Update in geriatrics

We have recently observed an abnormal pattern of protein tyrosine phosphoryl-ation in resting T lymphocytes obtained from peripheral blood of patients with systemic lupus erythematosus (SLE). To examine whether these findings may be related to dysregulated protein tyrosine kinase (PTK) function, we tested the relative amount and enzyme activity of the main PTKs involved in the earliest signalling steps triggered via the CD3 pathway. Cell lysates from peripheral blood T cells in SLE patients showed lower amounts of p59(fyn) and p56(lck) as shown by immunoblot. In contrast, the amount of ZAP-70, a PTK of the syk family, was comparable in both groups. However, p59(fyn) immuno-precipitates obtained from unstimulated peripheral blood SLE T cells showed enhanced PTK activity as compared to controls, whereas the PTK activity of p56(lck) and ZAP-70 molecules was comparable in both groups. The unchecked activity of the TCR/CD3-associated src kinase p59(fyn) may alter the balance needed for regulated T cell responses in SLE patients.     

Regulation of CTL by ecto-nictinamide adenine dinucleotide (NAD) involves ADP-ribosylation of a p56lck-associated protein

Receptor-mediated activation of T lymphocytes involves protein phosphorylation by several protein tyrosine kinases, among those the src-related enzymes p56lck and p59fyn. Accumulating evidence supports the notion that these enzymes are regulated by tyrosine phosphorylation and dephosphorylation, but much is yet to be learned about regulation of their activity. Here we demonstrate that p56lck but not p59fyn exists as a complex with a 40-kDa protein, which in its ADP-ribosylated form inhibits p56lck kinase activity. ADP-ribosylation of this protein is mediated by an arginine-specific mono-ADP-ribosyltransferase, which makes use of extracellular nicotinamide adenine dinucleotide (NAD). This enzyme is a glycosyl-phosphatidylinositol-anchored protein releasable from the surface of cytotoxic T cells by glycosyl-phosphatidylinositol-specific phospholipase C. Release of arginine-specific mono-ADP-ribosyltransferase results in failure of extracellular NAD to downmodulate p56lck kinase activity. Concomitant to suppression of the kinase by NAD, CD8 mediated transmembrane signaling and p56lck kinase activation are inhibited.     

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.     

Zinc is essential for binding of p56(lck) to CD4 and CD8alpha

Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8alpha is necessary for T-lymphocyte development and activation. Association of p56(lck) with CD4 requires two conserved cysteine residues in the cytosolic domain of CD4 and two in the amino terminus of p56(lck), consistent with the notion that these four residues coordinate a single metal atom (1-5). Here we demonstrate that Zn2+ is essential for complex formation. In an in vitro binding reaction, Zn2+ mediates p56(lck) association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of CD4 or CD8alpha; no other metals tested support binding. Treatment of preformed GST-CD4.p56(lck) dimers with the Zn2+ chelators 1,10-O-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid results in dissociation of GST-CD4 from p56(lck), consistent with the finding of Huse et al. (5) that Zn2+ is contained within similar complexes. Furthermore, we show that, within live cells, CD4.p56(lck) and CD8alpha.p56(lck) interactions occur in a zinc-dependent fashion. Specifically, pretreatment of the human Jurkat T-cell line with membrane permeable zinc chelators disrupts CD4.p56(lck) complexes, and treatment of COS cells co-expressing CD8alpha and p56(lck) with such chelators likewise leads to dissociation of CD8alpha.p56(lck) complexes. CD4. p56(lck) and CD8alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization.     

Tumor-induced suppression of T lymphocyte proliferation coincides with inhibition of Jak3 expression and IL-2 receptor signaling: role of soluble products from human renal cell carcinomas

The proliferative capacity of T cells infiltrating human tumors is known to be impaired, possibly through their interaction with tumor. Here we demonstrate that soluble products derived from renal cell carcinoma (RCC-S) explants but not normal kidney can inhibit an IL-2-dependent signaling pathway that is critical to T cell proliferation. A major target of the immunosuppression was the IL-2R-associated protein tyrosine kinase, Janus kinase 3 (Jak3). RCC-S suppressed basal expression of Jak3 and its increase following stimulation with anti-CD3/IL-2. Jak3 was most sensitive to suppression by RCC-S; however, reduction in expression of p56(lck), p59(fyn), and ZAP-70 was observed in some experiments. Expression of other signaling elements linked to the IL-2R (Jak1) and the TCR (TCR-zeta, CD3-epsilon, and phospholipase C-gamma) were minimally affected. In naive T cells, RCC-S also partially blocked induction of IL-2R alpha-, beta- and gamma-chain expression when stimulating via the TCR/CD3 complex with anti-CD3 Ab. To determine whether RCC-S suppressed IL-2-dependent signaling, primed T cells were employed since RCC-S had no effect on IL-2R expression but did down-regulate Jak3 expression and, to a lesser degree, p56(lck) and p59(fyn). Reduction in Jak3 correlated with impaired IL-2-dependent proliferation and signal transduction. This included loss of Jak1 kinase tyrosine phosphorylation and no induction of the proto-oncogene, c-Myc. These findings suggest that soluble products from tumors may suppress T cell proliferation through a mechanism that involves down-regulation of Jak3 expression and inhibition of IL-2-dependent signaling pathways.     

Two distinct pathways exist for down-regulation of the TCR

TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. Down-regulation of the TCR is induced by engagement of the TCR by specific ligands and/or by activation of protein kinase C (PKC). We report here that ligand- and PKC-induced TCR down-regulation is mediated by two distinct, independent mechanisms. Ligand-induced TCR down-regulation is dependent on the protein tyrosine kinases p56(lck) and p59(fyn) but independent of PKC and the CD3gamma leucine-based (L-based) internalization motif. In contrast, PKC-induced TCR down-regulation is dependent on the CD3gamma L-based internalization motif but independent of p56(lck) and p59(fyn). Finally, our data indicate that in the absence of TCR ligation, TCR expression levels can be finely regulated via the CD3gamma L-based motif by the balance between PKC and serine/threonine protein phosphatase activities. Such a TCR ligation-independent regulation of TCR expression levels could probably be important in determining the activation threshold of T cells in their encounter with APC.     

Differences in binding of PI 3-kinase to the src-homology domains 2 and 3 of p56 lck and p59 fyn tyrosine kinases

Two T cell-specific src-family tyrosine kinases, p56 lck (lck) and p59 fyn (fyn), are implicated in regulating PI 3-kinase activity in response to interleukin-2 (IL-2), a cytokine that induces T cell proliferation. The src- homology domains 3 (SH3) of src-family kinases can directly interact with the PI 3-kinase regulatory subunit p85 and this may be a mechanism to regulate PI 3-kinase activity. In order to understand the mode of PI 3-kinase activation by the IL-2 receptor, we examined the association of PI 3-kinase to SH2 and SH3 domains of lck and fyn in IL-2-dependent kit 225 cells. The fyn SH3 domain bound more PI 3-kinase and its p85 subunit than the lck SH3 domain, while the lck SH2 domain bound more PI 3-kinase than the fyn SH2 domain. None of these interactions were regulated by IL-2. Low binding of PI 3-kinase to the lck SH3 domain was not observed in IL-2-independent Jurkat T cells. Thus, SH3 and SH2 domains of lck and fyn bound different amounts of PI 3-kinase, a feature that was dependent on a T cell type, but was not influenced by IL-2.     

Surface IgM-stimulated proliferation, inositol phospholipid hydrolysis, Ca2+ flux, and tyrosine phosphorylation are not altered in B cells from p59fyn-1- mice

The surface immunoglobulin M (sIgM)-associated src family protein tyrosine kinases (PTKs) p55blk, p59fyn, and p53/56lyn become activated in B cells within seconds following sIgM cross-linking. Studies using protein tyrosine kinase (PTK) inhibitors have demonstrated that PTK activity is crucial for downstream events such as calcium flux, inositol phospholipid hydrolysis, and cell cycle entry. The roles that the individual src family PTKs play in sIgM signaling are largely unknown, however. In order to determine whether p59fyn plays a distinct role in sIgM signal transduction, the signaling capabilities of B cells isolated from fyn "knockout" mice were evaluated. We observed that in the absence of p59fyn, there was no demonstrable compromise of the sIgM-coupled signaling events measured (tyrosine phosphorylation, inositol phospholipid hydrolysis, and Ca2+ flux). We propose that either p59fyn is not involved in coupling sIgM to these specific signaling pathways or that other PTKs are able to compensate for the absence of p59fyn, indicating redundancy in the sIgM signaling pathways.     

CD4:p56lck association studied in vivo using antibody-induced capping and double indirect immunofluorescence microscopy

Accumulating data suggest that the T-cell surface antigen CD4 transduces an independent signal during antigen-mediated T-cell activation. In vitro studies which showed that the cytoplasmic protein tyrosine kinase p56lck is present in anti-CD4 immunoprecipitates led to the model that p56lck is associated with the cytoplasmic domain of CD4. In this report we have extended these studies and examined potential CD4:p56lck associations in vivo. We show here by double immunofluorescence microscopy a specific co-distribution of p56lck with antibody-induced CD4 caps in intact cells. Murine T-cell hybridoma lines expressing mutant forms of CD4 were used to demonstrate that the 31 carboxyterminal aminoacids of its cytoplasmic domain, in particular cysteine-420 and cysteine-422, are crucial for the formation of CD4:p56lck complexes in vivo. The potential of the method applied is discussed with regard to studies of other transmembrane signalling systems involving src-like kinases.     

Novel T cell antigen 4-1BB associates with the protein tyrosine kinase p56lck1

4-1BB is a 30-kDa inducible T cell Ag, and is expressed predominantly as a 55-kDa dimer on both CD4+ and CD8+ T lymphocytes. The cytoplasmic tail of 4-1BB contains the sequence Cys-Arg-Cys-Pro, which is similar to the sequence Cys-X-Cys-Pro, which mediates the binding of the CD4 and CD8 molecules to the protein tyrosine kinase p56lck. An anti-4-1BB mAb, 53A2, was used to determine whether 4-1BB may associate with p56lck. The 53A2 mAb specifically recognized 4-1BB on a CD8+ T cell line, CTLL-2, and coimmunoprecipitated a 56-kDa protein along with 4-1BB. Peptide mapping indicated that the 56-kDa phosphoprotein was identical to p56lck. The coimmunoprecipitation of p56lck with 4-1BB also occurred in nonlymphoid cells such as insect (Sf-21) and HeLa cells when the two recombinant proteins were coexpressed. Analysis of mutant p56lck recombinant proteins showed that two cysteine residues critical for p56lck-CD4 (or -CD8) complex formation are also required for the p56lck-4-1BB interaction. These studies establish that 4-1BB physically associates with p56lck.     

Contribution of tyrosine kinases to the selective orientation of human CD4+ bifunctional cloned T cells toward proliferation or cytolytic function

We show that T cell activation of human CD4+ cloned T cells through the CD2 molecule can induce either autocrine proliferation or cytolysis, depending on the pair of anti-CD2 mAbs used for stimulation, that is, D66/T11(1) or GT2/T11(1), respectively. As the earliest biochemical event after CD2 stimulation is likely the induction of tyrosine phosphorylation of various proteins, we investigated whether differential activation of protein tyrosine kinases (PTKs) could contribute to the selective induction of each function. Results show that herbimycin A, a potent PTK inhibitor, markedly decreased the induction of both proliferation and cytolysis. This implies a regulatory role for tyrosine phosphorylation in the induction of each function by CD2. However, that PTKs are differentially activated upon induction of proliferation by D66/T11(1) or cytotoxic function by GT2/T11(1) emanated from two different approaches. First, immunoblotting total cellular extracts with an anti-phosphotyrosine mAb showed different patterns of tyrosine phosphorylation depending on the pair of CD2 mAbs used for stimulation. Second, a differential activation of p56lck, a src-related PTK, was observed after stimulation with D66/T11, and GT2/T11(1). Although induction of proliferation by D66/T11(1) was correlated with increased Lck activity, this was not observed when cells were triggered to lyse by GT2/T11(1). Thus, by providing striking correlative evidences linking differences in PTK activation with induction of different functions in bifunctional cloned T cells, our results strongly suggest that PTKs may contribute to the selective orientation of T cell functions at a single-cell level.     

Interphase cytogenetic (in situ hybridization) analysis of astrocytomas using archival, formalin-fixed, paraffin-embedded tissue and nonfluorescent light microscopy

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.     

Differential requirement for p56lck in fetal and adult thymopoiesis

The protein tyrosine kinase p56lck is critical for the generation of mature thymocytes in adult mice. However its requirement during the maturation of thymocytes from the fetal to the adult stage has not been clearly defined. We analyzed prenatal and postnatal thymocyte maturation in mice deficient for p56lck (lck[-/-]). Before birth, lck appears to play a crucial role in the expansion and proliferation of CD4+CD8+ double positive thymocytes, whereas proliferation and absolute numbers of CD4-CD8- double negative thymocyte precursors remained within the normal range until the end of the second week postnatal. Three weeks after birth, the total numbers of double negative and immature single positive thymocytes underwent a dramatic reduction that correlated with a decrease in the double positive population. This ontogenic defect was associated with a significant decrease in the proliferation rates of thymocyte precursors. Our data suggest that signaling via p56lck kinase is differentially required within a given phenotypically defined thymocyte subpopulation, depending on its stage of thymocyte maturation.     

Phosphorylation and coupling of zeta-chains to activated T-cell receptor (TCR)/CD3 complexes from peripheral blood T-cells of elderly humans

Aging is often accompanied by altered T-cell signaling and functions. Signals mediated through the T-cell receptor (TCR)/CD3 complex are associated with tyrosine phosphorylations of zeta-chains by the regulated activities of protein tyrosine kinases p56(lck) and p59(fyn) as well as protein tyrosine phosphatases. In the present investigation, the coupling and phosphorylation of zeta-chains to TCR/CD3 immunocomplexes were examined in peripheral blood T-cells from 13 elderly and young humans stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. Western blots analyzing the non-covalent coupling of zeta-chains to TCR/CD3 immunocomplexes from Brij-96 detergent lysates of anti-CD3 ligated T-cells showed that the levels of zeta-chains within TCR/CD3 immunocomplexes from T-cells of elderly and young subjects did not significantly differ. By contrast, the levels of phosphorylated zeta-chains generated during in vitro phosphorylations of TCR/CD3 immunocomplexes from elderly subjects were significantly reduced and averaged 44% of those observed for anti-CD3epsilon ligated T-cells from young subjects. Analyses of the levels of zeta-chain coupling and phosphorylations in T-cells from each of the 13 elderly individuals also showed that the reductions in zeta-chain phosphorylations were heterogeneous and unrelated to modest reductions in coupling. Furthermore, the age-related decreases in zeta-chain phosphorylations were not due to diminished frequencies of CD3epsilon+ cells or densities of CD3epsilon surface receptors and could be observed without reductions in epsilon-chain phosphorylations. These results suggest that aberrancies of zeta-chain phosphorylations can occur in T-cells of elderly humans independent from any uncoupling of zeta-chains to activated TCR/CD3 complexes.     

The CD4-associated tyrosine kinase p56lck is required for lymphocyte chemoattractant factor-induced T lymphocyte migration

Lymphocyte chemoattractant factor (LCF) is a polypeptide cytokine which induces both cell motility and activation of T lymphocytes. These LCF-induced events demonstrate an absolute requirement for the cell surface expression of CD4. Because many CD4-mediated T lymphocyte activation events have been demonstrated to require the association of the src-related tyrosine kinase p56lck with the cytoplasmic domain of CD4, we examined the role of p56lck in LCF-induced lymphocyte migration in a murine T cell hybridoma line expressing transfected human CD4. LCF induces the catalytic activity of CD4 associated p56lck at chemoattractant concentrations of cytokine. Hybridoma cells that express CD4 with cytoplasmic point mutations which uncouple the CD4-lck association lack both lck enzymatic activity and chemotactic responses to LCF. The enzymatic activity of lck however does not appear to be required for CD4-mediated migratory signal. First, the protein tyrosine kinase inhibitor herbimycin A blocked LCF-induced p56lck activation but had no effect on the LCF-induced motile response. Second, T cell hybridomas expressing a chimeric receptor combining the extracellular domain of human CD4 and murine p56lck which lacked the kinase domain had a normal LCF-induced motile response. We conclude from these observations that CD4-lck coupling is essential for LCF-induced T lymphocyte migration but the motile response is independent of the enzymatic activity of CD4-associated p56lck.     

Prostaglandin E2 modulation of p59fyn tyrosine kinase in T lymphocytes during sepsis

Prostaglandin E2 (PGE2) has been implicated in the suppression of T cell IL-2 production and proliferation during burn and sepsis. The present study evaluated the potential intracellular mechanism of suppressed T cell responses by assessing the activation of p59fyn kinase in T cells from septic rats as well as the T cells incubated with PGE2. p59fyn is known to regulate T cell functions. Sepsis was induced in rats by implanting fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10(4) CFU) into the abdominal cavity. For the assessment of PGE2 role in sepsis, a group of septic rats were treated with indomethacin, which inhibits endogenous PGE2 synthesis. As assessed by immunoblotting or in vitro kinase assay, a more than 40% inhibition of p59fyn phosphorylation and kinase activity was observed in septic rat T cells compared with the T cells from sterile or control rats. A similar inhibition in p59fyn phosphorylation and kinase activity was observed in PGE2-treated T cells compared with the T cells incubated in the absence of PGE2. The septic-related suppression in p59fyn phosphorylation and kinase activity in T cells was prevented in rats treated with indomethacin. We observed that the inhibition in p59fyn activation in septic or PGE2-treated T cells was due primarily to a suppression in p59fyn phosphorylation and not due to alterations in p59fyn protein expression. These findings suggest that PGE2 released during sepsis could contribute to the sepsis-related suppression in T cell proliferation by attenuating p59fyn phosphorylation and its kinase activity.     

Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes

Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin ("caveolae"). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low-density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes. We provide a detailed analysis of the two most highly expressed NRPTK, p53/p56lyn and p56lck, which are involved in the transduction of signals for proliferation and differentiation of monocytes/B lymphocytes and T lymphocytes, respectively. We show that lyn is selectively recovered in LDTI complexes isolated from human leukemic cell lines (promyelocytic [HL-60], erythroid [K562] and B-lymphoid [697]) and from normal human granulocytes, and that lck is recovered from LDTI fractions of leukemic T- and B-lymphoid cell lines (CEM, 697). In LDTI fractions of leukemic cells, lck and lyn are enriched 100-fold as compared with the total cell lysates. Analysis of these fractions by electron microscopy shows the presence of 70- to 200-nm vesicles: lyn and lck are homogenously distributed in the vesicles, as revealed by an immunogold labeling procedure. These novel results propose a role for these vesicles in signal transduction mechanisms of normal and neoplastic hematopoietic cells. In support of this hypothesis, we further observed that molecules participating in B- and T-cell receptor activation cofractionate in the LDTI fractions, CD45/lyn (B cells) and CD45/lck/CD4 (T cells).     

The protein tyrosine kinase p56lck regulates cell adhesion mediated by CD4 and major histocompatibility complex class II proteins

The CD4 protein is expressed on a subset of human T lymphocytes that recognize antigen in the context of major histocompatibility complex (MHC) class II molecules. Using Chinese hamster ovary (CHO) cells expressing human CD4, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In T lymphocytes, CD4 can also function as a signaling molecule, presumably through its intracellular association with p56lck, a member of the src family of protein tyrosine kinases. In the present report, we show that p56lck can affect cell adhesion mediated by CD4 and MHC class II molecules. The expression of wild-type p56lck in CHO-CD4 cells augments the binding of MHC class II+ B cells, whereas the expression of a mutant p56lck protein with elevated tyrosine kinase activity results in decreased binding of MHC class II+ B cells. Using site-specific mutants of p56lck, we demonstrate that the both the enzymatic activity of p56lck and its association with CD4 are required for this effect on CD4/MHC class II adhesion. Further, the binding of MHC class II+ B cells induces CD4 at the cell surface to become organized into structures resembling adhesions-type junctions. Both wild-type and mutant forms of p56lck influence CD4-mediated adhesion by regulating the formation of these structures. The wild-type lck protein enhances CD4/MHC class II adhesion by augmenting the formation of CD4-associated adherens junctions whereas the elevated tyrosine kinase activity of the mutant p56lck decreases CD4-mediated cell adhesion by preventing the formation of these structures.     

Lineage-specific control of superantigen-induced cell death by the protein tyrosine kinase p56(lck)

Cell fate decisions in developing T cells depend on signal transduction via the Ag-specific TCR. Although the same TCR can signal for survival or cell death, specific signals that lead to cellular activation or death have not been identified. To study the role of the src tyrosine kinase p56(lck) in cell death of developing T cells, we introduced endogenous mouse mammary tumor retroviruses encoding superantigens (SAG) into p56(lck)-deficient mice. We show that clonal deletion of SAG-reactive CD4+ T cells does occur in p56(lck) -/- mice. Clonal deletion was also evident in CD4+ cells expressing TCRVbeta7, which has low affinity for Mls-1a. However, clonal deletion did not occur in SAG-reactive CD8+ T cells from p56(lck) -/- mice. Deletion of cells expressing SAG-reactive TCRVbeta chains was apparent in CD4+ single-positive but not in CD8+ single-positive thymocytes. Both CD4+ and CD8+ peripheral T cells from Mls-1b p56(lck) -/- mice responded to Mls-1a in vitro. However, CD8+ T cells from Mls-1a p56(lck) -/- mice that did not undergo deletion could not respond to Mls-1a, indicating that these cells are functionally unresponsive. These data show that p56(lck) is not required for clonal deletion of SAG-reactive CD4+ lymphocytes, including CD4+ cell expressing TCRs with low affinity for the SAG. However, p56(lck) appears to be an important signal transduction molecule involved in deletion of SAG-reactive CD8+ T cells.