RESUMEN
Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection.
Asunto(s)
Proteínas 14-3-3/inmunología , Proteínas 14-3-3/metabolismo , Anticuerpos Fosfo-Específicos/inmunología , Técnicas Biosensibles/métodos , Estructura Molecular , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Sensibilidad y EspecificidadRESUMEN
Ntcp is a phosphoprotein, and its translocation by cAMP to the plasma membrane is associated with dephosphorylation. However, the phosphorylation site(s) of Ntcp is not known. Thus, the aim of the present study was to determine the potential Ntcp phosphorylation sites and whether any of these phosphorylation sites is involved in Ntcp translocation. To determine the potential phosphorylation sites, metabolically labeled [32P]Ntcp isolated from hepatocytes was digested with clostripain and then subjected to SDS-PAGE followed by autoradiography. Clostripain digestion resulted in two phosphorylated peptides, and cAMP decreased phosphorylation of one of the peptides (7.8 K(d)), which contains the putative third cytoplasmic loop with three serine (Ser-213, Ser-226, and Ser-227) and two threonine (Thr-219 and Thr-225) residues. To determine whether any one of these serine/threonine residues is phosphorylated and/or is involved in Ntcp translocation, each of these serine/threonine residues were mutated to alanine. HuH-7 cells were transiently transfected with the wild-type and the mutated Ntcps followed by determination of taurocholate uptake and Ntcp expression, translocation and phosphorylation. Mutation of only Ser-226 resulted in 30% decrease in Ntcp phosphorylation and in 2.5 and 3.2-fold increases in taurocholate uptake and Ntcp retention in the plasma membrane, respectively. Cyclic AMP failed to further decrease phosphorylation and increase translocation of S226A-Ntcp. Taken together, these results suggest that the Ser-226 in the third cytoplasmic loop of Ntcp is phosphorylated and cAMP may increase Ntcp translocation to the plasma membrane by dephosphorylating Ntcp at this site.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Serina/metabolismo , Alanina/metabolismo , Sustitución de Aminoácidos , Animales , Autorradiografía , Sitios de Unión , Línea Celular , Línea Celular Tumoral , AMP Cíclico/metabolismo , Cisteína Endopeptidasas/farmacología , Electroforesis en Gel de Poliacrilamida , Hepatocitos/química , Humanos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Peso Molecular , Transportadores de Anión Orgánico Sodio-Dependiente , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Radioisótopos de Fósforo , Mutación Puntual , Estructura Secundaria de Proteína , Transporte de Proteínas , Ratas , Serina/química , Simportadores , Ácido Taurocólico/metabolismo , TransfecciónRESUMEN
The major histocompatibility complex nonrestricted cytotoxic leukemic T cell line T acute lymphoblastic leukemia (TALL)-104 is being pursued as a therapeutic agent for cancer. However, the receptors and effector mechanisms responsible for its broad tumoricidal function remain undefined. Here, we examined the roles played by natural cytotoxicity receptors (NCR), killer cell immunoglobulin-like receptors, cytolytic granule components, and tumor necrosis factor (TNF) family members in tumor recognition and lysis by TALL-104 cells. The perforin-granzyme pathway, TNF-related apoptosis-inducing ligand (TRAIL), and Fas were each involved in the lysis of particular tumor targets by TALL-104. Furthermore, phorbol 12-myristate 13-acetate/ionomycin treatment induced surface expression of Fas-L and TRAIL. In addition, supernatants from CD3-stimulated TALL-104 cultures exhibited antiproliferative activity, which was blocked 50-90% by anti-TNF-alpha monoclonal antibody (mAb). Although negative for the NCR natural killer (NK)p44, this cell line was found to express NKp46. An anti-NKp46 antibody strongly blocked TALL-104-mediated lysis of certain targets and directly induced cytokine production, granule release, and redirected lysis responses. Anti-NKG2D and anti-2B4 also stimulated redirected cytotoxicity by TALL-104. By contrast, anti-NKG2A mAb did not stain the cells or inhibit killing responses. Alternatively, KIR3DL2 was detected on TALL-104, and expression of its reported ligand, human leukocyte antigen (HLA)-A, on target cells provided protection from cytotoxicity. Thus, NKp46, NKG2D, and 2B4 are activating receptors, and KIR3DL2 is an inhibitory receptor on TALL-104. The data demonstrate the ability of TALL-104 cells to recognize a wide variety of tumors with NK cell receptors and kill them with a broad arsenal of cytolytic effector mechanisms, including cytolytic granules and TNF family ligands.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Proteínas Reguladoras de la Apoptosis , Complejo CD3/inmunología , Carcinógenos/farmacología , Degranulación de la Célula/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Proteína Ligando Fas , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Proteínas Inmediatas-Precoces/inmunología , Ionomicina/farmacología , Ionóforos/farmacología , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Proteínas de Unión al GTP Monoméricas/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptor 1 Gatillante de la Citotoxidad Natural , Neoplasias/inmunología , Neoplasias/terapia , Receptores Inmunológicos/inmunología , Receptores KIR , Receptores KIR3DL2 , Receptores de Células Asesinas Naturales , Transducción de Señal/efectos de los fármacos , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Linfocitos T Citotóxicos/citología , Ligando Inductor de Apoptosis Relacionado con TNF , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/inmunología , Factores de Necrosis Tumoral/inmunología , Células U937RESUMEN
Data presented here demonstrate that vaccine-induced CD8(+) T cells can eliminate their specific tumor-target with a two-staged attack. First, they release interferon-gamma that results in growth arrest of the tumor cells via induction of antiangiogenic mediators. Then, during the latter stages of the immune response, CD8(+) effector T cells eradicate the remaining tumor cells through perforin-mediated lysis. A combination of these two mechanisms is highly effective in the described model, while either pathway alone fails to completely achieve tumor rejection.