RESUMEN
The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.
Asunto(s)
Actinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Receptor fas/metabolismo , Androstadienos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Cromonas/farmacología , Proteína Ligando Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Citometría de Flujo , Humanos , Células Jurkat , Microdominios de Membrana/metabolismo , Morfolinas/farmacología , Complejos Multiproteicos/metabolismo , Mutación , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , WortmaninaRESUMEN
Lectin-like transcript 1 (LLT1) encoded by CLEC2D gene is a C-type lectin-like molecule interacting with human CD161 (NKR-P1A) receptor expressed by natural killer cells and subsets of T cells. Using RT-PCR and sequencing, we identified several CLEC2D alternatively spliced transcript variants generated by exon skipping. In addition to the reported transcript variants 1 (LLT1) and 2, we identified a novel splice variant 4 and transcripts coding for putative soluble proteins. CLEC2D transcripts were detected primarily in hematopoietic cell lines and were found to be co-induced by the same activation signals. Although very low amounts of putative soluble CLEC2D protein isoforms could be produced by transfectants, CLEC2D isoforms 2 and 4 were efficiently expressed. By contrast to LLT1, which was detected on the cell surface, isoform 2 and 4 remained in the endoplasmic reticulum where they formed homodimers or heterodimers with LLT1. They failed to interact with CD161, leaving LLT1 as the sole ligand for this receptor. CLEC2D therefore uses gene splicing to generate protein isoforms that are structurally distinct and that have different biological activities.
Asunto(s)
Empalme Alternativo , Lectinas Tipo C/genética , Receptores de Superficie Celular/genética , Transcripción Genética/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Retículo Endoplásmico/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Células Jurkat , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Subfamilia B de Receptores Similares a Lectina de Células NK/química , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Constitutive activation of the PI3K/mTOR signaling pathway contributes to carcinogenesis and metastasis in most, if not all, breast cancers. From a chromene backbone reported to inhibit class I PI3K catalytic subunits, several rounds of chemical syntheses led to the generation of a new collection of chromologues that showed enhanced ability to kill PI3K-addicted cancer cells and to inhibit Akt phosphorylation at serine 473, a hallmark of PI3K/mTOR activation. This initial screen uncovered a chromene designated DHM25 that exerted potent antitumor activity against breast tumor cell lines. Strikingly, DHM25 was shown to be a selective and covalent inhibitor of mTOR using biochemical and cellular analyses, modeling, and a large panel of kinase activity assays spanning the human kinome (243 kinases). Finally, in vivo, this novel drug was an efficient inhibitor of growth and metastasis of triple-negative breast cancer cells, paving the way for its clinical application in oncology.