Lck Signaling Research Articles

CAML loss causes anaphase failure and chromosome missegregation

Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed cytoplasmic protein that is implicated in the EGFR and LCK signaling pathways and required for early embryonic and thymocyte development. To further define the critical biological functions of CAML at the cellular level, we generated CAML-deleted mouse embryonic fibroblasts (MEFs) using an in vitro Cre-loxP mediated conditional knockout system. We found that CAML–/– MEFs have severely impaired proliferation and a strong reduction of normal anaphases. The primary mitotic defect of CAML–/– MEFs is that duplicated chromosomes fail to segregate in anaphase, resulting in nuclear bisection by the cleavage furrow as cells decondense their DNA and exit mitosis, highly reminiscent of the “cut” phenotype in fission yeast. This phenotype is due to spindle dysfunction rather than inability to resolve physical connections between sister chromatids. Furthermore, CAML–/– MEFs display defects often seen in cells with mitotic checkpoint gene deficiencies, including lagging and misaligned chromosomes and chromatin bridges. Consistent with this, we found that CAML–/– MEFs have a modestly weakened spindle assembly checkpoint (SAC) and increased aneuploidy. Thus, our data identify CAML as a novel chromosomal instability gene and suggest that CAML protein acts as a key regulator of mitotic spindle function and a modulator of SAC maintenance.

Role of STAT3 in CD4+CD25+FOXP3+ Regulatory Lymphocyte Generation: Implications in Graft-versus-Host Disease and Antitumor Immunity

Immunological tolerance is maintained by specialized subsets of T cells including CD4(+)CD25(+)FOXP3(+) regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. Moreover, foxp3 gene transfer in murine CD4(+)CD25(-) T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4(+)CD25(-) T lymphocytes leads to STAT3 Tyr(705) phosphorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4(+)CD25(-) T cell conversion into Treg through costimulation with TCR/CD28 and TGF-beta1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-gamma. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differentiated CD4(+)CD25(+) T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4(+)CD25(+) T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4(+) lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance.

Signaling Mechanisms in Ischemic Preconditioning

See related article, pages 878–883 The cardiac “warm up” phenomenon, described more than 50 years ago in patients with coronary artery disease, refers to improvement in cardiac symptoms and physical performance following exposure to short periods of ischemia.1 Several mechanisms, such as adaptive reduction in oxygen consumption by the ischemic myocardial region, improved oxygen supply via collateral recruitment or dilation of the stenotic vessel, and activation of an intrinsic phenomenon called ischemic preconditioning (IPC) have been proposed to account for this phenomenon. IPC refers to a process in which brief periods of ischemia improves the ability of the heart to tolerate subsequent prolonged ischemic periods.3 It was first identified in the heart in 1986 by Murry et al,2 and has since been demonstrated in various experimental and animal models.3 Several triggers have been proposed for IPC, including adenosine, bradykinin, protaglandins, opiod receptors, nitric oxide, and Ca2+.4 These triggers lead to the activation of several intracellular pathways that ultimately protect myocardial cells against injury. Although the details of these pathways have not been totally characterized, mitochondria have been shown to be key mediators of IPC. Specifically, the opening of a mitochondrial channel, called the mitochondrial ATP-sensitive potassium channel or mitoKATP is believed to be critical for the induction of IPC; drugs that activate this channel protect against ischemia and inhibitors of mitoKATP reverse these protective effects.5 The signaling pathways that lead to the activation of mitoKATP are still under investigation.5 In a recent article, Oldenburg et al demonstrated that in isolated rabbit adult cardiomyocytes, bradykinin increased the levels of reactive oxygen species (ROS) and this effect was reversed by inhibitors of both mitoK …

Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement

T cell polarization and redistribution of cellular components are critical to processes such as activation, migration, and potentially HIV infection. Here, we investigate the effects of CD4 engagement on the redistribution and localization of chemokine receptors, CXCR4 and CCR5, adhesion molecules, and lipid raft components including cholesterol, GM1, and glycosyl-phosphatidylinositol (GPI)-anchored proteins. We demonstrate that anti-CD4-coated beads (αCD4-B) rapidly induce co-capping of chemokine receptors as well as GPI-anchored proteins and adhesion molecules with membrane cholesterol and lipid rafts on human T cell lines and primary T cells to the area of bead–cell contact. This process was dependent on the presence of cellular cholesterol, cytoskeletal reorganization, and lck signaling. Lck-deficient JCaM 1.6 cells failed to cap CXCR4 or lipid rafts to αCD4-B. Biochemical analysis reveals that CXCR4 and LFA-1 are recruited to lipid rafts upon CD4 but not CD45 engagement. Furthermore, we also demonstrate T cell capping of both lipid rafts and chemokine receptors at sites of contact with HIV-infected cells, despite the binding of an HIV inhibitory mAb to CXCR4. We conclude that cell surface rearrangements in response to CD4 engagement may serve as a means to enhance cell-to-cell signaling at the immunological synapse and modulate chemokine responsiveness, as well as facilitate HIV entry and expansion by synaptic transmission.

Regulated expression of galectin-1 during T-cell activation involves Lck and Fyn kinases and signaling through MEK1/ERK, p38 MAP kinase and p70S6kinase

Recent evidence has implicated galectins and their carbohydrate ligands as novel regulators of T-cell homeostasis. Galectin-1 (Gal-1), a member of this family, inhibits clonal expansion, induces apoptosis of antigen-primed T lymphocytes and suppresses the development of T-cell-mediated autoimmune diseases in vivo. Because the beta-galactoside-binding protein is expressed in activated but not resting T cells, it has been hypothesized that Gal-1-induced apoptosis may constitute an autocrine suicide mechanism to eliminate activated T cells contributing to the termination of an effector immune response. We undertook this study to investigate the signals and intracellular pathways leading to Gal-1 expression during T-cell activation. When T cells were stimulated either with anti-CD3 or anti-CD28 monoclonal antibody plus PMA in the presence of accessory cells, a sustained up-regulation of Gal-1 was observed, reaching a plateau between days 3 and 5 following CD3 engagement or costimulation through CD28. Investigation of the signal transduction events involved in this process revealed a role for Lck and Fyn kinases, since the Src kinase inhibitor PP1 inhibited the up-regulated expression of Gal-1 following T-cell activation. Downstream signaling routes involve mitogen-activated protein kinase (MAPK) kinase (MEK)1/extracellular signal-regulated kinase (ERK) and p38 MAPK, as Gal-1 expression was prevented by U0126 and SB202190. In addition, expression of Gal-1 involves interleukin (IL)-2-dependent signaling routes triggered by p70S6 kinase, as it could be inhibited by rapamycin. This is the first demonstration of the intracellular pathways that control activation-induced expression of Gal-1, which may reveal potential targets for immune intervention to modulate expression of this beta-galactoside-binding protein in pathological disorders.

A model for antigen-specific T-cell anergy: displacement of CD4-p56(lck) signalosome from the lipid rafts by a soluble, dimeric peptide-MHC class II chimera.

Soluble, dimeric peptide-MHC chimeras were shown to induce Ag-specific T cell anergy in vitro and in vivo. In this study, we describe a mechanism by which a soluble, dimeric peptide MHC class II chimera (DEF) induces Ag-specific T cell anergy. The anergic cells showed a displacement of the CD4-p56(lck) signaling module from the GM1-rich plasma membrane microdomains (lipid rafts), and subsequently an increase in p59(fyn) kinase activity, a dominant expression of p21 inhibitory TCR zeta-chain, and a poor phosphorylation and recruitment of zeta-associated protein of 70 kDa kinase to the TCR's immunoreceptor tyrosine-based activation motifs. The Th1 and Th2 transcription was suppressed and the cells were arrested in the Th0 stage of differentiation. Recovery from DEF anergy occurred late and spontaneously at the expense of low thresholds for activation-induced cell death. In contrast to DEF, a combination of TCR and CD4 mAbs did not induce such alterations or anergy, indicating that the ligand-mediated topology of TCR and CD4 coengagement can differentially affect the T cell function. Our results argue for a model of anergy in which the defective partitioning of signaling molecules in lipid rafts is an early, negative signaling event in T cells. Physiological ligands like DEF chimeras may provide new tools for silencing the autoimmune processes, and may also help in deciphering new mechanisms of negative regulation in T cells.

Up-regulated expression of MICA on activated T lymphocytes involves Lck and Fyn kinases and signaling through MEK1/ERK, p38 MAP kinase, and calcineurin.

Major histocompatibility complex class I-related chain (MICA) is a cell stress-regulated molecule recognized by cytotoxic cells expressing the NKG2D molecule. MICA can be induced on T cells after CD3 or CD28 engagement. Here, we investigated the intracellular pathways leading to activation-induced expression of MICA. The Src kinase inhibitor PP1 inhibited up-regulated expression of MICA on anti-CD3-stimulated T cells. Downstream signaling routes involved mitogen-activated protein kinase (MAPK) kinase (MEK)1/extracellular signal-regulated kinase (ERK), p38 MAPK, and calcineurin, as MICA expression was prevented by U0126, SB202190, cyclosporin A, and FK506. Also, Lck and Fyn as well as MEK1/ERK and p38 MAPK were found to regulate MICA expression in anti-CD28/phorbol 12-myristate 13-acetate-stimulated T cells. Expression of MICA on activated T cells involved interleukin-2-dependent signaling routes triggered by Janus tyrosine kinases/signal transducer and activators of transcription and p70(S)(6) kinase, as it could be inhibited by AG490 and rapamycin. This is the first demonstration of the intracellular pathways involved in activation-induced expression of MICA, which may reveal potential targets for immune intervention to modulate MICA expression in pathological disorders.

SWAP-70-like Adapter of T Cells, an Adapter Protein that Regulates Early TCR-Initiated Signaling in Th2 Lineage Cells

We describe the isolation of a protein, SWAP-70-like adapter of T cells (SLAT), which is expressed at high levels in thymocytes and differentiated Th2 cells. SLAT expression was upregulated in differentiating Th2 cells and downregulated in Th1 cells. Ectopic SLAT expression exerted positive or negative effects on IL-4 versus IFNγ induction, respectively. TCR signaling induced translocation of SLAT to the immunological synapse and its association with ZAP-70 kinase. SLAT reduced the association of ZAP-70 with TCR-ζ and interfered with ZAP-70 but not Lck signaling. Consistent with these results, pharmacological inhibition of ZAP-70 also induced Th2 skewing. Thus, SLAT is a protein which plays a role in Th2 development and/or activation, perhaps by interfering with ZAP-70 signaling.

Commitment to the CD4 Lineage Mediated by Extracellular Signal-Related Kinase Mitogen-Activated Protein Kinase and Lck Signaling

The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.

Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors.

We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J. Corbeil, M. Tremblay, and D. D. Richman, J. Exp. Med. 183:39-48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56(lck) (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild-type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56(lck) or NF-kappaB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56(lck) signaling, and may involve a critical region for CD4 dimerization.