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Posttranslational modification of proteins by phosphorylation is involved in regulation of sperm function. Protein phosphatase 1 gamma isoform 2 (PPP1CC_v2) and protein YWHA (also known as 14-3-3) are likely to be key molecules in pathways involving sperm protein phosphorylation. We have shown that phosphorylated PPP1CC_v2 is bound to protein YWHAZ in spermatozoa. In somatic cells, protein YWHA is known to bind a number of phosphoproteins involved in signaling and energy metabolism. Thus, in addition to PPP1CC_v2, it is likely that sperm contain other YWHA-binding proteins. A goal of the present study was to identify these sperm YWHA-binding proteins. The binding proteins were isolated by affinity chromatography with GST-YWHAZ followed by elution with a peptide, R-11, which is known to disrupt YWHA complexes. The YWHA-binding proteins in sperm can be classified as those involved in fertilization, acrosome reaction, energy metabolism, protein folding, and ubiquitin-mediated proteolysis. A subset of these putative YWHA-binding proteins contain known amino acid consensus motifs, not only for YWHA binding but also for PPP1C binding. Identification of sperm PPP1CC_v2-binding proteins by microcystin-agarose chromatography confirmed that PPP1CC_v2 and YWHA interactomes contain several common proteins. These are metabolic enzymes phosphoglycerate kinase 2, hexokinase 1, and glucose phosphate isomerase; proteins involved in sperm-egg fusion; angiotensin-converting enzyme, sperm adhesion molecule, and chaperones; heat shock 70-kDa protein 5 (glucose-regulated protein 78 kDa; and heat shock 70-kDa protein 1-like. These proteins are likely to be phosphoproteins and potential PPP1CC_v2 substrates. Our data suggest that in addition to potential regulation of a number of important sperm functions, YWHA may act as an adaptor molecule for a subset of PPP1CC_v2 substrates.
Immotile testicular sperm undergo a complex maturation process in the epididymis. Following passage through the epididymis, sperm attain the capacity for motility and the ability to bind to eggs. The ability to fertilize eggs further develops in the female reproductive tract, where sperm undergo capacitation before they undergo the acrosome reaction, penetrate, and fertilize eggs. Changes in protein phosphorylation at least partially mediate sperm motility, capacitation, acrosome reaction, and sperm-egg fusion . Protein phosphatases and kinases together control levels of protein phosphorylation. Numerous studies have explored the role of protein kinases in sperm function [2–5]. However, the role of protein phosphatases in spermatozoa is just beginning to emerge. We have shown that the PPP1C isoform, PPP1CC_v2, is a key enzyme involved in sperm motility and sperm morphogenesis. The role of PPP1C in sperm motility was first identified by the potent motility effects of the PPP1C inhibitors calyculin A and okadaic acid . Male mice lacking the gene for PPP1CC are infertile , and spermatozoa from these animals display defects in head shape, midpiece, and outer dense fibers . In somatic cells, PPP1C activity is regulated by phosphorylation and by its association with binding proteins [9, 10]. How exactly PPP1CC_v2 is regulated in the testis and sperm is not known. Purification of PPP1CC_v2 in sperm extracts showed that YWHAZ is one of its binding proteins . In sperm, YWHAZ binds to a distinct pool of phosphorylated PPP1CC_v2 .
The family of YWHA proteins are highly conserved acidic proteins. They are present in a wide variety of cells and organisms ranging from plants to mammals. Nearly 100 YWHA-binding partners have been identified in somatic cells using affinity chromatography coupled with proteomic analysis [12–15]. Many, but not all, YWHA-binding proteins contain phosphoserine and phosphothreonine residues within the amino acid sequence motifs RSXpSXP and RX(Y/F)XpSXP, where pS is phosphorylated serine and X is any amino acid [16, 17]. The protein phosphatases PPP1C and PPP2C are regulators of YWHA binding due to their ability to dephosphorylate its binding partners. Protein YWHA is thought to act as an adaptor protein in cellular signaling and metabolism. YWHA proteins appear to regulate and coordinate a diverse array of cellular processes, such as cell cycle progression, apoptosis, protein trafficking, cytoskeleton rearrangements, metabolism, and transcriptional regulation of gene expression . Homozygous null mutants for the Leonardo gene, 14-3-3ζleo in Drosophila melanogaster die as mature embryos with defects in synaptic vesicle dynamics , whereas low levels of expression of Leonardo in mushroom bodies result in olfactory learning defects . In yeast Saccharomyces cerevisiae, deletion of both homologues of YWHA, BMH1, and BMH2 results in lethality . Gene knockout analysis in mice showed that YWHAE is essential for normal brain development .
We first identified protein YWHA as a binding partner of sperm PPP1CC_v2 by column purification and microsequencing . Preliminary studies at that time showed that in addition to phopsho-PPP1CC_v2, sperm contain other possible YWHA-binding proteins. This raised the possibility that YWHA could be involved in regulation of signaling and metabolism by virtue of its binding to PPP1CC_v2 and other sperm phosphoproteins. We thus undertook a comprehensive proteomic analysis to identify the YWHA interactome in spermatozoa, the objective of study reported here. Since many of the binding partners of YWHA are phosphorylated, we also hypothesized that our approach to isolate YWHA-binding proteins also could result in the identification of phosphoproteins that could be potential substrates for sperm PPP1CC_v2 and phospho-proteins involved in the regulation male gamete function.
Testes with intact tunica from mature bulls were obtained from a local slaughterhouse. Spermatozoa were isolated from the caput and caudal epididymis and washed twice in a sperm diluent buffer (10 mM/l Tris-HCl [pH 7.2] containing 10 mM KCl, 120 mM NaCl, and 5 mM MgSO4). Sperm pellets were resuspended in RIPA buffer (0.05 M Tris-HCl [pH 7.4], 0.15 M NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, and 1 mM EDTA) containing Roche complete protease inhibitor tablet (one tablet in 50 ml RIPA buffer) and phosphatase inhibitors (10 mM NaF, 10 mM β-glycerolphosphate, 5 mM sodium pyrophosphate, and 50 mM activated sodium orthorvanadate) and kept on ice for 45 min. The sperm suspensions then were centrifuged at 16000 × g for 15 min. The supernatants were centrifuged further at 100000 × g for 60 min. The 100000 × g supernatants were used to isolate the YWHA-binding proteins.
Samples boiled in Laemmli sample buffer were separated by 12% SDS-PAGE and electrophoretically transferred to Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking nonspecific binding sites with 5% nonfat milk in Tris-buffered saline (TBS; 25 mM Tris-HCl [pH 7.4], 0.15 M NaCl, containing 0.1% Tween 20), blots were incubated with one of the following primary antibodies: mouse monoclonal anti-GSK3A/B (ab45383; 1:5000; Abcam), goat polyclonal anti-angiotensin-converting enzyme (anti-ACE; 1:10000; generous gift from Dr. Ganes C. Sen, Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH), anti-phospho GSK-3α/β serine 21/9 (1:1000; 9327; Cell Signaling Technology), rabbit polyclonal anti-PPP1CC_v2 (1:2000), rabbit polyclonal anti-glutathione S-transferase (anti-GST; 1:2000; 06332; Millipore), rabbit polyclonal anti-phosphoserine YWHA-binding motif (1:1000; 9601; Cell Signaling Technology), rabbit polyclonal anti PGK2 (1:5000; generous gift from Dr. Deborah A. O'Brien, Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC), and rabbit polyclonal anti-YWHA (1:2000; 51–0700; Invitrogen). Following incubation with the primary antibodies overnight at 4°C, the membranes were washed and incubated for 1 h at room temperature with anti-mouse (1:2000) for anti-GSK3A/B, anti-goat (1:1000) for ACE antibody, and anti-rabbit (1:2000) for all other antibodies. Blots were washed and developed by enhanced chemiluminescence.
Complementary DNA for YWHAZ subcloned in pcDNA 3.1/V5-His- TOPO was a gift from Helen M. Piwnica-Worms (Washington University, St. Louis, MO). The cDNA for YWHAZ was amplified by the forward primer containing a BamHI restriction site preceding the start codon and a reverse primer containing an EcoRI site following the stop codon. The PCR product following restriction enzyme digestion was subcloned into the pGEX 4T-2 vector (GE Healthcare Biosciences) containing a GST tag. Escherichia coli (BL21) cells were transformed with plasmid containing GST (empty vector pGEX 4T-2) or GST-YWHAZ. Bacterial cultures were incubated at 37°C until the optical density at 600 nm reached 0.5–2 units. Following addition of 75 mM isopropyl-1-thio-β-d-galactopyranoside, the incubation was continued for another 4 h. The cultures were transferred to appropriate centrifuge containers and centrifuged at 6000 × g at 4°C to sediment the cells. After resuspension in PBS containing protease inhibitors (Roche Applied Sciences), the pellet was disrupted by sonication. Lysed bacteria were centrifuged at 16000 × g for 10 min, and the supernatant was incubated with prewashed Glutathione Sepharose 4B beads (GE Healthcare Life Sciences) for 2 h. The beads along with supernatant were transferred to a disposable column (Bio-Rad Laboratories). The beads were washed with PBS (three times the bed volume) to remove proteins bound nonspecifically to the beads. Glutathione S-transferase and GST-YWHAZ bound to beads were eluted with GST elution buffer (20 mM glutathione in 10 mM Tris-HCl [pH 8.0]). The eluted proteins were dialyzed overnight in PBS containing 1 mM PMSF. Sodium dodecyl sulfate-PAGE followed by Coomassie blue staining of gels containing purified GST and GST-YWHAZ showed single protein bands migrating at 27 kDa and 55 kDa, respectively.
Bovine caput and caudal spermatozoa samples boiled in Laemmli sample buffer were separated by 12% SDS-PAGE and electrophoretically transferred to Immobilon-P PVDF membrane. Proteins were denatured by incubation in a denaturation buffer (50 mM Tris-HCl, 6 M guanidine HCl, 6.25 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.05% Tween 20) for 1 h and then renatured by incubating in renaturation buffer (denaturation buffer without guanidine HCl and Tween 20). Membranes were blocked with 5% nonfat milk in TTBS (0.1% Tween-20 TBS) to prevent nonspecific binding and were incubated with 0.1 μg/ml GST-YWHAZ and 1 mg/ml BSA overnight. Control samples were run with the same concentrations of proteins and incubated with equimolar concentrations of GST alone overnight. Membranes were washed three times for 10 min each and probed with anti-GST primary antibody as described above.
This protocol was adapted from Meek et al. . Purified GST or GST-YWHAZ was covalently coupled to activated CH-Sepharose (GE Healthcare Life Sciences). GST-YWHAZ or GST (18 mg each) was incubated at 4°C for 4 h with 6 ml CH-Sepharose in coupling buffer (0.1 M NaHCO3 [pH 8.0] and 0.5 M NaCl). Glutathione S-transferase or GST-YWHAZ protein/CH-Sepharose mixtures were transferred to disposable columns. The GST and GST-YWHAZ columns were washed with 36 ml (six bed volumes) of cold coupling buffer. To block the remaining active groups, 18 ml (three bed volumes) blocking buffer (0.1 M Tris-HCl [pH 8.0]) was passed through the beads, and the column was incubated further with 12 ml blocking buffer for 1 h. The column was washed with five bed volumes (30 ml) of low-pH wash buffer and then with 30 ml high-pH wash buffer to remove excess GST and GST-YWHAZ. This was followed by three more cycles of low- and high-salt washes. The columns were stored in storage buffer (1 M Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% NaN3 with protease inhibitors) for 24 h. Bovine caudal sperm extracts containing approximately 60–70 mg protein were incubated with the GST-sepharose for 1 h at 4°C to preclear proteins that may bind to GST or the sepharose matrix. The precleared extract was incubated with the GST-YWHAZ column for 3 h at 4°C. The flow-through fraction from the column was collected and the column was washed extensively with 60 ml low-salt buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl) and 60 ml high-salt buffer (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl). Proteins bound to the column were eluted with the R-11 peptide, RDLSWLDLEAN (0.75 mM), in 10 ml high-salt buffer. R-11 is a truncated form of the R-18 peptide and is able to disrupt YWHA interaction with its binding proteins . The eluate was concentrated to 500 μl using an Amicon-15 filter (Millipore). The concentrated proteins were subjected to 12% SDS-PAGE, stained with colloidal Coomassie blue (Proteome Systems), and microsequenced as described below.
Microcystin-agarose (Millipore) was washed three times with ice-cold PBS. Microcystin-agarose was incubated at 4°C for 1 h with 1 ml bovine sperm extracts (1 mg protein/ml) prepared in RIPA buffer with protease inhibitors. Microcystin-agarose then was centrifuged at 16000 × g for 1 min, and supernatant was removed. Nonspecifically bound proteins were removed by washing the beads five times with RIPA buffer. The proteins and complexes bound to microcystin were eluted by boiling the beads with 2× SDS sample buffer for 5 min. The extracted proteins were subjected to 12% SDS-PAGE, stained with colloidal Coomassie blue (Proteome Systems), and microsequenced as described below.
Bovine caudal sperm extracts were prepared in RIPA buffer in the presence of protease and phosphatase inhibitors as described above. Purified rabbit immunoglobulin G (IgG; Jackson ImmunoResearch) or anti-PPP1CC_v2 or YWHAB (628; Santa Cruz Biotechnology) antibodies at a final concentration of 2.5–4 μg were incubated with 250 μl sperm extracts for 2 h at 4°C. Protein A sepharose (35 μl; 50% slurry) was washed three times with TTBS solution and incubated with antibody-sperm extract mixture at 4°C for 1 h. The mixture was spun down at 900 × g, and beads were washed five times with buffer containing 50 mM Tris-HCl, 150 mM NaCl, and 0.1% Nonidet P-40. The beads were boiled in 2× sample buffer for 5 min, and the proteins were analyzed by Western blot analysis.
Sixteen bands from Coomassie blue-stained gels were cut, followed by in-gel proteolysis. The peptides released by proteolysis were extracted from the gel in two aliquots of 30 μl of 50% acetonitrile and 5% formic acid. The extracts were combined and evaporated to less than 10 μl in a Speedvac (Savant Instruments Inc.) and then resuspended in 1% acetic acid to a final volume of approximately 30 μl for liquid chromatography-mass spectrometry (LC-MS). The LC-MS system was a Finnigan LCQ ion trap mass spectrometer system (Thermo Scientific Inc.). The high-performance liquid chromatography column was a self-packed 12 cm × 75 μm inner diameter Phenomenex Jupiter C18 reversed-phase capillary chromatography column. The extracts (2 μl) were injected, and the peptides eluted from the column by an acetonitrile/0.05 M acetic acid gradient at a flow rate of 0.3 μl/min were introduced into the source of an on-line mass spectrometer. The microelectrospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent multitask capability of the instrument acquiring full-scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans. This mode of analysis produced approximately 2500 collision-induced dissociation (CID) spectra of ions ranging in abundance over several orders of magnitude. The data were analyzed using all CID spectra collected in the experiment to search the National Center for Biotechnology Information (NCBI) bovine RefSeq database with the search program Mascot. Additional searches against the NCBInr database with a mammalian taxonomy filter were also done. All matching spectra were verified by manual interpretation. The interpretation process was aided by additional searches using the program Sequest. The Eukaryotic linear motif server at www.elm.eu.org. was used for identification of consensus motifs 1, 2, and 3 for YWHA binding and RVXF motif for PPP1C binding.
The presence of YWHA-binding proteins was determined by Western blot analysis of caput and caudal sperm extracts probed with a phosphoserine YWHA-binding motif antibody. This antibody apparently recognizes motifs containing phosphoserine within a consensuses sequence containing proline and arginine . Immunoblotting revealed at least three distinct bands at 100 kDa, 50 kDa, and 20 kDa in both caput and caudal sperm extracts (Fig. 1A). Immunoreactivity of caudal sperm extracts appears stronger than caput sperm extracts, indicating that changes in phosphorylation levels of putative YWHA-binding proteins occur during epididymal sperm maturation.
Overlay analysis with recombinant GST-YWHAZ was used to further confirm the presence of YWHA-binding proteins. Caput and caudal sperm extracts following SDS-PAGE were transferred to PVDF membranes. The membranes were incubated with GST-YWHAZ or with GST alone as control. After washing, the membranes were probed with GST antibody. Data in Figure 1B show the presence of a number of proteins binding to GST-YWHAZ in both caput and caudal spermatozoa. The proteins apparently bound specifically to YWHAZ, since GST, used as control, did not show any immunoreactive bands. Results from overlay analysis showed the presence of a number of putative YWHA-binding proteins (20–100 kDa) in epididymal sperm extracts (Fig. 1B).
Next, we used an affinity chromatography procedure to isolate sperm YWHA-binding proteins (Fig. 2A). CH-Sepharose was coupled to GST-YWHAZ or GST alone. Covalent coupling was preferred to immobilization with glutathione, since the coupled matrix is more stable to stringent washes required to remove proteins bound nonspecifically. Bovine caudal sperm extracts (60–70 mg total protein) were prepared in RIPA buffer containing protease and phosphatase inhibitors, as outlined in Materials and Methods. We first incubated the extracts with GST-coupled Sepharose to remove proteins that may bind to the Sepharose matrix and to GST. Extracts precleared with the GST column were subsequently incubated with GST-YWHAZ sepharose. Proteins that may have bound nonspecifically to the column were removed by extensive washing with low- and high-salt buffers, as described in Materials and Methods. Proteins presumably bound to YWHAZ then were eluted with R-11 peptide. The peptide R-11 binds competitively to YWHA, thus displacing the bound proteins . Proteins eluted from the affinity column were concentrated and analyzed by SDS-PAGE. Affinity chromatography isolated a limited set of proteins compared with those present in the original sperm lysate (Fig. 2B).
Next, the eluate from the YWHAZ affinity column was subject to Western blot analysis probed with PPP1CC_v2 and phosphoserine YWHA-binding motif antibodies. Both PPP1CC_v2 (Fig. 3A) and proteins reacting with phosphoserine YWHA-binding motif antibodies (Fig. 3B) were eluted from the column. The presence of PPP1CC_v2 in the eluate is further validation of our observation that PPP1CC_v2 is bound to YWHA . Sperm contain the signaling enzyme GSK3 . The enzyme GSK3B is a known binding partner of YWHA in somatic cells . Therefore, it was of interest to see whether this binding also occurs in spermatozoa. Western blot analysis showed that both alpha and beta isoforms of sperm GSK3 were found in the YWHAZ affinity column eluate (Fig. 4A). No signal was detected in the YWHAZ eluate with phospho-GSKA/B antibody, which detects phosphorylated forms of GSK3A and GSK3B, although input contained both phosphorylated GSK3 isoforms (data not shown). In addition to PPP1CC_v2 and GSK3, the eluate fraction contained PGK2 (Fig. 4B) and radial spoke head 1 homologue (RSPH1), also known as testis-specific gene 2 (TSGA-2; Fig. 4C). PGK2 is a glycolytic enzyme, whereas RSPH1 is a testis-specific protein with a suspected role in sperm motility and sperm-egg interactions . We analyzed for PGK2, since it was one of the proteins detected by microsequencing (Table 1), and RSPH1, since this protein coeluted with YWHA during column fractionation of sperm extracts (data not shown).
Proteins from the eluate of the YWHAZ column separated by SDS-PAGE were analyzed by LC-MS. Microsequencing revealed the presence of 35 YWHA-interacting proteins. The proteins identified by microsequencing could be subdivided into six broad categories (Table 1). A majority of the proteins are involved in sperm-egg interactions and sperm metabolism. In addition, chaperones, proteins involved in the ubiquitin pathway, and antioxidants were detected. Proteins thought to be involved in sperm-egg interactions include ACE, sperm adhesion molecule 1 (SPAM1), and zona pellucida-binding protein (ZPBP) [28–30]. Acrosin and pro-acrosin-binding protein precursor sp32 are proteins likely to be involved in acrosome reaction [31, 32]. The sperm YWHA interactome also included glycolytic enzymes phosphoglycerate kinase 2 (PGK2), hexokinase 1 (HK1), and glucose phosphate isomerase (GPI). A number of mitochondrial enzymes, including ATP synthase H+ transporting complex O subunit (ATP5O), were also identified. Heat shock proteins—heat shock 70 kDa protein 5 (HSPA5), heat shock 70 kDa protein 9 (HSPA9), and heat shock 70 kDa protein 1-like (HSPA1L)—and proteins involved in ubiquitin pathway, such as kelch-like 10 (KLHL10) and cullin 3 (CUL3) , also were among the sperm YWHA-interacting proteins.
Next, we analyzed the amino acid sequences to examine whether consensus sequences for YWHA binding are present in the list of sperm YWHA-binding proteins. Approximately 68% of the proteins had at least one of the common YWHA-binding domains. Interestingly, as seen in Table 1, a number of YWHA-binding proteins also contained a consensus sequence (the RVXF motif) for PPP1C binding.
Microcystins are cyclic heptapeptides, known to bind to the catalytic subunit of the phosphatases PPP1C and PPP2C . Microcystin affinity chromatography can be used to isolate PPP1C-binding proteins . Bovine caudal sperm extracts containing protease inhibitors were incubated with microcystin agarose. To block nonspecific binding sites, the microcystin beads were preincubated with a synthetic peptide corresponding to the C-terminal sequence in PPP1CC_v2. After this, the microcystin beads were incubated with caudal sperm extracts. The proteins bound to the beads after extensive washing were eluted by boiling the beads with SDS sample buffer. Eluted proteins were subject to SDS-PAGE, followed by Coomassie blue staining and LC-MS. A total of 56 proteins were identified. The proteins that were common to the YWHA and PPP1CC_v2 interactomes are proteins presumed to be involved in sperm-egg interactions: ACE and SPAM1, the glycolytic enzymes PGK2, HK1, and GPI, chaperones, HSPA5 and HSPA1L, and the protein that is implicated in the ubiquitin pathway, CUL3 (Table 2).
It should be noted that the potential YWHA-binding proteins isolated during affinity purification likely represent endogenous interacting partners; however, some of these proteins may be proteins that associate with column-immobilized YWHA during affinity purification. Immunoprecipitation could be used to detect protein YWHA complexes in cell lysates. Since we had antibodies in hand for ACE and PGK2, we analyzed whether ACE and PGK2 are bound to YWHA and PPP1CC_v2 in sperm lysates. We immunoprecipitated YWHA and PPP1CC_v2 from sperm extracts using their respective antibodies. Western blot analysis showed that ACE and PPP1CC_v2 could be coprecipitated with YWHA antibodies (Fig. 5) and YWHA and ACE immunoprecipitated with PPP1CC_v2 antibodies from sperm extracts (Fig. 6). In contrast, PGK2 could be detected in PPP1CC_v2 immunoprecipitates but not in YWHA immunoprecipitates (Fig. 6A).
Spermatozoa are terminally differentiated cells with little RNA transcription and protein translation. Thus, posttranslational modifications of preexisting proteins, especially protein phosphorylation, play key roles in the development of motility and fertilization competence of spermatozoa. Protein YWHA is known to bind to a variety of phosphoproteins in somatic cells. Thus, YWHA participates in variety of cell regulatory processes. To date, more than 100 YWHA-binding proteins have been identified in somatic cells [12–15, 35]. Protein YWHAZ is a PPP1CC_v2-binding protein in bovine epididymal spermatozoa . Immunoreactivity to phosphoserine YWHA-binding motif antibody suggests the presence of other YWHA-binding proteins in spermatozoa (Fig. 1A). Interestingly, the phosphorylation status of putative YWHA-binding proteins in caudal spermatozoa appears higher than caput spermatozoa. Far-Western analysis (Fig. 1B) also confirmed the presence of YWHA-binding proteins in spermatozoa. These observations suggest that phosphorylation of sperm proteins increases during epididymal sperm maturation. It would be interesting to determine how these phosphorylation changes may relate to the development of sperm function in the epididymis.
We used GST-YWHAZ affinity chromatography for the isolation of YWHA-binding proteins in spermatozoa. The zeta isoform of YWHA was chosen in our experiments, since this was the isoform identified by microsequencing following purification of PPP1CC_v2 from bovine sperm extracts . However, the mouse proteome, published recently, includes zeta, beta, and epsilon isoforms of YWHA . It should be noted that YWHA has been shown to interact with its binding partners in an isoform nonspecific manner, with few exceptions [37, 38]. Thus, it is unlikely that there may be major differences in the binding profile for the different YWHA isoforms in sperm. However, we cannot rule out the possibility that some proteins with higher affinity binding for YWHAB beta and YWHAE isoforms could have been missed in our study. Another possibility is that heterodimers between the different YWHA isoforms also could bind unique proteins .
A notable feature of the affinity chromatography technique used in this study is that elution of the bound proteins to the column is accomplished by an R-11 peptide, which is a truncated form of R-18 peptide, a polypeptide isolated from a phage display screening . The interaction of R-18 with YWHA does not require phosphorylation of R-18. Peptides R-18 and R-11 bind to YWHA via the amphipathic sequence WLDLE, with its two acidic groups coordinating with the basic residue clusters on YWHA . Since R-18 shares a common binding site on YWHA with other ligands, R-18 competitively interferes with YWHA/ligand interactions . Affinity chromatography followed by R-18 elution for identification of YWHA-binding proteins has been used in studies with somatic cells [14, 15]. The column eluate contained proteins cross-reacting with the phosphoserine YWHA-binding motif antibody. In addition, the R-11 eluate contained PPP1CC_v2, a molecule we had shown to be complexed with YWHAZ by column chromatography and immunoprecipitation . Taken together these observations suggest that affinity chromatography indeed isolated YWHA-binding proteins.
The enzyme GSK3B in brain was shown to bind YWHA . Changes in tyrosine and serine phosphorylation of GSK3 occur in parallel with motility initiation and motility stimulation in sperm . It was therefore of interest to determine whether YWHA may also bind GSK3 in spermatozoa. It appears that GSK3 in sperm extracts is able to bind to YWHA (Fig. 4A). GSK3B phosphorylation at serine 9 has been shown to be required for its binding with YWHA . Although GSK3 is phosphorylated at serine 9 in caudal sperm, GSK3 bound to YWHA isolated by affinity chromatography did not appear to be phosphorylated. Whether this is due to the limit of detection of phosphorylated GSK3 or whether GSK3 binding to YWHA in sperm may be indirectly mediated by some other protein is not known. The possibility that GSK3 may regulate PPP1CC_v2 due to its ability to phosphorylate its inhibitor subunit 2 (PPP1R2)  and the fact that PPP1CC_v2 is itself a YWHA-binding protein suggest regulatory mechanisms in spermatozoa linking the kinase and the phosphatase.
Western blot analysis with radial spoke protein homologue 1 (RSPH1) antibody showed its presence in YWHA affinity column fractions of sperm extracts. Partial purification of PPP1CC_v2 by fast protein liquid chromatography and nondenaturing gel electrophoresis, Western blot analysis, and microsequencing of column fractions showed that YWHA and RSPH1 coelute (data not shown). The protein RSPH1 is highly expressed in testis and ciliated cells. The protein is localized to sperm tail and anterior acrosome . RSPH1 in t-complex mice has been suggested to be one of the genes associated with male sterility . Determination of whether TSGA-2 is bound to YWHA in spermatozoa may enable elucidation of its significance in sperm function.
Studies in somatic cells have implicated YWHA in a wide variety of cellular processes—metabolism, protein folding, ubiquitination, motility, cell cycle regulation, transcription, and regulation of protein translation [13–15]. In this study we have identified glycolytic enzymes—PGK2, HK1, and GPI as potential YWHA interactors in spermatozoa (Table 1). Interaction of the glycolytic enzymes, phosphoglycerate kinase 1, glyceraldehyde 3 phosphate dehydrogenase, and pyruvate kinase, with protein YWHA has been documented in somatic cells [14, 15]. It would be of great interest to determine whether the glycolytic enzymes we have identified are phosphoproteins, whether their binding to YWHA in vivo is direct or mediated by other proteins, and whether YWHA binding affects their catalytic activity. It is known that glycolytic activity increases in parallel with motility development during the passage of sperm through the epididymis . It is possible that YWHA binding could be part of the mechanisms involved in changes in sperm metabolism during epididymal sperm maturation. Our study also identified mitochondrial ATP synthase (ATP5O) as a potential YWHA-interacting protein. Binding of the mitochondrial ATP synthase to YWHA has been shown to decrease its catalytic activity . The intriguing possibility of whether changes in sperm mitochondrial respiration also depend on phosphorylation and YWHA binding will be revealed in future studies.
Sperm YWHA-binding proteins also include polypeptides known to be involved in acrosome reaction and sperm-egg interactions. The functional significance of this binding in uncapacitated caudal epididymal sperm is unknown. It is known that motile caudal spermatozoa need to undergo capacitation and acrosome reaction before they can penetrate and fertilize eggs. It is likely that the profile of YWHA-binding proteins might change and be different in capacitated and acrosome-reacted sperm compared with activated caudal epididymal spermatozoa. It will be interesting to determine what these capacitation and acrosome reaction-dependent changes are in YWHA binding.
One of the significant advances in YWHA research was the elucidation of YWHA-binding motifs in Raf kinase . Other studies that followed identified alternate binding domains, such as those present in H+ATPase . In silico analysis of the YWHA-binding proteins identified in the present study show that some but not all of the proteins contain one or more canonical YWHA binding motifs, suggesting that binding of some of these identified proteins to YWHA could be mediated by other proteins. Surprisingly, some of the YWHA-binding proteins also contained a hydrophobic RVXF sequence, which is a PPP1C-binding motif . Many but not all interactors of PPP1C contain an RVXF variant that binds to this hydrophobic channel . We have already shown that YWHAZ interacts with sperm-specific PPP1CC_v2 in vivo and in vitro . This raises the possibility that these two proteins might be part of trimeric or multimeric complexes.
Microcystin pulldown assay identified proteins ACE, PGK2, GPI, and HSPA5, etc., as proteins that are common to YWHA and PPP1CC_v2 interactomes (Table 2). PPP1C and PPP2C have been shown to play a key role in regulating binding interactions of YWHA with its binding partners. In somatic cells it has been shown that CDC25C binds to YWHA and PPP1C through its canonical YWHA- and PPP1C-binding motifs . Binding of phosphorylated CDC25C to YWHA restricts it to the cytoplasm in resting cells. During mitosis, dephosphorylation of CDC25C at the YWHA-binding site by PPP1C disrupts YWHA binding, resulting in the translocation of CDC25C to nucleus . Notably, PPP1CC_v2 was suggested to exist as a heterodimer with HSPA5 . Our results suggest that YWHA, PPP1CC_v2, and HSPA5 might exist as a complex. It is possible that YWHA may act as a cochaperone or control the binding of PPP1CC_v2 to substrates or inhibitors. It would be interesting to determine whether ACE, which contains both YWHA- and PPP1C-binding motifs, forms a trimeric complex with YWHA and PPP1CC_v2.
Previous studies from our lab have shown that YWHA is localized mainly to the postacrosomal region of sperm, whereas PPP1CC_v2 and GSK3 are present in both the postacrosomal region of the sperm head and in the flagellum, and phosphorylated PPP1CC_v2 appears predominant in postacrosomal region [6, 11, 25]. Thus, intrasperm localization of these polypeptides is consistent with the possibility that they may be bound in vivo. Electron microscopic studies have shown that both ACE and HK1 in human and mouse sperm are present both in the postacrosomal region of the sperm head and in the flagellum, regions where YWHA and PPP1CC_v2 also are found [53, 54]. Intrasperm localization of PGK2 is not known.
Immunoprecipitation analysis revealed that ACE can be immunoprecipitated with both YWHA and PPP1CC_v2 antibodies, and YWHA and PPP1CC_v2 can be immunoprecipitated with each other from sperm extracts. PPP1CC_v2 could immunoprecipitate PGK2, but we could not detect PGK2 in YWHA immunoprecipitate. This could be due to the inability of YWHA antibodies to quantitatively immunodeplete YWHA from sperm extracts. We have used transgenic mice expressing tandem affinity purification tag YWHA in mouse testis to purify YWHA complexes. Our preliminary results have shown association of YWHA with PPP1CC_v2, GSK3, and PGK2.
We emphasize that our biochemical approaches used in the present study identify potential sperm YWHA-binding proteins in spermatozoa. The binding interactions of selected proteins were further confirmed by immunoprecipitation. It should be emphasized that pulldown studies, coimmunoprecipitation, and cellular colocalization suggest, but do not prove, that these proteins may exist as complexes in vivo. The answers to questions of whether the proteins shown in Tables 1 and and22 indeed interact with YWHA and/or PPP1CC_v2 in vivo and what the physiological significance of these interactions are must await further studies.
It may be noted that signaling enzymes PPP1CC_v2 and GSK3, although detected by Western blot analysis, were not identified in mass spectrometry. Although tandem mass spectrometry is a sensitive method with the ability to detect femtomolar levels of proteins, detection of other low-abundance proteins may not occur. It is also possible that the use of bovine sperm extracts may have limited identification, since many proteins are not yet represented in the bovine protein database. Since R-11 competes for YWHA binding with proteins that contain the amino acid sequence motifs RSXpSXP and RX(Y/F)XpSXP, it is possible that R11 may not be able to elute proteins bound to YWHA through other sequence motifs . Thus, the list of sperm YWHA-binding proteins is likely to expand.
In summary, our study is the first identification of potential YWHA-binding proteins in caudal bovine spermatozoa. An intriguing finding here is the suggestion that some of the sperm YWHA-binding proteins may also bind PPP1CC_v2, a signaling enzyme essential for spermatogenesis and sperm function. It appears that YWHA may play a key role in regulating metabolic enzymes involved in glycolysis and respiration in spermatozoa, similar to its role in regulating metabolism in plants and somatic cells in mammals. The identification of proteins unique to sperm also suggests a specific role for YWHA in male gamete function. In this regard, it may be noted that YWHA is present in sperm from diverse species . Elucidation of the exact role of YWHA in regulating the function of a number of sperm proteins and a determination how PPP1CC_v2 may modify this function are the foci of ongoing research in our laboratory.
We thank Dr. Helen Piwnica-Worms (The Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO) for her generous gift of protein YWHAZ plasmid. We also thank Dr. Deborah A. O'Brien (Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC), Dr. Ganes C. Sen (Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH), and Dr. Stephen Pilder (Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA) for their generous gifts of PGK2, ACE, and RSPH1 antibodies. We also thank Lina Cheng, David Soler, and Dr. Saurabh Chattopadhay for their help in results and discussion.
1Supported by National Institute of Health grant HD38520 to S.V.