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Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1; and function in T cell
activation. Our results showed that the five membrane-distal tyrosines
were phosphorylated upon T cell activation. Grb2, Gads, and PLC-1
associated with LAT preferentially via different sets of tyrosine
residues; however, they failed to interact with LAT mutants containing
only one tyrosine. We also determined the minimal requirement of LAT
tyrosine residues in T cell activation and thymocyte development. Our
results showed that a minimum of three tyrosines is required for LAT to
function in T cell activation and thymocyte development. LAT mutants
that were capable of binding Grb2 and PLC-1 could reconstitute T
cell activation in LAT-deficient cells and thymocyte development in
LAT-deficient mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LAT has a short extracellular domain, a transmembrane domain, and a
cytosolic tail with multiple tyrosines (9, 10). It is
palmitoylated via two cysteine residues near its transmembrane domain
(11). Palmitoylation of LAT is required for its
localization in lipid rafts. Upon T cell activation, LAT is
phosphorylated by ZAP-70 tyrosine kinase and associates with Grb2,
Grb2-related adapter downstream of Shc (Gads), and phospholipase C
(PLC)-1. Of the 10 tyrosines in human LAT, Y110, Y127, Y171, Y191,
and Y226 are within a Grb2-binding motif, and Y132 is within a
PLC-1-binding motif. These motifs allow LAT to interact with the Src
homology 2 (SH2) domains of Grb2 and PLC-1 directly. Mutation of
three Grb2 binding sites (Y171, Y191, and Y226) simultaneously
abolishes the interaction of LAT with Grb2 and Gads, and mutation of
Y132 in LAT abolishes PLC-1 binding, leading to defective Ras-MAPK
activation and Ca2+ flux (12). These
data indicate that recruitment of Grb2 and PLC-1 is essential for
LAT function.
In addition to its role in the TCR signaling pathway, LAT is also
essential during thymocyte development. Disruption of LAT in mice has a
profound effect on thymocyte development (13).
LAT-deficient mice lack mature T cells in the periphery, while they
have normal B cells. Even though thymocytes from these mice have a
normal TCR
chain rearrangement, they are arrested at the
CD25+CD44- subset of the
CD4-CD8- stage. These
cells cannot be rescued by injection of anti-CD3 Abs, suggesting
that LAT is required in the pre-TCR signaling pathway. Recent data show
that mice expressing LAT with mutations in the four membrane-distal
tyrosines also exhibit defective thymocyte development, indicating that
these tyrosines are essential for LAT function in thymocyte development
(14).
In this study, we made a series of LAT mutants with Y to F mutations to
define the minimal tyrosines that are required for LAT binding to Grb2,
Gads, and PLC-1. We also examined the function of these mutants in
TCR-mediated Ras-MAPK activation, Ca2+ flux,
NF-AT activation, and thymocyte development. Our results showed that
LAT binding of Grb2, Gads, and PLC-1 requires multiple tyrosines.
LAT binding to Grb2 and PLC-1 is necessary for T cell activation and
thymocyte development. LAT with a minimal three of four membrane-distal
tyrosines can function in T cell activation and thymocyte
development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Site-directed mutagenesis was performed to mutate tyrosine
residues in LAT using the Quick-Exchange kit from Stratagene (La Jolla,
CA). LAT mutants with combinations of different tyrosine residues were
constructed by using restriction endonuclease sites present in
LAT cDNA or created during mutagenesis. These LAT mutants
were cloned into two retroviral vectors, pLXIN (Clontech, Palo Alto,
CA) and pMSCV-IRES (internal ribosomal entry site)/GFP (green
fluorescent protein). LAT expression is driven by the 5' LTR. The
NeoR and GFP genes were placed after LAT under
the control of an internal ribosomal entry site element (Fig. 1
A). The LAT mutants used in this study were listed in Fig. 1B. The pLXIN constructs were used to generate stable cell
lines expressing LAT mutants in the J.CaM2.5 cell line, while the pMSCV
constructs were mainly used to transduce bone marrow cells from
LAT-/- mice in the adoptive transfer
experiments.
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LAT-deficient cells (J.CaM2.5) were maintained in RPMI 1640 medium supplemented with 10% FBS. Phoenix-ecotropic virus-packaging cells were maintained in DMEM medium supplemented with 10% FBS. A total of 10 µg of each retroviral construct was used to transfect Pheonix-ecotropic packaging cells using calcium phosphate. Forty-eight to seventy-two hours after transfection, the cell culture supernatant containing recombinant retroviruses was harvested for immediate viral transduction or frozen at -80°C.
To make J.CaM2.5 cells transducible by ecotropic viruses, the ecotropic receptor MCAT-1 was introduced. A cDNA fragment from MCAT-1 was cloned into pMSCVpuro (Clontech). The resulting construct was then used to pack pantropic retroviruses using VSV-G as the envelope glycoprotein (Clontech). Retroviruses containing the MCAT-1 gene were subsequently used to transduce J.CaM2.5 cells. These transduced J.CaM2.5 cells were selected in the presence of puromycin and further subcloned. Each clone was tested for the expression of the ecotropic receptor by its ability to be transduced by a retrovirus expressing GFP. One designated as J.CaM2.5-MCAT was chosen for further studies. J.CaM2.5 cells expressing various LAT mutants were generated by transducing J.CaM2.5-MCAT cells with recombinant retroviruses containing different LAT mutants in pLXIN. Stable transductants were selected for in the presence of G418 (1.2 mg/ml).
Abs, immunoprecipitation, and Western blotting
The following Abs were used for immunoprecipitation and Western
blotting: anti-TCR (C305), anti-myc (9E10), and
rabbit polyclonal anti-LAT Abs (10);
anti-phosphotyrosine (PY20) and anti-Grb2 mAbs were from
Transduction Laboratories (Lexington, KY); monoclonal anti-PY
(4G10), anti-PLC-1, and rabbit anti-Gads Abs were from
Upstate Biotechnology (Lake Placid, NY); rabbit anti-Grb2
polyclonal Ab was from Santa Cruz Biotechnology (Santa Cruz, CA); an
anti-active MAPK mAb was purchased from New England Biolabs
(Beverly, MA).
For immunoprecipitation, J.CaM2.5 cells expressing different LAT mutants were either stimulated with C305 (1:50 tissue culture supernatant) for 1 min and 30 s or left untreated. A total of 1 x 107 cells were lysed in 500 µl of ice-cold lysis buffer (1% Brij97, 25 mM Tris-Cl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4) with protease inhibitors. Cell lysates were subjected to immunoprecipitation with different Abs, as indicated in each figure. Immunoprecipitated proteins were resolved on SDS-PAGE, transferred to Immobilon-P transfer membrane (Millipore, Bedford, MA), and blotted with different Abs, as indicated in each figure. Immunoreactive proteins were detected with an HRP-coupled secondary Ab (Amersham, Arlington Heights, IL), followed by detection with SuperSignal West Pico Luminol/Enhancer Solution (Pierce, Rockford, IL).
MAPK activation, Ca2+ flux, and luciferase assay
Twelve to sixteen hours after transduction with retroviruses containing LAT mutants and GFP, GFP+ cells were sorted by FACS and cultured for another 24 h before harvest. These sorted cells were either left untreated or stimulated with C305 for 5 min. Cell lysates were resolved on SDS-PAGE and further analyzed by an anti-active MAPK Western blot.
Intracellular free Ca2+ measurement was performed
as described (15). Twelve to sixteen hours after
retrovirus transduction, J.CaM2.5 cells were loaded with Indo-1.
Anti-CD3
Ab (OKT3) was used to activate those cells.
Ca2+ flux in GFP+ cells was
analyzed using a flow cytometer (FACScan; BD Biosciences, San Jose, CA)
by monitoring the fluorescence emission ratio at 405–495 nm.
For luciferase assays, 1.6 x 106 J.CaM2.5 cells were transfected with 1.5 µg pNFAT-luciferase plasmid and 0.5 µg of different LAT plasmids or an empty vector using Superfect transfection reagent (Qiagen, Valencia, CA). Twenty to twenty-four hours after transfection, J.CaM2.5 cells were stimulated with OKT3 (1:500 ascites) coated on tissue culture plates, PMA (10 ng/ml) plus ionomycin (1.5 µM), or left untreated for 6 h. Luciferase activity was assayed according to the manufacturer’s protocol (Promega, Madison, WI).
Transduction of bone marrow cells, and adoptive transfer
Bone marrow cells were taken from the femurs of LAT knockout mice that were treated with 5-fluorouracil (150 mg/kg) for 48 h. After removal of RBCs, they were cultured in IMDM with 15% FBS in the presence of IL-3 (20 ng/ml), IL-6 (20 ng/ml), and stem cell factor (50 ng/ml) for 3–4 days. For retroviral transduction, bone marrow cells were then mixed with retroviral supernatant in the presence of polybrene (8 µg/ml) and centrifugated at 1300 x g for 3 h at 22°C. These cells were cultured in the presence of IL-3, IL-6, and stem cell factor for an additional 24 h and then transduced with retroviruses again. The efficiency of retroviral transduction of bone marrow cells was examined by monitoring GFP expression using fluorescence microscopy. Before adoptive transfer, transduced bone marrow cells were harvested and washed once with IMDM without FBS. A total of 0.5–1 x 106 cells were injected i.v. via a tail vein into LAT knockout mice that were irradiated at 5 Gy.
Flow cytometric analysis
Five to six weeks after adoptive transfer, thymocytes and
splenocytes were prepared from adoptively transferred mice. After
removal of RBCs, these cells were stained with the following Abs:
biotin-conjugated anti-TCR and B220; PE-conjugated anti-CD4,
CD25, and IgM; APC-conjugated anti-CD4 and CD8; and
PE-Cy5-conjugated anti-CD44. Each sample was further analyzed on the
FACStar cytometer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The LAT-deficient Jurkat cell line, J.CaM2.5, is defective in
Ca2+ flux, Ras-MAPK activation, and AP-1- and
NF-AT-mediated transcriptional activation in response to anti-TCR
or anti-CD3 stimulation. Reconstitution of this cell line with
wild-type (WT) LAT corrects these signaling defects (7).
We chose to use this cell line to analyze LAT function in TCR signaling
pathways by reconstituting it with different LAT mutants containing Y
to F substitutions. In the cytoplasmic domain of human LAT, there are
nine conserved tyrosines. Five of these tyrosines, Y110, Y127, Y171,
Y191, and Y226, are within a Grb2-binding motif and are likely to bind
Grb2 upon phosphorylation. Y171 and Y191 also probably interact with
Gads. Y132 is crucial for interaction with PLC-1, as demonstrated in
our previous studies (12). Although previous studies had
shown that Y132, Y171, Y191, and Y226 are critical for TCR-mediated
signaling (12, 14), it has not been formally demonstrated
which of these tyrosines are phosphorylated in vivo and which of these
are required for the formation of LAT-mediated signaling complexes.
To answer these questions, we generated a LAT mutant with all tyrosines
mutated (10YF) and then made a series of additional mutants in which
each of these tyrosines was singly added back. We also made a mutant
with the four membrane-distal tyrosines (LAT-4Y), mutants with either
two or three of four membrane-distal tyrosines, and a mutant with
mutations at five membrane-distal tyrosines (Fig. 1
B). We used recombinant
ecotropic retroviruses encoding LAT mutants to transduce LAT-deficient
cells. Stable transductants were selected in the presence of G418 and
were used for further analysis without subcloning to avoid differences
among each subclone, such as levels of LAT and TCR expression.
For biochemical analysis of LAT mutants, J.CaM2.5 cells reconstituted
with different LAT mutants were stimulated with an anti-TCR Ab,
C305. Because all LAT mutants had a Myc tag, LAT was immunoprecipitated
from cell lysates with an anti-myc Ab. Tyrosine
phosphorylation of LAT was determined by Western blotting with a
combination of two anti-PY mAbs, 4G10 and PY20. As shown in Fig. 2A, different LAT mutants were
expressed at similar levels in J.CaM2.5 cells. LAT-WT was heavily
phosphorylated after C305 stimulation. Among those mutants with one
tyrosine, only the mutants with a tyrosine at 127, 171, 191, or 226
were phosphorylated upon TCR cross-linking (Fig. 2B). There
was no tyrosine phosphorylation of LAT mutants with any of the four
membrane-proximal tyrosines, Y36, Y45, Y64, and Y110 (data not shown),
and the LAT mutant with a tyrosine at 132. Lack of phosphorylation of
four membrane-proximal tyrosines was supported by analysis of another
mutant, 5YF, which had mutations at five membrane-distal tyrosines.
Although this mutant still had either four membrane-proximal tyrosines,
it was not phosphorylated upon stimulation (Fig. 2B).
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1-binding and MAPK activation (12). It
is likely that phosphorylation of LAT Y132 is dependent on prior
phosphorylation of four membrane-distal tyrosines, although we could
not rule out the possibility that phosphorylated Y132 could not be
recognized by these two anti-PY Abs. These data, together with our
previous data, indicate that five membrane-distal tyrosines, Y127,
Y132, Y171, Y191, and Y226, are phosphorylated upon T cell
activation.
Interaction of LAT with Grb2, Gads, and PLC-1 requires multiple
tyrosines
The results from Fig. 2B indicated that the LAT mutants
with a tyrosine at 127, 171, 191, or 226 could be phosphorylated. Next
we examined the association of these mutants with Grb2, Gads, and
PLC-1. After stimulation with C305, reconstituted J.CaM2.5 cells
were lysed, and detergent extracts were immunoprecipitated with
anti-Grb2, Gads, or PLC-1 Abs, respectively. Due to the presence
of nonspecific proteins comigrated with LAT on SDS-PAGE in anti-LAT
Western blotting, association of LAT with Grb2, Gads, or PLC-1 could
only be determined by anti-PY Western blotting. As shown in Fig. 2C, none of the LAT mutants with one tyrosine was capable of
interacting with Grb2, Gads, or PLC-1, despite the fact that four of
these mutants were phosphorylated. A weak band detected by anti-PY
Western blotting in anti-Gads or PLC-1 immunoprecipitation was
not LAT, because it migrated slightly faster than LAT-myc on
SDS-PAGE. These data suggest that LAT interactions with these signaling
proteins require the phosphorylation of more than one tyrosine.
However, it is also possible that phosphorylation of these single
tyrosine mutants was too weak to detect in anti-Grb2, Gads, and
PLC-1 immunoprecipitation. However, this conclusion was supported by
the results in Fig. 3, as discussed
below.
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1 bind LAT via different sets of tyrosines
To further determine which tyrosines in LAT are required for its
interaction with Grb2, Gads, and PLC-1, we made another series of
LAT mutants with combinations of two or three of the four
membrane-distal tyrosines (Fig. 1B). We concentrated on
these four tyrosines because the LAT mutant with only these four
tyrosines (LAT-4Y) was well phosphorylated, capable of interacting with
Grb2, Gads, and PLC-1 (Fig. 3, lane 12), and functional
in TCR signaling, as discussed below (Fig. 4). In addition, a LAT mutant with
mutations of these four tyrosines failed to function in thymocyte
development (14), suggesting that these four tyrosines are
critical in LAT function. These mutants were introduced into J.CaM2.5
with retroviruses and expressed at similar levels (Fig. 3). The
interactions of these mutants with Grb2, Gads, and PLC-1 were
examined by immunoprecipitation of each protein, followed by
anti-PY blotting.
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, these mutants were all phosphorylated after C305
stimulation. Of the six mutants with two tyrosines (lanes
2-7), only LATY171Y191, Y171Y226, and Y191Y226 mutants
were able to associate with Grb2 (Fig. 3, lanes 5–7).
Tyrosines 171, 191, and 226 are all within a consensus Grb2-binding
motif (YXN). Because LAT mutants with only one Grb2-binding motif were
not able to bind Grb2, it is possible that two Grb2 molecules bind two
phosphorylated tyrosines on LAT cooperatively. In contrast to Grb2,
Gads only interacted with the LAT mutant Y171Y191, two tyrosines that
are both within the YVNV context (Fig. 3, lane 5). Other
two-tyrosine mutants were well phosphorylated similarly to the Y171Y191
mutant, with an exception of the Y132Y191 mutant, but they failed to
interact with Gads. Different from Grb2 and Gads, PLC-1 could only
associate with the Y132Y191 mutant (Fig. 3, lane 3). This
mutant was least phosphorylated compared with other two-tyrosine
mutants. These results indicated that failure of those two-tyrosine
mutants to interact with Grb2, Gads, or PLC-1 was not due to weak
phosphorylation and further supported the conclusion from Fig. 2 that
Grb2, Gads, and PLC-1 interaction with LAT requires multiple
tyrosines. These data suggest that Grb2, Gads, and PLC-1 interact
with LAT via different sets of tyrosines.
Because Y127 was also phosphorylated in our single mutants, we wanted
to determine whether it was involved in the interaction of LAT with
Grb2, Gads, and PLC-1. We made mutants with a tyrosine at 127 in
combinations with one of the four membrane-distal tyrosines. None of
these mutants was able to interact with Grb2, Gads, or PLC-1 (data
not shown), suggesting that Y127 is less likely involved in binding
these signaling molecules.
Grb2 stabilizes the interaction between LAT and Gads
To ascertain whether an additional phosphorylated tyrosine could
recruit another protein, perhaps involved in stabilization of the LAT
signaling complex, we examined the interaction of Grb2, Gads, and
PLC-1 using LAT mutants with combinations of three of four
membrane-distal tyrosines (LAT-3Ya, b, c, and d; Fig. 1B).
The addition of Y132 to any of the two-tyrosine mutants did not enhance
the interaction of LAT with Grb2 and Gads (lane 8 vs
5, lane 9 vs 6, and lane 10
vs 7). This result was expected because Y132 is most likely
involved in PLC-1 binding. As discussed previously, of the six
two-tyrosine mutants, Gads only associated very weakly with
LAT-Y171Y191 (lane 5) compared with LAT-WT
(lane 1) and LAT-4Y (lane 12).
Strikingly, addition of a Grb2 binding site at 226 (LAT-3Yd)
dramatically increased the interaction between Gads and LAT
(lane 11 vs 5). This increased association
was not due to increased phosphorylation of LAT-3Yd, because this
mutant was phosphorylated to a similar extent as LAT-Y132/171/191 (3Ya)
and LAT-Y171Y191 (lane 11 vs 5 and
8). This result suggests that binding of Grb2 to
phosphorylated Y226 could stabilize a Gads-LAT interaction.
Compared with the LAT mutant with two tyrosines at 132 and 191 (Fig. 3, lane 3), the addition of a tyrosine at 171
(lane 8) or 226 (lane 10) did not
significantly increase the LAT-PLC-1 interaction. The apparent
increased association with phosphorylated LAT (lanes
8 and 10 vs 3) was most likely due to
increased tyrosine phosphorylation of these mutants (lanes
8 and 10 vs 3). It is interesting that in
the absence of Y191, the LAT mutant with tyrosines at 132, 171, and 226
(3Yb) could also interact with PLC-1 to some extent
(lane 9). This result is in agreement with our
previous finding that mutation of Y191 alone reduced PLC-1 binding
(12).
Interaction of LAT with both Grb2 and PLC-1 is essential for T
cell activation
We also examined whether these two- and three-tyrosine mutants
could correct signaling defects in LAT-deficient cells. Recombinant
retroviruses were used to deliver different mutants into J.CaM2.5 cells
transiently. None of the two-tyrosine mutants was able to restore
Ca2+ mobilization in those cells (data not
shown). Cells reconstituted with 3Yb and 3Yc were capable of fluxing
some Ca2+ in response to receptor stimulation
(Fig. 4A), although not as well as LAT-WT and LAT-4Y (Fig. 4A). Cells with 3Ya and 3Yd had weak
Ca2+ flux. We also determined whether these
mutants could restore MAPK activation. LAT-3Ya could reconstitute MAPK
activation similar to LAT-4Y, although this mutant only weakly
reconstituted Ca2+ flux in J.CaM2.5 cells.
LAT-3Yb and LAT-3Yc could partially reconstitute MAPK activation, while
LAT-3Yd failed completely (Fig. 4B). We also transiently
transfected these LAT mutants together with an NF-AT-luciferase
reporter construct to determine whether NF-AT activation in J.CaM2.5
cells could be restored. As shown in Fig. 4C, LAT-WT and
LAT-4Y restored NF-AT activation in J.CaM2.5 cells. LAT mutants with
only two tyrosines were unable to restore NF-AT activation when
introduced into J.CaM2.5 cells (Fig. 4C). LAT-3Ya, 3Yb, and
3Yc could partially restore NF-AT activation. LAT-3Yd was unable to
correct the defective NF-AT activation in J.CaM2.5 cells. LAT-3Ya, 3Yb,
and 3Yc, which are all capable of binding Grb2 and PLC-1, could
partially restore signaling defects in LAT-deficient cells, suggesting
that Grb2 and PLC-1 binding to LAT is sufficient for its function in
NF-AT activation. However, Gads binding to LAT is required for full
activation of T cells.
Rescue of thymocyte development with LAT-WT
To study LAT function in thymocyte development, we used recombinant retroviruses to introduce LAT-WT and various LAT mutants into bone marrow cells from LAT-/- mice in vitro. These transduced cells were then transferred into irradiated LAT-/- mice by injection via a tail vein. Five to six weeks after injection, thymocytes and splenocytes from these reconstituted mice were analyzed by FACS. Because these recombinant viruses carried both LAT and GFP genes, all lymphocytes derived from transduced bone marrow cells should be GFP+ and express LAT.
We first tested whether introduction of LAT-WT into bone marrow cells
from LAT-/- mice could rescue thymocyte
development. Thymocytes from LAT knockout and B6 mice were used as
controls. As shown in Fig. 5A,
GFP+ thymocytes were observed in LAT knockout
mice reconstituted with LAT-/- bone marrow
cells transduced with viruses containing LAT-WT. Thymocytes from these
reconstituted mice were further analyzed for CD4, CD8, TCR, CD44,
and CD25 expression. As shown in previous studies (13),
LAT knockout mice had no CD4 and CD8 double-positive (DP) and
single-positive (SP) thymocytes, and thymocyte development was blocked
at the CD25+CD44- subset
of the double-negative (DN) stage (Fig. 5, B and
D). In mice reconstituted with
LAT-/- bone marrow cells transduced with
viruses containing WT-LAT, the majority of GFP+
thymocytes, which should be derived from transduced bone marrow cells,
were CD4+CD8+ DP, similar
to thymocytes from B6 mice (Fig. 5B). Some
GFP+ cells were either CD4+
or CD8+ SP. These thymocytes expressed the TCR
chain on their cell surface (Fig. 5C). To determine whether
thymocyte development at the DN stage was normal in these reconstituted
mice,
GFP+CD4-CD8-
thymocytes were analyzed for CD44 and CD25 expression. As shown in Fig. 5D, introduction of LAT into LAT-/-
thymocyte could drive thymocyte development beyond the
CD25+CD44- stage. CD44 and
CD25 expression in thymocytes from reconstituted mice was very similar
to that in B6 thymocytes.
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, B220, or IgM Abs. SP T cells were absent in
LAT knockout mice, as previously described (Fig. 5F).
Approximately 22.6% of splenocytes from the LAT-WT-reconstituted
knockout mouse were GFP+ (Fig. 5E).
Among GFP+ cells, 61.1% were
CD4+ and 13.6% were CD8+.
These SP T cells expressed the TCR chain, as indicated by
anti-TCR chain staining (Fig. 5G). A small percentage
of GFP+ cells were B cells, as revealed by
staining with anti-B220 and IgM Abs (Fig. 5H),
suggesting that transduced bone marrow cells could also develop into B
cells. These data indicated that thymocyte development in
LAT-/- mice was successfully rescued by
introduction of LAT-WT into LAT-/- bone marrow
cells. We also performed the same experiment with the LAT-10YF mutant
(with mutations at all tyrosines). This mutant failed to rescue
thymocyte development of bone marrow cells from
LAT-/- mice (data not shown).
Binding of LAT to both Grb2 and PLC-1 is important during
thymocyte development
Because LAT-4Y was able to associate with Grb2, Gads, and PLC-1
and rescue Ras-MAPK activation as well as Ca2+
flux in LAT-deficient cells, we determined whether this mutant was also
able to rescue thymocyte development in LAT knockout mice. As shown in
Fig. 6, in the mice reconstituted with
LAT-4Y, a large percentage of thymocytes were
GFP+, and most of these
GFP+ cells were DP or SP (Fig. 6B).
Although it appeared that there were less DP cells in thymuses from
mice reconstituted with LAT-4Y than in those in thymuses from mice
reconstituted with LAT-WT in this experiment, this subtle difference
was also seen in different mice reconstituted with LAT-WT most likely
due to variation in viral transduction and adoptive transfer. These
GFP+ thymocytes expressed the TCR chain (Fig. 6C). We also stained splenocytes from mice reconstituted
with LAT-4Y with anti-CD4, CD8, and TCR Abs.
CD4+ and CD8+ SP cells were
present in mice reconstituted with LAT-4Y (Fig. 6E), and
these cells expressed the TCR chain. These data indicated that the
four membrane-distal tyrosines were sufficient in TCR-mediated
signaling and thymocyte development.
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, some of these
two-tyrosine mutants could bind either Grb2 or PLC-1, but not both
Grb2 and PLC-1. We were not able to detect any
GFP+ DP or SP thymocytes in mice reconstituted
with any of the two-tyrosine mutants (data not shown). We also
transduced LAT-/- bone marrow cells with LAT
mutants with combinations of three of the four membrane-distal
tyrosines to determine the minimal LAT tyrosine residues required for
thymocyte development. As shown in Fig. 6, LAT-3Ya, 3Yb, and 3Yc, which
could bind both Grb2 and PLC-1 (Fig. 2), were capable of efficiently
rescuing thymocyte development of bone marrow cells from
LAT-/- mice. Mice reconstituted with
LAT-/- bone marrow cells transduced with
LAT-3Ya, 3Yb, and 3Yc retroviruses had a high percentage of
GFP+ thymocytes (Fig. 6A). Most
GFP+ thymocytes were either DP or SP. SP
thymocytes from these mice expressed the TCR chain on their surface
(data not shown). When GFP+ splenocytes from
those mice were examined, CD4+ and
CD8+ cells were present in spleens from the mice
reconstituted with three-tyrosine mutants (Fig. 6D).
Anti-TCR staining of these splenocytes showed that they expressed
the TCR chain (Fig. 5E). In mice reconstituted with
LAT-3Yd, which only associated with Grb2 and Gads, but not PLC-1,
there were fewer GFP+ thymocytes, and most of
these thymocytes were DN. In the spleens from these mice, 5% of
splenocytes were GFP+. Although these
GFP+ cells were either CD4+
or CD8+, they expressed a very low level of TCR
on their surface. We also transduced bone marrow cells from
LAT-/- mice with the LAT mutant-10YF and
transferred these cells back into LAT-/- mice.
As shown in Fig. 5, there was no significant number of DP or SP T cells
in the thymuses and spleens from these mice. The differences in the
percentage of DP cells in thymuses reconstituted with LAT-4Y, 3Ya, 3Yb,
and 3Yc could be due to either variation in viral transduction and
efficiency of adoptive transfer or differences in signal strength of
various LAT mutants. Future studies with transgenic mice or knockin
mice will be done to examine these possibilities.
Results from biochemical analysis and thymocyte reconstitution were
summarized in Fig. 7. Three LAT mutants
that were competent to efficiently rescue thymocyte development were
all capable of binding both Grb2 and PLC-1, suggesting that LAT
binding to both Grb2 and PLC-1, not Grb2 or PLC-1 alone, is
important for LAT function. Of these three mutants, LAT-3Yb and 3Yc
failed to associate with Gads, but were able to rescue thymocyte
development, indicating that Gads binding to LAT is dispensable for
thymocyte development. This is in agreement with the data from
Gads-deficient mice (16). In summary, our data indicated
that LAT interaction with both Grb2 and PLC-1 is important in
thymocyte development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1
possesses two SH2 domains. These three proteins most likely interact
with phosphorylated LAT directly via their SH2 domains. Previous
studies showed that mutation of the four membrane-distal tyrosines,
Y132, Y171, Y191, and Y226, in human LAT affects LAT binding to these
molecules (12). In this study, we constructed a series of
LAT mutants with Tyr to Phe substitutions. We showed that in addition
to Y132, Y171, Y191, and Y226, Y127 was also phosphorylated upon
stimulation via the TCR. Interestingly, none of the mutants with one
phosphorylated tyrosine associated with Grb2, Gads, and PLC-1.
Instead, binding of LAT to these signaling proteins required
phosphorylation on multiple tyrosines of LAT. Grb2, Gads, and PLC-1
interacted with LAT preferentially via different sets of tyrosine
residues. We also examined the function of these LAT mutants in the TCR
signaling pathway by using LAT-deficient cells, and in thymocyte
development by reconstituting bone marrow cells from
LAT-/- mice. Our results indicated that LAT
function in T cell activation and thymocyte development requires a
minimum of three membrane-distal tyrosines.
Human LAT has 10 tyrosine residues in its cytoplasmic domain, 9 of
which are conserved between murine and human LAT. Our results obtained
using LAT mutants with one tyrosine showed that Y127, Y171, Y191, and
Y226 were phosphorylated upon T cell activation. Surprisingly, the
mutant LAT with one tyrosine at 132 was not phosphorylated. Y132 was
demonstrated to be required for PLC-1 binding and activation of
Ras-MAPK (12), implying that this residue must be
phosphorylated after TCR engagement. Based on published data and our
data in this study, it is likely that phosphorylation of Y132 is
dependent on the phosphorylation of other tyrosines. Phosphorylation of
those tyrosines could change the conformation of LAT so that Y132 could
be further phosphorylated. It is also possible that other
phosphorylated tyrosines might recruit tyrosine kinases, such as
IL-2-inducible T cell kinase to LAT to phosphorylate Y132. Because the
LAT mutant with tyrosines at 132 and 191 could associate with PLC-1,
Y191 might be one of the tyrosines required for phosphorylation of
Y132. We could not exclude the possibility that any of the four
membrane-proximal tyrosines could be phosphorylated in a way similar to
Y132. Y110 might be phosphorylated during T cell activation, as
suggested by recent data (17). In addition, studies
described in this work mainly focused on TCR-mediated tyrosine
phosphorylation of LAT. It is possible that costimulation via CD28
might induce phosphorylation of these membrane-proximal tyrosines.
Because LAT-deficient cells are also deficient in CD28 and other
surface molecules (7), future studies need to be done to
address this possibility after reconstitution of LAT-deficiency cells
with CD28.
Our data showed that Y127 was phosphorylated, possibly more than any
other tyrosine in LAT. This tyrosine was also found to be
phosphorylated in vitro by ZAP-70 (18). Although this
tyrosine is within a Grb2-binding motif
(Y127HNP), this tyrosine after phosphorylation
was not involved in binding of Grb2, Gads, or PLC-1. LAT mutants
with Y127 in combination with any one of these tyrosines, Y132, Y171,
Y191, or Y226, failed to associate with Grb2, Gads, and PLC-1 (data
not shown). When we examined the sequence between Y127 and Y132 in
human LAT (Y127HNPGY132),
we found that it contains a binding motif
(NPGY132) for proteins containing a PTB domain.
However, this motif is not present in murine LAT. It is not clear
whether this motif in LAT binds any PTB-containing proteins. It is
possible that phosphorylation of Y127 might change the local structure
of LAT and affect the binding of PLC-1 to Y132. It is also possible
that this tyrosine after phosphorylation is involved in association
with other signaling proteins.
One surprising finding from our studies is that the LAT mutants with one single tyrosine at 127, 171, 191, or 226 failed to associate with Grb2, although these tyrosines are all in the YXN motif recognized by Grb2 and were phosphorylated upon T cell activation. However, the LAT mutants with any two of these tyrosines (Y171, Y191, or Y226) could associate with Grb2. This result suggests that two Grb2 molecules might bind to LAT cooperatively to form a stable complex. In the solved three-dimensional structure of Grb2, two Grb2 molecules in one asymmetric unit form a large contact area over 4100 Å2 (19), suggesting that Grb2 could potentially form a dimer on LAT.
Our studies also revealed that PLC-1 preferentially binds to LAT
with tyrosines at Y132 and Y191. The LAT mutant with only these two
tyrosines could associate with PLC-1, although weakly compared with
LAT with four membrane-distal tyrosines. This mutant failed to
associate with Grb2 or Gads, suggesting that PLC-1 could bind LAT
directly as proposed in other studies (20, 21). Because
PLC-1 has two SH2 domains, it is possible that each SH2 domain binds
one phosphorylated tyrosine on LAT. The N-terminal SH2 domain of
PLC-1 might bind phosphorylated Y132, while the C-terminal SH2
domain binds Y191. Y191 might not be directly involved in interaction
with PLC-1. Phosphorylation of Y191 is simply required for
phosphorylation of Y132. Phosphorylation of Y191 might change LAT
conformation or recruit other tyrosine kinases so Y132 could be
phosphorylated.
The LAT mutant with four membrane-distal tyrosines could associate with
Grb2, Gads, and PLC- similar to LAT-WT. It could reconstitute
TCR-mediated Ras-MAPK and Ca2+ flux in
LAT-deficient cells, although not as well as LAT-WT. This result
suggests that these four tyrosines play a major role in LAT-mediated
signaling events, while other tyrosines, such as Y110 and Y127, might
also contribute to LAT function to some extent. The LAT mutants with
one or two of these four membrane-distal tyrosines failed to
reconstitute Ras-MAPK activation, Ca2+ flux, and
NF-AT activation. None of these two-tyrosine mutants could associate
with both PLC-1 and Grb2. However, three three-tyrosine LAT mutants,
LAT-3Ya, 3Yb, and 3Yc, were capable of correcting these signaling
defects to some extent. LAT-3Yb and -3Yc, which did not associate with
Gads, could partially reconstitute Ras-MAPK, Ca2+
flux, and NF-AT activation in LAT-deficient cells. This result is
different from the data from Gads knockout mice. Peripheral T cells
from Gads knockout mice are defective in TCR-induced
Ca2+ mobilization (16). It is
possible that Gads might play some role in TCR-mediated signaling
independent of its interaction with LAT.
We used retroviruses to deliver various LAT mutants into
LAT-/- bone marrow cells, followed by adoptive
transfer to study the importance of LAT binding to Grb2, Gads, and
PLC-1 in thymocyte development. Compared with the procedure used to
generate transgenic mice and knockin mice, this method is expedient and
simple. The disadvantage of this retroviral system is the variations in
retroviral transduction and adoptive transduction. Due to these
variations, it is difficult to address whether LAT mutants have
quantitative differences in TCR signaling during thymocyte development.
Our experiments showing successful reconstitution of
LAT-/- bone marrow cells with WT-LAT
demonstrated that this system could be used to study LAT function in
thymocyte development. Our results showed that the LAT mutant with four
membrane-distal tyrosines was capable of binding to Grb2, Gads, and
PLC-1 and correcting the defective TCR signaling events, such as the
Ras-MAPK activation, Ca2+ mobilization, and NF-AT
activation in LAT-deficient cells. This mutant was also sufficient for
rescuing thymocyte development. This is in agreement with results from
a study of knockin mice with mutation of these four tyrosines. Mutation
of these four tyrosines abolishes LAT function in thymocyte development
(14). Among these four tyrosines, Y132 in LAT has been
shown to be responsible for binding of PLC-1. Y171, Y191, and 226
are the binding sites for Grb2. Y171 and Y191 are also the binding
sites for Gads (12, 17). When we examined the function of
LAT mutants with combinations of three of these four membrane-distal
tyrosines, we found that only LAT-3Ya (Y132/171/191), LAT-3Yb
(Y132/171/226), and LAT-3Yc (Y132/191/226) efficiently rescued
thymocyte development. Of these three LAT mutants, LAT-Y132/171/226 and
Y132/191/226 could not associate with Gads, suggesting that LAT binding
to Gads is not required for thymocyte development. This conclusion is
in agreement with the data from Gads knockout mice. In Gads knockout
mice, there is a severe block in proliferation of
CD4-CD8- thymocytes, but
those thymocytes can still differentiate into mature T cells
(16). Because recruitment of SLP-76 to the membrane might
be important for SLP-76 function (22), it is possible that
SLP-76 might be recruited to LAT complexes via adaptor proteins other
than Gads, such as Grb2. SLP-76 was originally identified as a protein
that interacts with Grb2. It is possible that Grb2 recruits SLP-76 to
LAT in the absence of Gads or the Gads-LAT interaction.
It was unexpected that LAT-3Yd with tyrosines at 171, 191, and 226
could rescue thymocyte development, although it did so less
efficiently. This mutant could only bind to Grb2 and Gads, not
PLC-1. This mutant failed to reconstitute MAPK and NF-AT activation
like the LAT-Y132F mutant. T cells from mice reconstituted with this
mutant have no or low TCR expression on their cell surface similar to
those from mice reconstituted with the LAT Y132F mutant (data not
shown) and mice with a knockin mutation at this tyrosine (Y136F in
mouse LAT). Interestingly, these mutant mice developed a
lymphoproliferative disorder and autoimmune disorder (23, 24). Our results showed that LAT with four membrane-distal
tyrosines, Y132, Y171, Y191, and Y226, could function in thymocyte
development like LAT-WT, while the mutant with three tyrosines, Y171,
Y191, and Y226, functioned similarly to the Y132F mutant, suggesting
that Y171, Y191, and Y226 are the ones that cause lymphoproliferation
in mice by interacting with Grb2, Gads, or other unidentified proteins.
How these tyrosines cause lymphoproliferative disorder and further
autoimmune disease in the absence of PLC-1 interaction remains to be
determined in the future.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Weiguo Zhang, Department of Immunology, Jones Building/Room 112, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address: zhang033{at}mc.duke.edu
3 Abbreviations used in this paper: LAT, linker for activation of T cells; DN, double negative; DP, double positive; Gads, Grb2-related adapter downstream of Shc; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PY, phosphotyrosine; SH2, Src homology 2; SP, single positive; WT, wild type.
Received for publication August 22, 2002. Accepted for publication October 23, 2002.
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  M. Hundt, Y. Harada, L. De Giorgio, N. Tanimura, W. Zhang, and A. Altman Palmitoylation-Dependent Plasma Membrane Transport but Lipid Raft-Independent Signaling by Linker for Activation of T Cells J. Immunol., August 1, 2009; 183(3): 1685 - 1694. [Abstract] [Full Text] [PDF]
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O. Malbec, M. Malissen, I. Isnardi, R. Lesourne, A.-M. Mura, W. H. Fridman, B. Malissen, and M. Daeron Linker for Activation of T Cells Integrates Positive and Negative Signaling in Mast Cells J. Immunol., October 15, 2004; 173(8): 5086 - 5094. [Abstract] [Full Text] [PDF]
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