Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility.
Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well.
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D004285:Dogs; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*; D045162:Thiazolidinediones
D000098:Acetophenones; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D045162:Thiazolidinediones; C042737:axokinin; C001995:5-carboxymethyl-3-p-tolylthiazolidine-2,4-dione-2-acetophenone; D011494:Protein Kinases