Differential T-cell antigen receptor signaling mediated by the Src family kinases Lck and Fyn

Src family tyrosine kinases play a key role in T-cell antigen receptor (TCR) signaling. They are responsible for the initial tyrosine phosphorylation of the receptor, leading to the recruitment of the ZAP-70 tyrosine kinase, as well as the subsequent phosphorylation and activation of ZAP-70. Molecular and genetic evidence indicates that both the Fyn and Lck members of the Src family can participate in TCR signal transduction; however, it is unclear to what extent they utilize the same signal transduction pathways and activate the same downstream events. We have addressed this issue by examining the ability of Fyn to mediate TCR signal transduction in an Lck-deficient T-cell line (JCaM1). Fyn was able to induce tyrosine phosphorylation of the TCR and recruitment of the ZAP-70 kinase, but the pattern of TCR phosphorylation was altered and activation of ZAP-70 was defective. Despite this, the SLP-76 adapter protein was inducibly tyrosine phosphorylated, and both the Ras-mitogen-activated protein kinase and the phosphatidylinositol 4, 5-biphosphate signaling pathways were activated. TCR stimulation of JCaM1/Fyn cells induced the expression of the CD69 activation marker and inhibited cell growth, but NFAT activation and the production of interleukin-2 were markedly reduced. These results indicate that Fyn mediates an alternative form of TCR signaling which is independent of ZAP-70 activation and generates a distinct cellular phenotype. Furthermore, these findings imply that the outcome of TCR signal transduction may be determined by which Src family kinase is used to initiate signaling.     

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.     

Evidence that Llck-mediated phosphorylation of p56dok and p62dok may play a role in CD2 signaling

The Lck tyrosine kinase is involved in signaling by T cell surface receptors such as TCR/CD3, CD2, and CD28. As other downstream protein-tyrosine kinases are activated upon stimulation of these receptors, it is difficult to assign which tyrosine-phosphorylated proteins represent bona fide Lck substrates and which are phosphorylated by other tyrosine kinases. We have developed a system in which Lck can be activated independently of TCR/CD3. We have shown that activation of an epidermal growth factor receptor/Lck chimera leads to the specific phosphorylation of Ras GTPase-activating protein (RasGAP) and two RasGAP-associated proteins, p56(dok) and p62(dok). Activation of the chimeric protein correlates with an increase in cellular Ca(2+) in the absence of ZAP-70 and phospholipase Cgamma1 phosphorylation. Furthermore, we have found that p62(dok) co-immunoprecipitates with the activated epidermal growth factor receptor/LckF505 and that phosphorylated Dok proteins bind to the Src homology 2 domain of Lck in vitro. In addition, we have shown that activation via the CD2 but not the TCR/CD3 receptor leads to the phosphorylation of p56(dok) and p62(dok). Using JCaM1.6 cells, we have demonstrated that Lck is required for CD2-mediated phosphorylation of Dok proteins. We propose that phosphorylation and Src homology 2-mediated association of p56(dok) and p62(dok) with Lck play a selective function in accessory receptor signal transduction mechanisms.     

Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function.

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 up-regulated 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 lck. 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.     

Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319 phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.     

A weak Lck tail bite is necessary for Lck function in T cell antigen receptor signaling

Src family kinases are suppressed by a "tail bite" mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)(505) affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.     

The SH3 domain of Lck modulates T-cell receptor-dependent activation of extracellular signal-regulated kinase through activation of Raf-1

Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-γ, and Ras. We determined that the signaling lesion in W97ALck-expressing cells lies at the level of Raf-1 activation and is dependent on the presence of tyrosines 340/341 in the Raf-1 sequence. These data demonstrate a second function for Lck in TCR-mediated signaling to ERK. Additionally, we found that a significant fraction of Lck is localized to the Golgi apparatus and that, compared with wild-type Lck, W97ALck displays aberrant Golgi membrane localization. Our results support a model where under conditions of weak stimulation through the TCR, in addition to activated Ras, Golgi apparatus-localized Lck is needed for the full activation of Raf-1.     

The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM.     

Functional analysis of Csk in signal transduction through the B-cell antigen receptor.

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.     

Activation of Zap-70 tyrosine kinase due to a structural rearrangement induced by tyrosine phosphorylation and/or ITAM binding

The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor (TCR) signaling. Binding of ZAP-70 to the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain through its two src-homology 2 (SH2) domains results in its activation coupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as well as by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still not completely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substrate phosphorylation assay. Under these conditions, we observed a lag phase of several minutes before full ZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggesting that it might be due to an intramolecular interaction involving the interdomain A and SH2 region. Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinant SH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation with ATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. The same results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findings are consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s) other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR, can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.     

Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proximal TCR signaling by targeting ZAP-70

The long cytoplasmic tail (CT) isoforms of carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1) are expressed on activated human T cells and possess two ITIM motifs in the CT. These isoforms of CEACAM1 are inhibitory for T cell responses initiated by the TCR/CD3 complex with the inhibition dependent upon the ITIMs of CEACAM1 and Src homology 2 domain-containing phosphatase 1 (SHP-1). However, the mechanism by which this inhibition occurs in T cells is unknown. We demonstrate here that the Src family kinase, Lck, and the ability of CEACAM1 to bind homophilically are required for the ITIM phosphorylation of CEACAM1 that is a prerequisite for CEACAM1 association with SHP-1. We further show that CEACAM1 associates with and recruits SHP-1 to the TCR/CD3 complex leading to decreased phosphorylation of CD3-zeta and ZAP-70 and consequently decreased activation of the elements downstream of ZAP-70. This is physiologically relevant because extinction of SHP-1 expression or blockade of homophilic binding by CEACAM1 using a Fab that specifically recognizes the homophilic binding region of human CEACAM1 increases the cytolytic function initiated by the TCR/CD3 complex. These studies show that long CT isoforms of CEACAM1 orchestrate an inhibitory program that abrogates extremely proximal events downstream of the TCR/CD3 complex by focusing on the activation of ZAP-70.     

Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.     

Dexamethasone induces rapid tyrosine-phosphorylation of ZAP-70 in Jurkat cells

Steroid hormones are known to mediate rapid non-genomic effects occurring within minutes, besides the classical genomic actions mediated by the nuclear translocation of the cytoplasmic glucocorticoid receptor (GR). The glucocorticoid hormone (GC) has significant role in the regulation of T-cell activation; however, the cross-talk between the GC and T-cell receptor (TcR) signal transducing pathways are still to be elucidated. We examined the rapid effects of GC exposure on in vitro cultured human T-cells. Our results showed that Dexamethasone (DX), a GC analogue, when applied at high dose (10 microM), induced rapid (within 5 min) tyrosine-phosphorylation events in Jurkat cells. Short DX pre-treatment strongly inhibited the tyrosine-phosphorylation stimulated by CD3 cross-linking. Furthermore, we also investigated the phosphorylation status of ZAP-70, an important member of tyrosine kinase mediated signalling pathway of TcR-elicited T-cell activation. Here, we demonstrate that high dose DX induced a rapid ZAP-70 tyrosine-phosphorylation in Jurkat T-cells. DX-induced ZAP-70 phosphorylation could be inhibited by RU486 (GR antagonist), suggesting that this process was GR mediated. DX-induced ZAP-70 phosphorylation did not occur in the absence of active p56-lck as examined in the p56-lck kinase-deficient Jurkat cell line JCaM1.6. Our results show that DX, at a high dose, can rapidly influence the initial tyrosine-phosphorylation events of the CD3 signalling pathway in Jurkat cells, thereby modifying TcR-derived signals. Lck and ZAP-70 represent an important molecular link between the TcR and GC signalling pathways.     

ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain.

Protein-tyrosine kinases (PTKs) play an integral role in T cell activation. Stimulation of the T cell antigen receptor (TCR) results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR zeta chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. We have recently identified a 70 kd tyrosine phosphoprotein (ZAP-70) that associates with zeta and undergoes tyrosine phosphorylation following TCR stimulation. Here we report the isolation of a cDNA clone encoding ZAP-70. ZAP-70 represents a novel PTK and is expressed in T and natural killer cells. Moreover, tyrosine phosphorylation and association of ZAP-70 with zeta require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP-70 may interact to mediate TCR signal transduction.     

T cell activation stimulates the association of enzymatically active tyrosine-phosphorylated ZAP-70 with the Crk adapter proteins.

Engagement of the T cell antigen receptor initiates signal transduction involving tyrosine phosphorylation of multiple effector molecules and the formation of multimolecular complexes at the receptor site. Adapter proteins that possess SH2 and SH3 protein-protein interaction domains are implicated in the assembly of cell activation-induced signaling complexes. We found that Crk adapter proteins undergo activation-induced interaction with the zeta-chain associated protein (ZAP-70) tyrosine kinase in the human T cell line, Jurkat. Incubation of various glutathione S-transferase fusion proteins with a lysate of activated Jurkat cells resulted in selective association of ZAP-70 with Crk, but not Grb2 or Nck, adapter proteins. In addition, tyrosine-phosphorylated ZAP-70 co-immunoprecipitated with Crk from a lysate of activated Jurkat cells, and ZAP-70 association with GST-Crk was observed in a lysate of activated human peripheral blood T cells. Association between the two molecules was mediated by direct physical interaction and involved the Crk-SH2 domain and phosphotyrosyl-containing sequences on ZAP-70. The association required intact Lck, considered to be an upstream regulator of ZAP-70, because it could not take place in activated JCaM1 cells, which express normal levels of ZAP-70 but are devoid of Lck. Finally, glutathione S-transferase-Crk fusion proteins were found to interact predominantly with membrane-residing tyrosine-phosphorylated ZAP-70 that exhibited autophosphorylation activity as well as phosphorylation of an exogenous substrate, CFB3. These findings suggest that Crk adapter proteins play a role in the early activation events of T lymphocytes, apparently, by direct interaction with, and regulation of, the membrane-residing ZAP-70 protein tyrosine kinase.     

Differential requirement of ZAP-70 for CD2-mediated activation pathways of mature human T cells

This study addresses the role of the tyrosine kinase ZAP-70 in CD2-mediated T cell activation. Patients lacking ZAP-70 have few mature CD8+ T cells and high numbers of CD4+ T cells that are nonfunctional upon TCR triggering. Such a patient with a homozygous deletion in the zap-70 gene that resulted in the complete absence of ZAP-70 protein expression has been identified. Expression of the tyrosine kinases Lck, Fyn, and Syk was normal. The patient's T cells were activated with two different pairs of mitogenic mAbs. CD2-induced phosphorylation of the zeta-chain and influx of Ca2+ was defective in the ZAP-70-deficient T cells, whereas CD2-induced phosphorylation of several other proteins, including Syk, was not affected. CD2-induced proliferation as well as production of TNF-alpha and IFN-gamma was abrogated in ZAP-70-deficient T cells, whereas PMA plus ionomycin induced normal activation of these cells. Together, this study shows that CD2-activation triggers ZAP-70-dependent and -independent pathways. Deletion of ZAP-70 affected CD2- and CD3-mediated proliferation and cytokine production in a similar way, suggesting that one of the different CD2 pathways converges with a CD3 pathway at or upstream of the activation of ZAP-70.     

Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains.

T-cell receptors (TCRs) upon binding to peptide-MHC ligands transduce signals in T lymphocytes. Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (gammadeltaepsilon) and zeta subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP-70 kinase and other signaling components. We examined the role of the low-density detergent-insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine-phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM-associated receptor complexes are highly enriched in the hyperphosphorylated p23 zeta chains, contain most of the TCR/CD3-associated, phosphorylation-activated ZAP-70 kinases and seem to integrate into higher order, multiple tyrosine-phosphorylated substrate-containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.