(Received for publication, December 23, 1996, and in revised form, February 25, 1997)
From the § Graduate Program in Immunology,
¶ Department of Internal Medicine, and Department of
Physiology and Biophysics, University of Iowa College of Medicine,
Iowa City, Iowa 52242, the ** BASF Bioresearch Corporation,
Worcester, Massachusetts 01605, and the
Howard Hughes Medical Institute, University
of California, San Francisco, California 94143
Previous work has demonstrated that SLP-76, a Grb2-associated tyrosine-phosphorylated protein, augments Interleukin-2 promoter activity when overexpressed in the Jurkat T cell line. This activity requires regions of SLP-76 that mediate protein-protein interactions with other molecules in T cells, suggesting that SLP-76-associated proteins also function to regulate signal transduction. Here we describe the molecular cloning of SLAP-130, a SLP-76-associated phosphoprotein of 130 kDa. We demonstrate that SLAP-130 is hematopoietic cell-specific and associates with the SH2 domain of SLP-76. Additionally, we show that SLAP-130 is a substrate of the T cell antigen receptor-induced protein tyrosine kinases. Interestingly, we find that in contrast to SLP-76, overexpression of SLAP-130 diminishes T cell antigen receptor-induced activation of the interleukin-2 promoter in Jurkat T cells and interferes with the augmentation of interleukin-2 promoter activity seen when SLP-76 is overexpressed in these cells. These data suggest that SLP-76 recruits a negative regulator, SLAP-130, as well as positive regulators of signal transduction in T cells.
Engagement of the T cell antigen receptor (TCR)1 results in the activation of protein tyrosine kinases (PTK) and the subsequent tyrosine phosphorylation of numerous proteins in T cells (1). Our efforts to characterize substrates of the TCR-induced PTK activity led to the cloning of SLP-76, a tyrosine-phosphorylated hematopoietic cell-specific protein that associates with the SH3 domains of Grb2 (2, 3). A possible function of SLP-76 in T cells was suggested by experiments showing that overexpression of SLP-76 augments TCR-mediated signals that lead to the induction of IL-2 gene promoter activity (4, 5). We have shown that the activity of SLP-76 requires engagement of the TCR and that overexpression of SLP-76 results in increased activation of the mitogen-activated protein kinase cascade following TCR ligation.2 Interestingly, three distinct regions of SLP-76 that are responsible for protein-protein interactions in T cells are required for its ability to augment IL-2 promoter activity when overexpressed (6, 7).2 These data suggest that SLP-76 functions as a link between proteins that regulate signals generated by TCR ligation.
To investigate the function of SLP-76 in T cells further, we and others have begun to characterize SLP-76-associated proteins that may participate with SLP-76 in transducing signals from the TCR to the nucleus. These proteins include Vav, which associates with the amino-terminal acidic region of SLP-76 in a phosphotyrosine-dependent manner (5, 8, 9); the adapter protein Grb2, which interacts with a proline-rich motif of SLP-76 via its SH3 domains (3, 4); and two unidentified tyrosine-phosphorylated proteins of 64 and 130 kDa and a serine/threonine kinase, all of which associate with the carboxyl-terminal SH2 domain of SLP-76 (4). In this study, we report the cloning of the cDNA encoding a 130-kDa protein (SLAP-130 for P-76 ssociated hosphoprotein of 130 kDa) that associates with the SH2 domain of SLP-76. Additionally, we provide evidence that SLAP-130 may function as a negative regulator of TCR signals that activate IL-2 gene transcription.
Jurkat T cells were maintained as described (10). JA2/SLP-SH2, a Jurkat T cell variant expressing an A2/SLP-SH2 chimera, was maintained in medium supplemented with 2 mg/ml geneticin (Life Technologies, Inc.).
cDNA ConstructsThe SH2 domain of SLP-76 was amplified
by PCR and subcloned in frame with HLA-A2 in pcDNA3/A2/HCP (10) to
generate pcDNA3/A2/SLP-SH2. The amino-terminal 1350 nucleotides of
SLAP-130 were amplified from Jurkat RNA and ligated in frame with the
FLAG tag in pEF/SLP-76 (4). The remaining 3 cDNA was amplified
from Jurkat RNA by overlap extension PCR. The resulting fragment
was ligated in frame with the amino-terminal 1350 bp in pEF above to
create pEF/SLAP-130. pEF/A2, the expression vector containing HLA-A2
cDNA, was a gift of B. Shraven (University of Heidelberg, Germany).
NFAT-luc was a gift of G. Crabtree (Stanford University, Palo Alto,
CA). The expressed sequence-tagged (EST) clone (I.M.A.G.E. consortium
ID number 241254) was obtained from Genome Systems, Inc. (St. Louis, MO).
Anti-A2 mAb CR11-351 (gift of C. Lutz, University of Iowa, Iowa City, IA). Anti-TCR mAb C305 (gift of A. Weiss, University of California, San Francisco, CA). Anti-FLAG mAb M2 (International Biotechnologies Inc., New Haven, CT). Anti-phosphotyrosine mAb 4G10 (Upstate Biotechnologies Inc., Lake Placid, NY). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-sheep antiserum (Bio-Rad). Anti-SLP-76 sheep antiserum has been described previously (4). Anti-SLAP-130 sheep antiserum was generated against a GST fusion protein containing amino acids 1-340 of human SLAP-130.
ImmunoprecipitationsCells were left unstimulated or stimulated with anti-TCR mAb (C305 acites, 1:1000) for the indicated times or pervanadate (11) for 1 min and lysed in Nonidet P-40 lysis buffer (10). For immunoprecipitations, antibodies were conjugated to GammaBind Plus Sepharose (Pharmacia Biotech Inc., Uppsala, Sweden) for 2 h at 4 °C. Lysates were subjected to precipitation with the indicated antibodies or GST fusion protein for 2 h at 4 °C. Precipitated complexes were washed four times in high salt lysis buffer (500 mM NaCl), resolved by reducing SDS-PAGE, and subjected to immunoblot analysis.
Cloning of Human pp130 cDNAA large scale anti-A2
immunoprecipitation of pervanadate-stimulated JA2/SLP-SH2 cells was
subjected to SDS-PAGE, transferred to polyvinylidene difluoride
membrane (Millipore, Bedford, MA), and visualized by Ponceau S
staining. The protein band of 130 kDa was excised and subjected to
trypsin digestion and reverse phase high performance liquid
chromatography for protein sequencing. Individual peptides were
sequenced using a Procise 492 protein sequencer (Perkin-Elmer/ABD,
Foster City, CA). One peptide sequence, PPNVDLTK, was represented in an
EST clone containing an open reading frame of 1074 bp. Nucleotides
1088-1266 (Fig. 1) were amplified by PCR from the EST and used to
screen a human thymus gt10 cDNA library (#NL1127a, Promega,
Madison, WI). A
clone was identified containing 1008 bp 5
of the
EST. The remaining cDNA was amplified from Jurkat cDNA by 3
RACE (rapid amplification of cDNA ends) using the Marathon cDNA
Amplification Kit (CLONTECH, Palo Alto, CA).
Independent amplification of the entire coding sequence of SLAP-130
from Jurkat RNA was performed by RT-PCR using the GeneAmp kit
(Perkin-Elmer).
Northern Analysis
Northern blot analysis was performed
following the protocols included with the human multiple tissue
Northern Blots I and II (CLONTECH). A SLAP-130 PCR
product (nucleotides 1088-1266) was labeled with
[-32P]dCTP (Amersham) by random priming and hybridized
to poly(A)+ RNA from multiple tissues.
Cells were washed
in phosphate-buffered saline and suspended in cytomix (12) at a
concentration of 2 × 107 cells/400 µl of
cytomix/cuvette. Cells were electroporated at 250V, 960µF using a
Gene Pulser (Bio-Rad) with 15 µg of NFAT-luc, 5 µg of
cytomegalovirus--galactosidase, and 40 µg of either pEF/SLP-76 or
pEF/SLAP-130. The total amount of plasmid DNA was equilibrated to 100 µg with the vector control pEF/A2. After 24 h, 5 × 105 cells were stimulated in triplicate for 10 h with
media or anti-TCR mAb C305 (ascites 1:1000). Additionally, triplicate
samples of 5 × 105 unstimulated cells were assayed
for
-galactosidase activity using the Galacto-Light Plus Reporter
Gene Assay System (Tropix Inc., Bedford, MA). Luciferase activity was
determined as described previously (10). Luciferase light units were
normalized to
-galactosidase activity present in each transfectant
to standardize for transfection efficiency.
We have shown previously that the SH2 domain of SLP-76 associates with two unidentified tyrosine-phosphorylated proteins upon stimulation of Jurkat T cells with either anti-TCR mAb or the protein tyrosine phosphatase inhibitor pervanadate (4). To facilitate purification of these SLP-76-associated proteins, we established a variant of the Jurkat T cell line, JA2/SLP-SH2, which expresses a chimeric surface protein consisting of the extracellular and transmembrane domains of the HLA-A2 molecule in frame with the SH2 domain of SLP-76. The A2 epitope enabled the isolation of proteins associated with the SLP-76 SH2 domain by immunoprecipitation with anti-A2 mAb.
JA2/SLP-SH2 cells were stimulated with pervanadate for maximal tyrosine
phosphorylation of proteins and lysed in Nonidet P-40 lysis buffer.
Anti-A2 immunoprecipitates were resolved on SDS-PAGE and transferred to
polyvinylidene difluoride membrane. A single major species of
approximately 130 kDa was excised and subjected to tryptic digestion to
generate peptides for protein sequencing. One peptide sequence,
PPNDVLTK, was represented in an EST clone. Sequencing this clone
revealed an open reading frame of over 1000 bp. A region of this clone
was then used to probe a human thymus gt10 cDNA library to
obtain further sequence.
A cDNA clone containing 370 bp of the EST sequence and an
additional 1008 bp of coding sequence 5 of the EST was isolated and
found to contain a putative start site 27 bases downstream of a stop
codon. A cDNA containing the remaining 3
-coding sequence was
amplified from Jurkat cDNA by 3
RACE and contained 267 additional bases of coding sequence followed by a stop codon 2349 bases downstream of the putative start. A contiguous cDNA was generated by RT-PCR amplification of pp130 from Jurkat RNA (Fig. 1). This
sequence was confirmed by sequencing the product of multiple
independent RT-PCR reactions. We have designated this protein SLAP-130
for P-76 ssociated
hosphoprotein of 130 kDa.
The complete open reading frame of SLAP-130 consists of 2349 bp translating to a protein of 783 amino acids with an abundance of proline (12%) and charged (31%) residues. Putative nuclear localization sequences are found within amino acids 480-503 and 683-700 (Ref. 13 and see Fig. 1). We do not know yet whether SLAP-130 is present in the nucleus of Jurkat T cells. There are several potential tyrosine phosphorylation sites in the carboxyl-terminal region of the molecule that may be responsible for the interaction of SLAP-130 with the SH2 domain of SLP-76. In addition, there are four tyrosines (residues 462, 595, 651, and 771) present in motifs that may mediate binding to the SH2 domain of src family kinases (14), suggesting that SLAP-130 may interact with proteins other than SLP-76 in T cells.
Tissue Distribution of SLAP-130Northern blot analysis of
human mRNA from multiple tissues was performed to determine the
tissue distribution of SLAP-130 (Fig. 2). As shown,
SLAP-130 mRNA is expressed in hematopoietic tissues (peripheral
blood mononuclear cells, spleen, and thymus) but not in non-lymphoid
tissues. Additionally, SLAP-130 was amplified as a single product from
total Jurkat RNA (data not shown), demonstrating expression in this
cell line.
Expression of cDNA Encoding SLAP-130 Results in a Protein That Migrates Similarly to pp130
Translation of the open reading frame
of the SLAP-130 cDNA predicts a protein with a molecular mass of 86 kDa. However, the presence of stop codons flanking both the 5 and 3
ends of the putative coding sequence suggested that we had isolated the
full-length cDNA encoding a 130-kDa, SLP-76-associated protein.
This was confirmed by generating an epitope (FLAG)-tagged version of
SLAP-130 cDNA (pEF/SLAP-130) for transient expression in Jurkat T
cells. As shown in Fig. 3A, transfection of
Jurkat T cells with pEF/SLAP-130 results in the expression of a protein
reactive with anti-FLAG mAb that migrates with an apparent molecular
mass of 130 kDa (lane 2). This protein does not appear in
lysates of cells transfected with the control vector pEF (lane
1). Whether the apparent migration of SLAP-130 at 130 kDa instead
of the predicted 86 kDa results from post-translational modifications
or the abundance of charged amino acids present in the molecule is not
yet known.
To investigate further whether the protein encoded by the SLAP-130 cDNA is identical to the 130-kDa, SLP-76-associated phosphoprotein, we prepared a GST fusion protein containing the SH2 domain of SLP-76 (GST/SH2). As shown in Fig. 3B, this fusion protein precipitates epitope-tagged SLAP-130 from pervanadate-stimulated Jurkat cells (lane 4), but not from resting cells (lane 3) transfected with pEF/SLAP-130. The loss of function mutant of the SLP-76 SH2 domain when expressed as a GST fusion protein (GST/R448K) fails to associate with FLAG-SLAP-130 in either resting or pervanadate-stimulated cells (lanes 1 and 2). The protein precipitated by the wild type SLP-76 SH2 domain migrates identically to a protein detected by anti-FLAG immunoblot analysis of an anti-FLAG immunoprecipitate (lane 5) or whole cell lysates (lane 6) from Jurkat cells transfected with pEF/SLAP-130. Additionally, the FLAG-SLAP-130 protein migrates identically to an endogenous tyrosine-phosphorylated protein precipitated by the SLP-76 SH2 fusion protein from untransfected Jurkat cells stimulated with pervanadate (lane 7).
SLAP-130 and SLP-76 Associate in CellsThe association of
SLP-76 and SLAP-130 in Jurkat T cells was investigated using
anti-SLAP-130 sheep antiserum. The anti-SLAP-130 antiserum, but not
preimmune serum, precipitates and immunoblots a protein of 130 kDa
in Jurkat T cell lysates (Fig. 4A, lanes 1-3). To determine if SLAP-130 and SLP-76 associate within cells, lysates were prepared from resting and pervanadate stimulated Jurkat
cells. These lysates were then subjected to immunoprecipitation with
anti-SLP-76, followed by immunoblot analysis with anti-SLAP-130. As
shown, SLAP-130 is present in SLP-76 immunoprecipitates from resting
Jurkat cells (Fig. 4A, lane 4). These results are consistent with the low levels of tyrosine-phosphorylated SLAP-130 seen in unstimulated Jurkat cells (see Fig. 4B) and our previous
finding of a 130-kDa phosphoprotein associated with SLP-76 in resting cells (4). However, this interaction is not always demonstrated in the
resting state, as shown in Fig. 3B. The amount of SLAP-130 associating with SLP-76 increases following stimulation of Jurkat with
pervanadate (lane 5).
We next investigated whether SLAP-130 is a substrate of the TCR-induced PTKs. Jurkat T cells were left unstimulated or stimulated with anti-TCR mAb or pervanadate. At the indicated time points whole cell lysates were subjected to immunoprecipitation with anti-SLAP-130 antiserum followed by immunoblot analysis with anti-phosphotyrosine mAb (Fig. 4B, top panel). As noted above, there is a basal level of tyrosine phosphorylation of SLAP-130 in resting Jurkat. TCR-induced tyrosine phosphorylation of SLAP-130 occurs maximally at 1 min and diminishes to basal levels within 15 min of TCR stimulation. To demonstrate equal loading of SLAP-130 in each lane, the anti-phosphotyrosine blot was stripped and immunoblotted with anti-SLAP-130 antiserum (Fig. 4B, lower panel).
Overexpression of SLAP-130 Interferes with TCR-mediated Activation of NFATWe and others have shown that overexpression of SLP-76
augments TCR signals leading to IL-2 promoter activity (4, 5). Since a
functional SH2 domain is required for SLP-76 activity in Jurkat T cells
(4, 7), we speculated that proteins associated with the SLP-76 SH2
domain would also regulate signals through the TCR in a positive
manner. To determine the effect of SLAP-130 on signals generated by TCR
ligation, Jurkat cells were transiently transfected with pEF/SLAP-130
or a control vector and a luciferase reporter construct driven by the
NFAT response element. Fig. 5A demonstrates
that, in contrast to the effect of SLP-76 on T cell signaling,
overexpression of SLAP-130 results in diminished NFAT activity
following TCR ligation. Furthermore, co-transfection of SLAP-130 and
SLP-76 reveals that overexpression of SLAP-130 inhibits the
augmentation of TCR-stimulated IL-2 promoter activity by SLP-76.
Expression of the FLAG-tagged cDNAs was confirmed by immunoblotting
whole cell lysates with anti-FLAG mAb.
We were surprised to discover that overexpression of SLAP-130 appears to interfere with TCR-induced NFAT activation in Jurkat cells and, additionally, inhibits the ability of transfected SLP-76 to augment TCR responses. Unfortunately, the primary sequence of SLAP-130 does not provide insight into how it may function as an inhibitor of TCR-induced NFAT activation. Structure-function experiments are ongoing to determine the regions of SLAP-130 required for its inhibitory effects.
Northern blot analyses indicate that SLP-76 and SLAP-130 are expressed coordinately in hematopoietic tissues. Defining their role in other hematopoietic cells may provide insight into how these proteins regulate T cell signals. We have found an association between the SLP-76 SH2 domain and a 130-kDa protein in rat basophillic leukemia cells (15). Since engagement of the high affinity receptor for IgE on rat basophillic leukemia cells leads to tyrosine phosphorylation of SLP-76 (15), experiments are ongoing to determine if overexpression of SLP-76 augments signaling events downstream of receptor binding in these cells. Similarly, it will be of interest to determine if overexpression of SLAP-130 impacts negatively on signal transduction via the IgE receptor.
It will be important to determine if the down-regulation of TCR-induced NFAT activation by overexpression of SLAP-130 requires an interaction between SLP-76 and SLAP-130. Experiments are in progress to determine the site on SLAP-130 responsible for its interaction with SLP-76. Manipulation of the SLAP-130 cDNA prior to transfection into Jurkat cells expressing wild type or overexpressed levels of SLP-76 will facilitate our understanding of how these two molecules may interact to regulate signals downstream of TCR engagement.