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Amnon Altman

Publications and source records attributed to Amnon Altman.

30 records · Page 2Linked to original sources
PubMed2003

A single amino acid alteration in cytoplasmic domain determines IL-2 promoter activation by ligation of CD28 but not inducible costimulator (ICOS).

The CD28 family molecules, CD28, and inducible costimulator (ICOS) all provide positive costimulatory signals. However, unlike CD28, ICOS does not costimulate IL-2 secretion. The YMNM motif that exists in the CD28 cytoplasmic domain is a known binding site for phosphatidylinositol 3-kinase (PI3-K) and Grb2. ICOS possesses the YMFM motif in the corresponding region of CD28 that binds PI3-K but not Grb2. We postulated that the reason that ICOS does not have the ability to induce IL-2 production is because it fails to recruit Grb2. To verify this hypothesis, we generated a mutant ICOS gene that contains the CD28 YMNM motif and measured IL-2 promoter activation after ICOS ligation. The results indicated that ICOS became competent to activate the IL-2 promoter by this single alteration. Further analysis demonstrated that Grb2 binding to ICOS was sufficient to activate the NFAT/AP-1 site in the IL-2 promoter and that the cytoplasmic domain of CD28 outside of the YMNM motif is required for activation of the CD28RE/AP-1 and NF-kappaB sites. Together, these observations lead us to believe that the difference of a single amino acid, which affects Grb2 binding ability, may define a functional difference between the CD28- and ICOS-mediated costimulatory signals.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading.

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. Beta1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on beta1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and beta1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.

Animals↗
PubMed2003

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 IFNgamma 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-zeta 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.

Amino Acid Sequence↗
PubMed2003

Protein kinase C-theta (PKCtheta): it's all about location, location, location.

Much progress has been made in understanding the function of protein kinase C-theta (PKCtheta) in the immune system since this Ca2+-independent PKC isotype was isolated in 1993 as an enzyme that is highly expressed in T lymphocytes and in muscle cells. Biochemical and genetic approaches revealed that, while dispensable for T-cell development, PKCtheta is required for the activation of mature T cells and for interleukin (IL)-2 production. This deficiency results from impaired receptor-induced stimulation of the transcription factors AP-1 and NF-kappaB. PKCtheta integrates T-cell receptor (TCR)/CD28 costimulatory signals, which are essential for productive T-cell activation and, most likely, for prevention of T-cell anergy. A unique property of PKCtheta is its highly selective recruitment to the central supramolecular activation complex (cSMAC) region of the immunological synapse (IS) in antigen-stimulated T cells. Our work revealed that this highly selective localization is not entirely dependent on phospholipase C (PLC) activity and diacylglycerol (DAG) production. Instead, a novel signaling pathway that requires functional Vav1, phosphatidylinositol 3-kinase (PI3-K), the small GTPase Rac and actin cytoskeleton reorganization regulates the localization and, perhaps, activation of PKCtheta. PKCtheta also provides a survival signal, which protects T cells from apoptosis. Additional work is required to identify the immediate targets of PKCtheta and its immune functions in vivo. This work is likely to validate PKCtheta as an attractive drug target.

Animals↗
PubMed2002

Pleiotropic defects in TCR signaling in a Vav-1-null Jurkat T-cell line.

The Rac/Rho-specific guanine nucleotide exchange factor, Vav-1, is a key component of the T-cell antigen receptor (TCR)-linked signaling machinery. Here we have used somatic cell gene-targeting technology to generate a Vav-1-deficient Jurkat T-cell line. The J.Vav1 cell line exhibits dramatic defects in TCR-dependent interleukin (IL)-2 promoter activation, accompanied by significant reductions in the activities of the NFAT(IL-2), NFkappaB, AP-1 and REAP transcription factors that bind to the IL-2 promoter region. In contrast, loss of Vav-1 had variable effects on early TCR-stimulated signaling events. J.Vav1 cells display a selective defect in sustained Ca(2+) signaling during TCR stimulation, and complementation of this abnormality by exogenously introduced Vav-1 is dependent on the Vav-1 calponin homology domain. While JNK activation was severely impaired, the stimulation of Ras, ERK and protein kinase C-theta activities, as well as the mobilization of lipid rafts, appeared normal in the J.Vav1 cells. Finally, evidence is presented to suggest that the alternative Vav family members, Vav-2 and Vav-3, are activated during TCR ligation, and partially compensate for the loss of Vav-1 in Jurkat T cells.

Animals↗
PubMed2002

Translocation of PKC[theta] in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C.

PKCtheta plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-kappaB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCtheta to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCtheta, we addressed here the question of whether the translocation and activation of PKCtheta in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCgamma1-deficient T cell line, or a dominant negative PLCgamma1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCtheta are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCalpha. Membrane or lipid raft recruitment of PKCtheta (but not PKCalpha) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCtheta;, but did not associate with it. These results provide evidence that a nonconventional PI3-K- and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCtheta in T cells.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

Vav-induced activation of the human IFN-gamma gene promoter is mediated by upregulation of AP-1 activity.

The role of Vav in the transcriptional regulation of the human interferon-gamma (IFN-gamma) promoter was investigated. Overexpression of Vav in Jurkat-TAg cells enhanced T cell receptor (TCR)-induced activation of a luciferase (Luc) reporter gene construct driven by cis-regulatory element of the IFN-gamma gene (-346 to +7). Electrophoresis mobility shift and Luc reporter assays demonstrated that the DNA-binding and transcriptional activity of the proximal AP-1-dependent NFAT site (positions -172 to -138), the AP-1/Ying-Yang 1 (YY1)-binding site (-209 to -184), and a consensus AP-1-binding site were upregulated by Vav. Vav enhanced TCR-induced activation of c-Jun N-terminal kinase (JNK) and its upstream regulator, Rho family GTPases. Finally, coexpression of a dominant-negative Rac1 mutant suppressed Vav-mediated upregulation of the transcriptional and DNA-binding activity of the proximal NFAT/AP-1 site and the AP-1/YY1 site, as well as the complete IFN-gamma promoter activity. Vav activates the IFN-gamma promoter via upregulation of AP-1-binding through a Rac1/JNK pathway.

DNA-Binding Proteins↗
PubMed2002

Ordered just so: lipid rafts and lymphocyte function.

Immunologists have long been occupied with the description of cellular activation signaling events that originate with the stimulation of multichain immunoreceptors at the cell surface. These signals are transmitted by a protein-partner-signaling cascade through the cytoplasm to the nucleus, where they culminate in changes in gene expression, metabolic state, and entry into cell cycle. For T cells and B cells, these signaling cascades start with the ligation of the T cell receptor (TCR) and B cell receptor (BCR), respectively, and result in the recruitment and activation of related families of signaling molecules at the cell surface. Until recently, this gathering of signaling proteins was thought to occur within the featureless plasma membrane, a cellular organ that was envisioned as a boundary between the inner and outer components of the cell, but which contributed little to the signaling process. However, the past few years have seen the gradual realization that activation of signaling in lymphocytes takes place in and around specialized membrane subdomains called lipid rafts (also known as DIGs and GEMs). Here, we provide a brief overview of the analogous structures and compositions of lipid raft-associated signaling complexes in T cells and B cells, and the ways in which lymphocytes--and their pathogen adversaries--use lipid rafts to their benefit.

Humans↗
PubMed2002

Protein kinase C-theta (PKC theta): a key enzyme in T cell life and death.

The novel protein kinase C (PKC) isoform, PKC theta, is expressed in a relatively selective manner in T lymphocytes (and muscle). Recent analysis of this PKC isotype in T cells and the characterization of PKC theta-deficient mice revealed important clues about its function and regulation. PKC theta does not have an obvious role in T cell development, but it is essential for the activation of mature T cells. The requirement of PKC theta for T cell activation, proliferation and cytokine production reflects the essential role of this isotype in inducing signaling pathways leading to the activation of the transcription factors AP-1 and NF-kappa B in a T cell-specific manner. A unique feature of PKC theta is its highly selective translocation to the central region of the immunological synapse (IS) in antigen-stimulated T cells, a property apparently important for its proper signaling functions. This localization implies unique pathway(s) that regulate the translocation and/or activation of this enzyme. Our work suggests that sustained PKC theta membrane translocation and phosphorylation are relatively independent of phospholipase C (PLC) activation and diacylglycerol (DAG) production. Instead, a pathway that requires Vav, phosphatidylinositol 3-kinase (PI3-K), Rac1 and actin cytoskeleton reorganization mediates these events. Additionally, PKC theta provides an important survival signal to T cells. Nevertheless, several questions regarding the function and regulation of PKC theta and the identity of its immediate targets/substrates remain open. Resolution of these questions could open the way to the development of selective PKC theta inhibitors, which may have therapeutic potential in immunological diseases and in cancer.

Animals↗
PubMed2002

Protein kinase C(theta) in T cell activation.

The novel protein kinase C (PKC) isoform, PKC theta, is selectively expressed in T lymphocytes and is a sine qua non for T cell antigen receptor (TCR)-triggered activation of mature T cells. Productive engagement of T cells by antigen-presenting cells (APCs) results in recruitment of PKC theta to the T cell-APC contact area--the immunological synapse--where it interacts with several signaling molecules to induce activation signals essential for productive T cell activation and IL-2 production. The transcription factors NF-kappa B and AP-1 are the primary physiological targets of PKC theta, and efficient activation of these transcription factors by PKC theta requires integration of TCR and CD28 costimulatory signals. PKC theta cooperates with the protein Ser/Thr phosphatase, calcineurin, in transducing signals leading to activation of JNK, NFAT, and the IL-2 gene. PKC theta also promotes T cell cycle progression and regulates programmed T cell death. The exact mode of regulation and immediate downstream substrates of PKC theta are still largely unknown. Identification of these molecules and determination of their mode of operation with respect to the function of PKC theta will provide essential information on the mechanism of T cell activation. The selective expression of PKC theta in T cells and its essential role in mature T cell activation establish it as an attractive drug target for immunosuppression in transplantation and autoimmune diseases.

Animals↗
PubMed2002

Protein kinase C-theta (PKCtheta), a potential drug target for therapeutic intervention with human T cell leukemias.

The link between apoptosis and malignant cell growth is firmly established, and various forms of therapy in cancer, e.g., the use of DNA-damaging chemotherapeutic drugs, are based on the principle of inducing apoptosis in malignant cells. However, in many known instances, tumor cells develop resistance to apoptosis through various mechanisms. Thus, interventions designed to facilitate tumor cells apoptosis are likely to have a therapeutic benefit. PKCtheta, which is expressed relatively selectively in T cells, plays an important role in mature T cell activation and proliferation upon its translocation to the plasma membrane. PKCtheta is necessary for induction of the interleukin-2 (IL-2) gene because the transcription factors AP-1 and NF-kappaB, which are essential for IL-2 gene promoter activation, are main targets of PKCtheta. Recent studies revealed that PKCtheta provides an important survival signal that protects leukemic T cells from Fas- or UV-induced apoptosis. These findings and the constitutive localization of PKCq in the membrane of some leukemic T cells suggests that it plays a role in leukemic T cell survival and/or proliferation, and that selective PKCtheta-inhibitory strategies may facilitate elimination of malignant T cells. The high-affinity IL-2 receptor (IL-2Ralpha) is a major target of receptor-directed therapy in several human diseases, and it is constitutively expressed by the malignant cells in some T cell leukemias, suggesting an autocrine IL-2/IL-2R loop that participates in the expansion of leukemic IL-2R(+) cells. Therefore, given the essential role of PKCtheta in IL-2 production, IL-2 gene regulation by PKCtheta could also be of therapeutic interest.

Animals↗