Regulatory Mechanisms of the Activity of Pax6 that Controls Cell Differentiation

利用与Pax6蛋白直接结合的靶DNA片段LE9增强子元件，我们研究了Pax6蛋白通过磷酸化对Pax6蛋白的调控机制。我们的结果表明，ERK_1/2或p38MAPK可显著提高Pax6对LE9的活性。我们在其羧基末端结构域确定了四个磷酸化位点，其中三个位点在体外能够被p38MAPK直接磷酸化。此外，其中一个残基的Ser-to-Ala突变导致了对激酶途径激活的转录反应的抑制。因为Pax6的磷酸化对Pax6蛋白的稳定性影响很小，所以磷酸化的位点似乎是通过与其他因素相互作用而作用于LE9的转录激活的。通过制备针对含有磷酸化残基的氨基酸序列的特异性单抗，可以对体内Pax6的磷酸化模式进行分析。虽然生长信号诱导的核Pax6的磷酸化增加支持ERK通路的意义，但其持续的磷酸化表明另一种激酶参与其中。以上结果表明，Pax6的功能受其C-末端结构域的磷酸化调控，这一调控依赖于MAP激酶通路。在LE9增强子被认为真正发挥作用的眼睛分化过程中，阐明磷酸化信号转导的机制将是一个重要的问题。

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.