(Received for publication, March 7, 1997)
From the a Howard Hughes Medical Institute, and the Departments of c Immunology, d Molecular Biotechnology, h Biochemistry, and i Medicine (Medical Genetics), University of Washington, Seattle, Washington 98195 and the e Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201
Stimulation of the T cell antigen receptor (TCR)
activates a set of non-receptor protein tyrosine kinases that assist in
delivering signals to the cell interior. Among the presumed substrates
for these kinases, adaptor proteins, which juxtapose effector enzyme systems with the antigen receptor complex, figure prominently. Previous
studies suggested that Lnk, a 38-kDa protein consisting of a single SH2
domain and a region containing potential tyrosine phosphorylation
sites, might serve to join Grb2, phospholipase C-1, and
phosphatidylinositol 3-kinase to the TCR. To elucidate the
physiological roles of Lnk in T cell signal transduction, we isolated
the mouse Lnk cDNA, characterized the structure of the mouse
Lnk gene, and generated transgenic mice that overproduce Lnk in thymocytes. Here we report that although Lnk becomes
phosphorylated during T cell activation, it plays no limiting role in
the TCR signaling process. Moreover, we have distinguished
p38Lnk from the more prominent 36-kDa tyrosine phosphoproteins
that appear in activated T cells. Together these studies suggest that Lnk participates in signaling from receptors other than antigen receptors in lymphocytes.
The earliest detectable biochemical event following stimulation of
the T cell receptor (TCR)1 is the
activation of several protein-tyrosine kinases, resulting in transient
tyrosine phosphorylation of numerous intracellular substrates. These
substrates include the ,
, and
components of the CD3 complex
(1), the
subunit of the TCR (2), phospholipase C-
1 (PLC-
1)
(3-5), the Vav proto-oncogene product (6, 7), the
Ras-GTPase-activating protein (Ras-GAP) (8), ezrin (9), several
cytoskeletal proteins, and the non-receptor protein-tyrosine kinases
themselves, Lck, Fyn, and ZAP-70 (10-13). Phosphorylation of Lck (14,
15), ZAP-70 (16), and PLC-
1 (17) alters the catalytic activity of
each protein, but a detailed explication of the machinery that links
ligand occupancy of the TCR to changes in cell behavior remains
unapproachable. In part, this process remains mysterious because of an
incomplete appreciation of the total set of enzymes that associate
with, and respond to Lck, Fyn, and ZAP-70 as they interact with the TCR
complex.
Among the substrates that become rapidly phosphorylated on tyrosine following T cell stimulation is a set of so-called adaptor proteins, molecules which lack catalytic function (insofar as is known) but which possess interaction domains, e.g. SH2 domains that bind tightly to phosphotyrosine residues or SH3 domains that bind proline-rich sequences (18). An appreciation for the importance of these proteins derives from studies of growth factor receptor kinases, where initial binding and phosphorylation of the Shc adaptor creates a binding site for a second adaptor, Grb2, that then directs Sos, a guanine nucleotide exchange factor, to the membrane where it can act upon Ras (19, 20). In a similar way, the adaptor proteins of T cells, by binding to tyrosine-phosphorylated components of the TCR, should serve to juxtapose other SH2 domain-containing proteins with the TCR, thereby regulating the interaction of downstream signaling pathways with the ligand-engaged receptor. A thorough enumeration of the normal complement of adaptor proteins, and an analysis of how these proteins behave, should therefore help to illuminate mechanisms of signal transduction in lymphoid cells.
Lymphocytes themselves contain Shc, which becomes phosphorylated and
binds both TCR subunits and the Grb2-Sos complex following TCR
stimulation (21). Although several observations support the view that
Shc may help to link Grb2 and perhaps other proteins to the TCR
complex, the importance of this interaction remains controversial, in
part because only a small proportion of total cellular Grb2 binds to
phosphorylated Shc after TCR stimulation (22, 23). SLP-76 (SH2
domain-containing leukocyte protein of 76 kDa) contains many
tyrosine-phosphorylation sites, an SH2 domain, and a proline-rich
region that may interact with SH3-containing proteins (24). TCR
ligation stimulates SLP-76 phosphorylation, and augmented expression of
SLP-76 in the Jurkat cell line improves the nuclear translocation of
the NFAT transcription factor (a key regulatory event in IL-2
expression) following TCR cross-linking (25). Moreover, SLP-76 has been
shown to bind to the Vav oncoprotein, a putative exchange factor for
Rho/Rac GTP-binding proteins (26), which is also phosphorylated by
protein tyrosine kinases upon TCR stimulation. Coexpression of SLP-76
with Vav in Jurkat cells produces synergistic increases in NFAT
activation following TCR stimulation (27). Yet another adaptor protein,
the 120-kDa product of the cbl gene, becomes tyrosine
phosphorylated following TCR activation and thereafter associates with
a variety of signaling molecules including Crk, Grb2, Fyn, Lck, ZAP-70,
Ras-GAP, PLC-1, and the phosphatidylinositol 3-kinase (28-34).
While the significance of these interactions remains obscure, the
documented transforming ability of v-cbl, in which normal
cbl-encoded sequences are partially truncated, supports the
view that this molecule plays an active role in TCR-mediated signal
transduction (35). A fourth presumed adaptor protein, HS1, which was
recently shown to behave as an Lck-binding protein (36), contains an
SH3 domain and also becomes tyrosine phosphorylated following antigen
receptor cross-linking (37, 38). T lymphocytes from mice lacking HS1
exhibit impaired proliferative responses and a disturbance in normal
repertoire selection (38). At a minimum, then, the adaptor proteins
Shc, Grb2, SLP-76, Cbl, and HS1 all behave as molecules that may link activation of kinase components of the T cell antigen receptor complex
to subsequent biochemical responses.
One prominent substrate of the TCR-activated protein tyrosine kinase(s)
is a 36-38-kDa protein (pp36) that associates with the SH2 domains of
numerous signaling molecules, including Grb2, PLC-1,
phosphatidylinositol 3-kinase, GAP, and the Src family kinases (22,
39-42). Although other phosphoproteins, notably Shc, have been shown
to associate with the Grb2 SH2 domain, pp36 is the predominant species
that binds the SH2 domain of Grb2 in anti-TCR-stimulated T lymphocytes.
Moreover, tyrosine phosphorylation of pp36 correlates well with
inositol phosphate production, a measure of PLC-
1 activity (43).
Transfection of a chimeric CD45 phosphotyrosine phosphatase containing
the Grb2 SH2 domain into Jurkat T lymphoblasts yielded cells in which
TCR stimulation failed to induce tyrosine phosphorylation of pp36,
while phosphorylation of other proteins remained intact (43). These
results were interpreted to suggest that phosphorylated pp36, once
bound to the chimeric Grb2/CD45 protein, became rapidly
dephosphorylated. Interestingly, inositol phosphate production and
calcium mobilization were compromised in the transfected cells, further
suggesting a role for pp36 in coupling the TCR to phosphoinositide
metabolism. Efforts have therefore focused on biochemical
characterization of pp36.
Recently, cDNA clones encoding a 38-kDa molecule called Lnk, which
shares many properties with pp36, were isolated (44). Lnk consists of a
single SH2 domain juxtaposed with a sequence of unknown function in
which are embedded potential tyrosine-phosphorylation sites. In an
initial report, Lnk expression was documented in lymphoid tissues and
the Lnk protein was shown to become tyrosine phosphorylated after TCR
cross-linking. Intriguingly, phosphorylated Lnk appeared to interact
(as judged by coimmunoprecipitation) with Grb2, PLC-1, and
phosphatidylinositol 3-kinase. These observations suggested that Lnk
was in fact the pp36 protein, the phosphorylation of which
correlates so well with phosphoinositide turnover (43, 44).
To investigate the role of Lnk in TCR signal transduction and in T cell development, we have characterized the structure of mouse Lnk, the gene that encodes it, and its expression in various tissues and lymphocyte subsets. In addition, we generated transgenic mice expressing Lnk at very high levels in thymocytes. These studies demonstrate that the availability of Lnk protein does not restrict normal T cell development or responsiveness. Thymocytes from transgenic mice proliferated normally and showed an equivalent pattern of tyrosine-phosphorylated proteins when compared with normal thymocytes stimulated via TCR cross-linking. By studying the behavior of proteins bound by GST-Grb2 fusion proteins, and by anti-Lnk heteroantisera, we learned that pp36 and Lnk are distinct entities, although both become phosphorylated during T cell activation. Thus Lnk represents a novel adaptor protein expressed in lymphocytes, which almost certainly interacts with phosphoproteins other than those that comprise the T cell antigen receptor.
A mouse Lnk
cDNA fragment was amplified from thymocyte cDNA by PCR using
the following primers, which were synthesized based on the published
rat Lnk cDNA sequence: forward (FWD) primer: 5-TCACTTCCTGTCCTGCTACCCCTG, reverse (REV) primer:
5
-GCACAGCTGTGAGAGAGGGGTGTA. The cDNA fragment was labeled using
the random-primer method with [
-32P]dATP (DuPont NEN),
and used as a probe to screen a library previously constructed with
mouse thymocyte cDNA in the
ZAP II vector (Stratagene, La Jolla,
CA) by plaque hybridization. Five positive plaques were isolated from
1 × 106 plaques screened, and phage DNAs were excised
in vivo to rescue phagemids containing the cDNA
fragment. The DNA sequences of these clones were determined using the
dideoxy chain termination method employing Sequenase 2.0 (U. S. Biochemical Corp.). Genomic clones were isolated from a
FIX II
library of 129SV liver DNA partially digested with MboI
(Stratagene) using [
-32P]dATP-labeled cDNA
fragments as probes. A total of 1 × 106 plaques were
screened and 28 positive plaques were isolated. Five independent clones
of these were expanded and each NotI restriction fragment
was subcloned into pBluescript KS(+) (Stratagene). Physical maps of
subcloned plasmids were determined by restriction enzyme digestion,
Southern blotting, PCR, and DNA sequencing.
The cDNA fragments encoding the
N-terminal domain (Lnk-N: amino acids 1-96), and the C-terminal domain
(Lnk-C: amino acids 197-307) of mouse Lnk, and the bulk of mouse Grb2
(amino acids 2-217) were synthesized by PCR using the following
BamHI site-containing primers: Lnk-N FWD,
5-AAGGATCCATGCCTGACAACCTCTACACC; Lnk-N REV, 5
-ATGGATCCTAGGGGTAGCAGGATAGGAAG; Lnk-C FWD,
5
-TTGGATCCGTCCTCTCTCAGGCACCAG; Lnk-C REV,
5
-TAGGATCCATTCACACGTCTGCCTCT; Grb2 FWD,
5
-TTGGATCCGAAGCCATCGCCAAATATGAC; Grb2 REV,
5
-ATGGATCCTTAGACGTTCCGGTTCACTGG. PCR fragments were inserted into the
BamHI cloning site of pGEX-2T (Pharmacia Biotech Inc.), and
the resulting plasmids were used to transform Escherichia coli BL21(DE3)/pLysS (Novagen, Madison, WI). Recombinant proteins were induced with 1 mM
isopropyl-
-D-thiogalactopyranoside and purified by
adsorption to glutathione-coupled Sepharose-4B (Pharmacia). Purified
GST-Grb2 fusion proteins were dialyzed against phosphate-buffered saline (154 mM NaCl, 10 mM PO4, pH
7.4) and conjugated to activated Affi-Gel-10 agarose (Bio-Rad)
according to the manufacturer's instructions.
Rabbit polyclonal antisera were raised against a synthetic peptide spanning amino acids 281-309 of mouse Lnk, or against the GST-Lnk fusion proteins. The antibodies were purified by chromatography on Affi-Gel-10 conjugated with either the synthetic peptide, GST-Lnk-N, or GST-Lnk-C fusion protein, followed by elution using 0.1 M glycine, pH 2.5. Antibodies against GST were absorbed from sera against GST-Lnk fusion proteins using GST-coupled glutathione-Sepharose-4B. Affinity-purified antibodies to GST-Lnk fusion proteins were coupled to Affi-Gel-10, and used for immunoprecipitation studies. Antibodies against the Lnk peptide were used for immunoblotting.
Flow Cytometric AnalysisFlow cytometry was performed by
staining 106 freshly isolated thymocytes and splenocytes
from 5-7-week-old mice (45) using the following reagents: fluorescein
isothiocyanate (FITC)-coupled anti-CD8 (Pharmingen, San Diego, CA),
phycoerythrin (PE)-conjugated anti-CD4 (Pharmingen), FITC-anti-B220
(Caltag Laboratories, Burlingame, CA), PE- or biotin-conjugated
anti-CD3 (Pharmingen), biotin-anti-CD69 (Pharmingen), PE- or
TRI-COLOR-conjugated streptoavidin (Caltag). Cells were analyzed with a
FACScan instrument (Becton-Dickinson, Mountain View, CA) using Lysis II
(Becton-Dickinson) and Reproman software (Truefacts Software, Seattle,
WA). To obtain purified lymphocyte subpopulations, a single-cell
suspension of thymocytes from 6-week-old C57BL/6J mice was stained with
FITC-anti-CD8 and PE-anti-CD4, and splenocytes were stained with
FITC-anti-B220 and PE-anti-CD3
. Purified
CD4
8
, CD4+8+,
CD4+8
, and CD4
8+
thymocytes, or B220+ and CD3+ splenocytes were
obtained using a FACStar (Becton-Dickinson) cell sorter.
Poly(A)+ RNA was isolated from
mouse tissues using the Micro-Fast Track kit (Invitrogen, Carlsbad,
CA), fractionated on a 1% agarose/formaldehyde gel, and transferred
onto nitrocellulose membranes (Amersham). These membranes were
hybridized with 32P-labeled cDNA fragments encoding
mouse Lnk (amino acids 36-268) or mouse -actin (amino acids
160-375). For quantitative reverse transcriptase-PCR analysis,
poly(A)+ RNA was isolated from purified cells using the
Micro-Fast Track kit, and first strand cDNA template was
synthesized using avian myeloblastosis virus reverse transcriptase
(Stratagene) with random primers. Serial dilutions of these cDNA
templates were subjected to PCR amplification using sets of primers
spanning several introns of Lnk (FWD primer, 5
-ATGCCTGACAACCTCTACAC;
REV primer, 5
-ATTCACACGTCTGCCTCTCT) or
-actin (FWD primer,
5
-ACACTGTGCCCATCTACGAG; REV primer, 5
-CTAGAAGCACTTGCGGTGCA). Cycling
parameters were 1 min at 94 °C, 2 min at 64 °C, and 3 min at
72 °C for 34 cycles to detect Lnk mRNA, and 27 cycles for
-actin. PCR products were separated by electrophoresis on a 1.0% agarose gel and visualized by staining with ethidium bromide.
Southern blot filters of C57BL/6J × Mus spretus backcross DNA panels digested with HindIII were purchased from the Jackson Laboratory (Bar Harbor, ME). The filters were hybridized with a 32P-labeled 2-kb BglII-BamHI fragment of the Lnk gene containing exon 1. The probe detected a restriction fragment length polymorphism, previously defined using test digestions, that distinguishes C57BL/6J mice from the M. spretus strain (a 10.5-kb fragment in C57BL/6J DNA, and 8-kb and 2.5-kb fragments in M. spretus DNA, respectively). The results of these hybridizations were scored blindly, and forwarded to the Jackson Laboratory Backcross DNA Panel Map Service, which used these data to position the Lnk gene.
In Vitro Phosphorylation AssayBaculovirally expressed murine p56lck was prepared as described (46). High Five cells containing baculovirally expressed affinity-tagged ZAP-70 (47) were generously provided by Dr. L. E. Samelson (National Institutes of Health, Bethesda, MD). ZAP-70 was isolated via immunoprecipitation with the 9E10 monoclonal antibody, which recognizes the Myc affinity tag, as described elsewhere (48).
Phosphorylation of GST-Lnk-N with recombinant p56lck and 9E10
precipitates of ZAP-70 was performed for 1 h at 30 °C in 50 mM Tris, pH 7.5, 10 mM MnCl2, 0.1%
Nonidet P-40, 250 µM ATP with [-32P]ATP
added to 4400 dpm/pmol. Phosphoproteins were separated, and unreacted
[
-32P]ATP removed, by SDS-PAGE on 12% gels, and
transferred to nitrocellulose. Phosphoproteins of interest were
identified by autoradiography. Tryptic digestion was performed on the
membrane. The resulting peptides were separated on two-dimensional
phosphopeptide maps and then subjected to IMAC (immobilized metal
affinity chromatography)-HPLC (high performance liquid
chromatography)-ESI (electrospray ionization)-MS (mass spectrometry)
analysis as described elsewhere (48) with the following modification:
the second dimension ascending chromatography buffer for the
two-dimensional phosphopeptide mapping was 55:45:40:9, butan-1-ol,
pyridine, water, acetic acid (v/v/v/v).
The DNA sequences surrounding the
initiation codon of the mouse Lnk cDNA were modified by PCR to
provide a better consensus sequence (49) using the following two
primers: FWD primer (containing a BamHI site),
5-AAGGATCCACCATGCCTGACAACCTCTACACC, and the Lnk-N REV primer. A
BamHI-SacI fragment of the modified cDNA was
used to replace a SacI fragment containing the initiation
codon of the original Lnk cDNA. The Lnk transgene was produced by
ligation of the combined Lnk cDNA (containing 8 base pairs of
modified 5
-untranslated region, the entire coding region and 50 base
pairs of 3
-untranslated region) into the BamHI cloning site
of the p1017 vector (50), which consists of the murine lck
proximal promoter and a human growth hormone gene cassette. The Lnk
transgene, purified as a NotI fragment, was injected into
(C57BL/6J × DBA/2J) F2 mouse zygote pronuclei as described
previously (51). Transgenic founders were detected by hybridization of
genomic DNA with an human growth hormone probe, and stable lines of
mice were generated by backcrossing founders with C57BL/6J mice.
Thymocytes were stimulated
with anti-CD3 (2C11) and anti-CD4 (GK1.5) mAbs in combination with
anti-rat IgG (Sigma) and anti-hamster IgG (Sigma) secondary antibodies
as described previously (21). Following stimulation, the cells were
lysed with lysis buffer (150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, 1% Nonidet P-40, 10 mM sodium fluoride,
1 mM Na3VO4, 2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin) and the lysates were clarified by centrifugation. The
lysates were incubated with agarose beads conjugated with anti-Lnk-N
and anti-Lnk-C antibodies or GST-Grb2 at 4 °C for 1 h. The
beads were then washed three times with lysis buffer. Lysates of 2 × 106 cells, or precipitated proteins from lysates of
4 × 107 cells were resolved by SDS-12.5% PAGE under
reducing conditions, and transferred to a nitrocellulose membrane
(Schleicher & Schull). After blocking with 5% skim
milk/phosphate-buffered saline, blots were probed with
anti-phosphotyrosine mAb (4G10, Upstate Biotechnology, Lake Placid,
NY), or anti-Lnk peptide antibodies, and incubated with horseradish
peroxidase-conjugated secondary antibodies (Cappel, Durham, NC).
Filters were washed in 0.05% Tween 20/phosphate-buffered saline and
developed by enhanced chemiluminescence (DuPont NEN).
To pursue the importance
of Lnk in immune physiology, we determined the structure of mouse Lnk,
thereby providing entree to the set of immunological reagents available
in this species. A full-length cDNA encoding mouse Lnk was isolated
from a thymocyte cDNA library and its nucleotide sequence
determined using standard techniques. Not surprisingly, Lnk is well
conserved between mouse and rat. The mouse Lnk cDNA sequence is
95% identical with that of rat (data not shown; the mouse Lnk cDNA
sequence is available through GenBank, accession number U89992[GenBank]). The
deduced amino acid sequences of mouse and rat Lnk share 96% identity
and 98% similarity overall, with complete identity within the SH2 domain (Fig. 1A). Possible
tyrosine-phosphorylation sites suggested in this paper (see below) and
by Huang et al. (44) are also perfectly conserved. By
comparing the Lnk amino acid sequence with the GenBank data base
(translated in all possible reading frames), we identified several
proteins with significant similarities to either the N-terminal domain
of Lnk or to its SH2 domain (Fig. 1B). Among these, the
recently characterized SH2-B protein, which was isolated in a yeast
"tribrid" screen by virtue of its ability to bind the
immunoreceptor tyrosine-based activation motif (ITAM) of the FcRI
subunit (52), was especially similar. Although no functional data have
yet been reported regarding SH2-B, simple structural comparison
suggests that Lnk might also interact with phosphorylated ITAM-like
motifs to link signal-transducing molecules to cell surface receptors.
Besides SH2-B, the Lnk SH2 domain also appears related to those of the
protein-tyrosine phosphatase corkscrew, the adaptor proteins Shc and
Nck, and the protein-tyrosine kinases Srm and Abl.
Expression of Lnk mRNA
RNA blotting was performed to
characterize the tissue distribution of the Lnk mRNA (Fig.
2A). Two transcripts of 5 and 3 kb were
strongly expressed in spleen, lymph node, and bone marrow, and found at
low levels in thymus. A fair amount of Lnk mRNA was also detected
in lung, liver, and kidney. Lnk has been reported to be expressed
preferentially in lymphocytes (44); however, the level of transcript
accumulation in lung and liver is consistent with the existence of
other populations of Lnk-expressing cells in those organs. To assess
the pattern of Lnk expression during lymphocyte development, we
purified thymocyte subpopulations based on their discrete patterns of
CD4 and CD8 expression (Fig. 2B). CD48
, CD4+8+,
CD4+8
, and CD4
8+
thymocytes were purified by sorting, and expression of Lnk in each
subpopulation was analyzed using a semi-quantitative reverse transcriptase-PCR approach. The PCR primers employed in amplification spanned five introns of the mouse Lnk gene (see below), and
hence the products amplified from cDNA (1.0-kb) could be easily
distinguished from those arising through amplification of contaminating
genomic DNA (1.6-kb). In each case, the amount of template cDNA
present was normalized between samples using PCR detection of
-actin cDNA. Interestingly, Lnk transcripts were present at essentially equivalent levels throughout thymocyte development, including in the
CD4
8
population where few cells express the
T cell antigen receptor. Analysis of splenic subpopulations revealed
that Lnk mRNA accumulates maximally in B lymphocytes
(B220+ cells), reaching levels nearly 5-fold greater than
those seen in mature CD3
+ T cells (Fig. 2B).
As was noted in studying rat Lnk (44), several alternatively spliced
transcripts of unknown significance were found at low levels in
lymphoid tissues. To gain an appreciation for these transcripts we also
obtained clones of the mouse Lnk gene.
Organization of the Mouse Lnk Gene
We screened a 129SV
genomic library in bacteriophage using the Lnk cDNA fragments as
probes. The restriction map of a 30-kb region containing the mouse
Lnk gene was constructed by analysis of five overlapping
bacteriophage clones (Fig. 3A). Restriction maps for BamHI, EcoRI, HindIII, and
BglII were determined and analyzed at greater resolution by
Southern blotting with cDNA probes and specific oligonucleotides,
or by PCR. In all cases, the sizes of subcloned restriction fragments
corresponded with the sizes of cross-hybridizing bands seen on genomic
blots analyzed under high stringency conditions (data not shown). In
addition, we found no clones harboring intron-containing or processed
pseudogenes. These results suggest that Lnk exists as a
single copy in the haploid mouse genome. All exons and introns spanning
the translated region of the Lnk gene were sequenced (Fig.
3B). For convenience, the most 5 exon, which contains
untranslated region sequences mapped by PCR and DNA blotting, has been
tentatively designated as exon 1, however, the in vivo
transcription start site has not yet been defined. Comparison of
genomic and cDNA sequences revealed that the Lnk gene is
divided into at least 7 exons and 6 introns. The coding region of Lnk
resides on 6 exons (exons 2-7), encompassing just 1.6 kb of the mouse
genome. The sequences surrounding all exon-intron boundaries fulfilled
typical AG/GT rules (53). In previous studies of rat Lnk, several
rarely expressed cDNAs termed Lnk3 and
Lnk42 (GenBank accession numbers U24654[GenBank] and
U24655[GenBank], respectively) were identified. Alignment of these sequences
demonstrated that each contains one or more of the small introns which
punctuate the mouse sequence, confirming that these rare rat sequences
represent partially processed transcripts (data not shown). We were
unable to adduce evidence that these rare transcripts direct the
synthesis of a protein product.
Chromosomal Localization of the Mouse Lnk Gene
We determined
the chromosomal localization of the mouse Lnk gene using an
interspecific backcross DNA panel (54). Restriction fragment length
polymorphisms within Lnk that distinguish C57BL/6J DNA from
M. spretus DNA were scored by blotting and hybridization using a probe containing exon 1, and used to follow the segregation of
Lnk alleles within an interspecific backcross that has been characterized at the Jackson Laboratory. This analysis permitted assignment of Lnk to mouse chromosome 5. Complete
co-segregation was observed with Tbx3 (T/omb homologous
domain containing gene 3 (55)) and NOS1 (neuronal nitric
oxide (NO) synthase (56)); all three genes lie approximately 17 cm
centromeric to Epo, the gene encoding erythropoietin (Fig.
4). There are no known mouse mutants, other than those
bearing targeted disruptions of NOS1, that map to this
precise region (57). Interestingly, the human NOS1 gene has
been reported to localize within the 12q24.2 region (58, 59), which
adjoins a site of frequent chromosomal abnormalities in some malignant
diseases (see "Discussion").
In Vitro Tyrosine Phosphorylation of Lnk by Lck and ZAP-70
Lnk is reported to become rapidly
tyrosine-phosphorylated upon TCR stimulation. Among those kinases
that might catalyze this event, Lck and ZAP-70 are especially
prominent. Both of these kinases associate physically and functionally
with the TCR, and both become rapidly activated after TCR cross-linking
(10-13). We therefore examined whether purified GST-Lnk fusion
proteins containing the N-terminal (GST-Lnk-N) or the C-terminal domain of Lnk (GST-Lnk-C) can serve as substrates for Lck and Zap-70 in
vitro. As shown in Fig. 5A, GST-Lnk-N
was phosphorylated by both purified Lck and ZAP-70. Two-dimensional
phosphopeptide mapping showed that both kinases phosphorylated the same
peptide from GST-Lnk-N (Fig. 5B). Further analysis using the
IMAC-HPLC-ESI-MS system (48) identified tyrosine 6 as the residue
phosphorylated by Lck and ZAP-70 (Fig. 5C and data not
shown). GST-Lnk-C also appeared to be phosphorylated by Lck (data not
shown), however, we were unable to identify the phosphorylated tyrosine
residue unambiguously in this case. Tyrosine 297 would appear to be an attractive target for phosphorylation within the C-terminal domain, since this residue lies embedded within a sequence that resembles the
consensus sequence of ITAMs (Y-X-X-L/I), a known in
vitro target for Lck. Moreover, a synthetic phosphotyrosine
297-containing peptide bound efficiently to Grb2, PLC-1, and
phosphatidylinositol 3-kinase, molecules that also bind rat Lnk
(44).
Consequences of Lnk Overexpression in Thymocytes
To
investigate the function of Lnk in vivo, we generated
transgenic mice expressing high levels of Lnk in thymocytes using the
thymocyte-specific lck proximal promoter (50). In all, we examined 7 independent transgenic founder animals and 13 lines of mice
expressing transgene-encoded Lnk protein. To assess the importance of
Lnk abundance on lymphocyte behavior, we used antisera directed against
a peptide to quantitate the level of Lnk expression by immunoblotting,
and compared the values obtained in this way with a standard immunoblot
prepared using serial dilutions of heterologously expressed GST-Lnk-C
fusion protein. Whereas normal mouse thymocytes contain 2.5 × 101 Lnk molecules per cell, the levels in transgenic
animals ranged from 1.2 × 102 to 1.5 × 104 molecules per cell. As expected, expression of the
p1017lnk transgene was confined almost entirely to
thymocytes, with some modest expression also observed in peripheral T
cells. Thymuses from p1017lnk transgenic mice exhibited
normal cellularity, and the proportion of thymocyte subpopulations was
in most cases also grossly normal as assessed by CD4 and CD8
expression. However, two lines of transgenic mice, expressing
especially high levels of transgene-derived Lnk, showed a decreased
number of single-positive thymocytes as compared with normal littermate
control mice. Data obtained from a representative individual of one of
those transgenic lines (the highest-expressing line) are shown in Fig.
6, and our experience with such mice is summarized in
Table I. This reduction in single-positive thymocytes was only observed in mice expressing >1.2 × 104 Lnk
molecules per cell.
|
Thymocytes from all transgenic mice proliferated as well in response to anti-CD3 stimulation as did those from normal mice, and the levels of CD3 and CD69 were comparable with those seen in normal thymocytes (data not shown). In the highest expressing line, Lnk levels were 600-fold elevated over normal on a per cell basis. It should be noted, however, that the molar abundance of Grb2 in such thymocytes (2.3 × 104 molecules per cell) exceeded that of transgenic Lnk (1.5 × 104 molecules per cell) by an appreciable margin (data not shown). Hence we would not expect the Lnk SH2 domain to compete effectively for binding sites ordinarily occupied by Grb2. As was seen in thymocytes, splenocytes expressing transgene-derived Lnk were present in normal numbers, and responded normally to anti-CD3 stimulation (data not shown).
Lnk Is Not a Major Tyrosine-phosphorylated Protein upon TCR StimulationIt has been shown that Lnk becomes
tyrosine-phosphorylated upon TCR/CD4 stimulation in rat lymph node T
cells. We investigated whether the overexpression of Lnk in thymocytes
affects TCR-induced phosphorylation of endogenous substrates on
tyrosine. Thymocytes were stimulated with a combination of anti-CD3 and
anti-CD4 mAbs, and phosphotyrosine-containing proteins were visualized
by SDS-PAGE and immunoblotting (Fig. 7A).
Augmented expression of Lnk did not measurably affect the induced
tyrosine phosphorylation of cellular proteins. Of particular note,
although the transgenic animals contain extraordinarily high levels of
Lnk, no increase in the abundance of phosphotyrosine-containing
proteins was observed in the 36-38-kDa region where Lnk migrates.
Immunoprecipitation using anti-Lnk antibodies revealed that Lnk is
rapidly (albeit weakly) phosphorylated on tyrosine following TCR
stimulation of thymocytes (Fig. 7B). However, the abundance
of this phosphotyrosine-containing species was not increased in the
p1017lnk transgenic thymocytes. Since the pattern of
accumulation of phosphotyrosine-containing species in the 36-kDa range
remained unaffected despite massive increases in the intracellular Lnk
concentration, we suspected that Lnk and pp36 might be different
proteins. To examine this hypothesis, we separated Lnk from pp36 using
anti-Lnk antibodies or a GST-Grb2 fusion protein. Antibodies raised
against Lnk immunoprecipitated Lnk effectively, but did not absorb or
immunoprecipitate pp36 from cell lysates. Reciprocally, a GST-Grb2
fusion protein bound pp36 and absorbed pp36 proteins from lysates, but
did not bind Lnk at all (Fig. 7C). Moreover, Lnk was
phosphorylated very weakly under the conditions that we employed while
pp36 was strongly phosphorylated, and Lnk migrated more slowly in
SDS-PAGE than did pp36. These results demonstrate that although Lnk is
an SH2 domain-containing protein that becomes phosphorylated on
tyrosine during T cell activation, it is not the easily identified,
phosphotyrosine-containing species in activated T cells that migrates
at 36 kDa.
It has been proposed that Lnk, a cellular adaptor protein
expressed preferentially in lymphoid tissues, becomes tyrosine- phosphorylated upon TCR stimulation and participates in TCR signaling by associating with Grb2, PLC-1, and phosphatidylinositol
3-kinase (44). We therefore sought to elucidate the function of Lnk in lymphocytes by isolating the mouse Lnk cDNA, clarifying the genomic structure and chromosomal localization of the Lnk gene, and
defining the basic features of Lnk phosphorylation through in
vitro studies and the analysis of transgenic mice. Our studies
suggest that although Lnk may participate in TCR signaling, its
functions are in no way limiting during T cell development or
activation.
The overall structure of Lnk is well maintained between mouse and rat. The SH2 domain and possible phosphorylation sites are especially closely conserved. These characteristic sequences are also conserved in human Lnk.2 In addition, the putative phosphorylation sites of Lnk served as satisfactory substrates in vitro for two protein-tyrosine kinases known to be involved in TCR signaling, Lck and ZAP-70. These observations support the hypothesis that Lnk participates in a TCR-coupled signaling pathway and that tyrosine phosphorylation is crucial for its functions. However, TCR cross-linking induced only very modest tyrosine phosphorylation of Lnk in both normal thymocytes and in thymocytes expressing high levels of transgene-derived Lnk protein. Phosphorylation of Lnk was also not prominent in splenic T cells from normal or Lnk transgenic mice.3 In light of these results, we believe that Lnk abundance in no way limits T cell responsiveness, perhaps because Lnk ordinarily participates in relaying signals other than those which we tested. It is also conceivable that the protein-tyrosine kinases responsible for Lnk phosphorylation, or binding sites that provide access to these protein-tyrosine kinases, are limited, such that only a fraction of endogenous Lnk protein can sustain phosphorylation.
Interestingly, the SH2 and N-terminal domains of Lnk are most
homologous to another 80-kDa adaptor-like molecule, SH2-B, that was
identified by virtue of its ability to bind a tyrosine phosphorylated ITAM of the FcRI
chain (52). Although SH2-B mRNA is not
expressed in splenocytes and the function of the SH2-B protein is
unknown, the structural similarity between SH2-B and Lnk, particularly in their unique N-terminal domains, suggests that these proteins participate in related signaling pathways. The availability of mouse
GST-Lnk fusion proteins should provide a basis for the identification of cellular proteins with which Lnk, and perhaps SH2-B, interact.
Our experiments also help to resolve the relationship between Lnk and
the long sought after pp36 protein, the prominent
tyrosine-phosphorylated species in TCR-stimulated T cells. These
proteins share similar molecular weights, become phosphorylated on
tyrosine, and bind (at least in vitro in the case of Lnk) to
the SH2 domains of Grb2, PLC-1, and phosphatidylinositol 3-kinase.
However, our results clearly demonstrate that Lnk and pp36 are
distinct. Anti-Lnk antibodies precipitate Lnk, but not pp36. On the
other hand, GST-Grb2 fusion proteins bind pp36 efficiently but barely
interact with Lnk. Finally, Lnk and pp36 differ with respect to
mobility in SDS-PAGE, and the extent to which each is phosphorylated
after TCR cross-linking. Enumeration of those proteins phosphorylated
during T cell activation has led to the discovery of ZAP-70 and SLP-76,
both of which play important roles in TCR signaling. Our results should
encourage renewed efforts aimed at the purification of pp36.
In general, previously studied
adaptor proteins, when expressed in cells at high levels, stimulate
dramatic changes in cell behavior. For example, augmented expression of
Shc triggers substrate-independent growth (i.e.
transformation) in NIH3T3 cells (60). Similarly, the magnitude of NFAT
translocation following TCR stimulation increases substantially in
Jurkat lymphoblasts expressing high levels of SLP-76, especially when
the Vav protein is also present at high levels (25, 27). We therefore
expected that augmented expression of Lnk in the thymocytes of
transgenic mice would influence the outcome of TCR stimulation, and
thereby perhaps perturb T cell development. In fact, Lnk expression in
transgenic thymocytes proved surprisingly benign. Massive accumulation
of Lnk, to levels 600-fold that ordinarily present, did not alter the
proliferative response of thymocytes to TCR stimulation, the pattern of
proteins phosphorylated on tyrosine, or the mobilization of
intracellular calcium (Fig. 7 and data not shown). In transgenic mice
expressing especially high levels of Lnk, the proportion of
CD4+8 mature thymocytes was slightly
decreased. This observation may provide a clue to the normal biological
function of Lnk, suggesting that it acts to relay signals received
during thymocyte differentiation. This effect cannot be explained by
proposing that accumulation of an SH2 domain-containing protein masks
important phosphotyrosine sequences indiscriminately. For example, the
endogenous level of Grb2 protein (about 2.3 × 104
molecules per cell) exceeds that of the transgene-encoded Lnk (at best
1.5 × 104 molecules per cell). The isolated SH2
domain of Grb2, but not that of Shc, is capable of inhibiting signal
transduction of Jurkat T cell transfectants (61). Regardless of the
specific basis for this phenotype, most Lnk transgenic mice manifest no
abnormalities, and in view of the very high levels of Lnk protein
required to provoke disturbances in thymocyte maturation, we cannot
comfortably conclude that T cell development ordinarily requires Lnk.
This question can be better addressed through the generation of mice bearing mutations in the Lnk gene.
Interestingly, Lnk is expressed in all thymocyte subsets, and the
abundance of Lnk mRNA remains constant throughout T cell development. Hence differential expression cannot serve to regulate thymocyte development. Lnk mRNA proved to be more abundant in B
cells than in T cells, which presumably explains why peripheral lymphoid organs and bone marrow contain more Lnk mRNA than does the
thymus (Fig. 2). A tyrosine-phosphorylated protein of 38 kDa that binds
to the Ig- chain cytoplasmic domain has been observed in a B
lymphoma line (62). Our experiments do not permit resolution of whether
this protein represents Lnk itself, or yet another protein migrating at
a similar apparent molecular weight. If a family of
phosphotyrosine-containing proteins of approximately 38 kDa exists in
lymphoid cells, the genes encoding these proteins differ very
substantially, since exhaustive screening of both cDNA and genomic
libraries with the Lnk cDNA yielded only Lnk sequences.
We mapped Lnk to mouse chromosome 5 at a position coincident with the NOS1 and Tbx3 gene. Given the close proximity of the Lnk gene with the NOS1 gene in the mouse genome, the human Lnk gene is probably located on 12q24 of chromosome 12 where the NOS1 gene has been mapped (58, 59). Interestingly, a t(5;12)(q21-q22:q22-q24) has been reported in Richter's syndrome, the most common type of acute transformation seen in patients with chronic lymphocytic leukemia (63), and trisomy 12 is the most common numerical anomaly in chronic lymphocytic leukemia (64). A dup(12)(q13-qter) has been reported in B cell-derived non-Hodgkin's lymphomas that do not carry the IgH/BCL2 recombination often seen in this disease (65). While numerous genes positioned on the long arm of chromosome 12 might contribute to the pathogenesis of these diseases, it is of interest that Lnk transcripts accumulate to high levels in B cells. Hence translocation of another gene into the Lnk locus could result in inappropriate B cell-specific transcription. Alternatively, although augmented expression of Lnk did not affect T cell proliferation, it remains possible that B cell signaling responds more emphatically to the presence of increased levels of Lnk.
Our studies indicate that Lnk, although expressed in T cells, does not subserve an important adaptor function in TCR signaling. Lnk has characteristics of what we take to be a large class of adaptor proteins, at a minimum including Shc, Grb2, SLP-76, Cbl, HS1, and almost certainly SH2-B, that must serve to organize signal transduction complexes from a variety of receptor structures in lymphoid cells. Since massive overexpression of Lnk does not perturb the function of the TCR pathway that recruits other, related adaptor proteins, proper interaction of adaptor proteins with their cognate receptors will require substantial specificity. This in turn suggests that genetic and biochemical strategies will ultimately prove effective in defining the signaling processes mediated by Lnk.
We are indebted to Xiao-Cun Pan for assistance in maintaining our mouse colony, to Sylvia Chien and Richard Peet for technical assistance, to Kathi Prewitt for expert secretarial assistance, to Kathryn J. Allen and David Coder for assistance in flow cytometry analysis, and to our colleagues for helpful discussions. We are also grateful to Lawrence E. Samelson (National Institutes of Health, Bethesda, MD) for ZAP-70 expressing cells, and to Ming Gu for MS analysis.