Distinct Regulation of Integrin-Dependent T Cell Conjugate
Formation and NF-?B Activation by the Adapter
Brandon J. Burbach,* Rupa Srivastava,* Ricardo B. Medeiros,2* William E. O’Gorman,*
Erik J. Peterson,†and Yoji Shimizu3*
Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation-promoting adapter protein
(ADAP) to control both integrin adhesive function and NF-?B transcription factor activation. We have investigated the molecular
basis by which ADAP controls these events in primary murine ADAP?/?T cells. Naive DO11.10/ADAP?/?T cells show impaired
adhesion to OVAp (OVA aa 323–339)-bearing APCs that is restored following reconstitution with wild-type ADAP. Mutational
analysis demonstrates that the central proline-rich domain and the C-terminal domain of ADAP are required for rescue of T:APC
conjugate formation. The ADAP proline-rich domain is sufficient to bind and stabilize the expression of SKAP55 (Src kinase-
associated phosphoprotein of 55 kDa), which is otherwise absent from ADAP?/?T cells. Interestingly, forced expression of
SKAP55 in the absence of ADAP is insufficient to drive T:APC conjugate formation, demonstrating that both ADAP and SKAP55
are required for optimal LFA-1 function. Additionally, the ADAP proline-rich domain is required for optimal Ag-induced acti-
vation of CD69, CD25, and Bcl-xL, but is not required for assembly of the CARMA1/Bcl10/Malt1 (caspase-recruitment domain
(CARD) membrane-associated guanylate kinase (MAGUK) protein 1/B-cell CLL-lymphoma 10/mucosa-associated lymphoid tis-
sue lymphoma translocation protein 1) signaling complex and subsequent TCR-dependent NF-?B activity. Our results indicate
that ADAP is used downstream of TCR engagement to delineate two distinct molecular programs in which the ADAP/SKAP55
module is required for control of T:APC conjugate formation and functions independently of ADAP/CARMA1-mediated NF-?B
activation. The Journal of Immunology, 2008, 181: 4840–4851.
grams that promote proliferation and the gain of effector function.
Cytosolic adapter proteins and enzymes control the initiation and
transmission of these TCR-specific signals (1–3). One key adapter,
the linker for activated T cells (LAT),4is rapidly tyrosine phos-
phorylated following TCR stimulation and provides docking sites
for the Src homology 2 domain-containing leukocyte-specific
aive T cells respond to Ag engagement of the TCR by
rapidly increasing the functional activity of their integrin
adhesion receptors, and by initiating molecular pro-
phosphoprotein of 76 kDa (SLP-76) and phospholipase C (PLC-
?1) (4). These proteins are critical for proximal signals generated
by the TCR, as loss of LAT or SLP-76 expression severely disrupts
T cell development (5–7). In contrast, disruption of proteins down-
stream of the LAT signalosome results in more selective defects in
T cell activation.
Adhesion and degranulation promoting adapter protein (ADAP;
formerly known as Fyb or SLAP-130) is expressed by T cells and
other hematopoietic lineages except B cells. ADAP was originally
identified by its TCR-dependent tyrosine phosphorylation and in-
teraction with SLP-76 and the tyrosine kinase Fyn (8, 9). Like
other adapters, ADAP contains a number of protein-protein and
protein-lipid interaction domains (1, 10, 11). The C terminus of
ADAP contains several tyrosine residues that are involved in TCR-
dependent binding to SLP-76 and Fyn (12–14), as well as FPPP
domains that have been implicated in interaction with the Ena/
Vasp family of actin regulatory proteins (15). A central proline-
rich domain between aa 326–426 of murine ADAP constitutively
binds the Src homology 3 (SH3) domain of another adapter,
SKAP55 (Src kinase-associated phosphoprotein of 55 kDa) (16–
18). Immediately adjacent to this domain, aa 426–541 in ADAP con-
tains an E/K-rich domain and part of an atypical helical SH3 domain
(hSH3-N) that comprise a binding site for the adapter CARMA1
(caspase-recruitment domain (CARD) membrane-associated guany-
late kinase (MAGUK) protein 1) that is critical for formation of the
CARMA1/Bcl10 (B-cell CLL-lymphoma 10)/Malt1 (mucosa-associ-
ated lymphoid tissue lymphoma translocation protein 1) signaling
*Department of Laboratory Medicine and Pathology and†Department of Medicine,
Center for Immunology, Masonic Cancer Center, University of Minnesota Medical
School, Minneapolis, MN, 55455
Received for publication November 27, 2007. Accepted for publication August
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by National Institutes of Health Grants R01AI038474 (to Y.S.),
R01AI031126 (to Y.S.), R01AI056016 (to E.J.P.), and T32DE007288 (to B.J.B.).
Y.S. is supported in part by the Harry Kay Chair in Biomedical Research at the
University of Minnesota.
2Current address: R&D Systems, 614 McKinley Place NE, Minneapolis, MN 55413.
3Address correspondence and reprint requests to Dr. Yoji Shimizu, Department of
Laboratory Medicine and Pathology, University of Minnesota Medical School, MMC
334/Room 6-112 NHH, 312 Church Street SE, Minneapolis, MN 55455. E-mail
4Abbreviations used in this paper: LAT, linker for activated T cells; ADAP, adhesion
and degranulation-promoting adapter protein; Bcl10, B-cell CLL/lymphoma 10;
CARMA1, caspase-recruitment domain (CARD) membrane-associated guanylate ki-
nase (MAGUK) protein 1; CBM, CARMA1/Bcl10/Malt1; HA, hemagglutinin;
hCAR, human coxsackievirus and adenovirus receptor; hSH3, helical SH3; KO,
knockout; Malt1, mucosa-associated lymphoid tissue lymphoma translocation protein
1; OVAp, OVA aa 323–339; RIAM, Rap1-GTP-interacting adapter molecule; SH3,
Src homology 3; SKAP55, Src kinase-associated phosphoprotein of 55 kDa; SLP-76,
Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa;
WT, wild type; wtADAP, wild-type ADAP.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
complex and NF-?B activation (19). This hSH3-N domain and a
highly homologous hSH3-C located at the extreme C terminus of
ADAP bind to phospholipids in vitro (20, 21). Finally, the N-terminal
?300 aa of ADAP binds HIP-55 (hematopoietic progenitor kinase
1-interacting protein of 55 kDa) (22), although the significance of this
interaction is unclear.
Although initial overexpression studies reported both positive
and negative roles for ADAP in T cell activation (8, 9, 12, 14), the
production of ADAP?/?mice demonstrated that ADAP positively
regulates T cell activation (23, 24). While ADAP?/?T cells mice
show normal proximal TCR-dependent responses, including Ca2?
mobilization and ERK phosphorylation, they exhibit impaired
TCR and Ag-dependent integrin-mediated adhesion and subse-
quent T cell activation and survival (23–25). ADAP or SKAP55
overexpression enhances LFA-1-dependent T cell interactions
with APCs in a manner dependent on the SKAP55 SH3 domain
(26). Similarly, T cells from SKAP55 knockdown (27) or from
SKAP55?/?mice (28) exhibit defects in LFA-1 function compa-
rable to ADAP?/?mice. The ADAP proline-rich and SKAP55
SH3 domains are critical for TCR-dependent membrane targeting
of Rap1 (29), a small GTPase that is important for T cell integrin
activation and T:APC conjugate formation (30–32). Additionally,
SKAP55 constitutively associates with RIAM (Rap1-GTP-inter-
acting protein), which binds to the active form of Rap1 (29, 33).
Thus, an ADAP/SKAP55/RIAM/Rap1 signaling arm appears to be
required for control of T cell integrin activation. However, the
identification of precise functions for ADAP has been complicated
by the observation that stable SKAP55 expression requires a con-
stitutive SH3 domain-mediated interaction of SKAP55 with
ADAP (17, 34). The role of the ADAP/SKAP55 interaction in
NF-?B activation, as well as the function of SKAP55 independent
of ADAP expression, has not been investigated.
In the present study, we used adenovirus to express ADAP and
ADAP mutant constructs in resting naive T cells from ADAP?/?
mice expressing the hCAR (human coxsackievirus and adenovirus
receptor) adenovirus receptor (35, 36) and the OVA aa 323–339
(OVAp)-specific DO11.10 transgenic TCR (25, 37). Using naive
mouse T lymphocytes, we show that a small region of the ADAP
proline-rich domain is required for rescue of SKAP55 expression
by ADAP?/?T cells, as well as for optimal Ag-dependent T:APC
conjugate formation and downstream T cell activation. However,
SKAP55 expression alone in the absence of ADAP is not sufficient
to restore T:APC conjugate formation. Furthermore, we demon-
strate that formation of the ADAP/SKAP55 complex is not re-
quired for control of NF-?B activation and that SKAP55 is not
found in the CARMA1/Bcl10 complex, suggesting differential
control of integrin and NF-?B pathways by ADAP.
Materials and Methods
DO11.10/ADAP?/?and ADAP?/?mice on the BALB/c background have
been previously described (25) and were crossed to the hCAR transgenic
mice (35) (provided by Dr. C. Weaver, University of Alabama-Birming-
ham). Mice were housed in specific pathogen-free facilities at the Univer-
sity of Minnesota and were used between 8 and 12 wk of age. All exper-
imental protocols involving the use of mice were approved by the
Institutional Animal Care and Use Committee at the University of
The Jurkat E6-1 human T cell line was obtained from the American Type
Culture Collection and maintained at 37°C in RPMI 1640 supplemented
with FCS (Atlanta Biologicals), L-glutamine, and penicillin/streptomycin
and grown in 5% CO2.
Abs and reagents
The DO11.10 TCR was detected with FITC- or biotin-conjugated mAb
KJ1-26 (Caltag Laboratories). Anti-Thy1.1 allophycocyanin or PE, anti-
B220-PE-Cy5.5, anti-CD69-FITC, and anti-CD25 allophycocyanin were
from eBioscience. Sheep anti-murine ADAP has been previously described
(38). The following Abs were purchased: rabbit anti-SKAP55 (Upstate
Biotechnology), rabbit anti-CARMA1 (Alexis Biochemicals), goat anti-
hemagglutinin (HA) agarose (Bethyl Laboratories), mouse anti-HA (clone
16B12) (Covance), and mouse anti-Bcl10 (331.3) and mouse anti-NF-?B
p65 (F-9) (Santa Cruz Biotechnology). Alexa 488-conjugated donkey anti-
sheep and goat anti-rabbit IgG were used for intracellular staining detection.
goat anti-rabbit, as well as IRDye 800-conjugated goat anti-rabbit, goat
anti-mouse, and donkey anti-sheep IgG (Rockland Immunochemicals)
The pENTR-HA-ADAP construct for adenovirus production encodes for
full-length murine ADAP (130-kDa isoform) and is derived from pENTR-
UP-IT (19). ADAP mutants were generated as previously described (19)
using the QuickChange II XL site-directed mutagenesis kit (Stratagene) by
introducing a stop codon at amino acid position 426 (mutants ?426–819)
or by using primers flanking both sides of the deleted site (mutants ?1–327,
?326–426, ?338–358, and ?426–541). FLAG-tagged SKAP55 was ob-
tained from Dr. B. Schraven (Otto-von-Guericke University, Magdeburg,
Germany) and was subcloned into pENTR-UP-IT by blunt ligation. GFP-
Rap1 was generated by inserting Rap1 in frame in pEGFP-C1 (Clontech)
and subcloning the GFP-Rap1 cassette into the EcoRV site of pENTR-UP-
IT. All constructs were verified by DNA sequencing at the University of
Minnesota Microsequencing Facility.
Adenovirus production and transduction
Adenovirus expression plasmids were generated as previously described
(19) using Gateway recombination between pAD/PL-DEST (Invitrogen)
and either control pENTR-UP-IT or pENTR-HA-ADAP. Adenovirus was
produced in AD-293 packaging cells (Stratagene), and viral particles were
purified and titered as described previously (35, 39, 40). Freshly isolated,
resting ADAP?/?and ADAP?/?lymph node T cells expressing the hCAR
receptor were transduced with control, ADAP, or SKAP55 adenoviruses as
described (35) and incubated at 37°C for 3 days in complete T cell medium
containing 5 ng/ml mouse IL-7 (R&D Systems). Preliminary experiments
were performed to optimize the length of incubation required to achieve
peak ADAP or SKAP55 expression. Expression of HA-ADAP or FLAG-
SKAP55 was confirmed in all experiments by intracellular flow cytometry
for ADAP (38), HA, or SKAP55. Transduction of Jurkat T cells was per-
formed similarly to the primary T cells (19). Briefly, 10 ? 106Jurkat cells
at a density of 50 ? 106/ml in DMEM containing 10 mM HEPES (pH 7.4)
were incubated with 500 ? 106infectious units of adenovirus (multiplicity
of infection of 50) for 30 min at 37°C. Cells were washed and cultured in
T cell medium at 0.5–1 ? 106cells/ml for 2 days. Flow cytometric analysis
(data not shown) indicated that 80–90% of cells in these cultures were
transduced and expressed the indicated ADAP construct.
Conjugate and activation assays
Flow cytometry-based conjugate assays were performed as previously de-
scribed (25). Briefly, control wild-type (WT) and ADAP?/?DO11.10/
hCAR bulk lymph node T cells were transduced with adenovirus as de-
scribed above. Fresh nontransgenic BALB/c splenocytes were labeled with
Cell Tracker Orange (Molecular Probes) and then left unpulsed or pre-
pulsed for 30 min at 37°C with OVAp (Invitrogen) at the indicated con-
centrations. Transduced KJ1-26?Thy1.1?T cells were then combined at a
1:4 ratio with the peptide-loaded splenocytes, pelleted in a 96-well round-
bottom plate, incubated for 10 min at 37°C, mixed for 20 s in a plate
shaker, fixed with 1% paraformaldehyde, and stained for flow cytometry.
Conjugates were defined as KJ1-26?Thy1.1?T cell events costaining with
Cell Tracker Orange and B220. In vitro activation was performed similar
to the conjugate assays except that 0.1 ? 106KJ1-26?Thy1.1?T cells
were seeded into 96-well flat-bottom plates and activated by the addition of
0.2 ? 106unstained, peptide-loaded splenocytes. Cells were harvested at
the indicated times and KJ1-26?Thy1.1?T cells were stained for CD25,
CD69, and Bcl-xLas previously described (25).
Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting for Bcl10, CARMA1, and en-
dogenous ADAP was performed as previously described (19, 41). Briefly,
10 ? 106cells per condition in PBS containing 0.5% BSA were stimulated
4841The Journal of Immunology
with PMA (50 ng/ml), lysed with an equal volume of 2? lysis buffer (2%
Nonidet P-40, 100 mM Tris-HCl (pH 7.6), 300 mM NaCl, 4 mM EDTA,
2 mM sodium vanadate, 10 ?g/ml leupeptin, 10 ?g/ml aprotonin, 2 mM
PMSF), and then cleared by centrifugation at 12,000 relative centrifugal
force. For immunoprecipitation, individual Abs were adsorbed to protein
A-Sepharose for 2 h at 4°C, washed with 0.2 M NaBO4(pH 9.0), resus-
pended in 20 mM dimethyl pimelimidate, and incubated for 30 min at room
temperature, washed with 0.2 M ethanolamine (pH 8.0), and incubated for
2 h in 0.2 M ethanolamine. The cleared lysates were then incubated over-
night at 4°C with the crosslinked bead/antibody complexes. The immune
complexes were washed twice with 1? lysis buffer containing 0.1% Tri-
ton-X 100 and separated by SDS-PAGE. After Western transfer of the
proteins, polyvinylidene difluoride membrane was blocked with 0.2% ca-
sein for 1 h, primary Ab was incubated overnight in PBS/0.2% casein/0.2%
Tween 20, washed with PBS containing 0.2% Tween 20, incubated for 1 h
in secondary Ab in PBS/0.2% casein/0.2% Tween 20, and finally washed
with PBS/0.2% Tween 20/0.02% SDS. The membrane was imaged with an
Odyssey infrared imager (LI-COR Biosciences). Coimmunoprecipitation
of SKAP55 with HA-ADAP constructs was performed by lysing cells as
described above, incubating the soluble lysate with anti-HA agarose for 3 h
at 4°C, and immunoblotting for ADAP, HA, or SKAP55 as described
NF-?B nuclear translocation
Lymph node T cells isolated from ADAP?/?or ADAP?/?mice were
transduced and cultured as described above. At the time of harvest, cells
were washed into PBS/0.2% BSA and stimulated with 10 ?g/ml anti-CD3
(2C11) and 1 ?g/ml anti-CD28. Cells were then fixed in 2% paraformal-
dehyde, stained with anti-Thy1.1-PE, anti-p65 FITC, and 7-aminoactino-
mycin D (eBioscience), and then analyzed for NF-?B nuclear translocation
using the ImageStream 100 multispectral imaging flow cytometer (Amnis)
lymphocytes from transgenic DO11.10/hCAR/ADAP?/?(WT) or ADAP?/?(knockout (KO)) mice were transduced with adenovirus encoding Thy1.1
alone (Ctrl), Thy1.1 plus WT murine ADAP (wtADAP), or mock transduced (No Virus) and cultured ex vivo for 3 days as described in Materials and
Methods. A, Cells were stained for Thy1.1 and either the DO11.10 TCR (KJ1-26) or CD69 and analyzed by flow cytometry. B, Cells were fixed and
intracellular staining was performed with a sheep anti-ADAP Ab. C, T:APC conjugate formation between KJ1-26?Thy1.1?T cells and BALB/c spleno-
cytes prepulsed with the indicated concentrations of OVAp (left panel) was performed as described in Materials and Methods. D, The same KJ1-
26?Thy1.1?population of T cells was gated into three subpopulations expressing either low, medium, or high levels of Thy1.1, and T:APC conjugate
efficiency with 10 ?M OVAp was determined. Results are representative of at least three independent experiments performed.
Rescue of Ag-dependent T:APC conjugate formation in ADAP?/?T cells following reconstitution of wtADAP. Freshly harvested, naive
4842 DISTINCT INTEGRIN AND NF-?B REGULATION BY ADAP
as described previously (42). A minimum of 10,000 cells were collected
and analyzed under each experimental condition. NF-?B p65 nuclear trans-
location was specifically assessed in cells expressing similar levels of
Five-micrometer latex beads (Interfacial Dynamics) were coated with anti-
CD3 (2C11) in PBS for 3 h at 37°C, blocked with 3% BSA, and pelleted
briefly at a 1:1 ratio with transduced T cells. After 2 min, the pellets were
gently resuspended and incubated at 37°C for an additional 10 min. The T
cell/bead complexes were then fixed with paraformaldehyde and processed
for anti-SKAP55 intracellular staining or analyzed directly for GFP-Rap1
using the ImageStream cytometer as described above. Amnis Ideas 3.0
software was used to quantify the percentage of GFP-Rap1 or SKAP55 at
the T cell/bead contact. First, a mask encompassing the bead area was
dilated to include the adjacent T cell surface. Then, the percentage of re-
cruited protein was defined as the intensity of GFP or SKAP55 within this
mask, divided by the total intensity within the entire T cell/bead event
Measures of statistical significance were determined with GraphPad Prism
5.0 software using an unpaired Student’s t test. As indicated, ???, p ?
0.001; ??, p ? 0.01; ?, p ? 0.05; ns, not significant.
numbering is given for the murine ADAP p130 isoform. Abbreviations: PRO, proline-rich domain; E/K, glutamic acid and lysine-rich domain; hSH3,
N-terminal (N) or C-terminal (C) helical SH3 domain; EVH1, Ena/Vasp homology domain. Asterisks are used to denote the position of tyrosines 547,
549, 584, 615, and 687 found in phosphorylation consensus motifs. B, Freshly harvested naive DO11.10/hCAR/ADAP?/?(WT) or ADAP?/?(KO)
lymphocytes were transduced with adenoviruses encoding the indicated constructs or Thy1.1 control adenovirus (Ctrl) and fixed and stained with
an anti-Thy1.1 Ab and either anti-ADAP Ab or nonimmune sheep serum (IgG) and analyzed by flow cytometry. C, Same as in B except the indicated
constructs were stained with an anti-hemagglutinin (HA) Ab. Expression profiles are representative of at least three independent analyses performed
for each construct shown.
Schematic and expression of ADAP mutant constructs. A, Scale diagram of ADAP expression constructs used in this study. Amino acid
4843 The Journal of Immunology
Rescue of T:APC conjugate formation in naive ADAP?/?
T cells by expression of WT ADAP (wtADAP)
To investigate the ADAP functional domains that control integrin
activation in naive primary murine T cells, we analyzed Ag-de-
pendent T:APC conjugate formation by DO11.10/ADAP?/?T
cells expressing either wtADAP or ADAP deletion mutants. We
expressed the hCAR transgene with a T cell-specific promoter (35)
on the DO11.10/ADAP?/?background (25) to permit efficient ad-
enovirus-mediated transduction of T cells from bulk lymph node
preparations (data not shown) (35). Recombinant adenoviruses en-
coding the WT murine ADAP gene and an IRES (internal ribo-
some entry site)-driven Thy1.1 (CD90.1) cell surface reporter (19)
were used to reconstitute ADAP expression in freshly harvested,
resting hCAR?/D011.10/ADAP?/?T cells (Fig. 1). Thy1.1 ex-
pression was observed following adenovirus transduction (Fig. 1A,
no virus vs control (Ctrl; Thy1.1 only) virus). Both ADAP?/?and
ADAP?/?cells expressed similar levels of the DO11.10 TCR fol-
lowing incubation with adenovirus, as judged by equivalent inten-
sity of the KJ1-26 staining (Fig. 1A, upper panels). There was no
up-regulation of expression of either CD69 (Fig. 1A, lower panels)
or CD25 (data not shown, see also Fig. 7B) in Thy1.1-expressing
cells across these treatment groups, indicating that the cells were
not being activated while resting in culture following adenovirus
transduction. Intracellular staining with an anti-ADAP Ab (Fig.
1B) indicated the presence and absence of endogenous ADAP in
the majority of Thy1.1?ADAP?/?and ADAP?/?cells, respec-
tively. Flow cytometric analysis of ADAP?/?cells transduced
with wtADAP demonstrates that Thy1.1 expression correlated
with rescue of ADAP expression to levels approaching, but not
exceeding, those of endogenous ADAP expression (Fig. 1B, right
DO11.10/ADAP?/?T cells show an ?30–50% impairment in
LFA-1-dependent T cell conjugate formation with OVAp-pulsed
APCs at all Ag doses examined (19, 25). A similar defect in
T:APC conjugate formation was observed following OVAp stim-
ulation in this system. Background levels of T:APC conjugates in
the absence of OVAp were low (?2%; Fig. 1C, 0 ?M OVAp).
Following incubation with APCs loaded with 10 ?M OVAp,
?35% of control adenovirus-transduced ADAP?/?T cells inter-
acted with APCs. In contrast, this conjugate efficiency was reduced
to ?25% for control ADAP?/?T cells (Fig. 1C). Importantly,
reconstitution of ADAP?/?T cells with wtADAP restored conju-
gate formation to a level similar to that found in ADAP?/?cells
(Fig. 1C) (19). Because ADAP detection by intracellular staining
jugate formation. Naive DO11.10/hCAR/ADAP?/?(KO) or ADAP?/?
(WT) lymphocytes expressing the indicated ADAP constructs were ana-
lyzed for T:APC conjugate formation between KJ1-26?Thy1.1?T cells
and BALB/c splenocytes prepulsed with the indicated concentrations of
OVAp. Results are shown for a single representative experiment and are
representative of at least four independent experiments performed for each
The ADAP proline-rich domain is required for T:APC con-
rich domain controls SKAP55 ex-
pression in ADAP?/?T cells. Naive
ADAP?/?(WT) lymphocytes ex-
pressing the indicated ADAP con-
structs, WT SKAP55, or the control
adenovirus (Ctrl) were fixed and in-
tracellular staining was performed
with rabbit anti-SKAP55 Ab or con-
trol rabbit Ig (IgG) and analyzed by
flow cytometry. Results are shown for
a single representative experiment
and are representative of at least four
independent experiments performed
for each construct.
The ADAP proline-
4844 DISTINCT INTEGRIN AND NF-?B REGULATION BY ADAP
was sensitive enough to detect differences in ADAP expression
based on the transduced level of Thy1.1 staining (43), we analyzed
conjugate formation in ADAP?/?T cells gated for increasing
ranges of Thy1.1 expression and found that the magnitude of this
rescue depended on the level of ADAP reconstitution (Fig. 1D).
For example, KJ1-26?cells falling within the lower third of
Thy1.1 expression showed only modest rescue of adhesion, while
cells within the medium or high Thy1.1 gate showed highly sig-
nificant increases in adhesion compared with the ADAP?/?vector
control (Fig. 1D). Subsequent analyses are presented using the top
approximate third of Thy1.1?(Thy1.1high) cells expressing the
wtADAP construct, with this gate being applied identically to all
samples in the experiment to ensure consistency between the
The ADAP C terminus and proline-rich domains are required
for efficient T:APC conjugate formation
We next prepared a panel of ADAP mutants to identify the mo-
lecular domains in ADAP that are required for control of T:APC
conjugate formation (Fig. 2A). The expression of constructs con-
taining the N terminus of ADAP was demonstrated by intracellular
staining with an anti-ADAP Ab that recognizes the N terminus
(Fig. 2B). The ADAP?1–327 and ADAP (?326–426) constructs,
which lack the N terminus, were detected by staining with an anti-
HA Ab (Fig. 2C). As in Fig. 1B, Thy1.1 expression correlated with
increased expression of all the ADAP constructs we examined.
Wild-type ADAP and an ADAP mutant removing the first 327 aa
(ADAP?1–327) both rescued conjugate formation (Figs. 1C and
3), indicating that the N terminus of ADAP is not essential for this
response. However, deletion of the proline-rich domain of ADAP
(?326–426) completely abolished rescue of conjugate formation,
down to levels of control virus alone. Similarly, a restricted dele-
tion within this proline-rich domain (?338–358) also failed to res-
cue T:APC adhesion, indicating that this 20-aa segment between
aa 338–358 of ADAP is critical for control of integrin function
(Fig. 3). However, expression of the proline-rich domain alone
(?326–426) failed to rescue conjugate formation, indicating the
requirement for a second domain in ADAP (Fig. 3). Indeed, re-
moval of the C-terminal ?400 aa of ADAP (?426–819) also re-
stricted rescue of conjugate formation, indicating that the C ter-
minus of ADAP is also required for efficient conjugate formation.
The ADAP proline-rich domain controls SKAP55 expression in
naive T cells
Analysis of ADAP-deficient Jurkat T cells (34) and ADAP?/?
murine T cells (44) has demonstrated that SKAP55 expression is
also severely impaired in the absence of ADAP, due to caspase
and/or proteosome-mediated destabilization of free SKAP55 pro-
tein when it cannot interact with ADAP. We have confirmed this
finding using intracellular flow cytometry of WT or ADAP?/?
lymphocytes using an anti-SKAP55 Ab (Fig. 4) and Western blot-
ting (data not shown). Wild-type T cells infected with control
ADAP?/?T cells infected with the same control adenovirus dem-
onstrated very low expression, consistently just above the baseline
signal derived from control Ig (Fig. 4). Similar results were ob-
served using a monoclonal anti-SKAP55 Ab for flow cytometry
and Western blotting, and in freshly isolated, non-adenovirus-
transduced lymphocytes (data not shown). Adenoviral-mediated
reconstitution of ADAP expression allowed SKAP55 levels to ac-
cumulate (Fig. 4), with peak expression observed after ?3 days of
infection (data not shown). This effect was positively correlated
with the level of Thy1.1 expression, indicating that SKAP55 ac-
cumulation mirrors the level of ADAP reconstitution (see Fig. 1D).
Analysis of the ADAP domains required for SKAP55 stability
demonstrated that neither the N terminus (aa 1–327) nor the C
terminus (aa 426–819) of ADAP is essential. In contrast, removal
of the central proline-rich domain of ADAP (?326–426) or the
restricted deletion (?338–358) within this region precluded accu-
mulation of SKAP55 (Fig. 4). Expression of the proline-rich do-
main alone (?326–426) was sufficient to stabilize SKAP55, even
though this domain does not rescue conjugate formation (Fig. 4).
We next performed coimmunoprecipitation analysis to confirm
that the interaction of ADAP with SKAP55 is dependent on the
ADAP proline-rich domain. Jurkat T cells were transduced with
the control virus, or with HA-tagged wtADAP, ?426–541,
T:APC conjugate formation. A, Jurkat T cells were transduced with control
adenovirus (Ctrl) or with the indicated HA-tagged ADAP constructs. After
2 days, 106cells were left unstimulated or stimulated for 5 min with anti-
TCR mAb OKT3, lysed, and immunoprecipitated with agarose-conjugated
anti-HA and Western blots for ADAP and SKAP55 were performed. The
relative molecular mass of size standards is shown on the right. Similar
results were observed in four independent experiments. B, Naive hCAR/
ADAP?/?lymphocytes expressing endogenous levels of SKAP55 were
transduced with either full-length murine ADAP (wtADAP) or ADAP
lacking the restricted proline-rich domain (?338–358). Cells were
lysed as described in Materials and Methods, immunprecipitated with
agarose-conjugated anti-HA Abs, and Western blots were performed
with anti-HA or anti-SKAP55 Abs. C, Naive DO11.10/hCAR/ADAP?/?
(KO) or ADAP?/?(WT) lymphocytes expressing the indicated constructs
were analyzed for T:APC conjugate formation between KJ1-26?Thy1.1?
T cells and BALB/c splenocytes prepulsed with the indicated concentra-
tions of OVAp. Results are shown for a single representative experiment
and are representative of at least four independent experiments performed
for each construct.
SKAP55 expression in ADAP?/?T cells fails to rescue
4845 The Journal of Immunology
?426–819, or ?326–426, and stimulated with anti-TCR mAb
OKT3 or left unstimulated. Following lysis and anti-HA immu-
noprecipitation, SKAP55 constitutively interacted with all con-
structs except the proline-rich domain mutant (?326–426) (Fig.
5A) and with the restricted proline deletion (?338–358, data not
shown). To assess the dependence on this proline-rich domain
in primary murine T cells, we transduced ADAP?/?T cells,
which maintain endogenous SKAP55, with either HA-tagged
wtADAP or HA-tagged ADAP?338–358. Input whole-cell ly-
sates from each sample contained equivalent levels of SKAP55,
and the epitope-tagged ADAP construct was pulled down
equally following anti-HA immunoprecipitation (Fig. 5B). Im-
portantly, SKAP55 was detected in the HA immunoprecipitate
from the wtADAP-transduced sample but not from the
ADAP?338–358 sample (Fig. 5B). Thus, the ADAP proline-
rich motif is critical for association with and stability of
SKAP55 in primary murine T cells.
SKAP55 expression is not sufficient for T:APC conjugate
Since cells expressing the ADAP proline-rich domain mutants lack
normal levels of SKAP55, it remains possible that stable SKAP55
expression alone might be sufficient for T:APC conjugate forma-
tion. To directly test this model, we infected ADAP?/?T cells
with adenovirus expressing SKAP55. Following transduction
with the virus, SKAP55 was detected by intracellular flow cy-
tometry (see Fig. 4, final panel) at levels approaching that of
endogenous SKAP55 found in ADAP?/?cells. This level of
SKAP55 is at or above the level of SKAP55 that accumulated
following expression of wtADAP. Furthermore, the exog-
enously supplied SKAP55 was able to interact with ADAP
when expressed in ADAP?/?T cells (data not shown). How-
ever, SKAP55 expression in DO11.10/ADAP?/?T cells failed
to rescue T:APC conjugate formation (Fig. 5C). This result sug-
gests that SKAP55 is not sufficient for this function and that
both ADAP and SKAP55 are required for control of Ag recep-
tor-dependent integrin function in naive T cells.
We additionally tested the dependence on ADAP for recruit-
ment of Rap1 and SKAP55 to TCR-coated beads, as a measure of
the membrane recruitment potential of SKAP55 and Rap1. ADAP
has been has been shown to be important for TCR-induced mem-
brane recruitment of Rap1 (29 and R. B. Medeiros and Y. Shimizu,
unpublished observations). Due to limitations in cell numbers ob-
tained from our ex vivo adenovirus cultures, we were unable to
perform biochemical fractionation assays as previously described
(29). Instead, we expressed GFP-Rap1 in WT or ADAP?/?T cells
and monitored targeting of this construct to the interface of anti-
TCR-coated beads using image-scanning cytometry. Although we
did not see striking organization of Rap1 at the contact site (Fig.
6A), ?40% of the T cell Rap1 was within the bead contact site in
WT T cells (Fig. 6B). Surprisingly, ADAP?/?T cells did not show
a defect in Rap1 targeting to the TCR contact site (Fig. 6B, p ?
0.96), and there were no differences in this measure of Rap1 re-
cruitment between ADAP?/?cells reconstituted with wtADAP,
ADAP?426–819, or ADAP?326–426 (data not shown). The re-
cruitment of SKAP55 was also monitored in ADAP?/?or
ADAP?/?T cells expressing wtADAP, ?426–819, or SKAP55 in
the absence of ADAP (Fig. 6C). In contrast to Rap1, SKAP55 was
tightly recruited to the T cell/bead interface and frequently adopted
a bimodal staining as the T cell wrapped around the edges of the
bead (Fig. 6C). Quantification of these observations indicated that
?40% of the SKAP55 in the cell was recruited to the bead inter-
face in this assay. However, no differences in SKAP55 recruitment
were observed between ADAP?/?cells reconstituted with wt-
ADAP or ADAP?426–819 (36 vs 39%; Fig. 6D). Similarly, we
also noticed that 40% of exogenous SKAP55 expressed in the
absence of ADAP was also recruited to the bead contact. This
suggests that determinants within SKAP55 may be sufficient to
drive membrane targeting in these assays. Interestingly, ADAP?/?
cells were somewhat more efficient in their overall ability to recruit
SKAP55 to the bead contact (48% of cellular SKAP55) compared
with ADAP?/?cells expressing wtADAP, ADAP?426–819, or
SKAP55 alone (p ? 0.0001).
Rap1 or SKAP55 to the contact site between T cells and
anti-TCR beads. A, Naive DO11.10/hCAR/ADAP?/?
(WT) or ADAP?/?(KO) lymphocytes were transduced
with GFP-Rap1 and conjugates with anti-TCR (2C11)-
coated beads were formed as described in Materials and
Methods. GFP-Rap1-expressing cells were gated and
photographed by image-scanning cytometry and a rep-
resentative image from each sample is shown. An ex-
ample of GFP-Rap1 expression in a cell absent of bead
stimulation is also shown (Unstim). B, Graphical dis-
play of the percentage of total GFP-Rap1 signal in each
cell that is concentrated against the anti-TCR-coated
bead, quantified as described in Materials and Methods.
C, Naive DO11.10/hCAR/ADAP?/?(WT) T cells ex-
pressing the control virus (Ctrl) or ADAP?/?(KO) lym-
phocytes expressing wtADAP, ADAP?426–819, or
SKAP55 were stimulated with anti-TCR-coated beads
as described in A, fixed, and stained for SKAP55 as
described for Fig. 4. D, Graphical display of the per-
centage of total SKAP55 signal in each cell that is con-
centrated against the anti-TCR-coated bead.
ADAP is not required for recruitment of
4846DISTINCT INTEGRIN AND NF-?B REGULATION BY ADAP
ADAP/SKAP55 interaction is required for optimal T cell
In addition to impaired integrin-mediated adhesion, ADAP is also
important for TCR- and Ag-dependent T cell activation and clonal
expansion, especially at low Ag concentrations (25). However, it is
not known whether these reported defects in Ag-dependent T cell
activation trace to impaired integrin-mediated adhesion at the on-
set of Ag stimulation, or to other ADAP-dependent signaling path-
ways. To determine which ADAP functional domains are impor-
tant for T cell activation, we monitored expression of the early
activation marker CD69, the IL-2 receptor (CD25), and the pro-
survival protein Bcl-xLin ADAP?/?T cells expressing the wt-
ADAP or ADAP mutants. Using primary naive DO11.10/hCAR/
ADAP?/?or ADAP?/?cells reconstituted ex vivo with control
adenovirus, we consistently observed a reduction in the percentage
of ADAP?/?cells displaying CD69 and CD25 expression follow-
ing 18 h of stimulation with OVAp (Fig. 7, A and B). Bcl-xL
expression was also impaired in ADAP?/?cells after 48 h of stim-
ulation (Fig. 7C), consistent with previous results (25). Reconsti-
tution of ADAP?/?cells with wtADAP but not with the SKAP55-
binding mutant ADAP?338–358 restored CD69, CD25, and
Bcl-XLactivation to levels at or above ADAP?/?T cells (Fig. 7),
suggesting that the ADAP/SKAP55 interaction is important for
optimal Ag-dependent T cell activation. Similarly, ADAP?/?cells
expressing the ADAP?426–819 C-terminal mutant, or expressing
SKAP55 alone in the absence of ADAP, were unable to activate
CD69, CD25, or Bcl-xLas robustly as when intact wtADAP is
expressed (data not shown). In contrast, ADAP?/?cells express-
ing the CARMA1-binding mutant ADAP?426–541 did not show
appreciable defects in CD69, CD25, and Bcl-xLexpression, espe-
cially when compared with ADAP?/?cells. These results indicate
that ADAP-dependent conjugate formation influences the func-
tional activation state of T cells.
ADAP/SKAP55 is not required for NF-?B activation
ADAP also regulates TCR-mediated activation of the transcription
factor NF-?B in T cells via TCR-regulated binding of the NF-?B
regulatory protein CARMA1 to aa 426–541 of ADAP, and sub-
sequent assembly of the CARMA1/Bcl10/MALT1 complex (19).
However, the ADAP mutant lacking CARMA1 binding capacity
(ADAP?426–541) can restore efficient T:APC conjugate forma-
tion (19). In line with this finding, primary ADAP?/?T cells ex-
pressing ADAP?426–541 showed efficient accumulation of
SKAP55, suggesting that the CARMA1-binding domain within
ADAP is dispensable for SKAP55 stabilization (Fig. 4). This sug-
gests that ADAP forms physically and/or functionally distinct
complexes with SKAP55 and with CARMA1 in the cell. However,
it is unclear whether the ADAP/SKAP55 complex is required for
NF-?B activity. To address this question, ADAP?/?T cells ex-
pressing wtADAP, ADAP?426–541, or ADAP?338–358 were
stimulated, and then cell lysates were prepared and subjected to
CARMA1 and ADAP. Consistent with previous results (19), stim-
ulation with PMA enhanced the association of CARMA1 with
Bcl10 in control ADAP?/?cells and in ADAP?/?cells expressing
wtADAP, but not in ADAP?/?cells expressing either control ad-
enovirus or ADAP?426–541 (Fig. 8A). When ADAP?338–358
was expressed in ADAP?/?T cells, CARMA1 also associated
with Bcl10 following PMA stimulation (Fig. 8A), indicating that
neither the ADAP proline-rich domain nor SKAP55 is required for
and Westernblotting for
lymphocytes expressing the indicated ADAP constructs were stimulated with fresh BALB/c splenocytes pulsed with the indicated doses of OVAp as
described in Materials and Methods. A and B, Cells were stimulated for 18 h, stained for KJ1-26, Thy1.1, CD69, and CD25 as indicated, fixed, and analyzed
by flow cytometry. C, Cells were stimulated for 48 h with OVAp, stained for KJ1-26 and Thy1.1, fixed, permeablized with saponin, stained for anti-Bcl-xL,
and analyzed by flow cytometry. A–C, The percentage of CD69?, CD25?, or Bcl-xL
Results are shown for a single representative experiment and are representative of at least four independent experiments performed for CD69 and CD25
and three experiments for Bcl-xL.
The ADAP proline-rich domain is required for Ag-dependent T cell activation. Naive DO11.10/hCAR/ADAP?/?(WT) or ADAP?/?(KO)
?cells within the KJ1-26?Thy1.1?gate is shown on each histogram.
4847 The Journal of Immunology
this complex to assemble. Furthermore, ADAP was only found in
the Bcl10 immunoprecipitates where the Bcl10/CARMA1 associ-
ation formed (Fig. 8A).
To assess whether the cellular pool of ADAP assembled with
Bcl10/CARMA1 is separate from that found with SKAP55, iden-
tical aliquots of stimulated lysates from ADAP?/?cells were sub-
jected to immunoprecipitation with either anti-Bcl10 or anti-
ADAP Abs. Assembly of Bcl10, CARMA1, and ADAP was
readily observed by either immunoprecipitation strategy (Fig. 8B).
Interestingly, while anti-ADAP immunoprecipitation showed
SKAP55 association (Fig. 8B, left panels), SKAP55 was not de-
tectable in Bcl10 immunoprecipitates (Fig. 8B, right panels). This
suggests that SKAP55 and its binding domains in ADAP are not
involved in TCR-dependent NF-?B activation. Consistent with
this model, expression of the ADAP?338–358 mutant rescued
TCR-dependent NF-?B nuclear translocation to levels similar to
that observed when wtADAP is expressed (Fig. 8C). In contrast,
ADAP?426–541, which fails to bind Bcl10 and CARMA1, did
not rescue NF-?B activation (Fig. 8C). Taken together, these find-
ings indicate that the ADAP proline-rich domain and assembly of
the ADAP/SKAP55 complex are not important for ADAP-depen-
dent NF-?B activity.
We have investigated the molecular mechanism of ADAP-depen-
dent integrin and NF-?B activation in naive primary murine T
lymphocytes. The development and analysis of ADAP?/?mice
clearly demonstrated that ADAP positively regulates T cell acti-
vation, ?1and ?2integrin-dependent adhesion (23, 24), and pep-
tide Ag-dependent T:APC conjugate formation (19, 25). However,
initial analysis of ADAP function before the development of
ADAP?/?mice utilized overexpression approaches and yielded
results that were consistent with both a positive and negative func-
tion for ADAP in regulating TCR-dependent signaling (8, 9, 12,
14). Several recent investigations of ADAP function have also
utilized overexpression approaches and Ab-mediated TCR stimu-
lation in Jurkat T cells (20, 29, 33) or retroviral-mediated trans-
duction and overexpression in activated primary T cells (26, 45).
Given the concerns with interpreting functional effects of muta-
tions under conditions where ADAP is overexpressed in cells, we
reasoned that structure/function analysis of ADAP would be most
informative under conditions where mutant ADAP constructs
could be expressed in the absence of endogenous ADAP. To over-
come the technical challenges of gene delivery into naive primary
murine ADAP?/?T cells, we crossed the OVAp Ag-specific
DO11.10/ADAP?/?mice (25) to mice bearing the hCAR trans-
gene (35), which allows efficient adenoviral-mediated gene deliv-
ery into resting, naive T lymphocytes.
Using this system, we show that impaired peptide Ag-dependent
T:APC conjugate formation in ADAP?/?T cells (25, 46) is res-
cued following expression of wtADAP to levels approaching that
of endogenous ADAP. Our mutational analysis indicates that while
the N-terminal 327 aa of ADAP is not required for the rescue of
conjugate formation, the central proline-rich domain is necessary
but not sufficient for rescue. We also found that this ADAP pro-
line-rich domain is important for optimal functional T cell activa-
tion as measured by CD69, CD25, and Bcl-xLexpression follow-
ing Ag stimulation. Furthermore, the C-terminal half of ADAP (aa
426–819) is a second region critical for Ag-dependent integrin
adhesive function. Thus, our results are consistent with recent
work demonstrating a requirement for the ADAP proline-rich and
C-terminal domains for TCR-induced adhesion to immobilized ?1
and ?2integrin ligands (29, 33).
Several previous studies have outlined the physical and func-
tional relationship between the T cell adapter proteins ADAP and
bly of the CARMA1/Bcl10 complex or TCR induced NF-?B activation. A,
Naive DO11.10/hCAR/ADAP?/?(KO) or ADAP?/?(WT) lymphocytes
expressing the indicated ADAP constructs or the control (Ctrl) were left
untreated (?) or stimulated with PMA (?) and lysed as described in
Materials and Methods. Lysates were subjected to immunoprecipitation
with an anti-Bcl10 Ab, and Western blots were performed with Abs to
CARMA1, ADAP, or Bcl10. B, Freshly harvested resting ADAP?/?
(WT) or ADAP?/?(KO) lymphocytes were left unstimulated or stim-
ulated with PMA and lysed as in A and immunoprecipiated in parallel
with Abs to either ADAP (left panel) or Bcl10 (right panel). Western
blots were performed with Abs to ADAP, CARMA1, Bcl10, and
SKAP55 as indicated between the panels. Results are representative of
three (A) or two (B) independent experiments. C, Naive DO11.10/
hCAR/ADAP?/?(KO) or ADAP?/?(WT) lymphocytes expressing the
indicated constructs were stimulated with anti-CD3 plus anti-CD28 Abs
as described in Materials and Methods or left untreated. The samples
were fixed and stained with Abs to Thy1.1 and NF-?B (p65), and nuclei
were stained with 7-aminoactinomycin D. Cells were analyzed on a
multispectral image-scanning flow cytometer as described in Materials
and Methods. Nuclear localization of p65 in unstimulated T cells was
set to 1, and results show the increase in p65 nuclear translocation in
stimulated relative to unstimulated Thy1.1?cells from three indepen-
The ADAP/SKAP55 interaction is not required for assem-
4848DISTINCT INTEGRIN AND NF-?B REGULATION BY ADAP
SKAP55. A direct association between the central proline-rich do-
main of ADAP and the SH3 domain of SKAP55 was first identi-
fied using two-hybrid screens and coimmunoprecipitation experi-
ments with Jurkat T cells (17, 18). The link between the ADAP/
SKAP55 complex and promotion of LFA-1 integrin function was
demonstrated by overexpressing either protein in an Ag-specific T
cell hybridoma line, or by retroviral-mediated overexpression of
SKAP55 in activated primary murine T cells (26). Conversely, loss
of SKAP55 by small interfering RNA-mediated gene knockdown
(27, 29, 33) or in SKAP55?/?mice (28) decreased LFA-1-medi-
ated adhesion. Detailed mechanistic studies on the role of ADAP
and SKAP55 in T cell activation and integrin function have re-
cently been confounded by the observation that ADAP?/?T cells
are severely deficient in SKAP55 (28, 29, 34). This loss of
SKAP55 in the absence of ADAP traces to the constitutive inter-
action between ADAP and the SH3 domain of SKAP55, which
acts to protect SKAP55 from caspase- and proteasome-mediated
degradation (34). Our studies show that the SKAP55 deficiency in
ADAP?/?primary murine T cells is reversible upon reintroduc-
tion of intact ADAP protein. Furthermore, we show that a 20-aa
region between aa 338–358 in the ADAP proline-rich domain is
necessary and sufficient for stabilization of endogenous SKAP55.
This region of murine ADAP is analogous to a 24-aa site defined
in human ADAP (aa 340–364) (29), and contains a 12-aa LGPP-
PPKPNRPP sequence that includes a canonical core PxxPxP motif
capable of binding to several types of SH3 domains (47). There is
high sequence identity in this region of ADAP isolated from hu-
man, mouse, monkey, dog, cow, chicken, and zebrafish, suggesting
a conserved function for ADAP that is dependent on this proline-
Since cells expressing ADAP proline-rich domain mutants si-
multaneously fail to rescue conjugate formation and to reexpress
SKAP55, it is unclear whether ADAP, SKAP55, or both are re-
quired for control of Ag-dependent integrin function. Indeed, we
are unaware of any experiments to date that have examined the
function of SKAP55 independent of ADAP expression. In our ex-
periments, expression of the isolated proline-rich domain of ADAP
(?326–426) or of the C-terminal deletion (?426–819) permits
significant recovery of endogenous SKAP55 expression, while still
failing to rescue conjugate formation. To directly test the model
that free SKAP55 expression in the absence of any ADAP inter-
action could be sufficient for T:APC conjugate formation, we spe-
cifically expressed SKAP55 in ADAP?/?T cells. Our results
show that even in ADAP?/?T cells containing SKAP55 similar to
endogenous levels, T:APC conjugate formation was not enhanced.
Thus, SKAP55 expression in the absence of ADAP is not sufficient
to regulate TCR signaling to integrins. The presence of the proline-
rich domain of ADAP, as well as the C terminus of ADAP, in
combination with SKAP55 is necessary for optimal integrin
Regulation of ?1and ?2integrin function in T cells is dependent
on both the activation and the membrane/synapse targeting of the
small GTPase Rap1 (30, 41, 48). Recently, ADAP?/?T cells have
been shown to be defective for membrane targeting of the active
(GTP-bound) form of Rap1 (29), a finding consistent with the de-
fects in integrin-mediated adhesion observed in ADAP?/?T cells.
We attempted to test the role of our ADAP C-terminal mutations
in controlling Rap1 membrane targeting, but we were unable to
obtain enough transduced cells to perform these biochemical com-
parisons. To test whether ADAP controls recruitment of Rap1 to
the TCR signaling complex, we transduced primary WT or
ADAP?/?cells with GFP-Rap1 and monitored the recruitment of
GFP-Rap1 to anti-TCR-coated beads. Using this approach, we did
not detect any gross differences in the percentage of Rap1 recruited
to the T cell/bead interface. This apparent discrepancy between our
results and the published results may trace to the ability of Rap1 to
localize or become activated on intracellular membranes (49, 50),
to rapid kinetics of Rap1 activation that we were unable to capture
during imaging, or to the inherent differences between soluble anti-
TCR stimulation vs T cell activation against an anti-TCR-coated
bead, which is somewhat more similar to an APC.
Elucidation of the molecular pathways downstream of the
ADAP/SKAP55 complex have implicated the involvement of the
constitutive SKAP55-binding protein, RIAM (also known as
PREL1), which contains a central RA-PH domain capable of bind-
ing to active Rap1 (33). RIAM has also been shown to control ?1
and ?2integrin activation in T cells following TCR stimulation
(51), and it has been implicated in binding Ena/Vasp proteins and
the regulation of actin dynamics in T cells (52). RNA interference-
mediated depletion of SKAP55 in Jurkat T cells results in impaired
membrane localization of RIAM and Rap1 following TCR stim-
ulation (33), consistent with the role of SKAP55 as a critical ef-
fector in TCR-mediated integrin signaling (28). By contrast,
ADAP membrane targeting following TCR stimulation is unaf-
fected by SKAP55 depletion (29, 33). While we were unable to
detect the ADAP/SKAP55/RIAM complex in the present study
(data not shown), we were able to monitor the targeting of
SKAP55 to anti-TCR-coated beads. ADAP?/?cells expressing
wtADAP or ADAP?426–819 were equally able to recruit their
rescued SKAP55 to the T cell/bead interface. Interestingly,
SKAP55 was also recruited even when expressed in the absence of
ADAP. These previous results and our findings suggest that both
ADAP and SKAP55 may contain membrane targeting information
that has the capacity to recruit the proposed ADAP/SKAP55/
RIAM/Rap1-GTP complex to the immune synapse following TCR
activation by APCs. Alternatively, the entire complex itself may
complete a quaternary structure that promotes membrane targeting
and/or integrin activation.
The precise role of the ADAP C terminus (aa 426–819) in pro-
moting integrin function remains unclear. Although the extreme
C-terminal SH3-like domain (hSH3c) of ADAP has been impli-
cated in a secondary low-affinity interaction with SKAP55 that is
released following TCR activation and SKAP55 phosphorylation
(53, 54), this domain of ADAP is not absolutely required for en-
hanced integrin-mediated adhesion in mast cells (55). Indeed, we
were able to detect robust TCR-independent interaction between
ADAP and SKAP55 following removal of the entire ADAP C
terminus (ADAP?426–819), suggesting that the ADAP C termi-
nus is not absolutely required for ADAP interaction with SKAP55.
Additionally, the ADAP domain between aa 426 and 541, which is
critical for interaction with CARMA1 and for NF-?B activation, is
not required for T:APC conjugate formation (19) or for constitu-
tive binding to SKAP55. However, since the ADAP C terminus
still controlled the ultimate outcome of T:APC conjugate forma-
tion and T cell activation, it is thus likely that an ?200-aa segment
between murine ADAP aa 542 and 750 is a secondary domain that
controls T:APC conjugate formation. This region contains several
tyrosine residues implicated in binding SLP-76 (12, 13). Presum-
ably, ADAP tyrosine phosphorylation in this region is responsible
for its recruitment to LAT-associated SLP-76 at the plasma mem-
brane following TCR stimulation (1, 3, 11). Although a reduction
in integrin activation has been reported following treatment with a
SLP-76 inhibitor peptide (56), and an ADAP construct containing
mutations in the ADAP tyrosine residues implicated in binding to
the SH2 domain of SLP-76 shows impaired overexpression-in-
duced T:APC conjugate formation (45), a direct role for SLP-76 in
T cell integrin function has not yet been carefully defined. The
4849 The Journal of Immunology
ADAP C terminus also contains EVH1 (Ena/Vasp homology do-
main 1) homology motifs that have been implicated in binding
members of the actin-regulatory Ena/Vasp family proteins (15,
57), which could affect T cell integrin activity. Interestingly, the
helical extension of the two noncanonical hSH3 domains of ADAP
has been reported to influence integrin-dependent adhesion (20).
These domains bind phospholipids in vitro (21) and are predicted
to influence the overall conformation of ADAP in response to ox-
idative stress following T cell activation (58). Additionally, part of
the N-terminal hSH3 domain of ADAP overlaps with the
CARMA1 binding site in ADAP (19). Thus, future experiments
will be required to further distinguish molecular signatures in the
C terminus of ADAP required for T:APC conjugate formation.
In addition to controlling TCR-dependent integrin function,
ADAP regulates TCR-dependent NF-?B activation (19). This
novel function for ADAP is dependent on the ability of the central
aa 426–541 in ADAP to bind the adapter CARMA1, which in turn
allows the assembly of the CARMA1/Bcl10/Malt1 (CBM) signal-
ing complex. This ADAP-CBM signaling complex is critical for
phosphorylation and degradation of I?B, liberating NF-?B to
translocate to the T cell nucleus and promote gene transcription.
We previously reported that the CARMA1-binding domain in
ADAP is dispensable for T:APC conjugate formation (19). In the
present study we found that the CARMA1 binding function of
ADAP was also not required for rescue of downstream activation
markers of T cell function including CD69, CD25, and surpris-
ingly the NF-?B-regulated gene Bcl-xL. This suggests that the ini-
tial integrin-dependent adhesion during T cell priming is a critical
event in determining the activation status 1–2 days following Ag
The role of the ADAP proline-rich domain and the ADAP/
SKAP55 complex in regulating NF-?B activation has not been
previously investigated. We show herein that reconstitution of
ADAP?/?T cells with ADAP lacking its proline-rich domain
(which negates SKAP55 expression) is sufficient to rescue assem-
bly of the CBM complex and NF-?B activation. Indeed, while
ADAP immunoprecipitates from activated T cells contain Bcl10,
CARMA1, and SKAP55, Bcl10 immunoprecipitates from acti-
vated T cells contain ADAP and CARMA1, but not SKAP55. This
is consistent with reports that TCR-induced activation of a NF-?B
reporter is unaffected by RNA interference-mediated suppression
of SKAP55 expression (59). In summary, our data support a model
where ADAP coordinates two distinct and physically segregated
signaling pathways following TCR stimulation. One pathway in-
volves recruitment of the ADAP/SKAP55/RIAM/Rap1-GTP com-
plex to the membrane and leads to LFA-1 activation and cluster-
ing. A second pathway involves TCR-mediated protein kinase C ?
activation that promotes ADAP-dependent assembly of the CBM
complex and subsequent NF-?B activation.
The relative contributions of ADAP-dependent T:APC conju-
gate formation and NF-?B activation toward T cell activation are
currently not clear. While we found that the ADAP/CARMA1 in-
teraction is not absolutely required for early T cell activation
events in vitro, we were unable to assess Ag-dependent T cell
clonal expansion, which occurs 2–3 days following activation, be-
cause of the transient nature of our adenovirus expression assay.
Furthermore, it is clear that these ex vivo activation assays do not
accurately recapitulate the in vivo microenvironment. In particular,
there are defects in clonal expansion of DO11.10 ADAP?/?T
cells in response to Ag challenge in vivo that are particularly pro-
nounced when naive T cells are present at physiologically low
precursor frequencies (25). Our results and those of others (28)
suggest that impaired interactions of ADAP?/?T cells with APCs
in vivo may lower TCR sensitivity to Ag, resulting in inefficient
delivery of activation signals required for activation and clonal
expansion. Thus, our finding that ADAP also controls a separate T
cell activation pathway involving NF-?B suggests that impaired
clonal expansion of ADAP?/?T cells in vivo in response to Ag
may also be due to impaired induction of NF-?B-dependent genes
critical for T cell activation and survival. The combined functions
of ADAP may explain the dramatically impaired clonal expansion
of ADAP?/?T cells at low precursor frequencies in vivo, despite
the fact that ADAP?/?T cells still exhibit some level of T:APC
conjugate formation and activation in vitro. A role for ADAP in
NF-?B signaling suggests that T cells capable of forming conju-
gates with APCs in the absence of ADAP may still not receive the
proper array of signals necessary for optimal T cell activation.
Indeed, a recent report found that while ADAP?/?CTLs have no
defects in target cell killing, they exhibited impaired allograft-me-
diated rejection, consistent with the presence of underlying non-
adhesion-dependent activation defects in the absence of ADAP
(60). Future mechanistic studies in vivo will need to distinguish
between concurrent defects in both ADAP-dependent integrin-me-
diated adhesion and NF-?B signaling.
We thank S. Highfill and M. Schwartz for mouse genotyping and colony
maintenance and Drs. C. Weaver and B. Schraven for mice and reagents.
We thank the University of Minnesota Flow Cytometry Core for FACS and
ImageStream instrumentation and technical assistance.
The authors have no financial conflicts of interest.
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4851 The Journal of Immunology