Agnes Scott College, Department of Chemistry, 141 E. College Ave., Decatur, GA 30030, USA.
Agnes Scott College, Department of Chemistry, 141 E. College Ave., Decatur, GA 30030, USA.
Cells Dev. 2022 Mar;169:203757. doi: 10.1016/j.cdev.2021.203757. Epub 2021 Nov 24.
A common bridge between a linear cytoplasmic signal and broad nuclear regulation is the family of MAP kinases which can translocate to the nucleus upon activation by the cytoplasmic signal. One pathway which functions to activate the ERK family of MAP kinases is the Ras signaling pathway which functions at multiple times and locations during the development of Caenorhabditis elegans including the development of the excretory cell, germ cells, male tail, and vulva. It has been most extensively characterized during the development of the vulva which is formed from the vulval precursor cells (VPCs), a set of six equivalent, epithelial cells designated P3.p - P8.p. Although LIN-1 appears to be a primary target of ERK MAP kinase during vulval development, it is likely that other developmentally important molecules are also regulated by ERK-mediated phosphorylation. The identification of physiological substrates of MAP kinases has been aided by the identification of docking site domains in substrate proteins that contribute to high-affinity interactions with kinases. Our laboratory has identified the C. elegans protein, T08D10.1/NFYA-1, as a potential ERK MAP kinase substrate in this manner, and we have initiated a characterization of its role during Ras-mediated development. T08D10.1 possesses significant homology to the CCAAT-box DNA-binding domain of the vertebrate nuclear transcription factor-Y, alpha (NF-YA) family of proteins. NF-Y proteins act as part of a complex to regulate the transcription of a large number of genes, in particular, genes that function in the G1/S cell cycle transition. T08D10.1/NFYA-1 is predicted to code for a protein containing multiple potential phosphorylation sites for ERK MAP kinase and a D-domain docking site. We demonstrate through biochemical analysis of purified NFYA-1 protein that it can act in vitro as a high affinity substrate for activated ERK MAP kinase. Growth factor activation of the Ras pathway in a tissue culture system has negligible effect on the protein's transactivation potential, however, the DNA-binding activity of the protein is reduced after treatment with activated ERK-MAP kinase. We demonstrate through mutant analysis that nfya-1 acts to inhibit vulval development and functions downstream or in parallel to let-60/ras. Both the NF-Y complex and the Ras signaling pathway play a fundamental role in cell proliferation and oncogenesis and the connection between the two is an important insight into the mechanisms of cell fate specification and cellular response.
一种将细胞质信号与广泛的核调节联系起来的常见桥梁是 MAP 激酶家族，它可以在细胞质信号激活后转位到细胞核。激活 ERK 家族 MAP 激酶的途径之一是 Ras 信号途径，它在秀丽隐杆线虫的发育过程中多次和多个位置起作用，包括排泄细胞、生殖细胞、雄性尾巴和阴门的发育。它在阴门的发育过程中得到了最广泛的描述，阴门是由阴门前体细胞（VPCs）形成的，一组六个相等的、上皮细胞指定为 P3.p - P8.p。尽管 LIN-1 似乎是 ERK MAP 激酶在阴门发育过程中的主要靶标，但其他对发育很重要的分子也可能受到 ERK 介导的磷酸化调节。通过鉴定底物蛋白中的对接位点域，促进与激酶的高亲和力相互作用，从而有助于 MAP 激酶的生理底物的鉴定。我们的实验室以这种方式鉴定了秀丽隐杆线虫蛋白 T08D10.1/NFYA-1 作为潜在的 ERK MAP 激酶底物，并且我们已经开始表征其在 Ras 介导的发育过程中的作用。T08D10.1 与脊椎动物核转录因子-Y，alpha（NF-YA）家族的蛋白质的 CCAAT 框 DNA 结合域具有显著同源性。NF-Y 蛋白作为复合物的一部分起作用，调节大量基因的转录，特别是在 G1/S 细胞周期过渡中起作用的基因。T08D10.1/NFYA-1 预计编码一种含有多个潜在 ERK MAP 激酶磷酸化位点和 D 结构域对接位点的蛋白质。我们通过对纯化的 NFYA-1 蛋白的生化分析证明，它可以在体外作为激活的 ERK MAP 激酶的高亲和力底物起作用。在组织培养系统中，生长因子激活 Ras 途径对蛋白质的反式激活潜力几乎没有影响，然而，在用激活的 ERK-MAP 激酶处理后，蛋白质的 DNA 结合活性降低。我们通过突变分析证明 nfya-1 抑制阴门发育，并且作用于 let-60/ras 的下游或平行。NF-Y 复合物和 Ras 信号途径在细胞增殖和致癌作用中起着基本作用，两者之间的联系是深入了解细胞命运特化和细胞反应机制的重要见解。
