Testis-specific serine/threonine protein kinase 4 (Tssk4) phosphorylates Odf2 at Ser-76

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.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 ...

Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella

“…6h). Furthermore, TSSK4 was reported to influence sperm flagellar formation by enhancing the phosphorylation state of ODF2, which is a prominent protein of the sperm tail outer dense fibers 13,30–33 . Because no commercial antibody was available, we did not confirm that TSSK4 can increase the phosphorylation of ODF2.…”

Testis-specific serine kinase protein family in male fertility and as targets for non-hormonal male contraception†

“…Using a yeast two-hybrid approach with the cAMP response element-binding protein (CREB; previously known as AMP-responsive transcription factor) as bait, Chen and coworkers found that TSSK4 can phosphorylate CREB and may stimulate the CRE/CREB pathway by phosphorylation of CREB at Ser-133 [ 31 ]. More recently, TSSK4 was proposed as one of the kinases involved in phosphorylation of outer dense fiber 2 (Odf2), an essential protein in the sperm flagellum [ 56 ]. Using a phospho-specific antibody, in vitro phosphorylation of Odf2 in Ser76 by Tssk4 was verified by co-transfection of Tssk4 and Odf2 serine/tyrosine/lysine-to-alanine point mutants in 293 T cells.…”

“…As described above, further experimentation showed that Tssk4 and Odf2, a major cytoskeletal protein, are both localized in the principal piece of the sperm flagellum; it should be noted that Tssk4–Odf2 co-immunolocalization studies could not be performed because both antibodies were raised in rabbits. Based on the similar patterns of distribution in sperm flagellum, it was proposed that Tssk4 and Odf2 could interact in vivo, probably affecting the phosphorylation status of one another [ 56 ], which would in turn explain the abnormal morphology and functionality of sperm flagellum observed in the Tssk4 knockout mice.…”

A homozygous missense variant in VWA2, encoding an interactor of the Fraser-complex, in a patient with vesicoureteral reflux

“…A subsequent literature research revealed that TSSK4 and ATP10D have not been implicated in pathways related to kidney development to date. TSSK4 appears to have a rather specialized role for male fertility [ 28 ] while sequence variants in ATP10D have been demonstrated to significantly associate with metabolic disorders including insulin resistance [ 29 ]. NELL2 and TTLL12 however, remotely appear connected to pathways that have been previously discussed in the development of CAKUT.…”