RESUMEN
The Hippo pathway regulates a complex signalling network which mediates several biological functions including cell proliferation, organ size and apoptosis. Several scaffold proteins regulate the crosstalk of the members of the pathway with other signalling pathways and play an important role in the diverse output controlled by this pathway. In this study we have identified the scaffold protein IQGAP1 as a novel interactor of the core kinases of the Hippo pathway, MST2 and LATS1. Our results indicate that IQGAP1 scaffolds MST2 and LATS1 supresses their kinase activity and YAP1-dependent transcription. Additionally, we show that IQGAP1 is a negative regulator of the non-canonical pro-apoptotic pathway and may enable the crosstalk between this pathway and the ERK and AKT signalling modules. Our data also show that bile acids regulate the IQGAP1-MST2-LATS1 signalling module in hepatocellular carcinoma cells, which could be necessary for the inhibition of MST2-dependent apoptosis and hepatocyte transformation.
Asunto(s)
Apoptosis , Transducción de Señal , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular , Ácido Quenodesoxicólico/farmacología , Vía de Señalización Hippo , Humanos , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/metabolismo , Serina-Treonina Quinasa 3 , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína Tumoral p73/metabolismo , Proteínas Señalizadoras YAP , Proteínas Activadoras de ras GTPasa/químicaRESUMEN
In cell line development the identification of stable Chinese hamster ovary cells for production is a critical but onerous task. The stability trial focus upon high-level attributes can mask profound underlying cellular changes, leading to unstable clones mistakenly being chosen for production. The challenge is to assay underlying cell pathways and subsystems without pushing up cell line development costs. ChemStress® cell function profiling is a simple, multiwell plate-based assay that uses a panel of active chemicals to mimic known bioprocess stresses and challenge key pathways. After 3 days of static culture on the plate, functional responses are assayed, for example, titer and growth. Here this approach is used to monitor 131 clones as they change over real stability trials. A novel stability metric is defined over the data to identify stable clones that remain unperturbed across many components of cell function. This allows stability trials to look beneath the titer to identify clones that are internally more stable.
Asunto(s)
Células Clonales/citología , Animales , Biotecnología , Células CHO , Técnicas de Cultivo de Célula , Células Clonales/metabolismo , Cricetulus , FenotipoRESUMEN
Ras GTPases convey signals from different types of membranes. At these locations, different Ras isoforms, interactors and regulators generate different biochemical signals and biological outputs. The study of Ras localisation-specific signal transduction networks has been hampered by our inability to specifically activate each of these Ras pools. Here, we describe a new set of site-specific tethered exchange factors, engineered by fusing the RasGRF1 CDC25 domain to sub-localisation-defining cues, whereby Ras pools at specific locations can be precisely activated. We show that the CDC25 domain has a high specificity for activating HRas but not NRas and KRas. This unexpected finding means that our constructs mainly activate endogenous HRas. Hence, their use enabled us to identify distinct pathways regulated by HRas in endomembranes and plasma membrane microdomains. Importantly, these new constructs unveil different patterns of HRas activity specified by their subcellular localisation. Overall, the targeted GEFs described herein constitute ideal tools for dissecting spatially-defined HRas biochemical and biological functions.
Asunto(s)
Ingeniería de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ras-GRF1/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Chlorocebus aethiops , Humanos , Ratones , Transducción de SeñalRESUMEN
Signal transduction requires the coordination of activities between different pathways. In mammalian cells, Raf-1 regulates the MST-LATS and MEK-ERK pathways. We found that a complex circuitry of competing protein interactions coordinates the crosstalk between the ERK and MST pathways. Combining mathematical modelling and experimental validation we show that competing protein interactions can cause steep signalling switches through phosphorylation-induced changes in binding affinities. These include Akt phosphorylation of MST2 and a feedback phosphorylation of Raf-1 Ser 259 by LATS1, which enables Raf-1 to suppress both MST2 and MEK signalling. Mutation of Raf-1 Ser 259 stimulates both pathways, simultaneously driving apoptosis and proliferation, whereas concomitant MST2 downregulation switches signalling to cell proliferation, transformation and survival. Thus, competing protein interactions provide a versatile regulatory mechanism for signal distribution through the dynamic integration of graded signals into switch-like responses.
Asunto(s)
Genes de Cambio/fisiología , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Animales , Apoptosis/fisiología , Proliferación Celular , Células Cultivadas , Embrión no Mamífero , Células HEK293 , Células HeLa , Vía de Señalización Hippo , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Células MCF-7 , Ratones , Células 3T3 NIH , Fosforilación , Unión Proteica , Serina-Treonina Quinasa 3 , Pez Cebra/embriologíaRESUMEN
Epithelial to mesenchymal transition (EMT) is a fundamental cell differentiation/dedifferentiation process which is associated with dramatic morphological changes. Formerly polarized and immobile epithelial cells which form cell junctions and cobblestone-like cell sheets undergo a transition into highly motile, elongated, mesenchymal cells lacking cell-to-cell adhesions. To explore how the proteome is affected during EMT we profiled protein expression and tracked cell biological markers in Madin-Darby kidney epithelial cells undergoing hepatocyte growth factor (HGF) induced EMT. We were able to identify and quantify over 4000 proteins by mass spectrometry. Enrichment analysis of this revealed that expression of proteins associated with the ubiquitination machinery was induced, whereas expression of proteins regulating apoptotic pathways was suppressed. We show that both the mammalian Hippo/MST2 and the ISG15 pathways are regulated at the protein level by ubiquitin ligases. Inhibition of the Hippo pathway by overexpression of either ITCH or A-Raf promotes HGF-induced EMT. Conversely, ISG15 overexpression is sufficient to induce cell scattering and an elongated morphology without external stimuli. Thus, we demonstrate for the first time that the Hippo/MST2 and ISG15 pathways are regulated during growth-factor induced EMT.
Asunto(s)
Transición Epitelial-Mesenquimal , Factor de Crecimiento de Hepatocito/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Ubiquitinas/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular , Perros , Factor de Crecimiento de Hepatocito/farmacología , Integrinas/metabolismo , Células de Riñón Canino Madin Darby , Proteoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
K-Ras is frequently mutated in human cancers. Mutant (mt) K-Ras can stimulate both oncogenic transformation and apoptosis through activation of extracellular signal-regulated kinase (ERK) and AKT pathways and the MST2 pathway, respectively. The biological outcome is determined by the balance and cross talk between these pathways. In colorectal cancer (CRC), a K-Ras mutation is negatively correlated with MST2 expression, as mt K-Ras can induce apoptosis by activating the MST2 pathway. However, wild-type (wt) K-Ras can prevent the activation of the MST2 pathway upon growth factor stimulation and enable transformation by mt K-Ras in CRC cells that express MST2. Here we have investigated the mechanism by which wt and mt K-Ras differentially regulate the MST2 pathway and MST2-dependent apoptosis. The ability of K-Ras to activate MST2 and MST2-dependent apoptosis is determined by the differential activation kinetics of mt K-Ras and wt K-Ras. Chronic activation of K-Ras by mutation or overexpression of Ras exchange factors results in the activation of MST2 and LATS1, increased MST2-LATS1 complex formation, and apoptosis. In contrast, transient K-Ras activation upon epidermal growth factor (EGF) stimulation prevents the formation of the MST2-LATS1 complex in an AKT-dependent manner. Our data suggest that the close relationship between Ras prosurvival and proapoptotic signaling is coordinated via the differential regulation of the MST2-LATS1 interaction by transient and chronic stimuli.