Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human and monkey ejaculated sperm contain protein phosphatase-1 (PP1), PP1 inhibitor 2 (12), and glycogen synthase kinase-3 (GSK-3). Inhibition of ejaculated human sperm protein phosphatase (PP) activity with calyculin-a (CL-A) significantly stimulates motility, implicating protein dephosphorylation in motility regulation. The present experiments were conducted to characterize and compare PP and GSK-3 activity in monkey caput and caudal epididymal sperm, to determine the cellular distribution of these enzymes, and to test the thesis that epididymal sperm PP activity is inversely related to motility. Caput epididymal sperm populations, (8.8% motile) contained levels of PP activity that were >3 times as high as those of caudal spermatozoa. This PP activity was further identified by inhibitor response profiles as PP1. In both caput and caudal sperm, the majority of this PP1 activity was localized in 100,000 x g soluble fractions. Western blot analysis indicated that a portion of this difference was the result of elevated amounts of PP1 in caput compared with caudal epididymal sperm. The presence of GSK-3 activity was undetectable in 100,000 x g insoluble fractions of epididymal sperm, whereas both caput and caudal sperm soluble fractions contained GSK-3 activity, which was approximately threefold higher in caput sperm compared with caudal populations. Treatment of caput epididymal sperm from the rhesus macaque with the PP inhibitor CL-A resulted in a significant, dose-dependent increase from 8 to 38% motile cells (without any effect on their path velocity). In contrast, CL-A had no significant influence on either percent motility or path velocity of caudal epididymal sperm. Cytosolic PP1 and GSK-3 activities appear to be inversely related to the motility of monkey epididymal sperm and may have a regulatory role in the development of the potential for motility in epididymal sperm.
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PMID:Motility potential of macaque epididymal sperm: the role of protein phosphatase and glycogen synthase kinase-3 activities. 1010 Apr 73

Plasma membranes of caput and cauda epididymal spermatozoa of hamster exhibited protein phosphatase activity. This membrane-associated protein phosphatase was identified as a protein tyrosine phosphatase based on its ability to hydrolyse a substrate specific for PTPase, by inhibition of its activity with a specific inhibitor of PTPase (sodium orthovanadate) and by the inability to inhibit its activity with calyculin, okadaic acid, trifluoperazine, calcium, EGTA, and EDTA, which are specific inhibitors of other protein phosphatases, namely PP-1, PP-2A, PP-2B, and PP-2C respectively. The specific activity of the protein tyrosine phosphatase both in the caput and cauda epididymal sperm plasma membranes was similar, implying that the enzyme may not be solely responsible for the differential phosphorylation of membrane proteins observed during maturation (Uma Devi et al. 1997. Mol Reprod Dev 47:341-350). Thus the significance of the PTPase activity in epididymal maturation still remains to be determined. The membrane-associated PTPase may not be essential for acquisition of motility. However, it appears that the activity is essential for the sustenance of motility since sodium orthovanadate, which specifically inhibits PTPase activity, also inhibits motility of spermatozoa and decreases the overall velocity of the spermatozoa by decreasing the average path velocity, straight line velocity, curvilinear velocity, and amplitude of lateral head displacement of the treated spermatozoa.
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PMID:Plasma membrane-associated protein tyrosine phosphatase activity in hamster spermatozoa. 1023 Aug 15

The effect of genistein on anion secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in cultured rat cauda epididymal epithelia was studied by short-circuit current (Isc) technique. Genistein added apically stimulated a concentration-dependent rise in Isc due to Cl(-) and HCO(3)(-) secretion. The genistein-induced Isc was observed in basolaterally permeabilized monolayers, suggesting that the Isc response was mediated by the apical anion channel. The response could be blocked by the nonspecific Cl(-) channel blocker, diphenylamine-2-carboxylate (DPC), but not by the Ca(2+)-activated Cl(-) channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Genistein did not increase intracellular cAMP, but H-89, a protein kinase A inhibitor, completely abolished the Isc response to genistein. Moreover, pretreatment of the tissues with MDL-12330A, an adenylate cyclase inhibitor, markedly attenuated the Isc response to genistein, but the response was restored upon the addition of exogenous cAMP. Ca(2+), protein kinase C, tyrosine kinase, and protein phosphatase signalling pathways were not involved in the action of genistein. It is speculated that genistein stimulates anion secretion by direct interaction with CFTR. This requires a low level of phosphorylation of CFTR by basal protein kinase A activity. It is suggested that genistein may provide therapeutic benefit to male infertility associated with cystic fibrosis.
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PMID:Activation of cystic fibrosis transmembrane conductance regulator in rat epididymal epithelium by genistein. 1061 Oct 78

A phosphoprotein phosphatase (PPase M-I) that dephosphorylates serine and threonine residues of histones was isolated from the goat cauda-epididymal sperm plasma membrane and partially characterized. The PPase was solubilized from the sperm membrane by treating it with 0.1 N NaOH at pH 11.4 and the solubilized enzyme was partially purified by concanavalin A-sepharose affinity chromatography and high-performance liquid chromatography (HPLC), revealing it to be a 520-kDa protein. The PPase gave a single protein band in native polyacrylamide gel electrophoresis (PAGE), but in the presence of SDS it resolved into multiple proteins (35-170 kDa) showing that the isolated enzyme contained a few contaminating proteins. The enzyme is a glycoprotein because it binds with high affinity to concanavalin A. It was maximally active at pH 8.0 and its activity was not dependent on bivalent metal ions. The enzyme is a specific phosphatase as it displayed higher affinity for dephosphorylation of large molecular weight phosphate esters. The PPase showed broad substrate specificity for the dephosphorylation of a variety of proteins. The membrane-associated PPase was strongly (70-80%) inhibited by detergents (0.5%) such as Nonidet P-40, Lubrol PX, Triton X-100 and Tween-20. Pyrophosphate (5 mm) and orthovanadate (400 microM) had no significant effect on the activity of the isolated PPase whereas polyamines such as spermine (10 mM) and spermidine (10 mM) slightly inhibited (20%) the enzymatic activity. Inorganic phosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolic PPases, had no appreciable effect on the activity of PPase M-I, indicating that the membrane-bound PPase is distinct from the cytosolic PPases. The enzyme was radiolabelled when the intact spermatozoa were subjected to lactoperoxidase-mediated radioiodination reaction. The results show that the PPase M-I is an ecto-enzyme that may play an important role in sperm physiology by causing the dephosphorylation of the sperm outer surface phosphoproteins.
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PMID:Partial purification and characterization of a phosphoprotein phosphatase from sperm plasma membrane. 1097 6

Idiopathic azoospermia, characterized by abnormal spermatogenesis, is commonly treated by performing intracytoplasmic sperm injection (ICSI) with sperm retrieved from testicular biopsies. However, no controlled experiments have been performed using an animal model to assess the efficacy or safety of the procedure. We have performed ICSI with testicular sperm obtained in a similar manner from testes of male mice homozygous for a null mutation in the protein phosphatase 1cgamma gene (PP1cgamma) or those of their wild-type littermates. PP1cgamma mutant testicular sperm are less resistant to sonication than are wild-type sperm and display a range of morphological abnormalities, similar to those reported for testicular sperm from idiopathic azoospermic men. PP1cgamma mutant sperm are unable to support development to the blastocyst stage, resulting in arrested development either before or just after compaction. A comparison of testicular and epididymal sperm from wild-type males revealed that the epididymal sperm caused embryos to fragment at an elevated rate. These results suggest that ICSI with any kind of testicular sperm carries an increased risk of embryo fragmentation and that abnormal testicular sperm has an added risk of embryo wastage at later preimplantation stages.
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PMID:Development to blastocyst is impaired when intracytoplasmic sperm injection is performed with abnormal sperm from infertile mice harboring a mutation in the protein phosphatase 1cgamma gene. 1260 45

Testis- and sperm-specific protein phosphatase, PP1gamma2, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP1gamma2 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP1gamma2 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1gamma2 in motile caudal spermatozoa (Huang Z, et al. Biol Reprod 2002; 67:1936-1942). The objectives of this study were to determine: 1) stoichiometry of PP1gamma2-sds22 binding and 2) whether PP1gamma2 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP1gamma2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP1gamma2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP1gamma2 coeluted in the gradient fraction. In contrast, caput sperm sds22 and PP1gamma2 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP1gamma2-sds22 complex from caudal sperm was 88 kDa in size. Caput sperm sds22 and PP1gamma2 eluted at 60 kDa and 39 kDa, respectively. SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP1gamma2 (39 kDa), suggesting a 1:1 complex between these two proteins. PP1gamma2 bound to sds22 in this complex was inactive. Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17). This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP1gamma2. Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP1gamma2 during epididymal sperm maturation.
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PMID:Binding and inactivation of the germ cell-specific protein phosphatase PP1gamma2 by sds22 during epididymal sperm maturation. 1282 76

The enzyme PP1gamma2 is a testis- and sperm-specific isoform of type 1 protein phosphatase (PP1), and it is the only isoform of PP1 in spermatozoa. The enzyme PP1gamma2 is essential for spermatogenesis and is also a key enzyme in the development and regulation of sperm motility. The carboxy terminus of the enzyme contains a consensus amino acid sequence for phosphorylation by cyclin-dependent kinases. Using antibodies specific to this phosphorylated amino acid sequence domain, we found that phosphorylated PP1gamma2 is present in bovine epididymal spermatozoa. The level of phosphorylated PP1gamma2 is significantly higher in motile caudal compared to immotile caput epididymal spermatozoa. A number of treatments, such as 2-chloro adenosine, cAMP analogues, cAMP phosphodiesterase inhibitors, and calcium, which stimulate sperm motility, did not alter the level of phosphorylated PP1gamma2. However, calyculin A, which is an inhibitor of protein phosphatase subtypes PP1 and PP2A, significantly increases the level of phosphorylated PP1gamma2 in both caput and caudal epididymal spermatozoa. Partial purification by column chromatography showed that phosphorylated PP1gamma2 is catalytically active. Phosphorylated PP1gamma2 is the only spontaneously catalytically active form of the enzyme in caudal sperm extracts. Western blot analysis shows that the enzyme cyclin-dependent kinase 2, one of the enzymes that phosphorylates the consensus domain at the carboxy terminus in PP1 isoforms, is present in spermatozoa. Western blot analysis of proteins extracted from purified head and tail fragments of spermatozoa showed that phosphorylated PP1gamma2 is present predominantly in the sperm head. Fluorescence immunocytochemistry also showed that phosphorylated PP1gamma2 is present predominantly in the posterior region of the sperm head. The distinct subcellular localization and changes in its level during sperm maturation suggest a possible role for sperm phosphorylated PP1gamma2 in signaling events during fertilization.
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PMID:Increased phosphorylation of a distinct subcellular pool of protein phosphatase, PP1gamma2, during epididymal sperm maturation. 1456 12

The protein phosphatase PP1gamma2 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 PP1gamma2 is phosphorylated and that the amount of phosphorylated PP1gamma2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439-447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1gamma2 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 PP1gamma2 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 PP1gamma2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1gamma2 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 PP1gamma2 is one of its binding partners.
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PMID:Protein 14-3-3zeta binds to protein phosphatase PP1gamma2 in bovine epididymal spermatozoa. 1502 37

We have identified for the first time the presence of chloride intracellular channel (CLIC) proteins in bovine epididymal spermatozoa. CLIC1 was discovered during microsequencing of proteins that co-purified with protein phosphatase 1, PP1gamma2, in sperm extracts. In addition to CLIC1, Western blot showed that two additional CLIC family members, CLIC4 and CLIC5, are also present in spermatozoa. CLIC fusion proteins, GST-CLIC1, GST-CLIC4 and GST-CLIC5, were all able to bind to PP1gamma2 in sperm extracts during pull-down assays. Immunofluorescence microscopy revealed that each of the three isoforms occupies a distinct location within the cell. Given that PP1gamma2 is a key enzyme regulating sperm motility, PP1gamma2-binding proteins, such as the CLIC proteins, are likely to play significant roles in sperm function.
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PMID:Identification of chloride intracellular channel proteins in spermatozoa. 1514 83

The intracellular mediators cyclic AMP, calcium and pH regulate sperm function through changes in protein phosphorylation. Protein phosphorylation is the net result of the actions of protein kinases and phosphatases. The protein phosphatase isoform, PPlgamma2, with a unique C-terminus extension is highly enriched in spermatozoa and testis. Changes in PPlgamma2 catalytic activity, its phosphorylation, and binding to its regulatory proteins change during epididymal maturation. Thus PPgamma2 is a key protein in sperm motility regulation; decreased enzyme activity is associated with increased motility. This review summarizes the current knowledge of this sperm protein phosphatase. The biochemical properties of its regulatory proteins, sds22 and protein 14-3-3, among others, are discussed. Future studies will elucidate sperm signalling pathways involving PP1gamma2 and determine if the unique structure of PP1gamma2 is critical to normal male gamete development and function. Understanding the role of PP1gamma2 will not only contribute to the basic understanding of male gamete functions but also has practical applications in clinical andrology and in the development of male contraceptives.
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PMID:Regulation of sperm function by protein phosphatase PP1gamma2. 1756 66


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