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

G-CSF介导的中性粒细胞生长和分化的分子机制

基本信息

批准号：
10680669
负责人：
FUKUNAGA Rikiro
金额：
$ 2.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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, 50 amino-acid residues) in the middle of the molecule, and a DNA-binding zinc-finger domains in the C-terminal region. Overexpression of a mutant protein containing only the TA domain inhibited the MZF-2-mediated transcriptional activation, suggesting that the TA domain recruits a myeloid-specific transcriptional coactivator. G-CSF is known to activate the MAP kinase pathway. To elucidate the role of the MAP kinase pathway in G-CSF signalling, we analysed in vivo function of the protein kinase MNK1, one of the MAP … More kinase targets. Expression of a constitutively active MNK1 mutant or a dominant-negative mutant resulted in constitutive phosphorylation or constitutive dephosphorylation of eukaryotic translation initiation factor 4E (eIF-4E) at Ser209, respectively, indicating that eIF-4E is a physiological target for MNK1. Treatment of cells with various growth or stress stimuli rapidly induces phosphorylation of eIF-4E through MNK1, suggesting a physiological function of MNK1 in the regulation of protein synthesis. Cell growth is regulated by a family of cell-cycle regulating protein kinases called Cdks. By a phosphorylation screen for cyclin E/Cdk2 substrates, we have identified a novel cyclin E/Cdk2 substrate, PRC1, which has sequence homology to the budding yeast protein Ase1p. PRC1 protein localized to the cell midbody during cytokinesis. Microinjection of anti-PRC1 antibodies into HeLa cells blocked cellular cleavage, but not nuclear division, indicating a functional role for PRC1 in the process of cytokinesis. Less

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结构域(TA结构域，50个氨基酸残基)，在C末端有一个与DNA结合的锌指结构域。只含有TA结构域的突变蛋白的过表达抑制了MZF-2介导的转录激活，表明TA结构域招募了髓系特异的转录辅助激活因子。已知G-CSF可激活MAP激酶通路。为了阐明MAP激酶通路在G-csf信号通路中的作用，我们分析了MAP…之一的蛋白激酶MNK1在体内的功能更多的激酶靶点。真核细胞翻译起始因子4E(eIF-4E)在Ser209位发生结构性磷酸化或结构性去磷酸化，表明eIF-4E是MNK1的一个生理靶点。用不同的生长或应激刺激处理细胞后，迅速通过MNK1诱导eIF-4E的磷酸化，提示MNK1在调节蛋白质合成方面具有生理功能。细胞生长受细胞周期调节蛋白激酶家族的调控。通过对Cyclin E/CDK2底物的磷酸化筛选，我们鉴定了一种新的Cyclin E/CDK2底物Prc1，它与发芽酵母蛋白Ase1p具有序列同源性。在胞质分裂过程中，Prc1蛋白定位于细胞中体。向HeLa细胞中微量注射抗Prc1抗体可以阻止细胞分裂，但不能阻止核分裂，这表明Prc1在细胞质分裂过程中发挥了作用。较少