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Gamete Biology; |
Department of Obstetrics & Gynecology,3 Virginia Commonwealth University, Richmond, Virginia 23298
Center for Research on Reproduction and Women's Health,4 University of Pennsylvania, Philadelphia, Pennsylvania 19104
Department of Cell Biology,5 University of Virginia Medical School, Charlottesville, Virginia 22908
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.
kinase, phosphorylation, SPAG16L, sperm motility
The mammalian Spag16 gene is orthologous to the Chlamydomonas PF20 gene. In Chlamydomonas, a single PF20 protein is translated from the gene, and this protein is localized to the flagellar axoneme microtubule doublets. Pf20 mutants have paralyzed flagella and an associated absence of the entire central pair [1]. In mammals, two SPAG16 proteins, 71-kDa SPAG16L and 35-kDa SPAG16S, are translated from a single Spag16 gene [2]. SPAG16L is translated from a 2.5-kb mRNA, and both mRNA and protein are expressed in tissues with flagellated cells or cilia, such as testis, brain, and trachea [3–5]. SPAG16S is translated from a 1.4-kb mRNA that is only present in the testis, indicating that the formation of this message is driven by a testis-specific promoter. The two SPAG16 isoforms play different roles: SPAG16L is incorporated into the axonemal central pair and is essential for flagellar motility [6], serving a homologous function to the Chlamydomonas PF20 protein. However, SPAG16S is localized in the nuclei of germ cells and is required for normal spermatogenesis [3]. Our recent studies suggest that SPAG16S is a transcription factor that can transactivate the promoter of the L isoform of Spag16 (Zhang et al., unpublished observation).
Both of the SPAG16 proteins contain seven WD repeats, a conserved motif known to be involved in protein-protein interactions [7, 8]. WD repeats typically contain a GH dipeptide 11–24 residues from the N-terminus, and the WD dipeptide at the C-terminus. Proteins with WD repeats have diverse functions, such as signal transduction, RNA processing, transcription, cytoskeleton assembly, mitotic spindle formation, regulation of vesicle formation and vesicular trafficking, control of various aspects of cell division, and the regulation of sulfur metabolism in fungi [9–18].
To search for potential binding partner(s) of SPAG16, a yeast two-hybrid screen was performed previously, with the WD repeat as the bait identifying several proteins, including SPAG6 and MEIG1, as potential binding partners [2, 3]. The same screen identified another potential binding partner, TSSK2, a testis-specific serine/threonine kinase.
TSSK2 belongs to a testis-specific serine/threonine kinase family. Using degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase catalytic domain and a PCR-based cloning strategy, TSSK1 was the first member identified [19]. By screening a testis cDNA library using Tssk1 cDNA as probe under low-stringency strategy, Tssk2 and Tssk3 were subsequently cloned [20–22]. Tssk6, also known as SSTK, was cloned by searching public databases with conserved amino acid sequences [23]. Later, TSSK5 was discovered [24] and added to this family of testis-specific kinases.
Little has been known about the functions of this kinase family. Mouse TSSK1 is abundant in germ cells during meiotic metaphase [25], whereas mouse TSSK2 is localized in the cytoplasm of male germ cells at the late stage of spermatogenesis, and also in the flagella, acrosomal region of mature sperm, which is consistent with SPAG16L localization. In human sperm, TSSK2 was found in the neck region, equatorial region, and midpiece of the flagella [20, 25, 26]. It has been shown that TSKS is a substrate for both TSSK1 and TSSK2 [19, 21]. TSSK3 is expressed in the Leydig cells of sexually mature mice [21]. TSSK6 shares a similar expression pattern as TSSK2 [22]. TSSK6 can phosphorylate histones and plays a role in postmeiotic chromatin remodeling and male fertility [23]. TSSK5 can phosphorylate cAMP-response element binding (CREB) protein and stimulate the CRE/CREB-responsive pathways [24]. In this study, we demonstrated that TSSK2 associates with SPAG16L, and SPAG16L can be phosphorylated by TSSK2. Thus, SPAG16L is a new substrate for TSSK2.
The present study was approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania and Virginia Commonwealth University. Procedures involving animals were conducted with the approval of the Institutional Animal Care and Use Committee of the Virginia Commonwealth University in accordance with the Guide for Care and Use of Laboratory Animals (protocol no. 06013417).
A mouse testis cDNA library was screened previously using the C-terminus domain of SPAG16 as the bait [2].
Two-Dimensional Western Blotting
Epididymal sperm were collected from male mice (CD1 retired breeders; Charles River Laboratories, Wilmington, MA) by mincing the caudae epididymides and allowing the sperm to swim out in PBS. The sperm were collected by centrifugation at 800 x g for 5 min at room temperature. Two-dimensional (2D) Western blotting was performed in the Proteomics Core Facility of University of Pennsylvania. The sample was dissolved in 2D sample buffer (40 mM Tris-HCl, pH 9; 8 M urea; 4% [w/v] CHAPS; 100 mM dithiothreitol; 1x protease inhibitor mixture [Roche Applied Science]) and centrifuged at 10 000 x g for 5 min, and the pellet was discarded. The amount of protein was determined by the Bradford method. For phosphatase treatment, samples were pretreated with 1 IU calf intestinal alkaline phosphatase (CIP; New England Biolabs) for 30 min at 37°C. A total of 100 µg protein, treated or not with CIP, was used for 2D gel electrophoresis following the same procedure described by Cao et al. [27] and Arakane et al. [28].
Generation of an Anti-Mouse TSSK2 Antibody
A cDNA encoding the full length of mouse Tssk2 was amplified by RT-PCR with the following primers: forward 5'-GGCATATGATGGACGATGCGGCGGTCC-3' (NdeI) and reverse 3'-CTGAAAGCTTCGGTACTTGCTTTCTCC-3' (HindIII). The PCR product was cloned into the pET28a vector for protein expression in bacteria. The fusion protein was purified, and the rabbit polyclonal antibody was generated as described previously [2, 3]. Antibodies against N-terminus and C-terminus of SPAG16 were generated in our laboratory previously [2, 3].
Constructs for Mammalian Cell Expression
Full-length Tssk2 cDNA was cloned into mammalian expression vectors pTarget, DsRed-N1, and pEGFP-N2. The PCR primers are as follows: for pTarget, forward primer: 5'-GAATTCATGGACGATGCGGCGGTCCTA-3' (EcoRI) and reverse primer: 5'-GTCGACCTAGGTACTTGCTTTCTCCAC-3' (SalI); for DsRed-N1, forward primer: 5'-CGAGAATTCTGATGGACGATGCGGCGGTCCTA-3' (EcoRI) and reverse primer 5'-CGCGGATCCCGGGTACTTGCTTTCTCCACCTC-3' (BamHI); and for pEGFP-N2, forward primer: the same as for pTarget, and reverse primer 5'-CGCGGATCCCGGTACTTGCTTTCTCCACCTC-3' (BamHI). Polymerase chain reactions were performed with a mouse testis cDNA as template. The PCR products were cloned into pCR2.1 TOPO vector; after sequencing, the inserts were subcloned into the mammalian expression vectors. SPAG16L/pEGFP-N2, SPAG16S/pEGFP-N2, SPAG16L/pTarget, and SPAG16S/pTarget were generated previously [2]. The DNA sequence encoding N-terminal SPAG16L was amplified with the same forward primer and diverse reverse primers to create N-SPAG16/pcDNA3 and N-SPAG16/pEGFP-N2. The forward primer: the same as for pTarget, and reverse primers: 5'-CTCGAGTCAATCTACAGGAAATTCTGAATC-3' (XhoI, for pcDNA3) and 5'-GGATCCCATCTACAGGAAATTCTGAATC-3' (BamHI, for pEGFP-N2).
TSSK2/pTarget, TSSK2/DsRed-N1, TSSK2/pEGFP-N2, N-SPAG16/pcDNA3, and N-SPAG16/pEGFP-N2 plasmids were transfected into Chinese hamster ovary (CHO) cells. At 48 h after transfection, the cells were lysed with RIPA buffer, and Western blotting was performed to verify TSSK2 and N-SPAG16 protein translation. A 1:2000 dilution of anti-TSSK2 antibody and 1:1000 dilutions of anti-green fluorescent protein (anti-GFP) and N-SPAG16 antibodies were used in our studies.
Cell Culture and Transfection for Confocal Microscopy
Chinese hamster ovary cells were cultured in two-well chamber slides. The cells were transfected with SPAG16L/pEGFP-N2 or TSSK2/DsRed-N1, or a combination of TSSK2/DsRed-N1 and SPAG16L/pEGFP-N2 using FuGENE (Roche). As controls, empty DsRed-N1 was cotransfected with empty pEGFP-N2 or SPAG16/pEGFP-N2 plasmid. Forty-eight hours after transfection, the cells were visualized by confocal microscopy.
Coimmunoprecipitation from Transfected Cell Lines and Testicular Extracts
Chinese hamster ovary cells were cotransfected with indicated TSSK2 and SPAG16 plasmids. Forty-eight hours later, the cells were harvested into immunoprecipitation buffer (150 mM NaCl; 50 mM Tris·HCl, pH 8.0; 5 mM EDTA; 1% Triton X-100; 1 mM PMSF; and proteinase inhibitor mixture), and the lysates were passed through a 20-gauge needle. After centrifugation at 11 600 x g for 5 min, the supernatants were precleared with protein A beads at 4°C for 30 min. The supernatants were then incubated with 1 µl (1 µg/µl) of indicated antibodies or preimmune serum at 4°C for 2 h, and protein A beads were added with a further incubation at 4°C overnight. The beads were washed with immunoprecipitation buffer three or four times; 1x protein loading buffer was then added to the beads, which were boiled at 100°C for 10 min; and the samples were then processed for Western blotting using monoclonal anti-GFP, or anti-TSSK2 and SPAG16 polyclonal antibodies.
Coimmunoprecipitation (Co-IP) with testis extracts was performed with a Co-IP kit from Roche. Briefly, 1 mg testis extract was precleared with protein A beads, the precleared extract was added with preimmune serum or indicated antibody, and the complex was incubated at 4°C with rotation for 3 h, followed by addition of protein A beads, and the whole complex was incubated overnight at 4°C with rotation. After washing, the samples were subjected to 10% PAGE, and Western blotting was performed with indicated antibody.
COS-1 cells were cotransfected with TSSK2/pTarget and SPAG16/pTarget. Forty-eight hours after transfection, the cells were harvested into immunoprecipitation buffer, and Co-IP was performed as previously described [3] with C- or N-terminal (negative control) anti-SPAG16 polyclonal antibodies. After washing, the complexes were resuspended in 20 mM Tris-HCl, pH 7.5; 10 mM MnCl2; and 10 mM MgCl2 containing 5 µCi [
-32P]ATP and incubated at 37°C for 15 min. Labeled proteins were resolved by SDS-PAGE, and dried gels were exposed directly to x-ray films. To determine whether N-SPAG16 and full-length SPAG16 can be phosphorylated by purified recombinant TSSK2, N-SPAG16/pTarget and SPAG16/pTarget plasmids were transfected into COS-1 cells, and the two proteins were immunoprecipitated with an anti-N-terminus SPAG16 antibody. Half of the sample was subjected to Western blotting for the confirmation of the presence of the two proteins, and the rest was incubated with 2 µg purified mouse TSSK2 protein (Invitrogen) at 37°C for 15 min in the same buffer as described above containing labeled ATP, labeled proteins were resolved by SDS-PAGE, and the dried gels were exposed to x-ray films.
Total testicular RNA was isolated from sexually mature wild-type and Spag16L-null mice. Northern blots were performed with specific mouse Tssk2, Spag16L, and Akap4 probes.
A knockout model, Spag16tm2Jfs, referred to as Spag16L, was generated previously in our laboratory [6]. Five- to six-month-old male mice were used for the study.
Potential Phosphorylation Sites of SPAG16L Proteins
Identification of TSSK2 in a yeast two-hybrid screen with SPAG16 as bait suggested that SPAG16 proteins might be phosphorylated in vivo. To locate potential phosphorylation sites, the SPAG16L protein sequence (Fig. 1, upper panel) was analyzed by NetPhos2.0 Server (http://www.cbs.dtu.dk/services/NetPhos/) [29]. SPAG16L potentially contains 30 serine, 11 threonine, and 7 tyrosine phosphorylation sites (Fig. 1, lower panel). These potential phosphorylation sites are distributed throughout the protein. To predict potential kinases that might phosphorylate these sites, the SPAG16L protein sequence was analyzed using the NetPhosK1.0 Server (http://www.cbs.dtu.dk/services/NetPhosK/) [30]. Potential kinases are listed in Supplemental Table 1 (available online at www.biolreprod.org). Besides TSSK2, we also identified another kinase in our yeast two-hybrid screen (Table 1), GSK3B, but we failed to confirm the interaction of this kinase with SPAG16 in coimmunoprecipitation experiments (data not shown).
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SPAG16L Is Phosphorylated In Vivo
To investigate whether SPAG16L protein undergoes posttranslational modification, especially phosphorylation in vivo, epididymal sperm extracts were subjected to 2D gel electrophoresis followed by Western blotting (2D Western) with an antibody against N-terminus of SPAG16L. A major signal was observed at a spot of 71 kDa and isoelectric point (pI) of 8.0, the predicted size for SPAG16L, whereas a more acidic 71-kDa immunoreactive species was also identified at around pI 4.0 (Fig. 2, upper panel), indicating that SPAG16L undergoes posttranslational modification. After the protein samples were treated with CIP, the 71-kDa signal at pI 4.0 disappeared (Fig. 2, lower panel), suggesting that this acidic isoform of SPAG16L is phosphorylated in vivo.
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Interaction of SPAG16L and TSSK2
From our original yeast two-hybrid screen with the C-terminus of SPAG16 as bait, a number of candidates were identified. Some have been confirmed by Co-IP experiment, such as MEIG1 and SPAG6 (Table 1). TSSK2 was another potential molecule that might associate with SPAG16. To further study the interaction between the two proteins, TSSK2 mammalian expression plasmids were constructed. When TSSK2/DsRed-N1 or TSSK2/pEGFP-N2 was transfected into CHO cells, TSSK2 protein was expressed (Fig. 3Aa), and the protein was localized in the cytoplasm (Fig. 3Ab). To explore the interaction of TSSK2 and SPAG16L, SPAG16L/pTarget and TSSK2/pTarget plasmids were cotransfected into COS-1 cells, and Co-IP was performed. The cell lysates were pulled down with an anti-TSSK2 antibody, and Western blotting was carried out with an anti-N-terminus SPAG16L antibody. SPAG16L was immunoprecipitated by the anti-TSSK2 antibody (Fig. 3B).
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The association of TSSK2 and SPAG16L in vivo was also investigated. Immunoprecipitates were isolated from testis extracts with anti-TSSK2 or preimmune serum and analyzed on Western blots with anti-N-terminus SPAG16L antibody, revealing that 71-kDa SPAG16L was present in the TSSK2 immune but not the preimmune precipitates (Fig. 3C). Even though 35-kDa SPAG16S also contains the same WD repeats as 71-kDa SPAG16L, the protein was not pulled down by anti-TSSK2 antibody (data not shown).
As described previously, SPAG16L protein was also localized in the cytoplasm of SPAG16L/pEGFP-N2 plasmid-transfected CHO cells [2], similar to the distribution pattern of TSSK2. To further study the association of the two proteins, TSSK2/DsRed-N1 and SPAG16L/pEGFP-N2 were cotransfected into CHO cells, and the localization of each protein was examined by confocal microscopy. The two proteins colocalized in the cytoplasm (Fig. 4, upper two panels). As controls, empty DsRed-N1 plasmid was cotransfected with empty pEGFP-N2 or SPAG16L/pEGFP-N2 plasmid. Both GFP and DsRed proteins were distributed throughout the cells (Fig. 4, panel 3). Even though SPAG16L/GFP and DsRed are coexpressed in cells, they had different localizations. SPAG16L/GFP was present in the cytoplasm, whereas DsRed was in the cytoplasm and nuclei (Fig. 4, panel 4).
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C-Terminal WD Repeats Mediate the Association of TSSK2 and SPAG16L
To further investigate the domain that mediates the interaction, the cDNA encoding the 36-kDa N-terminus of the L isoform of Spag16 was cloned into pEGFP-N2 and pcDNA3 to create expression plasmids. Both plasmids expressed the N-terminus SPAG16L protein in CHO cells, as evaluated by Western blotting (Fig. 5Aa).
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Chinese hamster ovary cells were cotransfected with TSSK2/pTarget and N-SPAG16/pEGFP-N2 plasmids, and Co-IP was performed. The anti-TSSK2 antibody immunoprecipitated the TSSK2 protein (Fig. 5Ab) but not the N-SPAG16 protein (Fig. 5Ac), suggesting that TSSK2 does not associate with the N-terminus of SPAG16L.
The C-terminus domain of SPAG16L contains seven WD repeats, which mediate protein-protein interactions. To investigate whether TSSK2 interacts with this domain, TSSK2/pEGFP-N2 or TSSK2/pTarget and C-SPAG16/pTarget were cotransfected into CHO cells, and Co-IP experiments were carried out. The complex was pulled down with an anti-C-terminus SPAG16 antibody, and Western blot was performed with an anti-GFP antibody (Fig. 5Ba) or anti-TSSK2 antibody (Fig. 5Bb). In Figure 5Ba, 66-kDa TSSK2/pEGFP fusion protein, but not the 26-kDa GFP protein, was selectively pulled down from the complex, indicating an interaction of TSSK2 with the C-terminus domain of SPAG16L. Similarly, as shown in Figure 5Bb, when the cells were cotransfected with TSSK2/pTarget and C-SPAG16/pTarget, the TSSK2 protein, although lacking a tag, can still be pulled down with anti-C-terminus SPAG16 antibody.
SPAG16 Is Phosphorylated by TSSK2 In Vitro
To examine whether SPAG16L can be phosphorylated by TSSK2, CHO cells were cotransfected with TSSK2/pTarget and SPAG16L/pTarget plasmids. After immunoprecipitation with anti-TSSK2 antibody, a Western blot was performed with half of the complex using anti-N-terminus of SPAG16L antibody (Fig. 6, upper panel), and another half of the complex was incubated with kinase buffer and [-32P]ATP (Fig. 6, lower panel). Immunoprecipitates of the labeled cells with anti-TSSK2 antibody revealed a major band corresponding to 71-kDa SPAG16L, indicating that SPAG16L is phosphorylated in this in vitro model (Fig. 6).
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The phosphorylation of SPAG16L and N-SPAG16L by recombinant TSSK2 protein was also investigated. Chinese hamster ovary cells were transfected with SPAG16L/pTarget or N-SPAG16/pTarget plasmids. After immunoprecipitation with an anti-N-terminus SPAG16 antibody, a Western blot was performed with half of the complex using anti-N-terminus of SPAG16L antibody (Fig. 7, A and B), and half the complex was incubated with purified recombinant TSSK2 protein in the presence of kinase buffer and [-32P]ATP (Fig. 7C). SPAG16L but not N-SPAG16 was phosphorylated by TSSK2.
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TSSK2 Is Reduced in the Testis of Most Spag16L-Null Mice
A knockout model, Spag16tm2Jfs (referred to as Spag16L), previously generated in our laboratory, was employed to study TSSK2 expression in Spag16L mutant testes. Of the 12 null mice analyzed, TSSK2 protein was undetectable or markedly diminished in five of them, reduced by approximately 50% in four, and unaltered in three. However, TSSK2 was present in all eight wild-type mice analyzed (Fig. 8A). Even though the TSSK2 protein was missing in some of the null mice, Tssk2 mRNA was observed in all Spag16L-null mice analyzed (Fig. 8B).
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Protein phosphorylation is the most common posttranslational protein modification in eukaryotes and a fundamental mechanism for the direct or indirect control of all cellular processes. For example, protein phosphorylation is involved in the control of cell division, metabolic activity, cell adhesion and migration, cell-to-cell communication, and signal transduction [31, 32]. During spermatogenesis, protein phosphorylation has been shown to play a role in several processes, including gene transcription [33] and chromatin remodeling [34]. During fertilization, spermatozoa undergo a series of changes before and during egg binding that are related to acquisition of the ability to fuse with the oocyte, including major changes in the phosphorylation of spermatozoa proteins. Increased protein phosphorylation is associated with capacitation, hyperactivated motility, zona pellucida binding, acrosome reaction, and sperm-oocyte binding and fusion [35–39]. Several flagellar proteins are known to be phosphorylated during capacitation, including the fibrous sheath proteins CABYR and AKAPs 3 and 4 [40–43].
Phosphorylation of flagellar proteins is linked to hyperactivated motility in spermatozoa [37]. SPAG16L is localized in the central apparatus of the axoneme, and this protein could be one of the proteins phosphorylated in vivo in preparation for fertilization. Through searching the potential sites for phosphorylation, it was found that SPAG16L contains multiple phosphorylation sites, and 2D Western blotting confirmed that SPAG16L is phosphorylated in vivo.
Proteins are phosphorylated by protein kinases, which include serine/threonine or tyrosine kinases. From previous yeast two-hybrid screening, TSSK2 was identified preliminarily as a potential SPAG16L binding partner, and the present study confirmed that TSSK2 and SPAG16L are coimmunoprecipitated from the testis as well as from cotransfected cells in culture. Moreover, TSSK2 is able to phosphorylate SPAG16L in vitro. Taken together, the yeast two-hybrid results and direct biochemical coisolation of these proteins in complex lead to the conclusion that SPAG16L is a TSSK2 substrate.
TSSK2 appears to bind SPAG16L via the C-terminus domain, which contains WD repeats. Two lines of evidence support this conclusion. First, TSSK2 and SPAG16L were initially identified by a yeast two-hybrid screening with C-terminus WD repeats domain as the bait. Second, TSSK2 associates with C-SPAG16 rather than N-SPAG16 in Co-IP experiments. The fact that TSSK2 interacts with the C-terminus of SPAG16L and is capable of phosphorylating it, at least under in vitro conditions, raises the possibility that TSSK2 also can phosphorylate SPAG16S. SPAG16S is localized to the nucleus of germ cells, where it is thought to function as a transcription factor. If SPAG16S is phosphorylated by TSSK2, this event would likely occur in the cytoplasm. However, SPAG16S was not pulled down in the Co-IP experiment with testis extract using anti-TSSK2 antibody. Despite this negative result, future studies should investigate the possibility of TSSK2 phosphorylation of SPAG16S and its impact upon the distribution and function of SPAG16S.
Even though TSSK1 and TSSK2 share 83% identity in protein sequences, and TSSK2 also shares a homolog with other testis-specific kinases, we did not identify other testis-specific kinases from an initial yeast two-hybrid screen. This could reflect the fact that SPAG16L is not a substrate of those kinases, or the screen failed to detect them as potential binding partners. Thus, we cannot exclude the possibility at this juncture that other members of the TSSK family also phosphorylate SPAG16L.
Interestingly, in the testes of 75% of SPAG16L-deficient mice, TSSK2 protein was either absent or significantly reduced, although TSSK2 transcripts were present at normal levels. This may suggest that SPAG16L protein either feedbacks on TSSK2 translation by some as-yet undiscovered mechanism, or that SPAG16L is required to sustain TSSK2 translation or protein integrity. The variation in this phenotype may reflect the mixed background (SV129 and C57) of the mutant mice. We have observed such variation in other knockout models that we have created targeting other central apparatus protein genes [44]. In any case, the absence of TSSK2 in more than half of these SPAG16L-null animals indicates that SPAG16L/TSSK2 coexpression is not a constant and that the association between these proteins may not be stable in vivo.
It has been shown that a 65-kDa protein, TSKS, is a substrate of TSSK2 [26]. TSKS localizes predominantly to the centrioles of human sperm; however, TSSK2 is localized to the sperm neck, midpiece of the sperm tail, and the equatorial segment (J.C. Herr, personal communication). The broader localization of TSSK2 compared to TSKS implies that additional substrates for the enzyme may exist [45]. The fact that both TSSK2 and SPAG16L are present in the sperm tail is consistent with the notion that SPAG16L is another substrate for TSSK2 in vivo, and that TSSK2 might, through phosphorylation of SPAG16L, regulate sperm motility. Tssk1/2 mutant mice have been generated recently, in addition to defect in spermatogenesis, another major phenotype is severe sperm motility defects in the mutant mice (J.C. Herr, personal communication).
Even though it is not clear which protein, TSSK1 or TSSK2, is responsible for these phenotypes, phosphorylation of some sperm proteins, such as SPAG16L, might be impaired, which might be predicted to result in sperm motility defects.
In conclusion, we have identified a new substrate of TSSK2 in the sperm tail, leading to the conclusion that TSSK2 might regulate sperm motility through phosphorylation of sperm tail protein(s), one of which is SPAG16L.
FOOTNOTES
1Supported by National Institutes of Health grants HD37416, HD06724, and TW06223-01. 
Correspondence: 2Zhibing Zhang, Department of Obstetrics & Gynecology, Virginia Commonwealth University, 11028A Sanger Hall, 1101 East Marshall St., Richmond, VA 23298. FAX: 804 828 0573; e-mail: zzhang4{at}vcu.edu
Received: 26 October 2007.
First decision: 7 December 2007.
Accepted: 17 March 2008.
REFERENCES
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