Pearl River, NY, USA 3Corresponding author ZAP-70 is a protein tyrosine kinase (PTK) required for T-cell development and T-cell antigen receptor (TCR) function. ZAP-70 is associated with the phosphorylated antigen receptor and undergoes tyrosine phosphorylation following receptor activation. We demonstrate here that tyrosine phosphorylation of ZAP-70 results in an increase in its catalytic activity and that this activation is mediated by the phosphorylation of tyrosine residue 493 by the src family of PTKs. The activity of baculoviral expressed ZAP-70 was upregulated 10-fold when ZAP-70 was co-infected and phosphorylated by the src family PTK, lck. Mutation of Y493 alone abrogated the ability of ZAP-70 to be activated by Ick. Moreover, we demonstrate that phosphorylation of Y493 and activation of ZAP-70 is required for antigen receptor-mediated induction of interleukin-2 (IL-2) secretion in lymphocytes.

The threshold at which antigen triggers lymphocyte activation is set by the enzymes that regulate tyrosine phosphorylation. Upon T cell activation, the protein tyrosine phosphatase SHP-1 was found to bind to the protein tyrosine kinase ZAP-70. This interaction resulted in an increase in SHP-1 phosphatase activity and a decrease in ZAP-70 kinase activity. Expression of a dominant negative mutant of SHP-1 in T cells increased the sensitivity of the antigen receptor. Thus, SHP-1 functions as a negative regulator of the T cell antigen receptor and in setting the threshold of activation.

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Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Srcand Syk-PTKs, in T-and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70-and Syk-deficient lymphocytes (Syk ؊ DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T-and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.Stimulation of T and B lymphocytes through the T-cell receptor (TCR) and B-cell receptor (BCR), respectively, activates a cascade of protein tyrosine kinases (PTKs) that is required for lymphocyte cellular responses (5, 43). T and B cells appear to utilize analogous proximal signal transduction pathways in mediating these responses. Two distinct families of cytoplasmic PTKs, the Src-and Syk-PTK families, have been implicated in the proximal activation events for both BCRs and TCRs (6). In T cells, the Src-PTKs Lck and/or Fyn are thought to phosphorylate the two tyrosine residues within a 16-aminoacid motif (YXXLX 6-8 YXXL, the immunoreceptor tyrosinebased activation motif [ITAM]) located in the cytoplasmic domains of CD3 and chains. In B cells, the Src-PTKs Lyn, Fyn, and/or Blk are thought to phosphorylate the immunoglobulin ␣ chain (Ig␣) and Ig␤ ITAM sequences. Phosphorylation of the ITAM provides docking sites for the two SH2 domains within the Syk-PTKs (ZAP-70 and Syk) which associate with the phosphorylated TCR and BCR complexes. The phosphorylation and activation of ZAP-70 and Syk following TCR and BCR engagement, respectively, are in part mediated by the Src-PTKs. Coexpression of Lck or Fyn with ZAP-70 or Syk in insect and COS cells results in catalytic activation of the SykPTKs (7,9,10,12,19,24,38,41). Recent evidence also suggests that binding of Syk to the Ig␣, Ig␤, or FcεRI␥ ITAMs can mediate Syk activation and phosphorylation presumably by an allosteric mechanism (33, 34).Expression of dominant negative forms or deletion of Src or Syk-PTKs abrogates thymocyte development and/or antigen receptor function (2,3,8,11,14,21,28,29,35,36,39). Activation of both families of PTKs is thought to be required to activate at least two distinct pathways, ras and calcium, to mediate transcriptional activation of cytokine genes. Recent evidence in anergic T cells has demonstrated that activation of these two pathways may be differentially regulated (15, 25). We examine here the role of ZAP-70 phosphorylation and activation in regulating the divergent pathways activated by the l...

Assembly of intracellular macromolecular complexes is thought to provide an important mechanism to coordinate the generation of second messengers upon receptor activation. We have previously identified a B cell linker protein, termed BLNK, which serves such a scaffolding function in B cells. We demonstrate here that phosphorylation of five tyrosine residues within human BLNK nucleates distinct signaling effectors following B cell antigen receptor activation. The phosphorylation of multiple tyrosine residues not only amplifies PLCgamma-mediated signaling but also supports 'cis'-mediated interaction between distinct signaling effectors within a large molecular complex. These data demonstrate the importance of coordinate phosphorylation of molecular scaffolds, and provide insights into how assembly of macromolecular complexes is required for normal receptor function.