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Updated: Jul 6, 2026
Phosphopeptide Analysis of Rodent Epididymal Spermatozoa
Published on: December 30, 2014
Phosphorylation of mouse sperm axoneme central apparatus protein SPAG16L by a testis-specific kinase, TSSK2
Zhibing Zhang1, Xuening Shen, Brian H Jones
1Department of Obstetrics & Gynecology, Virginia Commonwealth University, Richmond, Virginia 23298, USA. zzhang4@vcu.edu
Abstract:
The mammalian protein SPAG16L, the ortholog of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites, and the protein was confirmed to be phosphorylated in vivo. A yeast two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by coimmunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their colocalization was also noted by confocal microscopy in Chinese hamster ovary cells, where they were coexpressed. TSSK2 associates with SPAG16L via its C-terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L-null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.
Insights
Sperm motility relies on the SPAG16L protein, an axoneme component. This study reveals SPAG16L is phosphorylated by TSSK2, a testis-specific kinase, highlighting a crucial interaction for flagellar function.
Area of Science:
- Cell Biology
- Reproductive Biology
- Biochemistry
Background:
- SPAG16L is an axoneme central apparatus protein essential for mammalian flagellar motility.
- SPAG16L is known to be phosphorylated in vivo, suggesting regulatory mechanisms.
- The interaction partners and specific kinases involved in SPAG16L regulation were not fully elucidated.
Purpose of the Study:
- To identify binding partners of SPAG16L.
- To investigate the functional relationship between SPAG16L and its interacting kinase.
- To determine if SPAG16L is a substrate for TSSK2.
Main Methods:
- Yeast two-hybrid screening to identify SPAG16L binding partners.
- Coimmunoprecipitation assays to confirm protein-protein interactions.
- Confocal microscopy to visualize protein colocalization.
- In vitro kinase assays to assess phosphorylation.
- Analysis of SPAG16L-null mice.
Main Results:
- TSSK2 was identified as a SPAG16L binding partner.
- SPAG16L and TSSK2 interact and colocalize in cells.
- TSSK2 phosphorylates SPAG16L in vitro.
- TSSK2 is significantly reduced in SPAG16L-null mouse testes.
Conclusions:
- SPAG16L is a substrate of the testis-specific kinase TSSK2.
- The interaction and phosphorylation of SPAG16L by TSSK2 are critical for its function in flagellar motility.
- This study elucidates a key regulatory pathway in sperm development.
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