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A Altman

Publications and source records attributed to A Altman.

At least 55 records · Page 3Linked to original sources
PubMed1997

Differential T cell signaling induced by antagonist peptide-MHC complexes and the associated phenotypic responses.

Certain changes in TCR contact residues have been shown to have profound effects on the capacity of a peptide Ag to stimulate a T cell response. Although some of these changes apparently lead to a complete loss of the ability to interact with the TCR, others result in partial agonist activity (e.g., cytokine production without proliferation) or antagonist activity (i.e., the capacity to inhibit the engagement to the TCR by Ag). We show MHC class II-restricted antagonist activity was associated with a differential pattern of early tyrosine phosphorylation events that was characterized by a preponderance of phosphorylation of low molecular mass TCRzeta and the failure to phosphorylate Zap-70. These early tyrosine phosphorylation patterns are the same as those previously described for partial agonists. Thus, a partial agonist phenotype such as anergy induction cannot be ascribed in a causal manner to this pattern of tyrosine phosphorylation. We further extend the studies of signal transduction elicited by agonist and antagonist peptides by characterizing differential recruitment of Zap-70 associated with TCRzeta isoforms and differential phosphorylation of p120 proto-oncogene c-Cbl. Another early event following TCR engagement by Ag, down-modulation of the TCR, was studied with antagonist peptides. We show that antagonist peptides do not cause TCR down-modulation. This failure may represent a mechanism by which antagonists inhibit antigen-mediated stimulation of T cells.

Amino Acid Sequence↗
PubMed1997

Ras-dependent, Ca2+-stimulated activation of nuclear factor of activated T cells by a constitutively active Cbl mutant in T cells.

T cell receptor (TCR) stimulation induces rapid tyrosine phosphorylation of cellular proteins, including Cbl, a protooncogene product whose function remains unclear. As a first step toward elucidating the function of Cbl in TCR-initiated signaling, we evaluated the ability of wild-type Cbl or a transforming Cbl mutant (70Z/3) to induce transcriptional activation of a nuclear factor of activated T cells (NFAT) element derived from the interleukin 2 (IL2) promoter in transiently cotransfected Jurkat-TAg T cells. 70Z/3, but not Cbl, caused NFAT activation which was significantly enhanced by stimulation with calcium ionophore, and was drastically reduced by cyclosporin A pretreatment. A point mutation of a potential phosphatidylinositol 3-kinase (PI3-K) binding site (Y731EAM to Y731EAC) in 70Z/3 disrupted the association of PI3-K with 70Z/3, but did not reduce the induction of NFAT activity, suggesting that the interaction between Cbl and PI3-K is not required in the 70Z/3-mediated induction of NFAT. Additional mapping studies indicated that defined deletions of C-terminal 70Z/3 sequences affected to a variable degree its ability to stimulate NFAT activity. Strikingly, deletion of 346 C-terminal residues augmented this activity, whereas removal of 20 additional residues abolished it. Coexpression of dominant negative Ras abrogated the basal or ionomycin-stimulated, 70Z/3-mediated NFAT activation, suggesting a functional Ras is required for this activation. These results implicate Cbl in Ras-dependent signaling pathways which lead to NFAT activation.

Animals↗
PubMed1997

Views on Vav.

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Animals↗
PubMed1997

Association of immunophenotype with cerebrospinal fluid involvement in childhood B-lineage acute lymphoblastic leukemia.

The records of 71 pediatric patients who had B-lineage acute lymphoblastic leukemia (ALL) were studied retrospectively to document the correlation between antigenic phenotype of leukemic blasts in bone marrow (BM) and cerebrospinal fluid (CSF) involvement. Immunofluorescence assay for terminal deoxynucleotidyl transferase (TdT-IF) was used to examine specimens of CSF for the presence (CSF+) or absence (CSF-) of leukemic blasts at diagnosis and at various times after the induction of chemotherapy. The results of immunophenotyping of the leukemic blasts in the BM of the CSF+ and CSF patient groups were then compared, and the relative risk ratio and positive predictive value were calculated for each marker. In addition, other possible prognostic factors, such as gender, race and ethnicity, age, and initial leukocyte count were compared between the two groups. Thirty-eight percent of all patients, including those who had cytologically confirmed cases of CNS leukemia, had TdT+ cells in the CSF at diagnosis or during the course of the disease. No differences were observed between the CSF+ and CSF- groups with respect to clinical and demographic factors. However, the cases of CSF+ leukemia had the almost exclusive expression of cytoplasmic immunoglobulin heavy chain (c mu) or surface CD22 (Leu-14), CD23 (B6), and/or IgM markers, which normally characterize the most developmentally mature members of the B-cell series in BM. The expression of any of these antigenic markers at diagnosis appears to identify at least 88% of cases of B-lineage ALL that have or ultimately may have TdT+ cells in CSE The results of this study therefore may have both basic and clinical implications: The former concerns the mechanisms by which these phenotypically defined forms of ALL invade and persist in the CNS; the latter concerns the utility of routine immunophenotyping of BM blasts at diagnosis to assess the biologic predisposition to CNS involvement in individual cases of ALL.

Adolescent↗
PubMed1997

Hypertonic saline activates protein tyrosine kinases and mitogen-activated protein kinase p38 in T-cells.

OBJECTIVES: In previous in vitro studies, we have found that hypertonic saline (HTS) can augment T-cell proliferation and restore the function of suppressed T-cells. Our animal models have shown that HTS resuscitation reverses immunosuppression after hemorrhage and reduces mortality from sepsis. In the present study, we investigated if and how HTS may influence T-cell signaling and function on a subcellular level. DESIGN: Human peripheral blood mononuclear cells (PBMC) were used to determine the effect of HTS on T-cell interleukin 2 (IL-2) production and proliferation. Human Jurkat T-cells were used to study the effects of HTS on T-cell signal transduction, IL-2 mRNA transcription, and IL-2 expression. MATERIAL AND METHODS: The effect of HTS on T-cell proliferation and IL-2 production was measured with PBMC and Jurkat T-cells. IL-2 mRNA transcription in HTS-treated Jurkat cells was measured by reverse transcriptase polymerase chain reaction. HTS-induced protein tyrosine phosphorylation in Jurkat T-cells was determined by immunoblotting with anti-phosphotyrosine antibodies. Expression in Jurkat cells of the mitogen-activated protein kinase p38 (MAPK p38), a signal transduction protein that is activated by osmotic stress, was determined by immunoblotting with anti-MAPK p38 antibodies. HTS-induced MAPK p38 activation in Jurkat cells was measured with an immune-complex kinase assay using ATF-2 as a substrate. MEASUREMENTS AND MAIN RESULTS: Proliferation of activated human PBMC increased significantly upon addition of HTS to the culture medium. This effect of HTS was paralleled by enhanced IL-2 production of activated PBMC and Jurkat cells and IL-2 mRNA transcription of Jurkat cells. HTS exposure of Jurkat cells caused tyrosine phosphorylation of a number of cellular proteins. We found that Jurkat T-cells expressed MAPK p38 and that it was activated in the presence of HTS. All these effects of HTS on T-cell signaling and function were observed at NaCl concentrations that were within physiologically relevant levels (20-100 mmol/L hypertonicity). CONCLUSIONS: In T-cells, HTS triggers a signaling pathway that includes increased tyrosine phosphorylation of several cellular proteins and activation of MAPK p38. HTS alone does not result in IL-2 mRNA transcription, IL-2 expression, or T-cell proliferation. However, in combination with other stimuli, HTS augments T-cell IL-2 expression and proliferation. We speculate that HTS could "resuscitate" suppressed T-cells in trauma patients by circumvention of, or substituting for, blocked signaling pathways.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Hypertonic saline resuscitation: a tool to modulate immune function in trauma patients?

Hypertonic saline (HS) resuscitation has recently gained attention from trauma physicians because it may benefit the immune system of trauma patients. We have found that HS augments in vitro and in vivo immune function of healthy T-cells. In addition, HS restored the function of suppressed T-cells in vitro and in vivo and reduced immunosuppression after hemorrhage, protecting mice from subsequent sepsis. These effects of HS are based on its direct influence on cellular signaling events through specific signaling pathway(s) that include protein tyrosine kinase and mitogen-activated protein kinase p38 activation. HS provides a costimulatory signal that enhances the proliferation of activated T-cells. HS may be able to substitute signals lost through blockage as a result of trauma induced suppressive factors, thereby restoring the function of suppressed T-cells. Although further work is needed to determine the optimal conditions and possible risks of HS resuscitation, the data presented in this short review of our recent work shed a favorable light on HS as a simple but effective tool to modulate cellular immune function after trauma.

Adjuvants, Immunologic↗
PubMed1996

Identification of the site in the Syk protein tyrosine kinase that binds the SH2 domain of Lck.

The Syk protein tyrosine kinase (PTK) is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. Upon cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues and enzymatically activated. We and others have found that the Lck kinase, a member of the Src family of PTKs, binds through its Src homology (SH) 2 domain to tyrosine phosphorylated Syk and to the related Zap kinase. Here we report that this interaction is direct and identify the two tandem tyrosines at the autophosphorylation site of Syk, Tyr518, and Tyr519, as the binding site for the SH2 domain of Lck. Mutation of either or both tyrosines to phenylalanines abrogated binding, while mutation of a second repetition of the motif at Tyr539 and Tyr540, or of the three tyrosines in the C terminus of Syk, did not. The SH2 domain of Lck bound the autophosphorylation site only when both Tyr518 and Tyr519 were phosphorylated. In intact cells the binding of the SH2 domain of Lck correlated with the ability of Syk to induce tyrosine phosphorylation of cellular proteins.

Amino Acid Sequence↗
PubMed1996

Activation-modulated association of 14-3-3 proteins with Cbl in T cells.

14-3-3 proteins have recently been implicated in the regulation of intracellular signaling pathways via their interaction with several oncogene and protooncogene products. We found recently that 14-3-3 associates with several tyrosine-phosphorylated proteins and phosphatidylinositol 3-kinase (PI3-K) in T cells. We report here the identification of the 120-kDa 14-3-3tau-binding phosphoprotein present in activated T cell lysates as Cbl, a protooncogene product of unknown function which was found recently to be a major protein-tyrosine kinase (PTK) substrate, and to interact with several signaling molecules including PI3-K, in T lymphocytes. The association between 14-3-3tau and Cbl was detected both in vitro and in intact T cells and, in contrast to Raf-1, was markedly increased following T cell activation. The use of truncated 14-3-3tau fusion proteins demonstrated that the 15 C-terminal residues are required for the association between 14-3-3 and three of its target proteins, namely, Cbl, Raf-1, and PI3-K. The findings that 14-3-3tau binds both PI3-K and Cbl, together with recent reports of an association between Cbl and PI3-K, suggest that 14-3-3 dimers play a critical role in signal transduction processes by promoting and coordinating protein-protein interactions of signaling proteins.

14-3-3 Proteins↗
PubMed1996

Inhibition of phosphatidylinositol 3-kinase blocks T cell antigen receptor/CD3-induced activation of the mitogen-activated kinase Erk2.

The production of 3-phosphorylated inositol phospholipids is implicated in regulation of cell growth and transformation. To explore the role of these lipids in T cell antigen receptor (TCR)/CD3-induced signaling, we have examined the effects of a specific phosphatidylinositol 3-kinase (PtdIns3K) inhibitor, wortmannin, and overexpression of two PtdIns3K constructs on the activation of down-stream effectors in anti-CD3 treated T cells. We report that treatment of cells with wortmannin blocked anti-CD3-induced activation of the mitogen-activation kinase Erk2 while not affecting phorbol-ester-induced Erk2 activation. An inactive analog of wortmannin, WM12, did not affect TCR/CD3-induced Erk2 activation, and wortmannin had no effect on the activity of Erk2 when added directly to the in vitro assays. Expression of a disruptive PtdIns3K construct also reduced Erk2 activation, while a construct that stimulates PtdIns3K enhanced the activation of Erk2. Receptor-induced activation of other Ser/Thr kinases, such as c-Raf, B-Raf, Mek1, Mek2, Mekk, was not affected by wortmannin. Our results suggest that the production of 3-phosphorylated inositol phospholipids is involved in the activation of Erk2, but does not regulate the enzymes that are thought to be upstream of Erk2.

Amino Acid Sequence↗
PubMed1996

Vav: function and regulation in hematopoietic cell signaling.

Vav, a 95 kDa proto-oncogene product expressed specifically in hematopoietic cells, was originally isolated as a transforming human oncogene. Vav contains an array of functional domains that are involved in interactions with other proteins and, possibly, with lipids. These include, among others, a putative guanine nucleotide exchange domain, a cysteine-rich region similar to the phorbol ester/diacylglycerol-binding domain of protein kinase C, a pleckstrin-homology domain, and Src-homology 2 and 3 (SH2 and SH3, respectively) domains. The presence of these domains, the transforming activity of the vav oncogene, and the rapid increase in tyrosine phosphorylation of Vav induced by triggering of diverse receptors indicate that it plays an important role in hematopoietic cell signaling pathways. Such a role is supported by recent studies using "knockout" mice and transiently transfected T cells, in which Vav deletion or overexpression, respectively, had marked effects on lymphocyte development or activation. The presence of a putative guanine nucleotide exchange domain, the prototype of which is found in the dbl oncogene product, implies that Vav functions as a guanine nucleotide exchange factor (GEF) for one (or more) members of the Ras-like family of small GTP-binding proteins. In support of such a role, Vav preparations were found in some (but not other) studies to mediate in vitro-specific GEF activity for Ras. Additional studies are required to identify the physiological regulators and targets of Vav, and its exact role in hematopoietic cell development and signaling.

Amino Acid Sequence↗
PubMed1996

Functional and physical interactions of Syk family kinases with the Vav proto-oncogene product.

Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.

B-Lymphocytes↗
PubMed1996

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.

14-3-3 Proteins↗
PubMed1996

Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

Animals↗
PubMed1995

Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting and enzymatic assays as one of the 14-3-3-binding proteins in resting or activated cells. Moreover, endogenous 14-3-3 and PI3-K were coimmunoprecipitated from intact T cells. Far-Western blots of gel-purified, immunoprecipitated PI3-K with a recombinant 14-3-3 fusion protein revealed direct binding of 14-3-3 to the catalytic subunit (p110) of PI3-K. Finally, anti-phosphotyrosine immunoprecipitates from activated, 14-3-3-overexpressing cells contained lower PI3-K enzymatic activity than similar immunoprecipitates from control cells. These findings suggest that association of 14-3-3 with PI3-K in hematopoietic (and possibly other) cells regulates the enzymatic activity of PI3-K during receptor-initiated signal transduction.

14-3-3 Proteins↗
PubMed1995

The role of p56lck in CD4-mediated suppression of CD3-induced early signaling events in T lymphocytes.

Crosslinking of the CD4 coreceptor can inhibit subsequent T-cell activation via the T-cell antigen receptor (TCR)/CD3 complex. The ability of the human immunodeficiency virus (HIV) envelope protein, gp 120, to cause similar inhibition has implicated this inhibitory signal in the induction of T-cell anergy and apoptosis observed in the acquired immunodeficiency syndrome (AIDS). In order to clarify the biochemical basis of this inhibition, we analyzed the effect of CD4 ligation on early signaling events induced by subsequent CD3xCD4 co-crosslinking. By comparison with CD3 crosslinking alone, CD3xCD4 co-crosslinking of a CD3+CD4+ human T-cell leukemia line (SupT1) resulted in an enhanced increase in free intracellular calcium concentration and tyrosine phosphorylation of several cellular substrates, including the prominent phosphorylation of an unidentified 120-kDa protein (p120). Prior CD4 ligation inhibited these responses. Similar results were obtained with A3.01, another CD3+CD4+ T leukemic line. However, P120 was only minor phosphorylated on tyrosine upon receptor crosslinking in A2.01/CD4(-cyt401), a derivative line expressing a truncated CD4 coreceptor lacking its cytoplasmic domain which binds the p56lck protein tyrosine kinase (PTK). Furthermore, prior CD4 ligation failed to inhibit in this line the increased tyrosine phosphorylation induced by subsequent CD3xCD4 co-crosslinking. Thus, prior CD4 crosslinking, or expression of truncated CD4, are both associated with reduced p120 phosphorylation. These results suggest that p120 is a p56lck substrate playing an important role during T-cell activation.

CD3 Complex↗
PubMed1995

Regulation of the p70zap tyrosine protein kinase in T cells by the CD45 phosphotyrosine phosphatase.

Two classes of protein tyrosine kinases (PTK) are utilized by the T cell antigen receptor (TcR)/CD3 complex for initiation of the signaling cascade, the Src-family PTK p56lck and p59fyn, and the Syk-family PTK p70zap and p72syk. In addition, the CD45 phosphotyrosine phosphatase (PTPase) is required for the induction of tyrosine phosphorylation by the TcR/CD3, presumably by positively regulating Src-family PTK. Here we report that CD45 also regulates the Syk-family PTK p70zap (or ZAP-70). In CD45-negative T cells, p70zap was constitutively phosphorylated on tyrosine and co-immunoprecipitated with the TcR-zeta chain. In resting wild-type CD45-positive cells, p70zap was mainly unphosphorylated, but it was rapidly phosphorylated on tyrosine upon treatment of the cells with anti-CD3 or PTPase inhibitors. Finally, p70zap co-distributed with CD45 in intact T cells, and tyrosine phosphorylated p70zap was dephosphorylated by CD45 in vitro. These findings suggest that CD45 plays an important role, direct or indirect, in the regulation of p70zap and its function in TcR/CD3 signaling.

Cell Line, Transformed↗