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

我们的目标是了解粒细胞集落刺激因子(G-CSF)介导的中性粒细胞生长和分化的分子机制。为此，我们研究了髓系特异的锌指蛋白MZF-2，它在粒细胞分化中发挥作用。对小鼠MZF-2的突变分析表明，MZF-2蛋白在N端有一个转录抑制区，在分子中间有一个反式激活域(TA区)，在C端区有一个与DNA结合的锌指域。我们还发现，TA结构域被ERK MAP激酶磷酸化。为了确定ERK的磷酸化位点，我们构建了GST-MZF-2融合蛋白，并用不同的丙氨酸取代了可能的ERK磷酸化位点。利用突变底物进行的体外磷酸化分析表明，ERK能磷酸化TA结构域的三个丝氨酸残基(Ser257、Ser275和Ser295)。一个含有…的突变型MZF-2蛋白在LGM-1髓系细胞中表达时，这些丝氨酸残基的多个丙氨酸取代比野生型蛋白显示出更高的反式激活活性，这表明ERK MAP激酶在体内负调控MZF-2的转录活性。为了确定参与MZF-2依赖基因激活的核因素，我们利用ras募集双杂交系统筛选小鼠MZF-2结合蛋白的CDNA文库，并分离到编码一个巨大的(3035个氨基酸残基)蛋白的CDNA，该蛋白与MZF-2结合蛋白具有显著的同源性。果蝇多米诺蛋白。克隆的蛋白命名为哺乳动物Domino(MDomino)，具有SWI/SNF型ATP/解旋酶结构，C-末端含有两个聚谷氨酰胺区。利用mDomino的缺失突变体进行突变分析，发现MZF-2与C末端的富含谷氨酰胺的结构域相互作用。此外，当mDomino C末端结构域共表达时，依赖于MZF-2的报告基因的激活被增强。这些结果提示，mDomino/MZF-2复合体引起的染色质重塑可能调节粒细胞发育过程中髓系特异性基因的激活。较少

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