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Biomedical subjects

A Altman

Publications and source records attributed to A Altman.

At least 19 recordsLinked to original sources
PubMed2001

Protein kinase C-theta mediates a selective T cell survival signal via phosphorylation of BAD.

Protein kinase C (PKC)-activating phorbol esters protect T cells from Fas-induced apoptosis. However, the mechanism of this protective effect and the identity of the relevant PKC isoform(s) are poorly understood. Here, we show that PKCtheta plays a selective and important role in this protection. Fas triggering led to a selective caspase-3-dependent cleavage of the enzyme and proteasome-mediated degradation and inactivation of its catalytic fragment. These events preceded the onset of apoptosis. Pharmacological inhibition of PKCtheta promoted Fas-mediated apoptosis in three different types of T cells. Conversely, constitutively active PKCtheta (and, to a lesser degree, PKCepsilon) selectively protected T cells from Fas-induced apoptosis. We provide evidence that the distant Bcl-2 family member, BAD, is a PKCtheta substrate, is phosphorylated by TCR stimulation, and can mediate at least in part the anti-apoptotic effect of PKCtheta.

Apoptosis↗
PubMed2001

Vav2 activates c-fos serum response element and CD69 expression but negatively regulates nuclear factor of activated T cells and interleukin-2 gene activation in T lymphocyte.

Vav1 and Vav2 are members of the Dbl family of guanine nucleotide exchange factors for the Rho family of small GTPases. Although the role of Vav1 during lymphocyte development and activation is well characterized, the function of Vav2 is still unclear. In this study, we compared the signaling pathways regulated by Vav1 and Vav2 following engagement of the T cell receptor (TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR stimulation and by co-expressed Src and Syk family kinases. Using glutathione S-transferase fusion proteins, we observed that the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase extracellular signal-regulated kinase activation and to promote c-fos serum response element transcriptional activity. Moreover, both proteins displayed a similar action in increasing the expression of the early activation marker CD69 in Jurkat T cells. However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals leading to nuclear factor of activated T cells (NF-AT)-dependent transcription and induction of the interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase calcineurin because a constitutively active form of calcineurin rescued the effect of Vav2 by restoring TCR-induced NF-AT activation. Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were necessary for its inhibitory effect on NF-AT activation and for induction of serum response element transcriptional activity. Taken together, our results indicate that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. The negative regulatory pathway elicited by Vav2 might play an important role in regulating lymphocyte activation processes.

Antigens, CD↗
PubMed2001

Membrane lipid microdomains and the role of PKCtheta in T cell activation.

Productive T cell activation depends on the assembly of a highly ordered and compartmentalized immunological synapse or supramolecular activation complex (SMAC). Reorganization of the actin cytoskeleton and clustering of specialized membrane microdomains, or lipid rafts, occur early following TCR/CD3 and costimulatory receptor ligation. Many key signaling molecules localize in lipid raft patches during T cell activation. Lipid raft reorganization is required for T cell activation, where it plays an apparently important role in stabilizing the T cell synapse. Here we review recent evidence supporting the role of lipid rafts in T cell activation. Particular emphasis is placed on the coupling of protein kinase C-theta(PKCtheta), which is selectively expressed in T cells and is known to function as an essential signal for T cell activation, and lipid rafts.

Animals↗
PubMed2001

Antigen-induced translocation of PKC-theta to membrane rafts is required for T cell activation.

Protein kinase C-theta (PKC-theta) is essential for mature T cell activation; however, the mechanism by which it is recruited to the TCR signaling machinery is unknown. Here we show that T cell stimulation by antibodies or peptide-major histocompatibility complex (MHC) induces translocation of PKC-theta to membrane lipid rafts, which localize to the immunological synapse. Raft translocation was mediated by the PKC-theta regulatory domain and required Lck but not ZAP-70. In addition, PKC-theta was associated with Lck in the rafts. An isolated PKC-straight theta catalytic fragment did not partition into rafts or activate the transcription factor NF-kappa B, although addition of a Lck-derived raft-localization sequence restored these functions. Thus, physiological T cell activation translocates PKC-theta to rafts, which localize to the T cell synapse; this PKC-theta translocation is important for its function.

Antigen-Presenting Cells↗
PubMed2001

Spatial dependence of the phase in localized bioelectrical impedance analysis.

The variety of phase functions, theta(z) = arctan X(z)/R(z), observed earlier on the thighs of healthy and seriously ill subjects via localized bioelectrical impedance analysis, can be represented by a model which combines realistic thigh shapes with homogeneous, axially symmetric conductivity tensors. While quantitative results depend sensitively on the way current is injected, it appears to be generally true that d theta/d(z) < 0 whenever phi(r) > phi(z) (and vice versa), where phi(r) and phi(z) are the phases of the radial and longitudinal conductivity components.

Adult↗
PubMed2001

Vav-Rac1-mediated activation of the c-Jun N-terminal kinase/c-Jun/AP-1 pathway plays a major role in stimulation of the distal NFAT site in the interleukin-2 gene promoter.

Vav, a hematopoiesis-specific signaling protein, plays an important role in T-cell development and activation. Vav upregulates the expression of the interleukin-2 (IL-2) gene, primarily via activation of the distal NFAT site in the IL-2 gene promoter (NFAT-IL-2). However, since this site cooperatively binds NFAT and AP-1, the relative contribution of Vav to NFAT versus AP-1 activation has not been determined. Here, we studied the respective roles of the AP-1 and NFAT pathways in the T-cell receptor (TCR)-mediated, Vav-dependent activation of NFAT-IL-2. Although Vav stimulated the transcriptional activity of an NFAT-IL-2 reporter gene, it failed to stimulate the transcriptional or DNA-binding activities of an AP-1-independent NFAT site derived from the human gamma interferon gene promoter. Vav also did not stimulate detectable Ca(2+) mobilization and nuclear translocation of NFATc or NFATp. On the other hand, Vav induced the activation of Rac1 or Cdc42 and c-Jun N-terminal kinase (JNK), enhanced the transcriptional and DNA-binding activities of AP-1, and induced increased phosphorylation of c-Jun. Dominant-negative Vav and/or Rac1 mutants blocked the TCR-mediated stimulation of these events, demonstrating the physiological relevance of these effects. Vav also associated with Rac1 or Cdc42 in T cells, and anti-CD3 antibody stimulation enhanced this association. These findings indicate that a Rac1-dependent JNK/c-Jun/AP-1 pathway, rather than the Ca(2+)/NFAT pathway, plays the predominant role in NFAT-IL-2 activation by Vav.

Active Transport, Cell Nucleus↗
PubMed2000

T-cell receptor antagonists induce Vav phosphorylation by selective activation of Fyn kinase.

T cell receptor (TCR) antagonists inhibit antigen-induced T cell activation and by themselves fail to induce phenotypic changes associated with T cell activation. However, we have recently shown that TCR antagonists are inducers of antigen-presenting cell (APC)-T cell conjugates. The signaling pathway associated with this cytoskeleton-dependent event appears to involve tyrosine phosphorylation and activation of Vav. In this study, we investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in antagonist-induced signaling pathway. Antagonist stimulation increased tyrosine phosphorylation and kinase activity of Fyn severalfold, whereas little or no increase in Lck and ZAP-70 activity was observed. Second, TCR stimulation of Lck(-), Fyn(hi) Jurkat cells induced strong tyrosine phosphorylation of Vav. In contrast, minimal increase in tyrosine phosphorylation of Vav was observed in Lck(hi), Fyn(lo) Jurkat cells. Finally, study of T cells from a Fyn-deficient TCR transgenic mouse also showed that Fyn was required for tyrosine phosphorylation and activation of Vav induced by both antagonist and agonist peptides. The deficiency in Vav phosphorylation in Fyn-deficient T cells was associated with a defect in the formation of APC-T cell conjugates when T cells were stimulated with either agonist or antagonist peptide. We conclude from these results that Vav is a selective substrate for Fyn, especially under conditions of low-affinity TCR-mediated signaling, and that this signaling pathway involving Fyn, Vav, and Rac-1 is required for the cytoskeletal reorganization that leads to T cell-APC conjugates and the formation of the immunologic synapse.

Animals↗
PubMed2000

NF-kappa B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-theta.

Protein kinase C-theta (PKCtheta) is a Ca(2+)-independent member of the PKC family that is selectively expressed in skeletal muscle and T lymphocytes and plays an important role in T cell activation. However, the molecular basis for the important functions of PKCtheta in T cells and the manner in which it becomes coupled to the T cell receptor-signaling machinery are unknown. We addressed the functional relationship between PKCtheta and CD28 costimulation, which plays an essential role in T cell receptor-mediated IL-2 production. Here, we provide evidence that PKCtheta is functionally coupled to CD28 costimulation by virtue of its selective ability to activate the CD28RE/activator protein-1 (AP-1) element in the IL-2 gene promoter. First, CD28 costimulation enhanced the membrane translocation and catalytic activation of PKCtheta. Second, among several PKC isoforms, PKCtheta was the only one capable of activating NF-kappaB or CD28RE/AP-1 reporters in T cells (but not in 293T cells). Third, wild-type PKCtheta synergized with CD28/CD3 signals to activate CD28RE/AP-1. In addition, PKCtheta selectively synergized with Tat to activate a CD28RE/AP-1 reporter. Fourth, CD3/CD28-induced CD28RE/AP-1 activation and NF-kappaB nuclear translocation were blocked by a selective PKCtheta inhibitor. Last, PKCtheta-mediated activation of the same reporter was inhibited by the proteasome inhibitor MG132 (which blocks IkappaB degradation) and was found to involve IkappaB-kinase beta. These findings identify a unique PKCtheta-mediated pathway for the costimulatory action of CD28, which involves activation of the IkappaB-kinase beta/IkappaB/NF-kappaB-signaling cascade.

Biological Transport↗
PubMed2000

Regulation of protein kinase Ctheta function during T cell activation by Lck-mediated tyrosine phosphorylation.

Protein kinase C theta (PKCtheta) is a novel Ca(2+)-independent PKC isoform, which is selectively expressed in skeletal muscle and hematopoietic cells, especially T cells. In T cells, it colocalizes with the T cell antigen receptor (TCR).CD3 complex in antigen-stimulated T cells and is involved in the transcriptional activation of the interleukin-2 gene. In the present study, we report that PKCtheta is tyrosine phosphorylated in Jurkat T cells upon TCR.CD3 activation. The Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKCtheta. Lck phosphorylated and was associated with the regulatory domain of PKCtheta both in vitro and in intact cells. This association was constitutive, but it was enhanced by T cell activation, with both Src-homology 2 and Src-homology 3 domains of Lck contributing to it. Tyrosine 90 (Tyr-90) in the regulatory domain of PKCtheta was identified as the major phosphorylation site by Lck. A constitutively active mutant of PKCtheta (A148E) could enhance proliferation of Jurkat T cells and synergized with ionomycin to induce nuclear factor of T cells activity. However, mutation of Tyr-90 into phenylalanine markedly reduced (or abolished) these activities. These results suggest that Lck plays an important role in tyrosine phosphorylation of PKCtheta, which may in turn modulate the physiological functions of PKCtheta during TCR-induced T cell activation.

Humans↗
PubMed2000

Inhibition of the c-Jun N-terminal kinase/AP-1 and NF-kappaB pathways by PICOT, a novel protein kinase C-interacting protein with a thioredoxin homology domain.

Protein kinase C-theta (PKCtheta) is a Ca(2+)-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 PKCtheta, we have employed the yeast two-hybrid system to identify PKCtheta-interacting proteins. We report the isolation and characterization of a cDNA encoding a novel 335-amino acid (37. 5-kDa) PKCtheta-interacting protein termed PICOT (for PKC-interacting cousin of thioredoxin). PICOT is expressed in various tissues, including in T cells, where it colocalizes with PKCtheta. 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-kappaB. 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.

Amino Acid Sequence↗
PubMed2000

TCR antagonist peptides induce formation of APC-T cell conjugates and activate a Rac signaling pathway.

T cell receptor antagonists inhibit T cell activation by antigen, and by themselves fail to induce phenotypic changes associated with T cell activation. However, they can induce limited tyrosine phosphorylation of TCRzeta chain. Here we show that TCR antagonists are potent inducers of APC-T cell conjugates, cytoskeletal reorganization, and capping of certain T cell proteins. These events are associated with a signaling pathway involving tyrosine phosphorylation of Vav and SLP-76, activation and capping of Rac-1, a protein previously linked with cytoskeletal reorganization, and activation of JNK. The finding that antagonist peptides stimulate this pathway, while failing to stimulate other TCR-mediated signaling pathways, indicates the presence in T cells of a hierarchy of signaling that is sensitive to the avidity of Ag / MHC-TCR interaction.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Vav modulation of the Ras/MEK/ERK signaling pathway plays a role in NFAT activation and CD69 up-regulation.

Vav is expressed exclusively in hematopoietic cells and becomes phosphorylated on tyrosine in response to antigen receptor ligation. Although Vav can act as a Rac-specific guanine nucleotide exchange factor in vitro and as a c-Jun N-terminal kinase (JNK) activator in ectopic expression systems, its physiological functions in lymphocytes remain unclear. Indirect evidence suggests that Vav interacts with the Ras/ERK pathway in T cells. Here, we analyzed the effects of Vav on three known downstream targets of Ras, i. e. activation of ERK and NFAT, and up-regulation of the activation antigen CD69. The MEK inhibitor PD90859 inhibited Vav-induced activation of ERK, and Vav- or anti-CD3-induced activation of NFAT, suggesting that MEK and ERK are involved in Vav-mediated NFAT activation. Similarly to Ras, Vav cooperated with constitutively active calcineurin and with ERK to activate NFAT, and was capable of up-regulating CD69 expression in T cells. Moreover, these Vav-mediated functions were all inhibited by a dominant negative Ras mutant. Conversely, however, dominant negative Vav did not inhibit NFAT and ERK activation or CD69 expression induced by an active Ras mutant. These findings indicate that Ras functions as an important downstream target of Vav in signaling pathways that lead to NFAT and ERK activation, and to CD69 expression. Moreover, the finding that Vav- (or Ras-) induced CD69 expression was not inhibited by a dominant negative Rac mutant indicates that Vav mediates some Ras-dependent, but Rac-independent, functions in T cells.

Antigens, CD↗
PubMed2000

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

Recent studies have identified protein kinase C&theta; (PKCtheta), 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 PKCtheta involved in the activation and survival of T cells are reviewed herein. Among these, the nuclear factor-kappaB (NF-kappaB) signaling cascade appears to be the most critical target of PKCtheta in the T-cell receptor/CD28 costimulatory pathway that leads to T-cell activation.

Animals↗
PubMed2000

A novel functional interaction between Vav and PKCtheta is required for TCR-induced T cell activation.

Vav and PKCtheta play an early and important role in the TCR/CD28-induced stimulation of MAP kinases and activation of the IL-2 gene. Vav is also essential for actin cytoskeleton reorganization and TCR capping. Here, we report that PKCtheta function was selectively required in a Vav signaling pathway that mediates the TCR/CD28-induced activation of JNK and the IL-2 gene and the upregulation of CD69 expression. Vav also promoted PKCtheta translocation from the cytosol to the membrane and cytoskeleton and induced its enzymatic activation in a CD3/CD28-initiated pathway that was dependent on Rac and on actin cytoskeleton reorganization. These findings reveal that the Vav/Rac pathway promotes the recruitment of PKCtheta to the T cell synapse and its activation, essential processes for T cell activation and IL-2 production.

Actins↗
PubMed2000

Neoadjuvant hormonal ablative therapy before radical prostatectomy: a review. Is it indicated?

PURPOSE: Neoadjuvant hormonal ablation therapy has been used to decrease the rate of positive surgical margins in patients treated with radical prostatectomy. We reviewed the available literature to determine whether this therapy is indicated and beneficial. MATERIALS AND METHODS: We performed a MEDLINE key word search and assessed randomized prospective articles. Data were analyzed for the rate of positive surgical margins, seminal vesicle invasion and lymph node metastasis as well as surgical characteristics, including operative time, blood loss, hospital stay, rate of complications and difficulty of surgical dissection. In addition, these data were evaluated for prostate specific antigen-free survival. RESULTS: Neoadjuvant hormonal therapy decreased the rate of positive margins in 6 of the 7 randomized prospective studies. In none of 4 randomized prospective series was there an improved rate of seminal vesicle invasion with neoadjuvant hormonal therapy. Of 4 studies 3 showed no improvement in the rate of lymph node metastasis after neoadjuvant hormonal therapy compared with that in controls. Similarly there was no improvement in prostate specific antigen-free survival and no significant difference in operative time, operative blood loss, transfusion or hospital stay in patients treated with neoadjuvant hormonal therapy and controls. In addition, in 2 of 3 studies there was no difference in the complication rate. CONCLUSIONS: Analysis of the available literature revealed no significant improvement in outcome to support the routine administration of neoadjuvant hormonal therapy before prostatectomy.

Androgen Antagonists↗
PubMed1999

Protein kinase ctheta cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death.

Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKCtheta, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKCtheta mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKCtheta mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKCtheta synergized with calcineurin to provide a potent stimulus for FasL promoter activation. Full activation of the promoter required its binding sites for the transcription factors NF-AT, AP-1, and NF-kappaB. The biological significance of these findings is implicated by the finding that rottlerin, a selective PKCtheta inhibitor, blocked FasL induction by anti-CD3 or PMA plus ionomycin stimulation and, consequently, protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from activation-induced cell death.

Acetophenones↗