a
PTB domain. When phosphorylated by proximal tyrosine
kinases, Shc interacts with the SH2 domain of Grb2. One
of its functions is to enhance the interaction of Grb2 with
Sos [33,34]. Similar to Gads and Grb2, Shc has an impor-
tant function in pre-TCR signaling and thymocyte devel-
opment, Shc-deficient thymocytes have a partial block in
the transition to the CD4þCD8þ double-positive stage of
development [35]. In addition, Shc-deficient Jurkat cells
are unable to produce IL-2 in response to TCR stimula-
tion. Shc is important in Erk activation as well as c-Rel
nuclear translocation [36]. It is possible that Shc enhance-
ment of the Grb2 and Sos interaction may be partly
responsible for the high efficiency of Grb2 in Erk activa-
tion as noted above.
Adaptors as negative regulators of
lymphocyte activation
Although adaptors provide an important function in cou-
pling activation through the BCR and TCR to downstream

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effectors, they play an equally crucial part in downmodu-
lating this response. Recent insights into the roles of these
negative adaptors and the identification of new players
have significantly impacted how we view ‘turning off’ the
immune response.
Cbp/PAG
Cbp (for csk-binding protein)/PAG (for phosphoprotein
associated with glycolipid-enriched microdomains) is loca-
lized to lipid rafts and phosphorylated by the Src kinase Fyn
[37,38]. It is believed that, in the absence of stimulation,
phosphorylated Cbp recruits the kinase Csk to the vicinity
of Src family kinases, where it phosphorylates negative
regulatory residues [39]. In addition to its role in kinase
regulation, Cbp is implicated in controlling immune
synapse formation. In one model, Cbp binding to the newly
identified adaptor EBP (also known as Naþ/Hþ exchanger-
regulatory factor [NHE-RF]) serves as a link in a ‘chain’
between lipid rafts and the cytoskeleton. One function of
this ‘chain’ may be to retain the separateness of small lipid
rafts under basal conditions. Correspondingly, overexpres-
sion of Cbp has been found to limit synapse formation
with antigen stimulation whereas a mutant unable to bind
EBP has no effect on synapse formation [40]. Cbp is
a prime example of the multiple roles that adaptor pro-
teins may take on to adjust the immune response.
LAX
We have characterized a novel membrane-associated
adaptor related to LAT. Linker for activation of X cells
(LAX) is expressed in hematopoietic cells and contains
multiple tyrosine motifs that, when phosphorylated, bind
Grb2, Gads and PI3K. Unlike LAT, LAX is not found in
lipid rafts, and its overexpression in Jurkat T cells reduces
p38 and AP-1 activation [41]. It remains to be seen how
LAX preferentially inhibits p38 while Erk and Jnk acti-
vation remain normal. It is possible that LAX may act as a
‘sink’, recruiting signaling molecules away from the LAT
signaling complex in T cells.
SLAPs and Cbls
Similar to Cbp and LAX, SLAP (for Src-like adaptor
protein) is also expressed in both B and T cells [42].
Although SLAP lacks a kinase domain, it has SH3 and
SH2 domains as well as a unique carboxyl terminus.
SLAP interacts with Cbl, LAT, ZAP-70, Syk and the
TCRz chain in T cells [43]. Data from SLAP overexpres-
sion in Jurkat cells indicate that SLAP downregulates
signals that lead to IL-2 production [42]. Thymocytes
from SLAP-deficient mice have increased surface expres-
sion of the TCR and enhanced positive selection [44].
Several groups have identified a SLAP-related protein,
designated SLAP-2 [45–47]. SLAP-2 contains a putative
myristoylation site that is not only important for its mem-
brane localization but also its inhibitory effects [45–47].
Overexpression of SLAP-2 in B and T cell lines diminishes
www.current-opinion.com
Lymphocyte adaptor proteins Janssen and Zhang 273
upregulation of CD69 in response to antigen receptor
ligation [45]. A decrease in calcium flux as well as activa-
tion of NFAT and the IL-2 promoter can also be observed
in Jurkat T cells overexpressing SLAP-2 [45–47].
Multiple explanations have been offered to explain the
inhibitory effects of SLAP-2. As SLAP-2 resembles a Src
kinase, it may sequester their normal Src binding partners
through its SH2 domain [45]. Alternatively, these finding
may be explained by the association of the carboxyl
terminus of SLAP-2 with c-Cbl (for casitas B-lineage
lymphoma;[45–47]). In addition to their adaptor func-
tions, c-Cbl and Cbl-b have ring fingers and act as E3
ubiquitin ligases (reviewed in [48,49]). Recent reports have
shown that the ubiquitin ligase activities of c-Cbl and Cbl-
b play a role in the negative regulation of upstream kinases,
such as Lck, Syk and PI3K, in T and B cells [50–54].
Consistent with this notion, the carboxyl terminus of
SLAP-2 is necessary for its full inhibitory effect, and co-
transfection of SLAP-2 with Syk family kinases leads to a
dramatic reduction in kinase levels [46]. Thus, one poten-
tial scenario is that SLAP-2 acts as bridge between c-Cbl
and proximal kinases directing their degradation.
The function of the Cbl proteins may not be solely
negative. Mice with T cells deficient in both c-Cbl and
Cbl-b have an autoimmune phenotype with sustained
T-cell activation. Although they exhibit an increase in
ZAP-70 phosphorylation in comparison to wild-type mice,
a decrease in PLC-g1 phosphorylation and calcium flux
can be observed in the double-knockout T cells [55].
This finding is consistent with the observation that Cbl-b
positively regulates the calcium-signaling pathway.
Cbl-b-deficient DT40 B cells have reduced PLC-g2
activation [56]. Cbl-b may have both positive and nega-
tive regulatory function in T-cell activation.
Dok
Dok (for downstream of tyrosine kinases) proteins are
expressed in various hematopoietic cells and are impli-
cated in the negative regulation of signaling through cyto-
kine and immunoreceptors. These proteins contain PH,
PTB, and PR domains as well as numerous tyrosines. In B
cells, Dok-1 is tyrosine phosphorylated upon co-ligation of
the BCR and FcgRIIB. Phosphorylated Dok-1 associates
with RasGAP (for RasGTPase-activation protein), an
interaction which is dependent on the phosphatase, SHIP
(for SH2 domain-containing inositol 50 -phosphatase) [57].
Dok-1 also interacts with the SH2 domains of Nck, Csk,
and the adaptor SAP (for SLAM-associated protein)
[58,59]. Through these interactions Dok-1 is believed to
downregulate the Ras-MAPK pathway. In addition, thy-
mocytes, bone marrow and mast cells from Dok-1/ mice
show increased proliferation in response to stimulation
[60]. These findings suggest an important role for Dok-1
in downmodulating the activation and proliferation
responses of lymphocytes.
Current Opinion in Immunology 2003, 15:269–276
274 Lymphocyte activation
Another Dok family member, Dok-2, appears to play a
role in the negative regulation of T-cell maturation and
activation. T-cell proliferation in response to TCR liga-
tion and cytokines is markedly enhanced in mice expres-
sing only low levels of Dok-2 [61]. In addition,
overexpression of Dok-2 in the bone marrow reduces
its capacity to restore lymphoid tissues in irradiated hosts.
Specifically, it inhibits the transition of CD4CD8 to
CD4þCD8þ T cells. Its RasGAP and Nck binding sites
were found to be important for this activity [62].
Unlike Dok-1 and Dok-2, Dok-3 is not expressed in T
cells and is unable to bind RasGAP [63]. It is found in B
cells and phosphoryated with immunoreceptor ligation.
When phosphorylated, Dok-3 binds SHIP and Csk [64].
In contrast to other family members, Dok-3 is unable to
block MAPK activation caused by activated Ras; how-
ever, it does downregulate MAPK in response to expres-
sion of the Abl oncogene [63]. Also, when overexpressed
in B cells, Dok-3 leads to a decrease in NFAT activation
and cytokine secretion [64].
Recently, two new Dok family members have been iden-
tified, Dok-4 and Dok-5. Both are expressed in peripheral
blood T cells. Interestingly, Dok-5 is found in activated
and not resting T cells [65]. Perhaps its expression in
activated cells correlates with an ability to downregulate
cellular activation, similar to other Dok proteins.
Conclusions
Recent developments in the field of adaptor proteins
serve to highlight the importance of this group of proteins
in lymphocyte activation. Many of these proteins have
multiple roles and act in both positive and negative
regulatory pathways. In addition, newly discovered inter-
actions, together with the characterization of several
novel adaptors, have significantly affected our current
view of lymphocyte activation.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
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Current Opinion in Immunology 2003, 15:269–276
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