Molecular mechanism of growth and differentiation of neutrophilic granulocyte mediated by G-CSF

我们的目标是了解由粒细胞集落刺激因子（G-CSF）介导的嗜中性粒细胞生长和分化的分子机制。为此，我们研究了骨髓特异性锌指蛋白MZF-2，它在粒细胞分化中起作用。对小鼠MZF-2的突变分析表明，MZF-2蛋白在N-末端携带转录抑制区，在分子中间携带反式激活结构域（TA结构域），在C-末端区域携带DNA结合锌指结构域。我们还发现TA结构域被ERK MAP激酶磷酸化。为了确定ERK磷酸化的位点，我们构建了GST-MZF-2融合蛋白，其中假定的ERK磷酸化位点被各种丙氨酸取代。使用突变底物的体外磷酸化分析显示，ERK磷酸化TA结构域中的三个丝氨酸残基（Ser 257，Ser 275和Ser 295）。一种突变的MZF-2蛋白， ...更多信息 当在LGM-1骨髓细胞中表达时，这些丝氨酸残基的g丙氨酸取代显示出比野生型蛋白更高的反式激活活性，表明ERK MAP激酶在体内负调节MZF-2的转录活性。为了鉴定与MZF-2依赖性基因激活相关的核因子，我们利用ras募集双杂交系统从小鼠cDNA文库中筛选MZF-2结合蛋白，并分离到一个编码3035个氨基酸残基的巨大蛋白质的cDNA，该蛋白质与MZF-2具有显著的同源性。果蝇多米诺蛋白。克隆的蛋白质，命名为哺乳动物多米诺（mDomino），具有SWI/SNF型ATP /解旋酶结构域，在C-末端区域含有两个聚谷氨酰胺束。使用mDomino缺失突变体的突变分析显示，MZF-2与C-末端富含谷氨酰胺的结构域相互作用。此外，MZF-2依赖的报告激活增强时，mDomino C-末端结构域共表达。这些结果表明，由mDomino/MZF-2复合物引起的染色质重塑可能调节粒细胞发育中的骨髓特异性基因激活。少

Our goal is to understand the molecular mechanism of growth and differentiation of neutrophilic granulocytes, which are mediated by granulocyte colony-stimulating factor (G-CSF). For this purpose, we have investigated the myeloid-specific zinc-finger protein MZF-2 that plays a role in granulocyte differentiation. Mutational analysis of mouse MZF-2 demonstrated that MZF-2 protein carries a transcription-inhibitory region at the N-terminus, a transactivation domain (TA domain) in the middle of the molecule, and a DNA-binding zinc-finger domains in the C-terminal region. We also found that the TA domain was phosphorylated by ERK MAP kinase. To determine sites phosphorylated by ERK, we constructed GST-MZF-2 fusion proteins with various alanine substitution of putative ERK phosphorylation sites. An in vitro phosphorylation analysis using the mutant substrates revealed that ERK phosphorylated three serine residues (Ser257, Ser275 and Ser295) in the TA domain. A mutant MZF-2 protein containin … More g alanine substitutions of the these serine residues showed higher transactivation activity than the wild-type protein when expressed in LGM-1 myeloid cells, suggesting that ERK MAP kinase negatively regulates the transcriptional activity of MZF-2 in vivo. To identify nuclear factors involved in the MZF-2-dependent gene activation, we screened mouse CDNA library for MZF-2 binding protein by using the ras recruitment two-hybrid system, and isolated a CDNA encoding a huge (3035 amino acid residues) protein, which showed a significant homology to. Drosophila Domino protein. The cloned protein, designated as mammalian Domino (mDomino), has a SWI/SNF-type ATP /helicase structure with a domain containing two polyglutamine tracts in the C-terminal region. Mutational analyzes using deletion mutants of mDomino revealed that MZF-2 interacted with the C-terminal, glutamine-rich domain. Moreover, the MZF-2-dependent reporter activation was enhanced when the mDomino C-terminal domain was co-expressed. These results suggest that chromatin remodeling caused by mDomino/MZF-2 complex may regulate myeloid-specific gene activation in granulocyte development. Less