Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Aug 1;103(2):649–655. doi: 10.1083/jcb.103.2.649

Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility

PMCID: PMC2113824  PMID: 3733884

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (781.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson N. G., Anderson N. L. Analytical techniques for cell fractions. XXI. Two-dimensional analysis of serum and tissue proteins: multiple isoelectric focusing. Anal Biochem. 1978 Apr;85(2):331–340. doi: 10.1016/0003-2697(78)90229-4. [DOI] [PubMed] [Google Scholar]
  2. Anderson N. L., Anderson N. G. Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis. Anal Biochem. 1978 Apr;85(2):341–354. doi: 10.1016/0003-2697(78)90230-0. [DOI] [PubMed] [Google Scholar]
  3. Brandt H., Hoskins D. D. A cAMP-dependent phosphorylated motility protein in bovine epididymal sperm. J Biol Chem. 1980 Feb 10;255(3):982–987. [PubMed] [Google Scholar]
  4. Brokaw C. J. Cyclic AMP-dependent activation of sea urchin and tunicate sperm motility. Ann N Y Acad Sci. 1984;438:132–141. doi: 10.1111/j.1749-6632.1984.tb38282.x. [DOI] [PubMed] [Google Scholar]
  5. Garbers D. L., First N. L., Lardy H. A. Properties of adenosine 3',5'-monophosphate-dependent protein kinases isolated from bovine epididymal spermatozoa. J Biol Chem. 1973 Feb 10;248(3):875–879. [PubMed] [Google Scholar]
  6. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  7. Hoskins D. D., Stephens D. T., Hall M. L. Cyclic adenosine 3':5'-monophosphate and protein kinase levels in developing bovine spermatozoa. J Reprod Fertil. 1974 Mar;37(1):131–133. doi: 10.1530/jrf.0.0370131. [DOI] [PubMed] [Google Scholar]
  8. Ishiguro K., Murofushi H., Sakai H. Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa. J Cell Biol. 1982 Mar;92(3):777–782. doi: 10.1083/jcb.92.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lee M. Y., Iverson R. M. An adenosine 3':5' monophosphate dependent protein kinase from sea urchin spermatozoa. Biochim Biophys Acta. 1976 Mar 11;429(1):123–136. doi: 10.1016/0005-2744(76)90035-8. [DOI] [PubMed] [Google Scholar]
  10. Lindemann C. B., Lipton M., Shlafer R. The interaction of cAMP with modeled bull sperm. Cell Motil. 1983;3(2):199–210. doi: 10.1002/cm.970030208. [DOI] [PubMed] [Google Scholar]
  11. Mariash C. N., Seelig S., Oppenheimer J. H. A rapid, inexpensive, quantitative technique for the analysis of two-dimensional electrophoretograms. Anal Biochem. 1982 Apr;121(2):388–394. doi: 10.1016/0003-2697(82)90498-5. [DOI] [PubMed] [Google Scholar]
  12. Morisawa M., Okuno M., Suzuki K., Morisawa S., Ishida K. Initiation of sperm motility in teleosts. J Submicrosc Cytol. 1983 Jan;15(1):61–65. [PubMed] [Google Scholar]
  13. Tash J. S., Kakar S. S., Means A. R. Flagellar motility requires the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble 56 kd protein, axokinin. Cell. 1984 Sep;38(2):551–559. doi: 10.1016/0092-8674(84)90509-9. [DOI] [PubMed] [Google Scholar]
  14. Tash J. S., Means A. R. Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility. Biol Reprod. 1983 Feb;28(1):75–104. doi: 10.1095/biolreprod28.1.75. [DOI] [PubMed] [Google Scholar]
  15. Tash J. S., Means A. R. Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium. Biol Reprod. 1982 May;26(4):745–763. doi: 10.1095/biolreprod26.4.745. [DOI] [PubMed] [Google Scholar]
  16. Treetipsatit N., Chulavatnatol M. Effects of ATP, cAMP and pH on the initiation of flagellar movement in demembranated models of rat epididymal spermatozoa. Exp Cell Res. 1982 Dec;142(2):495–499. doi: 10.1016/0014-4827(82)90397-4. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES