RESUMO
The RING-finger domain is a novel zinc-binding Cys-His protein motif found in a growing number of proteins involved in signal transduction, ubiquitination, gene transcription, differentiation, and morphogenesis. We describe a novel muscle-specific RING-finger protein (MURF) expressed specifically in cardiac and skeletal muscle cells throughout pre- and postnatal mouse development. MURF belongs to the RING-B-box-coiled-coil subclass of RING-finger proteins, characterized by an NH(2)-terminal RING-finger followed by a zinc-finger domain (B-box) and a leucine-rich coiled-coil domain. Expression of MURF is required for skeletal myoblast differentiation and myotube fusion. The leucine-rich coiled-coil domain of MURF mediates association with microtubules, whereas the RING-finger domain is required for microtubule stabilization and an additional region is required for homo-oligomerization. Expression of MURF establishes a cellular microtubule network that is resistant to microtubule depolymerization induced by alkaloids, cold and calcium. These results identify MURF as a myogenic regulator of the microtubule network of striated muscle cells and reveal a link between microtubule organization and myogenesis.
Assuntos
Microtúbulos/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Diferenciação Celular , Linhagem Celular , Biblioteca Gênica , Células HeLa , Coração/fisiologia , Humanos , Leucina , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/citologia , Miocárdio/metabolismo , Especificidade de Órgãos , Biossíntese de Proteínas , Transcrição Gênica , TransfecçãoRESUMO
The chimeric gene EWS/FLI-1, the hallmark of the Ewing's sarcoma and primitive neuroectodermal tumor family, encodes a fusion protein with enhanced transcriptional activation properties and preserved recognition of canonical ETS binding sites. Although EWS/FLI-1 alters the expression of various genes, the precise mechanism by which EWS/FLI-1 acts as an oncogene remains to be defined. In this study we report that members of the mitogen-activated protein kinase (MAPK) signaling pathway, ERK1 and ERK2, are constitutively activated in NIH 3T3 cells expressing EWS/FLI-1. Interference with ERK activation by either highly specific inhibitors of MEK1 or a dominant negative ras mutant profoundly impaired the ability of EWS/FLI-1 to transform NIH3T3 cells to growth in semi-solid medium. An EWS/FLI-1 mutant defective in DNA-binding and transcriptional activation failed to activate ERK and was also defective in 3T3 cell transformation. Constitutive ERK activation was also evident in several human Ewing's sarcoma tumor-derived cell lines. Interestingly, cells expressing the type II EWS/FLI-1 fusion, recently demonstrated more potent in transcriptional activation, showed even greater MAPK activation than cells expressing the more common type I fusion. These results implicate ERK activation in EWS/FLI-1 transformation and suggest that this signaling pathway may be important in the pathogenesis of Ewing's sarcoma. Oncogene (2000) 19, 4523 - 4530.