Baek Sungmin, Seeling Joni M
Department of Biology, City University of New York, Queens College, 65-30 Kissena Blvd,, Flushing, NY 11367, USA.
BMC Dev Biol. 2007 Dec 19;7:139. doi: 10.1186/1471-213X-7-139.
Wnt signaling is a key regulator of development and tumorigenesis. Protein phosphatase 2A (PP2A), which consists of a catalytic C, a structural A, and a regulatory B subunit, plays diverse roles in Wnt signaling through its B56 subunits. B56 is a multigene family encoding for proteins with a conserved core domain and divergent amino- and carboxy-termini. Ectopic B56alpha and B56gamma reduce beta-catenin abundance and B56alpha reduces Wnt-dependent transcription, suggesting that B56alpha and B56gamma inhibit Wnt signaling. In contrast, B56epsilon is required for Wnt signaling. Knowledge of where and when B56 subunits are expressed during Xenopus development will aid in our understanding of their roles in Wnt signaling.
We have undertaken expression analyses of B56alpha and B56gamma in Xenopus laevis. We cloned Xenopus B56alpha; it is 88% identical to human B56alpha. Xenopus B56gamma is 94% identical with human B56gamma, however, a novel evolutionarily conserved mixed-isoform transcript was identified that contains a B56delta-like amino-terminal domain and a B56gamma core domain. The B56delta-like variable domain exon is located upstream of the B56gamma variable domain exon at the human B56gamma locus, suggesting that the mixed-isoform transcript is due to alternative splicing. B56gamma transcripts with different 3' ends were identified that lack or possess a 35 base pair sequence, resulting in either a transcript similar to human B56gamma1, or an uncharacterized evolutionarily conserved sequence. Real time RT-PCR analyses revealed that B56alpha is expressed at moderate levels before the midblastula transition (MBT), at reduced levels during gastrulation and neurulation, and at high levels during organogenesis, while B56gamma is expressed at low levels until organogenesis. B56alpha is enriched in the ventral hemisphere pre-MBT, while B56gamma is ventrally enriched post-MBT. Aalpha, Abeta, Calpha and Cbeta are expressed in early Xenopus development, suggesting the presence of a functional heterotrimer.
Our data suggest that B56 functional diversity is achieved in part through the synthesis of a novel mixed-isoform B56delta/gamma transcript. Our data also suggest that B56alpha functions pre-MBT, inhibiting Wnt signaling on the ventral side of the embryo, and again during organogenesis, while B56gamma functions primarily post-MBT.
Wnt信号通路是发育和肿瘤发生的关键调节因子。蛋白磷酸酶2A（PP2A）由催化亚基C、结构亚基A和调节亚基B组成，通过其B56亚基在Wnt信号通路中发挥多种作用。B56是一个多基因家族，编码具有保守核心结构域以及不同的氨基末端和羧基末端的蛋白质。异位表达的B56α和B56γ可降低β-连环蛋白丰度，B56α可降低Wnt依赖的转录，这表明B56α和B56γ抑制Wnt信号通路。相比之下，B56ε是Wnt信号通路所必需的。了解非洲爪蟾发育过程中B56亚基的表达位置和时间，将有助于我们理解它们在Wnt信号通路中的作用。
我们对非洲爪蟾中的B56α和B56γ进行了表达分析。我们克隆了非洲爪蟾的B56α；它与人类B56α的同源性为88%。非洲爪蟾的B56γ与人类B56γ的同源性为94%，然而，我们鉴定出一种新的进化上保守的混合异构体转录本，其包含一个B56δ样氨基末端结构域和一个B56γ核心结构域。在人类B56γ基因座上，B56δ样可变结构域外显子位于B56γ可变结构域外显子的上游，这表明混合异构体转录本是由可变剪接产生的。我们鉴定出具有不同3'末端的B56γ转录本，这些转录本缺失或拥有一个35个碱基对的序列，从而产生一个类似于人类B56γ1的转录本，或者一个未鉴定的进化上保守的序列。实时RT-PCR分析显示，B56α在囊胚中期转变（MBT）之前中等水平表达，在原肠胚形成和神经胚形成期间表达水平降低，在器官发生期间高水平表达，而B56γ在器官发生之前低水平表达。B56α在MBT前在腹侧半球富集，而B56γ在MBT后在腹侧富集。Aα、Aβ、Cα和Cβ在非洲爪蟾早期发育中表达，表明存在功能性异源三聚体。
我们的数据表明，B56的功能多样性部分是通过合成一种新的混合异构体B56δ/γ转录本实现的。我们的数据还表明，B56α在MBT前发挥作用，抑制胚胎腹侧的Wnt信号通路，在器官发生期间再次发挥作用，而B56γ主要在MBT后发挥作用。
