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Regulatory Mechanisms of the Activity of Pax6 that Controls Cell Differentiation

控制细胞分化的Pax6活性的调控机制

基本信息

批准号：
16370093
负责人：
OKAZAKI Kenji
金额：
$ 9.73万
依托单位：
Kyoto University (2005)Biomolecular Engineering Research Institute (BERI) (2004)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

By using LE9 enhancer element, a target DNA fragment directly bound by Pax6 protein, that is present in the head surface ectoderm-specific enhancer region of the pax6 gene and is responsive to the autoreguration of the pax6 gene expression, we studied regulatory mechanisms of Pax6 protein through its phosphorylation. Our result showed that the activity of Pax6 toward LE9 is highly elevated by ERK_<1/2> or p38MAPK. We identified four phosphorylation sites in the carboxy terminal domain, among which the three sites were able to be directly phosphorylated by p38MAPK in vitro. Furthermore, a Ser-to-Ala mutation of one of these residue resulted in a suppression of the transcriptional response to the activation of the kinase pathways. Because the phosphorylation of Pax6 had little influence to the stability of Pax6 protein, the phosphorylated sites seems to operate by interacting with other factors to contribute to the transcriptional activation of LE9. Analyses of the phosphorylation pattern of in vivo Pax6 became possible by preparation of monoclonal antibodies specific for an amino acid sequence containing the phosphorylated residue. Whereas a growth signal-induced increase of the phosphorylation of nuclear Pax6 supported the significance of ERK pathway, its sustained phosphorylation suggested an involvement of another kinase. Above results demonstrated that the function of Pax6 is regulated by the phosphorylation of its C-terminal domain and that this control is dependent on the MAP kinase pathways. It Will be an important issue to clarify the mechanisms for phosphorylation signaling in the differentiation process of eyes, in which LE9 enhancer is considered to really function.

LE9增强子元件是Pax6蛋白直接结合的靶DNA片段，存在于Pax6基因头表面外胚层特异性增强子区，对Pax6基因表达的自调节有应答作用，我们利用LE9增强子元件研究了Pax6蛋白通过磷酸化调控的机制。结果表明，ERK_<1/2>或p38MAPK可显著提高Pax6对LE9的活性。我们在羧基末端区域发现了4个磷酸化位点，其中3个位点能够在体外被p38MAPK直接磷酸化。此外，这些残基之一的Ser-to-Ala突变导致激酶途径激活的转录反应受到抑制。由于Pax6的磷酸化对Pax6蛋白的稳定性影响不大，磷酸化位点似乎是通过与其他因子相互作用来促进LE9的转录激活。通过制备含有磷酸化残基的氨基酸序列特异性的单克隆抗体，可以分析体内Pax6的磷酸化模式。尽管生长信号诱导的核Pax6磷酸化的增加支持了ERK途径的重要性，但其持续磷酸化表明另一种激酶的参与。以上结果表明Pax6的功能受其c端结构域磷酸化的调控，并且这种调控依赖于MAP激酶途径。阐明LE9增强子在眼睛分化过程中的磷酸化信号机制将是一个重要的问题。