Joint study on regulation of cell profferation by cytokines

本合作项目主要研究生长因子介导的哺乳动物细胞增殖和分化过程中DNA复制、基因表达和其他各种细胞反应的诱导机制，取得了以下研究成果：1）分离得到了一个可能的裂殖酵母Hsk 1激酶调控亚基。在几个编码Hsk 1相互作用分子的粟酒裂殖酵母克隆中，Him 1（Hsk 1与m__-分子1相互作用）与Hsk 1形成复合物，刺激其激酶活性，并被Hsk 1特异性磷酸化。him 1 ^+对裂殖酵母细胞的生存力至关重要，其转录受细胞周期调控，在G1/S期边界达到最大值，尽管它不受cdc 10 ^+的控制。2）我们还分离出一种新的人类cDNA H37，编码一种与huCdc 7相互作用的分子。当在COS 7细胞或昆虫细胞中与huCdc 7共表达时，H37和huCdc 7形成复合物，复合物的形成激活huCdc激酶， 关于我们 导致huCdc和H37的广泛磷酸化。这表明H37是huCdc激酶的调节亚基。显微注射抗H37蛋白的抗体可抑制原代成纤维细胞的DNA合成，表明H37-huCdc 7的功能是S期启动所必需的。3）利用竞争性PCR方法定位了人5 q区GM-CSF/IL-3位点的复制起点。我们的初步结果表明，在基因间区的可诱导DNaseI超敏感位点附近存在一个活跃的复制起点。4）我们已经定位了GM-CSF受体β链胞质区的酪氨酸残基，其磷酸化是DNA复制、诱导早期反应基因、防止凋亡、等等。5）我们已经表明Jak 2单独的二聚化可以诱导c-myc的诱导，STAT 5激活和非依赖于腺嘌呤的增殖。6）我们已经分离出了新的克隆，这些克隆是由突变型GM-1的细胞因子刺激诱导的。6）向人GM-CSF受体转基因小鼠施用人GM-CSF刺激小鼠中的骨髓生成以及红细胞生成和巨核细胞生成。它还影响了转基因小鼠中自然杀伤细胞的发育。少

In this collaborative project, we are studying on the mechanisms of induction of DNA replication, gene expression and other various cellular responses in growth factor-mediated proliferation and differentiation of mammalian cells.This year, we have obtained the following results.1) We have isolated a putative regulatory subunit for fission yeast Hsk1 kinase. Among several S.pombe clones encoding Hsk1-interacting molecules, Him1 (H__-sk1 i__-nteracting m__-olecule 1) forms a complex with Hsk1, stimulates its kinase activity and is specifically phosphorylated by Hsk1. him1^+ is essential for viability of fission yeast cells and its transcription is cell cycle-regulated, reaching maximum at the G1/S boundary, although it is not under the control of cdc10^+.2) We have also isolated a novel human cDNA,H37, encoding a huCdc7-interacting molecule. H37 and huCdc7, when coexpressed with huCdc7 in COS7 cells or in insect cells, forms a complex and the complex formation activates huCdc kinase, re … More sulting in extensive phosphorylation of both huCdc and H37. This indicates that H37 is a regulatory subunit for huCdc kinase. Microinjection of antibodies against H37 protein inhibited DNA synthesis in primary fibroblast cells, indicating that the function of H37-huCdc7 is required for S phase initiation.3) We have mapped replication origins in the GM-CSF/IL-3 locus of the human 5q region by using the competitive PCR method. Our preliminary results indicate the presence of an active replication origin near the inducible DNaseI-hypersensitive site within the intergenic region.4) We have mapped tyrosine residues of the cytoplasmic region of beta-chain of GM-CSF receptore whose phosphorylation is required for DNA replication, induction of early response genes, prevention of apoptosis, and so on.5) We have shown that dimerization of Jak2 alone can induce induction of c-myc, STAT5 activation and cytokine-independent proliferation.6) We have isolated novel clones whish are induced by cytokine stimulation of mutant GM-CSF receptors capable specifically of promoting proliferaton.6) Administration of human GM-CSF to human GM-CSF receptor transgenic mice stimulated myelopoiesis as well as erythropoiesis and megakaryopoiesis in mice. it also affected the development of natural killer cells in the transgenic mice. Less