Distinct Tyrosine Phosphorylation Sites in ZAP-70 Mediate Activation and Negative Regulation of Antigen Receptor Function

Abstract

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...

Quantitative Analysis of Phosphotyrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells

“…It is likely that activation of an alternate threonine-specific phosphatase could occur during CD3/CD28 costimulation, potentially delaying full activation of MAPKs while accelerating cell cycle entry by enhancing singly phosphorylated Cdc2 via decreased threonine phosphorylation. One outcome of T cell activation is cell cycle entry (39), and, at the G 2 -M boundary, Cdc2 becomes dephosphorylated at both Thr 14 and Tyr 15 , resulting in entry into mitosis (40). Complementary information on phosphoserine/threonine protein networks and time-dependent phosphotyrosine activation may answer these questions, but network level quantification for low abundant phosphoserine/threonine proteins remains a challenging task due to the complexity and dynamic range of the serine/threonine phosphoproteome (41).…”

“…3, B and C). In combination with phosphorylation of other downstream proteins, it seems that phosphorylation at Tyr 292 in Zap70 corresponded with activation of the TCR pathway in this system, although it has been previously implicated in negative regulation using a mutated construct (15). Hierarchical clustering and selforganizing maps clustered Zap70 Tyr 292 with CD3 ITAMs and PLC-␥ (Fig.…”

Immunohistochemical detection of ZAP-70 in 341 cases of non-Hodgkin and Hodgkin lymphoma

“…Subsequent phosphorylation of ZAP-70 triggers downstream signaling events that culminate in transcriptional induction of genes that regulate cell function, proliferation and death. 5,6 Similar proximal events including phosphorylation of ITAMS on the immunoglobulin a/b dimer by Src protein tyrosine kinases, and subsequent activation of Syk protein tyrosine kinases have been implicated in the B cell receptor signaling pathway. Mature B cells, however, generally lack ZAP-70 and use the related protein tyrosine kinase, Syk.…”

“…Mature B cells, however, generally lack ZAP-70 and use the related protein tyrosine kinase, Syk. 5 Recent investigations suggest that ZAP-70 is expressed in early B-cell development and plays a role in the transition of pro-B to pre-B-cell lymphocytes. 1 Additional studies demonstrated increased tyrosine phosphorylated proteins and association of ZAP-70 with IgM and CD79b after ligation of the Bcell receptor in ZAP-70-positive CLL/SLL.…”

Molecular characterization and expression of ZAP-70 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus

“…In mammal, full catalytic activity of ZAP-70 requires phosphorylation of Y 493 in the activation loop of the kinase domain (Chan et al 1995), and phosphorylated Y 315 and Y 319 in interdomain B are positive regulators of ZAP-70 activity, with the function of stabilization of the active conformation (Di Bartolo et al 2002Gelkop et al 2005;Williams et al 1999). In addition, phosphorylation of specific tyrosine residues, such as Y 292 in interdomain B and Y 492 in the kinase domain, has been implicated in negative regulation of ZAP-70 function (Kong et al 1996).…”