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1.
Dal Molin F Zornetta I Puhar A Tonello F Zaccolo M Montecucco C 《Biochemical and biophysical research communications》2008,376(2):429-433
The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed. 相似文献
2.
Structural and kinetic analyses of the interaction of anthrax adenylyl cyclase toxin with reaction products cAMP and pyrophosphate 总被引:3,自引:0,他引:3
Guo Q Shen Y Zhukovskaya NL Florián J Tang WJ 《The Journal of biological chemistry》2004,279(28):29427-29435
Anthrax edema factor (EF) raises host intracellular cAMP to pathological levels through a calcium-calmodulin (CaM)-dependent adenylyl cyclase activity. Here we report the structure of EF.CaM in complex with its reaction products, cAMP and PP(i). Mutational analysis confirmed the interaction of EF with cAMP and PP(i) as depicted in the structural model. While both cAMP and PP(i) have access to solvent channels to exit independently, PP(i) is likely released first. EF can synthesize ATP from cAMP and PP(i), and the estimated rate constants of this reaction at two physiologically relevant calcium concentrations were similar to those of adenylyl cyclase activity of EF. Comparison of the conformation of adenosine in the structures of EF.CaM.cAMP.PP(i) with EF.CaM.3.dATP revealed about 160 degrees rotation in the torsion angle of N-glycosyl bond from the +anti conformation in 3.dATP to -syn in cAMP; such a rotation could serve to distinguish against substrates with the N-2 amino group of purine. The catalytic rate of EF for ITP was about 2 orders of magnitude better than that for GTP, supporting the potential role of this rotation in substrate selectivity of EF. The anomalous difference Fourier map revealed that two ytterbium ions (Yb(3+)) could bind the catalytic site of EF.CaM in the presence of cAMP and PP(i), suggesting the presence of two magnesium ions at the catalytic site of EF. We hypothesize that EF could use a "histidine and two-metal ion" hybrid mechanism to facilitate the cyclization reaction. 相似文献
3.
Protein kinase A (PKA), in yeast, plays a major role in controlling metabolism and gene expression in connection with the available nutrient conditions. We here measure, for the first time, a transient change in the in vivo PKA activity, along a cAMP peak produced by 100 mM glucose addition to glycerol-growing cells as well as a change in the phosphorylation state of its catalytic subunit (Tpk1p) following PKA activation. PKA activity was measured in situ in permeabilized cells, preserving its intracellular localization. Comparison of total PKA activity, measured in situ in permeabilized cells with data obtained from in vitro assays in crude extracts, underscores the inhibitory potency of the regulatory subunit within the cell. Tpk1p phosphorylation was detected through non-denaturing gel electrophoresis. Phosphorylation of Tpk1p increases its specificity constant toward kemptide substrate. The use of mutants of the cAMP pathway showed that phosphorylation depends on the activation of PKA via the G-protein coupled receptor pathway triggered by glucose. The phosphorylation state of Tpk1p was followed during the diauxic shift. Tpk1p phosphorylation is dynamic and reversible: its up-regulation correlates with a fully fermentative metabolism, while its down-regulation with stationary phase or respiratory metabolism. Reversible phosphorylation can thus be considered a new control mechanism possibly pointing to a fine-tuning of PKA activity in response to environmental conditions. 相似文献
4.
Irene Zornetta Lucia Brandi Blythe Janowiak Federica Dal Molin Fiorella Tonello R. John Collier Cesare Montecucco 《Cellular microbiology》2010,12(10):1435-1445
To investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C‐terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin‐1 containing compartments and then endosomes marked by phoshatidylinositol 3‐phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time‐course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes. 相似文献
5.
Changes in the activities of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinases in response to prostaglandin (PG)E2-induced elevation of intracellular cAMP level were investigated with a murine macrophage-like cell line, P388D1. Photoaffinity labeling with 8-azido-[32P]cAMP showed that untreated P388D1 cells possess two types of cAMP-binding proteins of m.w. 49,000 and 52,000, respectively, in the cytosol fraction in a ration of 1:8. They must represent regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinases, because affinity chromatography on a column of omega-aminohexyl-agarose of the cytosol fraction clearly separated two fractions that exhibited the enzymatic activities and cAMP-binding activities. Photoaffinity labeling of these fractions with 8-azido-[32P]cAMP confirmed the separation of two types of isoenzymes, because each cAMP-dependent protein kinase active fraction was associated with only one type of regulatory subunit. The exposure of P388D1 cells to exogenously added PGE2 (1 microM) caused about 7.5-fold increase in the intracellular cAMP level within 30 sec. The cAMP level then sharply dropped to about 100 pmol/10(7) cells, remained at this level for about 20 min, and then gradually increased to 200 pmol/10(7) (about fivefold over the control). The enzyme assay of the cytosol demonstrated that the activation of cAMP-dependent protein kinases closely follows the kinetics of the intracellular cAMP level. The activation of the enzyme was specific for PGE2 and was not triggered by 1 microM PGF2 alpha or PGD2 which have been shown to be unable to activate adenylate cyclase of P388D1 cells. The PGE2-induced increase in the intracellular cAMP level appeared to activate preferentially the type I isoenzyme, inasmuch as the enzymatic activity of this type separated by the affinity chromatography of the cytosol of PGE2-exposed cells was lower in the presence than in the absence of cAMP, whereas the type II enzyme activity remained responsive to exogenously added cAMP. 相似文献
6.
Goraya TA Masada N Ciruela A Willoughby D Clynes MA Cooper DM 《Cellular signalling》2008,20(2):359-374
Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species. 相似文献
7.
Kotera J Sasaki T Kobayashi T Fujishige K Yamashita Y Omori K 《The Journal of biological chemistry》2004,279(6):4366-4375
8.
Cultured MDCK cells were treated with various gangliosides and sialylated compounds and their effects on the intracellular level of cAMP were compared to those of arginine vasopressin (AVP) and other substances which elevate cAMP. Since all those agents could stimulate dome formation, its correlation with cAMP production is discussed. Most gangliosides increased intracellular cAMP 3-4-fold, the increase being dose-dependent up to 25 microM ganglioside. AVP and cAMP analogs increased intracellular cAMP 3-40-fold. A unique feature of the ganglioside-induced cAMP increase was its extremely long time course (70 h), as compared to that induced by other agents which show much faster and less prolonged effects in other biological systems. This might indicate that gangliosides differ from AVP and other agents in the mechanisms by which they stimulate intracellular cAMP increase. The time course and the level of cAMP increase induced by GM3 or AVP did not correlate with those of dome formation. Furthermore, the ability of some gangliosides and other agents to induce dome formation did not correspond to their ability to elevate cAMP. It is suggested that although the remarkable dome-stimulating activity of gangliosides may be induced in part by a cAMP-dependent mechanism, gangliosides also act directly on the cellular components influencing dome formation, without involving changes in intracellular cAMP. 相似文献
9.
W H Fletcher S M Van Patten H C Cheng D A Walsh 《The Journal of biological chemistry》1986,261(12):5504-5513
Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization. 相似文献
10.
J A Alberta L F Epstein L A Pon N R Orme-Johnson 《The Journal of biological chemistry》1989,264(4):2368-2372
We have reported previously that a phosphoprotein, ib, is present in adrenal cortex, corpus luteum, and Leydig cells stimulated with either tissue-specific peptide hormone or with cAMP. The accumulation of protein ib in each of these cell types has been found to parallel the stimulation of steroid synthesis with respect to both time course and stimulant dose response. Thus, protein ib is a potential mediator in the acute stimulation of steroidogenesis by peptide hormone or cyclic AMP. A second protein, pb, the unphosphorylated form of ib, is synthesized constitutively in unstimulated but not stimulated cells and is not converted post-translationally to ib upon stimulation. Using two-dimensional gel electrophoresis of subcellular fractions isolated from rat adrenal cortex cells labeled with [35S] methionine, we have determined the intracellular localization of proteins p and i. We demonstrate that proteins ib and pb are localized predominantly in the mitochondria and are tightly associated with that organelle. We also find that inhibition of mitochondrial protein synthesis by chloramphenicol affects neither the accumulation of these proteins nor the stimulation of steroidogenesis. Thus, protein pb and its phosphorylated counterpart, ib, are synthesized in the cytosol and transported to the mitochondria, the site of the rate-limiting step in steroid hormone biosynthesis. 相似文献
11.
The intracellular second messenger cyclic AMP (cAMP) is degraded by phosphodiesterases (PDE). The knowledge of individual families and subtypes of PDEs is considerable, but how the different PDEs collaborate in the cell to control a cAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL and have compared intracellular cAMP measurements with it to measurements with the cytosolic BRET sensor CAMYEL in HEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET) sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitors and have identified PDE3 and PDE10A as the major membrane-bound PDEs and PDE4 as the major cytosolic PDE. Inhibition of membrane-bound or cytosolic PDEs can potentiate the cAMP response to adenylyl cyclase activation, but we see no significant difference between the potentiation of the cAMP response at the plasma membrane and in cytosol when membrane-bound and cytosolic PDEs are inhibited. When different levels of stimulation were tested, we found that PDEs 3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations. 相似文献
12.
Nguyen BT Yang L Sanborn BM Dessauer CW 《Molecular endocrinology (Baltimore, Md.)》2003,17(6):1075-1084
The G protein-coupled receptors LGR7 and LGR8 have recently been identified as the primary receptors for the polypeptide hormone relaxin and relaxin-like factors. RT-PCR confirmed the existence of mRNA for both LGR7 and LRG8 in THP-1 cells. Whole cell treatment of THP-1 cells with relaxin produced a biphasic time course in cAMP accumulation, where the first peak appeared as early as 1-2 min with a second peak at 10-20 min. Selective inhibitors for phosphoinositide 3-kinase (PI3K), such as wortmannin and LY294002, showed a dose-dependent inhibition of relaxin-mediated increases in cAMP, specific for the second peak of the relaxin time course. Adenylyl cyclase activation by relaxin in purified plasma membranes from THP-1 cells was not inhibited by LY294002, consistent with a mechanism involving direct stimulation by a Galphas-coupled relaxin receptor. However, reconstitution of membranes with cytosol from THP-1 cells enhanced adenylyl cyclase activity and restored LY294002 sensitivity. In addition, relaxin increased PI3K activity in THP-1 cells. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 was mediated by the activity of phosphodiesterases. Taken together, we show that PI3K is required for the biphasic stimulation of cAMP by relaxin in THP-1 cells and present a novel signal transduction pathway for the activation of adenylyl cyclase by a G protein-coupled receptor. 相似文献
13.
重组炭疽水肿因子的表达与生物活性分析 总被引:1,自引:0,他引:1
炭疽毒素包括3种蛋白因子,即保护性抗原(PA)、致死因子(LF)和水肿因子(EF)。EF是钙调蛋白依耐的腺苷酸环化酶,可使细胞cAMP浓度升高,导致宿主防御能力下降。为深入研究炭疽毒素的作用机理,构建了原核表达质粒,在大肠杆菌中表达出重组EF(rEF)。经鉴定,rEF以可溶形式表达于细菌胞质中。经过金属螯和层析、阳离子交换层析和凝胶层析,每升诱导培养物可获得约5mg 重组蛋白。用重组蛋白免疫家兔获得了兔多抗,能够在细胞试验中中和rEF,体外细胞试验显示rEF具有很好的生物活性,在J774A.1和CHO细胞试验中,能与LF共同竞争和PA的结合位点,相互抑制。上述工作为深入研究炭疽毒素的作用机理,开发针对EF的毒素抑制剂打下基础 相似文献
14.
Yassenko M Thérond P Evain-Brion D Keryer G 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》2001,324(1):23-31
During the G1/S transition of the cell cycle variations in the labelling by 8-N3-[32P]cAMP of the protein kinase A regulatory subunits RI and RII, used as a probe to monitor post-translational modifications that may regulate cAMP binding, were observed in synchronized HeLa cells. A decrease in 8-N3-[32P]cAMP labelling of RI, RII and RII phosphorylated by the catalytic subunit of PKA was correlated with the increased percentage of cells in phases G1. An increase in 8-N3-[32P]cAMP incorporated into the 54-kDa RII subunit during progression from G1 to S was correlated with an increase in intracellular cAMP. A transient increase in Mn-SOD activity was detected in cells arrested at the G1/S transition using two different techniques, suggesting that oxidative modulation of regulatory subunits by free radicals may modify cAMP binding sites during the cell cycle. Decreased photoaffinity labelling by 8-N3-[32P]cAMP of RI, RII and autophosphorylated RII subunits was found to be an inherent characteristic of PKA in the G1/S transition. 相似文献
15.
Warrier S Belevych AE Ruse M Eckert RL Zaccolo M Pozzan T Harvey RD 《American journal of physiology. Cell physiology》2005,289(2):C455-C461
-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by -adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of -adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types. adenovirus; protein kinase A; phosphodiesterase; L-type Ca2+ channel 相似文献
16.
The fate of the catalytic subunit of the Escherichia coli heat labile toxin (LTA(1)) was studied after expression in mammalian cells to assess the requirement for ADP-ribosylation factor (ARF) binding to localization and toxicity and ability to compete with endogenous ARF effectors. A progression in LTA(1) localization from cytosol to binding Golgi stacks to condensation of Golgi membranes was found to correlate with the time and level of LTA(1) expression. At the highest levels of LTA(1) expression the staining of LTA and both extrinsic and lumenal Golgi markers all became diffuse, in a fashion reminiscent of the actions of brefeldin A. Thus, LTA(1) binds to the Golgi and can alter its morphology in two distinct ways. However, point mutants of LTA(1) that are defective in the ability to bind activated ARF were also unable to bind Golgi membranes or modify Golgi morphology. Co-expression of mutants of ARF3 that regained binding to these same mutant LTA(1) proteins restored the localization and activities of the toxin. Thus, binding to ARF is required both for the localization of the toxin to the Golgi and for effects on Golgi membranes. A correlation was also seen between the ability of LTA mutants to bind ARF and the increase in cellular cAMP levels. These results demonstrate the importance of ARF binding to the toxicity and cellular effects of the ADP-ribosylating bacterial toxin and reveal that mutants defective in binding ARF retain basal ADP-ribosylation activity but are the least toxic LTA(1) mutants yet described, making them the best candidates for development as mucosal adjuvants. 相似文献
17.
Persistent elevations of cAMP levels are generally accompanied by an inhibition of granulocyte functions. Phosphodiesterases play a critical role in regulating intracellular levels of cAMP. The expression of three isoforms of type 4 cAMP-specific phosphodiesterase (PDE4) in neutrophils suggests diversity of isoform localization and targeting in regulating cell function. The sites of cAMP regulation in granulocytes by the PDE4A isoform were investigated by immunoelectron microscopy. PDE4A was localized uniformly in all granule classes of eosinophils, but was restricted in neutrophils to a subset of myeloperoxidase (MPO)-containing granules that were round or elongated with a central crystalloid core. Granulocytes were stimulated with fMLP to investigate the sites of PDE4A targeting during cell activation. In neutrophils, fMLP induced a rapid (1 min) translocation of granules containing PDE4A to the plasmalemma, where some PDE4A and MPO were exocytosed. In these cells, PDE4A labeling within granules was focal and no longer homogeneous. While immunogold labeling of PDE4A was reduced after fMLP stimulation, staining of MPO-containing granules remained high. Extracellular release of PDE4A was also observed in eosinophils stimulated with fMLP. Morphometry revealed that Au labeling was significantly reduced within 1 min, and that there was a shift in PDE4A localization within eosinophil granules from the crystalline core to the matrix. Fluctuations of cAMP levels and ectoprotein kinase activity with PKA properties occur in blood under normal and pathological conditions. The exclusive localization of PDE4A within granules of neutrophils and eosinophils suggests that PDE4A may function to downregulate cAMP signaling at the cell membrane and/or in the extracellular space at the time of granule release. 相似文献
18.
Dissociation and reassociation of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases I and II were studied in intact AtT20 cells. Cells were stimulated with 50 microM forskolin to raise intracellular cAMP levels and induce complete dissociation of R and C subunits. After the removal of forskolin from the incubation medium cAMP levels rapidly declined to basal levels. Reassociation of R and C subunits was monitored by immunoprecipitation of cAMP-dependent protein kinase activity using anti-R immunoglobulins. The time course for reassociation of R and C subunits paralleled the loss of cellular cAMP. Total cAMP-dependent protein kinase activity and the ratio of protein kinase I to protein kinase II seen 30 min after the removal of forskolin was the same as in control cells. Similar results were seen using crude AtT20 cell extracts treated with exogenous cAMP and Mg2+. Our data showed that after removal of a stimulus from AtT20 cells inactivation of both cAMP-dependent protein kinase isoenzymes occurred by the rapid reassociation of R and C subunits to form holoenzyme. Our studies also showed that half of the type I regulatory subunit (RI) present in control cells contained bound cAMP. This represented approximately 30% of the cellular cAMP in nonstimulated cells. The cAMP bound to RI was resistant to hydrolysis by cyclic nucleotide phosphodiesterase but was dissociated from RI in the presence of excess purified bovine heart C. The RI subunits devoid of C may function to sequester cAMP and, thereby, prevent the activation of cAMP-dependent protein kinase activity in nonstimulated AtT20 cells. 相似文献
19.
Activation of soluble guanylyl cyclase at the leading edge during Dictyostelium chemotaxis
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Veltman DM Roelofs J Engel R Visser AJ Van Haastert PJ 《Molecular biology of the cell》2005,16(2):976-983
Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell. 相似文献
20.
Akt is a protein serine/threonine kinase that plays an important role in the mitogenic responses of cells to variable stimuli. Akt contains a pleckstrin homology (PH) domain and is activated by phosphorylation at threonine 308 and serine 473. Binding of 3'-OH phosphorylated phosphoinositides to the PH domain results in the translocation of Akt to the plasma membrane where it is activated by upstream kinases such as (phosphoinositide-dependent kinase-1 (PDK1). Over-expression of constitutively active forms of Akt promotes cell proliferation and survival, and also stimulates p70 S6 kinase (p70S6K). In many cells, an increase in levels of intracellular cyclic AMP (cAMP) diminishes cell growth and promotes differentiation, and in certain conditions cAMP is even antagonistic to the effect of growth factors. Here, we show that cAMP has inhibitory effects on the phosphatidylinositol 3-kinase/PDK/Akt signaling pathway. cAMP potently inhibits phosphorylation at threonine 308 and serine 473 of Akt, which is required for the protein kinase activities of Akt. cAMP also negatively regulates PDK1 by inhibiting its translocation to the plasma membrane, despite not affecting its protein kinase activities. Furthermore, when we co-expressed myristoylated Akt and PDK1 mutants which constitutively co-localize in the plasma membrane, Akt activity was no longer sensitive to raised intracellular cAMP concentrations. Finally, cAMP was also found to inhibit the lipid kinase activity of PI3K and to decrease the levels of phosphatidylinositol 3,4,5-triphosphate in vivo, which are required for the membrane localization of PDK1. Collectively, these data strongly support the theory that the cAMP-dependent signaling pathway inhibits Akt activity by blocking the coupling between Akt and its upstream regulators, PDK, in the plasma membrane. 相似文献