Spatio-temporal regulation of ERK signaling by phosphatases in the female germline

项目摘要 RAS-细胞外调节激酶（ERK）通路控制女性生殖过程中广泛的细胞过程。 从蠕虫到哺乳动物的生殖细胞发育。ERK活性的任何改变都会破坏生殖细胞的发育 并导致生育力低下或不育。因此，ERK活性的调节在女性生殖细胞发育过程中至关重要。 发展在整个进化过程中，ERK的激活是由TEY上可逆的双重磷酸化控制的。 通过ERK激酶（MEK）在其活性位点中的基序和通过DUSPs（双特异性磷酸酶）的去磷酸化。 MEK（MAPK激酶）和DUSPs（双重磷酸化）对ERK的磷酸化和去磷酸化的平衡 特异性磷酸酶）分别紧密调节和调谐ERK活性的持续时间和幅度 在所有系统中的空间和时间上，这在秀丽隐杆线虫卵原生殖系中表现得最好。 C.线虫卵原生殖系，像大多数复杂的生物系统一样， ERK（MPK-1在C. elegans）活性。活性ERK MPK-1，如使用检测 在TEY基序的苏氨酸和酪氨酸处的双重磷酸化ERK，在 减数分裂I;去磷酸化，因此在晚粗线期和早双线期区域未检测到， 生殖系，对应于减数分裂轴的解体以触发双线期的染色体重塑 卵母细胞发育阶段。ERK MPK-1磷酸化在停止的终动期卵母细胞中再次可见。 这种模式的任何改变（通过mpk中的功能丧失和功能获得突变种系可视化）， 1 ERK途径）导致生育力低下或急性不育。因为总ERK蛋白在整个细胞中表达， 在卵源性生殖系中，这种引人注目的ERK激活的时空模式表明了局部激活， ERK MPK-1通过MEK和DUSPs失活。二十年来，DUSP 6/7同源物LIP-1（横向 信号诱导的磷酸酶-1）被认为调节卵子生殖系中的ERK MPK-1活性。然而，在这方面， 与此同时，包括我们在内的几个实验室观察到的表型和结果并不一致， 在生殖细胞发育过程中，LIP-1作为ERK MPK-1的DUSP发挥作用。通过仔细 重新评估LIP-1在卵子发生和ERK MPK-1调节中的作用，我们发现事实上LIP-1确实 在卵子生殖系中不起ERK DUSP的作用。沿着这条线，Tischer和Schuh，2016年，已经表明 哺乳动物DUSP 6/7同源物不调节ERK，而是靶向蛋白激酶C， 小鼠卵母细胞减数分裂成熟。总之，这些结果表明，LIP-1或DUSP 6/7不 在卵子发生过程中调节ERK MPK-1，这给我们在卵子发生的分子身份方面的知识留下了很大的空白。 控制女性生育能力的信号调节器。本提案的目标是鉴定磷酸酶， 制备试剂以检测其在蠕虫雌性生殖细胞发育过程中的表达和功能。 鉴定磷酸酶或磷酸酶的组合，所述磷酸酶或磷酸酶的组合起调节ERK活性和ERK-1的作用。 女性生育期间的依赖性事件填补了我们知识的重大空白，并为生殖医学提供了信息。

PROJECT SUMMARY The RAS-Extracellular regulated kinase (ERK) pathway controls wide-ranging cellular processes during female germ cell development from worms to mammals. Any alteration to ERK activity disrupts germ cell development and leads to sub-fertility or sterility. Thus, regulation of ERK activity is critical during female germ cell development. Across evolution, ERK activation is controlled by a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and, dephosphorylation by DUSPs (Dual Specificity Phosphatase). A balance of phosphorylation and dephosphorylation of ERK by MEK (MAPK kinase) and DUSPs (the Dual Specificity Phosphatase), respectively, tightly regulates and tunes the duration and magnitude of ERK activity in space and time across all systems and this is best visualized in Caenorhabditis elegans oogenic germline. C. elegans oogenic germline, like most complex biological systems, displays a controlled spatiotemporal pattern of ERK (MPK-1 in C. elegans) activity. Active ERK MPK-1, as assayed using an antibody that detects dual phosphorylated ERK at Threonine and Tyrosine of the TEY motif, is visualized in mid-pachytene stage of meiosis I; dephosphorylated and thus undetected in the late-pachytene and early-diplotene region of the germline, corresponding with disassembly of the meiotic axis to trigger chromosome remodeling in diplotene stage of oocyte development. ERK MPK-1 phosphorylation is again visible in the arrested diakinetic oocytes. Any alternation to this pattern (visualized via loss-of-function and gain-of-function mutant germlines in the mpk- 1 erk pathway) results in sub-fertility or acute sterility. Because total ERK protein is expressed throughout the oogenic germline, this striking spatiotemporal pattern of ERK activation suggests localized activation and inactivation of ERK MPK-1 through MEK and DUSPs. For two decades, the DUSP6/7 homolog LIP-1 (lateral signal induced phosphatase -1) was thought to regulate ERK MPK-1 activity in the oogenic germline. However, during the same time, several labs, including ours, observed phenotypes and results that were inconsistent with a role for LIP-1 to be the DUSP for ERK MPK-1 during germ cell development. Through careful reevaluation of the role of LIP-1 in oogenesis and regulation of ERK MPK-1, we showed that in fact LIP-1 does not function as an ERK DUSP in the oogenic germline. Along this line Tischer and Schuh, 2016, had shown that the mammalian DUSP6/7 homolog does not regulate ERK but rather targets Protein Kinase C during oocyte meiotic maturation in mouse. Together, these results demonstrate that LIP-1 or DUSP6/7 does not regulate ERK MPK-1 during oogenesis, leaving us with a big gap in our knowledge on molecular identity of signaling regulators that control female fertility. The goal of this proposal are to identify the phosphatase(s) and generate reagents to detect its expression and function during female germ cell development in worms. Identifying the phosphatase or a combination of phosphatases that function to regulate ERK activity and ERK- dependent events during female fertility fills a major gap in our knowledge and informs reproductive medicine.