Protein 14-3-3 zeta Binds to Protein Phosphatase PP1 gamma 2 in Bovine Epididymal Spermatozoa

• Published in: Biology of reproduction Vol. 71; no. 1; pp. 177 - 184
• Main Authors: Huang, Z, Myers, K, Khatra, B, Vijayaraghavan, S
• Format: Journal Article
• Language: English
• Published: 01.07.2004
• ISSN: 0006-3363
• DOI: 10.1095/biolreprod.104.027284

The protein phosphatase PP1 gamma 2 is critical in the regulation of sperm motility and fertility. Its activity is regulated by its binding proteins and by phosphorylation. We have recently shown that PP1 gamma 2 is phosphorylated and that the amount of phosphorylated PP1 gamma 2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439-447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1 gamma 2 during column chromatography of bovine sperm extracts. Western blot analyses confirmed the presence of protein 14-3-3 not only in bovine spermatozoa but also in spermatozoa of diverse species-bull, hamster, horseshoe crab, monkey, rat, turkey, and Xenopus. The binding between PP1 gamma 2 and protein 14-3-3 was confirmed by coimmunoprecipitation experiments and in pull-down assays with recombinant GST-14-3-3. Western blot analysis and protein 14-3-3 immunoprecipitates with antibodies against the consensus binding domain of protein 14-3-3 reveal that, in addition to PP1 gamma 2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1 gamma 2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of bovine spermatozoa. Together, these results provide conclusive evidence that protein 14-3-3 is present in mature spermatozoa and that PP1 gamma 2 is one of its binding partners.