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1.
S Sahara K Sato M Aoto T Ohnishi H Kaise H Koide K Ogita Y Fukami 《Biochemical and biophysical research communications》1992,182(1):105-114
Protein kinase C (PKC) was partially purified from Xenopus laevis oocytes by ammonium sulfate fractionation followed by DEAE-cellulose and hydroxyapatite column chromatography. In the latter chromatography, two distinct PKC activities were identified. Both PKC fractions contained an 80 kDa protein which was recognized by three antisera raised against the conserved regions of mammalian PKC. However, specific antisera against alpha, beta I, beta II, and gamma-subspecies of rat PKC did not recognize the protein. Kinetic properties of the Xenopus PKCs were very similar to those of the rat alpha PKC, and only a subtle difference was found in the mode of activation by arachidonic acid. When oocytes were treated with the tumor promoter, phorbol 12-myristate 13-acetate, one of the Xenopus PKCs was found to disappear very rapidly, while the other remained unchanged up to 2 hr. 相似文献
2.
D Singer-Lahat E Gershon I Lotan R Hullin M Biel V Flockerzi F Hofmann N Dascal 《FEBS letters》1992,306(2-3):113-118
L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role. 相似文献
3.
Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes. 总被引:12,自引:3,他引:9
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I Dominguez L Sanz F Arenzana-Seisdedos M T Diaz-Meco J L Virelizier J Moscat 《Molecular and cellular biology》1993,13(2):1290-1295
Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a large variety of cellular genes. However, the mechanism whereby this nuclear factor is activated remains to be determined. In this report, we present evidence that in oocytes from Xenopus laevis, (i) ras p21- and phospholipase C (PLC)-mediated phosphatidylcholine (PC) hydrolysis activates NF-kappa B and (ii) protein kinase C zeta subspecies is involved in the activation of NF-kappa B in response to insulin/ras p21/PC-PLC. Thus, the microinjection of either ras p21 or PC-PLC, or the exposure of oocytes to insulin, promotes a significant translocation to the nucleus of an NF-kappa B-like activity. This effect is not observed when oocytes are incubated with phorbol myristate acetate or progesterone, both of which utilize a ras p21-independent pathway for oocyte activation. These data strongly suggest a critical role of the insulin/ras p21/PC-PLC/protein kinase C zeta pathway in the control of NF-kappa B activation. 相似文献
4.
O Rey S H Young D Cantrell E Rozengurt 《The Journal of biological chemistry》2001,276(35):32616-32626
Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation. 相似文献
5.
We have combined a rapid cytoplasmic sampling technique with capillary electrophoresis to measure the activation of protein kinase C (PKC) in a small region (approximately 60 microm) of a Xenopus oocyte. The phosphorylation of a fluorescent PKC substrate was measured following addition of a pharmacological or physiological stimulus to an oocyte. When substrates for cdc2 kinase (cdc2K), PKC, and protein kinase A (PKA) were comicroinjected into an oocyte, all three substrates could be identified on the electropherogram after cytoplasmic sampling. With this new method, it should be possible to measure simultaneously the activation of multiple different kinases in a single cell, enabling the quantitative dissection of signal transduction pathways. 相似文献
6.
Olivier Haccard Catherine Jessus Helene Rime Jozef Goris Wilfried Merlevede Rene Ozon 《Molecular reproduction and development》1993,36(1):96-105
Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc. 相似文献
7.
Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase. 总被引:10,自引:1,他引:9
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Previous studies in this laboratory have shown that insulin treatment of Xenopus oocytes leads to an increase in phosphorylation of ribosomal protein S6. To investigate the mechanism of this increase, S6 kinase activity was measured in lysates of oocytes exposed to insulin. Insulin caused a rapid 4- to 6-fold increase in S6 kinase activity, which was maximal by 20 min and which could be reversed by removal of insulin prior to homogenization. Dose-response curves showed a detectable increase in specific activity at 1 nM insulin with a maximal effect at 100 nM. Treatment of oocytes with puromycin did not prevent this increase in S6 kinase activity, suggesting activation rather than synthesis of the enzyme. DEAE-Sephacel chromatography of extracts from insulin-treated oocytes revealed two peaks of S6 kinase activity, and the specific activity of the peak eluting at 300 nM NaCl was increased 3-fold in oocytes treated with insulin. The same peak of S6 kinase activity was increased 40% within 10 min in oocytes injected with highly purified insulin-receptor kinase. These results indicate that the insulin-dependent increase in S6 phosphorylation is due, at least in part, to activation of an S6 protein kinase, and this activation may result from the action of the insulin receptor at an intracellular location. 相似文献
8.
9.
MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom. 相似文献
10.
Though progesterone-induced maturation has been studied extensively in Xenopus oocytes, the mechanism whereby the prophase block arrest is released is not well understood. The current hypothesis suggests that a reduction in cAMP and subsequent inactivation of cAMP-dependent protein kinase is responsible for reentry into the cell cycle. However, several lines of evidence indicate that maturation can be induced without a concomitant reduction in cAMP. We show that the mass of diacylglycerol in whole oocytes and plasma membranes decreases 29% and 10% respectively, within the first 15 sec after the addition of progesterone. Diacylglycerol in plasma membranes further decreased 59% by 5 min. We also show that the protein kinase C inhibitors sphingosine and staurosporine can induce oocyte maturation. In addition, the synthetic diglyceride, DiC8, and microinjected PKC can inhibit or delay progesterone-induced maturation. These results together suggest that a transient decrease in protein kinase C activity may regulate entry into the cell cycle. The mechanism whereby DAG is decreased in response to progesterone is unclear. Initial studies show that progesterone leads to a decrease in IP3 suggesting that progesterone may act by reducing the hydrolysis of PIP2. On the other hand, progesterone caused a decrease in the amount of [3H]arachidonate labelling in DAG during the same time suggesting that progesterone may stimulate lipase activity. The relationship between postulated changes in the PKC pathway and those hypothesized for the PKA pathway are discussed. 相似文献
11.
Modulation of maturation and ribosomal protein S6 phosphorylation in Xenopus oocytes by microinjection of oncogenic ras protein and protein kinase C. 总被引:6,自引:4,他引:2
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Using Xenopus oocytes as a model system, we investigated the possible involvement of ras proteins in the pathway leading to phosphorylation of ribosomal protein S6. Our results indicate that microinjection of oncogenic T24 H-ras protein (which contains valine at position 12) markedly stimulated S6 phosphorylation on serine residues in oocytes, whereas normal ras protein (which contains glycine at position 12) was without effect. The S6 phosphorylation activity in the cell extract from T24 ras protein-injected oocytes was increased significantly. In addition, injection of protein kinase C potentiated the induction of maturation and S6 phosphorylation by the oncogenic ras protein. A similar potentiation was detected when T24 ras protein-injected oocytes were incubated with active phorbol ester. These findings suggest that ras proteins activate the pathway linked to S6 phosphorylation and that protein kinase C has a synergistic effect on the ras-mediated pathway. 相似文献
12.
Selective potentiation of N-methyl-D-aspartate-induced current by protein kinase C in Xenopus oocytes injected with rat brain RNA. 总被引:3,自引:0,他引:3
Glutamate receptors and protein kinase C (PKC) may play significant roles in long-term potentiation in hippocampus. To clarify the regulatory involvement of PKC in the functions of glutamate receptors, we examined the effects of PKC activation on current response induced by the activation of each subtype of glutamate receptor in Xenopus oocytes injected with rat brain RNA. Treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), potentiated N-methyl-D-aspartate (NMDA)-induced current by about 2.5-fold, although it did not affect kainate-induced current at all. Quisqualate-mediated oscillatory current was almost abolished by this treatment. The TPA-induced potentiation of NMDA current was suppressed by staurosporine, an inhibitor of protein kinases. Pretreatment with 4-O-methyl-TPA, an inactive phorbol ester, had no effect on NMDA current. Current response mediated by NMDA receptors would thus appear to be modulated by PKC. 相似文献
13.
The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced generation of superoxide anion and thromboxane B(2) (TXB(2)) in guinea-pig alveolar macrophages (AM). Genistein (3-100 muM) dose dependently inhibited FMLP (3 nM) induced superoxide generation in non-primed AM and TXB(2) release in non-primed or in lipopolysaccharide (LPS) (10 ng/ml) primed AM to a level > 80% but had litle effect up to 100 muM on phorbol myristate acetate (PMA) (10 nM) induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC(50) 0.21 +/- 0.10 muM) but had no effect on or potentiated (at 3 and 10 muM) FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 muM) inhibited primed TXB(2) release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism. 相似文献
14.
The hypothesis that specific protein kinase C (PKC) isoforms regulate dopamine transporter (DAT) function was tested in Xenopus laevis oocytes expressing human (h)DAT. Activation of conventional PKCs (cPKCs) and novel PKCs (nPKCs) using 10 nM phorbol 12-myristate 13-acetate (PMA) significantly inhibited DAT-associated transport currents. This effect was reversed by isoform-non-selective PKC inhibitors, selective inhibitors of cPKCs and deltaPKC, and by Ca2+ chelation. By contrast, the epsilonPKC translocation inhibitor peptide had no effect on PMA-induced inhibition of hDAT transport-associated currents. Thus, the primary mechanism by which PMA regulates hDAT expressed in oocytes appears to be by activating cPKC(s). 相似文献
15.
The mechanism of modulation of sodium channel alpha-subunits (Type IIA) by a protein kinase C (PKC) activator was studied on single channel level. It was found that: (i) time constants for channel activation were prolonged; (ii) inactivation remained virtually unchanged; (iii) peak sodium inward current was reduced as evidenced by calculation of average sodium currents; and (iv) time constants for current activation and decay were prolonged. (i), (iii) and (iv) were voltage dependent, being most prominent at threshold potentials. The data show that a voltage dependent action on the activation gate can account for the observed reduction of peak inward sodium current and prolongation of current decay in macroscopic experiments. 相似文献
16.
After a 60 min heat-shock at 36 degrees C, Xenopus oocytes are still able to accomplish a complete meiotic maturation in response to a progesterone treatment. The 36 degrees C heat-shock applied to maturing oocytes strongly enhances the synthesis of a single heat-shock protein of approx. 70 000 molecular weight (hsp70); after activation with the Ca2+-ionophore A 23187, matured oocytes still display the ability to synthesize hsp70 and to survive a heat-shock. A cycloheximide treatment combined with a heat-shock induces, during the recovery period, the synthesis of two heat-shock proteins, of approx. 70 000 and 83 000 molecular weight. 相似文献
17.
18.
Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes. 总被引:4,自引:1,他引:3
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I Dominguez M T Diaz-Meco M M Municio E Berra A García de Herreros M E Cornet L Sanz J Moscat 《Molecular and cellular biology》1992,12(9):3776-3783
A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways. 相似文献
19.
20.
R Devos J Tavernier G Plaetinck J Van der Heyden A Rolink W Fiers 《Biochemical and biophysical research communications》1990,172(2):570-575
In this study we describe the use of Xenopus laevis oocytes for the detection of mRNA coding for a murine interleukin-5 (mI15) receptor. When injected with sucrose gradient fractionated polyA+ RNA derived from the murine 115-dependent pre B cell line B13, these oocytes could specifically bind 35S-methionine labeled mI15. A size of approximately 4000 nucleotides (25S) was estimated for the mRNA corresponding to the mIL5-binding activity. This binding was not blocked by a monoclonal antibody R52 specific for the MI15-receptor, suggesting that the oocytes express a different form of this receptor. 相似文献