Regulation of GSK 3B in Xenopus

糖原合成酶激酶3β(GSK3b)是一种胞内丝氨酸激酶，存在于多种重要的信号转导通路中。这些包括阿尔茨海默病的胚胎模式形成、肿瘤抑制和神经原纤维缠结的调节。GSK3功能的一个戏剧性例子是在非洲爪蛙的轴形成过程中。通过GSK3b作用的分子的表达，如Wnt蛋白，或GSK3b本身的突变形式，会导致完整的异位背轴的形成。初步研究表明，内源GSK3b在胚胎的背侧没有腹侧丰富。这与异位表达Wnt的效果形成对比，在异位表达中，GSK3b的比活性降低，但丰度没有变化。本研究旨在分析非洲爪哇的GSK3b调控，比较其内源调控机制和经典的Wnt调控机制，以更好地了解这一重要蛋白质的调控模式。背侧GSK3b的内源性耗竭只能通过减少合成或稳定或通过蛋白质移位远离背侧而发生。最近证实的GSK3结合蛋白的作用暗示了死亡-翻译机制。因此，将通过测量表位标记的GSK3b在背侧和腹侧的半衰期来测试差异稳定性。预先存在的GSK3b蛋白的移位将通过可视化外源、标记的GSK3b与控制蛋白的定位或分散来监测。GSK3b蛋白上内源性和Wnt依赖性调节所需的位点尚不清楚。它们将被绘制成地图，利用非洲爪哇系统的优势来实现这一目的。背部特异的和依赖于Wnt的蛋白质修饰将通过胰多肽的质谱学得到粗略的映射。精细定位和功能关联将通过在体内进行定点突变和表达来完成。突变体将在候选的修饰位点上进行，并在体内测试有和没有表达Wnts的特定活性。初步的生化研究显示，在体内与GSK3b相关的蛋白质与已知的GSK3b结合蛋白并不对应。GSK3b相关蛋白显然很重要，因此为了鉴定这些新蛋白(并确定体内已知蛋白的结合与否)，将对与GSK3b免疫共沉淀的蛋白进行直接生化分析。将使用凝胶电泳法、蛋白质印迹法和显微测序法。

Glycogen synthase kinase 3 beta (GSK3b) is an intracellular serinethreonine kinase found at the heart of a number of important signalingpathways. These include embryonic pattern formation, tumor suppression andregulation of neurofibrillary tangles in Alzheimer's disease. A dramaticexample of GSK3 function is in axis formation in the frog Xenopus. Expressionof molecules that act via GSK3b, such as Wnt proteins, or of mutant forms ofGSK3b itself, result in formation of complete ectopic dorsal axes. Thissuggests that reduction in GSK3b activity is sufficient to cause "dorsalness".Preliminary studies indicate that endogenous GSK3b is less abundant on thedorsal side of the embryo than on the ventral side. This contrasts with theeffects of ectopic Wnt expression in which specific activity of GSK3b isreduced but abundance is not. This proposal aims to analyze GSK3b regulationin Xenopus, comparing endogenous mechanisms with classical Wnt regulation, tobetter understand the modes of regulation of this important protein. Endogenous dorsal depletion of GSK3b ccan only be by reduction ofsynthesis or stability or by translocation of protein away from the dorsalside. The recently demonstrated role for a GSK3-binding protein suggests apost-translational mechanism. Therefore, differential stability will be testedby measuring the half-life of epitope-tagged GSK3b in dorsal versus ventrallysates. Translocation of pre-existing GSK3b protein will be monitored byvisualizing localization or dispersion of exogenous, tagged GSK3b versuscontrol proteins. Sites on the GSK3b protein required for endogenous and Wnt-dependentregulation are unknown. They will be mapped, exploiting the advantages of theXenopus system for this purpose. Dorsal-specific and Wnt-dependent proteinmodifications will be coarsely mapped by mass spectrometry of trypticpeptides. Fine mapping and functional correlation will be done bysite-directed mutagenesis and expression in vivo. Mutants will be made atcandidate modification sites and tested in vivo for specific activity with andwithout expression of Wnts. Preliminary biochemical studies reveal proteins associated with GSK3b invivo that do not correspond to known GSK3b-binding proteins. GSK3b-associatedproteins are clearly important and so to identify these novel proteins (and todetermine the association or absence of the known proteins in vivo), directbiochemical analysis of proteins co-immunoprecipitated with GSK3b will becarried out. Gel electrophoresis, Western blotting and microsequencing will beused.