Date of Completion
Lisa M. Mehlmann, Laurinda A. Jaffe
Field of Study
Master of Science
1.5 million women in the U.S. are infertile, but assisted reproduction success rates are low for many reasons, including our limited understanding of oocyte maturation. Immature oocytes are arrested at prophase I of meiosis, regulated by transmembrane adenylyl cyclase 3 (AC3), which raises second messenger cyclic adenosine monophosphate (cAMP) levels. Elevated cAMP levels maintain meiotic arrest by activating protein kinase A (PKA), which ultimately inactivates cell division cycle 25B (CDC25B) protein and maturation-promoting factor (MPF). However, decreasing cAMP levels activates CDC25B, stimulates MPF, and induces meiosis. A-kinase anchoring proteins (AKAPs) localize PKA and other signaling molecules to facilitate local substrate phosphorylation events. Preliminary results have shown that, while its exact function is unknown, endogenous full length (FL) AKAP7 gamma is highly expressed in the nucleus and cytoplasm of oocytes, AC3 is the predominant AC in oocytes, and AKAP7 gamma is associated with AC3 activity. However, it is unknown whether AKAP7 gamma regulates meiotic arrest. Our hypothesis is that during meiotic arrest, AKAP7 gamma binds AC3 at the N-terminus (NT) of AC3 to localize anchored PKA to concentrated pools of cAMP. Our second hypothesis is that AKAP7 gamma also binds CDC25B to localize it to anchored PKA, which phosphorylates/inactivates CDC25B and ultimately inactivates MPF. Our long-term goal is to map the binding sites on AKAP7 gamma, AC3, and CDC25B and use competition assays to test the physiological relevance of CDC25B-AKAP7-AC3 complexes in arrested oocytes. Here, we demonstrate that AKAP7 gamma binds CDC25B/C and NT-AC3 (between amino acids 1 and 77), confirming these interactions with pure and transfected protein. Interestingly, CDC25B/C bound a disordered region between amino acids 1 and 60 of AKAP7 gamma. Most importantly, AC3 and CDC25C showed significant PKA activity, indicating direct interaction with AKAP7 gamma. Our work is important because defining the role of AKAP7 gamma interaction with AC3 and CDC25B in meiotic arrest may improve our understanding of meiotic resumption and oocyte maturation.
Plair, Akilah A., "A-Kinase Anchoring Protein (AKAP7) Interaction with Adenylyl Cyclase (AC3) and Cell Division Cycle Protein (CDC25)" (2016). Master's Theses. 932.
Kimberly L. Dodge-Kafka