Regulation of GSK3beta in Xenopus

0131755格林发育生物学的一个中心问题是脊椎动物胚胎中的身体轴是如何建立的。以前在非洲爪蟾中的研究表明，蛋白激酶GSK 3b在建立背腹不对称中起着关键作用。然而，据信GSK 3b比活性的不对称性，而不是GSK 3b蛋白丰度本身的不对称性，为胚胎提供了空间取向。 在初步研究中，绿色博士和同事们已经表明，背腹侧调节GSK 3 b涉及GSK 3 b的实际消耗，可能是通过蛋白质的差异稳定性。 本文所提出的研究建立在这一初步观察的基础上。 广泛的目标是确定建立背腹轴的GSK 3b调节机制。第一个目的是表征GSK 3b蛋白缺失的机制。将在早期胚胎中测量纯化的重组GSK 3b的稳定性。将检测泛素化/蛋白酶体抑制剂预防GSK 3b耗竭的能力。第二个目的是阐明Wnt信号转导在内源性背腹调控GSK 3b中的作用。将测量耗尽Wnt受体的胚胎中的GSK 3b丰度，并将母体Wnt与合子Wnt（已知仅降低GSK 3b比活性）比较其耗尽GSK 3b的能力。为了确定GSK 3b调节从消耗到比活性降低的可能的时间转变，将比较早期和晚期未受干扰的胚胎。第三个目的是研究GSK 3b定位在其调节中的作用。GSK 3b蛋白缺失紧密定位于细胞皮质，并且已显示参与Wnt转导蛋白Axin向Wnt跨膜受体LRP 5的Wnt依赖性募集。 将通过检查正常和操作胚胎皮质膜组分中的GSK 3b丰度和活性来检验膜相关GSK 3b形成高度Wnt和GBP-sensitive池的假设。这些项目将共同提供一个更精确的GSK 3b在非洲爪蟾早期发育调控的分子图像。

0131755GreenA central question in developmental biology is how the body axes are established in the vertebrate embryo. Previous studies in Xenopus have shown that the protein kinase GSK3b plays a pivotal role in establishing dorsoventral asymmetry. However, it was believed that asymmetry in GSK3b specific activity, rather than an asymmetry of GSK3b protein abundance per se, provided spatial orientation for the embryo. In preliminary studies, Dr. Green and colleagues have shown that dorsoventral regulation of GSK3b involves an actual depletion of GSK3b, perhaps through differential stability of the protein. The research proposed here builds upon this preliminary observation. The broad goal is to define the GSK3b regulatory mechanisms that establish the dorsoventral axis. The first aim is to characterize the mechanism of GSK3b protein depletion. The stability of purified recombinant GSK3b will be measured in early embryos. Ubiquitination/proteasome inhibitors will be tested for their ability to prevent GSK3b depletion. The second aim is to clarify the role of Wnt signaling in endogenous dorsoventral regulation of GSK3b. GSK3b abundance in embryos depleted for Wnt receptors will be measured and maternal Wnts will be compared with zygotic Wnts (that are known to reduce GSK3b specific activity only) for their ability to deplete GSK3b. To identify a possible temporal shift in GSK3b regulation from depletion to specific activity reduction, earlier and later stage unperturbed embryos will be compared. The third aim is to examine the role of GSK3b localization in its regulation. GSK3b protein depletion is tightly localized to the cell cortex, and has been shown to be involved in Wnt-dependent recruitment of the Wnt transduction protein Axin to the Wnt transmembrane receptor LRP5. The hypothesis that membrane-associated GSK3b forms a highly Wnt- and GBP-sensitive pool will be tested by examining GSK3b abundance and activity in cortical membrane fractions of normal and manipulated embryos. These projects will together provide a more precise molecular picture of GSK3b regulation in early Xenopus development.