Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When ram spermatozoa were treated with Ca2+ and the ionophore A23187 to induce acrosomal exocytosis, a rise in diacylglycerol (DAG) mass was observed, concomitant with a rapid breakdown of [32P]P1-labelled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and a rise in [32P]Pi-labelled phosphatidate. Inclusion of the DAG lipase inhibitor RHC 80267 resulted in further but biphasic increases in DAG; there was an increasing accumulation of DAG with concentrations of RHC 80267 up to 10 microM, whereas higher concentrations produced lessening accumulation. Inclusion of RHC 80267 in the ionophore induction system also resulted in significant accelerations of the onset of exocytosis. In spermatozoa stimulated with Ca2+/A23187 and the DAG kinase inhibitor R59022, a similar increase in DAG levels together with stimulation of acrosomal exocytosis were observed. Preincubation of spermatozoa with sn-1-oleoyl-2-acetylglycerol, rac-1-oleoyl-2-acetylglycerol, sn-1,2-dioctanoylglycerol and sn-1,3-dioctanoylglycerol before treatment with Ca2+/A23187 resulted in a dose-dependent stimulation of exocytosis by all these isomers. Neomycin inhibited Ca2+/A23187-induced generation of DAG together with polyphosphoinositide breakdown, as well as acrosomal exocytosis. Inclusion of exogenous DAG, however, overcame the inhibitory effect of neomycin on exocytosis. Our results suggest that DAG has a key role in acrosomal exocytosis and that it acts as a messenger rather than as a substrate from which other active metabolites are generated. The lack of stereospecificity shown by the exogenous DAGs implies that DAG does not act by stimulating protein kinase C, but the metabolite's actual target in the sperm cell is as yet unclear.
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PMID:The role of diacylglycerol in the exocytosis of the sperm acrosome. Studies using diacylglycerol lipase and diacylglycerol kinase inhibitors and exogenous diacylglycerols. 131 Nov 74

Mammalian spermatozoa undergo a Ca(2+)-dependent exocytotic event before fertilization which is known as the acrosome reaction. The process of exocytosis in several cell systems is mediated by a protein kinase C (PKC)-catalysed phosphorylation. Addition of phorbol 12-myristate-13-acetate or the membrane-permeant diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which are potent activators of PKC, to bovine spermatozoa resulted in stimulation of the acrosome reaction. This stimulation was inhibited by low concentrations (50% inhibition at 0.7 nM) of the PKC inhibitor staurosporine. PKC specific activity in bovine spermatozoa is extremely low in comparison with other cells; however, it is comparable with the activity found in human spermatozoa. Immunohistochemical analysis using anti-PKC antibodies revealed staining in the equatorial segment, the post-acrosomal region and the upper region of the head. We propose that PKC is involved in the mammalian sperm acrosome reaction.
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PMID:Role of protein kinase C in the acrosome reaction of mammalian spermatozoa. 173 94

The acrosome reaction of spermatozoa may be analogous to various somatic cell exocytotic events that incorporate cascade reactions. One such cascade system involves the hydrolysis of a membrane-bound phospholipid; generation of the intracellular second messenger, diacylglycerol; and activation of protein kinase C, followed by the phosphorylation of a number of intracellular proteins. Stimulators of protein kinase C, phorbol diesters and synthetic diacylglycerols, were evaluated to determine if this system functions in the human sperm acrosome reaction. Phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-didecanoate caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa. Conversely, an inactive phorbol diester had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. The synthetic diacylglycerols, 1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-dioleoyl-sn-glycerol caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa, and to a similar extent as the phorbol diesters. A nonactivating isomer of 1,2-dioleoyl-sn-glycerol, 1,3-diolein, had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. Protein kinase C activation is a diacylglycerol-dependent and Ca2(+)-dependent process, and stimulation of the acrosome reaction by 1,2-dioctanoyl-sn-glycerol required the presence of calcium ions in the capacitation medium. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), prevented the diacylglycerol-induced acrosome reaction (P less than 0.01). These results support the hypothesis that protein kinase C, via activation by the intracellular second messenger diacylglycerol, has a role in the human sperm acrosome reaction.
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PMID:Effect of phorbol diesters, synthetic diacylglycerols, and a protein kinase C inhibitor on the human sperm acrosome reaction. 184 29

At fertilization, the spermatozoon exocytoses its acrosomal granule in a Ca2(+)-dependent process known as the acrosome reaction. In mammalian spermatozoa, possibly because the acrosome is large and membrane fusion takes place between the outer acrosomal membrane and the overlying plasma membrane extensively over the anterior of the sperm head, the exocytotic process is slow and therefore amenable to biochemical dissection. By prelabelling sperm phospholipids with 32P and inducing the acrosome reaction with Ca2+ and the ionophore A23187, we have been able to show that membrane fusion occurs as the result of a sequence of events following Ca2+ entry; Ca2+ is required for at least 3 of these events. The process is initiated by a large-scale breakdown of polyphosphoinositides that is catalysed by a Ca2(+)-dependent phospholipase C. Of the resultant products, diacylglycerol is the essential one. Although its precise role remains to be established, this compound appears to stimulate a later process; it does not seem to act directly as a fusogen, nor does it act through a metabolite. However, it does not act through protein kinase C. At present we believe that diacylglycerol may simultaneously activate phospholipase A2 and inhibit lysophosphatide acyltransferase, to cause a large-scale build-up of fusogenic lysophospholipids in the acrosomal region; Ca2+ may bring about membrane fusion when the levels of these lipids have risen above a necessary threshold.
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PMID:Phosphoinositides and their products in the mammalian sperm acrosome reaction. 196 1

We have investigated phospholipase A2 (PLA2) activity in sonicates from ram spermatozoa and have analyzed the enzyme's susceptibility both to various inhibitors and to stimulation by diacylglycerols (DAGs). Ram sperm PLA2 activity was Ca2(+)-dependent and was inhibited by dexamethasone, chloracysine and compounds Ro 31-4493 and Ro 31-4639; mepacrine, however, did not inhibit PLA2 activity. Addition of three different 1,2-DAGs (dioctanoyl-sn-glycerol, oleoyl-acetyl-sn-glycerol and dioleoyl-sn-glycerol) markedly increased PLA2 activity; moreover, both 1,2- and 1,3-isomers enhanced enzyme activity. Since ram spermatozoa lack active protein kinase C, the target of DAG in most cells, our results suggest that stimulation of PLA2 activity by DAG may play an important role in intracellular signalling in the sperm cell.
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PMID:Diacylglycerol stimulates the Ca2(+)-dependent phospholipase A2 of ram spermatozoa. 201 23

We have investigated the production of diacylglycerol (DAG) and phosphatidate (PtdOH) during the exocytosis of the sperm acrosome. Ram spermatozoa treated with Ca2+ and the ionophore A23187 experienced a rapid breakdown of the polyphosphoinositides (PPIs), and a rise in [32P]Pi-labelled PtdOH and DAG mass; PtdOH mass, however, was unaffected. Treatment with Ca2+/A23187 and the DAG kinase inhibitor R59022 resulted in a dose-dependent increase in DAG mass and a concomitant decrease in [32P]PtdOH; such treatment showed a dose-dependent stimulation of acrosomal exocytosis. Pre-incubation with exogenous PtdOHs before stimulation with Ca2+/A23187 did not affect the time-course of exocytosis, whereas treatment with Ca2+/A23187 and exogenous DAGs (dioctanoylglycerol, oleoyl-acetyl-glycerol, or dioleoylglycerol) resulted in a dose-dependent stimulation of acrosomal exocytosis. Our results suggest that DAG, rather than PtdOH, is the important metabolite generated upon PPI hydrolysis; however, since spermatozoa lack protein kinase C, the target of DAG in most cells, a role for DAG in acrosomal exocytosis is as yet unclear.
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PMID:Diacylglycerol and phosphatidate production and the exocytosis of the sperm acrosome. 217 25

Phorbol ester, phorbol 12-myristate 13-acetate (PMA), induced a 20- to 50-fold increase (ED50: 2 microM) in cyclic adenosine 3', 5'-monophosphate (cAMP) levels in spermatozoa incubated in capacitation medium for short periods of time (30 min). Similar results were obtained with 1-oleoyl 2-acetylglycerol (OAG), whereas 1, 2 diolein, 1-oleoyl glycerol, or 4 alpha-phorbol 12, 13-didecanoate had no effect. When extracellular Ca2+ was complexed by [ethylenebis(oxyethyleneitrilo)] tetraacetic acid (EGTA), a 50% reduction of maximal stimulation was observed, and 90% inhibition was seen after chelation of both extra- and intracellular Ca2+ with EGTA and 2-[[2-[bis [(carbonyl) methyl] amino]-5-methylphenoxy] methyl]-6-methoxy-8-[bis[(carbonyl) methyl] amino] quinoline acetoxy methyl (Quin 2). The acrosome reaction was not affected by similar concentration of PMA or OAG at different periods of incubation. These results suggest the involvement of protein kinase C activity in the regulation of cAMP levels in sperm during capacitation. This stimulation is dependent on intracellular Ca2+ and probably is not linked to the process of the acrosome reaction.
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PMID:Phorbol esters stimulate cyclic adenosine 3', 5'-monophosphate accumulation in hamster spermatozoa during in vitro capacitation. 254 12

At fertilization, mammalian spermatozoa undergo a Ca2+-dependent exocytotic event known as the acrosome reaction. As protein kinase C (PKc) has been implicated in exocytosis in some other cell systems, we have searched for PKc in ram spermatozoa. We have found that: (a) no changes in protein phosphorylation pattern could be induced in the intact cells by phorbol dibutyrate (PDBu), a compound which binds to and stimulates PKc; (b) no changes in protein phosphorylation pattern could be detected during the course of the Ca2+/ionophore-induced acrosome reaction (when greater than 95% of the cells underwent exocytosis); (c) there was no effect of PDBu on the exocytotic response to various Ca2+ and ionophore levels; (d) no specific PDBu binding could be detected in the cells (this binding is considered to be indicative of the presence of active PKc). We conclude that potentially active PKc is not present in ram spermatozoa.
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PMID:Absence of active protein kinase C in ram spermatozoa. 313 97

Phosphorylation of demembranated fowl sperm proteins during incubation with [gamma-32P]ATP and various protein kinase substrate peptides at 30 degrees C was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A marked difference in phosphorylation was observed in a 30 kDa protein. This protein was strongly phosphorylated after the addition of Kemptide, a cAMP-dependent protein kinase (PKA) substrate peptide; Syntide 2, a calmodulin-dependent protein kinase II substrate peptide; a protein kinase C (PKC) substrate peptide; as well as control samples but only slightly phosphorylated in the presence of a myosin light chain kinase (MLCK) substrate peptide. The motility of demembranated spermatozoa at 30 degrees C remained high in control samples and following the addition of Kemptide, Syntide 2 and PKC substrate peptide, but decreased markedly following the addition of MLCK substrate peptide. These results suggest that the 30 kDa protein is identified as a substrate for MLCK or a MLCK-like protein in fowl spermatozoa and that phosphorylation-dephosphorylation of this protein is involved in the regulation of flagellar movement at 30 degrees C.
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PMID:Dephosphorylation of a 30-kDa protein of fowl spermatozoa by the addition of myosin light chain kinase substrate peptide inhibits the flagellar motility. 748 12

At the time of fertilization mammalian spermatozoa undergo a Ca(2+)-dependent exocytotic event, which is known as the acrosome reaction (AR). We describe here that EGF-receptor (EGFR) is localized in the head of bull spermatozoa and that epidermal growth factor (EGF) can induce the occurrence of the AR in its typical dose-dependent manner. Previously we showed that protein kinase C (PKC) is involved in the cascade leading to AR in bull spermatozoa. Here, we show that PKC is involved in the mechanism in which EGF exerts its effect on AR. These findings together with our results which show inhibition of AR by tyrosine-phosphorylation inhibitors, indicate that ejaculated bull sperm contain a typical 170-kDa EGFR which is active in the mechanism leading to AR.
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PMID:Epidermal growth factor induces acrosomal exocytosis in bovine sperm. 750 96


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