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* Department of Pathology and
Graduate Program in Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and
Department of Pathology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion. We have discovered
that 2B4 from transfected NK and T cell lines, as well as from primary
murine cells, coimmunoprecipitates with the phosphoprotein linker for
the activation of T cells (LAT), which is essential for TCR-mediated
signaling. This association is independent of both 2B4 phosphorylation
and the cytoplasmic tail of 2B4. We have found that, along with LAT,
2B4 is constitutively located in glycolipid-enriched microdomains of
the plasma membrane. In fact, 2B4 appears to associate with LAT only
when it localizes to glycolipid-enriched microdomains. This
localization of 2B4 occurs due to a CxC cysteine motif found in the
transmembrane region, as determined by mutagenesis studies.
2B4-mediated cytotoxicity is defective in the absence of LAT,
indicating that LAT is a required intermediate for 2B4 signal
transduction. However, we have also shown that LAT association alone is
not sufficient for maximal 2B4 activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2B4 is expressed on the surface of all murine NK cells, all dendritic
epidermal 
T cells, and the subset of 
T cells that exhibit
non-MHC-restricted killing after culture with IL-2 (4, 7).
2B4 belongs to the CD2 subfamily of the Ig superfamily (8)
and is a receptor for CD48 (9). The murine 2B4 gene
encodes two distinct polypeptides, as a result of alternative splicing,
that differ solely in their cytoplasmic regions (8).
However, only one isoform has been described for human 2B4
(10). The longer murine isoform, 2B4L, contains seven
potential phosphorylation sites, five of which are unique to 2B4L
(8). Several of these sites resemble immunoreceptor
tyrosine-based switch motifs found in the cytoplasmic region of a
fellow CD2 subfamily member, signaling lymphocytic activation molecule
(11). This motif has been named a "switch" motif
because it appears to control binding of a Src homology 2
(SH2)3
domain-containing protein tyrosine phosphatase vs an SH2
domain-containing inositol phosphatase through association with
signaling lymphocytic activation molecule-associated protein (SAP). The
shorter isoform, 2B4S, contains only one of these immunoreceptor
tyrosine-based switch motif sites, suggesting a possible difference in
signaling function (8). The phosphorylated 2B4L isoform
has been shown to associate with SH2 domain-containing protein tyrosine
phosphatase 2, whereas both isoforms associate with the defective
X-linked lymphoproliferative disease gene product, SAP (S. Stepp and J.
Schatzle, unpublished observations). Studies in transfected rat NK cell
lines suggest distinct functions for the two isoforms
(12). 2B4L appears to act as an inhibitory receptor, while
2B4S provides stimulatory functions (12). However, the
molecular mechanisms of signal transduction by 2B4 have yet to be
determined.
2B4 shows homology to CD48 and CD2 (7, 8), both of which
are signaling molecules resident in specialized subdomains of the
plasma membrane that contain a unique lipid composition of
sphingolipids, cholesterol, and GPI-anchored proteins (13, 14). These glycolipid-enriched microdomains (GEM) are nonionic
detergent insoluble, which allows them to be separated from all other
cellular components via density centrifugation. Studies in T cells have
led to the discovery that many signaling molecules are present in these
GEM fractions (15), suggesting that GEMs may concentrate
signaling components, thereby inducing more efficient pathway
activation. Linker for the activation of T cells (LAT) is a 36- to
38-kDa phosphoprotein resident within the GEM fractions of T cells
(16, 17). Palmitoylation of LAT appears to target this
transmembrane protein to GEMs (18). LAT is a critical
signaling molecule that couples proximal TCR-mediated activation events
with downstream activation of phospholipase C-1, Vav, SLP-76,
and other signaling molecules (16, 17). Less is known
about the signaling pathways of NK cell activation, but there is some
evidence to suggest that they may bear resemblance to T cell pathways.
In particular, LAT has not only recently been found to be essential for
T cell activation (17) but is also implicated in the FcR
activation pathway in NK cells (19). Recent studies
demonstrate the importance of GEMs in NK cell activation (20, 21). Because 2B4 is an activating receptor found in NK and T
cells, we examined whether it localizes in GEMs and associates with GEM
resident signaling molecules.
Studies using human NK cells have demonstrated an association between 2B4 and LAT (6, 22). Our studies in mice confirm this, but in addition we show that LAT associates with both the long and short isoforms of 2B4 in two transfected cell lines: RNK-16 (NK cells) and Jurkat (T cells). Our data also reveal that neither phosphorylation nor the cytoplasmic tail of 2B4 is necessary for its association with LAT. Furthermore, we show that 2B4 is resident in GEM fractions, where it associates with LAT. In fact, mutation of a CxC cysteine motif in the 2B4 transmembrane region impairs not only 2B4 localization to GEM fractions but also its association with LAT. Redirected lysis assays demonstrate that the 2B4 signaling pathway is defective in the absence of LAT, suggesting an important role for LAT in 2B4-mediated cytotoxicity. Even though LAT is required for 2B4 function, we have also shown that its association with 2B4 is not sufficient for maximal 2B4 activation to occur.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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293 T cells were grown in DMEM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All other cells and cell lines were grown in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. A total of 1000 U/ml IL-2 or 1 ng/ml IL-12 and 100 ng/ml IL-18 were provided for the lymphokine-activated killer (LAK) cultures as described (23). Transfected cell lines were selected for growth in complete RPMI 1640 or DMEM supplemented with 1 mg/ml G418.
Mice
C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred and maintained in a conventional colony at the University of Texas Southwestern Medical Center (Dallas, TX). C57BL/6 SCID and 129 mice were obtained from The Jackson Laboratory and LAT-/- mice were obtained from P. Love and L. Samelson (National Institutes of Health, Bethesda, MD). Both were maintained in a specific pathogen-free colony at the University of Texas Southwestern Medical Center. Mice used were 2–4 mo of age.
Antibodies
Anti-phosphotyrosine mAb and anti-Lck mAb were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LAT mAb was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-h2B4 mAb (C1.7) was obtained from Immunotech (Westbrook, ME). Purified, FITC-conjugated, and PE-conjugated anti-m2B4 mAb, anti-RT1A, and anti-LFA-1 mAb were obtained from BD PharMingen (San Diego, CA). All HRP-conjugated secondary Abs were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Polyclonal rabbit antiserum was prepared to an Ig fusion protein of 2B4 (S. Stepp, W. Lai, and J. Schatzle, unpublished results).
Flow cytometry
Surface expression of 2B4 on Jurkat cells, 293T cells, and LAKs was examined by staining with FITC-conjugated or PE-conjugated anti-2B4 mAb. For staining, cells were washed and resuspended in PBS supplemented with 2% FCS. Cells were incubated with mAb for 15 min at 4°C. A total of 10,000 events were analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA).
Immunoprecipitation and Western blot analysis
Surface proteins were biotinylated using the EZ Linker Sulfo-NHS-LC Biotinylation reagent system from Pierce (Rockford, IL) according to the manufacturer’s instructions. Where indicated, cells were treated with 0.03% hydrogen peroxide and 100 µm orthovanadate (pervanadate) for 10 min at 37°C as previously described (12). Approximately 1 x 108 cells were harvested for each condition and washed in PBS; cell pellets were then lysed at 4°C in lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM MgCl2, and 10% glycerol) containing 1% Brij-35. Immunoprecipitation and Western blot analysis were performed as previously described (12). Blots were developed with the SuperSignal chemiluminescence kit from Pierce.
Vector constructs, mutagenesis, transfections, and infections
2B4S or 2B4TMC>A cDNA was subcloned into the MigR1 retroviral
vector as described (24). 293T cells were transfected
using Effectene kit from Qiagen (Valencia, CA) following the
manufacturer’s protocol. LAKs were infected with MigR1:2B4S or
MigR1:2B4TMC>A retroviruses by spinfection on d1 as described
(25), with the following change: LAKs were incubated at
32°C for 24 h immediately after spinfection. cDNAs corresponding
to each of the 2B4 isoforms or various mutant forms of 2B4 were
subcloned into the pEMCVEN vector as previously described
(12). Mutagenesis was performed using the Altered Sites
Mutagenesis kit from Promega (Madison, WI). All constructs were
sequenced for confirmation before transfection. Transfections and
establishment of stable cell lines were performed as previously
described (12).
Sucrose density gradient centrifugation and GEM isolation
For each condition, 
1 x 108 cells
were harvested, washed in PBS, and lysed in lysis buffer with 1%
Brij-35 as described above. Lysates were adjusted to 4 ml of 40%
sucrose using a 60% sucrose stock dissolved in lysis buffer without
glycerol. The cell lysate was then placed on the bottom of 14 x
89 mm ultraclear centrifuge tubes from Beckman Coulter (Fullerton, CA).
Sequential 2-ml fractions (30, 20, 10, and 5% sucrose in lysis buffer)
were overlaid on top of the cell lysate. Samples were centrifuged at
39,000 rpm for 18 h at 4°C in a SW41 rotor. After
centrifugation, 1-ml fractions were collected from the top of the tube.
Aliquots of each fraction were then subjected to immunoblotting or
immunoprecipitation followed by immunoblotting as described above.
Cytotoxicity assays
Specific lysis of targets was determined by using a standard 4-h 51Cr release assay as previously described (23). Redirected lysis assays using P815 FcR+ targets were performed as previously described (12).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A, pervanadate-treated
RNK-16-2B4L transfectants were lysed and immunoprecipitated with
anti-m2B4. Immunoprecipitates were resolved by SDS-PAGE and
immunoblotted with anti-phosphotyrosine Abs. This process revealed a
66-kDa phosphoprotein corresponding to the molecular mass of 2B4L. In
addition, a 38-kDa phosphoprotein coimmunoprecipitated with 2B4L. Fig. 1B demonstrates that the 38-kDa phosphoprotein
immunoprecipitated by anti-m2B4 is indeed LAT. Immunoprecipitation
with anti-RT1A, a mAb against a rat class I molecule (Fig. 1B), and isotype control Ab (data not shown) failed to
coimmunoprecipitate the LAT protein, thereby serving as negative
controls.
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A
reveals that LAT associates with both the short and long 2B4 isoforms
(Fig. 2A, lanes 2 and 3,
respectively). Because 2B4 is also expressed in a subset of T cells, we
wondered whether this association would exist in a 2B4-transfected
Jurkat T cell line. Blotting of whole cell Jurkat lysates with
anti-LAT in Fig. 2B, lane 1, demonstrates the
migration of the LAT protein. No LAT protein is detected from m2B4
immunoprecipitates of untransfected Jurkat cells (Fig. 2B,
lane 2). However, the anti-LAT blots of m2B4
immunoprecipitates from Jurkat transfectants in Fig. 2B,
lanes 3 and 4, show that LAT associates with both
2B4S and 2B4L, respectively. Therefore, the LAT-2B4 association occurs
in both T and NK cell lines expressing exogenous murine 2B4. To
demonstrate the association between LAT and endogenous 2B4 in primary
murine cells, murine LAKs were immunoprecipitated with either
anti-m2B4 or anti-LFA-1 mAb followed by blotting with
anti-LAT. As shown in Fig. 2C, LAT coimmunoprecipitates
with 2B4, but not LFA-1, another surface protein found on NK cells,
illustrating the specific association between LAT and 2B4 from murine
NK cells.
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), we tested whether 2B4 phosphorylation was necessary
for this association. A mutant 2B4L construct (2B4LMY) was generated by
site-directed mutagenesis in which all cytoplasmic tyrosines were
mutated to phenylalanines. This mutant form of 2B4 exhibited no
phosphorylation in the presence of pervanadate (data not shown). RNK-16
transfectants expressing this 2B4LMY mutant, as well as those
expressing wild-type 2B4L, were immunoprecipitated with anti-m2B4
followed by immunoblotting with anti-LAT. These mutations do not
prevent the association of LAT with 2B4 (Fig. 3A, compare lanes 1
and 2). RNK-16 transfectants expressing a mutant 2B4S
protein (2B4SMY) in which all cytoplasmic tyrosines were mutated to
phenylalanines were also used in this assay. Again, 2B4 associates with
LAT (data not shown), confirming that 2B4 phosphorylation is not
required for this interaction to occur. Although LAT associates with
both isoforms of 2B4 independent of 2B4 phosphorylation status, the
nature of this association is unknown. The 2B4 isoforms have shared
cytoplasmic domains as well as domains unique to each isoform. To
examine which regions are required for LAT association, RNK-16 cells
were transfected with a previously described mutant 2B4 construct
(2B4D2) truncated at aa 301, leaving only regions shared by the two
isoforms (12). LAT was still found to associate with this
mutant 2B4 protein, indicating that this association does not require
any domains unique to either 2B4 isoform (data not shown). To determine
whether the shared domains of the 2B4 cytoplasmic tail were necessary
for LAT association, RNK-16 cells were transfected with 2B4D1, a mutant
form of 2B4 with the cytoplasmic tail removed by a truncation at aa 247
(12). Anti-LAT blots of m2B4 immunoprecipitates from
RNK-16 cells expressing 2B4S or 2B4D1 revealed the presence of LAT
(Fig. 3B, compare lanes 1 and 2),
indicating that the cytoplasmic tail of 2B4 is not necessary for LAT
association. It should be noted in Fig. 3B, lane
2, that the mutant 2B4 seems to elicit a stronger association with
LAT than the wild-type 2B4. However, this apparent increased
association is probably due to the higher expression of 2B4 in the
RNK-16-2B4D1 transfectants, as determined by FACS analysis (data not
shown). To test the function of 2B4 proteins with mutated cytoplasmic
domains, a redirected lysis assay was performed using 2B4SMY, 2B4D1,
and wild-type 2B4S RNK-16 transfectants as effectors. When murine 2B4
mAb was added to P815 FcR+ targets, all three
effectors (expressing similar levels of surface 2B4, as determined by
FACS analysis) demonstrate an increase in cytotoxicity, although the
two transfectants expressing mutant forms of 2B4 exhibit a decrease
relative to the wild-type 2B4 transfectants (Fig. 3C).
Therefore, the tyrosine motifs and the cytoplasmic tail of 2B4 are
required for maximal activation. Because these mutant forms of 2B4
still associate with LAT, this association alone is not sufficient for
maximum function of 2B4.
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A). To
determine whether the LAT-2B4 association occurs within GEMs, fractions
from the same lysates were subjected to immunoprecipitation with
anti-m2B4 followed by blotting with anti-LAT. Fig. 4B shows that LAT and 2B4 coimmunoprecipitate in GEM
fractions 7 and 8, suggesting that the LAT-2B4 interaction occurs
within GEMs. Similar results were observed for Jurkat transfectants as
well as primary NK cells (see Fig. 7 and data not shown).
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A shows
that GEMs are located in fractions 7–9. Immunoblotting with
anti-h2B4 indicates that 2B4 is also constitutively located within
GEM fractions from YT cells (Fig. 5B). Furthermore,
anti-LAT blots of h2B4 immunoprecipitates from YT whole cell
lysates demonstrate 2B4 and LAT association (data not shown). Thus, it
appears that 2B4 and LAT colocalize to GEMs within a variety of cell
types, including human NK and T cell lines and murine primary
cells.
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, where it mediates
association with LAT (28). However, using radiolabeled
palmitate, we were unable to demonstrate palmitoylation of 2B4 (data
not shown). To determine the importance of the CxC cysteine motif in
GEM localization, Jurkat cells were transfected with either wild-type
2B4 (J.2B4S) or with a 2B4 construct in which the cysteines of the CxC
motif were mutated to alanines (J.2B4TMC>A). Transfectants expressing
similar expression levels of 2B4 were selected for further study (Fig. 6, C and D). To
determine whether 2B4 is resident in GEMs of Jurkat cells, and to
compare the localization of wild-type and mutated 2B4 as well, we used
2B4 mAb to immunoprecipitate biotinylated surface proteins from density
gradient fractions of J.2B4S and J.2B4TMC>A lysates. These
immunoprecipitates were then blotted with streptavidin-HRP; Fig. 6A shows the location of wild-type 2B4 to exist
predominantly within GEM fractions 7–9. As an additional confirmation
that these buoyant fractions contain GEMs, we immunoblotted the
fractions from the same J.2B4S lysates with Lck, a protein known to be
disproportionately represented in GEMs (29). Lck was
predominantly located in fractions 7–10 (Fig. 6B),
indicating that the fractions containing the majority of wild-type 2B4
are indeed GEM fractions. Fig. 6A also suggests that the
majority of the 2B4 protein normally found in the GEM fractions is
shifted to the soluble fractions when the CxC motif is mutated. To more
directly compare the relative localization to GEMs of the wild-type and
mutant forms of 2B4, we chose to immunoblot fractionated lysates with
polyclonal m2B4 Ab rather than first immunoprecipitate with
anti-m2B4 mAb. This allows a direct comparison of the GEM
localization of the two proteins to be made. Despite having similar
expression levels (Fig. 6, C and D), the presence
of mutant 2B4 is much reduced in GEM fractions compared with wild-type
2B4 (Fig. 6E), suggesting that the mutation in J.2B4TMC>A
shifts the majority of 2B4 away from GEM fractions. These results
demonstrate that the CxC motif found in the transmembrane region of 2B4
is important for GEM localization.
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).
Furthermore, this association is not seen in any of the
detergent-soluble fractions, indicating that 2B4 localization to GEM
fractions is important to the 2B4-LAT interaction.
In an attempt to determine the functional significance of the
localization of 2B4 to GEMs, we planned to use LAKs retrovirally
transduced with either 2B4 or 2B4TMC>A constructs as effectors in a
redirected lysis assay. To obtain the viral supernatants needed to
perform the transduction, we first transfected 293T cells with
bicistronic retroviral constructs containing both 2B4 and green
fluorescent protein (GFP) open reading frames linked by an internal
ribosome entry site. This allows us to assess transduction efficiency
and sort transduced cells by flow cytometry. As seen in Fig. 8A, both 2B4 and 2B4TMC>A
proteins are expressed on the surface of transfected 293T cells (2B4
PE+GFP+). Splenocytes from
129 mice, which lack the allele of 2B4 recognized by murine 2B4 mAb,
were then used to generate the LAK cultures. However, extracellular
staining of these LAKs after retroviral transduction demonstrates that
only the wild-type 2B4 protein reaches the cell surface (Fig. 8B). Additionally, we were unable to obtain RNK-16
transfectants exhibiting surface expression of 2B4TMC>A (data not
shown). These data suggest that the CxC mutation found in 2B4TMC>A
negatively affects the capacity of 2B4 protein to reach the cell
surface of LAKs. Although it is unclear why 2B4TMC>A can be expressed
on the surface of 293T and Jurkat cells (Figs. 8A and
6C), this may be due to an abnormality in membrane
localization and composition, as is seen in some tumor cell lines
(30).
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B, Abs to m2B4 stimulate
lysis of P815 targets by C57BL/6 SCID LAKs but not by
LAT-/- LAKs, demonstrating that LAT is required
for 2B4-mediated NK cell cytotoxicity. It should be noted that there
appears to be a reduction in baseline lysis by
LAT-/- LAKs. However, as seen in Fig. 9A, LAT-/- LAK had fewer
2B4+ cells than SCID LAKs (27 vs 58%), which
probably accounts for the apparent reduction in baseline lysis by the
LAT-/- LAKs. Furthermore, Fig. 9A
demonstrates a slight reduction in 2B4 expression levels in
LAT-/- LAKs. The significance of this
reduction, which is also seen with another cell surface receptor, NK1.1
(data not shown), is unclear but would not account for the lack of
redirected lysis via 2B4 in these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies and others (6, 22) have determined that 2B4 associates with LAT, an important intermediate in both T cell and NK cell activation (17, 20). Furthermore, we have seen this association in both NK and T cell lines, as well as in primary murine NK cells. Because LAT is localized to GEMs (18), 2B4 membrane localization was also examined. GEMs are enriched in sphingolipids, cholesterol, and GPI-anchored proteins (35). All of these components have higher melting temperatures than glycerolipids due to the saturation of their fatty acyl chains. Lipids with saturated acyl chains can tightly pack together in a more ordered state due to favorable interactions among the acyl chains. This property creates clustering of sphingolipids, cholesterol, and GPI-anchored or fatty-acylated proteins in specialized areas of the membrane (GEMs) that are relatively detergent-insoluble. It is known that many signaling molecules important to both NK and T cell activation accumulate in GEMs (17, 19). Some of these molecules are GPI-anchored, some are palmitoylated, and some simply associate with other resident proteins. Clustering of signaling pathway intermediates results in amplification of signal transduction cascades, allowing a cell to more efficiently respond to an activation event. Therefore, GEM structures appear to be important to cellular activation (15, 18, 36). Unlike many signaling molecules, which are recruited to GEMs during activation, we have discovered that 2B4 is resident within GEMs. Residence within such an environment may allow 2B4 to respond more quickly to a ligand-induced interaction than a recruited receptor could respond. Through its localization to GEMs, 2B4 also has an increased likelihood of impacting the pathways of other GEM signaling receptors, either resident or recruited. In fact, recent studies in human NK cells suggest a coreceptor role for 2B4, in which 2B4 communicates with the NKp46 receptor (37). Therefore, GEM localization of 2B4 may impact the receptor pathways with which 2B4 can communicate.
We have determined that the 2B4-LAT interaction occurs within GEMs, which is not surprising given that both molecules reside there. Targeting of LAT to GEMs is dependent upon palmitoylation of a CxC motif (18). We discovered that a similar CxC motif found in the transmembrane region of 2B4 is also involved in targeting 2B4 to GEMs. Mutagenesis studies have shown that this CxC motif is also necessary to retain LAT association. However, unlike with LAT, we were unable to demonstrate palmitoylation of 2B4 (data not shown). Therefore, the mechanism by which this motif functions in 2B4 localization remains unclear.
Although Jurkat cells transfected with 2B4TMC>A clearly demonstrate a reduced capacity to reside in GEMs and associate with LAT, a small percentage of 2B4 protein shows residual association with GEMs. It is possible that 2B4 has other motifs or domains that aid in the localization to GEMs. Therefore, mutation of the CxC motif may only shift the balance away from GEM localization rather than abrogate it. Any remaining 2B4 present in GEMs may be free to associate with LAT.
Although we were unable to test the functional significance of the CxC mutation because of the inability of 2B4TMC>A to reach the cell surface in NK cells, we can conclude that LAT association alone is not sufficient for 2B4 function. Both mutation of cytoplasmic tyrosines (2B4SMY) and truncation of the cytoplasmic tail (2B4D1) lead to significant reductions in 2B4-mediated cytotoxicity. This is probably due to the fact that both mutations prevent the association of 2B4 with SAP (J. Schatzle, unpublished results), which has been shown to be essential to 2B4 function in human NK cells (38). However, the residual cytotoxicity seen with these mutations may be due to the remaining association with LAT.
Recent evidence, in fact, suggests that LAT is involved in 2B4
signaling (6, 22). When the 2B4 pathway in human NK cells
is stimulated, LAT is phosphorylated and LAT substrates phospholipase
C-1 and Grb2 are recruited to the 2B4 signaling complex.
Therefore, we examined the function of 2B4 in the absence of LAT using
NK cells from LAT-/- mice. We found that
redirected lysis triggered by cross-linking 2B4 is inhibited when LAT
is absent, although natural cytotoxicity is still intact. We conclude
that, under these circumstances, LAT is a required intermediate in the
2B4 signaling pathway. Future studies will focus on the establishment
of 2B4 function in T cells and the further characterization of the
molecules involved in the 2B4 signaling pathway in both NK and T
cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John D. Schatzle, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9072. E-mail address: schatzle.john{at}pathology.swmed.edu
3 Abbreviations used in this paper: SH2, Src homology 2; LAT, linker for the activation of T cells; GEM, glycolipid-enriched microdomain; SAP, signaling lymphocytic activation molecule-associated protein; GFP, green fluorescent protein; LAK, lymphokine-activated killer.
Received for publication May 29, 2001. Accepted for publication April 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release in human NK cells involves distinct pathways. J. Immunol. 167:6210.1 and the Ras pathway. Immunity 9:617.[Medline]
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F) were lysed and subjected to
immunoprecipitation with anti-m2B4 mAb. Immunoprecipitates were
resolved by SDS-PAGE under nonreducing conditions and immunoblotted
with anti-LAT mAb. Lane 1, RNK-16-2B4L
transfectants. Lane 2, RNK-16-2B4LMY transfectants.
B, RNK-16 cells transfected with 2B4S or 2B4D1 (mutant
with no cytoplasmic tail) were treated as described in
A. Lane 1, RNK-16-2B4S transfectants.
Lane 2, RNK-16-2B4D1 transfectants. C,
RNK-16 transfectants, as indicated, were used as effectors in a
redirected lysis assay against FcR+ P815 targets at an E:T
ratio of 100:1. As shown on the right, 10 µg/ml
anti-m2B4 mAb was added to effectors before addition of targets,
where indicated.
