We investigated the role of glycogen synthase kinase-3 (GSK-3), which is inactivated by AKT, for its role in the regulation of apoptosis. Upon IL-3 withdrawal, protein levels of MCL-1 decreased but were sustained by pharmacological inhibition of GSK-3, which prevented cytochrome c release and apoptosis. MCL-1 was phosphorylated by GSK-3 at a conserved GSK-3 phosphorylation site (S159). S159 phosphorylation of MCL-1 was induced by IL-3 withdrawal or PI3K inhibition and prevented by AKT or inhibition of GSK-3, and it led to increased ubiquitinylation and degradation of MCL-1. A phosphorylation-site mutant (MCL-1(S159A)), expressed in IL-3-dependent cells, showed enhanced stability upon IL-3 withdrawal and conferred increased protection from apoptosis compared to wild-type MCL-1. The results demonstrate that the control of MCL-1 stability by GSK-3 is an important mechanism for the regulation of apoptosis by growth factors, PI3K, and AKT.

Summary 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 pro-apoptotic 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 Tip60S86A 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.

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 prosurvival activities of GSK-3, which are observed in the context of nuclear factor kB (NFkB) signaling, and we discuss how GSK-3, by impacting on cell death and survival, might play a role in diseases such as cancer.

The BCR signals through conserved immunoreceptor tyrosinebased activation motifs (ITAMs) found within the cytoplasmic tails of its subunits. Ligation by antigen results in the activation of associated cytoplasmic protein tyrosine kinases (PTKs), including Syk, the Src-family PTKs Lyn and Fyn, and Btk. 2,3 The activated Lyn and Fyn kinases phosphorylate the ITAMs within the immunoglobulin alpha (Ig-␣) and Ig-␤ chains, resulting in the recruitment and activation of Syk. As a result, molecular scaffolds composed of adaptor proteins and key enzymes, such as phospholipase C ␥ (PLC-␥), phosphatidylinositol 3-kinase (PI3K), and guanine nucleotide exchange factors (GEFs) Vav1 and Vav2, are assembled and activated at the plasma membrane by Src and/or Syk PTKs. These scaffolds transduce signals to the cytoplasm and nucleus to activate gene expression and metabolic changes involved in B-cell homeostasis.One critical pathway stimulated by BCR triggers intracytoplasmic calcium movements leading to the activation of transcription factors, such as nuclear factor of activated T cells (NFATs). These nuclear factors coordinate the expression of cytokines and cellcycle proteins involved in B-cell growth and apoptosis. Calcium signals are triggered by activated PLC-␥ that produces inositol-1,4,5-triphosphate (IP3), followed by an increase of cytoplasmic calcium. 4 The regulation of PLC-␥ activity involves the membrane translocation and activation of Btk following PI3K-mediated production of phosphatidylinositol-3,4,5-triphosphate (PIP3). 5,6 Vav proteins also participate to the regulation of calcium signals and NFAT activation in lymphocytes. Vav proteins are activators of Rho family GTPases that control cytoskeletal reorganization and gene activation. 7,8 Analysis of Vav1-and Vav2-deficient mice showed the importance of these proteins in BCR signaling during development, maturation, and proliferation of B cells. [9][10][11] The major defect in vav1 Ϫ/Ϫ ϫ vav2 Ϫ/Ϫ B cells stimulated through antigen receptor appears to be a defect in calcium release. Although the basis for this defect is still unclear, it likely involves defects in PI3K, PLC-␥, and Tec-family kinases activation, since this phenomenon has been observed in Vav1-deficient T and mast cells. 12,13 Moreover, Vav3 has been shown to regulate PI3K activation in the DT40 chicken B-cell line. 14 The assembly of signaling complexes is regulated by adaptor proteins that mediate protein-protein or protein-lipid interactions. 15 In B cells, one such adaptor is the Syk substrate BLNK/SLP-65, which connects BCR to PLC-␥ activation by prodiving docking sites for Btk and PLC-␥2. 16,17 In a screen for Syk-kinase interacting proteins, we previously identified 3BP2, 18 a cytoplasmic adaptor originally identified as an Abl Src homology domain 3 (SH3) binding protein. 19 3BP2 (also known as SH3BP2) is preferentially expressed in hematopoietic tissues, 18,20 and positively regulates the activity of the transcription factors NFAT and AP-1 in T cells through calcineurin and Ras-dependent path...

SWAP-70-like adapter of T cells (SLAT) is a novel guanine nucleotide exchange factor for RhoGTPases 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.

SUMMARY SWAP-70-like adaptor of T cells (SLAT) is a guanine nucleotide exchange factor for Rho GTPases that regulates the development of T helper 1 (Th1) and Th2 cell inflammatory responses by controlling the Ca2+-NFAT signaling pathway. However, the mechanism used by SLAT to regulate these events is unknown. Here, we report that the T cell receptor (TCR)-induced translocation of SLAT to the immunological synapse required Lck-mediated phosphorylation of two tyrosine residues located in an immunoreceptor tyrosine-based activation motif-like sequence but was independent of the SLAT PH domain. This subcellular relocalization was coupled to, and necessary for, activation of the NFAT pathway. Furthermore, membrane targeting of the SLAT Dbl-homology (catalytic) domain was sufficient to trigger TCR-mediated NFAT activation and Th1 and Th2 differentiation in a Cdc42-dependent manner. Therefore, tyrosine-phosphorylation-mediated relocalization of SLAT to the site of antigen recognition is required for SLAT to exert its pivotal role in NFAT-dependent CD4+ T cell differentiation.

Transendothelial migration of activated lymphocytes from the blood into the tissues is an essential step for immune functions. The housekeeping chemokine CXCL12 (or stroma cell-derived factor-1␣), a highly efficient chemoattractant for T lymphocytes, drives lymphocytes to sites where they are highly likely to encounter antigens. This suggests that cross-talk between the T-cell receptor (TCR) and CXCR4 (the CXCL12 receptor) might occur within these sites. Here we show that the zeta-associated protein 70 (ZAP-70), a key element in TCR signaling, is required for CXCR4 signal transduction. The pharmacologic inhibition of ZAP- IntroductionLymphocyte migration from the blood to the tissues is an essential step for immune surveillance. Chemokines play a central role in this process by conferring specificity in lymphocyte trafficking. Those present at the surfaces of endothelial cells are responsible for the activation of lymphocyte integrins, which, within seconds, change affinity for their ligands from a low-to a high-affinity state, allowing lymphocytes to stop on the endothelial wall. Chemokines secreted by cells within the subendothelial matrix attract lymphocytes across the vascular endothelial cell wall into the extracellular matrix, where they can exert their immune functions. 1,2 Stroma cell-derived factor-1␣-or CXCL12, according to the new chemokine nomenclature 3 -belongs to the CXC chemokine subfamily and is a highly efficient chemoattractant for numerous cell types, 4,5 including T lymphocytes. 6 To date, CXCL12 is the only identified ligand for CXCR4, a 7-transmembrane domain G-protein-coupled receptor 7-9 that also serves as a coreceptor for T-cell tropic human immunodeficiency virus-1 strains. 6 Mice lacking CXCL12 or its receptor, CXCR4, exhibit lethal defects including cardiovascular, neurologic, and vascular deficiencies and severe impairments of lymphopoiesis and bone marrow myelopoiesis. [7][8][9] Recent reports have focused on the signal transduction pathway induced by the binding of CXCL12 to its receptor. CXCL12 induces the phosphorylation of several molecules that participate in the formation of focal adhesions and the reorganization of the cytoskeleton. 10 Moreover, CXCL12 activates protein kinase B, extracellular signal-regulated kinases (ERKs), 10,11 PI3 kinase,10,12 and the JAK/STAT pathway. 13 More recently, CXCL12 has been shown to induce the phosphorylation of the phosphatase SHP2 and Cbl and the activation of Fyn and Lyn protein tyrosine kinases (PTKs). 14 CXCL12 is a homeostatic chemokine, which means that it is produced within primary and secondary lymphoid tissues and in nonlymphoid tissues such as the skin and that it is involved in the physiologic traffic of cells of the immune system. [15][16][17] Within these localizations, T and B cells are highly susceptible to encounter antigen. Thus an important question is whether antigen presentation to T or B cells, that is, T-cell receptor (TCR) or B-cell receptor (BCR) occupancy, might regulate chemokine receptor-induced activation sign...

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.