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BOR - Papers in Press, published online ahead of print March 17, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027284
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BIOLOGY OF REPRODUCTION 71, 177–184 (2004)
DOI: 10.1095/biolreprod.104.027284
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Protein 14-3-3{zeta} Binds to Protein Phosphatase PP1{gamma}2 in Bovine Epididymal Spermatozoa1

Zaohua Huang3, Kimberley Myers3, Balwant Khatra4, and Srinivasan Vijayaraghavan2,3

Department of Biological Sciences,3 Kent State University, Kent, Ohio 44242 California State University,4 Long Beach, California 90840


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The protein phosphatase PP1{gamma}2 is critical in the regulation of sperm motility and fertility. Its activity is regulated by its binding proteins and by phosphorylation. We have recently shown that PP1{gamma}2 is phosphorylated and that the amount of phosphorylated PP1{gamma}2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439–447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1{gamma}2 during column chromatography of bovine sperm extracts. Western blot analyses confirmed the presence of protein 14-3-3 not only in bovine spermatozoa but also in spermatozoa of diverse species—bull, hamster, horseshoe crab, monkey, rat, turkey, and Xenopus. The binding between PP1{gamma}2 and protein 14-3-3 was confirmed by coimmunoprecipitation experiments and in pull-down assays with recombinant GST-14-3-3. Western blot analysis and protein 14-3-3 immunoprecipitates with antibodies against the consensus binding domain of protein 14-3-3 reveal that, in addition to PP1{gamma}2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1{gamma}2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of bovine spermatozoa. Together, these results provide conclusive evidence that protein 14-3-3 is present in mature spermatozoa and that PP1{gamma}2 is one of its binding partners.

epididymis, phosphatases, sperm, sperm maturation, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Research in our laboratory is devoted to understanding the biochemical basis underlying sperm motility and other biological functions. This includes studies on how a protein phosphatase, PP1{gamma}2, is regulated in spermatozoa. In somatic cells, at least four different types of serine-threonine phosphatases are known: PP1, PP2A, PP2B, and PP2C [1, 2]. Four different isoforms of PP1 (type 1 serine/threonine phosphatases), PP1{alpha}, PP1{gamma}1, PP1{gamma}2 and PP1ß are encoded by three different genes. The isoforms PP1{gamma}1 and PP1{gamma}2 are alternately spliced variants of a single gene [3, 4]. A unique 21-amino-acid carboxyl terminal in PP1{gamma}2, not found in PP1{gamma}1, is the only primary structural difference between the two isoforms. While PP1{gamma}2 is predominantly expressed in testis, PP1{gamma}1 is ubiquitous. Disruption of the gene for PP1{gamma}1 and PP1{gamma}2 in mice causes infertility by arresting spermatogenesis [5], suggesting that one or both of these isoforms are required in the final stages of sperm development.

Sperm motility and fertility are known to be regulated by numerous proteins and cellular conditions, including protein kinase C, tyrosine phosphorylation, intracellular cyclic AMP, calcium, and pH [6–16]. These intracellular mediators presumably act through changes in protein phosphorylation, resulting from the regulated actions of protein kinases and protein phosphatases. Sperm motility in immature spermatozoa can be initiated by pharmacological treatments that either stimulate a cAMP-dependent protein kinase or inhibit a serine/threonine protein phosphatase [9– 12, 17–19]. This suggests that the potential for motility already exists in immature epididymal spermatozoa and that low protein kinase and high protein phosphatase activities limit motility in immature spermatozoa. We have shown that high catalytic activity of sperm PP1{gamma}2 holds motility in check in immature caput epididymal spermatozoa [17– 19]. Protein phosphatase inhibitors, such as calyculin A and okadaic acid, initiate motility in caput spermatozoa and stimulate motility in caudal spermatozoa [17–19].

This study was prompted by our discovery that phosphorylated PP1{gamma}2 copurified with a protein identified by microsequencing to be protein 14-3-3{zeta}. Protein 14-3-3 is expressed in all eukaryotic cells and its amino acid sequence is highly conserved in species ranging from yeast to mammals [20–24]. Protein 14-3-3 has been shown to be essential to cell survival: deletion of protein 14-3-3 genes in Saccharomyces cerevisae and mutations in the Drosophila 14-3-3{zeta} gene Leonardo, are lethal [25, 26]. While protein 14-3-3 isoforms have been detected in testis and developing spermatocytes [27–29], their presence in mature epididymal spermatozoa has not been previously reported. The objectives of the study were to determine if protein 14-3-3 was present in spermatozoa of other species, to confirm binding interactions between sperm PP1{gamma}2 and protein 14-3-3, to document their intrasperm localization, and to determine whether there were other binding partners for protein 14-3-3 in spermatozoa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sperm Isolation and Extract Preparation

Testes from mature bulls with intact tunica were obtained from a local slaughter house. Spermatozoa were isolated from caput and caudal epididymis and washed as previously described [17] in buffer A containing 100 mM NaCl, 40 mM KCl, 10 mM Tris, and 5 mM MgSO4, pH 7.4. Sperm pellets were suspended in a homogenization buffer, buffer B (10 mM Tris, pH 7.2, 1 mM EDTA, and 1 mM EGTA), supplemented with protease inhibitors (10 mM benzamidine, 1 mM PMSF, and 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone [TPCK]) and 0.1% 2-mercaptoethanol. The sperm suspension was then sonicated on ice with three 10-sec bursts using a Biosonic II sonicator (Bronwell Scientific, Rochester, NY) at maximum setting. The sperm sonicate was centrifuged at 16 000 x g at 4°C for 10 min. The supernatants are referred to as sperm extracts in this report.

Purification of Protein 14-3-3-PP1{gamma}2 Complex

Bovine caudal epididymal sperm extract (150 ml prepared from 5 x 1011 spermatozoa) in buffer B was passed through a DEAE-cellulose (Amersham, Piscataway, NJ) column (0.5 cm x 13 cm) preequilibrated with buffer C (buffer B with 0.05 M KCl and additional protease inhibitors including pepstatin [1 µg/ml], leupeptin [0.5 µg/ml], and aprotinin [2 µg/ ml]). The column was washed with 20 ml buffer C, followed by elution with a linear gradient of 0.05–0.65 M KCl in buffer C. Flow-through and gradient fractions (0.185–0.35 M KCl) containing PP1{gamma}2 activity and/or immunoreactivity were pooled separately and concentrated using a Centricon 10 filter (Millipore Corp., Bedford, MA). DEAE-cellulose flow-through and gradient fractions containing PP1{gamma}2 were applied separately to a SP-Sepharose (5 ml prepacked; Amersham) column pre-equilibrated with buffer C. The column was washed with 10 ml buffer C, followed by elution with a linear gradient of 0.05–0.65 M KCl in buffer C. The flow-through and gradient fractions after SP-Sepharose column was concentrated and analyzed for PP1{gamma}2 activity and immunoreactivity. The concentrated PP1{gamma}2 of gradient fractions after SP-Sepharose column was also subjected to Superose 6 column for molecular weight determination and further purification. The fractions after Superose 6 column were concentrated and analyzed for PP1{gamma}2 activity and immunoreactivity. This preparation is called column-purified fraction in this report. All column procedures were conducted at 4°C. Total protein in caudal sperm extracts and in fractions obtained from column chromatography was measured with Coomassie brilliant blue dye reagent (Bio-Rad, Hercules, CA).

Protein Phosphatase Activity Assay

Preparation of radio-labeled phosphorylase a and protocols for its use as a substrate for measurement of PP1 activity have been described previously [17]. Briefly, aliquots of bovine caudal epididymal sperm extracts or column fractions or immunoprecipitates were incubated (in a total volume of 40 µl) at 30°C with 1 mM Mn2+ and with or without protein phosphatase inhibitor I2 for 10 min. At the end of this incubation, the reaction was terminated with 180 µl of 20% ice-cold trichloroacetic acid, after which the tubes were centrifuged for 5 min at 12 000 x g at 4°C. The supernatants were quantitated for 32PO4 released from phosphorylase a. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the release of 1 nmol of 32PO4/min under conditions of the enzyme assay. This assay is considered specific for the enzymes PP1 and PP2A [17] and PP1 activity is the activity sensitive to protein phosphatase inhibitor I2.

Electrophoresis and Western Blot Analysis

Bovine caudal epididymal sperm extracts (30–50 µg) and aliquots from fractions obtained from column chromatography (2–10 µg) were separated by SDS-PAGE through 12% polyacrylamide slab gels based on the protocol of Laemmli [30]. One milliliter of soluble extract (prepared from 109 cells) contain approximately 1 mg/ml or 1.9 mg/ml protein for caudal or caput sperm, respectively. After electrophoresis, proteins were electrophoretically transferred to Immobilon-P, polyvinylidene fluoride membrane (Millipore Corp.). Nonspecific protein-binding sites on the membrane were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS: 25 mM Tris-HCl, pH 7.4, 150 mM NaCl). The blots were washed twice for 15 min each with TTBS (TBS containing 0.1% Tween 20) and then incubated with primary antibody overnight, with shaking, at 4°C. Anti-PP1{gamma}2 (1: 5000) was raised against a synthetic carboxyl terminus extension of PP1{gamma}2 (22 amino acids of the carboxyl terminus). This antibody detects both nonphosphorylated and phosphorylated PP1{gamma}2. Anti-protein 14-3-3 is a commercially available rabbit polyclonal antibody (1:2000; Zymed Laboratories, San Francisco, CA). Phospho-PP1 antibody, prepared by using residues 316–323 of PP1 alpha as the antigen, was a generous gift from Dr. Greengard and Dr. Angus Nairn (Rockefeller University, New York City, NY). The efficacy of this antibody in detecting phosphorylated PP1{gamma}2 is documented [31]. Phospho-(Ser) 14-3-3 binding motif antibody was purchased from Cell Signaling (Charlottesville, VA). After washing, the blots were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (Amersham) at 1:2000 dilution for 1 h at room temperature. Blots were then washed with TTBS twice, 15 min each, and four times, 5 min each. Blots were developed with an ECL chemiluminescence kit (Amersham). For native PAGE runs, the protocol for SDS-PAGE was used except that SDS was omitted from the buffer as well as the gels and the sample was prepared in a nonreducing buffer without boiling.

Protein Microsequencing

The column-purified fractions as described above were resolved by SDS-PAGE electrophoresis. The gel was stained with Coomassie blue, destained, and a 28 Mr x 10–3 band was excised from each of the lanes. The excised band was washed once with HPLC-grade acrylonitrile (Sigma-Aldrich, St. Louis, MO) as per instructions from the Harvard microsequencing facility, Boston, MA. The protein was sequenced, after in-gel digestion, by microcapillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry on a Finnigan LCQ DECA quadrupole ion trap mass spectrometer (Thermo Finnigan, San Jose, CA).

Determination of Molecular Weight of the PP1{gamma}2-Protein 14-3-3 Complex

The column-purified fraction was applied in five batches to Superose 6 column (24 ml, prepacked high resolution Pharmacia FPLC column; Pharmacia, Piscataway, NJ) preequilibrated with buffer C. Calibration of the column for its void volume was performed by running blue dextran. Standardization of the column was done by resolving a mixture of aldolase (5 mg/ml), albumin (5 mg/ml), ovalbumin (10 mg/ml), and chymotrypsinogen (10 mg/ml). The elution was performed with buffer C. The eluted fractions were subjected to a dot blot assay and probed with antibodies against PP1{gamma}2 and protein 14-3-3.

Immunoprecipitation of PP1{gamma}2-Protein 14-3-3 Complex by Protein 14-3-3 and PP1{gamma}2 Antibodies

Bovine caudal epididymal sperm extracts were incubated for 1 h at 4°C with a protein 14-3-3 antibody (rabbit polyclonal antibody, from Zymed Laboratories, or mouse monoclonal antibody, from Neomakers, Fremont, CA) or PP1{gamma}2 antibody. Approximately 5 µg of each antibody was used. Protein G-Sepharose beads (Pharmacia) were washed once with distilled water and twice with TTBS. The extract/antibody solution was incubated with the beads by rocking for 1 h at 4°C. After incubation, the beads were washed once with TTBS and three times with buffer B. The pellets and supernatants were adjusted to equal volumes for PP1 activity assay and Western blot analysis.

GST Pull-Down Assay

Recombinant GST and GST-14-3-3{zeta} from cDNA inserts in pGEX plasmids were prepared in Esherichia coli. The GST-14-3-3{zeta} pGEX plasmid was a gift from Michael Yaffe, (Department of Biology, Massachusetts Institute of Technology, Cambridge, MA). Glutathione Sepharose 4B beads (Amersham) were prepared according to supplier instructions. The beads were resuspended in lysis buffer (20 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5% Tween-20, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 0.7 µg/ml pepstatin, 25 µg/ml PMSF) in a 50:50 slurry. Equal molar amounts of GST or GST-14-3-3 fusion proteins were added to 50 µl of the bead slurry and incubated by rocking at room temperature for 30 min. Beads were washed once in 1 ml lysis buffer and the supernatant was removed. Bovine caudal epididymal sperm extract prepared in homogenization buffer was added to the beads and incubated by rocking at 4°C overnight. Following four washes in 1 ml buffer B, the GST fusion proteins and any bound proteins were eluted with 50 µl of elution buffer (20 mM glutathione-reduced, 50 mM Tris, pH 8.0). Samples were separated through 12% SDS-PAGE and analyzed by Western blotting with PP1{gamma}2 (1:2000) and Phospho-(Ser) 14-3-3 binding motif (1:1000) antibodies.

Fluorescence Immunocytochemistry

Spermatozoa were isolated as described above, washed twice, and resuspended in PBS. Cells were fixed in 4% formaldehyde in PBS at 4°C for 30 min. The sperm solution was then treated with 0.2% Triton X-100. Fixed spermatozoa were attached to poly-L-lysine-coated cover slips. The cover slips were washed once with TTBS and three times with TTBS supplemented with 5% BSA and incubated for 1 h in a blocking solution containing 5% BSA and 5% normal goat serum in TTBS at room temperature. The cover slips were then incubated with primary antibody for 1 h at room temperature or overnight at 4°C, washed three times with TTBS, incubated with corresponding secondary antibody conjugated to indocarbocyanine (Cy3) (Jackson Laboratories, West Grove, PA) for 1 h at room temperature. The cover slips were washed five times with TTBS and examined by fluorescence microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Protein 14-3-3{zeta} in Bovine Sperm Extracts by Column Chromatography and Microsequencing

We used DEAE-cellulose, SP-Sepharose, and high-resolution Superose 6 columns for purification of PP1{gamma}2 from bovine caudal sperm extracts. This report concerns a fraction of PP1{gamma}2 that binds to DEAE-cellulose but elutes in the flow-through fraction in SP-Sepharose column (Table 1). This column-purified PP1{gamma}2 fraction, exhibiting an apparent molecular weight of 80 Mr x 10–3 in a Superose 6 molecular sizing column, was catalytically active (Table 1). Concentrated column-purified fractions of PP1{gamma}2 subjected to SDS-PAGE showed a number of bands. Two prominent bands were at 39 and 28 Mr x 10–3 (Fig. 1). The 39 Mr x 10–3 band was most likely PP1{gamma}2, as confirmed by Western blot analysis in experiments described below. The 28 Mr x 10–3 band was excised and microsequenced. The major protein in the 28 Mr x 10–3 band was protein 14-3-3 (Fig. 2). The peptides sequences covered 25.7% of the full sequence of protein 14-3-3{zeta} with 100% identity. Western blot analysis with protein 14-3-3 antibodies confirmed the presence of protein 14-3-3 in the column-purified fraction (Fig. 3A, lane 2). The calculated molecular weight of protein 14-3-3 is 28 kDa, it migrated at approximately 32 Mr x 10–3 in 12% SDS-PAGE (based on comparison of prestained protein markers). Figure 3A, lane 1, shows that the column-purified fraction containing protein 14-3-3 also contains immunoreactive PP1{gamma}2. Because protein 14-3-3 is known to bind to phosphorylated proteins, we next examined whether PP1{gamma}2 coeluting with protein 14-3-3 may be phosphorylated. We have previously shown that phosphorylated PP1{gamma}2 is present in spermatozoa and that its level increases during sperm maturation [31]. Western blot analysis with antibodies specific for phospho-PP1 showed that the column-purified fractions containing protein 14-3-3 also contain phospho-PP1{gamma}2 (Fig. 3A, lane 3). We also resolved the partially purified sample by nondenaturing gel electrophoresis followed by Western blot analysis with phospho-PP1{gamma}2 and protein 14-3-3 antibodies. Figure 3B, lanes 1 and 2, showed that phospho-PP1{gamma}2 and protein 14-3-3 comigrate in the nondenaturing gel.


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  		TABLE 1. Purification of PP1{gamma}2 from 150 ml caudal epididymal sperm extract.*



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  		FIG. 1. Partial purification of caudal sperm PP1{gamma}2. Bovine caudal epididymal sperm extracts were subjected to column chromatography as described in Material and Methods and Table 1. Superose 6 column fraction eluting at 80 Mr x 10–3 and containing PP1{gamma}2 was concentrated and subjected to SDS-PAGE. Approximately 5 µg protein was loaded and the gel was stained with Coomassie blue followed by silver stain



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  		FIG. 2. Amino acid sequence of protein 14-3-3{zeta}. The concentrated eluate from the Superose 6 column, as described in Figure 1 and Table 1, was resolved by SDS-PAGE and the 28 Mr x 10–3 band was microsequenced as described in Materials and Methods. The sequence showed that one of the proteins in this band is the zeta isoform of protein 14-3-3 (accession number NP_663723). The sequences obtained from the microsequencing are underlined. The underlined sequences cover 25.7% of the full sequence of protein 14-3-3{zeta} with 100% identity



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  		FIG. 3. Sperm PP1{gamma}2 coeluting with protein 14-3-3 is phosphorylated. A) The purified Superose 6 column fractions, as described in Figure 1, were subject to SDS-PAGE (2 µg protein per lane) in triplicate followed by Western blot analysis with PP1{gamma}2 (lane 1), protein 14-3-3 (lane 2), and phospho-PP1 antibodies (lane 3). B) The purified fraction as in A was subject to native gel electrophoresis followed by Western blot analysis with phospho-PP1 antibody (lane 1) and protein 14-3-3 antibody (lane 2)

Coimmunoprecipitation of Protein 14-3-3 and PP1{gamma}2 and Binding of PP1{gamma}2 to Recombinant GST-14-3-3

Protein 14-3-3 and PP1{gamma}2 coelute at approximately 80 Mr x 10–3 in a Superose 6 column. The individual molecular weights of protein 14-3-3 and PP1{gamma}2, based on their amino acid sequences, are 28 and 39, respectively. The calculated molecular weight of a complex between dimeric 14-3-3 and PP1{gamma}2 is 95, a value close to that observed for the elution of PP1{gamma}2 through Superose 6. Moreover, protein 14-3-3 and phospho-PP1{gamma}2 comigrate in nondenaturing PAGE. These data suggest, but do not prove, that PP1{gamma}2 may be bound to protein 14-3-3. To investigate further whether PP1{gamma}2 is bound to protein 14-3-3, immunoprecipitation studies with protein 14-3-3 and PP1{gamma}2 antibodies were performed. Data in Figure 4A shows Western blot analysis of immunoprecipitates obtained with protein 14-3-3 antibodies developed with phospho-PP1{gamma}2 and PP1{gamma}2 antibodies. Figure 4A shows the presence of phospho-PP1{gamma}2 (lane 2) and PP1{gamma}2 (lane 3) in the immunoprecipitation samples. Enzyme activity measurements show that PP1{gamma}2 immunoprecipitated by protein 14-3-3 antibodies is catalytically active (Fig. 4B). Control precipitates (obtained by incubating sperm extracts with purified rabbit IgG instead of 14-3-3 antibodies) have no PP1{gamma}2 or phospho-PP1{gamma}2 immunoreactivity (Fig. 4A, lane 1) and negligible PP1{gamma}2 activity (Fig. 4B) compared with the immunoprecipitation samples. Similar experiments were performed with PP1{gamma}2 antibodies for immunoprecipitation. Immunoprecipitates with PP1{gamma}2 antibodies contain protein 14-3-3 (Fig. 5, lanes 2 and 3). As expected, the immunoprecipitates also contain phospho-PP1{gamma}2 (lane 2) and PP1{gamma}2 (lane 3). Control precipitates with preimmune rabbit serum contain no detectable protein 14-3-3 or PP1{gamma}2 (Fig. 5, lane 1).



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  		FIG. 4. Immunoprecipitation of bovine caudal epididymal sperm extracts with protein 14-3-3 antibody. A) Western blot analysis of immunoprecipitates. The molecular mass markers are shown beside the blots. Lane 1: control pellet obtained with nonimmune rabbit serum; lanes 2 and 3: protein 14-3-3 immunoprecipitates obtained with 14-3-3 antibodies. Lane 2 was developed with phospho-PP1 antibody and lane 3 with PP1 {gamma}2 antibody. Lane 1 was developed with both antibodies. B) Protein phosphatase activity in the control and immunoprecipitate pellets were measured as described in Materials and Methods. The values of protein phosphatase activity are mean ± SD of activity from three different experiments, with each measurement performed in duplicate



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  		FIG. 5. Immunoprecipitation of bovine caudal epididymal sperm extracts with PP1{gamma}2 antibodies. Western blot analysis of immunoprecipitates. The molecular markers are shown beside the blots. Lane 1: control pellets obtained with nonimmune rabbit serum; lane 2: immunoprecipitation pellets obtained with PP1{gamma}2 antibody were analyzed with phospho-PP1 followed by protein 14-3-3 antibodies; lane 3: the same blot in lane 2 developed with PP1{gamma}2 antibody. Lane 1 was developed with all these antibodies

Next we used a pull-down assay to determine if recombinant protein 14-3-3 can bind to PP1{gamma}2 in sperm extracts. Caudal sperm extracts were incubated with GST-14-3-3 fusion protein bound to glutathione Sepharose beads. Proteins released from the beads with glutathione were analyzed by SDS-PAGE followed by Western blot analysis. Figure 6A shows that recombinant GST-14-3-3 is able to bind PP1{gamma}2 (lane 2), while GST alone is unable to bind PP1{gamma}2 (lane 1). Figure 6B is the same blot stained with Coomassie blue showing recombinant GST (27 Mr x 10–3 band in lane 1) and the GST-14-3-3 fusion protein (55 Mr x 10–3 band in lane 2).



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  		FIG. 6. Pull-down assay with recombinant GST-14-3-3. The pull-down samples, prepared as described under Materials and Methods, were subject to SDS-PAGE followed by Western blot analysis with PP1{gamma}2 antibody. A) Lane 1: pull-down sample from GST alone (control); lane 2: pull-down sample from GST-14-3-3 fusion protein; (B) is the blot in A stained with Coomassie blue showing equal molar amounts of GST and GST-14-3-3 protein were loaded

Protein 14-3-3 Binds to Other Phosphoproteins in Bovine Caudal Epididymal Spermatozoa

Results presented so far suggest that PP1{gamma}2 is a binding partner of protein 14-3-3. Because immunoprecipitation with PP1{gamma}2 antibody does not remove all of the protein 14-3-3 in sperm extracts (data not shown), it is likely that other protein 14-3-3 binding proteins are present in spermatozoa. A consensus sequence commonly found in phosphoproteins bound to protein 14-3-3 is RXXXS(p)/T(p)XP, where S(p)/ T(p) refers to phosphorylated serine or threonine. An antibody against one of the protein 14-3-3 binding motifs R-X-Y/Phe-X-S(p) is commercially available. This antibody binds to proteins containing this motif when the serine residue is phosphorylated. It should be emphasized that, in PP1{gamma}2, the sequence RXXT(p)XP (amino acid residue 308– 313), the suspected protein 14-3-3 binding region, is not recognized by the protein 14-3-3 domain-specific antibody. Western blot analysis with this protein 14-3-3 domain-specific antibody identified at least two prominent bands, at 114 Mr x 10–3 (p114) and 51 Mr x 10–3 (p51) in caudal sperm extracts (Fig. 7A). Pellets from protein 14-3-3 immunoprecipitation (Fig. 7B, lane 2), but not control pellets (Fig. 7B, lane 1), contain p114. The bands at 32 Mr x 10–3 in the control blot are most likely from light chains of mouse IgG. Whether p51 is also present in the 14-3-3 immunoprecipitation pellets is not known because the IgG heavy chain migrates at this position (Fig. 7B, lane 2). Significantly, p114 and p51 are also two of the proteins that specifically bind to recombinant GST-14-3-3 (Fig. 7C, lane 1). This binding is specific because GST alone could not bind these proteins (Fig. 7C, lane 2).



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  		FIG. 7. Proteins containing protein 14-3-3 binding motif are present in spermatozoa. A) Bovine caudal epididymal sperm extracts (approximately 20 µg protein) were subject to SDS-PAGE followed by Western blot analysis with protein 14-3-3 binding motif antibody. B) Immunoprecipitates obtained with protein 14-3-3 antibody were subject to SDS-PAGE followed by Western blot analysis with protein 14-3-3 binding motif antibody; lane 1 is the control sample and lane 2 is the immunoprecipitation sample. The two bands present at approximately 32–34 Mr x 10–3 in the control (lane 1) and one band at similar location in the IP (lane 2) are the light chains from the mouse antibodies. C) GST-14-3-3 pull-down assay samples were subjected to SDS-PAGE followed by Western blot analysis with protein 14-3-3 binding motif antibody. Lane 1: the GST-14-3-3 pull-down sample; lane 2: the GST pull-down sample

Protein 14-3-3 Is Present in the Spermatozoa from Diverse Species

Because protein 14-3-3 is a family of highly conserved protein, we investigated whether it is present in spermatozoa of other species. Western blot shows that a single immunoreactive band at 28 Mr x 10–3, most likely due to protein 14-3-3, is present in spermatozoa from bull, hamster, rhesus monkey, rat, turkey, horseshoe crab, and Xenopus (Fig. 8). The same blot probed with PP1{gamma}2 antibodies showed immunoreactive PP1{gamma}2 bands in mammalian spermatozoa but not in turkey, horseshoe crab, and Xenopus.



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  		FIG. 8. Presence of immunoreactive protein 14-3-3{zeta} and PP1{gamma}2 in spermatozoa from different species. Sperm extracts (20 µg each except for rat, where 5 µg protein was used) as indicated below were subject to SDS-PAGE followed by Western blot analysis with protein 14-3-3 antibody and PP1{gamma}2 antibody

Intrasperm Localization of Phospho-PP1{gamma}2 and Protein 14-3-3 in Bovine Caudal Epididymal Spermatozoa

Finally, immunofluorescence was used to localize phospho-PP1{gamma}2 and protein 14-3-3 within spermatozoa. As shown in Figure 9, intense staining for phosphorylated PP1{gamma}2 is mainly observed in the postacrosomal region of the head, including the equatorial segment. Staining is also observed in the principal piece (Fig. 9, C and D). Protein 14-3-3 antibodies stained the postacrosomal region of the sperm head and the principal piece (Fig. 9, A and B), a pattern similar to phosphorylated PP1{gamma}2 localization. No fluorescence was observed in control slides, where preimmune rabbit serum was used as primary (Fig. 9F) or secondary antibody alone was used (data not shown).



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  		FIG. 9. Localization of protein 14-3-3 and phospho-PP1{gamma}2 in bovine caudal epididymal spermatozoa using fluorescence immunocytochemistry. A, B) Single spermatozoon or a group of spermatozoa stained with protein 14-3-3 antibody. C, D) Phospho-PP1{gamma}2 labeling in a single spermatozoon and in a group of spermatozoa. Control spermatozoa (F) were incubated with nonimmune rabbit serum and the phase contrast image of the control is shown in (E). All the pictures were obtained with a 100x oil immersion lens with a total magnification of 1000; a 1-µm bar is shown in B


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated, for the first time, that protein 14-3-3 is present in spermatozoa from diverse species and that PP1{gamma}2 is one of the binding partners for protein 14-3-3 in bovine caudal epididymal spermatozoa. Protein 14-3-3{zeta} has been previously shown to be present in testis in late spermatids and Sertoli cells [27]. In addition, protein 14-3-3{zeta} has been shown to be present in developing spermatids and spermatocytes [28, 29], however, this isoform was not detected in either testicular or epididymal spermatozoa [29].

We also provide conclusive evidence for an interaction between PP1{gamma}2 and protein 14-3-3 in bovine caudal epididymal spermatozoa. Microsequencing of proteins obtained from purified sperm extract fractions containing PP1{gamma}2 revealed that protein 14-3-3 coeluted with phospho-PP1{gamma}2 during column chromatography, and Western blot analyses confirmed the presence of protein 14-3-3 in spermatozoa. We have shown that protein 14-3-3 binds to PP1{gamma}2 in coimmunoprecipitation experiments and in pull-down assays using recombinant GST-14-3-3. In addition, fluorescence immunocytochemistry results indicate that phospho-PP1{gamma}2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of spermatozoa. Together, these results provide conclusive evidence for an interaction between PP1{gamma}2 and protein 14-3-3 in spermatozoa.

While more than 100 binding partners have been identified for protein 14-3-3 [24], this is the first report that protein 14-3-3 binds PP1 in spermatozoa. An interaction between Reg1, a regulatory protein of yeast PP1 complex and yeast homologues of protein 14-3-3, has been previously reported [32]. Ours is the first demonstration of an association between PP1{gamma}2 and protein 14-3-3 in mammalian cells. Protein 14-3-3 binding normally involves interactions with target protein phospho-serine/threonine-containing motifs in a sequence-specific manner; two distinct 14-3-3 protein-binding motifs are RSXS(p)/T(p)XP and RXXXS(p)/T(p)XP [21, 22]. Sperm PP1{gamma}2 contains a similar sequence, RXXT(p)XP, which may be the site for protein 14-3-3 binding.

Because protein 14-3-3 binds to phosphorylated domains, we would expect that PP1{gamma}2 bound to protein 14-3-3 in sperm extracts to be phosphorylated. We have provided strong evidence that at least some of the PP1{gamma}2 bound to 14-3-3 is phosphorylated. These include the findings i) phospho-PP1{gamma}2 and protein 14-3-3 coelute in column chromatography, ii) phospho-PP1 and protein 14-3-3 comigrate in the nondenaturing gel, iii) protein 14-3-3 antibodies immunoprecipitate phospho-PP1{gamma}2, and iv) phospho-PP1{gamma}2 and protein 14-3-3 colocalized within bovine caudal epididymal spermatozoa in fluorescence immunocytochemistry. While the above evidence shows that phospho-PP1{gamma}2 is bound to protein 14-3-3, it does not prove that phosphorylation is essential for 14-3-3 binding. As noted earlier, PP1{gamma}2 does not contain the exact consensus sequence required for binding to protein 14-3-3. While immunoprecipitation with PP1{gamma}2 antibody precipitated protein 14-3-3 from sperm extracts, phospho-PP1{gamma}2 antibody did not (data not shown). It is possible that protein 14-3-3 binding to phosphorylated PP1 prevents access to the epitope required for phospho-PP1{gamma}2 antibody reaction. Furthermore, while we detected PP1{gamma}2 binding to GST-14-3-3 in pull-down assays (Fig. 6), we were unable to detect phospho-PP1{gamma}2. This may be because the low amounts of phospho-PP1{gamma}2 bound to GST-14-3-3 were below the limits of detection by the phospho-PP1{gamma}2 antibodies. A definitive answer to the question of whether phosphorylation is essential for PP1{gamma}2 binding to protein 14-3-3 should be provided by studies with recombinant PP1{gamma}2. However, bacterially expressed PP1{gamma}2 is catalytically inactive and could not be phosphorylated in vitro (data not shown) and could not bind to recombinant protein 14-3-3. This lack of interaction could be because PP1{gamma}2 expressed in bacteria is not folded in its proper conformation. It also remains to be conclusively determined whether phosphorylated PP1{gamma}2 directly binds to protein 14-3-3 or if this binding is mediated by another unidentified protein. Studies are in progress to express recombinant PP1{gamma}2 in eukaryotic cells and isolate the protein in sufficient amounts in its phosphorylated and nonphosphorylated forms for use in pull-down assays to resolve these questions.

The enzyme PP1{gamma}2 is apparently not the only binding partner of protein 14-3-3 in spermatozoa because a substantial amount of protein 14-3-3 remains in the supernatant following immunoprecipitation of PP1{gamma}2 (data not shown). Western blot analyses of protein 14-3-3 immunoprecipitates and of proteins bound to GST-14-3-3 with the protein 14-3-3 binding motif antibody showed that two proteins, p114 and p51 (Fig. 7), present in sperm extracts bind to protein 14-3-3. It should also be noted that nonmammalian spermatozoa contain protein 14-3-3 but no PP1{gamma}2 (Fig. 8). Investigations are underway to determine the identity of these and other sperm proteins that interact with protein 14-3-3.

Additional study is required to determine the biological significance of the interaction between protein 14-3-3 and PP1{gamma}2 in spermatozoa. The known regulatory actions of protein 14-3-3 binding include the alteration of the ability of the target protein to interact with other partners, the modification of the cytoplasmic/nuclear partition of the protein partner through increase or decrease of nuclear localization, the inhibition or the activation of the intrinsic catalytic activity of the target protein, the protection of the target protein from proteolysis and/or dephosphorylation [24]. Protein 14-3-3 binding does not appear to inhibit PP1{gamma}2 as noted in activity assays performed on the purified column fractions and immunoprecipitation samples containing both PP1{gamma}2 and protein 14-3-3. Therefore, we propose that the action of protein 14-3-3 does not directly affect the catalytic activity of PP1{gamma}2 but rather regulates PP1{gamma}2 by altering its ability to interact with other proteins. One possibility is that protein 14-3-3 in spermatozoa acts as either a bridge or an adaptor between PP1{gamma}2 and other sperm proteins that are involved in the regulation of sperm maturation and motility. Alternately, protein 14-3-3 may protect PP1{gamma}2 from degradation or dephosphorylation.

Given that a high level of PP1{gamma}2 catalytic activity suppresses motility in spermatozoa, that the inhibition of protein phosphatases stimulates motility, and that the level of PP1 phosphorylation is much higher in motile caudal spermatozoa compared with immotile caput spermatozoa [31], we speculate that the one of the functions of protein 14-3-3-PP1 binding in spermatozoa is to preserve the phosphorylated form of PP1{gamma}2. It is known that phosphorylation reduces PP1 catalytic activity [33–35]. Protein 14-3-3 may protect PP1{gamma}2 phosphorylation and thus maintain the low PP1{gamma}2 catalytic activity required for motility. In this regard, we have shown that epididymal sperm maturation is associated with increased phosphorylation of PP1{gamma}2 [31].

We have shown that protein 14-3-3 is present in spermatozoa from various species. Furthermore, we have also shown that, apart from PP1{gamma}2, other unidentified sperm phosphoproteins bind to protein 14-3-3. Molecular identification of these proteins and development of techniques to disrupt protein 14-3-3 function within spermatozoa will shed further light on the role of this evolutionarily conserved protein in spermatozoa.


   ACKNOWLEDGMENTS
 
We thank Drs. Paul Greengrad and Augus Nairn, Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY for their generous gift of phospho-PP1 antibody. We also thank Dr. Michael Yaffe (Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA) for his generous gift of protein 14-3-3{zeta} plasmid, Dr. Kline Douglas (Department of Biological Sciences, Kent State University, Kent, OH) for his assistance in microscopy and Dr. Payaningal Somanath, John Ferrara, and Mauris Nnamani for their assistance in the research and discussions.


   FOOTNOTES
 
1 Supported by NIH grant RO1 HD38520. Back

2 Correspondence: FAX: 330 672 3713; svijayar{at}kent.edu Back

Received: 9 January 2004.

First decision: 25 January 2004.

Accepted: 26 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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