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*
School of Pharmacy, University of Maryland, Baltimore, MD 21201; and
Boehringer Ingelheim Research and Development Center, Ridgefield, CT 06877
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, hLnk associated with tyrosine-phosphorylated TCR -chain
through its Src homology 2 domain. The overexpression of Lnk in Jurkat
cells led to an inhibition of anti-CD3 mediated NF-AT-Luc
activation. Our study reveals a potentially new mechanism of T
cell-negative regulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimer, which recognizes the Ag-MHC complex, and
six invariant chains (CD3
, CD3
, two copies of CD3
, and a
homodimer) that play an important role in mediating the TCR signal
transduction (4). The cytoplasmic domains of CD3 and
contain the immunoreceptor tyrosine-based activation motif
(ITAM)3
(5), consisting of two tandem YXXL/I motifs separated by
7–8 aa. The src family protein tyrosine kinases (PTK)
p56lck and p59fyn,
activated by TCR ligation, can phosphorylate tyrosine residues within
ITAMs. The doubly tyrosine-phosphorylated ITAMs serve as docking sites
for the Syk family PTK -associated protein-70 (ZAP-70) to help its
phosphorylation and activation by p56lck or
p59fyn (6). These PTKs
phosphorylate additional substrates, leading to downstream events
including activation of phospholipase C-1 (PLC-1), Ras, and
phosphatidylinositol 3-kinase (PI3-kinase). All of these events
contribute to the activation and binding of transcription factors, such
as NF-AT and AP-1, to the promoter regions of specific genes such as
IL-2, the production of which is a prelude for clonal expansion and
lymphokine secretion.
Extensive investigation has been focused on resolving how the early
tyrosine-phosphorylation events are coupled to downstream biochemical
cascades. It has been suggested that the transduction mechanism
involves assembly of multimolecular signaling complexes at and near the
TCR (2). A group of molecules called adaptor proteins,
which possess no intrinsic enzymatic function but can mediate
protein-protein interactions, have been characterized and shown to play
a crucial role in this process (reviewed in Refs. 7 and
8). For example, the transmembrane protein linker for
activation of T cells (LAT) can serve as a substrate of ZAP-70 upon TCR
stimulation and act as a central adaptor by direct association with
Grb-2 (through Grb2’s Src homology 2 (SH2) domain) and PLC-1
(9, 10). Other proteins also found in the complex include
Vav, Cbl, the p85 subunit of PI3-kinase, and SH2 domain-containing
leukocyte protein of 76 kDa (SLP-76), and their interaction with LAT
may occur in an indirect manner. The interaction of LAT with other
signaling molecules has been demonstrated to be necessary for T cell
activation (11). Another ZAP-70 substrate SLP-76, which
contains a proline-rich region, an SH2 domain and multiple
tyrosine-phosphorylation sites, can also help to form a multimolecular
complex by binding to Grb-2 and Vav (12, 13). A mutant T
cell line lacking SLP-76 expression shows impaired coupling of the TCR
to the Ras pathway and reduced PLC-1 tyrosine phosphorylation, which
can be restored by the reexpression of SLP-76 (14).
Adaptor proteins can also play a role as negative regulators of signaling. Cbl, a widely expressed adaptor molecule with a phosphotyrosine-binding domain, a proline-rich region, and multiple tyrosine-phosphorylation sites, can interact with Vav, Crk, Grb2, PI3-kinase, p59fyn, ZAP-70, and Syk (15, 16, 17, 18, 19, 20). Recent evidence suggests that Cbl acts as a negative regulator in lymphocytes (21). Overexpression of Cbl in Jurkat T cells inhibits AP-1 activity after TCR ligation (20). Cbl has also been found to negatively regulate Syk tyrosine kinase in mast cells (22).
Here we report that another adaptor protein, Lnk, can also play a role
as a negative regulator in T cells. Lnk was originally cloned from a
rat lymph node cDNA library (23). The deduced amino acid
sequence of Lnk reveals the presence of an SH2 domain and a putative
tyrosine-phosphorylation site. Lnk mRNA is preferentially expressed in
lymph node and spleen lymphocytes. Mouse Lnk cDNA has also been cloned,
and recombinant mouse Lnk protein was able to serve as a substrate of
p56lck and ZAP-70 (24), although it
was only weakly tyrosine-phosphorylated upon TCR stimulation. We
recently cloned human Lnk (hLnk) by screening a Jurkat cell cDNA
library and found that compared with the reported rat and mouse
sequences it has an extended coding region at the 5' end resulting in
an additional 267 amino-terminal amino acids. The additional coding
sequence reveals a pleckstrin homology (PH) domain and a proline-rich
region. We show here that hLnk can bind to tyrosine-phosphorylated
-chain ITAM residues via its SH2 domain.
p56lck phosphorylates hLnk and binds to the
tyrosine-phosphorylated hLnk through its SH2 domain. In addition, we
have found that when overexpressed in Jurkat cells, hLnk inhibits
anti-CD3-mediated activation of NF-AT transcription activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rabbit Ab generated against His-tagged rat Lnk, which also recognizes human Lnk, has been previously described (23). Mouse anti-p56lck and rabbit anti-CD8 were obtained from Santa Cruz Laboratories (Santa Cruz, CA). Anti-phosphotyrosine mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). OKT3 ascites used for stimulation of Jurkat cells was generated from hybridoma acquired from the American Type Culture Collection (Manassas, VA).
Constructs
The cDNA of constitutively active p56lck
(LckY505F) (25), a gift from Dr. Kristin M. Abraham
(University of Maryland School of Medicine, Baltimore, MD), was cloned
into pCDNA3 (Invitrogen, Carlsbad, CA). The kinase inactive form of
p56lck (LckR273) and another variant carrying
complete deletion of the SH2 domain (Lck
SH2) (25) were
kind gifts from Dr. André Veillette (McGill University,
Montréal, Canada). The Syk expression vector was kindly provided
by Dr. Edward Clark (University of Washington, Seattle, WA). The
chimeric receptor CD8-, originally described by Dr. Arthur Weiss
(University of California, San Fransisco, CA) (26), was a
gift form Dr. Cox Terhorst (Beth Israel Hospital, Harvard Medical
School, Cambridge, MA) and inserted into
pCDNA3.1- (Invitrogen). The reporter construct
NF-AT-Luc was kindly provided by Dr. Gerald Crabtree (Stanford
University Medical School, Stanford, CA).
Conjugation of phospho-peptides and binding assay
Five tyrosine-phosphorylated peptides representing sequences of
half ITAMs within TCR were obtained. The five peptides were:
NQLpYNELNL ( ITAM1 amino), RREEpYDVLDK ( ITAM1 carboxyl),
EGLpYNELQK ( ITAM2 amino), (Nle)AEApYSEIG(Nle) ( ITAM2 carboxyl),
and HDGLpYQGLST ( ITAM3 amino). The residue norleucine (Nle) was
substituted for methionine in the appropriate peptides to prevent
oxidation. Next, 100 µg of each peptide was dissolved in 100 µl of
coupling buffer (0.1 M NaHCO3 plus 0.5 M NaCl, pH
8.3) and added to 750 µl of normal human serum-activated Sepharose 4
Fast Flow (Amersham Pharmacia Biotech, Piscataway, NJ). Coupling was
done at room temperature for 3 h. Nonreacted groups on the gel
were blocked with 1 M Tris, pH 7.4. The gel was washed alternatively
with 0.1 M NaHCO3 plus 0.5 M NaCl, pH 8.0, and
0.1 M Na acetate plus 0.5 M NaCl, pH 4.0. Then, 30 µl of peptide
conjugated beads were added to the lysate of COS cells transfected with
Lnk or Lnk(R392K) and incubated at 4°C for 3 h. Unconjugated
beads with their reaction groups blocked were used as negative control.
After washing three times with PBS containing 0.1% Nonidet P-40, beads
were boiled together with 30 µl of 2x SDS sample buffer. The
supernatant was used for Western blot analysis with anti-Lnk
Ab.
cDNA cloning of hLnk
Primers corresponding to various regions of rat Lnk cDNA
sequence were synthesized. PCR was conducted using Jurkat cDNA prepared
with random primer. Partial hLnk sequence (
600 bp) was obtained with
the forward primer (5'-CATGCTCGAGATCACTTCCTGTCCTGCTAC-3') and the
reverse primer (5'-GATGGATCCGGTGTACTGGTTGTCAATGG-3') and was cloned
into SK vector at sites of BamHI and XhoI. The
insert was regenerated, labeled with
[-32P]dATP (ICN Pharmaceuticals, Costa Mesa,
CA) using a random primed DNA labeling kit (Boehringer Mannheim,
Indianapolis, IN), and used as a probe to screen a 
gt11 Jurkat
library (Clontech, Palo Alto, CA). Phage DNA from positive plaques was
purified with Nucleobond AX (The Nest Group, Southboro, MA). Sequence
analysis was done by dideoxy chain terminator and automated fluorescent
DNA sequencer.
Northern blot
Total RNA from Jurkat cells was isolated using TRIZOL reagent (Life Technologies, Gaithersburg, MD). Poly(A)+ RNA was isolated using Dynabeads Oligo(dT)25 (Dynal, Lake Success, NY). About 1.5 µg of Poly(A)+ RNA was electrophoretically separated on a 1% agarose gel and transferred onto Zeta-Probe Blotting Membrane (Bio-Rad, Hercules, CA). The membrane was hybridized with the same 32P-labeled cDNA fragment that was used in the library screening. The size of the mRNA was measured using an RNA ladder as standard (Life Technologies).
Construction of hLnk expression vector and mutagenesis
The full coding region plus 17 bp of noncoding sequence at the
5' end of hLnk cDNA was generated by PCR and inserted into pCDNA3. The
same sequence without the stop codon was inserted into pEGFP-N1
(Clontech) to express a Lnk-green fluorescent protein (GFP) chimeric
protein. The point mutation in the SH2 domain of Lnk(R392K) was
generated using a method previously described (27).
Briefly, two mutagenic primers were designed to encode the R
K
mutation as well as a silent mutation to introduce a flanking
SacII restriction site. Two separated PCR were conducted. An
upstream primer and the antisense mutagenic primer
(5'-TTTTTTCCGCGGCGCGTCTCGCTCTGCTTCACCAGGAACAC-3') were used in the
PCR to generate the upstream fragment, while the sense mutagenic primer
(5'-TTTTTTCCGCGGGGAATACGTGCTCACTTT-3') and a downstream primer were
used in another PCR to generate the downstream fragment. The two
fragments were purified and ligated after SacII digestion
and then ligated into the pCDNA3 expression vector. The mutation was
confirmed by sequencing.
Confocal microscopy
COS-7 cells transfected with the Lnk-GFP construct were grown on sterile glass coverslips. Then, 48 h after transfection, cells were viewed using a Zeiss laser scanning 410 confocal microscope (Zeiss, Oberkochen, Germany). The GFP molecules were excited using a 488-nm laser and imaged using a 515- to 540-nm bandpass filter.
COS cell transfection, immunoprecipitation, and Western blotting
COS-7 cells were cultured in DME-F12 medium containing 10% iron
supplemented calf serum. Cells were transfected with 10 µg each of
expression vector containing either Lnk or Lnk(R392K) with or without
Lck(Y505F), Lck(R273), Lck(SH2), or Syk using the DEAE-dextran
method (28). Some of these cells were cotransfected with
10 µg of CD8- together with or without 10 µg of Lck(Y505F).
Total amount of DNA was equalized with pCDNA3 empty vector. Then,
48 h after the transfection, cells were lysed in RIPA buffer
containing 1 mM Na3VO4, 10
mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml
pepstatin A, and 1 mM 4-(2-aminoethyl)benzene-sulfonyl fluoride
hydrochloride. Immunoprecipitation was conducted using 2 µg of rabbit
anti-CD8 Ab followed by protein A-Sepharose 4 Fast Flow
(Pharmacia). Immunoprecipitates were resolved on a SDS/10% PAGE under
reducing conditions and transferred to Immobilon-P membranes
(Millipore, Bedford, MA). After blocking with 5% skim milk in TBST (10
mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20), blots were probed with
primary Abs (anti-Lnk, anti-p56lck,
anti-phosphotyrosine Abs) followed by incubation with
HRP-conjugated secondary Abs (Cappel, Durham, NC) and developed using
enhanced chemiluminescent substrate (Pierce, Rockford, IL). Western
blot analyses were also conducted using aliquots of cell lysates from
each transfection with anti-Lnk, anti-CD8, and
anti-p56lck to detect the expression of each
construct.
Jurkat cell transfection, stimulation, and luciferase assay
Jurkat cells were grown in RPMI 1640 supplemented with 10% FBS. Transient transfection was performed by electroporating (at 250V and 960 µF) 107 cells in 0.4 ml RPMI 1640 in a Gene Pulse cuvette (Bio-Rad) with 10 µg of Lnk or the Lnk(R392K) mutant together with 10 µg of NF-AT-Luc. Transfected cells were cultured in 10 ml of growth medium for 24 h and then seeded at 106 cells/ml into 24-well plates. Cells were either left unstimulated or stimulated with OKT3 ascites at 1:500 dilution or with PMA (10ng/ml) plus ionomycin (1 µg/ml) for 8 h. Luciferase assays were performed according to the manufacturer’s protocol (Promega, Madison, WI). Luciferase activities, determined using a Microplate Luminometer (Packard, Meriden, CT), were shown as fold increases compared with levels from control unstimulated cells. In some experiments, the relative luciferase activities were normalized against the expressed protein levels of hLnk and hLnk(R392K). The levels of these proteins expressed in the transfected cells were determined by Western blot analyses using anti-hLnk Ab and [125I]-goat anti-rabbit Ab. The relative radiolabels associated with hLnk and hLnk(R392K) were measured using phosphoimager (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A partial hLnk sequence (600 bp) was obtained by PCR using cDNA
from Jurkat cells and primers designed from the rat Lnk sequence. The
partial hLnk sequence was used as a probe to screen a gt11 Jurkat
cDNA library. Five positive plaques were isolated from 360,000 plaques
screened. After combining all the sequence information, a signal open
reading frame encoding a protein with a predicted molecular mass of 63
kDa was obtained. The hLnk cDNA (GenBank accession no. NM005475) shows
high homology to both rat and mouse Lnk with 73% amino acid sequence
identity. However, we found that hLnk has a much larger 5'-coding
region, indicating that the previously reported rat and mouse sequences
were not complete. To confirm the new 5'-coding region sequence, PCR
was conducted using cDNA from other human cell lines including Daudi,
Raji, IM-9, and HepG2. All of the sequences obtained from the various
cell lines were 100% identical with the one from the Jurkat cDNA
library. The full coding region of Lnk contains an N-terminal
proline-rich region, a PH domain, and a SH2 domain (Fig. 1
). It is also consistent with the
sequence recently submitted to the GenBank by Bartholomew et al.
(GenBank accession no. AJ012793).
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The PH and SH2 domains of hLnk show similarity to those of APS
(adaptor molecule containing PH and SH2 domains) and SH2-B (Fig. 2, A and B). APS
was cloned from a human B cell cDNA library
(29), and SH2-B was cloned from a rat mast cell cDNA
library (30). The PH domain of hLnk is 35% identical with
that of APS and 29% to SH2-B, and the SH2 domain shares 65% and 68%
identity to APS and SH2-B, respectively. Each of these three proteins
has a proline-rich region at the N terminus. It has been suggested that
these three molecules form a new family of adaptor protein
(29).
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The hLnk cDNA probe hybridized to a 6.8-kb mRNA in Northern blot
analysis using poly(A) RNA from Jurkat cells. No signal was observed
even after long exposure when 20 µg of total RNA was used, indicating
the message level of mRNA was very low. The message was detected when
1.5 µg of poly(A)+ RNA was used in the assay
followed by overnight exposure (Fig. 3).
Similar results were also obtained from other human cell lines such as
Daudi, Raji, IM-9, and HepG2 (data not shown). The endogenous hLnk
protein could not be detected in Jurkat cells, presumably due to low
level of expression. When COS-7 and Jurkat cells were transfected with
an expression vector containing the hLnk cDNA, anti-Lnk Ab detected
a band with an apparent molecular mass of 75 kDa, which was absent from
the empty vector control cell extract (Fig. 4).
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To visualize the cellular localization of hLnk, COS-7 cells were
transfected with expression vector containing a chimeric protein of
Lnk-GFP. In the transfected cells, the majority of Lnk-GFP was found
around juxtanuclear region while some appeared to be localized to the
plasma membrane. In the control cells transfected with GFP only, GFP
was diffusely expressed throughout the cytosol (Fig. 5).
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COS-7 cells were cotransfected with expression vectors containing
hLnk, constitutively active p56lck kinase,
Lck(Y505F), or with Syk, which belongs to the same family of PTK as
ZAP-70. As shown in Fig. 6, hLnk can be
strongly phosphorylated by Lck(Y505F), while Syk had no effect on its
phosphorylation. The expressed Syk was active in the assay as judged by
its ability of autophosphorylation (data not shown). hLnk can also be
coimmunoprecipitated with Lck(Y505F) (Fig. 7A). A point mutation (R392K)
within the highly conserved FLVERS motif (31) in the SH2
domain of hLnk did not affect its association with Lck(Y505F). However,
when Lnk was cotransfected with a kinase inactive form of
p56lck (LckR273) or with
p56lck completely lacking the SH2 domain
(LckSH2), neither of the two mutated Lck proteins could be
coimmunoprecipitated with Lnk (Fig. 7B). These results
indicate that the interaction of hLnk and p56lck
requires hLnk to be tyrosine-phosphorylated by
p56lck and that the SH2 domain of
p56lck interacts with the phosphotyrosine
residue of hLnk.
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-chain
Because SH2-B, the protein that is structurally related to Lnk,
can bind to the ITAM sequences of IgE receptor FcRI
(30), we investigated the possibility that hLnk may be
able to associate with the TCR -chain. COS cells were cotransfected
with expression vectors containing hLnk and a chimeric receptor CD8-
with or without Lck(Y505F). The chimeric receptor CD8- has a
cytoplasmic domain of TCR -chain fused to the transmembrane and
extracellular domains of CD8. Lck(Y505F) can tyrosine-phosphorylate the
ITAMs in the -chain. As shown in Fig. 8, hLnk was able to bind to the -chain
in a p56lck-dependent manner. This association
was not detected with hLnk containing a R392K point mutation within the
SH2 domain. The cytoplasmic domain of -chain contains three ITAM
sequences, numbered ITAM1 through 3 in the order of its relative
position from N to C terminal. Within each ITAM, there are two tyrosine
residues that become phosphorylated by p56lck.
To determine whether hLnk can actually bind to the phosphotyrosine
residues of the -chain ITAM, nona- or decapeptides containing
phosphotyrosine corresponding to each ITAMs were constructed having
phosphotyrosine at the center, except for the ITAM3 carboxyl end
tyrosine residue. These peptides designated as ITAM1-N, ITAM1-C,
ITAM2-N, ITAM2-C, and ITAM3-N were conjugated to agarose beads and
incubated with lysate of COS cells transfected with hLnk. As shown in
Fig. 9, top panel, hLnk
preferentially bound to the ITAM1-N and ITAM2-N residues. The
association of hLnk with ITAM1-C, ITAM2-C, and ITAM3-N was minimal if
any. When agarose beads conjugated with ITAM1-N and ITAM2-N were
incubated with lysate of COS cells transfected with hLnk (R392K), its
association was significantly diminished, indicating that the
association of hLnk with ITAM1-N and ITAM2-N was SH2 domain dependent
(Fig. 9, middle panel). COS cells transfected with hLnk and
hLnk(R392K) expressed and contained equivalent amounts of hLnk proteins
in its lysates (Fig. 9, bottom panel).
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The endogenous expression of hLnk protein in Jurkat cell is very
low and could not be detected. Because hLnk can bind to the TCR
-chain via its SH2 domain, we examined the effect of hLnk on TCR
signaling in Jurkat cells overexpressing hLnk. The assay was conducted
by measuring transcriptional activation of NF-AT using a reporter
construct of IL-2 driven by the NF-AT response element (NF-AT-Luc). An
expression vector containing hLnk or a control empty vector was
transiently cotransfected into Jurkat cells with NF-AT-Luc. Upon
stimulation by OKT3-mediated CD3 cross-linking, cells transfected with
the control vector showed over a 5-fold increase in the luciferase
activity. In contrast, cells transfected with Lnk showed only about a
2-fold increase (***, p < 0.0003, two-tailed
Student t test, n = 3), indicating that the
TCR-mediated activation of NF-AT activity was inhibited by hLnk
expressed in Jurkat cells (Fig. 10A). The 60% inhibition is
comparable to the inhibition of AP-1 activity (measured by the
AP-1-seap reporter construct) by Cbl overexpression (20).
Interestingly, when cells were stimulated with PMA and ionomycin, which
can bypass the proximal TCR events and activate NF-AT-Luc activity, Lnk
had little or no inhibitory effect (p > 0.35,
not significant, two-tailed Student t test,
n = 3). The values between controls of vector- and
Lnk-transfected cells were also not statistically significant
(two-tailed Student t test, n = 3). The
transfection efficiencies between experiments were comparable. To test
whether the hLnk-mediated inhibition of OKT3-mediated activation of
NF-AT-Luc activation is due to the binding of hLnk to the -chain,
NF-AT-Luc activities were also examined in Jurkat cells transfected
with hLnk(R392K). hLnk with the SH2 point mutation (R392K) cannot bind
to the -chain ITAM (see above, Fig. 9
). The expressed protein levels
were quantitated using Western blot analyses of hLnk developed with
[125I]-goat anti-rabbit Ab on a
phosphoimager (Molecular Dynamics Storm System). The relative
luciferase activities were normalized to the expressed protein levels
of hLnk and hLnk(R392K). As shown in Fig. 10B, both hLnk
(p < 0.005, two-tailed Student t
test, n = 3) and hLnk(R392K) (p
< 0.001, two-tailed Student t test, n = 3)
significantly inhibited OKT3-mediated luciferase activity compared with
the vector control-transfected cells. However, the levels of inhibition
between hLnk and hLnk(R392K) were not significant
(p > 0.09, two-tailed Student t
test, n = 3). As in Fig. 10A, there were no
significant differences in the luciferase activities among control-,
hLnk-, and hLnk(R392K)-transfected cells when these cells were treated
with PMA plus ionomycin (p > 0.1, ANOVA test).
These results indicate that hLnk-mediated inhibition of the
OKT3-mediated stimulation of NF-AT-Luc activity is mediated by other
functional domain(s) besides the SH2 domain of hLnk. Furthermore, these
results indicate that the binding of hLnk to the
tyrosine-phosphorylated ITAM by itself have little effect on the
inhibition of OKT3-mediated NF-AT-Luc activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain through its SH2 domain. Results from
the experiments using synthetic peptides with phosphotyrosines
corresponding to each of the tyrosine residues within the -chain
ITAMs indicated that hLnk binds preferentially to the ITAM1-N and
ITAM2-N phosphotyrosines in a SH2 domain-dependent manner. Provided
that the amounts of peptides conjugated to the agarose beads were
equal, more hLnk appears to have bound to the ITAM1-N than to the
ITAM2-N. However, additional studies are needed to elucidate this
point. Our preliminary results indicated that both wild-type hLnk and
hLnk(R392K) with dysfunctional SH2 domain inhibited OKT-3-mediated
NF-AT-Luc activation to a comparable levels. Thus, it is possible that
other functional domains of hLnk other than the SH2 domain may
contribute to the inhibition of NF-AT-Luc activation.
The tyrosine-phosphorylation sites of hLnk is not presently clear. The
half ITAM motif (DNQYTPL, consensus
sequence in bold) found at the C terminus is a possible phosphorylation
site for p56lck. Previous studies of in vitro
kinase assays using recombinant hLnk indicated that both
p56lck and ZAP-70 kinases could phosphorylate
hLnk (24). In our experiments using COS cells
overexpressing p56lck and hLnk with YF
mutation at the half ITAM site (hLnk Y572F), tyrosine phosphorylation
by p56lck was observed indicating that hLnk can
be tyrosine phosphorylated at sites other than
Y572. The role of ZAP-70 kinase on hLnk
phosphorylation is difficult to assess in cells because the activation
of ZAP-70 requires active p56lck, which not only
can activate ZAP-70, but also can phosphorylate hLnk. In contrast to
ZAP-70, Syk kinase that belongs to the same family of kinase as ZAP-70
does not require the presence of p56lck for its
activation. In our experiments using COS cells overexpressing Syk and
hLnk, phosphorylation of hLnk did not take place while Syk activity was
clearly demonstrated by its autophosphorylation. It is not clear
whether this is due to the differences in substrate specificity of
these enzymes or to the experimental conditions (lack of TCR/CD3
receptor complex expression in COS cells) under which hLnk will not be
tyrosine-phosphorylated by Syk family kinases. It is also possible that
hLnk phosphorylation observed by in vitro kinase assay may not
necessarily reflect the role these enzymes play in the cells.
At present we do not know what other protein(s) hLnk can bind to with its functional domains. Collectively, hLnk has the potential to interact with multiple molecules through its functional domains.
The most interesting finding about hLnk is its ability to inhibit T
cell activation upon TCR stimulation. Considering the fact that hLnk
can bind to the ITAM of TCR -chain via its SH2 domain led to our
initial speculation that Lnk binding to the tyrosine-phosphorylated
ITAM within the -chain may prevent the association of other
molecules. ITAMs serve as docking sites to several key signal
transduction molecules such as ZAP-70, p56lck,
and Shc (33). However, due to the low level of binding
between Lnk and -chain observed in our experiments, it is not clear
whether this mechanism is solely responsible for the inhibitory role.
When hLnk- and hLnk(R392K)-mediated inhibition of NF-AT-Luc activation
was normalized to the respective levels of hLnk and hLnk(R392K) protein
expression, the inhibitory effects were comparable. These results
indicate that the binding of hLnk SH2 domain to the ITAM may not be the
mechanism for hLnk-mediated inhibition of NF-AT-Luc activation. We also
showed that Lnk could associate with p56lck with
the association being mediated by the SH2 domian of
p56lck and the kinase activity of
p56lck a requirement. It is possible that Lnk
may sequester p56lck from other molecules
through this interaction. It has also been reported that a peptide
corresponding to the C-terminal putative phosphorylation site of Lnk
inhibited association between two signaling molecules in T cells, Shb
and p36/LAT (34). Thus Lnk may act as an inhibitory
molecule by interaction with multiple proteins via its different
domains.
We have been puzzled by the fact that the expression level of hLnk is extremely low in all the cell lines we have examined so far. The expression of Lnk in human tissues has not been tested yet. Pervious studies on rat tissues showed that although it was preferentially expressed in lymphoid tissues such as lymph node and spleen, the expression level in these tissues was lower compared with other adaptor proteins such as Shc or Grb-2. In cell lines, it is difficult to detect the levels of endogenous Lnk even using 2 x 107 cells for immunoprecipitation. Thus it is not clear whether hLnk can exert an inhibitory role at such low levels. It is possible that Lnk is kept at a low expression level so the resting T cells can be activated upon stimulation. During certain states, when the response of T cells need to be inhibited, Lnk may be induced and play its inhibitory role. Future studies on the regulation of Lnk expression may tell us its physiological function.
The formation of multimolecular complexes has been a major focus in the study of signal transduction. Many adaptor proteins have been characterized and shown to mediate protein-protein association and thus facilitate transduction of signals. The study of adaptor proteins that act as negative regulators is still at an early stage, and not many molecules with such a function have been found. By forming or preventing the formation of a particular multimolecular complex, an adaptor protein may exert its inhibitory action. Further characterization of hLnk may reveal new mechanisms of negative regulation of T cell signal transduction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jun Hayashi, School of Pharmacy, University of Maryland, 20 North Pine Street, Baltimore, MD 21201.
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine kinase; GFP, green fluorescent protein; ZAP-70, -associated protein of 70 kDa; PLC-1, phospholipase C-1; PI3-kinase, phosphatidylinositol 3-kinase; LAT, linker for activation of T cells; SH2, Src homology 2; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa; hLnk, human Lnk; PH, pleckstrin homology; APS, adaptor molecule containing PH and SH2 domains.
Received for publication June 10, 1999. Accepted for publication March 7, 2000.
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References |
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