Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells.
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PMID:Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia. 869 88

The regulation of Ca2+-activated K+ channels (KCa channels) by cGMP-dependent protein kinase (cGMP kinase) and its molecular mechanism were investigated in Chinese hamster ovary (CHO) and tracheal smooth muscle cells. In CHO wild-type cells (CHO-WT cells) and in CHO cells stably transfected with cGMP kinase Ialpha (CHO-cGK cells), KCa channels with intermediate conductance (approximately 50 picosiemens) were identified. Due to the basal activity of cGMP kinase, Ca2+-activated K+ currents had a higher sensitivity toward the cytosolic Ca2+ concentration in CHO-cGK cells than in CHO-WT cells. Dialysis of the active fragment of cGMP kinase (300 n) into CHO-WT cells or of cGMP into CHO-cGK cells increased the Ca2+-activated K+ current, while the catalytic subunit of cAMP-dependent protein kinase (cAMP kinase) was without effect. In cell-attached patches obtained from freshly isolated bovine tracheal smooth muscle cells, the open state probability (NPo) of maxi-KCa channels (conductance of approximately 260 picosiemens) was enhanced by 300 microM 8-(4-chlorophenylthio)-cGMP, a specific and potent activator of cGMP kinase. In contrast, 1 microM isoprenaline, 20 microM forskolin, and 3 mM 8-bromo-cAMP failed to enhance KCa channel activity. In excised inside-out patches, only the active fragment of cGMP kinase (but not that of cAMP kinase) increased NPo when applied to the cytosolic side of the patch. The enhancement of NPo by cGMP kinase was inhibited in CHO cells as well as in tracheal smooth muscle cells by the cGMP kinase inhibitor KT 5823 (1 microM) and the protein phosphatase (PP) inhibitors microcystin (5 microM) and okadaic acid (10 nM). The catalytic subunit of PP2A (but not that of PP1) mimicked the effect of cGMP kinase on NPo in excised inside-out patches. The results show that cGMP kinase regulates two different KCa channels in two unrelated cell types by the same indirect mechanism, which requires the activity of PP2A. The regulation of the KCa channel is specific for cGMP kinase and is not mimicked by cAMP kinase.
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PMID:Protein phosphatase 2A is essential for the activation of Ca2+-activated K+ currents by cGMP-dependent protein kinase in tracheal smooth muscle and Chinese hamster ovary cells. 870 82

We have used the immunosuppressants cyclosporin A and FK506 to investigate the involvement of the Ca2+/CaM-dependent protein phosphatase calcineurin in Dictyostelium discoideum development. We found that CsA had little effect on cell growth, or on the aggregation of developing amoebae, suggesting that calcineurin does not play a significant role at these stages of the D. discoideum life cycle. In contrast, when cells were allowed to differentiate under buffer in the presence of cAMP, addition of CsA and FK506 strongly inhibited stalk cell formation in the wild-type and spore formation in a sporogenous derivative of D. discoideum strain V12. These agents also reduced the expression of prestalk-and prespore-specific transcripts in both strains. These results indicate a requirement for calcineurin activity in both pathways of cell differentiation. In addition, time-course experiments suggest that calcineurin is required early in the differentiation processes, but that the maturation of the two cell types is resistant to calcineurin inhibition. We also found that CsA and FK506 were unable to affect spore formation in rapidly developing/sporogenous rdeC mutants of strain NC4, showing that constitutive cAMP-dependent protein kinase activity renders the spore pathway resistant to calcineurin inhibition.
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PMID:A role for calcineurin in Dictyostelium discoideum development. 885 70

The involvement of cAMP in the process of sperm capacitation has been the subject of several studies. In addition, the importance of protein-tyrosine phosphorylation in this process has been investigated, although only a few studies have been reported in the human. Since agents regulating the intracellular concentrations of cAMP affect sperm capacitation rates, the role of cAMP on the expression of phosphotyrosine-containing proteins was investigated during human sperm capacitation. Fetal cord serum ultrafiltrate, a known capacitation inducer in human spermatozoa, caused an increase in the phosphotyrosine content of 105- and 81-kDa proteins (p105 and p81), the two major phosphotyrosine-containing proteins of human spermatozoa. Similar effects were observed when spermatozoa were incubated with phosphodiesterase inhibitors or cell-permeant cAMP analogs, suggesting that cAMP is involved in these two processes. Forskolin, an adenylyl cyclase activator, also caused an increase in both sperm capacitation rates and tyrosine phosphorylation of p105 and p81, while 12-O-tetradecanoyl phorbol 13-acetate stimulated both capacitation and tyrosine phosphorylation of p105 and p81 only when spermatozoa were incubated in the presence of bicarbonate, in agreement with its reported effects on cAMP production and hamster sperm capacitation. The inhibition of these phenomena by cAMP-dependent protein kinase inhibitors, and the stimulation by protein phosphatase inhibitors, suggest that Ser/Thr protein phosphorylation plays an important role in the regulation of both sperm capacitation and protein-tyrosine phosphorylation pathways. However, observations that both calyculin A and okadaic acid stimulated sperm capacitation, whereas only calyculin A increased p105 and p81 phosphotyrosine content and sperm velocity, suggest that protein phosphatase PP1 is involved in the two latter phenomena while PP2A mediates sperm capacitation. These results suggest that divergent pathways might regulate tyrosine phosphorylation of p105 and p81 and sperm capacitation after cAMP-dependent phosphorylation of an intermediate protein.
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PMID:Cyclic adenosine 3',5'monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. 886 88

This study identifies a 100-residue domain within the rabbit skeletal muscle regulatory subunit (PP1G) that binds both type-1 protein phosphatase (PP1C) and glycogen. An N-terminal portion of PP1G was cloned by RT-PCR, and different sized fragments were expressed in bacteria as glutathione S-transferase (GST) fusion proteins. A GST-PP1G fusion containing residues 51-240 bound both PPIC and glycogen, whereas GST alone or fusions containing residues 51-140 or 241-360 bound neither PP1C nor glycogen. The PPIC in whole cell lysates or partially purified PP1C from skeletal muscle, or a complex of PP1C-MCLR-biotin, all bound more effectively than Mn(2+)-activated, recombinant PP1C purified from bacteria. Binding was enhanced by increasing the ionic strength and was disrupted by ethylene glycol, consistent with hydrophobic interactions being critical for stable association. Phosphorylation of the GST-PP1G fusion by cAMP-dependent protein kinase prevented completely association of PP1C. This domain of PP1G, from residues 141-240, contains two sequence motifs of hydrophobic residues: Gx8FEKx10W and DxFxFxIxL, that are conserved among the known glycogen-binding PP1 regulatory subunits. These segments are predicted to form an alpha helix and a beta sheet, and we propose that they are the sites for association with PP1C and glycogen, respectively.
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PMID:Protein phosphatase type-1 and glycogen bind to a domain in the skeletal muscle regulatory subunit containing conserved hydrophobic sequence motif. 890 29

We have shown that ethanol inhibits uptake of adenosine by a specific nucleoside transporter in NG108-15 neuroblastoma x glioma cells and that cAMP-dependent protein kinase (PKA) activity is required for this inhibition. After chronic exposure to ethanol, adenosine uptake is no longer inhibited on rechallenge with ethanol, i.e. transport has become tolerant to ethanol. Here we show that protein kinase C (PKC) contributes to ethanol-induced tolerance of adenosine transport. Activation of PKC by phorbol esters in control cells results in an ethanol-tolerant phenotype, similar to that produced by chronic ethanol exposure. In addition, chronic exposure to ethanol increases the amounts of alpha, delta, and epsilon PKC. However, reducing PKC activity by inhibition with chelerythrine during chronic exposure to ethanol or down-regulation by phorbol esters prevents the development of ethanol-induced tolerance of adenosine transport. By contrast, the inhibition of PKA activity produces tolerance to ethanol inhibition of adenosine uptake. When protein phosphatase inhibitors are present, inhibiting PKA activity has no effect on ethanol sensitivity of adenosine uptake, suggesting a role for protein phosphatases in the regulation of ethanol sensitivity of uptake. Taken together, our results suggest that PKA and PKC have opposing effects on the ethanol sensitivity of adenosine transport; PKA activity is required for ethanol sensitivity, and PKC activation produces tolerance. Based on these data, we propose that chronic ethanol exposure increases PKC activity, leading to the activation of a protein phosphatase (1 or 2A). This phosphatase then dephosphorylates a PKA-phosphorylated site, which is required for ethanol to inhibit adenosine uptake. Therefore, the sensitivity of adenosine transport to ethanol appears to be maintained by a balance of PKA and protein phosphatase activities, and PKC may regulate phosphatase activity.
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PMID:The role of protein kinase C in cellular tolerance to ethanol. 891 Jun 14

We have examined the nature of signal transduction involving reversible protein phosphorylation in marine Prorocentrale species. Of particular interest is the marine dinoflagellate Prorocentrum lima in which the tumour promoter okadaic acid is produced and may interfere with signal transduction. We have identified cAMP-dependent protein kinase (PKA) activity in P. lima, P. micans, and P. minimum. The P. lima enzyme was characterized biochemically and appears to consist of two different isoforms in the R2C2 configuration. Whole cell extracts of P. micans and P. minimum treated with the specific PKA inhibitor peptide PKI (5-24) or cAMP demonstrated altered intensities of phosphopeptide 32P labeling, most likely involving regulation of a protein phosphatase via PKA activity. A primary candidate for PKA regulation is protein phosphatase-1 (PP-1), which in P. lima possesses a classical PKA consensus phosphorylation site. We demonstrate that a peptide fragment of PP-1 from P. lima corresponding to this PKA phosphorylation site can be effectively phosphorylated by PKA and dephosphorylated by calcineurin. We speculate that PP-1 activity among several lower eukaryotes may be mediated directly by reversible phosphorylation. Higher eukaryotes may have developed inhibitor proteins to provide more complex regulation of protein phosphatase activity.
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PMID:Identification and characterization of cAMP-dependent protein kinase and its possible direct interactions with protein phosphatase-1 in marine dinoflagellates. 896 Mar 62

Cyclic AMP regulates a variety of cellular responses through activation of cAMP-dependent protein kinase (PKA). The catalytic subunit of PKA, in turn, activate cAMP responsive element (CRE) and nuclear factor-kappa B (NF-kappa B) binding proteins. In this study, we demonstrated that binding activity to both CRE and kappa B sites in nuclear extracts from spleen cells is modulated by PKA in a time-dependent manner. Electrophoretic mobility shift assays showed that binding by transcription factors to either the CRE or kappa B motif was rapidly up-regulated by cAMP, with maximum binding detected at 30 min in response to forskolin stimulation of splenocytes. This was followed by a steady decline in CRE and kappa B thereafter reaching basal levels by 2 hr. This up-regulation in CRE and kappa B binding was closely associated with an enhancement of PKA activity which was maximum at 30 min following forskolin stimulation. However, unlike the binding of regulatory factors to CRE and kappa B motifs which was very transient, peak PKA activity was sustained for 2 hr. Interestingly, okadaic acid, a protein phosphatase inhibitor, prevented the decline in protein binding to CRE and kappa B motifs 2 hr following forskolin stimulation and actually produced a slight increase at 30 min. These data suggest that binding by transcription factors to CRE and kappa B sites are up-regulated concomitantly with PKA activation but subsequently down-regulated by a protein phosphatase.
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PMID:Transient CRE- and kappa B site-binding is cross-regulated by cAMP-dependent protein kinase and a protein phosphatase in mouse splenocytes. 903 89

The activity of Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) is not regulated by phosphorylation, presumably due to the absence of a suitable target serine and protein kinase recognition motif. We have engineered P. mevalonii HMG-CoA reductase to a form whose activity, like that of mammalian HMG-CoA reductases, is regulated by phosphorylation/dephosphorylation. We substituted serine for arginine 387, the residue that corresponds to the regulatory serine of the HMG-CoA reductases of higher eukaryotes. A recognition motif for cAMP-dependent protein kinase was added by replacing leucine 384 by histidine (enzyme L384H/R387S) and also valine 391 by leucine (enzyme L384H/R387S/V391L). The activity of P. mevalonii HMG-CoA reductase mutant enzymes L384H/R387S and L384H/R387S/V391L was attenuated by phosphorylation. Restoration of activity accompanied subsequent dephosphorylation catalyzed by lambda protein phosphatase. Incorporation and subsequent release of phosphate paralleled the attenuation and restoration of catalytic activity. Incorporation of 0.5 mol of phosphate per subunit was accompanied by an approximately 50% decrease in initial activity. As in the analogous Syrian hamster mutant enzyme S871D, P. mevalonii mutant enzyme R387D exhibited 10% wild-type activity, suggesting that the attenuation of activity that accompanies phosphorylation results at least in part from the introduction of negative charge. Engineering of P. mevalonii HMG-CoA reductase to forms whose activity is reversibly regulated by phosphorylation/dephosphorylation provides an attractive model for future structure-based mechanistic studies. Solution of the X-ray structure of phosphorylated and dephosphorylated forms of engineered P. mevalonii HMG-CoA reductase should then reveal interactions of the active site phosphoseryl residue that result in attenuation of catalytic activity.
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PMID:Protein engineering of the HMG-CoA reductase of Pseudomonas mevalonii. Construction of mutant enzymes whose activity is regulated by phosphorylation and dephosphorylation. 904 17

Among the cellular actions of vanadate ions are several that have the potential to be of significance in the regulation of protein phosphorylation. The effects of vanadate on adenosine 3',5' cyclic monophosphate (cAMP)-dependent and independent, alkali-resistant protein phosphorylation in a synaptosomal preparation from rat cortex were examined in this study. Three major vanadate-stimulated, cAMP-independent phosphoproteins (58-, 50-, and 39-kDa) and two cAMP-dependent species (37- and 32-kDa) were detectable. The potentiation between vanadate and cAMP in stimulating the phosphorylation of the latter two proteins is in contrast to the nonadditive combined effect of both on the phosphorylation of other synaptosomal proteins. The two cAMP-dependent, 32P-labeled proteins possess identical or very similar physicochemical properties to two previously cited neuronal phosphoproteins, namely, dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein-32 (DARPP-32) and inhibitor-1 (I-1). Such properties include phosphorylation by cAMP-dependent protein kinase, the presence of an alkali-resistant phosphothreonine residue, comigration on two-dimensional gel electrophoresis, dephosphorylation by type-2B protein phosphatase, and crossreactivity with specific antibodies. Costimulation by cAMP and vanadate of phosphorylation of the latter two proteins on threonine residues, at concentrations of vanadate consistent with the regulation of protein tyrosine phosphatase activity, indicates a unique interaction between these two regulators of protein phosphorylation at the nerve terminus.
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PMID:Adenosine 3',5'-cyclic monophosphate/vanadate-sensitive phosphorylation of DARPP-32- and inhibitor-1-immunoreactive proteins. 928 28


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