RESUMEN
Deleted-in-liver cancer 1 (DLC1) exerts its tumor suppressive function mainly through the Rho-GTPase-activating protein (RhoGAP) domain. When activated, the domain promotes the hydrolysis of RhoA-GTP, leading to reduced cell migration. DLC1 is kept in an inactive state by an intramolecular interaction between its RhoGAP domain and the DLC1 sterile α motif (SAM) domain. We have shown previously that this autoinhibited state of DLC1 may be alleviated by tensin-3 (TNS3) or PTEN. We show here that the TNS3/PTEN-DLC1 interactions are mediated by the C2 domains of the former and the SAM domain of the latter. Intriguingly, the DLC1 SAM domain was capable of binding to specific peptide motifs within the C2 domains. Indeed, peptides containing the binding motifs were highly effective in blocking the C2-SAM domain-domain interaction. Importantly, when fused to the tat protein-transduction sequence and subsequently introduced into cells, the C2 peptides potently promoted the RhoGAP function in DLC1, leading to decreased RhoA activation and reduced tumor cell growth in soft agar and migration in response to growth factor stimulation. To facilitate the development of the C2 peptides as potential therapeutic agents, we created a cyclic version of the TNS3 C2 domain-derived peptide and showed that this peptide readily entered the MDA-MB-231 breast cancer cells and effectively inhibited their migration. Our work shows, for the first time, that the SAM domain is a peptide-binding module and establishes the framework on which to explore DLC1 SAM domain-binding peptides as potential therapeutic agents for cancer treatment.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proliferación Celular , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Femenino , Proteínas Activadoras de GTPasa/química , Células HEK293 , Humanos , Modelos Moleculares , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Motivo alfa Estéril , Tensinas/química , Tensinas/metabolismo , Proteínas Supresoras de Tumor/química , Proteína de Unión al GTP rhoA/químicaRESUMEN
The cytoskeleton provides structural integrity to cells and serves as a key component in mechanotransduction. Tensins are thought to provide a force-bearing linkage between integrins and the actin cytoskeleton; yet, direct evidence of tensin's role in mechanotransduction is lacking. We here report that local force application to epithelial cells using a micrometer-sized needle leads to rapid accumulation of cten (tensin 4), but not tensin 1, along a fibrous intracellular network. Surprisingly, cten-positive fibers are not actin fibers; instead, these fibers are keratin intermediate filaments. The dissociation of cten from tension-free keratin fibers depends on the duration of cell stretch, demonstrating that the external force favors maturation of cten-keratin network interactions over time and that keratin fibers retain remarkable structural memory of a cell's force-bearing state. These results establish the keratin network as an integral part of force-sensing elements recruiting distinct proteins like cten and suggest the existence of a mechanotransduction pathway via keratin network.
Asunto(s)
Citoesqueleto/química , Células Epiteliales/química , Mecanotransducción Celular , Estrés Mecánico , Tensinas/química , Animales , Movimiento Celular , Perros , Humanos , Procesamiento de Imagen Asistido por Computador , Queratinas/química , Células de Riñón Canino Madin Darby , Proteínas de Microfilamentos/químicaRESUMEN
Regulation of tyrosine phosphorylation on insulin receptor substrate-1 (IRS-1) is essential for insulin signaling. The protein tyrosine phosphatase (PTP) C1-Ten/Tensin2 has been implicated in the regulation of IRS-1, but the molecular basis of this dephosphorylation is not fully understood. Here, we demonstrate that the cellular phosphatase activity of C1-Ten/Tensin2 on IRS-1 is mediated by the binding of the C1-Ten/Tensin2 Src-homology 2 (SH2) domain to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). We show that the role of C1-Ten/Tensin2 is dependent on insulin-induced phosphoinositide 3-kinase activity. The C1-Ten/Tensin2 SH2 domain showed strong preference and high affinity for PtdIns(3,4,5)P3. Using site-directed mutagenesis, we identified three basic residues in the C1-Ten/Tensin2 SH2 domain that were critical for PtdIns(3,4,5)P3 binding but were not involved in phosphotyrosine binding and PTP activity. Using a PtdIns(3,4,5)P3 binding-deficient mutant, we showed that the specific binding of the C1-Ten/Tensin2 SH2 domain to PtdIns(3,4,5)P3 allowed C1-Ten/Tensin2 to function as a PTP in cells. Collectively, our findings suggest that the interaction between the C1-Ten/Tensin2 SH2 domain and PtdIns(3,4,5)P3 produces a negative feedback loop of insulin signaling through IRS-1.
Asunto(s)
Proteínas Sustrato del Receptor de Insulina/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Tensinas/química , Tensinas/metabolismo , Dominios Homologos src , Animales , Escherichia coli , Células HEK293 , Humanos , Células L , Ratones , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Tensinas/genéticaRESUMEN
Competition to bind microRNAs induces an effective positive cross talk between their targets, which are therefore known as "competing endogenous RNAs" (ceRNAs). Although such an effect is known to play a significant role in specific situations, estimating its strength from data and experimentally in physiological conditions appears to be far from simple. Here, we show that the susceptibility of ceRNAs to different types of perturbations affecting their competitors (and hence their tendency to cross talk) can be encoded in quantities as intuitive and as simple to measure as correlation functions. This scenario is confirmed by extensive numerical simulations and validated by re-analyzing phosphatase and tensin homolog's cross-talk pattern from The Cancer Genome Atlas breast cancer database. These results clarify the links between different quantities used to estimate the intensity of ceRNA cross talk and provide, to our knowledge, new keys to analyze transcriptional data sets and effectively probe ceRNA networks in silico.
Asunto(s)
Algoritmos , Unión Competitiva , MicroARNs/metabolismo , Modelos Biológicos , Modelos Moleculares , Neoplasias de la Mama/metabolismo , Simulación por Computador , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Humanos , Cinética , MicroARNs/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Procesos Estocásticos , Tensinas/química , Tensinas/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Bone resorption by osteoclasts is mediated by a typical adhesion structure called the sealing zone or actin ring, whose architecture is based on a belt of podosomes. The molecular mechanisms driving podosome organization into superstructures remain poorly understood to date, in particular at the osteoclast podosome belt. We performed proteomic analyses in osteoclasts and found that the adaptor protein tensin 3 is a partner of Dock5, a Rac exchange factor necessary for podosome belt formation and bone resorption. Expression of tensin 3 and Dock5 concomitantly increase during osteoclast differentiation. These proteins associate with the osteoclast podosome belt but not with individual podosomes, in contrast to vinculin. Super-resolution microscopy revealed that, even if they colocalize in the x-y plane of the podosome belt, Dock5 and tensin 3 differentially localize relative to vinculin in the z-axis. Tensin 3 increases Dock5 exchange activity towards Rac, and suppression of tensin 3 in osteoclasts destabilizes podosome organization, leading to delocalization of Dock5 and a severe reduction in osteoclast activity. Our results suggest that Dock5 and tensin 3 cooperate for osteoclast activity, to ensure the correct organization of podosomes.