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Biomedical subjects

L E Samelson

Publications and source records attributed to L E Samelson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular basis of T cell inactivation by CTLA-4.

CTLA-4, a negative regulator of T cell function, was found to associate with the T cell receptor (TCR) complex zeta chain in primary T cells. The association of TCRzeta with CTLA-4, reconstituted in 293 transfectants, was enhanced by p56(lck)-induced tyrosine phosphorylation. Coexpression of the CTLA-4-associated tyrosine phosphatase, SHP-2, resulted in dephosphorylation of TCRzeta bound to CTLA-4 and abolished the p56(lck)-inducible TCRzeta-CTLA-4 interaction. Thus, CTLA-4 inhibits TCR signal transduction by binding to TCRzeta and inhibiting tyrosine phosphorylation after T cell activation. These findings have broad implications for the negative regulation of T cell function and T cell tolerance.

Abatacept↗
PubMed1998

ZAP-70 association with T cell receptor zeta (TCRzeta): fluorescence imaging of dynamic changes upon cellular stimulation.

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRzeta chain expressed as a chimeric protein (TTzeta and Tzetazeta), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tzetazeta fused to green fluorescent protein (ZAP-70 GFP and Tzetazeta-GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRzeta chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTzeta was indicated using mutant ZAP-70 GFPs and a truncated zeta chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tzetazeta- GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRzeta-ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.

Actins↗
PubMed1998

LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation.

Despite extensive study, several of the major components involved in T cell receptor-mediated signaling remain unidentified. Here we report the cloning of the cDNA for a highly tyrosine-phosphorylated 36-38 kDa protein, previously characterized by its association with Grb2, phospholipase C-gamma1, and the p85 subunit of phosphoinositide 3-kinase. Deduced amino acid sequence identifies a novel integral membrane protein containing multiple potential tyrosine phosphorylation sites. We show that this protein is phosphorylated by ZAP-70/Syk protein tyrosine kinases leading to recruitment of multiple signaling molecules. Its function is demonstrated by inhibition of T cell activation following overexpression of a mutant form lacking critical tyrosine residues. Therefore, we propose to name the molecule LAT-linker for activation of T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1998

LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation.

The linker molecule LAT is a critical substrate of the tyrosine kinases activated upon TCR engagement. Phosphorylated LAT binds Grb2, PLC-gamma1, and other signaling molecules. We demonstrate that human LAT is palmitoylated and that palmitoylated LAT predominantly localizes into glycolipid-enriched microdomains (GEMs). Although the LAT transmembrane domain is sufficient for membrane localization, palmitoylation at C26 and C29 is essential for efficient partitioning into GEMs. LAT palmitoylation is necessary for its tyrosine phosphorylation. After T cell activation, most tyrosine-phosphorylated LAT molecules and a fraction of PLC-gamma1 and other signaling molecules are present in GEMs. LAT is central to T cell activation and is a novel linker molecule shown to require targeting to membrane microdomains for signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1998

LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway.

In this study, we present the further characterization of a mutant Jurkat T cell line, J.CaM2, that is defective in TCR-mediated signal transduction. Although initial TCR-mediated signaling events such as the inducible tyrosine phosphorylation of the TCR-zeta chain and ZAP-70 are intact in J.CaM2, subsequent events, including increases in intracellular calcium, Ras activation, and IL-2 gene expression are defective. Subsequent analysis of J.CaM2 demonstrated a severe deficiency in pp36/LAT expression, a recently cloned adaptor protein implicated in TCR signaling. Importantly, reexpression of LAT in J.CaM2 restored all aspects of TCR signaling. These results demonstrate a necessary and exclusive role for LAT in T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.

T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs), including the Src family member, Lck, and the Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have demonstrated that ZAP-70 plays crucial roles in T-cell activation and development. However, progress toward a detailed understanding of the regulation and function of ZAP-70 during TCR signaling has been hampered by the lack of a suitable T-cell model for biochemical and genetic analyses. In this report, we describe the isolation and phenotypic characterization of a Syk- and ZAP-70-negative somatic mutant derived from the Jurkat T-cell line. The P116 cell line displays severe defects in TCR-induced signaling functions, including protein tyrosine phosphorylation, intracellular Ca2+ mobilization, and interleukin-2 promoter-driven transcription. These signaling defects were fully reversed by reintroduction of catalytically active versions of either Syk or ZAP-70 into the P116 cells. However, in contrast to ZAP-70 expression, Syk expression triggered a significant degree of cellular activation in the absence of TCR ligation. Transfection experiments with ZAP-70-Syk chimeric proteins indicated that both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional properties of these PTKs. These studies underscore the crucial role of ZAP-70 in TCR signaling and offer a powerful genetic model for further analyses of ZAP-70 regulation and function in T cells.

Clone Cells↗
PubMed1997

Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Animals↗
PubMed1997

ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis.

Activation-induced cell death (AICD) is initiated by the TCR-dependent up-regulation of Fas ligand (FasL) mRNA. The subsequently generated soluble or cell-associated FasL gene products bind Fas, leading to apoptosis of the T cells. Although TCR stimulation is essential to initiate AICD, little is known about which TCR-initiated second messengers are required for FasL expression. We provide evidence in this work that T cells lacking the tyrosine kinase ZAP-70 are unable to up-regulate FasL and undergo AICD. Transfection of wild-type ZAP-70 into the ZAP-70-deficient T cells restores their sensitivity to TCR-induced apoptosis, whereas transfection of catalytically inactive ZAP-70 does not. These results provide clear evidence that ZAP-70 tyrosine kinase is essential in up-regulating FasL for TCR-induced apoptosis.

Apoptosis↗
PubMed1997

The product of the proto-oncogene c-cbl: a negative regulator of the Syk tyrosine kinase.

Engagement of antigen and immunoglobulin receptors on hematopoietic cells is directly coupled to activation of nonreceptor protein tyrosine kinases (PTKs) that then phosphorylate critical intracellular substrates. In mast cells stimulated through the FcvarepsilonRI receptor, activation of several PTKs including Syk leads to degranulation and release of such mediators of the allergic response as histamine and serotonin. Regulation of Syk function occurred through interaction with the Cbl protein, itself a PTK substrate in this system. Overexpression of Cbl led to inhibition of Syk and suppression of serotonin release from mast cells, demonstrating its ability to inhibit a nonreceptor tyrosine kinase. Complex adaptor proteins such as Cbl can directly regulate the functions of the proteins they bind.

Animals↗
PubMed1996

Identification and characterization of a substrate specific for the T cell protein tyrosine kinase ZAP-70.

ZAP-70 is a protein tyrosine kinase (PTK) that plays a critical role in T cell activation. To study the role of ZAP-70 catalytic activity in this process, a substrate capable of distinguishing between the activities of ZAP-70 and other PTKs would be useful, especially since it has recently been shown that ZAP-70 interacts with another T cell PTK, Lck. We have thus identified a site of phosphorylation on the cytoplasmic fragment of the erythrocyte band 3 protein that is recognized by ZAP-70, but not Lck. A synthetic peptide based on this site has been demonstrated to be a good in vitro substrate for ZAP-70 and a poor substrate for the T cell PTKs Lck and Itk. This peptide molecule should thus prove useful to many investigators working in the field of T cell activation.

Amino Acid Sequence↗
PubMed1996

Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells.

Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.

Animals↗
PubMed1996

Regulation of the association of p120cbl with Grb2 in Jurkat T cells.

The c-cbl protooncogene product (p120(cbl)) is a known substrate of multiple tyrosine kinases. It is found in complexes with critical signal transduction molecules, including the linker protein Grb2. Here, we demonstrate using an immobilized Grb2-binding peptide that the Grb2-p120(cbl) complex dissociates in vivo following engagement of the T-cell antigen receptor in Jurkat T-cells. The early kinetics of this dissociation correlate with the known time course of tyrosine phosphorylation of p120(cbl) and other substrates. This dissociation persists in vivo even when p120(cbl) becomes dephosphorylated to basal levels. However, this decreased association is not observed in protein overlay assays on nitrocellulose membranes in which a Grb2 fusion protein is used to detect p120(cbl) from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120(cbl) does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120(cbl) to map the p120(cbl)-Grb2 interaction to amino acids 481-528 of p120(cbl); this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120(cbl) complex further supports the idea of a significant role for p120(cbl) in receptor-mediated signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Purification and characterization of human ZAP-70 protein-tyrosine kinase from a baculovirus expression system.

The ZAP-70 protein tyrosine kinase is essential for T cell antigen receptor (TCR)-mediated signaling. The absence of ZAP-70 results in impaired differentiation of T cells and a lack of responsiveness to antigenic stimulation. In order to study the characteristics of ZAP-70 in vitro, we overexpressed an epitopically tagged human ZAP-70 in a recombinant baculovirus expression system and purified it by column chromatography. The kinase activity of purified, recombinant ZAP-70 required cation and exhibited a strong preference for Mn2+ over Mg2+. The apparent Km of ZAP-70 for ATP was approximately 3.0 microM. The activity of the recombinant ZAP-70, unlike that of the homologous protein tyrosine kinase, Syk, was not affected by binding of TCR-derived tyrosine phosphorylated immunoreceptor tyrosine-based activation motif peptides. Several proteins were tested as potential in vitro substrates of ZAP-70. Only alpha-tubulin and the cytoplasmic fragment of human erythrocyte band 3 (cfb3), which have a region of sequence identity at the phosphorylation site, proved to be good substrates, exhibiting Kmvalues of approximately 3.3 and approximately 2.5 microM, respectively ([ATP] = 50 microM). alpha- and beta-Casein were poor substrates for ZAP-70, and no activity toward enolase, myelin basic protein, calmodulin, histone proteins, or angiotensin could be detected. In contrast to the T cell protein tyrosine kinase, Lck, ZAP-70 did not phosphorylate the cytoplasmic portion of the TCRzeta chain or short peptides corresponding to the CD3epsilon or the TCRzeta immunoreceptor tyrosine-based activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.

Amino Acid Sequence↗
PubMed1996

HIV envelope-directed signaling aberrancies and cell death of CD4+ T cells in the absence of TCR co-stimulation.

HIV-1 infection in CD4(+) T cells initiates a viral cytopathic effect (CPE) that is dependent on the activation of intracellular protein tyrosine kinases (PTK). PTK in T cells are also activated during the course of TCR or CD4 receptor engagement and the manner of receptor engagement may generate signals leading either to cell proliferation, tolerance induction (anergy) or programmed cell death (PCD). We have identified PTK triggered during the interaction of cells stably expressing surface HIV envelope (gp 120/gp41; HIVenv) and CD4(+)T cells, which leads to extensive and rapid individual cell death. We have found that killing is accompanied by tyrosine phosphorylation and activation of the CD4-associated p56(ICK) kinase, and by activation of a second member of the scr family of PTK, p59(fyn) kinase, normally associated with T cell stimulation through the TCR. Interestingly, in contrast with normal T cell signaling, the zeta subunit of the TCR fails to become tyrosine-phosphorylated during signaling accompanying HIV-directed cell killing. Downstream activation of the ZAP-70 PTK also does not occur. Unlike T cell apoptosis triggered by soluble HIVenv glycoproteins, which requires co-stimulation of CD4 and the antigen-specific TCR, T cell killing by membrane-associated HIVenv does not require TCR co-stimulation, because aberrant signaling and cell death are triggered by CD4(+) but TCR- cell lines. These results are the first report where dual activation of the Lck and Fyn PTK does not result in normal downstream signaling through the ZAP PTK, We suggest by analogy to SCID resulting from ZAP-70 mutations, that the dissociation of upstream PTK activation from ZAP-70 signaling contributes to T cell depletion by HIV and to the development of AIDS.

Apoptosis↗
PubMed1995

Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

Apoptosis↗
PubMed1995

Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70.

ZAP-70 is an 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that is is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.

Amino Acid Sequence↗