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

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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.