RESUMEN
Sphingolipids represent an important class of bioactive signaling lipids which have key roles in numerous cellular processes. Over the last few decades, the levels of bioactive sphingolipids and/or their metabolizing enzymes have been realized to be important factors involved in disease development and progression, most notably in cancer. Targeting sphingolipid-metabolizing enzymes in disease states has been the focus of many studies and has resulted in a number of pharmacological inhibitors, with some making it into the clinic as therapeutics. In order to better understand the regulation of sphingolipid-metabolizing enzymes as well as to develop much more potent and specific inhibitors, the field of sphingolipids has recently taken a turn toward structural biology. The last decade has seen the structural determination of a number of sphingolipid enzymes and effector proteins. In these terms, one of the most complete arms of the sphingolipid pathway is the sphingosine-1-phosphate (S1P) arm. The structures of proteins involved in the function and regulation of S1P are being used to investigate further the regulation of said proteins as well as in the design and development of inhibitors as potential therapeutics.
Asunto(s)
Lisofosfolípidos/metabolismo , Modelos Moleculares , Sistemas de Mensajero Secundario , Esfingosina/análogos & derivados , Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Animales , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Lisofosfolípidos/química , Moduladores del Transporte de Membrana , Conformación Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/química , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
The bioactive sphingolipid sphingosine-1-phosphate (S1P) mediates cellular proliferation, mitogenesis, inflammation, and angiogenesis. These biologies are mediated through S1P binding to specific GPCRs [sphingosine-1-phosphate receptor (S1PR)1-5] and some other less well-characterized intracellular targets. Ezrin-radixin-moesin (ERM) proteins, a family of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, are emerging as critical regulators of cancer invasion via regulation of cell morphology and motility. Recently, we identified S1P as an acute ERM activator (via phosphorylation) through its action on S1PR2. In this work, we dissect the mechanism of S1P generation downstream of epidermal growth factor (EGF) leading to ERM phosphorylation and cancer invasion. Using pharmacologic inhibitors, small interfering RNA technologies, and genetic approaches, we demonstrate that sphingosine kinase (SK)2, and not SK1, is essential and sufficient in EGF-mediated ERM phosphorylation in HeLa cells. In fact, knocking down SK2 decreased ERM activation 2.5-fold. Furthermore, we provide evidence that SK2 is necessary to mediate EGF-induced invasion. In addition, overexpressing SK2 causes a 2-fold increase in HeLa cell invasion. Surprisingly, and for the first time, we find that this event, although dependent on S1PR2 activation, does not generate and does not require extracellular S1P secretion, therefore introducing a potential novel model of autocrine/intracrine action of S1P that still involves its GPCRs. These results define new mechanistic insights for EGF-mediated invasion and novel actions of SK2, therefore setting the stage for novel targets in the treatment of growth factor-driven malignancies.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Comunicación Autocrina/genética , Proteínas del Citoesqueleto/genética , Factor de Crecimiento Epidérmico/genética , Células HeLa , Humanos , Lisofosfolípidos/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Fosforilación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
Mutations in the renal tumour suppressor protein, folliculin, lead to proliferative skin lesions, lung complications and renal cell carcinoma. Folliculin has been reported to interact with AMP-activated kinase, a key component of the mammalian target of rapamycin pathway. Most cancer-causing mutations lead to a carboxy-terminal truncation of folliculin, pointing to a functional importance of this domain in tumour suppression. We present here the crystal structure of folliculin carboxy-terminal domain and demonstrate that it is distantly related to differentially expressed in normal cells and neoplasia (DENN) domain proteins, a family of Rab guanine nucleotide exchange factors (GEFs). Using biochemical analysis, we show that folliculin has GEF activity, indicating that folliculin is probably a distantly related member of this class of Rab GEFs.