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Gamete Biology |
2 Is Regulated by a Homologue of the Yeast Protein Phosphatase Binding Protein sds221
a Biological Sciences Department, Kent State University, Kent, Ohio 44242
b California State University, Long Beach, California 90840
c Faculteit Geneeskunde, Katholieke Universiteit Leuven, Leuven, Belgium
d Veterans Affairs Medical Center, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 is a testis-specific protein phosphatase isoform in spermatozoa. This enzyme appears to play a key role in motility initiation and stimulation. Catalytic activity of PP12 is higher in immotile compared with motile spermatozoa. Inhibition of PP12 activity causes both motility initiation and motility stimulation. Protein phosphatases, in general, are regulated by their binding proteins. The objective of this article is to understand the mechanisms by which PP12 is regulated, first by identifying its regulatory proteins. We had previously shown that a portion of bovine sperm PP12 is present in the cytosolic fraction of sperm sonicates. We purified PP12 from soluble bovine sperm extracts by immunoaffinity chromatography. Gel electrophoresis of the purified enzyme showed that it was complexed to a protein 43 Mr x 10-3 in size. Microsequencing revealed that this protein is a mammalian homologue of sds22, which is a yeast PP1 binding protein. Phosphatase activity measurements showed that PP12 complexed to sds22 is catalytically inactive. The complex cannot be activated by limited proteolysis. The complex is unable to bind to microcystin sepharose. This suggests that sds22 may block the microcystin binding site in PP12. A proportion of PP12 in sperm extracts, which is presumably not complexed to sds22, is catalytically active. Fluorescence immunocytochemistry was used to determine the intrasperm localization of PP12 and sds22. Both proteins are present in the tail. They are also present in distinct locations in the head. Our data suggest that PP12 binding to sds22 inhibits its catalytic activity. Mechanisms regulating sds22 binding to PP12 are likely to be important in understanding the biochemical basis underlying development and regulation of sperm function.
epididymis, phosphatases, signal transduction, sperm maturation, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2, present in spermatozoa. Immotile spermatozoa contain higher activity levels of PP12 compared with motile spermatozoa. Inhibition of protein phosphatase activity by okadaic acid (OA) and calyculin A (CA) initiates motility in caput epididymal sperm without a requirement for a change in cAMP levels [2]. Based on this data, we speculated a key role for PP12 in sperm motility.
The sperm-specific phosphatase isoform, PP12, is expressed during germ cell differentiation in testis [4–6]. Of the four known isoforms (PP1
, PP1
, PP11, and PP12), PP12, with its unique carboxyl terminus, is the only PP1 isoform present in spermatozoa. We have shown that this protein phosphatase isoform is conserved and involved in the regulation of all mammalian spermatozoa studied so far, including human [2, 3, 7]. Since this discovery of its role in motility, other laboratories have shown that this phosphatase could also be involved in the onset of hyperactivated motility and acrosome reaction [8, 9]. A protein phosphatase homologue of PP1 also regulates rooster sperm motility [10, 11]. In Paramecium and Chlamydomonas, a serine/threonine phosphatase, tightly bound to the central doublet microtubules of the axoneme, regulates microtubule sliding velocity [12–16]. These studies show evolutionary conservation and the importance of serine/threonine phosphatases in regulating flagellar motility in spermatozoa and unicellular organisms.
In somatic cells, PP1 targeting and regulatory proteins regulate its activity. The catalytic unit (PP1C) of PP1 is generally associated with tissue-specific regulatory and targeting proteins [17]. Catalytic activity of PP1 could be further regulated by the phosphorylation state of the regulatory proteins. A number of protein inhibitors of PP1 have been identified—inhibitors I1 and I2, DARP32, and NIPP1 [18]. Inhibitor I1 and its brain-specific homologue DARP32 are activated by protein kinase A (PKA)-mediated phosphorylation and inactivated by calcineurin-induced dephosphorylation. In contrast, inhibitor I2 and NIPP1 are active in their dephosphorylated form but become inactive when phosphorylated by glycogen synthase kinase 3 (GSK-3) and PKA, respectively [18]. The objective of this article is to determine the identity and define the properties of the protein regulators of sperm PP12.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testes from mature bulls with intact tunica were obtained from a local slaughter house and spermatozoa were isolated from caput or caudal epididymis and washed as previously described [2] in a buffer containing 120 mM NaCl, 10 mM KCl, 10 mM Tris, pH 7.4. Sperm pellets were suspended in a homogenization buffer (HB buffer, containing 10 mM Tris, pH 7.2, 1 mM EDTA, and 1 mM EGTA) and sonicated with three 5-sec bursts of a Biosonic II (Bronwell Scientific, Rochester, NY) sonicator at maximum setting. The sperm sonicate was centrifuged at 16 000 x g for 10 min. The 16 000 x g supernatants are referred to as sperm extracts in this article.
Western Blot Analysis
Sperm extracts (usually 50 µg protein) were separated by SDS-gel electrophoresis through 10% or 12% acrylamide slab gels. After electrophoresis, proteins were electrophoretically transferred to Immobilon-P, PVDF membrane (Millipore Corp., Bedford, MA). 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 TBS containing 0.1% Tween 20 (TTBS) and then incubated with anti-PP12 (1:10 000) or anti-sds22 (1:2000). Both antibodies were commercially prepared (Zymed Laboratories, San Francisco, CA) using a synthetic carboxyl terminus extension of PP12 (22 amino acids of the carboxyl terminus) and amino acid residues 329–342 of sds22 as antigens. Antibodies were affinity purified with the synthetic peptides conjugated to a sulfo-link column (Pierce, Rockford, IL). After washing, the blots were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase at 1:2000 dilution for 1 h. The blots were then washed twice for 15 min each and four times for 5 min each before development with a ECL chemiluminescence kit (Amersham, Piscataway, NJ).
Phosphatase Activity
Procedures for the preparation of radiolabeled phosphorylase a and its use as a substrate for measurement of PP1 activity are previously reported [2]. The substrate and sperm extracts were incubated (in a total volume of 40 µl) at 30°C with or without inhibitors for 10 min. At the end of this incubation, the reaction was terminated with 180 µl 20% trichloro acetic acid (TCA), after which the tubes were centrifuged for 5 min at 16 000 x g at 4°C. The supernatants were analyzed for 32PO4 released from phosphorylase a. This assay is specific for the protein phosphatases PP1 and PP2A [2]. However, as previously reported, the predominant phosphatase in spermatozoa is PP1 [2]. The effect of limited proteolysis on phosphatase activity was performed by pretreating the samples with trypsin (50 µg/ml) (Sigma-Aldrich, St. Louis, MO) for 10 min at 30°C, and the reaction was stopped by 250 µg/ml trypsin inhibitor (Sigma-Aldrich). The samples were then assayed for phosphorylase phosphatase assay as described.
Immunoaffinity Purification
One milliliter of protein G sepharose suspension (Pharmacia, Piscataway, NJ) was first washed with distilled water followed by one wash with TTBS. The protein G sepharose was mixed with 0.6 mg of affinity-purified antibody and shaken for 2 h at 4°C. The slurry was transferred to a disposable column and washed with 15 ml of TTBS. Sperm 16 000 x g extract (7 ml prepared from 7 x 109 bovine caudal sperm extract) was passed through the column three times. After passing through the column, the extract is referred as the flow-through fraction or flow-through. The column was washed with 50 ml HB buffer supplemented with proteolytic inhibitors. The column matrix was transferred to two Eppendorf tubes and 0.5 ml of a 1 mg/ml PP12 carboxyl terminus peptide was added to each tube. The tubes were shaken for 2 h at 4°C. The gel slurry was transferred back to the column and the eluate (about 2 ml) was collected by a quick spin of the column. This eluate is referred as the complex or PP12 complex. The eluate was concentrated to about 0.1 ml through a Centricon-50 filter (Millipore Corp.).
Microsequencing
The concentrated eluate from the affinity column, as described above, was resolved by SDS gel electrophoresis. Twenty microliters each of the concentrated eluate was loaded in three lanes. The gel was stained with Coomassie blue, destained, and the 43 Mr x 10-3 band was excised from each of the lanes. The excised bands were washed once with HPLC-grade acrylonitrile (Sigma-Aldrich) as per instructions from the Harvard Microsequencing Facility. 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).
Microcystin-Agarose Chromatography
Microcystin-agarose (MC-agarose) (Upstate Biotechnology, Lake Placid, NY) was washed twice with HB buffer supplemented with 5% BSA to prevent nonspecific binding. Extracts of sperm sonicates (100 µl) were incubated with MC-agarose with shaking at 4°C for 2 h. After this incubation, the unbound or flow-through fraction was collected following centrifugation of the MC-agarose at 16 000 x g for 5 min. The pellet was washed five times with HB buffer. The flow-through fraction was supplemented with 10% glycerol and stored at -20°C until measurement of PP1 activity or Western blot analysis. Proteins bound to MC-agarose were analyzed by Western blot following boiling of the pellet with SDS sample buffer.
Fluorescence Immunocytochemistry
Sperm were isolated as described above and washed twice and resuspended in PBS. The cells were fixed in 4% formaldehyde in PBS at 4°C for 1/2 h. The sperm solution was then treated with 0.2% Triton X-100. The fixed sperm was attached to poly-L-lysine-coated coverslips. The coverslips 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 coverslips were then incubated with primary antibody for 1 h at room temperature or overnight at 4°C, washed three times with TTBS, and incubated with a secondary antibody conjugated to indocarbocyanine (Cy3), (Jackson Laboratories, West Grove, PA) for 1 h at room temperature. The coverslips were washed five times with TTBS and examined by fluorescence microscopy.
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RESULTS |
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2 by Affinity Chromatography
We had previously shown that sperm extracts contained a protein inhibitor of PP1 [2]. It was likely that a proportion of sperm PP12 was complexed to this inhibitor and other proteins. In initial attempts to isolate the PP12-inhibitor complex, we used column chromatography but failed to obtain sufficient yields and purity. We then tried an immunoaffinity column procedure for purification. We prepared polyclonal antibodies against a synthetic polypeptide corresponding to carboxyl-terminus of PP12. After affinity purification, these antibodies are high affinity and specific to PP12 [2]. Approximately 0.6 mg of this affinity-purified antibody immobilized with protein G sepharose was used to purify PP12 in sperm extracts. The extract, flow-through fraction, and the protein complex bound to the antibody column were analyzed by SDS gel electrophoresis followed by Western blot probed with PP12 antibody (Fig. 1A). Lanes 1 and 2 show that PP12 is present in the extract and the flow-through fraction. Lane 3 shows that immunoreactive PP12 is also present in the complex. Coomassie blue staining of the blot shows multiple protein bands in the extract and flow-through fraction but only two bands at 39 Mr x 10-3 and 43 Mr x 10-3 for the complex in lane 3. The 39 Mr x 10-3 band corresponds to PP12, the expected molecular weight of the protein [2]. The eluate containing the complex was concentrated to 0.1 ml with a Centricon-50 filter. The bands at 43 Mr x 10-3 and 39 Mr x 10-3 are seen better when this concentrated eluate was loaded (Fig. 1B). The 43 Mr x 10-3 band, the presumed PP12 binding protein, was excised from the acrylamide gel after Coomassie staining and sequenced at the Harvard Microsequencing facility.
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Microsequencing Shows That sds22 Is Present in Purified PP12
Microsequencing following in-gel proteolysis of the 43 Mr x 10-3 band showed that it was a protein resembling the human homologue of the yeast PP1 binding protein sds22. This human homologue has been previously cloned based on expressed sequence tag sequence similarities to yeast sds22 [19]. The amino acid sequence of human sds22 is shown in Figure 2. Peptide sequences obtained by microsequencing are underlined. The yeast protein sds22 is a prototypical member of a family of proteins containing repeats of a leucine-rich amino acid sequence motif [19]. Human sds22 contains 11 repeats of a leucine-rich 22 amino acid segment. The protein contains consensus sites for protein kinase A, calmodulin-dependant kinase II, and glycogen synthase kinase 3. All these protein kinases are present in spermatozoa [20–22]. In Figure 2, these consensus sequences are boxed and the serine/threonine residues that could be phosphorylated are in bold and underlined.
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Antibodies to sds22 Detect the Protein in the PP12 Complex and in Epididymal Sperm Extracts
We generated antibodies against sds22 using a synthetic polypeptide corresponding to amino acid residues 329–342 in sds22 protein. The antibodies were affinity purified. The antibodies reacted against the 43 Mr x 10-3 protein of the PP12 complex (Fig. 3), confirming the microsequencing data. Antibodies to sds22 raised against two other regions of the polypeptide (residues 338–350 and residues 346–360) also reacted against the same protein (data not shown). Data in Figure 3 also show that sds22 is present not only in soluble extracts of caudal epididymal spermatozoa but also in the pellet fraction of sperm sonicates.
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Sds22 Binding to PP12 Inhibits Its Catalytic Activity
We next investigated whether all of PP12 in sperm extract was complexed with sds22 and whether the PP12-sds22 complex was catalytically active. We prepared PP12 complex from caudal sperm extracts by the same immunoaffinity column procedure outlined under Figure 1. Phosphatase activity measurements and Western blot analysis of soluble sperm extracts, flow-through fractions, and the PP12-sds22 complex are shown in Figure 4. Enzyme activity in Figure 4B shows that PP12-sds22 complex isolated by immunoaffinity chromatography is responsible for less than 20% of the phosphatase activity present in the extracts, i.e., about 80% of the phosphatase activity of the soluble extracts is still present in the flow-through fraction of the immunoaffinity column. Surprisingly, however, Western blot analysis shows that the complex contains most of the immunoreactive PP12 while the flow-through fraction contains only a relatively small amount of PP12. The reason why this PP12, apparently responsible for most of the phosphatase activity in sperm extracts, cannot be removed by immunoaffinity column is not known. The PP12 complex isolated from the column contains both PP12 and sds22, further confirming data shown in Figure 3. Interestingly, a significant proportion of sds22 in sperm extracts is still present in the flow-through fraction, suggesting that there is an excess of sds22, which may or may not be bound to PP12. In experiments with protein G sepharose alone, without PP12 antibody, virtually no PP12 or sds22 was released from the column, showing that the immunoprecipitation data in Figure 4 were not due to nonspecific binding and release of PP12 and sds22. Immunoprecipitation experiments with sds22 antibodies were not successful, suggesting that the antibodies did not recognize sds22 in its native conformation in the extract.
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Sds22-Bound PP12 Does Not Bind> to Microcystin Agarose
To obtain independent verification of data with antibodies in Figure 4, we used another approach to isolate PP12 from sperm extracts—affinity chromatography with MC-agarose. Microcystin is an inhibitor of PP1. Microcystin-affinity chromatography has been used to isolate PP1 binding proteins. Bovine epididymal sperm extracts were incubated with MC-agarose that was prewashed with homogenization buffer supplemented with 5 mg/ml BSA. The unbound fraction (flow-through) and the proteins bound to MC-agarose were analyzed by Western blot (Fig. 5) probed with PP12 and sds22 antibodies. It can be seen in Figure 5A that only a proportion of PP12 present in the extract binds to MC. However, this MC-bound PP12 is not associated with sds22 because there is no immunoreactive sds22 detected in the MC-bound fraction. This implies that PP12-sds22 complex, the complex that could be isolated using immunoaffinity chromatography (Fig. 4), is unable to bind to microcystin. Alternatively, microcystin could have displaced sds22 from the PP12-sds22 complex. Because PP12-sds22 complex has little catalytic activity (Fig. 4), PP1 activity measurement in the flow-through fraction could tell us the activity state of PP12 not bound to microcystin. It can be seen in Figure 5B that less than 10% of the total PP1 activity in the extracts remains in the flow-through fraction. That PP12 not bound to microcystin is inactive. Activity measurements of the MC-bound PP12 were not feasible because the enzyme is bound to the inhibitor. Thus, MC-agarose is able to bind and remove almost all of the catalytically active phosphatase from the sperm extract. What remains in the extract following incubation with MC-agarose is most likely the inactive PP12-sds22 complex.
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Immunolocalization of sds22 and PP12
Immunofluorescence was used to localize PP12 and sds22 within spermatozoa. As seen in Figure 6, PP12 is present along the entire length of the flagellum including the mid-piece. In the head, staining is intense in the posterior region and in the equatorial segment. Staining is speckled in the anterior region of the sperm head. Sds22 antibodies stain the principal piece of the flagellum and the head-neck junction. Staining is relatively weak in the mid-piece. In the sperm head staining appears speckled through out with a more intense staining in the anterior region. Thus there are regions within spermatozoa where PP12 and sds22 appear to be co-localized. The staining with the two antibodies appear to be specific since there was no fluorescence observed when the primary antibodies were pre-incubated with the respective immunizing peptides or when sperm were incubated with secondary antibody alone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 in spermatozoa. At about the same time that we accomplished sequencing this PP12 regulatory protein from bovine spermatozoa, a group of researchers reported that sds22 is found in rat testis [23]. Because the expression of the messenger RNA for sds22 closely matched the expression of PP12 in developing rat testis, it was speculated that PP12 and testis sds22 are associated with each other [23]. That article did not present direct evidence for the association of the two proteins or activity measurements of the PP12-sds22 complex. Our data show that sds22 copurifies with sperm PP12 isolated by immunoaffinity chromatography. Our data also show that, in bovine spermatozoa, PP12 isolated as a complex with sds22 (Figs. 1 and 3) is catalytically inactive (Fig. 4).
Studies from the laboratory of one of the authors in this article were the first to characterize sds22 in somatic cells [19, 24, 25]. In rat liver cells, most of the sds22 is present in the nucleus. A proportion (about 10–15%) of protein phosphatase (PP1) in the nucleus is bound to sds22. Catalytic activity of PP1 bound to sds22 is inhibited but can be activated by limited proteolysis. The PP1-sds22 complex can also bind to microcystin. It has been proposed that nuclear sds22 could regulate PP1 activity during mitosis [26]. Our data show that, in spermatozoa, sds22 and its complex with PP12 differ substantially in their properties compared with somatic cells. In spermatozoa, most of the sds22 appears to be cytoplasmic because the protein can be relatively easily extracted under conditions not expected to affect the sperm nucleus and because fluorescence localization shows that a considerable proportion of the protein is present in the sperm tail (Figs. 3 and 6). It cannot be ruled out that a proportion of sds22 in the pellet fraction of sperm sonicates (Fig. 3) could be present in the nucleus. Proteolysis by trypsin fails to activate the PP12-sds22 complex. Finally, unlike in somatic cells, the PP12-sds22 complex fails to bind to MC-agarose. The reason for this could be that the microcystin-binding site is masked by sds22. Alternatively, it is possible that microcystin competes for the sds22 binding site on PP12. We consider this latter possibility unlikely in view of the activity data (Figs. 4 and 5), i.e., PP12 that is not bound to microcystin is inactive, consistent with data in Figure 4, which show that sds22-bound PP12 is inactive. These differences in the properties of the phosphatase-sds22 complex in somatic cells compared with sperm could be due to the unique properties of sperm-specific PP12. The fact that sds22 is a PP1 binding protein in yeast [27–29] lends strong support to our conclusion that its homologue in spermatozoa is also a PP1 regulatory protein. In yeast [30] and in somatic cells, sds22 is suggested to play a role in mitosis. A role for the protein in regulating PP12 in the terminally differentiated sperm cell is novel.
In yeast, sds22 binding apparently activates PP1 [26, 27]. In bull epididymal spermatozoa, catalytic activity of PP12 bound to sds22 is inhibited, as seen in Figure 4. However, PP12, which cannot bind to the immunoaffinity column, is catalytically active. Whether this PP12 is bound to sds22 is not known. It is possible that the PP12 that is catalytically active could also be bound to sds22 just as in yeast. This would imply that sds22 in spermatozoa exists in two states—an inhibitor and an activator form. It is possible that these two forms correspond to phosphorylated and dephosphorylated sds22. Data in Figure 5 could also be consistent with this interpretation, i.e., microcystin binds to active PP12 by displacing sds22 bound to it. A complete understanding of how sds22 binds to sperm PP12 may come from experiments with recombinant proteins. These studies are underway in our laboratory.
As noted above, most sperm sds22 appears cytoplasmic. The protein can be extracted by suspending spermatozoa in a hypotonic solution (HB buffer) followed by sonication (Fig. 3). The protein is also extracted, without sonication, when spermatozoa are suspended in HB buffer supplemented with 1% Triton X-100 (data not shown). Data in Figure 3 also show that a proportion of sds22 is still present in the pellet. It is possible that this sds22 could be bound to the insoluble fraction of PP12 in the pellet. Alternatively, this sds22 could be present in the sperm nucleus. Further studies are required to resolve this question. It should also be emphasized that a substantial excess of sds22 compared with PP12 appears to be present in the sperm extracts (Fig. 4). Analysis of the amino acid sequence of sds22 suggests that it could be a phospho-protein (Fig. 2). Whether this is the case in spermatozoa is of considerable interest. We have observed that sometimes sds22 can be resolved as a doublet in Western blot (data not shown), a possible indication that the slower migrating band could be phosphorylated sds22. It is interesting to note that there is a 46% identity between the amino acid sequence of the polypeptides in yeast and human. Antibodies used in this article, raised against a synthetic polypeptide in the carboxyl terminus region of the protein, cross reacts with a 43 Mr x 10-3 protein, presumably sds22, in extracts prepared from rat, hamster, and primate spermatozoa (data not shown). We have shown that PP12 is also present in a wide variety of mammalian species, including human [2, 3, 7]. It is quite likely that sds22 is a regulator of protein phosphatase in these species, as demonstrated in this article for bovine spermatozoa.
We have previously shown that bovine and primate sperm extracts contain a heat-stable inhibitor of PP12 [2]. We tested to see if sds22 was this heat-stable inhibitor. We found that it was not because sds22 was heat labile. The nature of the protein responsible for this inhibitory activity remains unknown. The stoichiometry of sds22 binding to PP12 is also not known. It should be emphasized that it is not possible to draw any conclusion about stoichiometry based on intensity of immunoreactivity to PP12 and sds22 antibodies in Western blots (Figs. 3 and 4A). The intensity of the Coomassie stain of the 43 Mr x 10-3 and 39 Mr x 10-3 bands in Figure 1B is also not a good indicator of the ratio of sds22 and PP12 in the complex. We have undertaken studies to isolate the different quaternary forms of PP12 by column chromatography. Size determination of the PP12 complexes by column chromatography could give a better indication of composition of the complexes. Studies are also underway to use recombinant PP12 and sds22 to further analyze the nature of interaction between these proteins.
A puzzling aspect of our observations is that a proportion of PP12 in sperm extracts does not bind to the immunoaffinity column (Figs. 1 and 4). It is unlikely that the reason for this is that the antibody binding capacity was limiting. An excess of antibodies was used in these experiments—up to 0.6 mg of affinity purified antibodies. However, the PP12 that fails to bind to the antibody in its native form in the extract is immunoreactive following denaturing gel electrophoresis and Western blot. A possible explanation for this observation is that the carboxyl terminus region of PP12, the region against which the antibodies were raised, is inaccessible to the antibodies, possibly due to the binding of a regulatory protein. This proportion of native PP12 that does not bind to antibodies is, however, responsible for almost all of the phosphorylase phosphatase activity present in the extracts. Based on these observations, we hypothesize that sperm PP12 exists in two quaternary structures, one that is inactive and bound to sds22 and another that is active and bound to an unidentified protein, which may be sds22 itself as noted above. Mechanisms regulating sds22 binding are likely to be responsible for the decline of PP12 activity during epididymal sperm maturation. Understanding what these mechanisms are will depend on the identification of the unknown regulatory protein associated with active PP12 and determination of the exact biochemical mechanisms that regulate sds22 binding to PP12. These studies are in progress in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 330 672 3713; svijayar{at}kent.edu
Received: 31 January 2002.
First decision: 19 February 2002.
Accepted: 3 July 2002.
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gene in rat hepatocellular carcinomas. Jpn J Cancer Res 1990 81:1272-1280[CrossRef][Medline]This article has been cited by other articles:
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 L. Pedelini, M. Marquina, J. Arino, A. Casamayor, L. Sanz, M. Bollen, P. Sanz, and M. A. Garcia-Gimeno YPI1 and SDS22 Proteins Regulate the Nuclear Localization and Function of Yeast Type 1 Phosphatase Glc7 J. Biol. Chem., February 2, 2007; 282(5): 3282 - 3292. [Abstract] [Full Text] [PDF]
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Z. Huang, P. R. Somanath, R. Chakrabarti, E. M. Eddy, and S. Vijayaraghavan Changes in Intracellular Distribution and Activity of Protein Phosphatase PP1{gamma}2 and Its Regulating Proteins in Spermatozoa Lacking AKAP4 Biol Reprod, February 1, 2005; 72(2): 384 - 392. [Abstract] [Full Text] [PDF]
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C. Hrabchak and S. Varmuza Identification of the Spermatogenic Zip Protein Spz1 as a Putative Protein Phosphatase-1 (PP1) Regulatory Protein That Specifically Binds the PP1c{gamma}2 Splice Variant in Mouse Testis J. Biol. Chem., August 27, 2004; 279(35): 37079 - 37086. [Abstract] [Full Text] [PDF]
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Z. Huang and S. Vijayaraghavan Increased Phosphorylation of a Distinct Subcellular Pool of Protein Phosphatase, PP1{gamma}2, During Epididymal Sperm Maturation Biol Reprod, February 1, 2004; 70(2): 439 - 447. [Abstract] [Full Text] [PDF]
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S. Mishra, P. R. Somanath, Z. Huang, and S. Vijayaraghavan Binding and Inactivation of the Germ Cell-Specific Protein Phosphatase PP1{gamma}2 by sds22 During Epididymal Sperm Maturation Biol Reprod, November 1, 2003; 69(5): 1572 - 1579. [Abstract] [Full Text] [PDF]
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 D. M. Katschinski, H. H. Marti, K. F. Wagner, J. Shibata, K. Eckhardt, F. Martin, R. Depping, U. Paasch, M. Gassmann, B. Ledermann, et al. Targeted Disruption of the Mouse PAS Domain Serine/Threonine Kinase PASKIN Mol. Cell. Biol., October 1, 2003; 23(19): 6780 - 6789. [Abstract] [Full Text] [PDF]
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