T cell receptor (TcR)͞CD3 ligation initiates a signaling cascade that involves src kinases p56 lck and -associated protein 70, leading to the phosphorylation of substrates such as TcR, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120͞130. FYN binding protein (FYB or p120͞130) associates with p59 fyn , the TcR͞ CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine͞glutamic acidrich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59 fyn , FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcR͞CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

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-gamma 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.

Engagement of the T cell antigen receptor (TCR)1 results in the sequential activation of the Src (p56 lck /p59 fyn ) and Syk (Syk/ZAP-70) families of protein-tyrosine kinases (PTKs) (1-3). Both families of PTKs are required for normal T cell development and function (4 -9). In resting T cells, the TCR chain is constitutively phosphorylated and associated with 11). Tyrosine phosphorylation of the receptor-associated ZAP-70 by p56 lck is a requisite modification resulting in the up-regulation of ZAP-70 catalytic activity (12, 13). Therefore, the recruitment of the CD4 and CD8 co-receptors into the TCR complex positions p56 lck to trans-phosphorylate ZAP-70. Phosphorylation and activation of ZAP-70, in turn, is required for both an increase in phosphoinositide metabolism and activation of the ras pathway.2 The integration of these downstream signals gives rise to transcriptional activation of cytokine genes and a resultant elevation in cytokine synthesis and secretion (15). While the up-regulation of ZAP-70 catalytic activity is required for TCR function, little is known about the cellular proteins which serve as substrates for this PTK.SLP-76 is a recently identified molecule which undergoes tyrosine phosphorylation upon TCR cross-linking (16 -19). This protein associates with both the SH3 domain of Grb2 and an SH2 domain of PLC␥. SLP-76 is structurally characterized by a C-terminal SH2 domain, a region enriched in proline residues which probably serves as the site for Grb2 binding, and an N-terminal motif which contains three tandemly repeated DYE(S/P)P sequences. We demonstrate here that SLP-76 is phosphorylated by ZAP-70 and that phosphorylation of these repeated tyrosine motifs is required for optimal generation of IL-2 in response to TCR ligation. In addition, overexpression of SLP-76 augments TCR-mediated transcriptional activation of the IL-2 gene, while mutation of the SH2 domain attenuates this response. Together, these studies identify SLP-76 as a physiologic substrate for ZAP-70 and suggest a mechanism by which TCR-induced activation of ZAP-70 regulates both the calcium and ras pathways. EXPERIMENTAL PROCEDURESCells and Antibodies-Jurkat and Sf9 cells (Pharmingen) were maintained as described previously (12). The mouse monoclonal antibody (mAb) H3 was generated against the SLP-76 SH2 domain. SLP-76 polyclonal antisera (22652) was generated against a peptide spanning amino acids 301-318 of human SLP-76. C305 is an anti-Jurkat Ti␣/␤-mAb (20); 4G10 (UBI) and PY20 (Santa Cruz) are anti-phosphotyrosine mAbs; 2F3.2 is an anti-ZAP-70 mAb (UBI), and 9E10 is an anti-myc mAb. Anti-GST mAb was purchased from Santa Cruz.Construction of Plasmids-A full-length SLP-76 cDNA was generated by reverse transcription PCR from murine T-cell mRNA. This cDNA was appended with a myc-epitope at the 3Ј end of the coding cDNA. Mutations were generated by PCR-directed mutagenesis and confirmed using standard dideoxy sequencing methods. These constructs were then subcloned into the pApuro vector (21) and the baculoviral vector pVL13...

The T cell receptor (TCR), 1 B cell receptor, and mast cell receptor (Fc⑀RI) are composed of multiple subunits, enabling the coupling of a variable antigen-recognition subunit to conserved signaling modules (1-3). Collectively, these receptors direct the antigen-specific responses of T cells, B cells, and mast cells. Although these antigen receptors do not contain intrinsic tyrosine kinases, the early events downstream of these antigen receptors are controlled by a relatively conserved series of tyrosine kinase-dependent events. In each case, Src family kinases initiate the signaling cascade by phosphorylating immunoreceptor tyrosine-based activation motifs present in the cytoplasmic tails of the signal-transducing receptor subunits. Complete immunoreceptor tyrosine-based activation motifs phosphorylation results in the recruitment of Syk/ZAP-70 family tyrosine kinases to the receptor. These kinases are subsequently activated by Src kinase-mediated transphosphorylation. The substrates of the Syk kinases include internal phosphorylation sites as well as transphosphorylation sites in LAT, SLP-76, and BLNK, a SLP-76-related adapter protein (1, 4 -7). These Syk kinase-dependent phosphorylations direct the recruitment of additional effector and adapter molecules to LAT, SLP-76, BLNK, and the Syk kinases themselves (7-13). In T cells LAT, which is recruited into lipid rafts by dual palmitoylation, nucleates a raft-associated complex containing multiple effectors, including Grb2 and Sos, Cbl, Vav, PLC␥1, and PI3K (5,14,15). The perturbation of this complex by the disruption of Lck, ZAP-70, LAT, or SLP-76 results in profound defects in the activation of calcium influxes and mitogen-activated protein kinases (MAPKs) downstream of the TCR (16 -20). Similar complexes may form in both B cells and mast cells.The Tec family kinases (Tec, Itk, Btk, Bmx, and Txk (21,22)) also participate in signal transduction pathways downstream of antigen receptors (3,(23)(24)(25)(26)(27)(28)(29)(30)(31). In particular, Tec kinases have been observed to contribute to the PI3K-dependent phosphorylation and activation of PLC␥ isoforms, the concomitant induction of sustained calcium influxes (32), and the activation of MAPKs (33-35). However, the mechanisms that integrate the Tec kinases into antigen receptor-dependent signaling pathways are not yet well understood. Structurally, these kinases share the conserved arrangement of SH3, SH2, and kinase domains found in Src kinases, but are distinguished by the absence of amino-terminal acylations, the absence of the carboxyl-terminal regulatory tyrosine, and by the presence of a conserved amino-terminal region containing a pleckstrin homology (PH) domain, a Btk homology motif, and a proline-rich region (PRR). Although the functions of the protein-binding domains of Tec kinases remain unclear, the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) binding pocket present in Tec kinase PH domains is clearly required for the cooperative activation of PLC␥ isoforms by PI3K and Tec kinases (32)...