RESUMO
Drosophila neural stem cells or neuroblasts undergo typical asymmetric cell division. An evolutionally conserved protein complex, comprising atypical protein kinase C (aPKC), Bazooka (Par-3) and Par-6, organizes cell polarity to direct these asymmetric divisions. Aurora-A (AurA) is a key molecule that links the divisions to the cell cycle. Upon its activation in metaphase, AurA phosphorylates Par-6 and activates aPKC signaling, triggering the asymmetric organization of neuroblasts. Little is known, however, about how such a positive regulatory cue is counteracted to coordinate aPKC signaling with other cellular processes. During a mutational screen using the Drosophila compound eye, we identified microtubule star (mts), which encodes a catalytic subunit of protein phosphatase 2A (PP2A), as a negative regulator for aPKC signaling. Impairment of mts function causes defects in neuroblast divisions, as observed in lethal (2) giant larvae (lgl) mutants. mts genetically interacts with par-6 and lgl in a cooperative manner in asymmetric neuroblast division. Furthermore, Mts tightly associates with Par-6 and dephosphorylates AurA-phosphorylated Par-6. Our genetic and biochemical evidence indicates that PP2A suppresses aPKC signaling by promoting Par-6 dephosphorylation in neuroblasts, which uncovers a novel balancing mechanism for aPKC signaling in the regulation of asymmetric cell division.
Assuntos
Divisão Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Neurônios/citologia , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/enzimologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Genes de Insetos , Modelos Biológicos , Neurônios/enzimologia , Folículo Ovariano/citologia , Fenótipo , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Transporte Proteico , Proteínas Supressoras de Tumor/metabolismoRESUMO
The SWI2/SNF2 family ATPase, p400/mDomino, is a core subunit of a large chromatin-remodeling complex, and is currently suggested to play a unique function in histone variant exchange, a process by which chromatin structure is altered. Here, we investigated the role of p400/mDomino in mammalian development by generating mutant mice with a targeted deletion of the N-terminal domain of p400/mDomino (referred to as mDom(DeltaN/DeltaN)). The mDom(DeltaN/DeltaN) mice died on embryonic day 11.5 (E11.5), and displayed an anemic appearance and slight deformity of the neural tube. DNA microarray and quantitative RT-PCR analyses revealed that all of the embryonic globin genes and a globin chaperone gene were poorly expressed in the mDom(DeltaN/DeltaN) embryo and yolk sac on E8.5, indicating that primitive erythropoiesis was impaired. A hematopoietic colony assay indicated that the hematopoietic activity of the yolk sac was significantly blocked in the mutant mice. We also found that the expression of a limited set of Hox genes, including Hoxa7, Hoxa9 and Hoxb9, was drastically enhanced in the mDom(DeltaN/DeltaN) yolk sacs. These results suggest that p400/mDomino plays a critical role in embryonic hematopoiesis by regulating the expression of developmentally essential genes such as those in the Hox gene cluster.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Hematopoese/fisiologia , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Animais , Sequência de Bases , Montagem e Desmontagem da Cromatina/genética , DNA Helicases , Primers do DNA/genética , Proteínas de Ligação a DNA , Eritropoese/genética , Eritropoese/fisiologia , Feminino , Desenvolvimento Fetal/genética , Desenvolvimento Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Hematopoese/genética , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Saco Vitelino/metabolismoRESUMO
The myeloid zinc finger protein (MZF)-2 is a C(2)H(2) zinc finger transcription factor that is expressed in myeloid cells and involved in the growth, differentiation, and tumorigenesis of myeloid progenitors. Here we describe a novel isoform of MZF-2, designated MZF-2A, and show that it is phosphorylated by the mitogen-activated protein (MAP) kinases. An in vitro phosphorylation experiment revealed that the transactivation domain (TAD) of MZF-2A was phosphorylated strongly by extracellular signal-regulated kinase (ERK) and phosphorylated weakly by p38 MAP kinase but not by Jun N-terminal kinase. Experiments using "add-back" mutants showed that three serine residues (Ser(257), Ser(275), and Ser(295)) in the TAD were phosphorylated in vitro by ERK. In myeloid LGM-1 cells, various extracellular stimuli induced the phosphorylation of these serine residues, which was differentially inhibited by the protein kinase inhibitors U0126 and SB203580. Substitution of these phosphorylation sites with alanines resulted in a strong enhancement of the ability of MZF-2A to activate transcription in a luciferase reporter assay. Taken together, these results indicate that MZF-2A is a novel target for the ERK and p38 MAP kinase signaling pathways, and its transactivation activity is negatively regulated by MAP kinase-mediated phosphorylation of the TAD.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Processamento Alternativo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Fatores de Transcrição Kruppel-Like , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Plasmídeos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera , Fatores de Transcrição/metabolismo , Transfecção , Dedos de ZincoRESUMO
BACKGROUND: The myeloid zinc finger protein 2A (MZF-2A) is a Krüppel-type C2H2 zinc finger transcription factor expressed in myeloid cells and involved in the growth, differentiation and tumorigenesis of myeloid progenitors. Previously we identified a 180 amino acid domain in MZF-2A which is responsible for the transcriptional activation of MZF-2A. To understand the mechanism of the MZF-2A-dependent transcriptional activation, we screened for molecules that interact with the transactivation domain (TAD) of MZF-2A. RESULTS: By using the yeast Ras recruitment two-hybrid screening, we identified a novel SWI2/SNF2-related protein, termed mammalian Domino (mDomino), as an MZF-2A-binding partner. The mDomino protein, which shows a marked similarity to the Drosophila Domino protein, contains a SWI2/SNF2-type ATPase/helicase domain, a SANT domain, and a glutamine-rich (Q-rich) domain. The C-terminal Q-rich domain of mDomino physically associates with the TAD of MZF-2A in mammalian cells as well as in yeast. Expression of the mDomino Q-rich domain, together with MZF-2A in myeloid LGM-1 cells, enhanced the MZF-2A-mediated activation of a reporter gene. CONCLUSIONS: These results strongly suggest that an ATP-dependent chromatin-remodelling complex containing mDomino interacts with MZF-2A to regulate gene expression in myeloid cells.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas Nucleares , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , DNA Helicases/genética , DNA Helicases/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Drosophila , Regulação da Expressão Gênica , Genes Reporter , Humanos , Fatores de Transcrição Kruppel-Like , Camundongos , Dados de Sequência Molecular , Células Mieloides/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica , Ativação Transcricional , Leveduras/genéticaRESUMO
DLG, the mammalian homolog of the Drosophila Discs Large suppressor protein, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. In the present study, we attempted to identify partner proteins for DLG, and found that DLG interacts through its PDZ domains with the ribosome receptor. The ribosome receptor is an integral endoplasmic reticulum protein that has been suggested to be involved in secretion. Our finding raises the possibility that DLG plays a role in the regulation of secretion by interacting with the ribosome receptor.