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

Abstract

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␣) 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␣M120A mutant protein is expressed and the wild-type C␣ 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␣M120A mutant protein. For C␣M120A sperm, 10 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 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␣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␣M120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.cAMP ͉ mouse genetics ͉ protein phosphorylation ͉ male fertility ͉ hyperactivation M ammalian sperm undergo a series of maturational changes in the female reproductive tract and this process, termed capacitation, is essential for subsequent fertilization of the egg (1). Although multiple signal transduction pathways are involved in capacitation, a central role for cAMP as a regulatory mediator is underscored by the male-infertility phenotypes of mice that carry targeted disruptions of the sperm-specific C␣2 catalytic subunit of protein kinase A (PKA) or of the atypical HCO 3 Ϫ -stimulated sperm adenylyl cyclase, SACY (formerly known as sAC). Studies of the mutant sperm that lack C␣2 (2) or SACY (3-5) have provided definitive evidence that neither protein is required for the initiation of motility or for maintenance of a slow basal flagellar beat. In contrast, both C␣2 and SACY are required for the acceleration of the flagellar beat that characterizes the rapid activation of motility by the HCO 3 Ϫ anion, and for HCO 3 Ϫ to rapidly facilitate evoked entry of Ca 2ϩ . The capacitation sequence that prepares sperm for fertilization includes two additional components that also are HCO 3 Ϫ dependent but delayed in onset. These delayed increases in protein tyrosine phosphorylation and hyperactivation of motility do not occur in the C␣2 or SACY null sperm. However, it has remained unclear whether cAMP and PKA...

Flow cytometry analysis reveals a decrease in intracellular sodium during sperm capacitation

“…It is noteworthy that a maximum decrease in [Na + ] i occurred at 30 minutes, whereas maximum PKA activation occurred much earlier ( Fig. 3) (Morgan et al, 2008). This observation is consistent with the idea that a cAMP-PKA pathway is upstream of Na + transport regulation.…”

Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

“…Capacitation-Increased protein tyrosine phosphorylation is a marker for sperm capacitation (27). After incubation in capacitation medium for 1-3 h, we analyzed the levels of tyrosinephosphorylated proteins via immunoblotting using anti-phosphotyrosine antibody (Fig.…”

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

“…As demonstrated by McKnight and co-workers (4,5), mice lacking the sperm-specific PKA catalytic subunit ␣2 (C␣2) are sterile, and their sperm do not hyperactivate. In vertebrates, cAMP is synthesized by two types of adenylyl cyclases as follows: a ubiquitous family of transmembrane adenylyl cyclases (tmACs) 4 with nine members (Adcy1-9) and soluble adenylyl cyclase encoded by a single gene (Adcy10 also known as SACY or sAC), which is alternatively spliced into multiple isoforms (6 -8). Adcy10 was origi-nally thought to be restricted to testis and sperm (9), but more recently it has been identified in other cell types (reviewed in Ref.…”