Mechanisms of Signal Transduction:
Synergistic Assembly of Linker for
Activation of T Cells Signaling Protein
Complexes in T Cell Plasma Membrane
Domains
J. Biol. Chem. 2003, 278:20389-20394.
doi: 10.1074/jbc.M301212200 originally published online March 19, 2003
Access the most updated version of this article at doi: 10.1074/jbc.M301212200
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
This article cites 24 references, 7 of which can be accessed free at
http://www.jbc.org/content/278/22/20389.full.html#ref-list-1
Downloaded from http://www.jbc.org/ at University of Liverpool on October 23, 2014
Lorian C. Hartgroves, Joseph Lin, Hanno
Langen, Tobias Zech, Arthur Weiss and
Thomas Harder
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 22, Issue of May 30, pp. 20389 –20394, 2003
Printed in U.S.A.
Synergistic Assembly of Linker for Activation of T Cells Signaling
Protein Complexes in T Cell Plasma Membrane Domains*
Received for publication, February 4, 2003, and in revised form, March 7, 2003
Published, JBC Papers in Press, March 19, 2003, DOI 10.1074/jbc.M301212200
Lorian C. Hartgroves‡, Joseph Lin§, Hanno Langen¶, Tobias Zech‡, Arthur Weiss§,
and Thomas Harder‡储
From the ‡Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE,
United Kingdom, the §Departments of Medicine and of Microbiology and Immunology, Howard Hughes
Medical Institute, University of California, San Francisco, California 94143-0795, and ¶Hoffmann-La Roche Ltd.,
Grenzacherstrasse 124, CH-4070 Basel, Switzerland
Activation of the T cell antigen receptor (TCR)1 by peptide/
major histocompatibility complex ligands triggers tyrosine
phosphorylation cascades, which induce downstream signaling
events and eventually lead to a physiological T lymphocyte
response. These cascades involve sequential activation of Src
family protein tyrosine kinases Lck/Fyn and the Syk family
protein tyrosine kinase ZAP-70 (1–3). TCR activation signals
are transduced via signaling protein complexes, which assemble at the plasma membrane in close vicinity of TCR engagement and form a platform for the activation of signaling enzymes. Transmembrane protein LAT (linker for activation of T
cells) is a central scaffold for these multimolecular signaling
complexes. Phosphorylation of LAT tyrosines by ZAP-70 cre-
* The work was supported by the Medical Research Council, UK
(MRC) Cooperative Component G0100252 of MRC Cooperative Grant
G0000764 “Immune Recognition.” 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.
储 To whom correspondence should be addressed. E-mail: thomas.
harder@path.ox.ac.uk.
1
The abbreviations used are: TCR, T cell antigen receptor; LAT,
linker for activation of T cells; NFAT, nuclear factor of activated T cells;
PLC␥, phospholipase C ␥; wt, wild type; MALDI-MS, matrix-assisted
laser desorption ionization-mass spectrometry; TLA, TCR䡠LAT signaling assembly; DRM, detergent-resistant membrane; BAPTA-AM, bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester; SH, Src homology.
This paper is available on line at http://www.jbc.org
ates docking sites for SH2 domain-containing signaling proteins (4). This leads to the formation of LAT-based protein
assemblies, which contain the adaptor/scaffolding proteins
Grb2, Gads, and SLP-76, as well as the signaling enzymes
phospholipase C ␥ (PLC␥), phosphatidylinositol 3-kinase, and
Vav (5–7). PLC␥, when recruited into these complexes, becomes
activated to catalyze hydrolysis of PI4,5P2 leading to Ca2⫹
mobilization, protein kinase C and Ras-activation (8, 9). Moreover, mitogen-activated protein kinase cascades are triggered
via the Grb2/SOS/Ras GTPase cascade, and Vav activation
triggers reorganization of the actin cytoskeleton (5).
Disruption of the LAT gene in mice causes a block in early T
cell development, reflecting a severe inhibition of TCR-evoked
signaling (10). The Jurkat leukemic T-cell derivative JCaM2
lacks LAT expression and consequently is incapable of transducing TCR-evoked Ca2⫹ fluxes and Ras-dependent downstream signaling (7). Extensive mutational analysis of LAT
identified tyrosine-based docking sites for PLC␥ at Tyr-132
(Tyr-6) and for adaptor proteins Grb2 and Gads at Tyr-171 and
-191 (Tyr-7 and -8) (11–13). Reconstitution of LAT variants into
LAT-deficient JCaM2 cells showed that these three tyrosines
(Tyr- 6, -7, and -8) comprise a minimal set of docking sites for
transducing Ca2⫹ fluxes and Ras-dependent downstream signaling (11). Moreover, Tyr-110 and -226 (Tyr-4 and -9) reside in
YXN Grb2-binding consensus motifs and are required to elicit
a maximal level of NFAT-mediated transcription (11).
Although the binding sites of LAT for different signaling
proteins and their role in TCR downstream signaling has been
extensively characterized, it is not known how they contribute
to the formation of multimolecular signaling complexes in
plasma membrane subdomains. Previously, we developed a
procedure to immunoisolate native plasma membrane fragments that strongly enrich TCR signaling complexes (14). Biochemical analysis of these isolates led to the description of
LAT-based signaling complexes, which accumulate in the vicinity of activated TCR (15). Moreover, formation and disassembly of such TCR䡠LAT signaling assemblies over time have
been followed using confocal microscopy (16, 17). Here, we
studied in JCaM2 cells expressing LAT variants how different
signaling protein docking sites affect the formation of LAT
signaling complexes in T cell plasma membrane subdomains.
We found that different docking sites on LAT synergize to
mediate accumulation of LAT and LAT-associated signaling
protein in TCR signaling domains, suggesting a cooperative
assembly of LAT multimolecular signaling complexes.
EXPERIMENTAL PROCEDURES
Cell Lines and Reagents—Jurkat derivative JCaM2 was maintained
in RPMI medium, 7% fetal calf serum, penicillin (100 mg/ml)/streptomycin (100 units/ml), 2 mM glutamax (Invitrogen). JCaM2 clones that
20389
Downloaded from http://www.jbc.org/ at University of Liverpool on October 23, 2014
Transmembrane adaptor molecule LAT (linker for activation of T cells) forms a central scaffold for signaling
protein complexes that accumulate in the vicinity of
activated T cell antigen receptors (TCR). Here we used
biochemical analysis of immunoisolated plasma membrane domains and fluorescence imaging of green fluorescence protein-tagged signaling proteins to investigate the contributions of different tyrosine-based
signaling protein docking sites of LAT to the formation
of LAT signaling protein assemblies in TCR membrane
domains. We found that the phospholipase C ␥ docking
site of LAT and different Grb2/Gads docking sites function in an interdependent fashion and synergize to accumulate LAT, Grb2, and phospholipase C ␥ in TCR signaling assemblies. Two-dimensional gels showed that
Grb2 is a predominant cytoplasmic adaptor in the isolated LAT signaling complexes, whereas Gads, Crk-1,
and Grap are present in lower amounts. Taken together
our data suggest a synergistic assembly of multimolecular TCR䡠LAT signal transduction complexes in T cell
plasma membrane domains.
20390
LAT Signaling Protein Complexes
For depletion of intracellular Ca2⫹ stores, cells were incubated in
RPMI, 1% fetal calf serum, 1 mM EGTA, 50 M BAPTA-AM (Sigma) for
30 min at 37 °C as described previously (19). Cell/bead conjugates were
formed in RPMI, 1% fetal calf serum, 1 mM EGTA, 50 M BAPTA-AM,
and isolations were performed as described above.
Detergent-insoluble Membrane Fractions—1 ⫻ 107 JCaM2 cells stably expressing either LAT All Y 3 F or wt LAT were lysed in 300 l of
ice-cold HNE (10 mM Hepes, pH 7.0, 150 mM NaCl, and 5 mM EDTA),
homogenized by 1% Triton X-100 and passage through a 22-gauge
needle, and incubated on ice for 15 min. The extract was adjusted to
40% OptiprepTM (Nycomed, Pharma), transferred to an SW55 centrifuge tube (Beckman Coulter), and overlaid with 1 ml of 35, 30, 25, and
0% OptiprepTM in HNE. The gradients were spun at 40,000 rpm for 3 h,
and 600-l fractions were trichloroacetic acid-precipitated and analyzed by Western blotting.
Two-dimensional Gel Analysis and Identification of Proteins by
MALDI-MS—Immunoisolates of 4 ⫻ 108 Jurkat cells were prepared as
described above except that prior to the homogenization, bead/cells
conjugates were incubated with non-cell-permeable cross-linker
bis(sulfo succinimdyl suberate) (Pierce), 1 mg/ml, in H buffer on ice for
10 min at 4 °C in order to increase the yields of the isolation and to
cross-link the antibody chains to the beads. Two-dimensional gels of the
isolates were run using Bio-Rad mini two-dimensional gel apparatus
according to the instructions of the manufacturers, using carrier ampholine mixtures, pH ranges 5–7, 3.5–10 (Amersham Biosciences), and
5–7 (Serva) for the isoelectric focusing gels (20). Proteins were stained
using a colloidal Coomassie Blue staining kit (Novex).
MALDI-MS analysis was performed as described (21) with minor
modifications. Briefly, spots were excised, destained with 30% (v/v)
acetonitrile in 0.1 M ammonium bicarbonate, and dried in a Speedvac
evaporator. The dried gel pieces were reswollen with 5 l of 5 mM
ammonium bicarbonate, (pH 8.8) containing 50 ng of trypsin (Promega,
Madison, WI), centrifuged for 1 min, and left at room temperature for
about 12 h. After digestion, 5 l of water was added; 10 min later 10 l
of 75% acetonitrile containing 0.3% trifluoroacetic acid was added,
centrifuged for 1 min, and the content was vortexed for 20 min. For
MALDI-MS 1.5 l from the separated liquid was mixed with 1 l of
saturated ␣-cyano cinnamic acid in 50% acetonitril, 0.1% trifluoroacetic
acid in water and applied to the MALDI target. The samples were
analyzed in a time-of-flight Bruker mass spectrometer (Reflex III)
equipped with a reflector and delayed extraction. An accelerating voltage of 20 kV was used. Des-Arg-1 Bradykinin (Sigma) and ACTH
(18 –38) (Sigma) were used as standard peptides. Calibration was internal to the samples. The peptide masses were matched with the
theoretical peptide masses of all proteins from all species of the SWISSProt data base. For protein search, monoisotopic masses were used and
a mass tolerance of 0.0075% was allowed. The protein search was
performed with a software, developed by Roche (Basel, Switzerland),
which is similar to the Peptident software on the ExPASy server
(expasy.hcuge.ch/sprot/peptident.html).
RESULTS
LAT Lacking Tyrosine-based Protein Docking Sites Fails to
Accumulate at the Site of TCR Engagement—Earlier work of
our laboratory showed that TCR䡠LAT signaling assemblies
(TLAs) form at the contact zone to ␣-CD3 antibody-coated
TCR-activating beads (15). To visualize LAT accumulation,
JCaM2 cells were reconstituted with fluorescent LAT䡠EYFP
and the cells were stimulated with ␣-CD3 dynabeads for 7 min.
LAT distribution was monitored by fluorescence microscopy. In
line with earlier reports using green fluorescence proteintagged LAT, we observed a strong accumulation of wt
LAT䡠EYFP in the plasma membrane contacting the TCR activating bead (Fig. 1A) (16, 17).
An EYFP fusion of a LAT mutant with all tyrosines replaced
by phenylalanine (LAT All Y 3 F䡠EYFP) was efficiently targeted into the plasma membrane. In contrast to wt LAT䡠EYFP,
we observed no enrichment of LAT All Y 3 F䡠EYFP at the
contact region to TCR-activating beads. This showed that LAT
accumulation at TCR activating beads depends on tyrosinebased docking sites for signaling proteins (Fig. 1B).
PLC␥ and Grb2 Binding Sites on LAT Contribute to TCR
Signaling Complex Formation in Membrane Domains—Next,
we studied which tyrosine-based protein docking motifs of LAT
Downloaded from http://www.jbc.org/ at University of Liverpool on October 23, 2014
stably express wt LAT, LAT All Y 3 F, LAT Y6F, LAT Y7F/Y8F, LAT
Y6F/Y7F/Y8F, and LAT F6Y/F7Y/F8Y were described previously (11)
and were maintained in full medium, 1 mg/ml G418. We generated new
JCaM2 cell lines stably expressing LAT Y7F/Y8F/Y9F and LAT F4Y/
F6Y/F7Y/F8Y/F9Y as described below. All the JCaM2 lines used in this
study were verified by Western blot and fluorescence-activated cell
sorter analysis to express the similar levels of LAT and CD3 (not
shown).
Anti-CD3 monoclonal antibody TR66 was from Antonio Lanzavecchia (Institute for Research in Biomedicine, Bellinzona, Switzerland).
Antibodies and reagents were purchased from the following sources:
monoclonal antibody against TCR -chain were from Santa Cruz Biotechnology; Grb2, PLC-␥, and ZAP-70 antibodies were purchased from
Transduction Laboratories. Rabbit polyclonal serum against CD3-⑀ was
obtained from Ed Palmer (University Hospital, Basel, Switzerland).
Phycoerythrin-conjugated ␣-human CD3 antibodies were from BD
PharMingen, and ␣-LAT rabbit antisera from Upstate Laboratories.
Secondary horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were obtained from Bio-Rad and Amersham Biosciences.
Expression Constructs—The expression construct of LAT F4Y/F6Y/
F7Y/F8Y/F9Y in pcdef was described previously (11). LAT Y7F/Y8F/
Y9F in pcdef was generated by PCR-mediated mutagenesis from wt
LAT cDNA clone without introducing a Myc tag, cloned into pcdef, and
verified by DNA sequencing. Wt LAT䡠EYFP and LAT All Y 3 F䡠EYFP
fusion constructs were generated by PCR using the Expand high fidelity
PCR system (Roche Diagnostics), introducing SalI and AgeI restriction
sites replacing the stop codon of LAT. The restriction-digested PCR
products were ligated in-frame into pEYFP N1 (Clontech). Sequences
were verified by sequencing, and all plasmids were purified using
Quiagen columns.
Transfections and Fluorescence Microscopy—JCaM2 cell lines were
transfected by electroporation in ice-cold cytomix electroporation buffers (120 mM KCl, 0.15 mM NaCl, 10 mM K-PO4 buffer, 25 mM Hepes, pH
7.6, 2 mM EGTA, 5 mM MgCl2, 5 mM reduced glutathione, 2 mM ATP) at
960 F, 280 mV in a 0.5-ml volume at a cell density of 1.5–2 ⫻ 107/ml
in a Bio-Rad gene pulser. After 5 min on ice, cells were collected,
transferred into full medium, and incubated overnight at 37 °C. To
select stable transfectants, cells were plated for selection in 2 mg/ml
G418 at a density of 8000 and 4000 cells/well in 96-well plates.
For immunofluorescence analysis, the cells were incubated with one
␣-CD3-coated dynabead per cell at 37 °C for 7 min in RPMI-Hepes, 1%
fetal calf serum. Subsequently the conjugates were washed twice in
phosphate-buffered saline, and 5 ⫻ 104 cells in 15 l of phosphatebuffered saline were placed on a poly(L)lysine (Sigma)-coated microscope slide for 30 s on ice. Subsequently the cells were fixed with 4%
paraformaldehyde in phosphate-buffered saline at room temperature
for 20 min. The cells were subsequently washed in phosphate-buffered
saline and mounted in fluoromount G (Southern Biotechnologies). Immunofluorescence was performed on a Zeiss Axioplan 2 fluorescence
microscope, and digital images were obtained using a 12-bit Spot camera (Diagnostic Instruments).
Immunoisolation—M-450 goat ␣-mouse magnetic beads (Dynal)
were coated with TR66 ␣-CD3 monoclonal antibody following the instructions of the manufacturers. 1– 4 ⫻ 107 Jurkat cells per data point
were pelleted at 2000 rpm, 10 s at 4 °C in an Eppendorf tabletop
centrifuge. Loose cell pellets were mixed with ␣-CD3 beads (1:2 ratio of
beads: cells), incubated at 0 °C for 2 min, and subsequently incubated at
37 °C for 0 min, 3 min, 7 min, respectively. Cell conjugates were washed
once in H-buffer (10 mM Na-Hepes, pH 7.2, 250 mM sucrose, 2 mM
MgCl2, 10 mM NaF, 1 mM vanadate) and suspended in 1 ml of H-buffer
containing CLAP protease inhibitors (100 M each of chymostatin,
leupeptin, antipain, pepstatin; Sigma) and 0.2 mM pervanadate. The
cells were N2-cavitated using a nitrogen cavitation bomb (model 4639,
Parr Instrument Company) equilibrated at 4 °C, 50 bar for 10 min. The
homogenate was filled to 10 ml with H-buffer; beads were subsequently
retrieved with a magnet (Dynal) and washed three times in 10 ml of
H-buffer for 3 min each at 0 °C. Beads were analyzed by Western
blotting using ECL chemiluminescence (Amersham Biosciences). In
some experiments non-reducing SDS-PAGE was used. ECL-exposed
x-ray films were scanned, and images were mounted using Photoshop™
(Adobe) software.
Pharmacological Treatments—For disruption of the actin cytoskeleton by latrunculin, bead cell conjugates were formed at 37 °C for 7 min
and subsequently treated with latrunculin (Calbiochem) at 12 M concentration for 3 min. This treatment causes complete disappearance of
phalloidin-actin staining in Jurkat cells (18) and leads to rapid rounding of the cells.
LAT Signaling Protein Complexes
20391
FIG. 1. Fluorescence microscopy of JCaM2 cells expressing
LAT䡠EYFP. JCaM2 cells transiently expressing wt LAT䡠EYFP and All
Y 3 F LAT䡠EYFP were conjugated with ␣-CD3-coated dynabeads (X)
and incubated for 7 min at 37 °C. Cells were adhered to glass microscope slides, fixed, and studied by fluorescence microscopy. Wt
LAT䡠EYFP strongly accumulates at the site of activation by the ␣-CD3coated dynabeads, whereas no accumulation of All Y 3 F LAT䡠EYFP
was detected. Bar, 10 m.
FIG. 2. Synergism of LAT Tyr-based protein docking sites. A,
scheme of LAT showing its tyrosine-based docking sites for signaling
proteins according to Refs. 11 and 12. B, Western blot analysis of
immunoisolated TCR signaling domains from JCaM2 cells expressing
LAT variants. Equivalent amounts of immunoisolated TCR signaling
domains after 0, 3, and 7 min of triggering with ␣-CD3-coated dynabeads at 37 °C were separated by SDS-PAGE and subsequently analyzed by Western blotting using the antibodies against PLC␥, ZAP-70,
LAT, Grb2, and TCR chain. LAT Y7F/Y8F/Y9F is not epitope-tagged
and hence runs with increased mobility. Stars indicate the position of
the heavy chain and light chain of the antibody used for immunoisolation. Docking sites for PLC␥ and Grb2/Gads synergistically contribute
to the accumulation of LAT, PLC␥, and Grb2 in immunoisolated TLAs.
C, density gradient analysis of TX-100 DRMs of JCaM2 cells stably
expressing wt LAT and LAT All Y 3 F. Fractions of the gradient were
analyzed by Western blot using LAT and CD3-⑀-specific antibodies. The
DRM float to fraction 2 of the gradient, whereas TX-100-solubilized
proteins reside in the heavy fraction 7 of the gradient. Please note that
similar amounts of wt and ALL Y 3 F LAT float with the DRM fraction,
whereas CD3-⑀ is solubilized in TX-100 and remains in the heavy
fraction 7.
based PLC␥ docking site are both disrupted. Interestingly, in
JCaM2 cells expressing the LAT Y6F/Y7F/Y8F variant not only
PLC␥ but also Grb2 incorporation into immunoisolated
LAT䡠TCR signaling complexes was strongly reduced. Moreover,
LAT recruitment was diminished. Taken together, these data
show that the Tyr-6 of LAT not only has an important role for
PLC␥ recruitment and activation of sustained Ca2⫹ fluxes but
that it also cooperates with tyrosine 7 and 8-based Grb2/Gads
binding sites to mediate LAT and Grb2 accumulation in immunoisolated TCR signaling domains.
Next, we analyzed signaling complexes formed by LAT Y7F/
Y8F/Y9F mutant, in which in addition to the Tyr-7 and -8
Grb2/Gads binding sites a Grb2 binding motif at Tyr-9 is dis-
Downloaded from http://www.jbc.org/ at University of Liverpool on October 23, 2014
are required for the formation of TLAs in plasma membrane
domains. We employed a biochemical analysis of TCR signaling
domains that were immunoisolated by a previously established
procedure (14). Briefly, ␣-CD3 antibody-coated magnetic beads
are used to trigger TCR in Jurkat T cells. Subsequently, bead/
cell conjugates are homogenized mechanically by nitrogen cavitation, and plasma membrane fragments attached to the beads
are retrieved. By Western blot analysis of the ␣-CD3-isolated
membranes we previously demonstrated signaling-dependent
accumulation of LAT-based signaling protein assemblies at the
site of TCR engagement (15). Here, we assayed formation of
TLAs mediated by LAT variants with specific mutations in
tyrosine-based protein docking sites (Fig. 2A). We compared
TCR signaling protein assemblies from JCaM2 cell lines stably
reconstituted with wt LAT, which fully restores TCR signal
transduction, and with LAT containing Tyr to Phe substitution
of all tyrosines (LAT All Y 3 F), which does not transduce TCR
signals (11). Western blots showed that similar amounts of
TCR chain and ZAP-70 are recruited into the immunoisolated
TCR signaling domains of all cell lines studied. LAT, Grb2, and
PLC␥ were enriched in immunoisolated TCR signaling complexes of wt LAT-reconstituted cells. In line with our fluorescence data, lower amounts of the LAT All Y 3 F mutant were
recovered in ␣-CD3 immunoisolates (Fig. 2B). In all our experiments the amounts of LAT All Y 3 F in ␣-CD3 immunoisolated signaling domains were significantly reduced. Occasionally, the residual LAT All Y 3 F reached relatively high levels
in the isolates, most likely because of higher plasma membrane
recovery in the isolated membrane fragments. Non-complexed
soluble Grb2 and PLC␥ are not associated with signaling complexes and are thus barely detected in the isolates from LAT All
Y 3 F-expressing cells.
We compared ␣-CD3 isolates of wt LAT and LAT All Y 3
F-expressing JCaM2 cells with JCaM2 lines expressing a LAT
variant carrying a Y7F/Y8F mutation deleting two Grb2/Gads
binding sites. This mutant was previously shown to exhibit a
mild reduction of the TCR signal transduction capabilities of
LAT (11, 12). Here, this mutant showed no reduction of LAT
and PLC␥ accumulation in TCR signaling domains, whereas
Grb2 appears to be slightly reduced (Fig. 2B).
Analysis of the JCaM2 cells reconstituted with LAT mutant
Y6F, which disrupts the PLC␥ binding motif of LAT, showed no
reduction of LAT accumulation and no significant change of
Grb2 recruitment into the isolates. In line with immunoprecipitation data, we observed a strong reduction of PLC␥ in the
isolates from LAT Y6F-expressing cells (11). Above-background
levels of PLC␥ were consistently observed in the isolates from
LAT Y6F-expressing cells (Fig. 2B). Consequently, we studied
␣-CD3 isolates formed by the LAT Y6F/Y7F/Y8F variant, in
which Tyr-7 and -8 Grb2/Gads docking sites and the Tyr-6-
20392
LAT Signaling Protein Complexes
FIG. 4. Western blot analysis of immunoisolated TCR signaling
domains from JCaM2 cells expressing LAT variants F6Y/F7Y/
F8Y and F4Y/F6Y/F7Y/F8Y/F9Y variants. Equivalent amounts of
immunoisolated TCR signaling domains after 0, 3, and 7 min of triggering with ␣-CD3-coated dynabeads at 37 °C were separated by SDSPAGE and subsequently analyzed by Western blotting using antibodies
against PLC␥, ZAP-70, LAT, Grb2, and TCR chain. Stars indicate the
position of the heavy chain and light chain of the antibody used for
immunoisolation. Tyrosines 4 and 9 contribute to recruitment of LAT
and Grb2 to immunoisolated TCR signaling domains.
rupted (Fig. 2B). Grb2 was absent from the isolated TLAs
formed by this LAT variant, whereas reduced amounts of PLC␥
were recruited into the immunoisolates. These data show that
the Grb2 binding site at Tyr-9 is involved in formation of
LAT䡠TCR signaling complexes and comprises an important motif for Grb2 recruitment into TLAs. However, as seen in the
strongly reduced Grb2 recruitment of LAT Y6F/Y7F/Y8F, Tyr-9
is by itself not sufficient to mediate Grb2 accumulation in
TCR/LAT signaling domains.
It is possible that the weak enrichment of LAT All Y 3 F in
anti-CD3 immunoisolate reflects a failure of raft targeting of
LAT All Y 3 F caused by defective LAT palmitoylation. Targeting of LAT into Triton X-100 detergent-resistant membranes (DRMs) strictly depended on its palmitoylation (22).
Thus we tested the palmitoylation status of LAT All Y 3 F of
TX-100 DRMs from JCaM2 expressing LAT All Y 3 F and wt
LAT. OptiprepTM gradient centrifugation of the TX-100-extracted cells showed that the DRM fraction (fraction 2) of wt
and All Y 3 F LAT contained very similar relative amounts of
LAT (Fig. 2C). Our results show that LAT All Y 3 F as well as
wt LAT are efficiently palmitoylated.
The Tyrosine 6-, 7-, and 8-based Docking Sites of LAT Are
Not Sufficient for Complete Signaling Complex Assembly—Our
data show that tyrosines 6, 7, and 8 are essential for the
formation of TCR䡠LAT signaling complexes. Moreover, these
sites have previously been shown to comprise the minimal
docking site requirement for LAT to be capable of transducing
Ca2⫹ fluxes and Ras-dependent Erk kinase and NFAT activation (11). Therefore, we asked whether these tyrosines are
sufficient to mediate the formation of TLAs. We employed
different LAT Y-F mutants in which specific tyrosines of the
LAT All Y 3 F variant have been restored.
First, we monitored the accumulation of Grb2, tagged with
an EYFP fluorophor, in TLA domains by fluorescence microscopy (Fig. 3). We transiently transfected different JCaM2 cell
lines expressing LAT variants with Grb2-EYFP. In line with
our biochemical analysis (15) and fluorescence imaging of TLAs
(16), we observed an accumulation of Grb2-EYFP at the contact
zone of JCaM2-wt LAT cells to ␣-CD3-coated beads. As expected, LAT All Y 3 F-expressing JCaM2 did not exhibit Grb2EYFP accumulation at the bead/cell contact zone, because of
the lack of Grb2 binding motifs in this LAT variant. LAT
F6Y/F7Y/F8Y mediated no detectable accumulation of Grb2YFP at the site of TCR activation, despite its two adjacent
Tyr-7 and -8 Grb2/Gads binding sites. In JCaM2 lines expressing LAT F4Y/F6Y/F7Y/F8Y/F9Y, which additionally restores
Grb2 binding motifs at Tyr-4 and -9, Grb2 accumulation at the
TCR activating bead was significantly restored.
These data were corroborated by our biochemical analysis of
immunoisolated TCR signaling domains (Fig. 4). Accumulation
of LAT F6Y/F7Y/F8Y in ␣-CD3 immunoisolates was clearly
detectable when compared with signaling complexes from All
Y 3 F LAT-expressing cells but significantly lower than in
isolates from wt LAT cells. Moreover, Grb2 recovery in the
isolated signaling domains from the LAT F6Y/F7Y/F8Y JCaM2
line was clearly reduced when compared with isolates from wt
LAT-expressing JCaM2 cells. Immunoisolates from F4Y/F6Y/
F7Y/F8Y/F9Y LAT cells accumulated LAT to wt LAT levels and
to a large extent reconstituted Grb2 accumulation. Despite
intense efforts it was not possible to resolve the individual
contributions of LAT Tyr-4- and -9-based Grb2 binding sites to
this enhancement, because the differences between signaling
complexes formed by LAT variants carrying the Tyr-4, -6, -7, -8,
Tyr-6, -7, -8, -9, and Tyr-4, -6, -7, -8, -9 docking sites were too
subtle and variable to be unambiguously resolved.
Taken together our data show that, albeit their important
contribution to signaling protein assembly, the protein docking
motifs of LAT comprising tyrosines 6, 7, and 8 are not sufficient
to fully reconstitute LAT-TCR signaling domain formation and
that additional Grb2 binding sites outside this motif contribute
to the recruitment of Grb2 and LAT into TCR signaling
domains.
Multiple SH2/SH3 Cytoplasmic Adaptors Are Present in Immunoisolated TCR䡠LAT Signaling Assemblies—Multiple cytoplasmic adaptors can be resolved on two-dimensional gels of
isolated Jurkat cell TLAs (Fig. 5). We identified SH2/SH3 adaptors Grb2, Gads, Grap, and Crk-1 by MALDI-MS on tryptic
digests of excised protein spots. Moreover, spots for Gads and
Grb2 were verified by Western blotting (data not shown). Importantly, Grb2 is a very abundant cytoplasmic adaptor,
whereas Gads is relatively minor and Grap and Crk-1 are
present in intermediate amounts, showing that cytoplasmic
adaptors strongly differ in their relative abundance in TLAs.
LAT was not resolved on our gels, possibly because of its highly
acidic isoelectric point or because of the hydrophobic nature of
its membrane anchor.
Downloaded from http://www.jbc.org/ at University of Liverpool on October 23, 2014
FIG. 3. Recruitment of Grb2-EYFP to TCR signaling domains
in LAT-reconstituted JCaM2 cells. JCaM2 cells stably express wt
LAT, LAT All Y 3 F, and LAT Y-F variants as indicated. The cells
transiently express Grb2-EYFP and were conjugated with ␣-CD3 beads
and subsequently incubated at 37 °C for 7 min. Cells were adhered to
microscope slides, fixed, and visualized by fluorescence microscopy. Wt
LAT-expressing JCaM2 cells (A) efficiently accumulate Grb2-EYFP at
the site of TCR activation, whereas no accumulation was detected in
LAT All Y 3 F (B) and LAT F6Y/F7Y/F8Y-expressing JCaM2 cells. LAT
F4Y/F6Y/F7Y/F8Y/F9Y reconstitutes Grb2-EYFP accumulation into
TLAs. Bar, 10 m.
LAT Signaling Protein Complexes
FIG. 5. Migration of cytoplasmic SH2/SH3 adaptors on a twodimensional gel of ␣-CD3 immunoisolated fragments. 4 ⫻ 108
Jurkat cells were used for immunoisolation after 7 min of triggering
with ␣-CD3 beads. Two-dimensional gels of isolated membranes were
stained with colloidal Coomassie Blue. Subsequently, the protein spots
were excised and tryptic digests were analyzed by MALDI-MS. SH2/
SH3 cytoplasmic adaptors identified are indicated. The x depicts actin.
DISCUSSION
Using imaging and biochemical analysis of TCR signaling
membrane domains, we studied the role of the tyrosine-based
protein docking sites of LAT in the accumulation of LAT and
cytoplasmic signaling proteins in the vicinity of activated TCR.
We analyzed signaling assemblies formed by LAT variants that
carry different combinations of tyrosine-based signaling protein docking sites.
Non-autonomous Signaling Protein Docking Sites on LAT:
Structural Implication—Our study showed that different docking sites for the signaling proteins PLC␥ and Grb2/Gads synergize in formation and/or stabilization of TCR䡠LAT signaling
FIG. 6. Western blot analysis of immunoisolated TCR signaling
domains after disruption of Ca2ⴙ fluxes and the actin cytoskeleton. A, JCaM2 cells stably expressing wt LAT were treated with
EGTA/BAPTA in order to deplete Ca2⫹ stores, conjugated to ␣-CD3
beads, and incubated at 37 °C in EGTA/BAPTA-containing medium for
0, 3, and 7 min. Immunoisolated proteins were separated by SDS-PAGE
and analyzed by Western blotting using the antibodies against PLC␥,
ZAP-70, LAT, Grb2, and TCR chain. Stars indicate the position of the
heavy chain and light chain of the antibody used for immunoisolation.
B, Jurkat cells were conjugated with ␣-CD3 beads and incubated for 7
min at 37 °C. Conjugates were treated either with latrunculin (latr.) or
left as control (ctr.) and incubated for a further 3 min at 37 °C. Immunoisolates were prepared as described. 2-fold more material from isolates of latrunculin-treated cells was loaded to correct for the reduction
in yields of TCR chain/ZAP-70.
complexes. This synergy may be caused by cooperative stabilization of these assemblies by networks of intermolecular interactions between signaling proteins. A prototypic model for such
multimolecular structures is the tetrameric complex formed by
LAT, Gads, SLP-76, and PLC␥ (6, 12, 24). However, Gads and
SLP-76 are present in the isolated complexes in relatively low
amounts (Fig. 5 and Ref. 15), possibly because of their early
dissociation from the TCR䡠LAT signaling complexes (19).
Therefore, other molecular interactions are also likely to play a
role in complex assembly.
Our two-dimensional gel analyses showed strong differences
in the relative amounts of different SH2/SH3 adaptor in the
immunoisolated signaling complexes. We identified the adaptors Crk-1, Grap, and, in particular, Gads as relatively minor
components, whereas Grb2 is by far the most abundant member of the SH2/SH3 adaptor family (for review on cytoplasmic
adaptors, see Ref. 25). Possibly, Grb2 has an important role,
beyond SOS recruitment and Ras activation, by organizing the
structure of LAT signaling complexes. This is corroborated by
the important contribution of multiple Grb2 binding motifs of
LAT to signaling complex formation and downstream NFAT
and Erk activation.
Moreover, our analysis showed that the PLC␥ docking site
Tyr-6 contributes to TLA assembly. It remains to be shown
whether the enzymatic activity of PLC␥ is required for this
process. However, our experiments blocking Ca2⫹ fluxes and
the efficient TLA formation mediated by the LAT F6Y, which
inefficiently transduces Ca2⫹ fluxes, suggest that Ca2⫹ fluxes
are not required for complex assembly. It remains to be tested
whether diacylglycerol production by PLC␥ plays a role in
complex formation, for example by activating protein kinase C,
and/or whether PLC␥ plays a role in the structure of signaling
assemblies. A role of Tyr-6 was also implicated by recent photobleaching experiments showing a stable anchoring of wt
LAT-GFP in TCR signaling assemblies, whereas LAT F6Y-GFP
and LAT F6Y/F7Y/F8Y/F9Y-GFP readily exchanged with their
environment (17).
It has recently been shown that phosphorylation of different
LAT tyrosines occurs in a non-autonomous fashion (13). In line
with the issues discussed above, tyrosine-phosphorylated docking motifs may become stabilized by tight association of their
specific binding partners upon complex assembly. Alternatively, phosphorylation and binding of signaling proteins may
lead to conformational changes on LAT or the whole signaling
Downloaded from http://www.jbc.org/ at University of Liverpool on October 23, 2014
Role of Ca2⫹ Fluxes and Actin in LAT Signaling Complex
Formation—The activation of PLC␥ leads to inositol 1,4,5, PI3
release and triggers intracellular Ca2⫹ fluxes. To test whether
intracellular Ca2⫹ fluxes influence the formation of TLAs we
performed immunoisolation using wt LAT-expressing JCaM2
cells that were treated with the intracellular Ca2⫹ chelator
BAPTA as described previously (19). Western blot analysis of
the respective immunoisolates from BAPTA-treated and control cells revealed no significant difference in the amount of
LAT, PLC␥, and Grb2 (Fig. 6A). This suggests that the formation of the TLAs does not require the induction of Ca2⫹ fluxes.
Moreover, we addressed the possibility that the formation of
TLAs is because of their mutual anchoring in close vicinity by
the actin cytoskeleton. We treated Jurkat cells with the drug
latrunculin A, which rapidly and efficiently disrupts the actin
cytoskeleton (23). It was not possible to form conjugates of
␣-CD3 beads with Jurkat cells treated with latrunculin A. This
is most likely because of the rounding of the cells following
addition of the latrunculin. However, we succeeded in obtaining immunoisolates from Jurkat cells that were treated with
latrunculin after conjugate formation and triggering for 7 min
(Fig. 6B). We observed a clear reduction of TCR and ZAP-70
yields in the isolates from latrunculin-treated cells. By loading
two times the equivalents of immunoisolated material from
latrunculin-treated cells on the Western blot, we normalized
the immunoisolates to essentially equal amounts of TCR and
ZAP-70. Under these conditions, the amounts of LAT, PLC␥,
and Grb2 in the isolates are very similar to the non-treated
cells. This showed that the ratio of TCR/ZAP-70 to LAT complexes is not affected by disruption of the actin cytoskeleton,
demonstrating that anchoring of LAT in the vicinity of TCR is
not directly mediated by the actin cytoskeleton. However, the
yields of signaling complexes were reduced upon latrunculin
treatment, possibly because of the strong reduction of bead/cell
contact area or reduced TLA anchoring in the plasma membrane domain TCR activating bead.
20393
20394
LAT Signaling Protein Complexes
formation reflects a mechanism to define a sequence and/or a
threshold of tyrosine phosphorylation on LAT required to trigger specific downstream responses.
Acknowledgments—We gratefully acknowledge the technical help of
Marina Kuhn in the initial phases of the work. We thank Karin
Engelhardt, Solveig Moré, and Gillian Griffiths for critically reading
the manuscript.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Weiss, A., and Littman, D. R. (1994) Cell 76, 263–274
Chan, A. C., and Shaw, A. S. (1996) Curr. Opin. Immunol. 8, 394 – 401
Wange, R. L., and Samelson, L. E. (1996) Immunity 5, 197–205
Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., and Samelson, L. E.
(1998) Cell 92, 83–92
Samelson, S. E. (2002) Ann. Rev. Immunol. 20, 371–394
Tomlinson, M. G., Lin, J., and Weiss, A. (2000) Immunol. Today 21, 584 –591
Finco, T. S., Kadlecek, T., Zhang, W., Samelson, L. E., and Weiss, A. (1998)
Immunity 9, 617– 626
Dower, N. A., Stang, S. L., Bottorff, D. A., Ebinu, J. O., Dickie, P., Ostergaard,
H. L., and Stone, J. C. (2000) Nature Immunol. 1, 317–321
Wilde, J. I., and Watson, S. P. (2001) Cell. Signal. 13, 691–701
Zhang, W., Sommers, C. L., Burshtyn, D. N., Stebbins, C. C., DeJarnette, J. B.,
Trible, R. P., Grinberg, A., Tsay, H. C., Jacobs, H. M., Kessler, C. M., Long,
E. O., Love, P. E., and Samelson, L. E. (1999) Immunity 10, 323–332
Lin, J., and Weiss, A. (2001) J. Biol. Chem. 276, 29588 –29595
Zhang, W., Trible, R. P., Zhu, M., Liu, S. K., McGlade, C. J., and Samelson,
L. E. (2000) J. Biol. Chem. 275, 23355–23361
Zhu, M., Janssen, E., and Zhang, W. (2003) J. Immunol. 170, 325–333
Harder, T., and Kuhn, M. (2001) Science’s STKE http://stke.sciencemag.
org/cgi/content/full/OC_sigtrans;2001/71/pl1
Harder, T., and Kuhn, M. (2000) J. Cell Biol. 151, 199 –208
Bunnell, S. C., Hong, D. I., Kardon, J. R., Yamazaki, T., McGlade, C. J., Barr,
V. A., and Samelson, L. E. (2002) J. Cell Biol. 158, 1263–1275
Tanimura, N., Nagafuku, M., Minaki, Y., Umeda, Y., Hayashi, F., Sakakura,
J., Kato, A., Liddicoat, D. R., Ogata, M., Hamaoka, T., and Kosugi, A. (2003)
J. Cell Biol. 160, 125–135
Harder, T., and Simons, K. (1999) Eur. J. Immunol. 29, 556 –562
Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W., and Samelson, L. E. (2001)
Immunity 14, 315–329
Celis, J. E., Ratz, G., Basse, B., Lauridsen, J. B., Celis, A., Jensen, N. A., and
Gromov, P. (1998) in Cell Biology (Celis, J. E., ed) Vol. 4, pp. 375–386,
Academic Press, New York
Antelmann, H., Bernhardt, J., Schmid, R., Mach, H., Volker, U., and Hecker,
M. (1997) Electrophoresis 18, 1451–1463
Zhang, W., Trible, R. P., and Samelson, L. E. (1998) Immunity 9, 239 –246
Schatten, G., Schatten, H., Spector, I., Cline, C., Paweletz, N., Simerly, C., and
Petzelt, C. (1986) Exp. Cell Res. 166, 191–208
Yablonski, D., Kadlecek, T., and Weiss, A. (2001) Mol. Cell. Biol. 21,
4208 – 4218
Leo, A., Wienands, J., Baier, G., Horejsi, V., and Schraven, B. (2002) J. Clin.
Invest. 109, 301–309
Downloaded from http://www.jbc.org/ at University of Liverpool on October 23, 2014
assemblies, causing exposure and availability of tyrosines for
further phosphorylation by kinases. Therefore, it is also possible that the phosphorylation events on LAT occur in an ordered
fashion, explaining the interdependence of the different tyrosine-based docking sites in TLA formation.
Further evidence for a structural basis of a cross-talk between signaling protein docking sites of LAT in the signaling
response stems from the observation that different tyrosine
protein docking sites had to reside on the same LAT molecule
in order to restore signaling function in JCaM2 cells (11). To
resolve the mechanisms for the synergistic interactions of tyrosine-based docking sites of LAT, LAT signaling complex
structure and temporal sequence of events leading to complex
formation have to be resolved.
Non-autonomous Signaling Protein Docking Sites on LAT:
Functional Implications—Formation of the LAT䡠TCR signaling
assemblies by respective LAT mutants correlated well with
their capability to transduce different downstream signaling
reactions. LAT tyrosines 6, 7, and 8 comprise a minimal set of
docking motifs to enable transduction of Ca2⫹ fluxes, Erk and
NFAT activation. However, the additional presence of the Grb2
docking sites tyrosines 4 and 9 was required to restore NFAT
and Erk activation to the same levels as transduced by wt LAT
(11). This correlated well with the incomplete accumulation of
LAT F6Y/F7Y/F8Y and Grb2 into ␣-CD3 immunoisolates,
whereas LAT signaling assembly formation was almost completely reconstituted by LAT F4Y/F6Y/F7Y/F8Y/F9Y. Disruption of the Tyr-6 PLC␥ binding motif of LAT has little effect on
Grb2 and LAT recruitment into TLAs but impedes induction of
Ca2⫹ fluxes and abolishes NFAT and Erk activation (12). A
residual amount of PLC␥ is recovered in isolates of LAT Y6Fexpressing cells, possibly caused by indirect association via
Gads/SLP 76. This residual PLC␥ may be sufficient for the
induction of weak or aberrant Ca2⫹ fluxes, which have been
described as JCaM2 cells expressing LAT Y6F (11, 12).
Because these experiments were performed in the JCaM2
leukemic T cell line, it will be very important to study the
physiological roles of TLA assembly in T lymphocytes. Possibly
the synergistic contribution of signaling protein docking to TLA