Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.
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PMID:G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells. 754 31

In mammalian cells, the guanine nucleotide exchange factor (GEF, eIF-2B) plays a major role in the regulation of initiation of protein synthesis. It catalyzes the exchange of eukaryotic chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the recycling of eIF-2 during polypeptide chain initiation. We used the Friend virus-transformed murine erythroleukemia (MEL) cell system to elucidate the translational regulatory processes that occur during growth and hexamethylene bisacetamide (HMBA)-induced cell differentiation. GEF activity is increased during growth and decreased during MEL cell differentiation, and this parallels the overall changes in protein synthesis during this period. Inhibition of GEF activity in induced cells may occur indirectly by phosphorylation of the alpha-subunit of eIF-2. However, the decrease in GEF activity in induced cells cannot be reversed by increasing the concentration of eIF-2-GDP added as a substrate in the GEF assay. This is diagnostic for the presence of eIF-2 alpha(P)-GDP in cell lysates and suggests that regulation of GEF activity may occur by one or more mechanisms other than eIF-2(alpha) phosphorylation. We have previously shown that the activity of GEF may be influenced directly by phosphorylation with casein kinase II (CK-II) of the 82-kD subunit of the factor. CK-II activity parallels the changes in GEF activity and the rate of protein synthesis during growth and differentiation of MEL cells. Addition of 1mM spermidine, a stimulator of CK-II but not of purified GEF, in induced MEL cell extracts enhances both CK-II and GEF activities approximately 48 and 32%, respectively. The results presented suggest that the inhibition of protein synthesis during MEL cell differentiation may be linked to the decreased CK-II and GEF activities.
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PMID:Hexamethylene bisacetamide-induced differentiation of Friend virus-transformed murine erythroleukemia cells is associated with parallel changes in casein kinase II and guanine nucleotide exchange factor activities. 755 31

We have previously reported that the calciotropic hormone 1,25(OH)2-vitamin D3 stimulates influx of Ca2+ into cultured rat and embryonic chick myoblasts via voltage sensitive Ca(2+)-channels. In the present study, we show that this effect of 1,25(OH)2D3 requires the mediation of the adenylylcyclase signalling system since the hormone-dependent Ca2+ influx is abolished by specific inhibitors of adenylylcyclase and protein kinase A and mimicked by forskolin and dibutyryl cAMP. 1,25(OH)2D3-stimulated elevations in cellular cAMP paralleled increases in Ca2+ uptake, further suggesting a coupling of adenylylcyclase activation and calcium influx. Fluoride and GTP gamma S mimicked 1,25(OH)2D3-stimulation of calcium influx while GDP beta S suppressed the effect of the hormone. Cholera toxin and Bordetella pertussis toxin both increased 45Ca2+ uptake in rat and chick myoblasts. The hormone further increased cholera toxin actions, but was unable to modify pertussis toxin-induced 45Ca2+ uptake, suggesting a similar target of action for pertussis toxin and 1,25(OH)2D3. Incubation of microsomal membranes with the sterol (10 nM, 2 min) markedly displaces (-32%) [35S]GTP gamma S binding to the membranes. ADP-ribosylation of the pertussis toxin-sensitive 41 kDa substrate was significantly increased (+40%) in 1,25(OH)2D3-pretreated cells. These results suggest that 1,25(OH)2D3-stimulated influx of Ca2+ into rat and embryonic chick cultured myoblasts sequentially requires inhibition of a pertussis toxin-sensitive G protein, accumulation of cAMP and activation of dihydropyridine-sensitive Ca(2+)-channels through PKA-mediated phosphorylation events.
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PMID:Modulation by 1,25(OH)2-vitamin D3 of the adenylyl cyclase/cyclic AMP pathway in rat and chick myoblasts. 757 77

In mammalian cells, the guanine nucleotide exchange factor (GEF or eIF-2B) is a key regulator of polypeptide chain initiation. The exchange of GDP bound to chain initiation factor 2 (eIF-2) for GTP by GEF is a rate limiting step in protein synthesis. The multisubunit characteristics of GEF suggest that this protein is composed of several distinct structural and functional domains, and is regulated by allosteric means and by phosphorylation. The activity of GEF may be regulated indirectly by the phosphorylation state of the smallest subunit of eIF-2 (alpha-subunit). On the other hand, phosphorylation of the largest subunit of GEF (82-kD subunit) by casein kinase (CK) I or II stimulates GDP/GTP exchange. GEF contains NADPH which is required for structural integrity of the protein. Upon stimulation of cells by insulin and growth factors, allosteric activation of GEF by sugar phosphates and other effector molecules may also play an important role in the regulation of polypeptide chain initiation. In this article, recent information about structure-function relationship of eIF-2 and GEF in nucleotide exchange and the regulatory mechanisms that influence the rate of polypeptide chain initiation under various physiological and pathological conditions are presented.
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PMID:Translational control of eukaryotic gene expression. Role of the guanine nucleotide exchange factor and chain initiation factor-2. 758 45

Eukaryotic initiation factor 2B, or guanine nucleotide-exchange factor, has been purified for the first time from the brain by a novel procedure that allows the purification of initiation factor 2 as well and uses a salt wash postmicrosomal supernatant as starting material. The procedure includes a three-part chromatographic step in heparin-Sepharose and in SP-5PW and diethylaminoethyl-5PW ion-exchange high-performance chromatographies. The purification of the factors was followed by measuring activity in the guanine nucleotide-exchange assay and the capacity of initiation factor 2 to form a ternary complex with the initiation form of methionyl-tRNA and GTP. The method yields guanine nucleotide-exchange factor (75%) and highly purified initiation factor 2 (> 95%), which are separated in the last step. The exchange factor from the brain is a multimeric protein with five subunits of molecular masses of 82, 65, 52, 42, and 30 kDa; it stimulates ternary complex formation in the presence of GDP, and this activity is inhibited by N-ethylmaleimide. A 37-kDa protein that copurifies with initiation factors is characterized in this study as a new calmodulin-binding protein (p37); it is highly phosphorylated by casein kinase activities and can comigrate with the alpha subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions.
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PMID:Purification and characterization of guanine nucleotide-exchange factor, eIF-2B, and p37 calmodulin-binding protein from calf brain. 761 32

We have characterized the regulation of the endogenous Na+/H+ exchanger in Xenopus laevis oocytes by G proteins and protein kinases by measuring the ethylisopropylamiloride-sensitive Li+ uptake. Injection of oocytes with the stable GTP analog GTP gamma S stimulated Li+ uptake up to almost 4-fold, an effect blocked by coinjection with the GDP analog, guanyl-5'-yl thiophosphate. Injection into oocytes of beta gamma subunits of the heterotrimeric G protein transducin enhanced Li+ uptake by about 3-fold. This stimulation was blocked by transducin alpha subunits, which by themselves did not influence Li+ uptake. Using various activators and inhibitors of protein kinases, it is demonstrated that the X. laevis oocyte Na+/H+ antiporter can be stimulated by activation of both protein kinase A and C. Stimulation of Na+/H+ exchanger activity by GTP gamma S but not that induced by transducin beta gamma subunits was blocked by the protein kinase A inhibitor H-89. On the other hand, transducin beta gamma subunit-stimulated activity was prevented by the protein kinase C inhibitor, calphostin C. The non-selective protein kinase inhibitor H-7 blocked both GTP gamma S- and transducin beta gamma subunit-stimulated Na+/H+ exchanger activity. The results suggest that the Na+/H+ exchanger of X. laevis oocytes can be activated by G proteins and that this activation is not direct but mediated by protein kinase A- and/or protein kinase C-dependent pathways.
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PMID:G protein regulation of the Na+/H+ antiporter in Xenopus laevis oocytes. Involvement of protein kinases A and C. 762 94

Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.
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PMID:Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets. 763 Jun 28

Inositol 2,4,5-trisphosphate irreversibly activated capacitative calcium entry in Xenopus oocytes, whereas guanosine thiotriphosphate (GTP[S]) and AIF4- only activated capacitative calcium entry transiently. Both GTP[S] and AIF4- inhibited capacitative calcium entry activated by thapsigargin pretreatment, but guanosine thiodiphosphate (GDP[S]), inositol 2,4,5-trisphosphate and dibutyryl cyclic GMP did not affect capacitative calcium entry. This suggests the involvement of heterotrimeric GTP-binding proteins in the regulation of capacitative calcium entry. Activation of protein kinase C or cyclic-AMP-dependent protein kinase had profound effects on capacitative calcium entry, which were consistent with the hypothesis that the effects of GTP[S] and AIF4- on capacitative calcium entry may be mediated via heterotrimeric GTP-binding protein stimulation of kinases. Further evidence for this hypothesis was derived from the result that the effects of GTP[S] on calcium entry could be inhibited by the application of the protein kinase inhibitor staurosporine.
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PMID:G-protein regulation of capacitative calcium entry may be mediated by protein kinases A and C in Xenopus oocytes. 774 94

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.
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PMID:R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism. 774 59

The regulation of a Cl- conductance in the choroid plexus by G proteins was investigated using whole-cell patch clamp methods. A transient Cl- current was evoked when GTP-gamma-S (0.1 mM) was included in the electrode solution. The GTP-gamma-S-evoked current was only observed when 2 mM ATP was present in the electrode solution, and was inhibited in the presence of 2 mM GDP-beta-S. The activated current was shown to be Cl(-)-selective in ion replacement studies, and exhibited inward-rectification. The current was not inhibited when intracellular Ca2+ was strongly buffered with either EGTA or BAPTA, suggesting that the current was not activated by a change in intracellular Ca2+. The effects of GTP-gamma-S were mimicked, however, when cyclic AMP or the catalytic-subunit of protein kinase A were included in the electrode solution. These data suggest that G proteins activate Cl- channels via a cAMP-dependent pathway in rat choroid plexus.
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PMID:Activation of chloride currents in choroid plexus by GTP-gamma-S and cyclic-AMP. 775 20


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