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

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.
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PMID:Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets. 765 73

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

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

To better understand the biochemical events accompanying lung alveolarization and development, we studied the specific activity of the cAMP-dependent protein kinase (PKA) and the type 2A protein phosphastase (PP2A), and the activity and protein content of the calcium- and lipid-dependent protein kinase (PKC) in cytosolic preparations of lungs. Lungs were obtained from rat pups on day 2 of life and on days 7, 14, and 27 from pups exposed to hyperoxia (> 95% O2, days 4-14; 65% O2 days 15-27) or normoxia from day 4 onwards. There were no significant changes in PKA specific activity with developmental age or hyperoxic exposure. PKC specific activity increased significantly (P < .05) in normoxic animals from day 2 (64 +/- 13.5 pmol phosphate released/min/mg protein) to day 14 (105 +/- 9). The increase was sustained to day 27. There was no effect on PKC activity due to hyperoxia alone (ANOVA). This increase in PKC activity was accompanied by an increase in the mass of the delta, epsilon and zeta isoforms of PKC in normoxic pups. The gamma isoform of PKC was undetectable in all samples whereas the alpha and beta isoforms were detectable but showed no changes with developmental age. PP2A specific activity increased significantly (P < .05) from 13.3 +/- 0.5 nmol phosphate released/min/mg protein on day 2 to 17.7 +/- 0.9 on day 7 in normoxic pups, then returned to day 2 level at advanced developmental age. Hyperoxia exposure prevented the increase in enzyme activity observed on day 7 in normoxic animals. These data suggest that protein phosphorylation may be one mechanism by which alveolarization is regulated in developing lungs.
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PMID:Effect of developmental age and hyperoxia exposure on kinase and phosphatase activities in newborn rat lungs. 963 55

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.
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PMID:Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase. 1098 83

N-Methyl D-aspartate (NMDA) receptor activation of extracellular-signal regulated kinase (ERK) was examined in primary cortical cultures. Tetrodotoxin, NMDA receptor antagonists, or reduced extracellular calcium (0.1 mm) greatly decreased basal levels of phospho-ERK2, indicating that activity-dependent activation of NMDA receptors maintained a high level of basal ERK2 activation. This activity-dependent activation of phospho-ERK2 was blocked by pertussis toxin and inhibition of calcium/calmodulin-dependent kinase II and phosphatidylinositol 3-kinase but not by inhibition of protein kinase C or cAMP-dependent protein kinase. Addition of a calcium ionophore or 100 microm NMDA decreased phospho-ERK2 in the presence of 1 mm extracellular calcium but enhanced phospho-ERK2 in 0.1 mm extracellular calcium. The reduction in basal phospho-ERK2 by 100 microm NMDA was also reflected as a decrease in phospho-cAMP response element-binding protein. Inhibition of tyrosine phosphatases and serine/threonine phosphatases protein phosphatase 1 (PP1), PP2A, and PP2B did not prevent the inhibitory effect of NMDA. In the presence of tetrodotoxin, NMDA produced a bell-shaped dose-response curve with stimulation of phospho-ERK2 at 10, 25, and 50 microm NMDA and reduced stimulation at 100 microm NMDA. NMDA (50 microm) stimulation of phospho-ERK2 was completely blocked by pertussis toxin and inhibitors of phosphatidylinositol 3-kinase and was partially blocked by a calcium/calmodulin-dependent kinase II inhibitor. These results suggests that NMDA receptors can bidirectionally control ERK signaling.
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PMID:N-methyl D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures. 1106 37

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.
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PMID:Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. 1129 21

Reversal of long term potentiation (LTP) may function to increase the flexibility and storage capacity of neuronal circuits; however, the underlying mechanisms remain incompletely understood. We show that depotentiation induced by low frequency stimulation (LFS) (2 Hz, 10 min, 1200 pulses) was input-specific and dependent on N-methyl-d-aspartate (NMDA) receptor activation. The ability of LFS to reverse LTP was mimicked by a brief application of NMDA. This NMDA-induced depotentiation was blocked by adenosine A(1) receptor antagonist. However, the reversal of LTP by LFS was unaffected by metabotropic glutamate receptor antagonism. This LFS-induced depotentiation was specifically prevented by protein phosphatase (PP)1 inhibitors, okadaic acid, and calyculin A but not by the PP2A or PP2B inhibitors. Furthermore, by using phosphorylation site-specific antibodies, we found that LFS-induced depotentiation is associated with a persistent dephosphorylation of the GluR1 subunit of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor at serine 831, a protein kinase C and calcium/calmodulin-dependent protein kinase II (CaMKII) substrate, but not at serine 845, a substrate of cAMP-dependent protein kinase. This effect was mimicked by bath-applied adenosine or NMDA and was specifically prevented by okadaic acid. Also, the increased phosphorylation of CaMKII at threonine 286 and the decreased PP activity seen with LTP were overcome by LFS, adenosine, or NMDA application. These results suggest that LFS erases LTP through an NMDA receptor-mediated activation of PP1 to dephosphorylate amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and CaMKII in the CA1 region of the hippocampus.
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PMID:Characterization of the mechanism underlying the reversal of long term potentiation by low frequency stimulation at hippocampal CA1 synapses. 1167 81

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.
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PMID:Protein kinase A and two phosphatases are components of the inositol 1,4,5-trisphosphate receptor macromolecular signaling complex. 1216 31

Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix-loop-helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-AB delta C by immunoblotting with specific antibodies. These results suggest that (ia) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (ii) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and long-term regulation of glucose metabolism and fat synthesis.
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PMID:Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver. 1272 58


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