In contrast, in the same assays, TSAd did augment IL-2 promoter activity induced by the same stimulus. a novel transcription-regulatory protein in T cells and illustrate the importance of the TSAd SH2 domain name in this role. strong class=”kwd-title” Keywords: transcription, T lymphocyte, interleukin 2, signal transduction, SH2 domain name Introduction Adapter proteins play important functions in mammalian cell signaling through the formation of intracellular signaling complexes with catalytically active molecules 1. In human T cells, one such adapter protein is the T cellCspecific adapter (TSAd) protein, whose expression is usually induced by TCR cross-linking 2 3. TSAd comprises of D-(+)-Xylose an NH2-terminal region of unknown function, a centrally located Src homology 2 (SH2) domain name with the potential to bind phosphotyrosine-containing protein ligands, and a COOH- terminal region which contains both a proline-rich stretch and tyrosine residues which, if phosphorylated, could function as docking sites for other signaling proteins. The murine ortholog of TSAd is known as LCK-associated adapter protein (LAD) or RLK/ITK-binding protein (RIBP), based D-(+)-Xylose on its ability to interact with these respective Src and Tec family kinases, at least in yeast hybrid systems 4 5. Like TSAd, LAD/RIBP is restricted in expression to T cells and is induced upon TCR engagement. The function of TSAd and LAD/RIBP in T cells is usually unknown. Constitutive expression of TSAd TLR1 in human Jurkat T leukemic cell line stable transfectants was previously shown to result in inhibition of TCR plus phorbol esterCinduced activation of the promoter for the T cell autocrine growth factor, D-(+)-Xylose IL-2. In addition, TSAd was shown to inhibit the tyrosine kinase activity of LCK when both were transfected into a fibroblast cell line 3. These findings, therefore, suggested that TSAd might function as a negative feedback regulator of T cell activation. However, in stark contrast to this, T cells from LAD/RIBP knockout mice were demonstrated to produce considerably reduced quantities of IL-2 and IFN- upon TCR challenge, compared with control T cells 5. This obtaining indicates that TSAd/LAD/RIBP in fact performs a net positive role in T cell signal transduction, in induction of Th1-type T cell cytokines. In these studies we D-(+)-Xylose show that TSAd has a predominant nuclear localization in T cells and can function as a potent activator of gene transcription in in vivo transactivation assays. Both TSAd nuclear import and transcription-activating function appear to be mediated by TSAd SH2 domain name recognition of a phosphotyrosine-containing ligand. We confirm that, under certain conditions in T cell transient transfection assays, TSAd can indeed activate gene transcription from an IL-2 promoterCreporter gene construct and that this activation requires an intact TSAd SH2 domain name. These findings illustrate the function of TSAd as a potential transcription-regulatory protein in T cells. Materials and Methods TSAd DNA Constructs. DNA corresponding to the TSAd full-length coding region or SH2 domain only was generated by PCR and inserted into the multiple cloning sites of pEF-FLAG 6, pSG424 7, or pGEX3X (Amersham Pharmacia Biotech). Resulting constructs encode for NH2-terminal FLAG, GAL4 DNA-binding domain name (dbd) (1C147), and glutathione em S /em -transferase (GST)-tagged TSAd proteins, respectively. Arginine to lysine 120 (R120K) mutations of the TSAd SH2 domain name were made with the use of a QuickChange site-directed mutagenesis kit (Stratagene). Intracellular Staining. Jurkat Tag C15 human T leukemia cells 8, COS-7 African Green Monkey kidney fibroblastClike cells (American Type Culture Collection), and 293T human kidney epithelial cells were D-(+)-Xylose transfected with pEF-FLAG or pSG424 constructs by electroporation (250 V, 960 F, 0.4-cm gap cuvettes). After 24 h, cells were cytospun, and fixed and permeabilized in 3.75% formaldehyde/0.1% Triton X-100, and stained with anti-FLAG M2 (Sigma-Aldrich) or anti-GAL4 (Santa Cruz Biotechnology, Inc.) antibodies followed by goat antiCmouse Ig (GAM)-Alexa 488 or Texas red-X (Molecular Probes). To detect endogenous TSAd, regular Jurkat cells (American Type Culture Collection) were.
