The Role of Zeste White 3 Kinase in Wg Signaling

无翅（Wingless，Wg）是Wnt家族的一员，是一种保守的分泌型生长因子，对无脊椎动物和脊椎动物的正常生长和分化都是必需的。 Wg和Wnt信号转导有一条共同的信号通路。 信号通路的一个关键组分是Zeste白色3（Zw 3），其是糖原合成酶激酶3（GSK-3）的果蝇同系物，GSK-3是丝氨酸苏氨酸蛋白激酶，其对Wg起拮抗作用。 已经表明，Wg信号传导是由Zw 3激酶的失活介导的。 本提案的长期目标是进一步了解Zw 3的体内活性，以及Wg信号的调节机制。 齐格弗里德博士将利用果蝇的强大技术，采取分子生物学、生物化学和遗传学的综合方法。 她的初步研究表明，Zw 3的过度表达导致内源性Wg信号通路的阻断，这表明高水平的Zw 3可以饱和Wg调节的正常机制，但这种机制尚不清楚。 然而，大鼠GSK-3b的表达可以拯救zw 3突变胚胎的事实表明Zw 3和GSK-3b之间存在共同的机制。 为了确定潜在调控的共同区域，她确定了Zw 3、脊椎动物GSK-3b和脊椎动物GSK-3a之间的系统发育关系，GSK-3a是GSK-3b的一种密切相关的蛋白质，尽管如此，它不能拯救zw 3突变胚胎。 这些分析表明，Zw 3的氨基末端结构域和激酶结构域中的7个残基在GSK-3b和Zw 3之间是保守的，但与GSK-3a不同，可能对体内正常活性很重要。 她建议通过定点突变和突变蛋白的表达来验证这一假设，以确定催化活性和调节的关键残基。 这些Zw 3的突变形式将在转基因果蝇中表达，突变蛋白的活性将通过拯救Zw 3突变胚胎和保留Wg调节的能力来测定。 她将确定突变蛋白质的激酶活性，测试它们是否可以调节体内的Armadillo蛋白水平，并确定它们的亚细胞定位。 最后，她将设计遗传筛选来鉴定Zw 3过表达表型的第二位点抑制子或增强子，这将鉴定与Zw 3相互作用的蛋白质，包括Wg信号传导的新组分。

Wingless (Wg) is a member of the Wnt family of conserved secretedgrowth factors which are essential for the normal growth anddifferentiation of both invertebrates and vertebrates. A common signalingpathway has been identified for both Wg and Wnt signaling. One keycomponent of the signaling pathway is Zeste White 3 (Zw3), the Drosophilahomolog of Glycogen Synthase Kinase 3 (GSK-3), a serine threonine proteinkinase, which acts antagonistically to Wg. It has been suggested that Wgsignaling is mediated by inactivation of Zw3 kinase. The long term goal ofthis proposal is to further our understanding of the activity of Zw3 invivo, and the mechanism of regulation by Wg signaling. Dr. Siegfried will takeadvantage of the powerful techniques available in Drosophila, and take acombined approach of molecular biology, biochemistry and genetics. Her preliminary studies have demonstrated that over-expression ofZw3 leads to a block in the endogenous Wg signaling pathway, suggestingthat high levels of Zw3 can saturate the normal mechanism of Wg regulation.What this mechanism is remains unclear. However, the fact that expressionof rat GSK-3b can rescue zw3 mutant embryos indicates that this mechanismis shared between Zw3 and GSK-3b. In an effort to identify shared regionsof potential regulation, she has determined the phylogenetic relationshipbetween Zw3, vertebrate GSK-3b and vertebrate GSK-3a, a closely relatedprotein to GSK-3b which, nonetheless, is unable to rescue zw3 mutantembryos. These analyses have suggested that the amino terminal domain ofZw3 and seven residues within the kinase domain which are conserved betweenGSK-3b and Zw3, but differ from GSK-3a, may be important for normalactivity in vivo. She proposea to test this hypothesis by site directed mutagenesis andexpression of mutant proteins to determine critical residues for catalyticactivity and regulation. These mutant forms of Zw3 will be expressed intransgenic flies and activity of mutant proteins will be assayed by theability to rescue zw3 mutant embryos and for retention of Wg regulation.The mutant forms of Zw3 will be also tested in cultured imaginal disc cellswhich are Wg sensitive and recapitulate the modulation of Armadillo seen invivo. She will determine the kinase activity of mutant proteins, test ifthey can modulate Armadillo protein levels in vivo, and determine theirsub-cellular localization. Finally, she will design genetic screens toidentify second site suppressors or enhancers of a Zw3 over-expressionphenotype which should identify proteins which interact with Zw3, includingnovel components of Wg signaling.