Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation

Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (C alpha) of PKA confers sensitivity to the pyrazolo[3,4-d] pyrimidine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the C alpha M120A mutant protein is expressed and the wild-type C alpha is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express C alpha M120A mutant protein. For C alpha M120A sperm, 10 mu M of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (< 90 s) evoked by the HCO3-anion. A continuous (90 min) inhibition with 10 mu M of 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in C alpha M120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional C alpha M120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.