RESUMO
The microphthalmia-associated transcription factor (Mitf) regulates gene expression required for osteoclast differentiation. Genes regulated by Mitf have been previously identified. However, proteins that interact and regulate Mitf's activity in osteoclasts are not well known. Here, we report that POH1, a subunit of the 19S proteasome lid is a regulator of Mitf. We show that POH1 and Mitf interact in osteoclasts and that this interaction is dependent on RANKL signaling. Overexpression of POH1 increased Mitf's activation of 5XGal4-TK and Acp5 promoters. The amino terminus of POH1 mediates the binding to Mitf and is sufficient to increase Mitf's transcriptional activity. Finally, we show that mutations in the JAMM motif of POH1 reduced Mitf activation of promoters. In summary, our results identify a novel mechanism of Mitf regulation in osteoclasts by POH1.
Assuntos
Fator de Transcrição Associado à Microftalmia/genética , Osteoclastos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Transativadores/metabolismo , Ativação Transcricional/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Diferenciação Celular , Humanos , Células Jurkat , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Transativadores/química , Técnicas do Sistema de Duplo-Híbrido , UbiquitinaçãoRESUMO
Microphthalmia-associated transcription factor, Mitf, has been shown to be necessary for regulating genes involved in osteoclast differentiation. Previously it was shown by others that Mitf translocates from the cytoplasm to the nucleus upon M-CSF/RANKL signaling in osteoclasts. Mitf's movement is regulated by its interaction with 14-3-3 and the kinase C-TAK1. Here we demonstrate that the related family member, Tfe3, does not shuttle from the cytoplasm to the nucleus and does not interact with C-TAK1. We also demonstrate that overexpression of C-TAK1 inhibits the expression of Acp5 while a kinase dead C-TAK1 or a Mitf mutant that cannot interact with C-TAK1 increased expression of Acp5. Finally, we show that the catalytic subunit of protein phosphatase 2A is up-regulated in osteoclasts with M-CSF/RANKL signaling, indicating a possible mechanism for dephosphorylating Mitf on its 14-3-3 binding site and allowing Mitf to be translocated to the nucleus of osteoclasts.