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R P Trible

Publications and source records attributed to R P Trible.

8 recordsLinked to original sources
PubMed2001

Down-modulation of the costimulatory molecule, CD28, is a conserved activity of multiple SIV Nefs and is dependent on histidine 196 of Nef.

In this study Nef proteins derived from simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) were compared to assess their abilities to down-modulate the cell surface levels of the T-cell costimulatory molecule CD28. We demonstrate that in addition to Nef derived from the prototypic SIVmac239, Nef proteins encoded by the pathogenic SIVsmmPBj molecular clone and the SIVsmmB670 isolate also down-modulate cell surface CD28. In contrast, Nef proteins derived from HIV failed to down-modulate CD28. We have also identified H196 as a critical residue which influences the capacity of SIVmac Nef to down-modulate CD28. Nef derived from SIVmacJ5 failed to down-modulate cell surface CD28, whereas a Q196H substitution mutant of SIVmacJ5 Nef was able to down-modulate cell surface CD28. Conversely, substitution of H196 to Q196 in SIVmac239 Nef resulted in a mutant that had minimal effect on cell surface CD28 expression, despite retaining the capacity to down-modulate cell surface CD3epsilon. H196 lies immediately adjacent to a documented di-leucine endocytic motif and mutation of this motif also abrogated the ability of SIVmac239 Nef to down-modulate CD28. These findings demonstrate that down-modulation of the costimulatory molecule, CD28, and clonotypic TCR/CD3 complex are conserved attributes of SIV Nef.

Animals↗
PubMed2001

Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT.

T cell activation induces functional changes in cell shape and cytoskeletal architecture. To facilitate the collection of dynamic, high-resolution images of activated T cells, we plated T cells on coverslips coated with antibodies to the T cell receptor (TCR). Using these images, we were able to quantitate the morphological responses of individual cells over time. Here, we show that TCR engagement triggers the formation and expansion of contacts bounded by continuously remodeled actin-rich rings. These processes are associated with the extension of lamellipodia and require actin polymerization, tyrosine kinase activation, cytoplasmic calcium increases, and LAT, an important hematopoietic adaptor. In addition, the maintenance of the resulting contact requires sustained calcium influxes, an intact microtubule cytoskeleton, and functional LAT.

Actins↗
PubMed2000

Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling.

The linker for activation of T cells (LAT) is a critical adaptor molecule required for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on tyrosine residues, and Grb2, Gads, and phospholipase C (PLC)-gamma1 bind LAT via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR engagement fails to induce ERK activation, Ca(2+) flux, and activation of AP-1 and NF-AT. We mapped the tyrosine residues in LAT responsible for interaction with these specific signaling molecules by expressing LAT mutants with tyrosine to phenylalanine mutations in LAT-deficient cells. Our results showed that three distal tyrosines, Tyr(171), Tyr(191), and Tyr(226), are responsible for Grb2-binding; Tyr(171), and Tyr(191), but not Tyr(226), are necessary for Gads binding. Mutation of Tyr(132) alone abolished PLC-gamma1 binding. Mutation of all three distal tyrosines also abolished PLC-gamma1 binding, suggesting there might be multiple binding sites for PLC-gamma1. Mutation of Tyr(132) affected calcium flux and blocked Erk and NF-AT activation. Since Grb2 binding is not affected by this mutation, these results strongly suggest that PLC-gamma activation regulates Ras activation in these cells. Mutation of individual Grb2 binding sites had no functional effect, but mutation of two or three of these sites, in combination, also affected Erk and NF-AT activation.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Essential role of LAT in T cell development.

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line.

The adaptor molecule LAT (linker for activation of T cells) is a palmitoylated integral membrane protein that localizes to the glycolipid-enriched microdomains in the plasma membrane. Upon TCR engagement, LAT becomes phosphorylated on multiple tyrosine residues and then binds several critical signaling molecules. Here, we describe the generation and characterization of a LAT-deficient cell line. Using this cell line, we demonstrate that LAT is required for TCR-mediated Ca2+ mobilization and optimal tyrosine phosphorylation of phospholipase C-gamma1, Vav and SLP-76. LAT is also required for Erk activation, CD69 up-regulation, and AP- and NFAT-mediated gene transcription. We also demonstrate, by reconstituting this cell line with LAT mutants, that the LAT transmembrane domain and palmitoylation at Cys26, but not Cys29, are required for LAT function and TCR signaling. These studies provide further evidence for the crucial role of the LAT molecule, and demonstrate the use of a LAT-deficient cell line for the analysis of LAT structure and function.

Adaptor Proteins, Signal Transducing↗
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↗