Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo.

Abstract

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l -/-; Prm2 S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l -/- mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.

• Authors: Katsuhiko Itoh, Gen Kondoh, Hitoshi Miyachi, Manabu Sugai, Yoshiyuki Kaneko, Satsuki Kitano, Hitomi Watanabe, Ryota Maeda, Akihiro Imura, Yu Liu, Chizuru Ito, Shigeyoshi Itohara, Kiyotaka Toshimori, Jun Fujita
• Journal: Science signaling (Sci Signal) Vol. 12 Issue 574 (03 26 2019) ISSN: 1937-9145 [Electronic] United States
• PMID: 30914484 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Chemical References

• Chromatin
• HSC70 Heat-Shock Proteins
• HSP70 Heat-Shock Proteins
• Hspa4l protein, mouse
• Hspa8 protein, mouse
• Protamines
• Protein Isoforms
• protamine 2
• Phosphoserine
• Ppp1cc protein, mouse
• Protein Phosphatase 1

Topics

• Animals
• Chromatin (metabolism)
• HSC70 Heat-Shock Proteins (metabolism)
• HSP70 Heat-Shock Proteins (metabolism)
• Infertility, Male (genetics)
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Mutation, Missense
• Phenotype
• Phosphorylation
• Phosphoserine (chemistry)
• Point Mutation
• Protamines (chemistry, genetics)
• Protein Isoforms (physiology)
• Protein Phosphatase 1 (physiology)
• Protein Processing, Post-Translational
• Sperm Head (ultrastructure)
• Sperm Maturation (physiology)