(Received for publication, December 15, 1994; and in revised form, January 12, 1995)
From the
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.
TCR ()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).
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.
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.
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].