Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases.

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 TCRinduced PTK activity led to the cloning of SLP-76, a tyrosinephosphorylated 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 SLP-76 associated phosphoprotein 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.

MATERIALS AND METHODS
Cells and Cell Culture-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 Constructs-The 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 3Ј 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 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.
Immunoprecipitations-Cells 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 cDNA-A 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 5Ј of the EST. The remaining cDNA was amplified from Jurkat cDNA by 3Ј 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 [␣-32 P]dCTP (Amersham) by random priming and hybridized to poly(A) ϩ RNA from multiple tissues.
Transfections and NFAT Luciferase Assays-Cells were washed in phosphate-buffered saline and suspended in cytomix (12) at a concentration of 2 ϫ 10 7 cells/400 l of cytomix/cuvette. Cells were electroporated at 250V, 960F using a Gene Pulser (Bio-Rad) with 15 g of NFAT-luc, 5 g of cytomegalovirus-␤-galactosidase, and 40 g of either pEF/SLP-76 or pEF/SLAP-130. The total amount of plasmid DNA was equilibrated to 100 g with the vector control pEF/A2. After 24 h, 5 ϫ 10 5 cells were stimulated in triplicate for 10 h with media or anti-TCR mAb C305 (ascites 1:1000). Additionally, triplicate samples of 5 ϫ 10 5 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.

RESULTS AND DISCUSSION
Cloning of cDNA Encoding SLAP-130 -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 se-quence, 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 5Ј 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 3Ј-coding sequence was amplified from Jurkat cDNA by 3Ј 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 SLP-76 associated phosphoprotein 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-130 -Northern 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.  (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).

Expression of cDNA Encoding SLAP-130 Results in a Protein
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 NFAT-We 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 FIG. 1. Amino acid and cDNA sequence of SLAP-130. The 130-kDa protein which co-precipitates with the SH2 domain of SLP-76 was subjected to tryptic digestion for protein sequencing. A peptide containing the sequence PPNDVLTK (underlined) was found to be encoded by an EST clone which was used to isolate coding sequence 5Ј of the EST from a human thymus cDNA library. The remaining cDNA was amplified by 3Ј RACE using total RNA from Jurkat T cells. The sequence shown represents a contiguous cDNA clone amplified from Jurkat RNA by PCR. SLAP-130, a Novel Substrate of the TCR-stimulated PTK 11676 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.