Noah Isakov

Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists

Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses to TCR occupancy by these variant ligands.

T-Lymphocyte Activation: The Role of Protein Kinase C and the Bifurcating Inositol Phospholipid Signal Transduction Pathway

T lymphocytes can be activated by interaction between a wide range of agonists and their respective cell surface receptors. Preceding activation, T cells are at Go, the resting stage of the cell cycle. After interaction with antigen or other ligands, such as mitogenic plant lectins or antibodies directed against various cell surface determinants, they undergo sequential differentiation steps that induce their entry into the d phase, rendering them responsive to additional signals which enable completion of the mitotic cycle. The first activation signal is provided by interaction between the antigen-specific receptor on T cells (TR) and nominal antigen associated with a membrane-bound major histocompatibility complex molecule. The monokine interleukin 1 seems to play a role in this event. As a result of this signal, ceil surface receptors for interleukin 2 (IL2) appear. Transition of T cells from Gi into the S phase (DNA synthesis) and progression into the mitotic cycle then occur as a result of IL2 production and binding to its high affinity receptors (IL2-R). Interactions of various agonists with their specific cell surface receptors in T cells have been studied extensively in the last few years. However, exactly how the membrane signals are transduced into the nucleus and regulate the cascade of activation and proliferation steps is unknown. Recent studies suggest that a signal transduction pathway involving inositol phospholipid hydrolysis is important in T-cell activation. In this pathway (Fig. 1), receptor occupation by a ligand stimulates a phosphoinositide-specific phosphodiesterase (phopholipase C) to hydrolyze phosphatidylinositol 4,5-bisphosphate {PIP2), a phospholipid normally

Inhibition of the c-Jun N-terminal Kinase/AP-1 and NF-κB Pathways by PICOT, a Novel Protein Kinase C-interacting Protein with a Thioredoxin Homology Domain

Protein kinase C-θ (PKCθ) is a Ca2+-independent PKC isoform that is selectively expressed in T lymphocytes (and muscle), and is thought to play an important role in T cell receptor-induced activation. To gain a better understanding of the function and regulation of PKCθ, we have employed the yeast two-hybrid system to identify PKCθ-interacting proteins. We report the isolation and characterization of a cDNA encoding a novel 335-amino acid (37.5-kDa) PKCθ-interacting protein termed PICOT (for PKC-interactingcousin of thioredoxin). PICOT is expressed in various tissues, including in T cells, where it colocalizes with PKCθ. PICOT displays an N-terminal thioredoxin homology domain, which is required for the interaction with PKC. Comparison of the unique C-terminal region of PICOT with expressed sequence tag data bases revealed two tandem repeats of a novel domain that is highly conserved from plants to mammals. Transient overexpression of full-length PICOT (but not its N- or C-terminal fragments) in T cells inhibited the activation of c-Jun N-terminal kinase (but not extracellular signal-regulated kinase), and the transcription factors AP-1 or NF-κB. These findings suggest that PICOT and its evolutionary conserved homologues may interact with PKC-related kinases in multiple organisms and, second, that it plays a role in regulating the function of the thioredoxin system.

Protein kinase Cθ: a new essential superstar on the T-cell stage

Abstract Recent studies have identified protein kinase Cθ (PKCθ), a member of the Ca 2+ -independent PKC family, as an essential component of the T-cell synapse that cooperates with calcineurin to activate the interleukin-2 (IL-2) gene. Several selective functions of PKCθ involved in the activation and survival of T cells are reviewed herein. Among these, the nuclear factor-κB (NF-κB) signaling cascade appears to be the most critical target of PKCθ in the T-cell receptor/CD28 costimulatory pathway that leads to T-cell activation.

Direct interaction between protein kinase C theta (PKC theta) and 14-3-3 tau in T cells: 14-3-3 overexpression results in inhibition of PKC theta translocation and function.

Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.

Signal transduction and intracellular events in T-lymphocyte activation

The important role of a novel signal transduction pathway has recently been established in the cellular activation and proliferation of many cell types. The key event in this pathway is the hydrolysis of a distinct membrane-associated inositol phospholipid and the resulting generation of two defined products that act as second messengers in cell activation. Sufficient evidence has accumulated to indicate that this signal transduction pathway plays an important role in T-cell activation. In this article, Noah Isakov and his colleagues review briefly the general characteristics of this pathway and describe in more detail experimental evidence that establishes its critical role in T-cell activation.

A motif in the V3 domain of the kinase PKC-[theta] determines its localization in the immunological synapse and functions in T cells via association with CD28

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. We found that the PKC-θ V3 domain is necessary and sufficient for IS localization mediated by Lck-dependent association with CD28. We identified a conserved proline-rich motif in V3 required for CD28 association and IS localization. CD28 association was essential for PKC-θ-mediated downstream signaling and TH2 and TH17, but not TH1, differentiation. Ectopic V3 expression sequestered PKC-θ from the IS and interfered with its functions. These results identify a unique mode of CD28 signaling, establish a molecular basis for the IS localization of PKC-θ, and implicate V3-based “decoys” as therapeutic modalities for T cell-mediated inflammatory diseases. The induction of an immune response depends on effective communication between antigen-specific T cells and antigen presenting cells (APCs). When a T cell expressing a cognate T cell receptor (TCR) encounters an activated APC, both cells actively redistribute their receptors and ligands to the interface, creating a platform for effective signaling, known as the immunological synapse (IS). At steady state, the mature IS is composed of concentric rings with a central core (cSMAC) containing clusters of TCR and costimulatory molecules, and a peripheral ring (pSMAC) of adhesion molecules1. The engagement of these surface molecules triggers signaling cascades resulting in the recruitment of *Correspondence: Amnon Altman, Ph.D., Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, California 92037, Tel.: (858) 752 6808, FAX: (858) 752 6986, amnon@liai.org. AUTHOR CONTRIBUTIONS K.-F.K. and A.A. designed the experiments and wrote the manuscript. K.-F.K. generated and analyzed the data. T.S. and T.Y. provided their expertise in cell imaging, T.S. participated in discussion of the data, and T.Y. generated the PKC-GFP and CD28-CFP fusion vectors. N.I. participated actively in discussions leading to this work and in experimental design. A.J.C.-B. performed various experiments and animal work. COMPETING FINANCIAL INTERESTS The authors have no competing financial interests. NIH Public Access

ITIMs and ITAMs. The Yin and Yang of antigen and Fc receptor-linked signaling machinery.

The initial stages of an immune response are regulated at the level of the cell-surface antigen and Fc receptors. The extracellular portions of these receptors provide immune specificity and determine the nature of the responding effector cells, whereas the intracellular portion transduces signals into the cell and determines the intensity and duration of the immune response. Recent studies led to the identification of two types of modules within the cytoplasmic region of receptor subunits that are critical for the activation and termination of signal transduction pathways. Phosphorylation of the conserved tyrosine residues within the two modules, the immunoreceptor tyrosine-based activation motif (ITAM) and the immunoreceptor tyrosine-based inhibition motif (ITIM), is followed by the recruitment of different sets of SH2-containing molecules to the receptor site. These proteins regulate the receptor-linked signal transduction pathways in a positive or a negative fashion, which is a reminiscent of the ancestral Yin-Yang principle.

PICOT-HD: a highly conserved protein domain that is often associated with thioredoxin and glutaredoxin modules

We thank our colleagues for helpful comments on the manuscript. The work reported herein was supported in part by a fellowship from the Deutsche Forschungsgemeinschaft to S.W., a grant from the Israel Science Foundation, the Israel Academy of Sciences and Humanities to N.I., an NIH grant CA35299 to A.A. and a USA–Israel Binational Science Foundation grant to N.I. and A.A.

New Perspectives on PKCθ, a Member of the Novel Subfamily of Protein Kinase C

Members of the protein kinase C (PKC) family of serine/threonine protein kinases have been implicated in numerous cellular responses in a large variety of cell types. Expression patterns of individual members and differences in their cofactor requirements and potential substrate specificity suggest that each isoenzyme may be involved in specific regulatory processes. The PKCθ isoenzyme exhibits a relatively restricted expression pattern with high protein levels found predominantly in hematopoietic cells and skeletal muscle. PKCθ was found to be expressed in T, but not B lymphocytes, and to colocalize with the T‐cell antigen receptor (TCR) at the site of contact between the antigen‐responding T cell and the antigen‐presenting cell (APC). Colocalization of PKCθ with the TCR was selective for this isoenzyme and occurred only upon antigen‐mediated responses leading to T‐cell activation and proliferation. PKCθ was found to be involved in the regulation of transcriptional activation of early‐activation genes, predominantly AP‐1, and its cellular distribution and activation were found to be regulated by the 14‐3‐3 protein. Other findings indicated that PKCθ can associate with the HIV negative factor (Nef) protein, suggesting that altered regulation of PKCθ by Nef may contribute to the T‐cell impairments that are characteristic of infection by HIV. PKCθ is expressed at relatively high levels in skeletal muscle, where it is suggested to play a role in signal transduction in both the developing and mature neuromuscular junction. In addition, PKCθ appears to be involved in the insulin‐mediated response of intact skeletal muscle, as well as in experimentally induced insulin resistance of skeletal muscle. Further studies suggest that PKCθ is expressed in endothelial cells and is involved in multiple processes essential for angiogenesis and wound healing, including the regulation of cell cycle progression, formation and maintenance of actin cytoskeleton, and formation of capillary tubes. Here, we review recent progress in the study of PKCθ and discuss its potential role in various cellular responses.