Céline Charvet

Phosphorylation of Tip60 by GSK-3 Determines the Induction of PUMA and Apoptosis by p53

Activation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60(S86A) mutant was less active to induce p53 K120 acetylation, histone 4 acetylation, and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86 phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53.

Proteolytic regulation of Forkhead transcription factor FOXO3a by caspase-3-like proteases

Forkhead family transcription factors are critical regulators of cell cycle progression and apoptosis in hematopoietic cells. Here, we show that FOXO3a (also known as FKHRL1) is a new substrate of caspase-3-like proteases during apoptosis in T lymphocytes. FOXO3a was cleaved in vivo by caspases in leukemic Jurkat cells following engagement of Fas (CD95) receptor, staurosporine, and etoposide treatment, but not following engagement of CD99, a caspase-independent cell death inducer. Caspase-mediated cleavage of FOXO3a was also observed in CD4+ peripheral T cells subjected to activation-induced cell death. The expression of the death adapter FADD and caspase-8 was required for Fas-induced FOXO3a cleavage, but activation of survival pathways by overexpression of FLICE-inhibitory protein or phorbol myristate acetate treatment prevented it. FOXO3a was cleaved in vitro by caspase-3-like proteases at the consensus sequence DELD304A, releasing the N-terminal DNA-binding domain of FOXO3a from its C-terminal transactivating domain. Whereas full-length FOXO3a enhanced Forkhead response element-dependent transcription and apoptosis in Jurkat cells, both fragments were inactive to promote gene activation and cell death. In contrast, a caspase-resistant FOXO3a mutant exhibited enhanced transcriptional and proapoptotic activities. Together, these results indicate that the proteolytic cleavage of FOXO3a by caspases may represent a novel regulatory mechanism of FOXO3a activity during death receptors signaling.

GSK-3 - at the crossroads of cell death and survival.

ABSTRACT Glycogen synthase kinase 3 (GSK-3) is involved in various signaling pathways controlling metabolism, differentiation and immunity, as well as cell death and survival. GSK-3 targets transcription factors, regulates the activity of metabolic and signaling enzymes, and controls the half-life of proteins by earmarking them for degradation. GSK-3 is unique in its mode of substrate recognition and the regulation of its kinase activity, which is repressed by pro-survival phosphoinositide 3-kinase (PI3K)–AKT signaling. In turn, GSK-3 exhibits pro-apoptotic functions when the PI3K–AKT pathway is inactive. Nevertheless, as GSK-3 is crucially involved in many signaling pathways, its role in cell death regulation is not uniform, and in some situations it promotes cell survival. In this Commentary, we focus on the various aspects of GSK-3 in the regulation of cell death and survival. We discuss the effects of GSK-3 on the regulation of proteins of the BCL-2 family, through which GSK-3 exhibits pro-apoptotic activity. We also highlight the pro-survival activities of GSK-3, which are observed in the context of nuclear factor &kgr;B (NF&kgr;B) signaling, and we discuss how GSK-3, by impacting on cell death and survival, might play a role in diseases such as cancer.

Vav1 Promotes T Cell Cycle Progression by Linking TCR/CD28 Costimulation to FOXO1 and p27kip1 Expression

Vav proteins play a critical role in T cell activation and proliferation by promoting cytoskeleton reorganization, transcription factor activation, and cytokine production. In this study, we investigated the role of Vav in T cell cycle progression. TCR/CD28-stimulated Vav1−/− T cells displayed a cell cycle block at the G0-G1 stage, which accounted for their defective proliferation. This defect was associated with impaired TCR/CD28-induced phosphorylation of Akt and the Forkhead family transcription factor, FOXO1. The cytoplasmic localization of FOXO1 and its association with 14–3-3τ were also reduced in Vav1−/− T cells. Consistent with the important role of FOXO1 in p27kip1 transcription, stimulated Vav1−/− T cells failed to down-regulate the expression of p27kip1, explaining their G0-G1 arrest. These defects were more pronounced in Vav1/Vav3 double-deficient T cells, suggesting partial redundancy between Vav1 and Vav3. Importantly, IL-2-induced p27kip1 down-regulation and cyclin D3 up-regulation and FOXO1 phosphorylation were similar in Vav1−/− and wild-type T lymphoblasts, indicating that defective FOXO1 phosphorylation and p27kip1 and cyclin D3 expression do not result from deficient IL-2 signaling in the absence of Vav1. Thus, Vav1 is a critical regulator of a PI3K/Akt/FOXO1 pathway, which controls T cell cycle progression and proliferation.

SLAT regulates Th1 and Th2 inflammatory responses by controlling Ca2+/NFAT signaling

SWAP-70-like adapter of T cells (SLAT) is a novel guanine nucleotide exchange factor for Rho GTPases that is upregulated in Th2 cells, but whose physiological function is unclear. We show that SLAT(-/-) mice displayed a developmental defect at one of the earliest stages of thymocyte differentiation, the double-negative 1 (DN1) stage, leading to decreased peripheral T cell numbers. SLAT(-/-) peripheral CD4(+) T cells demonstrated impaired TCR/CD28-induced proliferation and IL-2 production, which was rescued by the addition of exogenous IL-2. Importantly, SLAT(-/-) mice were grossly impaired in their ability to mount not only Th2, but also Th1-mediated lung inflammatory responses, as evidenced by reduced airway neutrophilia and eosinophilia, respectively. Levels of Th1 and Th2 cytokine in the lungs were also markedly reduced, paralleling the reduction in pulmonary inflammation. This defect in mounting Th1/Th2 responses, which was also evident in vitro, was traced to a severe reduction in Ca(2+) mobilization from ER stores, which consequently led to defective TCR/CD28-induced translocation of nuclear factor of activated T cells 1/2 (NFATc1/2). Thus, SLAT is required for thymic DN1 cell expansion, T cell activation, and Th1 and Th2 inflammatory responses.

Membrane Localization and Function of Vav3 in T Cells Depend on Its Association with the Adapter SLP-76

The Vav family of guanine exchange factors plays a critical role in lymphocyte proliferation, cytoskeletal reorganization, and gene transcription upon immunoreceptor engagement. Although the role of Vav1 in T cells is well documented, the role of Vav3 is less clear. We investigated the subcellular localization of Vav3 during T cell activation. We report here that phosphorylation of Vav3 on tyrosine residue Tyr173 is not required for T cell receptor (TCR)-induced Vav3 membrane translocation or immunological synapse (IS) recruitment, but mutation of this residue enhanced TCR-induced nuclear factor of activated T cells (NFAT) activation. However, Vav3 mutants either containing an Src homology 2 (SH2)-disabled point mutation (R697L) or lacking its SH3-SH2-SH3 domains were unable to bind SLP-76 did not translocate to the membrane or to the IS and furthermore failed to activate NFAT. Importantly, the membrane translocation of Vav3 was abrogated in Lck, ZAP-70, LAT, and SLP-76-deficient T cells, where Vav3 binding to SLP-76 was disrupted. Finally, we confirmed and underlined the critical role of Vav3 in NFAT activation by knocking down Vav3 expression in Vav1-deficient T cells. Altogether, our data show that TCR-induced association of Vav3 with SLP-76 is required for its membrane/IS localization and function.

Foxo1 Is a T Cell–Intrinsic Inhibitor of the RORγt-Th17 Program

An uncontrolled exaggerated Th17 response can drive the onset of autoimmune and inflammatory diseases. In this study, we show that, in T cells, Foxo1 is a negative regulator of the Th17 program. Using mixed bone marrow chimeras and Foxo1-deficient mice, we demonstrate that this control is effective in vivo, as well as in vitro during differentiation assays of naive T cells with specific inhibitor of Foxo1 or inhibitors of the PI3K/Akt pathway acting upstream of Foxo1. Consistently, expressing this transcription factor in T cells strongly decreases Th17 generation in vitro as well as transcription of both IL-17A and IL-23R RORγt-target genes. Finally, at the molecular level, we demonstrate that Foxo1 forms a complex with RORγt via its DNA binding domain to inhibit RORγt activity. We conclude that Foxo1 is a direct antagonist of the RORγt-Th17 program acting in a T cell–intrinsic manner.

Lipid raft targeting of hematopoietic protein tyrosine phosphatase by protein kinase C theta-mediated phosphorylation.

ABSTRACT Protein kinase C θ (PKC θ) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC θ-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC θ at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC θ−/− mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC θ and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.

Adaptive Immune-like γ/δ T Lymphocytes Share Many Common Features with Their α/β T Cell Counterparts

To better apprehend γ/δ T cell biological functions in the periphery, it appears crucial to identify markers highlighting the existence of distinct phenotypic and functional γ/δ T cell subsets. Interestingly, the expression of CD44 and Ly-6C subdivides murine peripheral γ/δ T cells into several subsets, with Ly-6C− CD44hi γ/δ T cells corresponding to the IL-17–producing CD27− γ/δ T cell subset exhibiting innate-like features. By comparing the other subsets to naive and memory CD8+ α/β T cells, in this study, we show that Ly-6C− or + CD44lo and Ly-6C+CD44hi γ/δ T cells greatly resemble, and behave like, their CD8+ α/β T cell counterparts. First, like memory CD8+ α/β T cells, Ly-6C+CD44hi γ/δ T cells are sparse in the thymus but largely increased in proportion in tissues. Second, similarly to naive CD8 α/β T cells, CD44lo γ/δ T cells are poorly cycling in vivo in the steady state, and their proportion declines with age in secondary lymphoid organs. Third, CD44lo γ/δ T cells undergo spontaneous proliferation and convert to a memory-like Ly-6C+CD44hi phenotype in response to lymphopenia. Finally, CD44lo γ/δ T cells have an intrinsic high plasticity as, upon appropriate stimulation, they are capable of differentiating nonetheless into Th17-like and Th1-like cells but also into fully functional Foxp3+ induced regulatory T cell–like γ/δ T cells. Thus, peripheral CD27+ γ/δ T cells, commonly considered as a functionally related T cell compartment, actually share many common features with adaptive α/β T cells, as both lineages include naive-like and memory-like lymphocytes with distinct phenotypic, functional, and homeostatic characteristics.

Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation

The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1wt or the phosphorylation-deficient mutant MCL-1S159A in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1S159A exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1S159A in Eμ-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with Eμ-Myc/MCL-1wt mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis.