Abstract
As a member of the testis-specific serine/threonine protein kinase (TSSK) family, Tssk4 is exclusively expressed in the testis and plays an essential role in male fertility. We previously reported that Tssk4 can associate with and phosphorylate Odf2, but the phosphorylation site is still unknown. Here we confirm that the C-terminal region (amino acids 214-638) of Odf2 is required for association with Tssk4. Furthermore, to identify the site at which Tssk4 phosphorylates Odf2, we generated several Odf2 point mutants (Ser/Thr/Lys to Ala) and identified serine 76 of Odf2 as one of the phosphorylation sites. In vivo, phosphorylated Odf2 was evaluated in mouse sperm using a specific phospho-Ser-76 Odf2 antibody and LC-MS/MS. These findings are the first to demonstrate the phosphorylation site in Odf2 by Tssk4, providing essential clues regarding the function of Tssk4 in regulating sperm motility and/or structure and thus male fertility.
Highlights
Reversible protein phosphorylation is critically significant in biology because of its influence on almost every important cellular process throughout phylogeny, including cell growth, cell differentiation, cell cycle, and cell migration[1]
We defined the C-terminal fragment of Odf[2], which is essential for the modification of Tssk[4], and we identified Ser-76 as a Tssk[4] phosphorylation site in Odf[2] both in vitro and in vivo
To investigate the connection between Tssk[4] and Odf[2] in detail, we co-transfected their plasmids into HEK-293T cells and found that the electrophoretic migration rates of both the Tssk[4] and Odf[2] proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels were altered (Fig. 1a)
Summary
Reversible protein phosphorylation is critically significant in biology because of its influence on almost every important cellular process throughout phylogeny, including cell growth, cell differentiation, cell cycle, and cell migration[1]. Tssk[4] belongs to the testis-specific serine/threonine protein kinase (TSSK) family, whose members play an important role in spermatogenesis and/or spermiogenesis. Five members of this family have been reported, including Tssk[1], Tssk[2], Tssk[3], Tssk[4] ( known as Tssk5), and Tssk[6] Due to their specific expression patterns during the processes of spermatogenesis and/or spermiogenesis, their physiological functions were explored by establishing gene knockout mouse models. The Tssk[1] and Tssk[2] double-deletion mouse was haploinsufficient and could not transmit the null genotype[19] Another group reported that the double-deletion mouse model displayed male sterility accompanied by chromatid body loss[20]. Tssk[4] can associate with and change the phosphorylation state of Odf[2], while ODF2 can potentiate the autophosphorylation activity of Tssk[4] at Ser-19721
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