Tssk4 belongs to the Testis Specific Serine/threonine protein Kinase (TSSK) family, members of which play an important role in spermatogenesis and/or spermiogenesis. Several Tssk family proteins have extensively been studied. However, the exact function of Tssk4 remains unclear. A Tssk4 knockout mouse model was generated and the males were subfertile due to seriously decreased sperm motility. The ultrastructure of the Tssk4(-/-)sperm tail is disorganized at the midpiece-principal piece junction, leading to a severe bend in the sperm flagellum. One or more axonemal microtubule doublets are absent and the midpiece is fused with the principal piece. Furthermore, we identified the association between Tssk4 and Odf2, a prominent cytoskeletal protein of the outer dense fiber (ODF) in sperm flagellum. Tssk4 can change the phosphorylation state of Odf2 and conversely Odf2 potentiates the autophosphorylation activity of Tssk4. These findings reveal that Tssk4 is required for maintaining the structural integrity of sperm flagellum and male fertility.

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.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 . In eukaryotic cells, protein kinases primarily phosphorylate serine (Ser), threonine (Thr), and tyrosine (Tyr) residues by transferring a phosphoryl group to an amino acid side chain 2 . Because phosphorylation is fast, reversible, and often highly specific, it is often employed for temporary modulation of protein function. Phosphorylation modification can alter the global protein structure and the local conformation in a specific peptide motif to change protein-protein interactions and modulate the enzymatic activity of phosphoproteins, resulting in the enhancement or inhibition of protein binding events and protein stability 3 . Over 500 kinases and 100 phosphatases are responsible for catalyzing protein phosphorylation and dephosphorylation and are themselves regulated by phosphorylation, revealing the interconnections among other cellular signaling pathways. Over 250,000 phosphorylation sites have now been mapped in the biological proteome.The outer dense fibers (ODFs) are prominent sperm tail-specific cytoskeletal structures. They function in forming "9+ 9+ 2" structures, together with nine outer doublet microtubules and a central pair of microtubules 4-7 . In the sperm tail, ODFs are thought to be contractile. In mammalian sperm, the ODFs consist of many proteins with a molecular mass ranging from approximately 11 kDa to approximately 87 kDa 4,8,9 . The ODF2 protein exhibits several isoforms, with a deduced molecular mass in the range of 60-96 kDa 5,10 . Secondary structure prediction indicated that ODF2 is an overall coiled-coil protein 11 . There are two leucine zipper motifs in the C-terminal region of the protein (aa 392-413 and aa 530-551 of rat ODF2), which are responsible for the interaction with the leucine zippers of ODF1 in yeast 5 . Nevertheless, the self-interacting capacity of rat ODF2 is independent of any of the leucine zipper ...

The testis specific serine/threonine protein kinase family (Tssk) members play important roles in spermatogenesis and/or spermiogenesis. Similar to other Tssk family members, Tssk4 protein shows exclusive expression in testis, but its biochemical and biological functions are still largely unknown. In present work, we generate a polyclonal antibody which specifically recognizes Tssk4 but not the other three Tssk family members (Tssk1, Tssk2 and Tssk3). By using the qualified antibody, we show that Tssk4 protein is constantly expressed in testis from haploid round spermatids to morphological mature spermatozoa. Further experiments reveal that Tssk4 has autophosphorylation activity and self-association character in vitro. Importantly, we find that autophosphorylation of Tssk4 at Thr-197 in the T-loop region is essential to its kinase activity. Taken together, these findings suggest that autophosphorylation at Thr-197 plays a critical role in maintaining Tssk4 kinase activity, which might be involved in spermiogenesis.

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Testis specific serine/threonine protein kinase 4 (TSSK4) belongs to the TSSK family, and its members play an important role in spermatogenesis and/or spermiogenesis. Mouse TSSK4 has been reported to be expressed exclusively in the testis and can maintain its kinase activity through autophosphorylation at Thr-197. However, its biological function remains poorly understood. Here we found that GFP-TSSK4-overexpressed HeLa cells showed apoptotic bodies, indicating TSSK4 can lead to apoptosis in vitro. Furthermore, TSSK4 induced apoptosis in different cell lines including HeLa, Cos-7 and H1299 tested by flow cytometry but not its kinase-dead mutant TSSK4-K54M. TSSK4 knockout mice showed increased testes weight and decreased apoptotic spermatogonia and spermatocytes at 21st day after birth tested by TUNEL technology. So TSSK4 was able to induce cell apoptosis in vitro depending on its kinase activity, which leads to abnormal testes weight and apoptosis, shedding light on its function in the process of spermatogenesis and/or spermiogenesis.