The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 ζ chain,FcϵRI γ chain and the p56lck kinase

The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183^?EB6⁺ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4^? C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183⁺/EB6⁺ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the ζ family (i.e. CD3 ζ and Fc?RI γ chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58⁺ NK cells indicated a preferential association with CD3 ζ chains either in the form of covalently linked ζ-γ homodimers or in the form of ζ-γ heterodimers, while γ-γ homodimers were detectable in low amounts. However, p58⁺ clones displaying a unique association with γ-γ homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56^lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56^lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 ζ chains, no tyrosine phosphorylation was detected in the p58-associated CD3 ζ, chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the γ chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompained by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 ζ chains.     

The protein interactions of the immunoglobulin receptor family tyrosine-based activation motifs present in the T cell receptor ζ subunits and the CD3 γ,δ and ϵ chains

Immunoglobulin family tyrosine-based activation motifs (ITAM), which define the conserved signaling sequence EX₂YX₂L/IX₇YX₂L/I, couple the T cell antigen receptor (TCR) to cellular proteins including protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3γ, δ and ? that each contain a single ITAM and the TCR ζ chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 γ, δ, ? ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP-70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb-2 and the p85 regulatory subunit of phosphoinositol 3′ kinase. The CD3 and ζ ITAM display a hierarchy of ZAP-70 binding: ζ1 = γ = δ > ζ3 > ζ2 = ?. Shc, Grb-2 and p85 could bind the ζ ITAM and the CD3 γ and δ ITAM, but not the CD3 ? ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 γ,δ and ζ ITAM that show that the ζ, CD3 γ, δ and ? ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.     

Association of the p56lck protein tyrosine kinase with the FCγRIIIA/CD16 complex in human natural killer cells

The multimeric FcγRIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50–70-kDa transmembrane glycoprotein Fcγ receptor (CD16), the T cell receptor (TCR)-ζ chain, and the FcεRIγ chain. Cross-linking FcγRIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the ζ, subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the FcγRIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human FcγRIIIA is complexed with p56^lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56^lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several FcγRIIIA-associated proteins are identified in immune complex kinase assays including the TCR-ζ, subunit, a p70–90 ζ-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56^lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56^lck is physically associated with FcγRIIIA(CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.     

Activation-induced proteolysis of the cytoplasmic domain of ζ in T cell receptors and Fc receptors

The CD3-T cell receptor (TCR) complex on T cells and the Fcγ receptor type III (FcγRIII)-ζ-γ complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits ζ and γ. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized ζ-p14 heterodimer. ζ-p14 is a component of both CD3-TCR and FcγRIII-ζ-γ. Peptide mapping analysis shows that p14 is structurally related to ζ, suggesting that it is either: (i) derived from ζ proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding ζ express ζ-p14 supports the former possibility. The expression of CD3-TCR complexes including ζ-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of ζ may contribute to receptor modulation or desensitization.     

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 ζ chain, a doubly tyrosine-phosphorylated synthetic peptide containing the membrane-proximal ζ chain ITAM (ζp ITAM) was used to characterize TCR ζ-associated PTPases. PTPase activity was detected in stable association with ζp 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 ζp 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.     

The glycosylphosphatidylinositol-anchored CD59 protein stimulates both T cell receptor ζ/ZAP-70-dependent and -independent signaling pathways in T cells

The glycosylphosphatidylinositol (GPI)-anchored CD59 protein (human protectin) protects cells against complement-induced lysis, binds to CD2 and also transduces activation signals within T cells. We have further examined the biochemical signals transduced by CD59 and addressed its role in regard to the CD3-mediated signaling cascade. We show here that CD59 cross-linking induces a time-dependent activation of p56^lck and of p70^zap (ZAP-70) in CD3-positive Jurkat cells, leading to the stimulation of the T cell receptor ζ/ZAP-70 signaling cascade and interleukin-2 (IL-2) synthesis. Cross-linking of CD59 on peripheral T cells and thymocytes induces tyrosine phosphorylations identical to those seen in Jurkat cells and this is followed by lymphokine production and proliferation. In contrast, only activation of CD59-associated p56^lck occurs in CD3-negative Jurkat cells, while IL-2 production is impaired, consistent with the lack of ZAP-70 tyrosine phosphorylation observed in these cells. CD59 triggers activation events even in the absence of CD3/T cell receptor expression in Jurkat cells. CD59 cross-linking synergizes with sub-optimal doses of phorbol ester for activation of the protein kinase C and of the p42^mapk, as shown by in vitro phosphorylation of histone HIIIS and myelin basic protein, respectively, and leads to CD25 but not CD69 expression. In conclusion, at least two signaling pathways are triggered through CD59, the first one involving ZAP-70 activation and leading to IL-2 secretion and a second pathway observed in the absence of ZAP-70 activation leading to CD25 expression. These two pathways are likely to be involved in the modulation of T cell activation by CD59 protein.     

The p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/CD3/ζ complex

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are thought to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56^lck SH2 domain with ζ-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/ζ complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8αβ and CD8αβ isoforms to the TCR. We demonstrate in vivo in association of CD8αα/p56^lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56^lck specifically impedes the association of ZAP-70 with CD8αα/p56^lck without affecting the ζ/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8αα or CD8αβ isoform with the TCR/CD3/ζ complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56^lck SH2 domain mediates recruitment of CD8/p56^lck to the activated TCR/CD3/ζ complex.     

Differential T cell receptor-mediated signaling in naive and memory CD4 T cells

Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3ζ subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3ζ in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72^syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.     

T cell receptor α-chain tail is required for protein kinase C-mediated down-regulation,but not for signaling

Antigen stimulation through the T cell receptor (TCR) induces phosphorylation of the associated CD3 γδσ- and ζ-chain cytoplasmic tails. These events lead to the induction of the intracellular signaling pathways with concomitant receptor down-regulation. The TCR is down-regulated from the cell surface by the activation of protein kinase C (PKC) and subsequent serine phosphorylation of the CD3 γ-chain. We report here that the TCR α-chain cytoplasmic tail is also necessary for PKC-mediated internalization of the TCR complex. The requirement for the TCR α-chain cytoplasmic tail is specific for internalization of the TCR complex, since down-regulation of CD4 is still intact in hybridoma cells expressing a tailless TCR α-chain. The absence of TCR internalization directly correlates with defective PKC-mediated phosphorylation of the CD3 γ-chain. Despite deficient PKC-mediated TCR down-regulation, the tailless αβ TCR still transduces antigenic signals resulting in the production of interleukin-2. Although the TCR tails are not obviously required for signal transduction, the TCR α-tail may serve as a targeting domain for PKC-mediated down-regulation of the TCR complex.     

The cytoplasmic tail of the T cell receptor ζ chain is dispensable for antigen-mediated T cell activation

The T cell antigen receptor consists of an antigen-binding αβ heterodimer and a group of invariant polypeptides denoted CD3-γ, CD3-δ, CD3-εand CD3-ζ. Whether antigen responsiveness is dependent on the expression of functional CD3-ζ subunit remains controversial. Forinstance, transfection of a ζ^? / n^? variant of the 2B4.11. T cell hybridoma with mutated ζ cDNA that encoded a ζ protein truncated at residue 108, restored the surface expression of T cell antigen receptor complexes with, however, impaired antigen responsiveness [Frank, S. J., Niklinska, B. B., Orloff, D. G., Mercep, M., Ashwell, J. D. and Klausner, R. D., Science 1990. 249: 174.]. In marked contrast, BW5147 transfectants that expressed T cell antigen receptors devoid of functional ζ subunits were still able to trigger the production of interleukin-2 in response to antigen [Wegener, A.-M. K., Letourneur, E, Hoeveler, A., Brocker, T., Luton, F. and Malissen, B., Cell 1992. 68: 83.]. To assess if the above discrepancies may have resulted from the use of different recipient T cells, we transfected a ζ/n-deficient variant of 2B4.11 (MA5.8) with the very same truncated ζ cDNA we previously used in BW5147. Consistent with our initial observations in BW5147, the cytoplasmic tail of the ζ polypeptide was found dispensable for antigenic responsiveness. Furthermore, a difference between the two recipient T cells was detected when cells were challenged via the Thy-1 and Ly-6 molecules. Once expressed in MA5.8, but not in BW5147, T cell antigen receptor complexes devoid of functional ζ subunits were able to sustain activation initiated via Thy-1 and Ly-6 molecules.     

The role of tyrosine phosphorylation in the interaction of cellular tyrosine kinases with the T cell receptor ζ chain tyrosine-based activation motif

Immunoglobulin receptor family tyrosine-based activation motifs (ITAM) define a conserved signaling sequence, EX₂ YX₂L/IX₇YX₂L/I, that mediates coupling of the T cell antigen receptor (TCR) to protein tyrosine kinases (PTK). In the present study, we explored the role of phosphorylation of the two ITAM tyrosine residues in the interactions of the motif with the PTK ZAP-70 and p59fyn. The data show that the phosphorylation of a single tyrosine within the motif enables binding of p59fyn, whereas phosphorylation of both tyrosines within the motif is required for maximal binding of the PTK ZAP-70. Quantitative binding experiments show that nanomolar concentrations of the doubly phosphorylated ζ1-ITAM are sufficient for ZAP-70 recruitment, whereas micromolar levels of singly phosphorylated ITAM are necessary for p59fyn binding. ZAP-70 binds with low efficiency to a singly phosphorylated ITAM, but shows preferential binding to the C-terminal phosphotyrosine in the ITAM, whereas p59fyn binds selectively to the N-terminal phosphotyrosine. The present data thus show that there is the potential for a singly phosphorylated ITAM to couple to cellular PTK. Moreover, the data suggest a mechanism for heterogeneity in signal transduction responses by the TCR, since ITAM could differentially couple the TCR to downstream signaling events depending on their phosphorylation state.     

Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorylations of zeta-chains and p59fyn/p56lck in human T cells

The expression and catalytic activity of the protein tyrosine kinase (PTK) ZAP-70 are needed for normal intracellular signaling through the T-cell receptor (TCR)/CD3 complex. However, the possible effect of aging on the catalytic activity of ZAP-70 in human peripheral blood T cells stimulated via the TCR/CD3 complex is unknown. The current studies show that T cells from a substantial proportion of elderly humans (12) exhibit significant reductions in the catalytic activity, but not expression of ZAP-70 when stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. In addition, the reduced catalytic activity of ZAP-70 in T cells from elderly subjects was not restored to the normal levels in response to ligation of CD4 receptors, suggesting defects in PTKs linked to both CD3 and CD4 receptors. Other experiments demonstrated that the age-related impairments of ZAP-70 activation in anti-CD3-stimulated T cells were accompanied by decreased tyrosine phosphorylations of zeta-chains and autophosphorylations of the PTKs p561ck/p59fyn. Moreover, the age-related defects in these early TCR/CD3-mediated phosphorylation events were readily detectable in both CD45RO+ memory and CD45RA+ naive T cells. Thus, these results suggest that defects in early TCR/CD3-mediated phosphorylation events among CD45RO+ memory and CD45RA+ naive T cells from certain elderly humans may con tribute to impaired induction of ZAP-70 catalytic activity.     

CD2 is functionally linked to the zeta-natural killer receptor complex.

Natural killer (NK) cells express two distinct surface receptors capable of triggering cytolytic effector function. The first is CD16, an immunoglobulin Fc receptor that allows NK cells to mediate antibody-dependent killing (ADCC). NK cells express CD16 in association with zeta, a signal-transducing subunit that is also a component of the T cell receptor complex. Activation of NK cells via CD16 results in tyrosine phosphorylation of zeta. The second NK cell triggering receptor is CD2, a 50-55-kDa cell surface molecule that is also expressed on T cells. Here we show that NK cell activation induced by mAb reactive with CD2 (either anti-T11.1 alone or with anti-T11.2 in combination) also results in the tyrosine phosphorylation of zeta. Our results indicate that CD2 is functionally linked to the CD16-zeta complex and suggest that the zeta subunit plays a central role in the signal transduction pathways utilized by NK cells.     

Alterations of CD2 association with T cell receptor signaling molecules in “CD2 unresponsive” human T lymphocytes

CD2-associated molecules were identified by means of in vitro kinase assays of CD2 immunoprecipitates obtained from nontransformed human T lymphocytes lysed in the detergent brij 58. Under these conditions CD2 was found to be associated with the protein tyrosine kinases p56^lck and p59^fyn as well as two low molecular weight phosphoproteins. The latter were identified as the ζ and ? chains, the major signaling components of the CD/7 TcR complex. Importantly, induction of T cell unresponsiveness towards CD2-mediated stimuli by means of CD3/Tcell receptor (TcR) modulation results in uncoupling of ζ and ? from the CD2 molecule, while its associations with p56^lck and p59^fyn remain unaffected. Moreover, despite the incapacity of T lymphocytes to undergo DNA synthesis in the CD3/TcR-modulated state, CD2 triggering still results in tyrosine phosphorylation of some unknown protein substrates. Thus, the same ζ and ? chains which are components of a functional TcR complex appear to also couple to the CD2 molecular complex. Moreover, dissociation of TcR and CD2 complexes in intact cells seems to block CD2-mediated Tcell growth but does not result in complete abolishment of the signal transducing capacity of the CD2 receptor.     

CD16-mediated activation of phosphatidylinositol-3 kinase (Pl-3K) in human NK cells involves tyrosine phosphorylation of Cbl and its association with Grb2, Shc,pp36 and p85 Pl-3K subunit

Phosphatidylinositol 3-kinase (Pl-3K) plays a key role in several cellular processes, including mitogenesis, apoptosis, actin reorganization and vesicular trafficking. The molecular events involved in its activation have not been fully elucidated and several reports indicate that a key event for enzyme activation is the interaction of the SH2 domains of the p85 regulatory subunit of Pl-3K with tyrosine-phosphorylated proteins. In this study, we investigated the involvement of the product of the proto-oncogene c-Cbl in the activation of Pl-3K triggered by CD16 in human NK cells and the possible mechanisms leading to Pl-3K recruitment to the plasma membrane. Our results indicate that stimulation of NK cells through CD16 results in a rapid tyrosine phosphorylation of Cbl, which is constitutively associated with Grb2 and forms an activation-dependent complex with the p85 subunit of Pl-3K. In addition, we detected the presence of the Grb2-associated tyrosine-phosphorylated p36 and Shc proteins in anti-Cbl and anti-p85 immunoprecipitates from CD16-stimulated NK cell lysates. Upon CD16 stimulation, Pl-3K activity was found associated with Cbl and to a lesser extent with Grb2 and Shc as well as with the ζ chain of the CD16 receptor complex. Overall these results suggest that the formation of a complex containing either Shc or pp36 associated with Grb2, Cbl and the p85 subunit of Pl-3K is one of the major mechanisms which might couple CD16 to the Pl-3K pathway in NK cells.     

The structure, regulation, and function of ZAP-70

Summary:  The tyrosine ZAP-70 (ζ-associated protein of 70 kDa) kinase plays a critical role in activating many downstream signal transduction pathways in T cells following T-cell receptor (TCR) engagement. The importance of ZAP-70 is evidenced by the severe combined immunodeficiency that occurs in ZAP-70-deficient mice and humans. In this review, we describe recent analyses of the ZAP-70 crystal structure, revealing a complex regulatory mechanism of ZAP-70 activity, the differential requirements for ZAP-70 and spleen tyrosine kinase (SyK) in early T-cell development, as well as the role of ZAP-70 in chronic lymphocytic leukemia and autoimmunity. Thus, the critical importance of ZAP-70 in TCR signaling and its predominantly T-cell-restricted expression pattern make ZAP-70 an attractive drug target for the inhibition of pathological T-cell responses in disease.     

Association of tissue factor with a γ chain homodimer of the IgE receptor type I in cultured human monocytes

Tissue factor (TF) is a high-affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the γ chain homodimer identified as a component of IgE receptor type I (FcϵRI). When 4-day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70-kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti-TF monoclonal antibody (mAb) HTF-K14, but not anti-TF mAb HTF-K180 to intact cultured monocytes. HTF-K14 blocked the binding of FVII/Vila to cell surface TF, whereas HTF-K180 did not. Anti-TF immunoprecipitates prepared from 1 % digitonin lysates of cultured human monocytes incorporated phosphate in a γ chain homodimer when incubated with [γ-³²P] ATP. The identity of the TF-associated structures as γ chains was established by immunoblot analysis of anti-TF mAb immunoprecipitates with anti-γ chain mAb. In addition, anti-TF immunoblot analysis showed that TF co-precipitated with anti-γ chain mAb. Our data suggest that γ chains may play an important role in signaling via TF in human monocytes/macrophages.     

Identification of a signaling complex involving CD2, ζ chain and p59fyn in T lymphocytes

CD2 is a cell surface receptor molecule which has been implicated in cell-cell adhesion and signaling functions in T lymphocytes and natural killer cells. The mechanism by which extracellular stimuli induce CD2-regulated signal transduction events is largely unknown. However, there is increasing evidence that in cells of hematopoietic origin several receptor-mediated signaling mechanisms involve transmembrane polypeptides related to the CD3 ζ chain and the activation of protein tyrosine kinases. We have therefore investigated the potential involvement of ζ chain and src family protein tyrosine kinases in signal transduction pathways initiated by CD2. Using in vitro kinase assays on CD2 immunoprecipitates from detergent lysates of T lymphocytes, we identified a complex consisting of CD2, ζ chain and the src family kinases p59^fyn and p56^lck. Furthermore, using double indirect immunofluorescence combined with capping techniques, we have revealed such complexes in viable T lymphocytes. These findings provide evidence for a multimolecular signaling complex consisting of at least CD2, ζ chain and p59^fyn in T lymphocytes and suggest a critical role for this complex in the initiation of CD2-mediated cellular activation by regulating the activation of intracellular signaling molecules.     

Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six–eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F_2P) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3ζ1 ITAM with a non-peptidic linker made up of γ-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.