©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of SLP-76, a 76-kDa Tyrosine Phosphoprotein Associated with Grb2 in T Cells (*)

(Received for publication, December 15, 1994; and in revised form, January 12, 1995)

Janet K. Jackman (§) David G. Motto(§) (2) Qiming Sun Masayuki Tanemoto Chris W. Turck (1) Gary A. Peltz Gary A. Koretzky (3)(¶) Paul R. Findell (**)

From the  (1)Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94304, theDepartment of Medicine, Howard Hughes Medical Institute, University of California, San Francisco, California 94143, and the Departments of (2)Physiology and Biophysics and (3)Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.


INTRODUCTION

TCR (^1)activation triggers a cascade of intracellular biochemical events that ultimately converge upon the nucleus to induce lymphokine gene expression and cell proliferation (1) . The earliest detectable biochemical event seen after TCR ligation is the activation of a set of cytoplasmic PTKs and the subsequent phosphorylation of a number of substrates(2, 3, 4) . TCR ligation also results in the activation of the Ras signaling pathway(5, 6, 7, 8) , which is essential for cytokine production by T cells(6) . Work by a number of laboratories studying various PTK receptors has demonstrated that Grb2, a highly conserved molecule consisting of two SH3 domains flanking a single SH2 domain, functions as an adaptor protein linking PTK activation with Ras signaling(9, 10, 11, 12, 13, 14, 15, 16, 17) . We and others have recently shown that Grb2, in addition to binding to SOS and Shc, also associates with several unidentified tyrosine phosphoproteins (pp36/38, pp76, and pp116) present in activated T cells(18, 19, 20, 21) . These phosphoproteins bind to different domains of Grb2, and it has been proposed that proteins bound to one domain of Grb2 influence interactions mediated through its other domains(19, 21) . Although both the in vitro and in vivo associations of Grb2 with each of these phosphoproteins is well documented, their molecular identities and function in TCR-mediated signal transduction remain unclear. In this report we describe the purification of the 76-kDa Grb2-associated tyrosine phosphoprotein from human T lymphocytes and the molecular cloning of its cDNA, and show by Northern analysis that pp76 is also expressed in B cell and monocytic cell lines. pp76 cDNA encodes a 533-amino acid protein with a single carboxyl-terminal SH2 domain and no putative enzymatic activity. We have termed this novel molecule SLP-76, for SH2 domain-containing Leukocyte Protein. Additionally, we provide evidence that Grb2, through its association with SLP-76, may play a role in the activation of phospholipase C-1 (PLC-1).


MATERIALS AND METHODS

Reagents, Antibodies, and GST Fusion Proteins

Anti-TCR mAb C305 was kindly provided by Dr. A. Weiss (UCSF, San Francisco, CA). Anti-phosphotyrosine (Tyr(P)) mAb and rabbit polyclonal antibody specific for human PLC-1 were generated at Syntex Discovery Research, Palo Alto, CA. Anti-Tyr(P) mAb 4G10 was purchased from Upstate Biotechnologies, Inc. cDNAs corresponding to the SH2 domain of pp76 (amino acids 422-514) or full-length GRB2 (amino acids 2-217; (9) ) were amplified by PCR and cloned into pGEX vectors (Pharmacia Biotech Inc.). Fusion proteins were expressed and affinity-purified using glutathione-agarose beads (Sigma) as described(22) . A GST fusion protein with SH3, C-SH2, and N-SH2 domains of PLC-1 (amino acids 530-850) was purchased from Santa Cruz Biotechnology, Inc.

Cells and Cell Stimulation

Human Jurkat, SKW3, HUT78, Ramos HL60, THP-1, U937, and JY cells were grown in RPMI supplemented with FCS and antibiotics. The culture of RAM and LIL cells was described previously(23) . IMR-32 and HeLa-Ohio cells were grown in Eagle's minimum essential medium supplemented with fetal calf serum. Jurkat cells were stimulated for 1 min with either anti-TCR mAb C305 or with 0.2 mM Na(3)VO(4), 8 mM H(2)O(2) (pervanadate)(21) , which mimics the effects of TCR ligation in Jurkat cells(24) .

Purification of pp76

40 times 10^9 Jurkat cells were stimulated with pervanadate, lysed in buffer containing 150 mM NaCl, 1% digitonin, 10 mM Tris-Cl, pH 7.4, 1 mM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and leupeptin and the lysates clarified by centrifugation. Lysates were precleared over protein A, and Tyr(P) proteins were immunoprecipitated with anti-Tyr(P) mAb. Tyrosine phosphoproteins were eluted with phenyl phosphate (0.1 M) and then incubated with the GST-GRB2 affinity support. The fusion protein complex was washed and bound proteins were released by heating in 0.5% SDS. The eluant was diluted into immunoprecipitation buffer and re-precipitated with anti-Tyr(P) mAb. Immunopreciptates were eluted with phenyl phosphate (0.1 M), concentrated, and subjected to SDS-PAGE with subsequent electroblotting onto a polyvinylidene difluoride membrane (Millipore). Proteins were visualized by Ponceau S staining and the excised protein band subjected to trypsin digestion. Tryptic peptides were then separated by reverse phase high performance liquid chromatography and individual peaks sequenced with a model 475A Protein Sequencer (Applied Biosystems).

Cloning of the Human and Murine pp76 cDNA

Degenerate oligonucleotides (sense: 5`-ACI GA(A/G) AA(T/C) GA(T/C) ATI CA(A/G) AAG-3`, antisense: 5`-(A/G)AA IGG (C/T)TC (A/G)TA (A/G)TC (A/G)AA IGG-3`; and sense: 5`-GCI CCI TT(T/C) GA(T/C) TA(T/C) GAA CC-3`, antisense: 5`-C(G/T) IAC IA(A/G) (G/A)AA IGT ICC (A/G)TC-3`) derived from tryptic peptides 2, 5, and 8 (shown in Fig. 2) were synthesized in a Millipore Expedite(TM) DNA synthesizer. Total cellular RNA was prepared from Jurkat cells using RNAzol (TEL-TEST, Inc.) and mRNA isolated using the Magnesphere(TM) PolyATtract® mRNA isolation system (Promega). cDNA was synthesized with an oligo(dT) primer using the Copy Kit(TM) (Invitrogen). cDNA was size-fractionated on a 1% agarose gel, and cDNAs 2-3 kb in size were isolated. Forty-cycle PCR amplification with a denaturing temperature of 94 °C for 1 min, an annealing temperature of 40 °C for 2 min with a slow 2-min rise to an extension temperature of 72 °C for 3 min for the first five cycles, followed by 35 cycles of 94 °C for 1 min, 45 °C for 2 min, and 72 °C for 3 min was performed using the Gene Amp PCR system (Perkin Elmer). Two 600-base pair PCR products, which contained sequences corresponding to pp76 peptides, were identified, labeled with [P]dCTP, and used as probes to screen a gt10 Jurkat cDNA library (Clontech) under high stringency hybridization conditions. Thirty-three clones were identified and their inserts subcloned into Bluescript SK(+) phagemid (Stratagene). Southern analysis of inserts was used to further screen clones. Seven hybridizing inserts (0.6-1.4 kb) were obtained and sequenced. All clones overlapped, had identical 3`-ends and generated a single, 1.0-kb open reading frame representing a partial coding region. The longest clone contained a 0.8-kb insertion of unidentified sequence, which was probably a cloning artifact. To obtain the 5`- and 3`-ends of pp76 cDNA, PCR amplifications were performed using the 5`- and 3`-Ampli-FINDER(TM) RACE (rapid amplification of cDNA ends) systems (Clontech) using anchor primers supplied by the manufacturer and three different nested gene-specific primers. Consensus sequences were determined from the three independent 5`- and 3`-end PCR amplifications. The combined sequences generated a cDNA of 2.1 kb. Oligonucleotides derived from this sequence were used in PCR reactions using Jurkat cell cDNA to obtain a full-length cDNA clone. DNA sequence was determined for both strands of the clone.


Figure 2: Predicted amino acid sequence of pp76. A, alignment of the predicted amino acid sequences of human (Hu) and murine (Mu) pp76. Dashes indicate identical amino acids; dots indicate gaps introduced for optimal alignment. Peptides (numbered 1-9) obtained from the tryptic digestion of pp76 are underlined. The darkshaded area outlines three 17-amino acid repeats with conserved tyrosine and acidic residues (boldtype). The lightershaded area outlines the SH2 domain of pp76, with boldtype indicating the invariant residues present in other SH2 domains. B, the 17-residue repeats with conserved tyrosine residues present in human and murine pp76 and in human p75(27) . The tyrosine residues are preceded by acid residues and are potential PTK phosphorylation sites.



The PCR fragments were also used to screen a Uni-ZAP XR E4L murine T cell library (Stratagene) under low stringency. Thirty clones were identified. Southern analysis of excised inserts was used to further screen clones. Six hybridizing inserts (1.6-2.3 kb) were chosen for further analysis. DNA sequence was determined for both strands of the longest clone, which represents a full-length cDNA. DNA sequencing of the remaining five clones indicated that they overlapped and were derived from the same cDNA.

In Vitro Transcription and Translation of pp76 cDNA

A cDNA for murine pp76 (nucleotides 1-2269) was transcribed and translated in vitro using the Stratagene mCAP(TM) RNA capping and In Vitro Express(TM) reticulocyte systems and labeled with [S]methionine (>1000 Ci/mmol) following procedures recommended by the supplier. Reaction products were resolved by 12.5% SDS-PAGE and the proteins detected by autoradiography.

Northern Blot Analysis

Total RNA was isolated from from SKW3, Jurkat, HUT78, JY, Ramos, RAM, LIL, U937, THP1, HL60, HeLa Ohio, and IMR32 human cell lines, size-fractionated on 1% agarose, 2.2 M formaldehyde gels, and transferred to nitrocellulose membranes. Nitrocellulose membranes containing poly(A)-enriched mRNA from human tissues was purchased from Clontech. Membranes were probed at high stringency with a 1.2-kb P-labeled cDNA fragment of pp76.

Protein Precipitation and Immunoblotting

Coprecipitation of proteins with GST-fusion proteins, immunoprecipitations, and immunoblotting were performed as described previously(21) . For immunoprecipitations, anti-PLC-1 antibody was used at a concentration of 5 µg/ml cell lysate. For immunoblotting, anti-Tyr(P) mAb was used at a 1:300 dilution.


RESULTS AND DISCUSSION

Stimulation of Jurkat cells with anti-TCR antibody results in the rapid tyrosine phosphorylation of 36/38-, 76-, and 116-kDa proteins, which coprecipitate with a GST/Grb2 fusion protein (Fig. 1A). These proteins were affinity-purified from a lysate of activated Jurkat cells, using anti-phosphotyrosine mAb and a GST-Grb2 fusion protein (Fig. 1B). After separation by SDS-PAGE, the tyrosine phosphoproteins were transferred to polyvinylidene difluoride membranes for microsequencing. Amino acid sequences were obtained from nine peptide fragments of pp76 (indicated in Fig. 2) and five peptide fragments of pp116 (data not shown). Analysis of the partial amino acid sequence of pp116 indicated that it is a novel protein distinct from the 120-kDa tyrosine phosphoprotein product of the c-cbl proto-oncogene, which is also expressed in Jurkat cells(25) .


Figure 1: Purification of Grb2-associated tyrosine phosphoproteins in TCR-activated Jurkat cells. A, Jurkat T cells were stimulated for 1 min with anti-TCR mAb or left unstimulated. Proteins in cell lysates coprecipitated by a GST-Grb2 fusion protein were subjected to SDS-PAGE and immunoblotted with anti-Tyr(P) mAb 4G10. The position of pp76 is indicated by an arrowhead. B, purified Grb2-associated proteins were analyzed by SDS-PAGE and anti-Tyr(P) (4G10) immunoblotting (lane1) or silver staining (lane2). The relative mobility of molecular size markers is shown in kilodaltons on the left.



A cDNA encoding human pp76 was isolated from a Jurkat cell cDNA library using oligonucleotides based on tryptic peptide sequences. This cDNA has a single predicted open reading frame, starting from an ATG codon with a consensus initiation sequence(26) , giving rise to a 533-amino acid polypeptide that contains all nine of the tryptic peptide sequences. The predicted amino acid sequence does not contain domains homologous to known tyrosine, lipid, or serine/threonine kinases. However, it does have an SH2 domain at its carboxyl terminus, with multiple amino acids identical to those invariantly present in SH2 domains of other proteins (indicated in Fig. 2A). We have termed this protein SLP-76. A 17-amino acid motif, in which 3 acidic amino residues (EDD) precede a conserved tyrosine-containing sequence (DYE(S/P)P), is tandemly repeated three times in this protein (amino acids 109-157) (Fig. 2B). A similar motif is also found in p75, a B-cell protein which has a carboxyl-terminal SH3 domain and is tyrosine-phosphorylated in response to cross-linking membrane-bound IgM(27, 28) . Phosphorylation of tyrosine residues within this motif may mediate interactions between SLP-76 and other T cell effector molecules.

A cDNA encoding the murine homologue of SLP-76 was isolated from a cDNA library prepared from a murine T cell line (EL4). The murine cDNA also encodes a predicted 533-amino acid protein (Fig. 2A). Alignment of murine and human SLP-76 nucleic acid (80% identity) and predicted protein sequences (84% identity) indicated a very high degree of sequence homology.

The SLP-76 cDNAs predict a 61-kDa polypeptide, significantly less than the protein's mobility of 76 kDa on SDS-PAGE. Translation of SLP-76 cDNA in vitro generates a single protein of approximately 76 kDa on SDS-PAGE (Fig. 3). Grb2, expressed as a GST fusion protein, precipitated this 76-kDa translation product. Precipitation was not seen with GST alone. The aberrantly slow mobility of SLP-76 by SDS-PAGE may result from the fact that SDS binds poorly to acidic proteins(29) , and SLP-76 has a highly acidic 61-amino acid region (residues 93-154).


Figure 3: Grb2 binds the in vitro translation product of pp76 cDNA. Murine pp76 cDNA was transcribed and and then translated with reticulocyte lysate in the presence of [S]methionine. Reaction products were resolved by 12.5% SDS-PAGE and proteins detected by autoradiography. Lane1, translated product in the absence of RNA; lane2, translated product using control RNA (the 15-kDa protein is not shown); lane3, translated product using pp76 RNA; lane4, pp76 RNA translation product purified with GST-Grb2 fusion protein; lane5, pp76 RNA translation product purified with GST alone.



Human and murine SLP-76 mRNA demonstrate an identical pattern of tissue-specific expression (murine data not shown). A 2.6-kb human SLP-76 mRNA transcript is abundantly expressed in human spleen, thymus, and peripheral blood leukocytes (Fig. 4A). Low level expression was noted in placenta and lung, probably due to the presence of leukocytes in these highly vascularized tissues. In the human cell lines examined, SLP-76 mRNA was abundant in T cell and monocytic cell lines, a low level of expression was noted in B cells, and was not found in fibroblast or neuroblastoma cell lines (Fig. 4B). Characterization of the human SLP-76 gene is continuing. Preliminary data from the Southern analysis of restriction enzyme-digested human genomic DNA indicate that SLP-76 is encoded by a single copy gene (data not shown).


Figure 4: pp76 expression in human tissues and cell lines. P-Labeled pp76 or G3PDH cDNA was hybridized with mRNA purified from the indicated human tissues (A) or with total RNA prepared from human T cell (SKW3, Jurkat, HUT78), B cell (JY, Ramos, RAM, LIL), monocytic (U937, THP1, HL60), fibroblast (HeLa), and neuroblastoma (IMR32) cell lines (B). DNA molecular weight markers are indicated on the left in each blot.



The SH2 domain of SLP-76 may interact with other cytoplasmic proteins involved in signal transduction. In cell lysates prepared from resting and TCR-activated Jurkat cells, two tyrosine phosphoproteins of 64 and 116 kDa coprecipitated with a GST fusion protein containing the predicted SH2 domain of SLP-76 (Fig. 5). These two coprecipitating proteins had the same electrophoretic mobilities as tyrosine phosphoproteins associating with Grb2 (Fig. 5).


Figure 5: pp76 is associated with other T cell effector molecules. Lysates were prepared from resting or anti-TCR mAb-activated Jurkat cells. Proteins coprecipitating with GST alone or GST fusion proteins containing either Grb2, the SH2 domain of pp76, or the SH3 and SH2 domains of PLC-1; proteins immunoprecipitating with an anti-PLC-1 antisera were separated by 12.5% SDS-PAGE and immunoblotted with anti-Tyr(P) mAb. The prominent 36/38-, 76-, and 116-kDa tyrosine phosphoproteins are indicated by arrowheads.



It was also of interest to determine if SLP-76 interacts with other cytoplasmic effector molecules. A tyrosine phosphoprotein of 74 kDa, present in lysates of activated T cells, has been reported to associate with the SH2 domains of PLC-1(30) . To investigate the possibility that SLP-76 associates with PLC-1, coprecipitation experiments were performed using PLC-1-specific antisera and a GST fusion protein containing SH2 and SH3 domains of PLC-1. Both reagents coprecipitate a tyrosine phosphoprotein from activated, but not resting Jurkat cell lysates, whose electrophoretic mobility was identical to that of Grb2-associated SLP-76 (Fig. 5). Similarly, proteins with electrophoretic mobilities identical to that of Grb2-associated pp36/38 and pp116 also coprecipitate with the PLC-1 reagents. These results are consistent with previous studies(20, 30) .

We have reported the molecular cloning of SLP-76, a cytoplasmic protein expressed solely in leukocytes, which is rapidly phosphorylated following TCR stimulation. In vitro translated SLP-76 associates with a GST/Grb2 fusion protein, demonstrating a direct interaction between these two molecules. Further experiments demonstrated that the SH2 domain of SLP-76 expressed as a GST fusion protein precipitates two tyrosine phosphoproteins of 64 and 116 kDa from lysates of stimulated Jurkat cells. The 116-kDa protein migrates by SDS-PAGE with an identical mobility to pp116, a protein we have recently identified that associates with Grb2 (Fig. 1A and (21) ). Experiments utilizing GST/PLC-1 fusion proteins and PLC-1-specific antisera demonstrated that PLC-1 also may exist complexed with proteins possessing electrophoretic mobilities identical to SLP-76, pp116, and a third protein, pp36/38, which also associate with Grb2 (Fig. 1A and Fig. 5and (20) ). Future experiments utilizing specific immunological reagents directed against SLP-76, pp116, and pp36/38 will be necessary to address the possibility of a large multiprotein complex consisting of some or all of these proteins. Our data, along with others (20) have raised the interesting possibility that in addition to potentially participating in Ras activation, Grb2, in association with SLP-76 (and possibly pp116 and pp36/38), may play a role in PLC-1 activation in T lymphocytes.


FOOTNOTES

*
This work was supported by Syntex Discovery Research and by National Institutes of Health Training Grant HL07638 (to D. G. M.) and the Carver Trust at the University of Iowa (to G. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U20158 [GenBank]and U20159[GenBank].

§
These authors contributed equally to this work.

Established Investigator of the American Heart Association.

**
To whom all correspondence should be addressed: Syntex Research, MS S3-10, 3401 Hillview Ave., Palo Alto, CA 94303. Tel.: 415-855-6080; Fax: 415-354-7554.

(^1)
The abbreviations used are: TCR, T cell antigen receptor; PTK, protein tyrosine kinase; GST, glutathione S-transferase; Tyr(P), phosphotyrosine; SH2, Src homology 2; SH3, Src homology 3; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PLC-1, phospholipase C-1; PCR, polymerase chain reaction; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Dr. Chinh Bach, Patricia Zuppan, and Calvin Yee for help with the DNA sequencing of pp76 clones. We also thank Dr. Arthur Weiss for the generous gift of anti-TCR mAb C305.


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