Interactions of 14-3-3 (YWHA) protein isoforms with CDC25B phosphatase in mouse oocytes
Annual Meeting of American Society for Cell Biology / San Francisco, California, USA
Immature mammalian oocytes are arrested at prophase I of meiosis by an inhibitory phosphorylation on Cyclin-Dependent Kinase I (CDK1). Release from this meiotic arrest is dependent on dephosphorylation of CDK1 by M-phase inducer phosphatase 2 (CDC25B). Evidence suggests that phosphorylated CDC25B is bound to 14-3-3 (YWHA) proteins in the cytoplasm and rendered inactive. We previously reported expression of all seven mammalian isoforms of 14-3-3 in mouse oocytes and eggs. To examine the interactions of 14-3-3 isoforms with CDC25B in oocytes and eggs, we performed an in situ Proximity Ligation Assay (PLA; Duolink in cell Co-IP, Olink Bioscience) that can detect protein-protein interactions at the single molecule level and allows visualization of the actual intracellular sites of the interactions. We observed prominent interaction of all seven 14-3-3 isoforms with CDC25B throughout cytoplasm and nuclei of mouse oocytes, along with reduced interactions for each individual isoform in eggs compared to oocytes. Co-immunoprecipitation studies with extracts of mouse oocytes also demonstrated interaction of CDC25B with six 14-3-3 isoforms. These results suggest that any of the 14-3-3 isoforms may have the potential to hold CDC25B inactive in oocytes to maintain the meiotic arrest. In preliminary experiments to explore if interactions of 14-3-3 with other proteins are important for maintaining meiosis I arrest, we microinjected oocytes with 0.5μg/μL R18, a synthetic non-isoform specific 14-3-3-blocking peptide. Injected oocytes were incubated overnight in media containing a threshold concentration of dibutyryl cAMP (0.05mg/mL) which normally holds oocytes arrested through activation of Protein Kinase A (PKA) and phosphorylation of CDC25B and CDK1. We observed a marked increase in germinal vesicle breakdown (GVBD), compared to control oocytes. To investigate which specific isoform(s) of 14-3-3 is/are responsible for maintaining the meiotic arrest, we reduced the synthesis of each 14-3-3 isoform in mouse oocytes by intracytoplasmic microinjection of 0.1mM translationblocking morpholino oligonucleotide against the corresponding isoform mRNA. Injected oocytes were held arrested at prophase I for 24 hours, and then incubated overnight in media containing the threshold concentration of dbcAMP. GVBD was observed in 70% of oocytes microinjected with morpholino against 14-3-3 eta, despite the presence of dbcAMP. Injection of morpholinos targeting other 14-3-3 isoforms caused little or no GVBD. Thus, reduction in 14-3-3 eta/CDC25B interaction releases the mouse oocyte from meiotic arrest. These results suggest that, while all 14-3-3 isoforms interact with CDC25B in mouse oocytes, 14-3-3 eta is essential for maintaining the prophase I meiotic arrest.
De, Santanu; Reese, Angela; and Kline, Douglas, "Interactions of 14-3-3 (YWHA) protein isoforms with CDC25B phosphatase in mouse oocytes" (2012). Biology Faculty Proceedings, Presentations, Speeches, Lectures. 330.