Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.     

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.     

Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number.

Engagement of the TCR initiates at least two transmembrane signaling pathways, the phosphatidylinositol pathway and a tyrosine kinase pathway. The T cell leukemic line Jurkat was used to study the relationship between the number of occupied TCR on the cell surface and the TCR-mediated activation of phosphatidylinositol-specific phospholipase C. We characterized a series of Ti beta-chain transfectants of the Jurkat mutant J.RT3-T3.5, in which surface expression of the TCR is limited by expression of the TCR beta-chain. Calibrated flow cytometry was used to determine the number of binding sites for anti-CD3 mAb on the surface of these cells, which was less than 1.2 x 10(3) to 1.2 x 10(4) sites/cell. In the presence of lithium chloride, the accumulation of inositol phosphates (InsP) in these cell lines in response to saturating concentrations of anti-CD3 mAb was proportional to the calculated surface TCR number. This result was consistent with dose-response studies using anti-CD3 mAb in Jurkat cells, in which ligand concentration, rather than number of binding sites, was limiting. Increase in intracellular free calcium concentration was a sensitive indicator of TCR engagement and correlated with the level of TCR expression, but less closely than did InsP levels. Induction of the early lymphocyte activation marker CD69 by anti-CD3 mAb also correlated with surface expression of TCR. In order to test whether limitation of this signaling pathway by TCR number may be relevant to signal transduction in the wild-type cell, we compared PLC activity in Jurkat cells during soluble anti-CD3 mAb-induced internalization of the TCR and also in response to immobilized mAb. The net accumulation of InsP per min decreased linearly with TCR number during the rapid phase of TCR internalization, confirming the limiting role of TCR number in this system. When internalization was prevented by immobilization of the stimulus, there was no decrease in the net accumulation of InsP per minute over time. In a Jurkat cell line transfected with the heterologous human muscarinic receptor, subtype 1, the InsP response to a muscarinic agonist was unaffected by TCR internalization, indicating that the distal phosphatidylinositol pathway was not affected by prolonged stimulation of the TCR. We conclude that transmembrane signaling through the TCR may be regulated by the number of surface TCR-ligand complexes. This observation has implications for transmembrane signaling in both mature T cells and thymocytes.     

Characterization of functional GTP binding proteins in Jurkat T cell mutants lacking either CD3-Ti or CD2 surface receptors

G proteins are membrane-bound molecules involved in coupling of surface receptors with signal transduction effector systems in multiple cell types including T lymphocytes. Given that mature T cells which lack antigen receptors (CDl-Ti) are refractory to stimulation through CD2 or other accessory molecules, T cell receptor components likely play a critical role in coupling surface receptors with signal transduction effectors. It has recently been proposed that modulation of T cell receptor components with MAbs results in a physical loss or functional inactivation of G protein(s). In view of the importance of the T cell activation process, we herein examined G proteins in untreated or antibody-modulated Jurkat T cells as well as in genetic variants lacking either CD3-Ti or CD2 surface receptors. 43- and 41-kDa G protein alpha chains are ADP ribosylated with cholera (CTX) and pertussis (PTX) toxins, respectively, in wild type and receptor minus cell populations. In the wild type Jurkat cell line as well as in CD3- and CD2- variants, AlF4- can activate the G protein(s) presumably associated with phospholipase C to generate polyphosphoinositide turnover as well as an increase in cytoplasmic free calcium ions. Furthermore, G protein(s) linked to adenylylcyclase, a pathway which inhibits T lymphocyte activation, can be directly activated with CTX in the absence of CD3-Ti or CD2 on the membrane. Importantly, AlF4- can also induce polyphosphoinositide turnover in Jurkat cells whose T cell receptor proteins have been modulated with anti-CD3 MAb. These data provide functional and biochemical evidence that at least certain G proteins are intact in the absence of surface expression of CD3-Ti or CD2 molecules and imply that CD3-Ti desensitization is not singularly due to G protein loss.     

Ultraviolet radiation rapidly induces tyrosine phosphorylation and calcium signaling in lymphocytes.

UV radiation is known to induce lymphocyte nonresponsiveness both in vitro and in vivo. We have found that UV radiation rapidly induced tyrosine phosphorylation and calcium signaling in normal human peripheral blood lymphocytes. In the leukemic T cell line Jurkat and the Burkitt's lymphoma cell line Ramos, UV rapidly induced tyrosine phosphorylation in a wavelength-dependent manner, giving strong signals after UVB and UVC, but not UVA, irradiation. Similarly, in Jurkat cells UV-induced calcium signals were dependent on the dose of UVB or UVC irradiation over a range of 150-1200 J/m2, but only a small signal was observed for UVA at a dose of 1200 J/m2. The UV-induced calcium signals were blocked by the tyrosine kinase inhibitor herbimycin A, indicating that they were dependent on tyrosine phosphorylation. Phospholipase C (PLC) gamma 1 was tyrosine phosphorylated in response to UV irradiation but to a lesser extent than observed after CD3 cross-linking. However, PLC gamma 1-associated proteins demonstrated to bind to the PLC gamma 1 SH2 domain were tyrosine phosphorylated strongly after UV irradiation. A similar dose response was observed for the inhibition by herbimycin A of UV-induced calcium signals and UV-induced tyrosine phosphorylation of PLC gamma 1 and associated proteins. We propose that in contrast to CD3/Ti stimulation, UV aberrantly triggers lymphocyte signal transduction pathways by a mechanism that bypasses normal receptor control.     

Spatial and functional heterogeneity of sphingolipid-rich membrane domains

Little is known about the organization of lipids in biomembranes. Lipid rafts are defined as sphingolipid- and cholesterol-rich clusters in the membrane. Details of the lipid distribution of lipid rafts are not well characterized mainly because of a lack of appropriate probes. Ganglioside GM1-specific protein, cholera toxin, has long been the only lipid probe of lipid rafts. Recently it was shown that earthworm toxin, lysenin, specifically recognizes sphingomyelin-rich membrane domains. Binding of lysenin to sphingomyelin is accompanied by the oligomerization of the toxin that leads to pore formation in the target membrane. In this study, we generated a truncated lysenin mutant that does not oligomerize and thus is non-toxic. Using this mutant lysenin, we showed that plasma membrane sphingomyelin-rich domains are spatially distinct from ganglioside GM1-rich membrane domains in Jurkat T cells. Like T cell receptor activation and cross-linking of GM1, cross-linking of sphingomyelin induced calcium influx and ERK phosphorylation in the cell. However, unlike CD3 or GM1, cross-linking of sphingomyelin did not induce significant protein tyrosine phosphorylation. Combination of lysenin and sphingomyelinase treatment suggested the involvement of G-protein-coupled receptor in sphingomyelin-mediated signal transduction. These results thus suggest that the sphingomyelin-rich domain provides a functional signal cascade platform that is distinct from those provided by T cell receptor or GM1. Our study therefore elucidates the spatial and functional heterogeneity of lipid rafts.     

CD3 receptor modulation in Jurkat leukemic cell line

CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low) (217.6), CD3+(217.9) or CD3(low) (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.     

Identification of a 43-kilodalton human T lymphocyte membrane protein as a receptor for pertussis toxin

Pertussis toxin (PTx), an exotoxin of Bordetella pertussis has been used as a molecular probe to study stimulus-response coupling in a wide variety of cells. We have previously shown that PTx activates the same signal transduction pathways as Ag or mAb directed against the CD3-T cell Ag receptor complex in human T cells. Because the EC50 for mitogenic stimulation by PTx was 1.7 nM, we suspected that the toxin was specifically interacting with a membrane protein or receptor. We have used both chemical cross-linking and Western blotting techniques to demonstrate that PTx shows specific binding to a 43 kDa-membrane protein on cells that respond to PTx by rapid second messenger production. The PTx receptor can be detected in both the E6-1 Jurkat cell line and a CD3-TCR-negative Jurkat line, demonstrating that it is not coordinately expressed with the Ag receptor complex. The 43 kDa-protein is also found in the HPB-ALL human T cell line and PBL, but not in a murine T cell hybridoma or human neutrophils, both of which are unresponsive to PTx activation. These data suggest that the biochemical basis for the mitogenic activity of PTx may lie in its binding to a specific membrane receptor that is capable of transmitting an activation signal.     

Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C

Signaling via the alpha-beta T cell Ag receptor (Ti)-CD3 complex is a complicated event that implicates several protein kinases, most notably protein kinase C (PKC). We have recently identified a serine kinase in T lymphocytes with the following characteristics: molecular mass 43 kDa, in vitro substrate affinity for microtubule associated protein 2 (MAP-2) with a preference for Mn2+ during the catalytic reaction, and elution from DEAE resin over a salt range 100 to 200 mM NaCl. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves prior phosphorylation; in vitro exposure of activated 43-kDa MAP-2 kinase (MAP-K) to an immobilized phosphatase abrogated its kinase activity. We now show that a MAP-2K response could also be obtained during treatment with mAb to Ti and the specific PKC agonist, PMA. Although the kinetics of the former response was rapidly reversible, PMA elicited a more prolonged response. The dose responsiveness for PMA was similar to the requirements for PKC activation in intact lymphocytes. Moreover, as with PKC, we found that the CD3-induced MAP-2K response could be further enhanced by using a second layer cross-linking antibody. The specificity of CD3/Ti in the Jurkat cell response is demonstrated by the fact that OKT-11(CD2) and anti-CD4 mAb did not stimulate a MAP-2K response. It was also not possible to elicit a response in a Jurkat cell mutant that lacks surface expression of CD3 and Ti. The specificity of PKC in these events was further explored with the cell permeant diacylglycerol, 1-oleoyl-2-acetylglycerol, and the nonagonist phorbol ester, 4 alpha-phorbol 12,13-didecanoate: whereas the former was an effective inducer of the MAP-2K response, the latter failed to yield any stimulation. Prior exposure of Jurkat cells to 100 mM PMA for 24 h eliminated greater than 60% of the MAP-2K response during anti-CD3 treatment. This response could also be inhibited in dose-dependent fashion by prior treatment of Jurkat cells with the potent PKC inhibitor 1-(5-isoquinolinesulfonyl) 2-methylpiperazine dihydrochloride. Although a Ca2(+)-ionophore failed to synergize with PMA at inducing a MAP-2K response, depletion of extracellular Ca2+ by EGTA abrogated anti-CD3 responsiveness. The events culminating in MAP-2K activation were slightly inhibited in the presence of cholera toxin but not pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenotypic effects of CD3zeta targeting into glycosphingolipid-enriched membrane microdomains (GEMs) of T cells

In the present study we tested whether the forced expression of the CD3zeta chain within detergent-resistant, glycosphingolipid-enriched membrane microdomains (GEMs) will result in a constitutively activated phenotype in human T cells. To this aim, a monomeric recombinant protein (LckSH4-CD3zeta), containing the intracellular part of human CD3zeta chain fused to N-terminal double-acylation motif (SH4 domain) of protein tyrosine kinase Lck, was expressed in Jurkat human T lymphoid cell line and its Lck-negative mutant, J. CaM1.6. The Lck SH4 domain indeed predominantly targeted the chimeric protein into GEMs. In transfectants derived from wild-type Jurkat cells, but not in those derived from the Lck-deficient mutant, the LckSH4-CD3zeta protein was constitutively tyrosine-phosphorylated. Tyrosine phosphorylation of a major Jurkat cell phosphoprotein (pp85) was diminished in the transfectants. However, the transfectants did not exhibit any features of constitutively activated T cells, and their responses to anti-CD3 treatment were very similar to the wild-type Jurkat cells. Thus, the constitutive expression of this form of CD3zeta chain in GEMs is not sufficient for eliciting an activated state in the Jurkat cells.     

CD3/T-cell receptor coupling to a pertussis and cholera toxin-insensitive G-protein

We have analyzed the effect of CD3/T-cell receptor stimulation on GTP hydrolysis and GTP binding. We show that stimulation of Jurkat, T-cell, membranes with OKT3 results in a 50% increase in GTP hydrolysis which is specifically inhibited by GDP. Pretreatment of the membranes with neither pertussis toxin nor cholera toxin inhibited the GTP hydrolysis. We also show that stimulation with OKT3 increases the binding of GTPγS to Jurkat membranes. These data strongly implicate the involvement of a G-protein in CD3/T-cell receptor signalling.     

Regulation of Pyk2 expression by p56(Lck) in Jurkat T lymphocytes

Lysates from the Jurkat T lymphocyte cell line were immunoblotted with anti-Pyk2, and two major forms of Pyk2 were identified. When lysates from the p56(Lck) negative (J.CaM1/Rep3) and CD45 negative Jurkat cell line derivatives were immunoblotted with anti-Pyk2, only the lower mobility form of Pyk2 was predominant. Transfection of J.CaM1 cells with p56(Lck) restored expression of the multiple forms of Pyk2. Using RT-PCR, we found that both species of the alternatively spliced mRNA for Pyk2 were present in all of the lines regardless of their ability to express CD45 or p56(Lck) protein. When p56(Lck) immunoprecipitates were immunoblotted with anti-Pyk2, only the higher mobility form of Pyk2 immunoprecipitated with p56(Lck). These data demonstrate that certain members of the Src family of kinases interact preferentially with the different isoforms of Pyk2 and may have a role in the regulation of the Pyk2 protein in lymphocytes.     

CD45 cross-linking regulates phospholipase C activation and tyrosine phosphorylation of specific substrates in CD3/Ti-stimulated T cells.

In lymphocytes, CD45 regulates the increase in cytoplasmic calcium concentration that occurs after receptor cross-linking. Here we show that T cell receptor complex (CD3/Ti)-mediated inositol phosphate production was inhibited by CD45 ligation in Jurkat cells. CD3/Ti signaling in normal T cells was also inhibited by CD45 ligation, but coupling of CD4 with CD3/Ti gave augmented calcium signals that were entirely resistant to the inhibitory effect of CD45. In contrast, CD3-induced T cell proliferation was suppressed by immobilized CD45 mAb even in the presence of CD4 mAb. The effect of CD45 and CD4 ligation on tyrosine phosphorylation during T cell activation was directly examined by immunoblotting with anti-phosphotyrosine. Using immobilized mAb, CD45 ligation suppressed the tyrosine phosphorylation of specific substrates induced by CD3/Ti stimulation, including almost complete suppression of 150-, 36-, and 35-kDa proteins and partial suppression of 76- and 80-kDa proteins. Other tyrosine-phosphorylated proteins induced by CD3/Ti stimulation, including 135- and 21-kDa proteins, were not suppressed by simultaneous ligation of CD3/Ti and CD45. Simultaneous ligation of CD3 and CD4 enhanced tyrosine phosphorylation of all substrates, but did not overcome the CD45-mediated suppression of tyrosine phosphorylation of the 35- and 36-kDa proteins. The CD45-mediated suppression of phospholipase C activation is therefore modulated by association with CD4 without altering the specific inhibition of tyrosine phosphorylation and T cell proliferation after co-ligation of CD45 and CD3/Ti.     

Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis

We previously showed that ceramide (Cer) formed during the execution phase of apoptosis is derived from plasma membrane sphingomyelin (SM), most likely by a neutral sphingomyelinase activity (Tepper et al., J. Cell Biol. 150, 2000, 155-164). In this study, we investigated the involvement of a cloned putative human neutral sphingomyelinase (nSMase1) in this process. Site-directed mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and His(272)) to Ala residues abolished the catalytic activity of nSMase1. Jurkat cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMase1. Cells overexpressing wildtype nSMase1 showed dramatically elevated in vitro nSMase activity. However, nSMase1 gene transduction (wildtype or mutant) did not alter steady-state levels of SM, Cer, or glucosylceramide. Moreover, the Cer response and apoptosis sensitivity to ligation of the CD95/Fas receptor in cells overexpressing wildtype or mutant nSMase1 were identical to vector-transduced cells. We conclude that not nSMase1 but a different, yet to be identified, nSMase accounts for the generation of Cer during the execution phase of death receptor-induced apoptosis.     

Docking protein Gab2 is phosphorylated by ZAP-70 and negatively regulates T cell receptor signaling by recruitment of inhibitory molecules

To maintain various T cell responses and immune equilibrium, activation signals triggered by T cell antigen receptor (TCR) must be regulated by inhibitory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cytokine receptors. We investigated the functional role of Gab2 in TCR-mediated signal transduction. Gab2 was phosphorylated by ZAP-70 and co-precipitated with phosphoproteins, such as ZAP-70, LAT, and CD3zeta, upon TCR stimulation. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybridomas resulted in the inhibition of NF-AT activation, interleukin-2 production, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-binding sites mostly abrogated the inhibitory activity of Gab2, but its inhibitory function was restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also impaired the inhibitory activity, and the pleckstrin homology domain-deletion mutant revealed a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a substrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecules to the TCR signaling complex.     

Synergy between the T3/antigen receptor complex and Tp44 in the activation of human T cells

In addition to the T3/antigen receptor complex (T3/Ti), other T cell surface molecules participate in early events involved in human T cell activation. In this report we document that monoclonal antibody 9.3, which recognizes a 90,000 dalton homodimer expressed on human T cells, synergizes with ligands reacting with T3/Ti to activate purified T cells and Jurkat, a human T cell leukemic line. Unlike phorbol myristate acetate (PMA), 9.3 was able to synergize only with anti-T3 or anti-Ti if these antibodies were immobilized. Moreover, 9.3 failed to synergize with the calcium ionophore ionomycin. At high concentrations only, 9.3 could synergize with PMA in the activation of Jurkat and a T3/Ti negative mutant of Jurkat. At such high concentrations of 9.3, small transient increases in cytoplasmic free calcium ((Ca++)i) were detected in quin 2-loaded Jurkat cells. This increase in (Ca++)i was the result of release of internal stores of calcium. 9.3 induced the hydrolysis of polyphosphoinositides, albeit the magnitude of inositol phosphates generated in response to 9.3 was substantially less than that observed with anti-Ti. No effect on pkC translocation was observed in Jurkat cells stimulated with 9.3. Although the small increase in (Ca++)i induced by 9.3 may account for its synergy with PMA, this effect is unlikely to account for the more potent synergistic effect observed with 9.3 and phytohemagglutinin or immobilized anti-T3 and anti-Ti antibodies.     

Cas-L is required for beta 1 integrin-mediated costimulation in human Tcells.

Beta 1 integrins provide a costimulus for TCR/CD3-driven T cell activation and IL-2 production in human peripheral T cells. However, this beta 1 integrin-mediated costimulation is impaired in a human T lymphoblastic line, Jurkat. We studied the molecular basis of this impaired costimulation and found that Cas-L, a 105-kDa docking protein, is marginally expressed in Jurkat T cells, whereas Cas-L is well expressed in peripheral T cells. Cas-L is a binding protein and a substrate for focal adhesion kinase and is tyrosine phosphorylated by beta 1 integrin stimulation. We here show that the transfection of wild-type Cas-L in Jurkat T cells restores beta 1 integrin-mediated costimulation. However, Cas-L transfection had no effect on CD28-mediated costimulation, indicating that Cas-L is specifically involved in the beta 1 integrin-mediated signaling pathway. Furthermore, transfection of the Cas-L Delta SH3 mutant failed to restore beta 1 integrin-mediated costimulation in Jurkat cells. Cas-L Delta SH3 mutant lacks the binding site for focal adhesion kinase and is not tyrosine phosphorylated after beta 1 integrin stimulation. These findings strongly suggest that the tyrosine phosphorylation of Cas-L plays a key role in the signal transduction in the beta 1 integrin-mediated T cell costimulation.     

Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies

Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.     

Partial deletions of the cytoplasmic domain of CD2 result in a partial defect in signal transduction

CD2 (T11, the T cell erythrocyte receptor or the SRBC receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, appears to play a role in T lymphocyte adhesion, signal transduction, and differentiation. Pairs of anti-CD2 mAb induce T cell proliferation, suggesting that CD2 may be an Ag-independent pathway of T cell activation. We have expressed the human CD2 and a number of cytoplasmic domain deletion mutants of CD2 in an Ag-reactive murine hybridoma. We have previously shown that a cytoplasmic domain deletion mutant, CD2 delta B, in which the carboxyl-terminal 100 amino acids have been deleted, is no longer capable of signaling through CD2. Here we have expressed a second cytoplasmic domain deletion mutant, CD2 delta S, in which the terminal 41 amino acids have been removed, including the region with greatest conservation between the mouse, rat, and human species. CD2 delta S+ hybridomas were able to respond to Ag and to LFA-3 plus an anti-CD2 mAb. Although the CD2 delta S+ hybridomas responded comparably to the wild-type CD2+ hybridomas to certain pairs of anti-CD2 mAb (e.g., MT110 + 9-1 mAb), these CD2 delta S+ hybridomas were markedly deficient in their ability to respond to other pairs of stimulatory anti-CD2 mAb (e.g., 9.6 + 9-1 mAb). These data suggest that the cytoplasmic domain may have several functional regions, as partial deletions of the cytoplasmic domain appear to result in partial defects in signal transduction.     

An activating combination of CD2 antibodies stimulates tyrosine phosphorylation in a T lymphocyte cell line.

The activating combination of CD2 antibodies Leu-5b plus 9.1 stimulates tyrosine phosphorylation in the human T cell line Jurkat. The tyrosine phosphorylation has the same molecular weight pattern as that seen when cells are stimulated on the CD3 receptor with OKT3 antibodies. These data provide evidence that signal transduction by the CD2 receptor is coupled to an increase in tyrosine phosphorylation that is similar to that coupled to the CD3 receptor.