Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240-10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold by incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.
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PMID:Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor 4E-binding protein 1 on the rapamycin-insensitive site (serine-111). 980 82

Highly purified plasma membranes, isolated by an aqueous two-phase polymer method from goat epididymal spermatozoa, were found to possess a kinase activity that causes phosphorylation of serine and threonine residues of several endogenous plasma membrane proteins. Cyclic AMP, cyclic GMP, Ca(2+)-calmodulin, phosphatidylserine-diolein, polyamines and heparin had no appreciable effect on this kinase. Autoradiographic analysis showed that the profile of the phosphorylation of membrane proteins by this endogenous cAMP-independent protein kinase underwent marked modulation during the transit of spermatozoa through the epididymis. In caput sperm plasma membrane, 18, 21, 43, 52, 74 and 90 kDa proteins were phosphorylated, whereas, in the corpus and cauda epididymal spermatozoa, a differential phosphorylation pattern was observed with respect to the 90, 74, 21 and 18 kDa proteins. The rate of phosphorylation of the 74 kDa protein decreased markedly during the early phase of sperm maturation (caput to distal corpus epididymides) whereas there was little change in kinase activity in sperm plasma membrane. In contrast, the rates of phosphorylation of the 18 and 21 kDa proteins increased during the terminal phase (distal corpus to distal cauda epididymides) of sperm maturity, although the kinase activity of membrane decreased significantly during this phase. The modulation of the phosphorylated states of these specific membrane proteins may play an important role in the maturation of epididymal spermatozoa.
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PMID:Maturation-dependent modification of the protein phosphorylation profile of isolated goat sperm plasma membrane. 1034 20

Sialoprotein "anti-agglutinin," previously shown to inhibit sperm head-to-head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti-agglutinin by mass spectrometry, by N-terminal amino acid sequence analysis, and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting techniques to assess phosphate content of the molecule. Anti-agglutinin had the SDS-PAGE mobility of approximately 25 kDa. By electrospray ionization-mass spectrometry, however, mass spectra of anti-agglutinin were characterized by two major peaks (19,379-19,382 Da and 19,395-19,397 Da) and several minor peaks. Mass spectrometry of tryptic peptide fragments of deglycosylated anti-agglutinin and amino acid sequence analysis revealed that the protein has a unique peptide-mass fingerprinting of fragments (12,668 Da, 5,209 Da, 1,226 Da, and 1,168 Da) and a novel N-terminal amino acid sequence (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally Western blot techniques, using commercially available monoclonal antibodies, were used to detect presence of phosphothreonine and phosphoserine substituents, but two different monoclonal antibodies did not detect phosphotyrosine. Moreover, treatment with two different alkaline phosphotases converted the molecule, as assessed by SDS-PAGE and detection by silver stain, from the parent form of about 25 kDa to forms of approximately 19 kDa (similar to that assigned by mass spectrometry) and/or 15 kDa. Original antiserum generated toward, and reacting with native anti-agglutinin, reacted only with 19 kDa form. These results are consistent with the conclusion that the native anti-agglutinin may be a novel protein that is phosphorylated at serine and/or threonine residues.
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PMID:Biochemical characterization of sialoprotein "anti-agglutinin" purified from boar epididymal and seminal plasma. 1060 79

Glycosylation is one of the important post-translational modifications of sperm plasma membrane proteins during the maturation of epididymal spermatozoa that results in the development of motility and fertilizing capability. The aim of the present study was to identify and characterize the maturation-dependent asparagine-linked (N-linked) and serine- and threonine-linked (O-linked) glycoproteins of the epididymal spermatozoa of rhesus monkeys. The presence of N- and O-linked glycoproteins was confirmed by treatment of sperm membranes with N-glycosidase F and O-glycosidase. The major maturation-dependent sperm membrane glycoproteins identified on blots of SDS-PAGE-fractionated proteins of purified sperm plasma membranes from five segments of epididymis, probed with biotinylated lectins and Vectastain-ABC reagent included O-linked 170, 150, 86 and 60/58 kDa glycoproteins; N-linked 68, 56, 48 and 38 kDa glycoproteins and N- and O-linked 116 kDa glycoprotein, all of which exhibited marked differences in the degree of glycosylation between immature and mature sperm surfaces. These glycoproteins can be used as markers of sperm maturation in the epididymis of rhesus monkeys, during the screening of antifertility agents acting at the epididymis, or may be developed as potential sperm antigens. The 100% inhibition of fertility in female rats and rabbits immunized with major maturation-dependent 116 kDa glycoprotein showed the significance of glycosylation changes in the maturation status of epididymal spermatozoa. This 116 kDa protein can be used as a marker parameter of sperm maturation in the rhesus monkey, which is often the preferred animal model for preclinical studies. These results will contribute to the identification of an appropriate animal model for the development of male contraceptives in humans.
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PMID:Maturation-dependent glycoproteins containing both N- and O-linked oligosaccharides in epididymal sperm plasma membrane of rhesus monkeys (Macaca mulatta). 1086 36

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

Differentiation of spermatids into spermatozoa is regulated via phosphorylated RNA-binding proteins that modulate the expression of stage-specific mRNAs. We demonstrate that the phosphoserine, -threonine or -tyrosine, interaction protein, Styx, complexes with a testicular RNA-binding protein and is essential for normal spermiogenesis. Ablation of Styx expression in mouse disrupts round and elongating spermatid development, resulting in a >1,000-fold decrease in spermatozoa production. Moreover, Styx(-/-) males are infertile because of structural head abnormalities in residual epididymal sperm. Immunoprecipitation of Styx with Crhsp-24, a phosphorylated RNA-binding protein implicated in translational repression of histone mRNAs, provides a strategy for regulating posttranscriptional gene expression.
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PMID:The archetype STYX/dead-phosphatase complexes with a spermatid mRNA-binding protein and is essential for normal sperm production. 1184 24

Adiponectin is an abundant adipocyte-derived plasma protein with anti-atherosclerotic and insulin-sensitizing properties that suppresses hepatic glucose production and enhances glucose uptake into skeletal muscle. To characterize the potential effects of adiponectin on glucose uptake into adipose cells, we incubated isolated epididymal rat adipocytes with the globular domain of recombinant adiponectin purified from an E. coli expression system. Globular adiponectin increased glucose uptake in adipocytes without stimulating tyrosine phosphorylation of the insulin receptor or insulin receptor substrate-1, and without enhancing phosphorylation of Akt on Ser-473. Globular adiponectin further enhanced insulin-stimulated glucose uptake at submaximal insulin concentrations and reversed the inhibitory effect of tumor necrosis factor-alpha on insulin-stimulated glucose uptake. Cellular treatment with globular adiponectin increased the Thr-172 phosphorylation and catalytic activity of AMP-activated protein kinase and enhanced the Ser-79 phosphorylation of acetyl CoA carboxylase, an enzyme downstream of AMP kinase in adipose cells. Inhibition of AMP kinase activation using two pharmacological inhibitors (adenine 9-beta-D-arabinofuranoside and compound C) completely abrogated the increase in glucose uptake stimulated by globular adiponectin, indicating that AMP kinase is integrally involved in the adiponectin signal transduction pathway. Coupled with recent evidence that the effects of adiponectin are mediated via AMP kinase activation in liver and skeletal muscle, the findings reported here provide an important mechanistic link in the signaling effects of adiponectin in diverse metabolically responsive tissues.
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PMID:Involvement of AMP-activated protein kinase in glucose uptake stimulated by the globular domain of adiponectin in primary rat adipocytes. 1276 44

Triacylglycerol synthesis in rat epididymal fat overshoots sedentary levels at 10, 29, and 53 h of physical inactivity after 21 days of wheel running. The purposes of the present study were to determine 1) whether this effect is also observed after an acute bout of physical activity and 2) what enzymatic changes might contribute to this effect. We show that more than one bout of physical activity, such as that which occurs with 21 days of wheel running, is necessary for palmitic acid incorporation into triacylglyceride (triglyceride synthesis) to overshoot sedentary values, which suggests that pretranslational mechanisms may be responsible for this overshoot effect. Ten hours after 21 days of wheel running, activity of the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform, a key regulator of triacylglycerol synthesis, overshot sedentary values by 48% and remained higher than sedentary values at 29 and 53 h of reduced physical activity. The overshoot in mtGPAT1 activity was accompanied by an increase in mtGPAT protein level. Cyclic AMP response element-binding protein-binding protein level was higher in sedentary 29 h after 21 days of wheel running. AMP kinase-alpha Thr(172) phosphorylation was increased immediately after treadmill running, but decreased to sedentary values by 5 h after activity. Casein kinase-2alpha protein level and activity were unchanged. We conclude that an increase in mtGPAT protein might contribute to the overshoot in triacylglycerol synthesis.
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PMID:Increased mitochondrial glycerol-3-phosphate acyltransferase protein and enzyme activity in rat epididymal fat upon cessation of wheel running. 1623 67

Crisp-1 is a member of the cysteine-rich secretory protein family. This family of proteins is characterized by the presence of 16 conserved cysteine residues, the characteristic from which the family name is derived. Members of the Crisp protein family are found in the secretions of the reproductive tract and salivary glands, including venom toxins from several species of snakes and lizards. The Crisp proteins are modular, each containing an amino terminal pathogenesis-related (PR)-like domain and a carboxyl terminal cysteine-rich domain (CRD) connected by a hinge region. Sequence and structural similarities to proteins with known functions suggest that the Crisp family of proteins may act by regulating cellular ion channels. Rat Crisp-1 is synthesized as two distinct isoforms (referred to as Proteins D and E) by the epididymal epithelium and both are secreted into the luminal fluid where they interact with spermatozoa. Our laboratory has correlated Crisp-1 binding to sperm with inhibiting the signaling cascades that initiate capacitation while others have shown that blocking Crisp-1 binding sites on oocytes interferes with sperm-egg fusion. We hypothesize that the D and E populations of rat Crisp-1 have different interactions with sperm that modulate these distinct biological activities. Through tandem mass spectrometry (MS/MS) and monosaccharide composition analyses, we have identified at least one difference between the D and E forms as an additional single O-linked N-acetyl galactosamine on an amino terminal threonine residue in Protein E. This post-translational modification appears to account for the unique 'E' epitope bound by monoclonal antibody 4E9 developed in our laboratory, and may also lead to differential processing and localization of Protein E on sperm, when compared to Protein D. These findings are the first step in distinguishing the molecular basis of the biological activities of the D and E forms of rat Crisp-1. The epididymal-specific expression of Crisp-1, combined with its role in regulation of sperm capacitation and oocyte interaction, make it an attractive target for post-testicular contraceptive development.
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PMID:Epididymal secreted protein Crisp-1 and sperm function. 1641 81

Passage of spermatozoa through the epididymis and emission of sperm during ejaculation are based on spontaneous and induced contractions of epididymal peritubular muscle layers. This study deals with the ejaculation-relevant factors noradrenaline (NA) and oxytocin (OT) and their contractile effects in the course of the bovine epididymal duct. Muscle tension recording revealed excitatory effects of NA in all duct regions. A peculiarity was found in a duct section between the mid-cauda and ductus deferens, where the responsiveness to NA was particularly faint in comparison with the adjacent regions. NA-induced contraction was primarily mediated by postjunctional alpha(2)-adrenoceptors (ADRA) in the caput and corpus regions, and by alpha(1)-ADRA in the cauda region. Contrary to NA, OT exerted regionally varying effects. The peptide induced contraction in intact and epithelium-denuded caput as well as in epithelium-denuded corpus segments but had a relaxant net effect in intact corpus and proximal cauda segments. Within the mid-cauda, OT evoked strong contraction, which progressively decreased distally. Receptor specificity of the epididymal OT effects was verified using the selective OT receptor (OTR) agonist [Thr(4),Gly(7)]OT and vasopressin. OTR immunoreactivity was detected in the epididymal peritubular muscle wall and epithelial principal cells. RT-PCR analysis confirmed the presence of OTR in all duct regions. In summary, different contractile responses to OT and NA occur in the course of the epididymal duct, possibly preventing excessive sperm transport through the corpus and serving orthograde emission of sperm during ejaculation.
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PMID:Differential modulation of bovine epididymal activity by oxytocin and noradrenaline. 1770 67


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