Immunoreceptors such as the high affinity IgE receptor, Fc⑀RI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated Fc⑀RI ITAM-containing subunits ␤ and ␥. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the Fc⑀RI ␤ or ␥ ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. Fc⑀RI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.The aggregation of immunoreceptors by antigen initiates a complex response leading to cellular activation (1). Receptors on T-, B-, and mast cells each contain subunits with similar primary amino acid sequence within their cytosolic domains, comprising the immunoreceptor-based tyrosine activation motif (ITAM), 1 whose consensus is (D/E)X 2 YXX(L/I)X 6 -8 YXX(L/I) (2, 3). Both tyrosine residues within the ITAM are rapidly phosphorylated by protein kinases after receptor aggregation. The bisphosphorylated ITAM then binds directly to cytosolic tyrosine kinases such as Syk in B-cells and mast cells and ZAP70 in T-cells, thereby activating their tyrosine kinase activity (4, 5). In mast cells, the Fc⑀RI subunits ␤ and ␥ each possess a single ITAM, which, when bisphosphorylated on tyrosine, binds to Syk (6 -8).We have used a novel genetic approach, the yeast tribrid system (8), to isolate cDNAs that encode proteins that interact with the tyrosine-phosphorylated Fc⑀RI ␥ ITAM. The yeast two-hybrid system facilitates the study of protein-protein interactions but is limited to the investigation of proteins that are properly expressed and modified in the host, Saccharomyces cerevisiae. S. cerevisiae does not employ tyrosine phosphorylation as a major regulatory modification of proteins (9, 10). This limits the utility of the two-hybrid system, especially in the area of signal transduction, where tyrosine phosphorylation is a critical component of the process. In order to study protein-protein interactions that are dependent on tyrosine phosphorylation or on other post-translational or allosteric modifications, the yeast tribrid system was developed (8, 11).In the yeast tribrid system, a third plasmid is introduced, which directs th...

Two classes of protein tyrosine kinases (PTK) are utilized by the T cell antigen receptor (TcR)/CD3 complex for initiation of the signaling cascade, the Src-family PTK p56lck and p59fyn, and the Syk-family PTK p70zap and p72syk. In addition, the CD45 phosphotyrosine phosphatase (PTPase) is required for the induction of tyrosine phosphorylation by the TcR/CD3, presumably by positively regulating Src-family PTK. Here we report that CD45 also regulates the Syk-family PTK p70zap (or ZAP-70). In CD45-negative T cells, p70zap was constitutively phosphorylated on tyrosine and co-immunoprecipitated with the TcR-zeta chain. In resting wild-type CD45-positive cells, p70zap was mainly unphosphorylated, but it was rapidly phosphorylated on tyrosine upon treatment of the cells with anti-CD3 or PTPase inhibitors. Finally, p70zap co-distributed with CD45 in intact T cells, and tyrosine phosphorylated p70zap was dephosphorylated by CD45 in vitro. These findings suggest that CD45 plays an important role, direct or indirect, in the regulation of p70zap and its function in TcR/CD3 signaling.

Activation of resting T lymphocytes through the T cell antigen receptor complex is initiated by critical phosphorylation and dephosphorylation events that regulate the function and interaction of a number of signaling molecules. Key elements in these reactions are members of the Src, Syk and Csk families of protein tyrosine kinases (PTKs) and the phosphotyrosine phosphatases (PTPases) that regulate and/or counteract them, such as CD45. The PTKs can autophosphorylate and phosphorylate each other at multiple sites and, as the result of these interactions, they are induced to phosphorylate other cellular proteins. These phosphorylation events lead to modulation of enzymatic activities and/or serve as binding sites for other signaling molecules having phosphotyrosine-binding Src homology 2 (SH2) domains. As a result, these proteins translocate to the receptor complexes and are juxtaposed to the kinases that phosphorylate them. Some of the SH2-domain-containing polypeptides lack enzymatic activities and, instead, serve as adapter molecules that couple the signal to downstream effectors, such as regulators of the Ras proteins, and further into serine/threonine-specific protein kinase cascades. Through largely unknown steps these reactions lead to the transcription of previously silent genes, activation of lymphocyte effector functions, progression through the cell cycle and cell proliferation.

A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated zeta-chain (TCR-zeta) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (Pl3K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-zeta may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering.