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Department of Microbiology, Immunology, and Molecular Genetics and
The Molecular Biology Institute, University of California School of Medicine, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have suggested that engagement of T cell costimulators
induces lipid raft recruitment to the TCR contact site and the
construction of a platform that facilitates TCR engagement and
processive and sustained signal transduction (2, 3). We
proposed that lipid rafts function as sites for the integration of TCR
engagement, early TCR signals, and actin cytoskeleton-mediated
reorganization of the TCR contact cap (2). These
suggestions were based, in part, on our observations that recruitment
of lipid raft-associated CD48 to the TCR contact site enhances
raft-dependent TCR-
chain tyrosine phosphorylation, :actin
cytoskeleton association, F-actin reorganization, morphology changes,
and Ag-induced functions (Ref. 2 ; and M. Moran and M.
C. Miceli, unpublished observations).
That lipid rafts might organize the TCR contact site for sustained and
processive signal transduction is further supported by a number of
other recent findings. First, several key TCR signal transducers
including Lck, Fyn, LAT, and GPI-linked CD48 preferentially partition
within lipid raft membranes (1, 4, 5), whereas putative
negative regulators of TCR engagement and activation, such as CD43 and
CD45, preferentially partition outside raft microdomains
(5, 6, 7). Second, TCR engagement triggers the increased
stability and/or concentration and tyrosine phosphorylation of TCR-
and signal transducers within lipid rafts (7, 8, 9).
Furthermore, perturbation of the structural integrity of lipid rafts
inhibits TCR-induced protein tyrosine phosphorylation and
Ca2+ flux (2, 8, 9). Finally,
TCR/CD28 or TCR/CD48 costimulation leads to lipid raft migration to and
coalescence at the site of TCR engagement and prolonged stability of
TCR-induced protein tyrosine phosphorylation (Ref. 3 ; data
presented here and M. Moran and M. C. Miceli, unpublished
data).
The relationship between the TCR-induced concentration and phosphorylation of transducers within lipid rafts and the coalescence of lipid rafts at the TCR contact cap has not been elucidated. Furthermore, how TCR/costimulator engagement induces raft membrane reorganization remains uncharacterized. Because Lck is a raft resident protein and a primary mediator of TCR signal transduction, we reasoned that Lck might function in regulating TCR/CD48-mediated lipid raft dynamics at the contact cap. Therefore, we analyzed TCR/CD48 and TCR/CD28 costimulatory activity in T cells expressing Lck Src homology 3 (SH3)3 mutants. Here, we present evidence that the Lck SH3 domain functions downstream of the initiation of TCR signal transduction to facilitate TCR/costimulator-induced lipid raft recruitment to the TCR contact cap, sustenance of TCR/costimulator-induced protein tyrosine phosphorylation, and IL-2 production.
By analyzing the Lck SH3 domain requirements for TCR/costimulator
raft-mediated events, we discriminate two distinctly regulated lipid
raft reorganization events required for T cell activation. In the first
step, TCR- and downstream transducers are concentrated and tyrosine
phosphorylated within lipid rafts. This step requires Lck kinase
activity and remains intact in cells expressing
kinase-active/SH3-impaired Lck mutants. The second step involves
subsequent recruitment of additional nonliganded lipid rafts to the TCR
contact cap and is dependent on costimulation and defective in cells
expressing kinase-active/SH3-impaired Lck mutants. Furthermore,
expression of these SH3 mutants disrupts sustained TCR signal
transduction and IL-2 production, but not apoptosis, shown only to
require "partial" TCR signals. These data correlate Lck SH3
activity and lipid raft recruitment with signals and functions that
rely on prolonged TCR engagement and processive TCR signal
transduction. Together with recent characterization of CD28 and CD48
costimulatory activities (3, 10, 11), our findings provide
a molecular and cell biological framework for two signal models of T
cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lck SH3 domain mutations were created using the QuickChange
Mutagenesis System (Stratagene, La Jolla, CA) using LckF505 as a
template. Mutants were sequenced to verify that only the intended
mutations were created. Stable transfections were performed by
electroporation as described (12). GST-LckSH3 fusion
constructs were created by subcloning fragments corresponding to amino
acids 1–149 from wild-type or mutant LckF505 into pGEX4T-1 (Pharmacia,
Piscataway, NJ) and fusion proteins were expressed and purified
according to manufacturer’s recommendations (Pharmacia). Hybridomas
producing Abs against TCR- (H146-968), CD28 (37.51), CD48 (5-8A10),
and CD3
(145-2C11, ATCC CRL-1975) were obtained from Dr. Kubo
(Cytel, San Diego, CA), Dr. Allison (University of California,
Berkeley), Dr. Reiser (Dana-Farber Cancer Institute, Boston, MA), and
American Type Culture Collection (Manassas, VA), respectively, and Abs
were produced and purified from hybridoma supernatants using standard
techniques. Goat anti-hamster (GAH) was purchased from Cappel
(Durham, NC). Abs directed against c-Cbl, Sam68, p85 were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Lck was
produced against a GST fusion protein comprising Lck amino acids 5-144.
Anti-phosphotyrosine Ab, 4G10 was purchased from Upstate Biotechnology
(Lake Placid, NY).
Cells and functional analysis
BI-141 is a T cell hybridoma that recognizes beef insulin in the
context of I-A
b
k.
FT5.7 is a derivative of the DAP cell line that has been transfected
with I-Abk (gift of
Dr. Germain, National Institutes of Health). Antigenic stimulation of
BI-141 T cells was performed as reported (13). For
experiments examining the requirements for duration of TCR engagement,
T cell transfectants were stimulated using immobilized anti-CD3
with or without anti-CD28 or anti-CD48 for various periods and
subsequently cultured in the absence of stimulus for the duration of
the 16- to 20-h experiment. IL-2 was quantitated either by bioassay or
by ELISA and cell death was detected using propidium iodide staining
and FACS analysis as described (13).
GST fusion protein precipitations
Resting BI-141 T cells (2.5 x 107) were lysed in 0.5% Triton X-100, 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, and 1 mM Na3VO4. Clarified lysates were incubated with 25 µg GST fusion protein coupled to 25 µl of GST-Sepharose 4B beads (Pharmacia) for 1 h at 4°C. Samples were washed in lysis buffer and separated on 10% SDS-PAGE, transferred to nitrocellulose, immunoblotted for c-Cbl, p85, and Sam68, and detected using ECL (Amersham, Arlington Heights, IL).
Lck kinase assay
Lck was transfected into COS cells using the Transfast Reagent (Promega, Madison, WI). Forty-eight hours posttransfection, cells were lysed in TNE buffer (50 mM Tris, 1% Nonidet P-40, 1 mM EDTA) containing 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM Na3VO4, and postnuclear lysates were immunoprecipitated with rabbit anti-Lck antiserum. Immunoprecipitations were split in half and used for in vitro kinase assay and determination of Lck expression levels by immunoblotting. Kinase assays were performed as previously described (12).
Raft fractionation
BI-141 T cells were incubated with 5 µg/ml of anti-CD3
(2C11), with or without 20 µg/ml anti-CD48 (5-8A10), or media for
30 min at 4°C, and cross-linked with GAH at 37°C for various times.
Anti-phosphotyrosine and anti-Lck immunoblots were performed as
described (2). For raft fractionation, T cells (4 x
107) were resuspended in buffer A (25 mM Tris,
pH7.5, 150 mM NaCl, 5 mM EDTA) containing 10 µg/ml aprotinin, 10
µg/ml leupeptin, 1 mM PMSF, and 1 mM
Na3VO4. Cell resuspensions
were briefly sonicated, centrifuged at 800 x g for 10
min and supernatants were incubated in 1% Brij58 for 1 h at
4°C. An equal volume of 80% sucrose in buffer A was added to the
Brij58 lysates and samples were placed in "Ultra-Clear" centrifuge
tubes (Beckman Coulter, Fullerton, CA). Samples were then overlaid with
2 volumes of 30% sucrose in buffer A and 1 volume of 5% sucrose in
buffer A and centrifuged at 200,000 x g in a SW55Ti
rotor (Beckman Coulter) for 16 h at 4°C. Fractions (400 µl)
were harvested from the top (9).
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) protection assay
Cells were stimulated with anti-CD3 (10 µg/ml) with or without anti-CD28 (40 µg/ml) or anti-CD48 (40 µg/ml) and GAH. After 5 min PP2 was added to a final concentration of 50 µM and aliquots sampled 0–5 min after PP2 addition were lysed in TNE buffer containing protease inhibitors, but without Na3VO4.
Confocal microscopy
T cells (5 x 104) were stimulated
with 5 x 104 Ab-coated beads (50 µg/ml
anti-CD3 with or without 200 µg/ml anti-CD48) in 25 µl
for 30 min at 37°C on poly-L-lysine-coated slides
(Carlson Scientific, Peotone, IL). Cells were fixed in 3.7%
formaldehyde for 10 min, stained with FITC-cholera toxin B subunit (8
µg/ml; Sigma, St. Louis, MO) for 45 min and analyzed by confocal
microscopy on a Carl Zeiss LSM 310 microscope using a x100 objective.
Sections (10–15 1 µm) were collected and overlaid to create the
composite images.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address the role of the Lck SH3 domain in TCR signal
transduction, we generated two clustered sets of mutations at sites
predicted to be important for intramolecular and intermolecular SH3
ligand binding (Fig. 1
, A and
B). We expressed wild-type and mutant Lck SH3 domains as GST
fusion proteins and determined their relative abilities to precipitate
known Lck SH3 ligands from T cell lysates. As shown in Fig. 1C, both SH3 mutants are defective at binding Lck SH3
ligands c-Cbl, Sam68, and the p85 subunit of phosphatidylinositol (PI)
3-kinase, relative to wild-type Lck SH3 GST-fusion protein. The YLDY
mutation is severely impaired, whereas the AF mutation maintains modest
residual binding activity (Fig. 1C).
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Lck SH3 mutants were transiently transfected into COS cells and the Lck
kinase activity was determined by in vitro kinase assay. The in
vitro kinase activity of LckAF/F505 is comparable to that of LckF505,
whereas LckYLDY/F505 activity is enhanced relative to LckF505 activity
(Fig. 1
D). To determine whether the measured in vitro Lck
kinase activity is relevant to protein tyrosine phosphorylation in
intact cells, levels of tyrosine phosphorylated proteins in COS cell
transfectants were measured by anti-phosphotyrosine
immunoblotting. The relative intensity of a prominent and
representative 40-kDa phosphorylated protein from COS cell
transfectants was quantitated and is shown in Fig. 1D. The
level of cellular protein tyrosine phosphorylation within COS cell
transfectants is coincident with the measured in vitro kinase activity
of the mutant expressed (Fig. 1, C and D).
Together, these data support models in which Lck SH3 intramolecular and
intermolecular interactions regulate kinase activity (16, 17). Furthermore, they provide us with two independent
kinase-active/SH3- impaired (AF/F505 and YLDY/F505) Lck mutants for use
in characterizing the contributions of Lck SH3 to T cell
activation.
The Lck SH3 domain is not required for the initiation of TCR signal transduction, once Lck is activated
To address the contribution of the Lck SH3 domain in TCR-induced
signal transduction and functions, LckF505 or SH3-impaired LckF505
mutants were stably expressed in the BI-141 T cell hybridoma. At least
four independent transfectants of each type were matched for Lck and
TCR expression levels and used for subsequent analysis (data not
shown). Transfectants were assessed for baseline levels of protein
tyrosine phosphorylation and for their ability to initiate protein
tyrosine phosphorylation in response to TCR engagement (Fig. 2). Both LckAF/F505 and LckYLDY/F505
transfectants seem to be less regulated by TCR engagement, having
higher baseline kinase activities than LckF505 transfectants (Fig. 2).
Nonetheless, TCR engagement induces additional protein tyrosine
phosphorylation (Fig. 2). Transfectants expressing
kinase-active/SH3-impaired mutants (LckAF/F505 or LckYLDY/F505)
initiate TCR-induced protein tyrosine phosphorylation as efficiently,
or slightly better than, LckF505 transfectants (Fig. 2). Analysis of
protein tyrosine phosphorylation at earlier time points ruled out the
possibility that LckAF/F505 or LckYLDY/F505 transfectants delay onset
of protein tyrosine phosphorylation relative to LckF505 transfectants
(data not shown). In keeping with a previous report (18),
these data indicate that the Lck SH3 domain is not essential for the
initiation of TCR signal transduction, once Lck is activated.
Discrepancies between relative LckAF/F505 and LckYLDY/F505 activity in
COS cells and T cells may reflect the presence of T cell-specific SH3
ligands that are able to activate LckAF/F505 through its residual site
3 mediated ligand binding activity (Fig. 1, C and
D vs Fig. 2).
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We next addressed whether LckF505 (kinase-active) and LckAF/F505
SH3 (kinase-active/SH3-impaired) transfectants were equally able to
sustain signals by maintaining TCR-induced protein tyrosine
phosphorylation (Fig. 3). LckF505 and
LckAF/F505 expressing transfectants were stimulated through CD3, CD3
plus CD28, or CD3 plus CD48 for 5 min. Protein tyrosine phosphorylation
was measured initially and at several time points after the addition of
PP2, an inhibitor of src family tyrosine kinases, to stop de
novo protein tyrosine phosphorylation. By monitoring levels of
phosphorylated proteins after PP2 addition, we are able to measure the
stability of phosphotyrosyl groups on proteins phosphorylated within
the first 5 min of activation (3). In LckF505
(kinase-active) transfectants, CD3 plus CD28 and CD3 plus CD48
costimulation stabilizes protein tyrosine phosphorylation beyond levels
observed in response to anti-CD3 alone (Fig. 3, A and
B). These findings indicate that, like CD28 costimulation
(3), CD48 costimulation can stabilize TCR-induced protein
tyrosine phosphorylation. LckAF/F505 (kinase-active/SH3-impaired)
transfectants are unable to stabilize protein tyrosine phosphorylation
through CD3 plus CD28 or CD3 plus CD48 costimulation (Fig. 3).
Consistent with reports in which CD28 is shown to stabilize protein
tyrosine phosphorylation using a similar assay, this effect is most
pronounced with regard to proteins migrating within the 110- to 125-kDa
and 36- to 40-kDa range (3). Under these assay conditions
in which kinase inhibitor is added before lysis, costimulation-mediated
enhancement of protein tyrosine phosphorylation is apparent after 5 min
of TCR/costimulation (Fig. 3). However, costimulatory effects become
increasingly dramatic when stability of protein tyrosine
phosphorylation is monitored after additional incubation at 37°C with
PP2 for 2 and 5 min before lysis. These data implicate the Lck SH3
domain in mediating costimulator stabilization of protein tyrosine
phosphorylation.
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Lck SH3 mutations discriminate two distinctly regulated lipid raft-dependent TCR activation events
Lipid rafts can be biochemically isolated based on their relative
detergent insolubility and buoyancy on sucrose gradients
(1). Recent data analyzing rafts purified in this manner
demonstrate that TCR engagement results in the relocalization and/or
tyrosine phosphorylation of transducers and adapters, including
TCR-, to lipid raft microdomains (4, 8, 9). Because
lipid rafts have been estimated to contain only 5% of the total plasma
membrane proteins (19), the accumulation of proteins in
raft fractions represents a dramatic concentration of those proteins
within rafts relative to the rest of the plasma membrane.
To determine whether LckAF/F505 efficiently compartmentalizes
TCR-induced phosphoproteins, LckF505 (kinase-active) and LckAF/F505
(kinase-active/SH3-impaired) transfectants were compared for their
ability to concentrate tyrosine phosphorylated and other signal
transducers within lipid rafts in response to stimulation with
anti-CD3 or anti-CD3 plus CD48 (Fig. 4B). Lysates from unstimulated
or stimulated LckF505 and LckAF/F505 transfectants were separated into
lipid raft and nonraft fractions by sucrose density centrifugation in
the absence of tyrosine kinase inhibitor. As shown in Fig. 4A, cellular Lck equally distributes in buoyant lipid raft
(1, 2, 3, 4) and nonraft (8, 9, 10, 11) fractions isolated
from either LckF505 or LckAF/F505 transfectants. This Lck distribution
is consistent with previous reports (6) and indicates that
mutation of the LckF505 SH3 domain does not affect its ability to
partition within lipid rafts. Aliquots of pooled raft
(1, 2, 3, 4) or nonraft (8, 9, 10, 11) fractions from
cells left unstimulated or stimulated through CD3 or CD3 plus CD48 for
5 min were separated on SDS-PAGE and immunoblotted with
anti-phosphotyrosine Ab reactive with phosphorylated pp21 and
pp23 isoforms. Both LckF505 and LckAF/F505 transfectants
successfully concentrate tyrosine phosphorylated and other
phosphoproteins in lipid rafts after TCR or TCR/CD48 engagement (Fig. 4B). Immunoblotting with an Ab (H146–968) reactive with
unphosphorylated and minimally phosphorylated TCR- demonstrates
increased levels of in raft fractions after TCR engagement in both
LckF505 and LckAF/F505 transfectants (Fig. 4C). Decreased
relative levels of p18 and pp19 in raft fractions from AF/F505
transfectants are offset by increased levels of phosphorylated pp21
and pp23 isoforms, consistent with the modest increase in efficiency
of protein tyrosine phosphorylation observed in LckAF/F505 relative to
LckF505 transfectants. Taken together, these data indicate that LckF505
and LckAF/F505 transfectants effectively tyrosine phosphorylate and
redistribute and other phosphoproteins within lipid rafts.
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A and M. Moran and M. C.
Miceli, unpublished observations). To investigate the role of Lck SH3
activity in raft redistribution to the TCR contact cap, we used
FITC-cholera toxin to visualize the lipid raft constituent GM1 after T
cell exposure to anti-CD3 or anti-CD3 plus CD48 coated
microspheres. Whereas both neo (control) and LckF505
(kinase-active) transfectants efficiently relocalize lipid rafts toward
the T cell:anti-CD3 plus CD48 coated microsphere interface,
transfectants expressing LckF505 SH3 mutants do not (Fig. 5).
Representative patterns of raft redistribution (clustering) and the
percentages of cells exhibiting polarized raft clustering are shown in
Fig. 5, A and B, respectively. Analysis of
single-section fluorescent images demonstrates redistribution of cell
surface, and not intracellular, GM1 (data not shown). Raft
redistribution is observed 30 min after CD3 + CD48 costimulation, but
not as early as 10 min. None of the transfectants redistribute rafts in
response to microspheres coated with anti-CD3 or anti-CD48
alone (data not shown). These data implicate Lck SH3 activity in
mediating signals required for the costimulation-dependent recruitment
of lipid rafts to the TCR contact site. Furthermore, they further
correlate migration of lipid rafts to the TCR contact site with the
stabilization of protein tyrosine phosphorylation (3).
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Because sustained TCR signal transduction and costimulation have
been implicated in regulating the functional outcome of TCR engagement,
we were interested in determining whether each of these raft-mediated
TCR activation events were equally important for all downstream
functions. To this end, we defined functions reliant on "sustained
and complete" or "partial" TCR signal transduction in BI-141 T
cells. We have previously reported that TCR-induced IL-2 production and
apoptosis have different requirements for Lck kinase and SH2 activities
(13). Indeed, BI-141 transfectants expressing
kinase-inactive or SH2 domain-impaired LckF505 are defective in
processively phosphorylating and downstream signal transducers and
producing IL-2 in response to Ag, although they remain fully competent
at undergoing apoptosis (12, 13). These data likely
reflect a differential dependence of downstream functions on processive
and sustained "complete" TCR/Lck signal transduction.
To further address this issue, we investigated the relationship between
duration of TCR engagement and functional outcome in BI-141 T cells. We
varied the duration of TCR engagement by stimulating BI-141 T cells
with plate-bound anti-CD3 and after a given time transferred them
to a fresh well in the absence of stimuli for the remainder of the
assay period. After 20 h of culture, supernatants were assayed for
IL-2 and cells were analyzed for viability. Significant levels of
apoptosis were detected within the first hour of TCR engagement. By
6 h of continual engagement as much as 60% of the maximal death
response was observed (Fig. 6A). In contrast, IL-2
production required more than 6 h of TCR engagement (Fig. 6A). Therefore, a shorter length of TCR engagement is
required for commitment to apoptosis than is required for commitment to
IL-2 production in BI-141 T cells. These data corroborate our
suggestion that apoptosis in BI-141 T cells requires "partial"
TCR/Lck signal transduction, whereas IL-2 production requires
"complete" TCR/Lck signal transduction. Furthermore, they are in
keeping with reports demonstrating a similar hierarchy of apoptosis and
IL-2 responses in the A.E7 and other T cell clones in response to
"partial" and "complete" agonist Ags and in primary T cells
under circumstances in which CD28 costimulation is or is not present
(20, 21). Together, these data contribute to the growing
body of evidence indicating that complete TCR activation requires
processive and sustained signal transduction and that the extent to
which this signal reaches "completion" can influence the functional
outcome of TCR engagement.
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, B–D).
Transfectants expressing Lck mutants that are either kinase inactive
(LckR273/F505) or SH3 impaired (LckAF/F505, LckYLDY/F505) are not
defective in apoptosis (Ref. 12 and Fig. 6B).
In contrast, transfectants expressing either of the SH3-impaired Lck
mutants produced significantly less IL-2 than LckF505 (kinase-active)
transfectants in response to anti-CD3 or Ag (Fig. 6, C
and D). LckAF/F505 or LckYLDY/F505 transfectants demonstrate
impaired IL-2 production relative to LckF505 transfectants despite
comparable or increased initiation of protein tyrosine phosphorylation
after TCR engagement (Figs. 2 and 4). These findings highlight the
importance of Lck SH3 domain contributions downstream or independent of
initiation of tyrosine kinase activity. Furthermore, these data
correlate Lck SH3 activity and lipid raft recruitment with signals and
functions that rely on prolonged TCR engagement and processive TCR
signal transduction. CD28 or CD48 TCR costimulation shortens the duration of TCR engagement required for commitment to IL-2 production
Previous reports indicate that CD28 functions to decrease the
duration of TCR engagement required for commitment to T cell activation
by increasing the potency of TCR engagement through a mechanism
involving raft mediated amplification and sustenance of TCR induced
signals (3). Therefore, we next investigated whether CD48
or CD28 costimulation decreases the duration of TCR engagement required
for commitment to IL-2 production in BI-141 T cells and what influence
LckSH3 mutant expression has on this costimulatory activity. To this
end, BI-141 transfectants expressing neo, LckF505 (kinase-active),
LckAF/F505 (kinase-active/SH3-impaired), or LckYLDY/F505
(kinase-active/SH3-impaired) were stimulated through their TCRs alone
or in the presence of CD48 or CD28 coengagement for varying times
ranging from 4 to 16 h. T cells were subsequently transferred to
wells in the absence of stimulation for the duration of the 16-h assay
and supernatants assayed for IL-2 production at the 16-h time point. As
shown in Fig. 7, either CD48 or CD28
costimulation can significantly shorten the duration of TCR engagement
required for commitment to IL-2 production in neo or LckF505
transfectants. Alternatively, transfectants expressing LckAF/F505 or
LckYLDY/F505 make very little IL-2 even after 16 h of continuous
CD3 or CD3 plus costimulator engagement. Low levels of residual
costimulatory activity in cells expressing SH3 mutants may result from
contributions of a subset of endogenous Lck not affected by expression
of mutant Lck SH3. These findings further support the concept that T
cell functions, such as IL-2 production, which require processive and
sustained TCR signal transduction are particularly sensitive to
costimulation and intact Lck SH3 activity. Furthermore, they correlate
requirements for raft clustering, sustenance of protein tyrosine
phosphorylation and decreased duration of engagement and provide
support for suggestions that raft clustering at the TCR contact cap
functions to increase the potency of TCR signal transduction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We expressed these Lck mutants in BI-141 T cells to determine the role of Lck SH3 in mediating TCR signal transduction. We demonstrated that once Lck is activated, Lck SH3 functions downstream of the initiation of TCR-induced protein tyrosine phosphorylation to regulate the costimulation-dependent migration of lipid rafts to the TCR contact cap, stabilization of protein tyrosine phosphorylation and induction of IL-2 production. These findings are consistent with previous reports implicating the Lck SH3 domain in generating TCR signal(s) downstream of the initiation of protein tyrosine phosphorylation that are required for Ag-induced IL-2 production (18, 27, 28). However, our studies extend previous findings by defining a role for Lck SH3 in controlling TCR/costimulation-induced lipid raft migration to the TCR contact cap, stabilization of TCR protein tyrosine phosphorylation, and shortening of the required duration of TCR engagement. Furthermore, our data establish critical relationships among Lck SH3 activity, raft coalescence at the contact cap, sustained signal transduction, and IL-2 production. Expression of kinase active/SH3 impaired mutants does not interfere with the concentration and phosphorylation of transducers within lipid rafts or with apoptosis, which only requires "partial" TCR signals. However, the expression of kinase-active/SH3-impaired mutants does disrupt costimulation-mediated recruitment of lipid rafts to the site of TCR engagement and stabilization of protein tyrosine phosphorylation, and IL-2 production. These data correlate Lck SH3 activity, lipid raft recruitment, and sustained signals with functions that rely on prolonged TCR engagement and processive and sustained TCR signal transduction. In keeping with data presented here, recent characterization of peripheral T cells from asmase-/- (raft-deficient) mice demonstrates that anti-CD3/anti-CD28 induced proliferation, but not anti-CD3-induced susceptibility to apoptosis, is impaired relative to wild type mice (29, 30).
Recent studies indicate that membrane and protein reorganization events
at the TCR contact cap are essential for successful T cell activation
(31). In response to APCs presenting antigenic peptide,
the T cell:APC contact site assembles into topologically and spatially
distinct regions (32, 33). Initial TCR engagement occurs
at the periphery of the APC:T cell contact area. Within 20 min of TCR
engagement, MHC/peptide-bound TCR complexes and membrane-associated
intracellular Lck, Fyn, and protein kinase C
(PKC) cluster in the
center of this specialized junction, referred to as the immunological
synapse (32, 33, 34). They are surrounded by a ring of
relatively taller (ICAM-bound) LFA-1 surface adhesion proteins and the
intracellular cytoskeletal protein talin. It has been suggested that
bulky CD43 and CD45 phosphatase need to be excluded from the T cell:APC
contact to accommodate TCR/Ag-MHC binding and sustained protein
tyrosine phosphorylation (13). The formation of a stable
central cluster at the heart of the synapse is determinative for the
induction of naive peripheral T cell activation (32, 33).
Therefore, the organized immunological synapse is the likely site in
which processive TCR signal transduction is orchestrated.
The molecular basis of synapse formation and function remains elusive, although related experiments have suggested that the liganded engagement of CD28 (3, 35), CD48 (2, 36), CD2 (37), and/or LFA-1 (35) costimulators may facilitate receptor recruitment, sorting, and cytoskeletal interactions at the TCR contact site and processive and sustained TCR signal transduction. Furthermore, inhibitor studies implicate PI 3-kinase and actin-myosin motor activities in the costimulation-induced flow of membrane lipids and proteins toward the contact cap and, thus, the formation of the immune synapse (3, 35). CD48 or CD28 costimulation-induced raft coalescence at the contact cap is temporally coincident with stable synapse formation (data presented here and (3, 33)). Furthermore, lipid rafts specifically include several proteins known to concentrate within the core of the immune synapse (i.e., Fyn, Lck, and TCR) and exclude proteins that segregate from the center of the synapse (CD45 and CD43) (1, 5, 6, 7). Together, these findings are suggestive of a role for rafts in immune synapse formation and stabilization.
We and others have previously suggested that raft recruitment to the TCR contact site might facilitate the stability of protein tyrosine phosphorylation through the exclusion of CD45 phosphatase activity (2, 3, 7). Raft/nonraft partitioning of CD45 phosphatase activity and TCR tyrosine phosphorylated substrates could explain why costimulators that induce raft recruitment to the TCR contact site also increase the stability of protein tyrosine phosphorylation. In keeping with this suggestion, Lck SH3 mutants that interfere with raft recruitment and coalescence also interfere with sustained TCR protein tyrosine phosphorylation and IL-2 production. Additionally, as TCR signal transducers are localized within growing lipid raft platforms, sustained Lck kinase activity may become increasingly reliant on SH3 intermolecular interactions to maintain kinase activity. Indeed, the liganded displacement of SH2 or SH3 intramolecular interactions may be the primary mechanism for Lck kinase activation within aggregated lipid rafts devoid of CD45. These suggestions are consistent with recent findings that the CD28 cytoplasmic tail activates Lck through direct interaction with Lck SH3 (10) and that CD28 engagement can induce lipid raft recruitment to the contact cap, prolong the stability of protein tyrosine phosphorylation, and sustain TCR signals (3).
We have yet to determine whether the requirement for Lck SH3 in
costimulation-mediated raft recruitment reflects its activity as an
adapter for localizing a particular SH3 ligand. The Lck SH3 has been
reported to associate with several TCR signal transducers, including
the p85 subunit of PI 3-kinase, PKC, HS1, CD2, CD28, Ras-GAP, Cdc2,
Sam68, c-Cbl, and extracellular signal-related kinase (ERK) (10, 38, 39, 40, 41, 42, 43, 44, 45, 46). The Lck SH3 ligands ERK, PI 3-kinase, and PKC have
been previously implicated in lipid raft reorganization, immune synapse
formation, actin cytoskeletal reorganization, costimulatory activity,
and/or downstream TCR signal transduction and, therefore, represent
prime effector candidates (3, 18, 34, 47). Although
previous studies have implicated Lck SH3 (18) and lipid
rafts (48, 49) in ERK activation, the relationship between
ERK activation and raft clustering has yet to be determined. Indeed, it
is possible that ERK activity is required for the migration of rafts to
the contact cap or that large raft platforms are required for efficient
and sustained ERK activation. Finally, initial ERK activation may
facilitate raft coalescence at the contact cap, which, in turn,
sustains ERK activity during T cell activation. Experiments addressing
the role of each of these proteins in TCR raft reorganization events
are underway.
Recent experiments have demonstrated an essential role for lipid raft
membrane compartmentalization in T cell activation (2, 3, 4, 8, 9). However, the relationship between the concentration and
phosphorylation of transducers within lipid rafts and the
costimulation-dependent migration of rafts to the contact cap has not
been previously established. Here we demonstrate that these events are
distinctly regulated and that both reorganization events are required
for "complete," but not "partial," TCR signal transduction.
Indeed, requirements for both the concentration and phosphorylation of
transducers within rafts and raft clustering for IL-2 production are
particularly evident under circumstances in which either the initial
signal strength (i.e, in neo vs LckF505 transfectants) or the duration
of TCR are limiting. Although neo transfectants can cluster rafts,
initial signal strength is lower than what is observed in LckAF/F505 or
LckF505 transfectants. Signal initiation is more intense in LckAF/F505
transfectants; however, signal amplification/sustenance is defective
due to impaired raft clustering. LckF505 transfectants have both strong
signal initiation and intact raft clustering mechanisms. That IL-2
production is lower in LckAF/F505 transfectants relative to neo or
LckF505 transfectants (Fig. 7) despite high signal initiation highlight
the importance of costimulation mediated raft clustering in processive
signal transduction. The fact that recruitment of lipid rafts is
impaired in cells expressing Lck SH3 mutants and that recruitment
affects lipid rafts extending beyond the area of direct Ab-coated
microsphere contact, indicate that signals generated through liganded
lipid raft-associated CD48/TCR lead to the active recruitment of
additional nonliganded rafts to the contact cap. Together with other
recent findings (3, 10), these data support a model in
which costimulators function by recruiting additional lipid rafts to
the contact cap and facilitating processive and sustained TCR
signals.
A "two (raft-mediated) signal model of T cell activation"
The two signal model of T cell activation is a central tenet of
cellular immunology proposed as a mechanism for regulating peripheral T
cell activation and tolerance induction. This model posits that two
independent signals are required for TCR activation: "signal one"
supplied by the TCR and "signal two" provided by a costimulator
(50). Generation of signal one without signal two results
in T cell inactivation (apoptosis or anergy), whereas generation of
both signals results in IL-2 production and T cell activation (and in
some instances rescue from apoptosis/anergy) (50, 51).
Whereas CD28 is the best-studied costimulator in this regard, the
recent description of dramatic T cell activation defects in
CD48-deficient mice highlight the importance of CD48 costimulatory
activity (11). Furthermore, similarities in the abilities
of CD48 and CD28 costimulators to recruit lipid rafts to the contact
cap stabilize TCR-induced protein tyrosine phosphorylation and decrease
the duration of TCR engagement required for IL-2 production and their
reliance on Lck SH3 activity indicate that they function similarly, but
likely not identically, to modulate TCR signals ((3, 10)
and data shown here). The two-signal model is well supported by
experiments analyzing the functional effects of engaging the TCR in the
presence or absence of costimulation. However, elucidation of a
costimulatory signal distinct from signal 1 has proven elusive. Recent
experiments indicate that costimulators may not send a second
independent signal, but rather, may function to enable the TCR to send
a sustained and processive signal (3, 10, 31). Here, we
propose a "two (raft-mediated) signal model of T cell activation"
reconciling these findings (Fig. 8).
|
and downstream
transducers are concentrated (or stabilized) and tyrosine
phosphorylated within lipid rafts (signal one). This step requires Lck
kinase activity, but remains intact in cells expressing
kinase-active/SH3-impaired Lck mutants. Completion of the first step
allows for the generation of early protein tyrosine phosphorylation and
the induction of functions reliant on partial TCR signals including
anergy and/or apoptosis. The second step involves the subsequent
costimulation-dependent recruitment of additional lipid rafts to the
TCR contact cap (signal two). In the case of CD48/TCR costimulation,
this second signal may result from CD48/TCR-mediated raft clustering
and actin cytoskeletal reorganization. How transmembrane CD28
interfaces with lipid rafts is unclear (52), thus how this
step is induced by CD28 costimulation remains speculative. This second
"raft recruitment" step is defective in T cells expressing
kinase-active/SH3-impaired Lck mutants and is required for the
generation of sustained TCR signals and functions dependent on
"complete" TCR signals such as IL-2 production. We propose that Lck
SH3-regulated/actin-cytoskeleton-dependent raft recruitment leads to
the concentration and raft-based sorting of TCR, costimulators, and
accessory molecules within the immune synapse. Furthermore, we propose
that the construction and stabilization of a raft-based platform(s) at
the TCR contact cap facilitates sustained and processive TCR signal
transduction. This likely results from a number of predicted outcomes
of raft recruitment and cross-linking at the TCR contact cap: 1) the
promotion of MHC peptide access to the TCR due to raft exclusion of
highly extending and glycosylated CD43 and CD45; 2) increased
interaction between apposing T cell:APC membrane protein pairs due to
increased lipid raft membrane rigidity and increased LFA-1 binding
(53); 3) the concentration and activation of
raft-associated tyrosine kinases through intermolecular displacement of
SH2 and SH3 kinase down-regulatory interactions; 4) the exclusion of
phosphatase activity; 5) the polarized recruitment and concentration of
downstream signal transducers to raft-associated proteins or lipids);
6) the integration of signal transduction pathways; and 7) the
induction of actin-cytoskeletal reorganization events that stabilize
the immune synapse. The studies presented here demonstrate that two distinctly regulated lipid raft reorganization events are required for complete T cell activation. They provide the molecular framework for a model of TCR/costimulator-mediated T cell activation. Future studies will test and further refine this model to determine the molecular mediators of each of these raft reorganization events and elaborate how different T cell costimulators modulate these events to facilitate TCR activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Carrie Miceli, Department of Microbiology, Immunology, and Molecular Genetics and The Molecular Biology Institute, University of California, Los Angeles School of Medicine, Los Angeles, CA 90095-1570.
3 Abbreviations used in this paper: SH3, Src homology 3; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine; ERK, extracellular signal-related kinase; GAH, goat anti-hamster; PI, phosphatidylinositol; PKC, protein kinase C.
Received for publication March 2, 2000. Accepted for publication October 11, 2000.
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