Interactions between the tyrosine kinases p56lck,p59fyn and p50csk in CD4 signaling in T cells

Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.     

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.     

HIV-1 glycoprotein gp120 disrupts CD4-p56lck/CD3-T cell receptor interactions and inhibits CD3 signaling

Using the CD4⁺ human T cell clone P28, we demonstrated that the HIV-1 glycoprotein gp120 inhibited CD3-induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56^lck from CD4 in CEM cells, with a concommittant inhibition of CD4-linked kinase activity. We have addressed the question whether disruption of CD4/p56^lck or CD4/CD3-T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti-CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4-associated p56^lck and CD4-linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti-CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3-induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide-specific phospholipase C-γ-1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3-induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4-p56^lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down-modulation of CD4-p56^lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3-linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.     

Expression of CD4-like structure on murine egg vitelline membrane and its signal transductive roles through p56lck in fertilization.

Expression of CD4-like molecule on vitelline membrane of murine eggs was demonstrated by indirect immunofluorescence (IIF) test and immunoprecipitation corresponding to the expression of major histocompatibility complex (MHC) class II molecule on murine sperm detected by immunoblotting. This molecule showed slightly larger size than that of the authentic CD4 molecule from T-cells on SDS-PAGE. This molecule was suggested to bind to MHC class II structure on sperm during fertilization because anti-CD4 monoclonal antibody (mAb) blocked in vitro fertilization (IVF). In addition, src-related tyrosine protein kinase (p56lck) was demonstrated in the inner vitelline membrane of eggs by means of IIF with anti-p56lck mAb and immune-complex kinase assay. This molecule was suggested to be associated with CD4-like molecule.     

Activation of phosphatidylinositol-3-kinase in Jurkat T cells depends on the presence of the p56lck tyrosine kinase

Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol-3-kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3-induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56^lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56^lck, JCaM1, these responses were absent. We also show that p56^lck directly activates PI3K purified from transfected COS-1 cells, indicating that other T cell-specific proteins are not required for the process. Finally, tryptic peptide maps show that p56^lck phosphorylates three tyrosine residues in the p85α subunit of PI3K and two in p110 of PI3K. Our results suggest that p56^lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.     

Lack of p56 lck expression correlates with CD4 endocytosis in primary lymphoid and myeloid cells

In cell lines the endocytic properties of CD4 are regulated through its association with the src -family tyrosine kinase p56 ^lck . In lymphoid cell lines expressing p56 ^lck , CD4 is restricted to the cell surface and undergoes only limited internalization. Phosphorylation of the cytoplasmic domain of CD4 causes p56 ^lck to dissociate and activates an endocytosis signal leading to the internalization of CD4 through clathrin-coated pits. In p56 ^lck -negative transfected cell lines CD4 is constitutively internalized, but internalization is inhibited when p56 ^lck is expressed in these cells. We now demonstrate that these endocytic properties of CD4 determined in transfected cell lines hold true for CD4 naturally expressed on myeloid cell lines (HL-60 and U937), as well as on primary lymphocytes, monocytes and macrophages isolated from human blood. CD4 showed limited internalization on p56 ^lck -positive lymphocytes, but was rapidly internalized in p56 ^lck -negative monocytes and macrophages. Surprisingly, rapid internalization of CD4 was seen with the lymphocytes from one unidentified donor. In these cells we failed to detect p56 ^lck expression by Western blotting.     

Allo-skin graft rejection,tumor rejection and natural killer activity in mice lacking p56lck

Mice lacking the p56^lck molecule (lck?/?) have a profound block in the maturation of thymocytes and a greatly reduced number of peripheral mature T cells. To analyze further the functions of the T cells developed in lck ?/? mice in vivo, we evaluated the ability of lck?/? mice to reject allo-skin grafts and methylcholanthrene (MCA)-induced syngeneic fibrosarcoma, and also examined the biological activity of lck ?/? natural killer (NK) cells. Mice lacking p56^lck failed to reject skin grafts from either MHC-disparate or minor-histocompatibility-different donors, even after they had been primed with donor spleen cells. They also failed to reject the MCA-induced immunogenic syngeneic fibrosarcoma, MC57X. NK activity in mice lacking p56^lck was normal, and there were no differences in the NK cell activation induced by poly(I) · poly(C) stimulation or interleukin-2 stimulation (lymphokine-activated killer induction) between mice lacking p56^lck and their immunocompetent heterozygous littermates. NK cells lacking p56^lck mediated a normal antibody-dependent cell-mediated cytotoxicity (ADCC) response. The results of this study indicate that the loss of p56^lck severely impairs the effectors of the immune system which mediate the rejection of allo-skin grafts and syngeneic tumors. The normal NK activity in lck ?/? mice suggests that p56^lck is not required for the development and activation of NK cells.     

The cytoplasmic domain of rat NKR-P1 receptor interacts with the N-terminal domain of p56lck via cysteine residues

NKR-P1 is a type II transmembrane protein which acts as an activation receptor on natural killer (NK) cells. The cytoplasmic domains of the CD4, CD8 and 4-1BB receptors contain the sequence Cys-X-Cys-Pro which is directly involved in coupling to another pair of cysteines in the N-terminal domain of the src family tyrosine kinase p56^lck. The cytoplasmic domain of NKR-P1 in rodents also contains the Cys-X-Cys-Pro sequence, but the capacity of the receptor to bind p56^lck is presently unknown. We tested for direct coupling between these proteins using both protein biochemistry and the yeast two-hybrid technique. Immunoprecipitation studies showed that p56^lck can be co-immunoprecipitated with NKR-P1 from a rat NK tumor cell line. In addition, the cytoplasmic domain of NKR-P1 interacted with the N-terminal domain of p56^lck in yeast as assessed by reporter gene activation. Integrity of the cysteine pairs in both proteins was critical in mediating the interaction. The experiments suggest that the association of p56^lck with NKR-P1 is somewhat weaker than the p56^lck association with CD8α, but of much lower avidity than between CD4 and p56^lck. This could reflect a higher activation threshold for the NKR-P1 and CD8 receptors, which are involved in cytolytic responses, compared to CD4 which is involved in T cell helper function.     

Co-capping studies reveal CD8/TCR interactions after capping CD8β polypeptides and intracellular associations of CD8 with p56lck

CD8 is a T cell surface glycoprotein that participates in recognition of peptide/MHC class I molecules by binding to their α3 domains. In addition, the cytoplasmic domain of CD8 associates with the intracellular tyrosine kinase p56^lck (lck) promoting recruitment of lck to the TCR signaling complex. Recent data have suggested also that CD8 may interact with the TCR to promote energetically favorable conformations which increase its ligand binding. We have used the techniques of co-capping and confocal microscopy to ask whether we can detect an association between CD8 and the TCR independently of their binding to MHC class I molecules. We show that capping CD8 heterodimers with antibodies to the CD8β polypeptide is significantly more efficient than antibodies to the CD8α polypeptide at inducing co-localization of TCR molecules with CD8, suggesting that there may be preferred conformations of CD8 which stabilize interactions with the TCR. In addition, we show by microscopy that intracellular lck redistributes very efficiently to the area of a CD8 cap, suggesting that there is a stronger association between lck and CD8 than has been proposed from immunoprecipitation analyses.     

Human immunodeficiency virus gp120 and derived peptides activate protein tyrosine kinase p56Ick in human CD4 T lymphocytes

Human immunodeficiency virus binds to CD4 T lymphocytes by interaction between its envelope glycoprotein gpl20 and the CD4 molecule. The latter is non-covalently associated with a src-related tyrosine kinase, p56^lck. CD4 cross-linking increases the activity of p56^lck, leading to phosphorylation of several cellular substrates. We report here that gpl60/120 increases both the autophos-phorylation of p56^lck and its enzymatic activity (reflected by phosphorylation of an exogeneous substrate) in normal T cells and the HUT78 CD4⁺ T cell line. This effect was detectable 5 min after activation and persisted for 40 min in normal T cells. It did not require gpl20 cross-linking and was associated with phosphorylation of tyrosine residue on several proteins, as shown by phosphotyrosine Western blot analysis. The pattern of proteins phosphorylated on tyrosine residues in response to gpl20 activation was distinct from that induced by anti-CD4 antibodies. p56^lck activation required its association with CD4, since p56^lck activity was not modified in HUT78 T cell lines expressing a truncated or mutated form of CD4 unable to associate with p56^lck. Peptides mimicking residues 418 to 434 and 449 to 464 of HIV-1 Bru gpl20, regions known to participate in gpl20 binding to CD4, also increased p56^lck activity and triggered phosphorylation of similar substrates. Taken together, these results show that gpl60/120 and derived peptides can transiently increase p56^lck activity without the need for CD4 cross-linking. This activation led to a specific pattern of tyrosine phosphorylation on cellular proteins that may be of significance in the biological effects of the gpl20/CD4 interaction, e.g. syncytium formation and inhibition of T cell activation.     

T cell receptor and CD8-dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein

Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56^lck kinase have now been examined in this two-step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56^lck. When CD8 bound to class I, phosphorylation of p56^lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56^lck activity.     

Anti-CD45 isoform antibodies enhance phagocytosis and gene expression of IL-8 and TNF-alpha in human neutrophils by differential suppression on protein tyrosine phosphorylation and p56lck tyrosine kinase

To determine the biological functions of membrane expressed CD45 isoforms on polymorphonuclear neutrophils (PMN), the monoclonal IgG F(ab')2 antibody against CD45, CD45RA or CD45RO was used as surrogate ligand for binding with these molecules on PMN. We found 99.5 +/- 3.2%, 42.3 +/- 5.8% and 96.7 +/- 2.6% PMN expressed CD45, CD45RA and CD45RO molecules on the cell surface, respectively. The interaction of CD45, CD45RA or CD45RO with its specific antibody on PMN enhanced phagocytosis markedly (34-83% increase), mainly via increased expression of complement receptor type 3 (CR3, CD11b) on the cells. The production of IL-8 by PMN was also increased significantly after binding with antibodies (anti-CD45 > anti-CD45RO > anti-CD45RA). Anti-CD45RA and anti-CD45RO, but not anti-CD45, enhanced TNF-alpha mRNA expression and decreased protein tyrosine phosphorylation of PMN. However, only anti-CD45RO suppressed Src family protein tyrosine kinase p56lck expression in the cells. These results suggest that the cross-linking of CD45 isoforms by their specific antibodies stimulated different PMN activities by differential suppression on protein tyrosine phosphorylation and Src family tyrosine kinase p56lck.     

CD4-dependent and -independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56^lck, p59^fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56^lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolées, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4⁺ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4⁺ T lymphocytes. Our results show that, in normal CD4⁺ T lymphocytes, as in CD4⁺ T cell lines, there is a stable and readily detectable association between CD4: p56^lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4⁺ lymphocytes have detectable amounts of p56^lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59^fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56^fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4⁺ T cells and CD4⁺ T cell lines in kinase association to the TcR/CD3 complex.     

蕈样肉芽肿患者外周血单一核细胞CD45RA及CD45RO表达的研究

目的探讨蕈样肉芽肿（Mycosis fungoides,MF）患者外周血单个核细胞CD45RA及CD45RO的表达及其与MF发病的关系。方法应用双荧光抗体标记、流式细胞仪检测15例MF患者外周血单个核细胞CD45RA及CD45RO的表达。结果（1）MF患者外周血CD3^＋、CD3^＋CD8^＋细胞与正常对照比较差异不显著（P〉0.05）。（2）MF患者外周血CD4^＋细胞低于正常对照,差异非常显著（P〈0.001）。（3）MF患者外周血T细胞CD3^＋CD4^＋/CD3^＋CD8^＋比值低于正常对照,差异显著（P〈0.001）。（4）MF患者外周血CD45RA^＋细胞低于正常对照,差异非常显著（P〈0.001）,CD45RO^＋细胞高于正常对照,差异非常显著（P〈0.001）。（5）MF患者外周血CD45RO^＋/CD45RA^＋比值高于正常对照,差异非常显著（P〈0.001）。（6）MF患者外周血CD4^＋CD45RA^＋细胞低于正常对照,差异非常显著（P〈0.001）。（7）MF患者外周血CD4^＋CD45RO^＋细胞及CD8^＋CD45RO^＋细胞均高于正常对照,差异非常显著（均P〈0.001）。（8）MF患者外周血CD4^＋CD45RO^＋/CD4^＋CD45RA^＋比值及CD8^＋CD45RO^＋/CD8^＋CD45RA^＋比值均高于正常对照,差异非常显著（P〈0.01及P〈0.001）。结论MF患者外周血中,不仅存在CD4^＋亚群失调和CD4^＋/CD8^＋比值降低,而且在CD4^＋和CD8^＋亚群中也存在CD45RA^＋、CD45RO^＋亚群失调和CD45RO^＋/CD45RA^＋比值升高,从而导致的机体免疫功能紊乱,可能与MF的发病或病情加剧有关。     

Interaction of human immunodeficiency virus glycoprotein 160 with CD4 in Jurkat cells increases p56lck autophosphorylation and kinase activity.

The tyrosine protein kinase p56lck, specifically expressed in lymphoid cells, undergoes modifications of its autophosphorylation and kinase activity when these cells are triggered by mAbs to the T cell determinants. The kinase activity and the autophosphorylation of p56lck were analysed following triggering Jurkat cells with the human immunodeficiency virus (HIV) glycoprotein gp160 which interacts with CD4: both the autophosphorylation and the kinase activity are increased within 1-5 min following addition of gp160, this increase is maximum at 5 min and is followed by a gradual return to the basal level within 2 h. Similar to observations made with anti-CD4 mAbs the increase in kinase activity of p56lck is not associated with changes in the gel mobility nor is it associated with T cell activation. Triggering of T cells with a combination of anti-CD3 mAbs which activate T cells but not p56lck and gp160 greatly potentiated the increase of p56lck autophosphorylation and kinase activity.     

CD45-associated protein promotes the response of primary CD4 T cells to low-potency T-cell receptor (TCR) stimulation and facilitates CD45 association with CD3/TCR and lck

Although it is clear that the CD45 tyrosine phosphatase is required for efficient T-cell activation and T-cell development, the factors that regulate CD45 function remain uncertain. Previous data have indicated that there is an association of CD45 with CD4 and the T-cell receptor (TCR) complex controlled by the variable ectodomain of CD45 and, following activation, by high- and low-potency peptides. This suggests that controlling substrate access to CD45 may be an important regulatory mechanism during T-cell activation. In the present study we have examined the role of the transmembrane adapter-like molecule CD45-associated protein (CD45-AP) in regulating the association of CD45 with CD3/TCR and lck, and in regulating primary CD4(+) T-lymphocyte activation. In CD4(+) T cells from CD45-AP-deficient mice, coimmunoprecipitation of CD45 with the CD3/TCR complex, in addition to lck, is significantly reduced compared with wild-type T cells. Functionally, this correlates with a decreased proliferative response, a decrease in interleukin (IL)-2 production, and a decrease in calcium flux upon stimulation with a low-potency altered peptide ligand. However, the response of CD45-AP-deficient T cells to stimulation with a high-avidity agonist peptide was largely intact, except for a modest decrease in IL-2 production. These data suggest that CD45-AP promotes or stabilizes the association of CD45 with substrates and regulates the threshold of T-cell activation.