Selective defect in antigen-induced TCR internalization at the immune synapse of CD8 T cells bearing the ZAP-70(Y292F) mutation

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3zeta degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.     

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.     

Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment.

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivo substrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement.     

(-)-Epigallocatechin gallate regulates CD3-mediated T cell receptor signaling in leukemia through the inhibition of ZAP-70 kinase

The zeta chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 micromol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cgamma1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells.     

Identification of a novel isoform of ZAP-70, truncated ZAP kinase

We identified a novel cDNA encoding truncated ZAP-70, which lacked the SH2 domain and a part of interdomain B, and named it truncated ZAP kinase (TZK). TZK was expressed in the thymus, spleen, and lymph nodes with ZAP-70. TZK was expressed in CD44⁺CD25⁻ thymocytes up to mature T cells, but ZAP-70 was not expressed in CD44⁺CD25⁻ or CD44⁺CD25⁺ thymocytes. ZAP-70 or TZK was transfected into P116 cells derived from a Jurkat T-cell line deficient in ZAP-70. The P116 cells with ZAP-70 induced the T-cell receptor-mediated signal transduction, but the cells expressing TZK did not. While ZAP-70 was accumulated at the immune synapse, TZK was not. Meanwhile, impaired phosphorylation of SLP-76, one of the substrates of ZAP-70, in P116 cells upon pervanadate stimulation was rescued in the cells expressing TZK. These findings show that TZK is a novel isoform of ZAP-70, which is expressed in pre-T-cell receptor-minus thymocytes and functions as a kinase not associated with T-cell receptor.     

A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with both the zeta chain and CD3 components of the activated T cell antigen receptor.

An early event in T cell antigen receptor (TCR)-mediated signal transduction is the activation of a protein tyrosine kinase (PTK) pathway. An unidentified PTK activity and a kinase substrate termed ZAP-70 have previously been shown to associate with TCR zeta upon cross-linking of TCR beta. Here we report that TCR activation, by antibody cross-linking of either TCR beta or CD3 epsilon, results in the association of a PTK activity with both CD3 and TCR zeta. A number of in vitro PTK substrates are also associated with CD3 and TCR zeta, including CD3 epsilon, TCR zeta, p60fyn, p62yes, and a predominant 70-kDa protein (ZAP-70). The shared PTK activity and PTK substrates suggest that both CD3 and TCR zeta are involved in signal transduction via a shared pathway. We used [alpha-32P]gamma-azidoanilido ATP, a photoreactive analogue of ATP, to detect CD3-associated proteins that bound ATP upon TCR activation, reasoning that such proteins could represent PTKs. A 70-kDa protein bound [alpha-32P]gamma-azidoanilido ATP only upon TCR activation, and we propose that this protein and the 70-kDa PTK substrate are the same protein. Furthermore, we propose that this protein is responsible for the PTK activity observed to be associated with TCR zeta and CD3 upon TCR activation.     

Inhibition of ZAP-70 kinase activity via an analog-sensitive allele blocks T cell receptor and CD28 superagonist signaling

ZAP-70 is a cytoplasmic protein tyrosine kinase that is required for T cell antigen receptor (TCR) signaling. Both mice and humans deficient in ZAP-70 fail to develop functional T cells, thus demonstrating its necessity for T cell development and function. There is currently no highly specific, cell-permeable, small molecule inhibitor for ZAP-70; therefore, we generated a mutant ZAP-70 allele that retains kinase activity but is sensitive to inhibition by a mutant-specific inhibitor. We validated the chemical genetic inhibitor system in Jurkat T cell lines, where the inhibitor blocked ZAP-70-dependent TCR signaling in cells expressing the analog-sensitive allele. Interestingly, the inhibitor also ablated CD28 superagonist signaling, thereby demonstrating the utility of this system in dissecting the requirement for ZAP-70 in alternative mechanisms of T cell activation. Thus, we have developed the first specific chemical means of inhibiting ZAP-70 in cells, which serves as a valuable tool for studying the function of ZAP-70 in T cells.     

The FcR gamma subunit and Syk kinase replace the CD3 zeta-chain and ZAP-70 kinase in the TCR signaling complex of human effector CD4 T cells

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3zeta-related FcRgamma signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3epsilon, but not CD3zeta. The TCR/CD3/FcRgamma complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.     

Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.     

Alternative antigen receptor (TCR) signaling in T cells derived from ZAP-70-deficient patients expressing high levels of Syk

ZAP-70-deficient patients present with nonfunctional CD4+ T cells in the periphery. We find that a subset of primary ZAP-70-deficient T cells, expressing high levels of the related protein-tyrosine kinase Syk, can proliferate in vitro. These cells (denoted herein as Syk(hi)/ZAP-70(-) T cells) provide a unique model in which the contribution of Syk to TCR-mediated responses can be explored in a nontransformed background. Importantly, CD3-induced responses, such as tyrosine phosphorylation of cellular substrates (LAT, SLP76, and PLC-gamma1), as well as calcium mobilization, which are defective in T cells expressing neither ZAP-70 nor Syk, are observed in Syk(hi)/ZAP-70(-) T cells. However, Syk(hi)/ZAP-70(-) T cells differ from control T cells with respect to the T cell antigen receptor (TCR)-mediated activation of the MAPK cascades: extracellular signal-regulated kinase activity and recruitment of the JNK and p38 stress-related MAPK pathways are diminished. This distinct phenotype of Syk(hi)/ZAP-70(-) T cells is associated with a profound decrease in CD3-mediated interleukin 2 secretion and proliferation relative to control T cells. Thus, ZAP-70 and Syk appear to play distinct roles in transducing a TCR-mediated signal.     

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.     

The Shc family protein adaptor, Rai, negatively regulates T cell antigen receptor signaling by inhibiting ZAP-70 recruitment and activation

Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps--recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol) or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction) prevent full triggering of the TCR signaling cascade.     

TCR and CD28 are coupled via ZAP-70 to the activation of the Vav/Rac-1-/PAK-1/p38 MAPK signaling pathway.

CD28 costimulation amplifies TCR-dependent signaling in activated T cells, however, the biochemical mechanism(s) by which this occurs is not precisely understood. The small GTPase Rac-1 controls the catalytic activity of the mitogen-activated protein kinases (MAPKs) and cell cycle progression through G1. Rac-1 activation requires the phospho-tyrosine (p-Tyr)-dependent recruitment of the Vav GDP releasing factor (GRF) to the plasma membrane and assembly of GTPase/GRF complexes, an event critical for Ag receptor-triggered T cell activation. Here, we show that TCR/CD28 costimulation synergistically induces Rac-1 GDP/GTP exchange. Our findings, obtained by using ZAP-70-negative Jurkat T cells, indicate that CD28 costimulation augments TCR-mediated T cell activation by increasing the ZAP-70-mediated Tyr phosphorylation of Vav. This event regulates the Rac-1-associated GTP/GDP exchange activity of Vav and downstream pathway(s) leading to PAK-1 and p38 MAPK activation. CD28 amplifies TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and linker for activation of T cells (LAT). These results favor a model in which ZAP-70 regulates the intersection of the TCR and CD28 signaling pathways, which elicits the coupling of TCR and CD28 to the Rac-1, PAK-1, and p38 MAPK effector molecules.     

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.     

Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins.

ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

CD43 regulates the threshold for T cell activation by targeting Cbl functions

T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and coreceptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Coreceptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of T cells by regulating TCR signals. In this report, we show that TC prestimulation by the CD43 coreceptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ-dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced mitogen-activated protein kinase activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function.     

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.