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1.
Colony-stimulating factor 1 activates protein kinase C in human monocytes. 总被引:13,自引:2,他引:13
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Colony-stimulating factor 1 (CSF-1) is required for the survival, proliferation and differentiation of monocytes. We previously demonstrated that the CSF-1 receptor is linked to a pertussis toxin-sensitive G protein and that the induction of Na+ influx by CSF-1 is a pertussis toxin-sensitive event. The present studies have examined activation of protein kinase C as a potential intracellular signaling event induced by the activated CSF-1 receptor. The results demonstrate that CSF-1 stimulates translocation of protein kinase C activity from the cytosol to membrane fractions. This activation of protein kinase C was sensitive to pretreatment of the monocytes with pertussis toxin. Lipid distribution studies demonstrated that phosphatidylcholine (PC) is the major phospholipid in human monocytes. Moreover, the results indicate that CSF-1 stimulation is associated with decreases in PC, but not in phosphatidylinositol (PI), levels. The absence of an effect of CSF-1 on PI turnover was confirmed by the lack of changes in inositol phosphate production. In contrast, CSF-1 stimulation was associated with increased hydrolysis of PC to phosphorylcholine and diacylglycerol (DAG) in both intact monocytes and cell-free assays. Furthermore, the increase in PC turnover induced by CSF-1 was sensitive to pertussis toxin. The results also demonstrate that the induction of Na+ influx by CSF-1 is inhibited by the protein kinase C inhibitors staurosporine and the isoquinoline derivative H7, but not by HA1004.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase 总被引:4,自引:0,他引:4
M Chedid F Shirakawa P Naylor S B Mizel 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(12):4301-4306
Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase. 相似文献
3.
S Takahashi K Hashida K Yatsunami T Fukui M Negishi T Katada M Ui Y Kanaho T Asano A Ichikawa 《Biochimica et biophysica acta》1991,1093(2-3):207-215
We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells. 相似文献
4.
Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins. 相似文献
5.
D A Ausiello J L Stow H F Cantiello J B de Almeida D J Benos 《The Journal of biological chemistry》1992,267(7):4759-4765
We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha i-3 subunit of the Gi-3 protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha i-3 subunit was a member of this Na+ channel complex. G alpha i structure and function were identified by Western blotting with specific G alpha i subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G alpha i proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha i-3 subunit of the Gi-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G alpha i-3 on Western blots of Golgi membranes which contain alpha i-3 but not Na+ channel proteins, nor do they immunoprecipitate alpha i-3 from solubilized Golgi membranes; however, alpha i-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha i-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha i-3, but not alpha i-2, stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha i-3 protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation. 相似文献
6.
ATP-induced Ca2+ influx is regulated via a pertussis toxin-sensitive mechanism in a PC12 cell clone.
E Clementi H Scheer M Raichman J Meldolesi 《Biochemical and biophysical research communications》1992,188(3):1184-1190
A PC12 cell clone that responds to ATP with polyphosphoinositide hydrolysis and with a marked, biphasic intracellular free Ca2+ concentration ([Ca2+]i) response (composed by release from intracellular stores accompanied by stimulated influx from the medium), was pretreated with pertussis toxin. In the pretreated cells the responses induced by ATP were differently modified. Polyphosphoinositide hydrolysis and Ca2+ release were moderately inhibited whereas Ca2+ influx was enhanced. Pharmacological experiments revealed the influx enhancement to be sustained by neither voltage-gated nor second messenger-operated Ca2+ channels. Rather, a channel of the receptor-operated type activated by ATP (P2w receptor) appears to work under the negative control of a pertussis toxin-sensitive G protein, acting presumably by direct interaction with the channel in the plane of the plasma membrane. 相似文献
7.
M Miwa H Tokuda K Tsushita J Kotoyori Y Takahashi N Ozaki O Kozawa Y Oiso 《Biochemical and biophysical research communications》1990,171(3):1229-1235
Prostaglandin F2 alpha (PGF2 alpha) stimulated the formation of inositol phosphates in a dose-dependent manner in cloned osteoblast-like MC3T3-E1 cells. This reaction was markedly inhibited dose-dependently by pertussis toxin. In the cell membranes, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGF2 alpha. These results suggest that pertussis toxin-sensitive GTP-binding protein is involved in the coupling of PGF2 alpha receptor to phospholipase C in these cells. 相似文献
8.
Y Fujinaga N Morozumi K Sato Y Tokumitsu K Fujinaga Y Kondo M Ui F Okajima 《FEBS letters》1989,245(1-2):117-121
Acute spontaneous c-myc gene expression and sustained increase of a GTP-binding protein(s) (G-protein) which is sensitive to islet-activating protein (IAP), pertussis toxin, occurred early during primary culture of adult rat hepatocytes. Following these earlier events, DNA synthesis was demonstrated in response to EGF and insulin. Addition of IAP immediately after plating of primary cultures inhibited c-myc expression and the hormone-induced DNA synthesis. Addition at 24 h or later following cell inoculation, however, produced only weak effects on DNA synthesis, even though the IAP-sensitive G-proteins were completely inactivated. We conclude that the IAP-sensitive G-protein(s) plays a role in the earlier process(es) of the G0-G1 transition, which is essential for the initiation of growth factor-dependent DNA synthesis. 相似文献
9.
Activation of Na+/Ca2+ exchange by adenosine in ewe heart sarcolemma is mediated by a pertussis toxin-sensitive G protein 总被引:1,自引:0,他引:1
V Brechler C Pavoine S Lotersztajn E Garbarz F Pecker 《The Journal of biological chemistry》1990,265(28):16851-16855
We studied the effect of adenosine on Na+/Ca2+ exchange activity in ewe heart ventricular sarcolemmal vesicles. Adenosine was found to stimulate Na+/Ca2+ exchange activity in a dose-dependent manner from 0.1 nM to 10 microM, with maximal stimulation (40%) at 0.1 microM adenosine. The Vmax of Na+/Ca2+ exchange was increased, but the Km for Ca2+ was not altered. The effect of adenosine was specific since 1 microM adenine, inosine, and guanosine led to less than 15% stimulation, and adenosine diphosphate had no effect. Caffeine antagonized the activation of Na+/Ca2+ exchange by adenosine, and the order of potency of adenosine analogs was N6-(L-2-phenylisopropyl)adenosine = N6-cyclohexyladenosine = 5'-(N- ethylcarboxamido)adenosine much greater than N6-(D-2-phenylisopropyl)adenosine, indicating the involvement of A1 subclass receptors. The effect of adenosine was mimicked by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and blocked by pertussis toxin treatment. Taken together, these results suggest that A1 subclass receptors coupled to a pertussis toxin-sensitive G protein mediate the activation of Na+/Ca2+ exchange activity by adenosine. We conclude that the negative inotropic effect of adenosine in ventricular muscle, antagonistic toward cyclic AMP, may involve activation of Na+/Ca2+ exchange. 相似文献
10.
Phorbol ester-induced changes in cytoplasmic Ca2+ in human neutrophils. Involvement of a pertussis toxin-sensitive G protein 总被引:2,自引:0,他引:2
Activation of neutrophils by most soluble stimuli is associated with a marked increase in intracellular free Ca2+ ([Ca2+]i). However, under physiological conditions (Na+-rich media), the potent activator 12-O-tetradecanoylphorbol-13-acetate (TPA) causes no change or a decrease in [Ca2+]i. We report here that the [Ca2+]i response to phorbol esters varies depending on the ionic composition of the medium. A marked increase in [Ca2+]i was detected in Na+-free solutions. Maximal effects were observed when N-methyl-D-glucammonium+ or choline+ were substituted for Na+, whereas an intermediate response was recorded in K+ medium. The increase in [Ca2+]i was substantially (approximately 65%) inhibited by removal of external Ca2+. A [Ca2+]i increase was also elicited by other beta-phorbol diesters and by diacylglycerol, but not by unesterified phorbol or by alpha-phorbol diesters, indicating involvement of protein kinase C. The increase in [Ca2+]i observed in Na+-free media is not due to inhibition of Na+/Ca2+ exchange, since no change in [Ca2+]i in response to TPA was observed in: 1) cells suspended in Li+, which is not countertransported for Ca2+; 2) cells preloaded with Na+ to eliminate the driving force for Na+/Ca2+ exchange; and 3) cells treated with 3',4'-dichlorobenzamyl, an inhibitor of Na+/Ca2+ exchange. Similarly, the [Ca2+]i increase in Na+-free media is not linked to the absence of Na+/H+ exchange and the associated cytoplasmic acidification since: 1) it was not observed in Na+ media in the presence of inhibitors of the Na+/H+ antiport and 2) it was not mimicked by inducing acidification with nigericin. Pretreatment with pertussis toxin largely inhibited the phorbol ester-induced change in [Ca2+]i, while activation of protein kinase C under these conditions was unaffected. It is concluded that in the absence of extracellular Na+ (or Li+), activation of protein kinase C leads to a net Ca2+ influx into the cytoplasm through a process mediated by a GTP-binding or G protein. Opening of a Na+-sensitive Ca2+ channel could partially explain these observations. Alternatively, the nature of the monovalent cation could conceivably affect the conformation of a G protein or of an associated receptor, inducing the appearance of a site susceptible to an activating phosphorylation by protein kinase C. 相似文献
11.
Y Kanaho K Takahashi U Tomita T Iiri T Katada M Ui Y Nozawa 《The Journal of biological chemistry》1992,267(33):23554-23559
In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils. 相似文献
12.
K Tsushita O Kozawa H Tokuda Y Oiso H Saito 《Prostaglandins, leukotrienes, and essential fatty acids》1992,45(4):267-274
PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cells. PGD2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 microM. The formation of inositol trisphosphate (IP3) induced by PGD2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGF2 alpha and both PGE2 and PGF2 alpha affected PGD2-induced IP3 formation additively. Pertussis toxin (PTX) inhibited both PGD2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 micrograms/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD2-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD2 was apparently different from that by PGE2 or PGF2 alpha. These results indicate that PGD2 activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2 alpha and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells. 相似文献
13.
H Tadenuma K Takahashi K Chiba M Hoshi T Katada 《Biochemical and biophysical research communications》1992,186(1):114-121
In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes, suggesting that a guanine nucleotide-binding protein (G protein) serving as the substrate of pertussis toxin is involved in the 1-MA receptor-mediated signal. We thus investigated properties of 1-MA receptors by means of binding of the radiolabeled ligand to the oocyte membranes. There were apparently two forms of 1-MA receptors with high and low affinities in the membranes. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. A 39-kDa protein, which had been identified as the alpha-subunit of the major substrate G protein for pertussis toxin, was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. The ADP-ribosylated 39-kDa alpha-subunit could be immunoprecipitated with antibodies raised against the carboxy-terminal site of mammalian inhibitory G-alpha. These results indicate that 1-MA receptors are functionally coupled with the 39-kDa pertussis toxin-substrate G protein in starfish oocyte membranes. 相似文献
14.
G protein subunit, alpha i-3, activates a pertussis toxin-sensitive Na+ channel from the epithelial cell line, A6 总被引:7,自引:0,他引:7
H F Cantiello C R Patenaude D A Ausiello 《The Journal of biological chemistry》1989,264(35):20867-20870
In nonpolar excitable cells, guanine nucleotide regulatory (G) proteins have been shown to modulate ion channel activity in response to hormone receptor activation. In polarized epithelia, hormone receptor-G protein coupling involved in the generation of cAMP occurs on the basolateral membrane, while the physiological response to this messenger is a stimulation of ion channel activity at the apical membrane. In the present study we have utilized the patch-clamp technique to assess if the polarized renal epithelia, A6, have topologically distinct G proteins at their apical membrane capable of modulating Na+ channel activity. In excised inside-out patches of apical membranes, spontaneous Na+ channel activity (conductance 8-9 picosiemens) was inhibited by the addition of 0.1 mM guanosine 5'-O-(2-thio)diphosphate to the cytosolic membrane surface without an effect on single channel conductance. In contrast, the percent open time of spontaneous Na+ channels increased from 6 to 50% following the addition of 0.1 mM GTP. The addition of preactivated pertussis toxin (100 ng/ml) to the cytosolic bathing solution of the excised patch inhibited spontaneous Na+ channel activity within a minute by 85% from approximately 47 to 7% open time and reduced the percent open time for Na+ channel activity to zero after approximately 3 min. The addition of 0.1 mM guanosine 5'-(3-O-thio)triphosphate or the addition of 20 pM purified human alpha i-3 subunit to pertussis toxin-treated membrane patches restored Na+ channel activity from zero to 35% open time. As little as 0.2 pM alpha i-3 subunit was capable of restoring Na+ channel activity. These data provide evidence for a role of pertussis toxin-sensitive G proteins in the apical plasma membrane of renal epithelia distal to signal transduction pathways in the basolateral membrane of these cells. This raises the possibility of a topologically distinct signal transducing pathway co-localized with the Na+ channel. 相似文献
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17.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine which produces diverse biological effects in target cells of myeloid origin. GM-CSF enhances the production of superoxide anion (O2-) by mature neutrophils in response to chemotactic peptides such as formyl-methionyl-leucyl-phenylalanine (fMLP), but alone it has no effect on this system. This process has been termed "priming." fMLP activates neutrophils via a pertussis toxin-sensitive GTP-binding protein, leading to the rapid production of the second messengers diacylglycerol (DAG) and inositol trisphosphate, via phosphatidylinositol turnover, and arachidonic acid (AA) by a presumptive phospholipase A2-mediated mechanism. All three second messengers may lead to the generation of O2-. We investigated the effect of priming of GM-CSF on these systems. GM-CSF had no effect on fMLP-stimulated DAG and inositol trisphosphate levels, nor did it amplify the response to exogenously added phorbol ester (to mimic the action of DAG) or calcium ionophore. Neutrophils primed with the cytokine showed a small, but significant, enhancement of fMLP-stimulated AA release. Compared with unprimed controls, primed neutrophils also showed a significant increase in O2- production when stimulated with either AA or the nonhydrolyzable GTP analogue, GTP-gamma-S. The magnitude of enhanced O2- production was similar to that observed after fMLP treatment of primed cells. All of these effects, including the increased sensitivity to AA treatment, were inhibited by pertussis toxin. These data show that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein coupled to a metabolic pathway that mobilizes and utilizes arachidonic acid. 相似文献
18.
C L Huang M G Cogan E J Cragoe H E Ives 《The Journal of biological chemistry》1987,262(29):14134-14140
The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C. 相似文献
19.
We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis. pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ari?o, J. (1997) Eur. J. Biochem. 250, 476-483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells. K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions. trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions. 相似文献
20.
Following the differentiation of 3T3-L1 fibroblasts by insulin/dexamethasone/methylisobutylxanthine, marked increases in cAMP levels by isoproterenol but not forskolin and in 2-deoxyglucose uptake by insulin occurred. Pertussis toxin-pretreatment prior to addition of insulin/dexamethasone/methylisobutylxanthine and exposure of cells to pertussis toxin during differentiation attenuated glycerophosphate dehydrogenase activity as a differentiation marker enzyme and the responses to isoproterenol and insulin by approximately 50% of those in pertussis toxin-untreated cells. On the other hand, insulin/dexamethasone/methylisobutylxanthine caused induction of c-fos proto-oncogene in confluent 3T3-L1 fibroblasts. This induction was also reduced in pertussis toxin-pretreated cells. These results suggested that pertussis toxin-sensitive GTP-binding protein(s) is involved in expression of c-fos mRNA accompanied by differentiation. In addition, accumulation of c-fos mRNA by insulin/dexamethasone/methylisobutylxanthine was enhanced in protein kinase C-depleted cells pretreated with phorbol 12-myristate 13-acetate, indicating that protein kinase C may negatively regulate c-fos expression induced by insulin/dexamethasone/methylisobutylxanthine. 相似文献