Sater Amy K, El-Hodiri Heithem M, Goswami Mousumi, Alexander Tara B, Al-Sheikh Oday, Etkin Laurence D, Akif Uzman J
Division of Molecular and Cell Biology, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA.
Differentiation. 2003 Sep;71(7):434-44. doi: 10.1046/j.1432-0436.2003.7107006.x.
We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.
我们之前已经表明，丝裂原活化蛋白（MAP）激酶活性对于非洲爪蟾的神经特化是必需的。在哺乳动物细胞中，BMP - 4效应因子Smad1在MAP激酶位点被磷酸化而受到抑制（克雷茨施马尔等人，1997年）。为了验证MAP激酶在非洲爪蟾早期发育过程中抑制BMP - 4/Smad1信号通路这一假说，我们构建了一个缺失MAP激酶磷酸化位点的Smad1突变体（M4A - Smad1），并比较了野生型（WT）- Smad1和M4A - Smad1对非洲爪蟾胚胎轴向模式和神经特化的影响。尽管WT - Smad1或M4A - Smad1的过表达都会产生腹侧化的胚胎，但在每个mRNA浓度下，M4A - Smad1的腹侧化作用都比WT - Smad1更强。有趣的是，在腹侧卵裂球中过表达任何一种形式的Smad1都会破坏后部模式和形态发生；同样，M4A - Smad1的表达比等量的WT - Smad1产生的缺陷更严重。M4A - Smad1的外胚层表达在体内破坏了前神经基因otx2的表达，并抑制了中胚层 - 外胚层重组体中对内源信号的神经特化。相比之下，相同水平的WT - Smad1过表达对神经特化或otx2表达几乎没有影响。对WT - Smad1或M4A - Smad1过表达后蛋白质水平的比较表明，WT - Smad1可能比M4A - Smad1稍微更稳定一些；因此，稳定性的差异不能解释M4A - Smad1有效性的增加。我们的结果表明，破坏MAPK磷酸化位点的突变共同作为Smad1的功能获得性突变起作用，并且Smad1的抑制性磷酸化可能是非洲爪蟾发育过程中调节BMP - 4/Smad1信号的一个重要机制。
