| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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
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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
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