Yost C, Torres M, Miller J R, Huang E, Kimelman D, Moon R T
Department of Biochemistry, University of Washington School of Medicine, Seattle, 98195-7370, USA.
Genes Dev. 1996 Jun 15;10(12):1443-54. doi: 10.1101/gad.10.12.1443.
The serine/threonine kinase Xgsk-3 and the intracellular protein beta-catenin are necessary for the establishment of the dorsal-ventral axis in Xenopus. Although genetic evidence from Drosophila indicates that Xgsk-3 is upstream of beta-catenin, direct interactions between these proteins have not been demonstrated. We demonstrate that phosphorylation of beta-catenin in vivo requires an in vitro amino-terminal Xgsk-3 phosphorylation site, which is conserved in the Drosophila protein armadillo. beta-catenin mutants lacking this site are more active in inducing an ectopic axis in Xenopus embryos and are more stable than wild-type beta-catenin in the presence of Xgsk-3 activity, supporting the hypothesis that Xgsk-3 is a negative regulator of beta-catenin that acts through the amino-terminal site. Inhibition of endogenous Xgsk-3 function with a dominant-negative mutant leads to an increase in the steady-state levels of ectopic beta-catenin, indicating that Xgsk-3 functions to destabilize beta-catenin and thus decrease the amount of beta-catenin available for signaling. The levels of endogenous beta-catenin in the nucleus increases in the presence of the dominant-negative Xgsk-3 mutant, suggesting that a role of Xgsk-3 is to regulate the steady-state levels of beta-catenin within specific subcellular compartments. These studies provide a basis for understanding the interaction between Xgsk-3 and beta-catenin in the establishment of the dorsal-ventral axis in early Xenopus embryos.
丝氨酸/苏氨酸激酶Xgsk - 3和细胞内蛋白β - 连环蛋白对于非洲爪蟾背腹轴的建立是必需的。尽管来自果蝇的遗传学证据表明Xgsk - 3位于β - 连环蛋白的上游，但尚未证实这些蛋白之间存在直接相互作用。我们证明，体内β - 连环蛋白的磷酸化需要一个体外氨基末端Xgsk - 3磷酸化位点，该位点在果蝇蛋白犰狳中是保守的。缺乏该位点的β - 连环蛋白突变体在诱导非洲爪蟾胚胎异位轴方面更活跃，并且在存在Xgsk - 3活性的情况下比野生型β - 连环蛋白更稳定，这支持了Xgsk - 3是β - 连环蛋白的负调节因子且通过氨基末端位点起作用的假说。用显性负性突变体抑制内源性Xgsk - 3功能会导致异位β - 连环蛋白的稳态水平增加，表明Xgsk - 3的功能是使β - 连环蛋白不稳定，从而减少可用于信号传导的β - 连环蛋白量。在存在显性负性Xgsk - 3突变体的情况下，细胞核内源性β - 连环蛋白的水平增加，这表明Xgsk - 3的作用是调节特定亚细胞区室内β - 连环蛋白的稳态水平。这些研究为理解早期非洲爪蟾胚胎背腹轴建立过程中Xgsk - 3与β - 连环蛋白之间的相互作用提供了基础。
