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 (Calpha) 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 CalphaM120A mutant protein is expressed and the wild-type Calpha 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 CalphaM120A mutant protein. For CalphaM120A sperm, 10 microM of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (<90 s) evoked by the HCO(3)(-) anion. A continuous (90 min) inhibition with 10 microM 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 CalphaM120A 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 CalphaM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.

Fig. 1.

Knockin of the CαM120A mutation. (A) Schematic diagram of the targeting vector that specified the CαM120A mutation in exon 5 of the Prkaca (Cα) gene. A NEO cassette was flanked by two loxP sites and a downstream minigene containing the wild-type form of exons 5–10. The NEO cassette was removed by partial recombination using EIIa-Cre transgenic animals to give CαLoxM120A mice. The wild-type minigene, also flanked by a downstream loxP site, was removed by complete Cre-mediated excision after mating with EIIa-Cre transgenic mice. Excision of NEO and the minigene by Cre caused expression of the CαM120A mutation. (B) Southern blot analysis of BamHI-digested genomic tail DNA using an external DNA probe identified correctly targeted ES cell clones that were used for microinjection. The mutant allele yielded a 7.0-kb band and the wild-type allele generated a 6.5-kb band. (C) PCR analysis of genomic DNA from the offspring of mice that carried the targeted allele and also an EIIa-Cre recombinase transgene. CαM120A is the product of complete recombination and was genotyped with a PCR product across the lox1,3 site. CαLoxM120A is the product of partial recombination between lox1 and lox2 and was genotyped by a PCR across the lox1,2 site.

Fig. 2.

1NM-PP1 inhibits the mutant CαM120A form of PKA in testis and sperm. PKA activity in homogenates of sperm (A and C) and testis (B and D) from wild-type (WT) and mutant (M120A) mice assayed in the presence or absence of 25 μM of cAMP. In C and D assays also contained the indicated concentrations of 1NM-PP1. Averaged values (± SEM) from triplicate assays are shown. PKA activity is expressed as units (pmoles of phosphate incorporated per min) per mg of protein. (E) Western blot analysis for Cα and RIIα proteins in extracts from the testes of WT or M120A homozygotes. Twenty micrograms of protein were loaded per lane.

Fig. 3.

Bicarbonate-evoked activation of sperm motility. (A) Averaged flagellar beat frequency was determined for wild-type (Left) and M120A (Right) sperm that were bathed in HS medium that lacked or contained 10 μM of 1NM-PP1 (1NM). Individual sperm were examined before and then again after 1 min of perfusion with the same media fortified with 15 mM of HCO₃⁻ (n = 19–39 cells. At least 9 cells were examined from each animal in 2–3 independent experiments.). *, P < 0.05 (M120A, HCO₃⁻ vs. HCO₃⁻ + 1NM-PP1). B and C compare averaged beat frequencies of wild-type (■) and M120A (●) sperm. In B, sperm were bathed in HS medium and sequentially perfused with HS medium containing 15 mM of HCO₃⁻ for 5 min, then with HS containing both 10 μM of 1NM-PP1 and 15 mM of HCO₃⁻ for 7 min (n = 19–49 cells. At least 9 cells were examined from each animal in 2–3 independent experiments.). In C, individual sperm were sequentially perfused with HS containing 10 μM of 1NM-PP1 for 1 min, then with HS containing 15 mM of HCO₃⁻ (n = 9–15 cells from one wild-type animal, 16–67 cells from two to four M120A mutant animals). Error bars for all experiments represent standard error of the mean.

Fig. 4.

PKA inhibition does not reverse the flagellar waveform asymmetry of hyperactivation. (A) Contrast-enhanced, stop-motion image of a sperm loosely tethered at the base of the head. The lines show d, the distance along the flagellum and θ_d, the tangent angle at point d (yellow dot). Scale bar, 25 μm. (B and C) Mean values of flagellar asymmetry (the time-averaged θ_d) obtained by waveform analysis of individual CαM120A mutant sperm examined before B and after C capacitating incubation to produce hyperactivation. For C each cell was sampled twice, once after transfer form capacitating medium to medium HS alone (black squares and line), then again at the end of a 5-min perfusion with HS plus 10 μM of 1NM-PP1 (red circles and line) (n = 11 cells from one wild-type animal, 23 cells from three M120A mutant animals).

Fig. 5.

Continuous PKA activity is required to initiate tyrosine phosphorylation during capacitating incubations. (A) Sperm from WT or M120A mutant mice were incubated at 37 °C in HS buffer containing 5 mg/ml of BSA. Thirty micromoles of 1NM-PP1 were added for a 5-min preincubation followed by addition of 15 mM of HCO₃⁻ at 0 time where indicated. Sperm were harvested by centrifugation at 90 s and subjected to Western blot analysis using anti-phospho PKA substrate antibody. (B) Protein tyrosine phosphorylation was assessed by Western blot analysis of extracts from WT and M120A sperm after incubation for 90 min in HS plus BSA in the presence of continuous 30 μM of 1NM-PP1 and HCO₃⁻ as indicated. (C) Same as B except that 1NM-PP1 was not added until 30 min after start of incubation. (D) Same as C except that 1NM-PP1 was added at 90 min and the sperm were harvested at 95 min for phosphotyrosine analysis on Western blots. Only the mutant M120A sperm samples are shown in D. The blots shown are representative of those obtained in three separate experiments.