Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Nov 1;107(5):1809–1816. doi: 10.1083/jcb.107.5.1809

Immunogold localization of the regulatory subunit of a type II cAMP- dependent protein kinase tightly associated with mammalian sperm flagella

PMCID: PMC2115342  PMID: 2972731

Abstract

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.

Full Text

The Full Text of this article is available as a PDF (4.0 MB).

Selected References

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

  1. Brokaw C. J. Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation. J Cell Biochem. 1987 Nov;35(3):175–184. doi: 10.1002/jcb.240350302. [DOI] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Cascieri M., Amann R. P., Hammerstedt R. H. Adenine nucleotide changes at initiation of bull sperm motility. J Biol Chem. 1976 Feb 10;251(3):787–793. [PubMed] [Google Scholar]
  4. De Camilli P., Moretti M., Donini S. D., Walter U., Lohmann S. M. Heterogeneous distribution of the cAMP receptor protein RII in the nervous system: evidence for its intracellular accumulation on microtubules, microtubule-organizing centers, and in the area of the Golgi complex. J Cell Biol. 1986 Jul;103(1):189–203. doi: 10.1083/jcb.103.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ey P. L., Prowse S. J., Jenkin C. R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  6. Flockhart D. A., Corbin J. D. Regulatory mechanisms in the control of protein kinases. CRC Crit Rev Biochem. 1982 Feb;12(2):133–186. doi: 10.3109/10409238209108705. [DOI] [PubMed] [Google Scholar]
  7. Frenkel G., Peterson R. N., Freund M. The role of adenine nucleotides and the effect of caffeine and dibutyryl cyclic AMP on the metabolism of guinea pig epididymal spermatozoa. Proc Soc Exp Biol Med. 1973 Nov;144(2):420–425. doi: 10.3181/00379727-144-37604. [DOI] [PubMed] [Google Scholar]
  8. Gray J. P., Drummond G. I., Luk D. W., Hardman J. G., Sutherland E. W. Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm. Arch Biochem Biophys. 1976 Jan;172(1):20–30. doi: 10.1016/0003-9861(76)90043-6. [DOI] [PubMed] [Google Scholar]
  9. Horowitz J. A., Toeg H., Orr G. A. Characterization and localization of cAMP-dependent protein kinases in rat caudal epididymal sperm. J Biol Chem. 1984 Jan 25;259(2):832–838. [PubMed] [Google Scholar]
  10. Horowitz J. A., Wasco W., Leiser M., Orr G. A. Interaction of the regulatory subunit of a type II cAMP-dependent protein kinase with mammalian sperm flagellum. J Biol Chem. 1988 Feb 5;263(4):2098–2104. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Milkowski A. L., Babcock D. F., Lardy H. A. Activation of bovine epididymal sperm respiration by caffeine. Its transient nature and relationship to the utilization of acetyl carnitine. Arch Biochem Biophys. 1976 Sep;176(1):250–256. doi: 10.1016/0003-9861(76)90163-6. [DOI] [PubMed] [Google Scholar]
  13. Nigg E. A., Schäfer G., Hilz H., Eppenberger H. M. Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes. Cell. 1985 Jul;41(3):1039–1051. doi: 10.1016/s0092-8674(85)80084-2. [DOI] [PubMed] [Google Scholar]
  14. Noland T. D., Abumrad N. A., Beth A. H., Garbers D. L. Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein. Biol Reprod. 1987 Aug;37(1):171–180. doi: 10.1095/biolreprod37.1.171. [DOI] [PubMed] [Google Scholar]
  15. Paupard M. C., MacLeod J., Wasco W., Orr G. A. Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase. J Cell Biochem. 1988 Jun;37(2):161–175. doi: 10.1002/jcb.240370204. [DOI] [PubMed] [Google Scholar]
  16. Sarkar D., Erlichman J., Rubin C. S. Identification of a calmodulin-binding protein that co-purifies with the regulatory subunit of brain protein kinase II. J Biol Chem. 1984 Aug 10;259(15):9840–9846. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Weinman S., Ores-Carton C., Rainteau D., Puszkin S. Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I. J Histochem Cytochem. 1986 Sep;34(9):1171–1179. doi: 10.1177/34.9.2426345. [DOI] [PubMed] [Google Scholar]

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

RESOURCES