T-cell Receptor Zeta Research Articles

The 21- and 23-kD forms of TCR zeta are generated by specific ITAM phosphorylations.

The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.

CD4+ T cell survival is not directly linked to self-MHC-induced TCR signaling.

T cell receptor (TCR) signaling triggered by recognition of self-major histocompatibility complex (MHC) ligands has been proposed to maintain the viability of naïve T cells and to provoke their proliferation in T cell-deficient hosts. Consistent with this, the partially phosphorylated state of TCR zeta chains in naïve CD4+ and CD8+ T cells in vivo was found to be actively maintained by TCR interactions with specific peptide-containing MHC molecules. TCR ligand-dependent phosphorylation of TCR zeta was lost within one day of cell transfer into MHC-deficient hosts, yet the survival of transferred CD4+ lymphocytes was the same in recipients with or without MHC class II expression for one month. Thus, despite clear evidence for TCR signaling in nonactivated naïve T cells, these data argue against the concept that such signaling plays a predominant role in determining lymphocyte lifespan.

Agonist peptide from a cytotoxic t-lymphocyte epitope of human carcinoembryonic antigen stimulates production of tc1-type cytokines and increases tyrosine phosphorylation more efficiently than cognate peptide

The identification of an agonist peptide (YLSGADLNL, designated CAP1-6D) to an immunodominant cytotoxic T-lymphocyte (CTL) epitope (designated CAP1) of human carcinoembryonic antigen (CEA) has previously been reported. The agonist peptide harbors a single amino acid substitution at a non-MHC anchor residue and is proposed to exert its effects at the level of the T-cell receptor (TCR). The type and magnitude of cytokines produced by CAP1-reactive CTL upon stimulation with the agonist peptide, CAP1-6D, were compared to those obtained upon stimulation with the cognate CAP1 peptide. In addition, early events in the TCR signaling pathway were examined for differences in tyrosine phosphorylation. Upon stimulation with the agonist peptide CAP1-6D, several different CEA-specific CTL lines exhibited a marked shift in the peptide dose response, which resulted in as much as a 1,000-fold increase in the levels of GM-CSF and gamma-IFN produced as compared with the use of the CAP1 peptide. However, levels of IL-4 and IL-10, which are associated with anti-inflammatory effects, were very low or non-existent. The cytokine profile of CAP1- and CAP1-6D-specific CTL is consistent with a Tc1-type CTL. Consistent with these findings, CEA-specific CTL showed increased tyrosine phosphorylation of TCR signaling proteins ZAP-70 and TCR zeta chains in response to both peptides. However, when CAP1-6D was compared with the wild-type peptide, the increase in ZAP-70 phosphorylation was greater than the increase in zeta phosphorylation. CTL generated with the CAP1-6D agonist were shown capable of lysis of human carcinoma cells expressing native CEA. The ability to upregulate the production of GM-CSF, gamma-IFN, TNFalpha and IL-2 with the agonist peptide, as compared with CAP1, may help in initiating or sustaining anti-tumor immune responses and thus potentially prove to be useful in the treatment of CEA-positive tumors.

Heat stress downregulates TCR zeta chain expression in human T lymphocytes.

After heat treatment, human T lymphocytes downregulate the T-cell receptor (TCR)/CD3-mediated [Ca(2+)](i) response and production of inositol triphosphate. Here we demonstrate that heat treatment of T lymphocytes at sublethal temperature decreases the expression of TCR zeta chain, which plays a critical role in the regulation of TCR/CD3-mediated signal transduction. Downregulation of TCR zeta chain in heat-treated T cells was observed at 8 h and reached a maximum at 16 h. Under these conditions, the expression of CD3 epsilon or TCR alphabeta chains was minimally affected. Consistent with the decrease in TCR zeta chain, a reduction in the level of TCR/CD3 induced tyrosine phosphorylation of several cellular protein substrates, and a delay in the kinetics of peak tyrosine phosphorylation was observed in heat-treated T cells. Interestingly, analysis of the TCR zeta chain content in the detergent-insoluble membrane fraction showed that heat treatment induces translocation of soluble TCR zeta chain to the cell membranes. In addition, the mRNA level of TCR zeta chain was reduced in heat-treated T cells. Correlative with the downregulation of TCR zeta chain mRNA, the level of the TCR zeta chain transcription factor Elf-1 was also reduced in heat-treated cells. We conclude that heat stress causes a decrease in the level of TCR zeta chain by increasing its association with the membranes and decreasing the transcription of the TCR zeta gene. Decreased expression of the TCR zeta chain is apparently responsible for the decreased TCR/CD3 responses of T cells.

Dual fatty acylation of p59(Fyn) is required for association with the T cell receptor zeta chain through phosphotyrosine-Src homology domain-2 interactions.

The first 10 residues within the Src homology domain (SH)-4 domain of the Src family kinase Fyn are required for binding to the immune receptor tyrosine-based activation motif (ITAM) of T cell receptor (TCR) subunits. Recently, mutation of glycine 2, cysteine 3, and lysines 7 and 9 was shown to block binding of Fyn to TCR zeta chain ITAMs, prompting the designation of these residues as an ITAM recognition motif (Gauen, L.K.T., M.E. Linder, and A.S. Shaw. 1996. J. Cell Biol. 133:1007-1015). Here we show that these residues do not mediate direct interactions with TCR ITAMs, but rather are required for efficient myristoylation and palmitoylation of Fyn. Specifically, coexpression of a K7,9A-Fyn mutant with N-myristoyltransferase restored myristoylation, membrane binding, and association with the cytoplasmic tail of TCR zeta fused to CD8. Conversely, treatment of cells with 2-hydroxymyristate, a myristoylation inhibitor, blocked association of wild-type Fyn with zeta. The Fyn NH2 terminus was necessary but not sufficient for interaction with zeta and both Fyn kinase and SH2 domains were required, directing phosphorylation of zeta ITAM tyrosines and binding to zeta ITAM phosphotyrosines. Fyn/zeta interaction was sensitive to octylglucoside and filipin, agents that disrupt membrane rafts. Moreover, a plasma membrane bound, farnesylated Fyn construct, G2A,C3S-FynKRas, was not enriched in the detergent insoluble fraction and did not associate with zeta. We conclude that the Fyn SH4 domain provides the signals for fatty acylation and specific plasma membrane localization, stabilizing the interactions between the Fyn SH2 domain and phosphotyrosines in TCR zeta chain ITAMs.

TCR kappa, a new splicing of the murine TCR zeta gene locus, is modulated by glucocorticoid treatment.

T-cell receptor (TCR) is a multichain receptor in which the TCRzeta subunit is important for membrane assembly and signal transduction. Four alternative splicings of the murine TCRzeta gene locus have been previously described. We here describe a new alternative splicing of murine TCRzeta gene, TCRkappa, cloned by RT-PCR, that is encoded by exons 1-7, a portion of exon 9 and the whole exon 10 of TCRzeta gene. The protein encoded by TCRkappa mRNA is identical to that encoded by TCReta mRNA, because the stop codon is present in the exon 9 before splicing with exon 10. RNAse protection assays carried out on total RNA from thymocytes indicate that TCRkappa mRNA is 1 half with respect to TCReta mRNA, suggesting that TCRkappa mRNA contributes to determine the TCReta protein levels. The 3' untranslated region of TCRkappa mRNA is different from that of TCReta and this might lead to different t(1/2) for each species in vivo. We also show that dexamethasone (DEX), a synthetic glucocorticoid hormone, increases the amount of TCRkappa in the hybridoma T-cell line 3DO (about 5-fold increase), as indicated by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNAse protection assays. This newly described effect of DEX may constitute a further molecular mechanism that contributes to its immunomodulating activity.

Crosslinking of Fas/CD95 suppresses the CD3-mediated signaling events in Jurkat T cells by inhibiting the association of the T-cell receptor zeta chain with src-protein tyrosine kinases and ZAP70.

Crosslinking of Fas (APO-1/CD95) on the surface of T cells initiates a biochemical cascade leading to programmed cell death. We have previously shown that crosslinking of Fas with an apoptosis-inducing IgM anti-Fas mAb results in suppression of the CD3-initiated cell signaling including Ca2+ mobilization and protein tyrosine phosphorylation. We conducted experiments to decipher the mechanisms whereby the cross talk between the Fas- and CD3 signaling pathways occur. We used lysates from Jurkat T and examined the composition of the TCR zeta chain-precipitated immune complexes using immunoblots. While crosslinking of Fas affected the association of p59fyn and p56lck tyrosine kinases with the TCR zeta chain to a limited degree, it dramatically inhibited the association of the protein tyrosine kinase ZAP70 with the zeta chain. In cells that were preincubated with an apoptosis-inducing anti-Fas mAb, the binding of the protein tyrosine phosphatases SHP-1 to the TCR zeta chain was increased. These experiments indicate that crosslinking of Fas interferes with early T cell signaling events by promoting the recruitment of SHP-1 and decreasing the association of protein tyrosine kinases with TCR zeta chain. Therefore, crosslinking of Fas antigen may regulate the antigen-induced T cell response and play an active role in the T cell anergy.

Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation.

The Nef protein of simian immunodeficiency virus (SIV) is dispensable for replication in established T-cell lines but essential for high level virus replication in the adult host, though the mechanism by which Nef contributes to this has remained unclear. We demonstrate here that SIV Nef binds to the zeta chain of the T-cell receptor (TCR). SIV Nef proteins that interact with TCR zeta in a yeast two-hybrid system also reduce T-cell surface expression levels of TCR alphabeta, CD3 and CD4. These findings are the first demonstration that Nef can bind directly to a component of the TCR-CD3 complex and modulate its surface expression.

Expression of signal transducing T-cell receptor zeta molecules after adoptive immunotherapy in patients with gastric and colon cancer.

We and others have shown decreased expression of T-cell receptor-CD3-associated signal transducing zeta molecules (TCRzeta) in tumor infiltrating and peripheral T cells of patients with advanced cancer. In the present study, we performed adoptive immunotherapy (AIT) with tumor-associated lymphocytes (TAL) in patients with gastric (n = 11) and colon (n = 3) cancer with stage IV and investigated whether the alteration of signal transducing molecules was observed with AIT, compared to an untreated control group (n = 13). Autologous TALs isolated from malignant ascites or pleural effusion were cultured with stimulation of autologous tumor in the presence of interleukin-2 (IL-2) and were transferred to the patients. TCR zeta expression in peripheral T cells was measured by flow cytometric analysis of permeabilized cells with anti-zeta monoclonal antibody (MAb) (TIA-2) before and after AIT. We confirmed the down-regulation of TCR zeta expression in peripheral blood lymphocytes (PBL) of patients with gastric and colon cancer with stage IV compared to healthy donors (n = 15). AIT induced up-regulation of TCR zeta expression in 2 of 14 treated patients, caused no significant change of TCR zeta expression in 7 patients and induced further down-regulation in 5 patients. The patients who achieved clinical responses (n = 3) with AIT showed no significant change of TCR zeta expression. On the other hand, in the control group without adoptive transfer, further down-regulation of TCR zeta expression was observed during the corresponding periods, paralleling disease progression. Taken together, TCR zeta expression in the patients was further down-regulated, corresponding to disease progression in individual cancer patients. In some patients, AIT could induce increased or stable TCR zeta expression. The quantitative analysis of TCR zeta expression might provide vital information that can be used to optimize therapy by preserving immune functions within cancer patients.

Impaired expression and function of signal-transducing zeta chains in peripheral T cells and natural killer cells in patients with prostate cancer.

Detection of functional, circulating T cells and NK cells may serve as a clinical test for the selection of individuals who can benefit from immunotherapy. Incidence of the T-cell receptor zeta (TCRdelta) chain within these populations appears to correlate with adequate effector cell function. In patients with advanced malignancy, the absence or reduced expression of delta chain has been documented. Flow cytometric analysis in the present study revealed a significant reduction in delta chain expression in peripheral blood lymphocytes (PBL) of 14 of 22 prostate cancer patients (P < 0.000001) as compared to normal donors, apparent in both T cells (CD3+, CD4+, CD8+), and NK (CD16+) cells. Compared to normal donor PBL, patient PBL cultured in the presence of CD3 and CD28, also demonstrated reduced expression of CD69 and/or CD25, and in some cases, failed to activate at all. Furthermore, evidence of cell proliferation in activation-stimulated patient PBL was muted: average PCNA positivity equaled 14%, a marked difference from what was observed in normal donors (P < 0.0002). In 8 of 16 samples of PBL, where delta expression was originally low, delta levels returned to the normal range after 48 hour culture in serum-free medium, suggesting that the loss of delta is reversible and may be caused by a tumor-derived substance. These data support the premise that monitoring the expression of delta in a cancer patient may provide a unique insight into the immune status and functionality of the individual, with the potential to redirect or augment therapies and ultimately alter prognosis.

Effect of dexamethasone on T-cell receptor/CD3 expression.

Glucocorticoid hormones (GCH) are anti-inflammatory and immunosuppressive agents that inhibit T-cell growth and activation. Since the T-cell receptor (TCR)/CD3 complex mediates T-lymphocyte activation, we studied the effect of in vitro dexamethasone (DEX), a synthetic GCH, on TCR/CD3 expression. DEX-treatment of a hybridoma T-cell line and normal un-transformed T-cell clones induced a decrease of the TCR/ CD3 membrane expression after 4 days. After 4 weeks, TCR/CD3 was undetectable. However, the amount of mRNAs coding TCR/CD3 chains, including TCR alpha, TCR beta, CD3 gamma, CD3 theta and CD3 epsilon, as well as the amount of CD3 epsilon protein, a major component of the complex, were unaltered. By contrast, a decrease of the mRNAs deriving from the TCR zeta gene locus, as well as of the TCR zeta protein which is responsible for the membrane expression of the TCR/CD3 complex, was induced. These data suggest that the down-modulation of TCR expression is due to the diminution of TCR zeta gene products in DEX-treated cells.

Differential association of protein tyrosine kinases with the T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation.

When stimulated through their antigen receptor, without costimulation, T cells enter a state of antigen-specific unresponsiveness, termed anergy. B7-mediated costimulation, signaling via CD28, is sufficient to prevent the induction of anergy. Here we show that ligation of T cell receptor (TCR) by alloantigen alone, which results in anergy, activates tyrosine phosphorylation of TCR zeta and its association with fyn. In contrast, TCR ligation in the presence of B7 costimulation, which results in productive immunity, activates tyrosine phosphorylation of TCR zeta and CD3 chains, which associate with activated lck and zeta-associated protein (ZAP) 70. Under these conditions, CD28 associates with activated lck and TCR zeta. These data suggest that the induction of anergy is an active signaling process characterized by the association of TCR zeta and fyn. In addition, CD28-mediated costimulation may prevent the induction of anergy by facilitating the effective association of TCR zeta and CD3 epsilon with the critical protein tyrosine kinase lck, and the subsequent recruitment of ZAP-70. Strategies to inhibit or activate TCR-associated, specific protein tyrosine kinase-mediated pathways may provide a basis for drug development with potential applications in the fields of transplantation, autoimmunity, and tumor immunity.

T cells from patients with Hodgkin's disease have a defective T-cell receptor zeta chain expression that is reversible by T-cell stimulation with CD3 and CD28

To investigate the mechanisms underlying the deficiency of T lymphocytes from patients with Hodgkin's disease, we investigated the expression of the T-cell receptor (TCR) zeta chain in patients with Hodgkin's disease. By flow cytometry using an anti-zeta chain monoclonal antibody, peripheral blood T lymphocytes from patients with untreated Hodgkin's disease were shown to express decreased levels of the TCR zeta chain. After stimulation by combined CD3 and CD28 cross-linking, T cells from Hodgkin's disease patients upregulated zeta chain protein expression to normal values within 48 hours and achieved a cytolytic potential and levels of interleukin (IL)-2 secretion that were not different from T cells obtained from healthy controls. These results show that downregulation of the TCR zeta chain in Hodgkin's T lymphocytes is a reversible event. Costimulation of CD3 and CD28 is a novel approach for overcoming the T-cell deficiency in Hodgkin's disease and might be exploited clinically. As upregulation of the zeta chain can also be achieved using bispecific monoclonal antibodies (BI-MoAbs) with specificity for tumor antigens and CD3 and CD28, respectively, an immunotherapy with CD3/CD30 and CD28/CD30 Bi-MoAbs may overcome and should therefore, not be jeopardized by the inherent T-cell deficiency in patients with Hodgkin's disease.

T cells from patients with Hodgkin's disease have a defective T-cell receptor zeta chain expression that is reversible by T-cell stimulation with CD3 and CD28

Abstract To investigate the mechanisms underlying the deficiency of T lymphocytes from patients with Hodgkin's disease, we investigated the expression of the T-cell receptor (TCR) zeta chain in patients with Hodgkin's disease. By flow cytometry using an anti-zeta chain monoclonal antibody, peripheral blood T lymphocytes from patients with untreated Hodgkin's disease were shown to express decreased levels of the TCR zeta chain. After stimulation by combined CD3 and CD28 cross- linking, T cells from Hodgkin's disease patients upregulated zeta chain protein expression to normal values within 48 hours and achieved a cytolytic potential and levels of interleukin (IL)-2 secretion that were not different from T cells obtained from healthy controls. These results show that downregulation of the TCR zeta chain in Hodgkin's T lymphocytes is a reversible event. Costimulation of CD3 and CD28 is a novel approach for overcoming the T-cell deficiency in Hodgkin's disease and might be exploited clinically. As upregulation of the zeta chain can also be achieved using bispecific monoclonal antibodies (BI- MoAbs) with specificity for tumor antigens and CD3 and CD28, respectively, an immunotherapy with CD3/CD30 and CD28/CD30 Bi-MoAbs may overcome and should therefore, not be jeopardized by the inherent T- cell deficiency in patients with Hodgkin's disease.

A tyrosine-phosphorylated 110-120-kDa protein associates with the C-terminal SH2 domain of phosphotyrosine phosphatase-1D in T cell receptor-stimulated T cells.

The role of cytosolic phosphotyrosine phosphatases (PTPase) in T cell receptor (TCR)-mediated signaling was investigated. PTPase activity was detected in a purified immunocomplex comprising aggregated TCR from the cell surface of Jurkat T cells. Since TCR aggregation results in phosphorylation of critical immunoreceptor tyrosine-based activation motifs (ITAM) in the TCR zeta chain, a doubly tyrosine-phosphorylated synthetic peptide containing the membrane-proximal zeta chain ITAM (zeta p ITAM) was used to characterize TCR zeta-associated PTPases. PTPase activity was detected in stable association with zeta p ITAM and the SH2 domain-containing PTPase PTP-1D (Syp, SH-PTP2) was identified in this complex. TCR stimulation resulted in increased total PTPase activity and PTP-1D protein in zeta p ITAM precipitates. TCR stimulation did not result in the tyrosine phosphorylation of PTP-1D but caused the rapid and transient tyrosine phosphorylation of a 110-120-kDa protein which associated selectively with the C-terminal SH2 domain of PTP-1D. This currently unidentified phosphotyrosine protein may be involved in localizing PTP-1D to the TCR following receptor stimulation.

Characterization of the Roles of SH2 Domain-Containing Proteins in T-Lymphocyte Activation by Using Dominant Negative SH2 Domains

Activation of the T-cell antigen receptor initiates a complex signaling cascade leading to changes in cytokine gene transcription. Several proteins containing Src homology 2 (SH2) domains, capable of interacting with phosphotyrosine-containing sequences within other proteins, are involved in the activation of signaling intermediates such as p2l(ras) and phospholipase Cgamma1. In this study, we used dominant negative SH2 domains to determine the importance of SH2 domain-containing proteins for T-cell activation. We show that tandem SH2 domains of either Zap70 or Syk tyrosine kinase are potent inhibitors of signaling initiated by the T-cell receptor zeta chain in vivo while individual SH2 domains are ineffective. Thus, while only the C-terminal SH2 domains appear to have significant affinity for immunoreceptor tyrosine-based activation motifs in vitro, the N-terminal SH2 domains are necessary in vivo. We find the spacing between the tandem SH2 domains of Zap70 to be critical for in vivo interactions. The SH2 domain of the adapter protein Grb2 is an effective inhibitor in our dominant negative assay, although it has little affinity for immunoreceptor tyrosine-based activation motifs. A single point mutation that abolishes phosphotyrosine binding renders the Grb2 SH2 domain incapable of this inhibition. In contrast, the SH2 domain of Shc does not inhibit this signaling cascade. We conclude that Grb2, but not Shc, is involved in T-cell receptor signaling.

Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes.

The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR-zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck.

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.

Role of TCR zeta chain in T cell development and selection.

Signals mediated by the T cell receptor (TCR) are required for thymocyte maturation and selection. To examine the role of TCR zeta chain signals in development, TCR expression was restored in zeta-deficient mice with transgenic zeta chains that partially or completely lacked sequences required for signal transduction. The zeta chain played a role in thymic development by promoting TCR surface expression, but zeta-mediated signals were not essential because TCRs that contained signaling-deficient zeta chains promoted T cell maturation and transduced signals associated with thymic selection.

The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration.