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)

gamma-PAK, originally designated PAK I and subsequently identified as a member of the p21-activated protein kinase family, has been shown to have cytostatic properties and to be involved in maintaining cells in a nondividing state [Rooney, R. D., et al., (1996) J. Biol. Chem. 271, 21498-21504]. The determinants for phosphorylation of substrates by gamma-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by gamma-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for gamma-PAK has been shown to contain two basic amino acids in the -2 and -3 positions, as represented by (K/R)RXS, in which the -2 position is an arginine, the -3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the -1 or -4 position improves the rate of phosphorylation by increasing the Vmax and decreasing the Km. An acidic amino acid in the -1 position increases the rate (2.5-fold), as does an acidic residue in the -4 position, although to a lower extent (1.6-fold). Proline in the -1 or +1 position has a deleterious effect and inhibits phosphorylation by gamma-PAK. The substrate requirements of protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue such as cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) have been compared with gamma-PAK using the same peptides. An acidic residue in the -1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used to identify gamma-PAK.
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PMID:Determinants for substrate phosphorylation by p21-activated protein kinase (gamma-PAK). 940 39

The neuropeptide oxytocin can depolarize parasympathetic preganglionic neurons in the dorsal motor nucleus of the vagus nerve of the rat by generating a sustained inward current, which is sodium-dependent and tetrodotoxin-insensitive. The second messenger activated by oxytocin receptor binding is, however, not yet known. In the present study, we attempted to characterize it by using the whole-cell recording technique and brainstem slices. When loaded with GTP-gamma-S, a non-hydrolysable analogue of GTP, vagal neurons generated a persistent inward current in the absence of agonist and the oxytocin effect was suppressed, suggesting that the peptide-evoked current was mediated by G-protein activation. Loading vagal neurons with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA) suppressed a calcium-dependent, slowly decaying potassium aftercurrent but did not affect the oxytocin response, suggesting that the latter was not mediated by an agonist-induced increase in the intracellular calcium concentration. Protein kinase C (PKC) activation was probably not involved, since the peptide-evoked current was not modified by loading neurons with the PKC inhibitor H7. Thus, the oxytocin-evoked current in vagal neurons was probably not mediated by phospholipase C-beta (PLC-beta) activation. Loading neurons with 8-Br-cAMP or with an adenylyl cyclase activator (forskolin) reduced the oxytocin-evoked current by about half. SQ 22536, an adenylyl cyclase inhibitor, reduced this current by a similar amount. However, the peptide-evoked current was unaffected by Rp-cAMPS and Sp-cAMPS, an inhibitor and an activator, respectively, of cAMP-dependent protein kinase (PKA). We suggest that oxytocin activates two distinct signalling pathways in vagal neurons: one which is cAMP-dependent, but PKA-independent, and one, unidentified, which is PLC-beta-and cAMP-independent. Each pathway accounts for about half of the peptide effect and both appear to involve G-protein activation.
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PMID:The oxytocin-induced inward current in vagal neurons of the rat is mediated by G protein activation but not by an increase in the intracellular calcium concentration. 951 66

We have reported recently a high density of transient A-type K+ channels located in the distal dendrites of CA1 hippocampal pyramidal neurons and shown that these channels shape EPSPs, limit the back-propagation of action potentials, and prevent dendritic action potential initiation (). Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. We investigated the effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette resulted in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also resulted in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation affected the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, we found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites.
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PMID:Downregulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons by activation of PKA and PKC. 957 Jul 83

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

Myosin binding protein C (MyBP-C) is a major myofibril-associated protein in cardiac muscle which is subject to reversible phosphorylation. Cardiac MyBP-C is a substrate in vivo and in vitro for cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase (PKC). Chicken cardiac MyBP-C was phosphorylated by PKA to 3.0 mol phosphate/mol and by PKC to 2.0 mol phosphate/mol. Tryptic phosphopeptides from MyBP-C were purified by successive iron iminodiacetate column chromatography and reversed-phase high-performance liquid chromatography. Three phosphopeptides purified from PKA-phosphorylated MyBP-C contained phosphoserine [T1, (RTS[P]LAGGGR) and T2, (KRDS[P]FLR)] or phosphothreonine (CT3, MT[P]SAFL). PKC phosphorylated two of the same sites (T1 and T2) as PKA and an additional site [T2a (TGTTYKPPS[P]YK)]. PKA phosphorylation sites corresponding to peptides T1, T2, and T3 were identified in the N-terminus of the cDNA deduced amino acid sequence (S265, S300, and T274, respectively). The PKC-specific site in peptide T2a was at position S1169. cDNA clones encoding rat cardiac MyBP-C were isolated, and the segment corresponding to PKA and major PKC phosphorylation sites was sequenced. Chicken cardiac MyBP-C has a threonine at position 274 (CT3), whereas rat cardiac MyBP-C has a serine at the corresponding position. Only chicken cardiac MyBP-C had a phosphorylatable residue at the position corresponding to S1169. All of the cardiac MyBP-C phosphorylation sites are absent in known sequences of skeletal muscle MyBP-C isoforms.
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PMID:Cardiac myosin-binding protein C (MyBP-C): identification of protein kinase A and protein kinase C phosphorylation sites. 978 45

Mammalian spermiogenesis is characterized by replacement of somatic histones by a set of basic nuclear transition proteins thought to be actively involved in the chromatin remodeling process. The two major transition proteins of the elongating spermatids, namely TP1 and TP2, were expressed and purified using a bacterial expression system. Both topoisomerase and ligase-mediated supercoiling assays demonstrated that TP1, as well as TP2, did not produce detectable changes in the twist and/or writhe of DNA molecules upon binding. Ligase-mediated circularization assay further demonstrated that neither of the transition proteins under study produced bends in linear DNA but that they both have the capacity to stimulate oligomerization of linear DNA fragments. We further established that the transition proteins are in vitro substrates for the Ca+2-phospholipid-dependent protein kinase (PKC) as well as the cAMP-dependent protein kinase (PKA). PKC phosphorylation was found to strongly weaken the DNA-condensing ability of TP2. These results suggest that the major transition proteins represent architectural factors able to stabilize DNA in a nonsupercoiled state, thereby promoting DNA condensation.
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PMID:Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2. 983 53

Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAA receptor subunits. Yet, the effect of phosphorylation on GABAA receptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAA receptors of adult CNS neurons are poorly understood. We used whole-cell patch-clamp recordings of GABAA receptor-mediated miniature IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices to determine the effects of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) activation on the function of synaptic GABAA receptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs were differentially affected by PKA and PKC. In pyramidal cells, PKA reduced mIPSC amplitudes and enhanced the fraction of events decaying with a double exponential, whereas PKC was without effect. In contrast, in GCs PKA was ineffective, but PKC increased the peak amplitude of mIPSCs and also favored double exponential decays. Intracellular perfusion of the phosphatase inhibitor microcystin revealed that synaptic GABAA receptors of pyramidal cells, but not those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This differential, brain region-specific phosphorylation of GABAA receptors may produce a wide dynamic range of inhibitory synaptic strength in these two regions of the hippocampal formation.
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PMID:Modulation of synaptic GABAA receptor function by PKA and PKC in adult hippocampal neurons. 988 May 88

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K+ channel have been described in vascular smooth muscle: Voltage-activated K+ channels (Kv) are encoded by the Kv gene family, Ca(2+)-activated K+ channels (BKCa) are encoded by the slo gene, inward rectifiers (KIR) by Kir2.0, and ATP-sensitive K+ channels (KATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4-1.6, Kv2.1, Kv9.3, Kv beta 1-beta 4, slo alpha and beta, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K+ channels are modulated by physiological vasodilators, which increase K+ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate KATP channels. These include adenosine, calcitonin gene-related peptide, and beta-adrenoceptor agonists. beta-adrenoceptors can also activate BKCa and Kv channels. Several vasoconstrictors that activate protein kinase C inhibit KATP channels, and inhibition of BKCa and Kv channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BKCa channels, and possibly KATP channels. Hypoxia leads to activation of KATP channels, and activation of BKCa channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of Kv channels. Vasodilation to increased external K+ involves KIR channels. Endothelium-derived hyperpolarizing factor activates K+ channels that are not yet clearly defined. Such K+ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.
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PMID:K+ channel modulation in arterial smooth muscle. 988 77

The release of histamine from mast cells and basophils during allergic reactions can regulate functions of T cells and may influence the nature of the immune response to a given antigen. The effects of histamine on T lymphocytes are associated with its binding to H2-receptors linked with adenylate cyclase, elevation of cAMP levels and activation of cAMP-dependent protein kinase (PKA). In this report we explore the role of PKA in histamine-mediated effects on IL-2 mRNA expression and IL-2 protein secretion. Fresh isolated mouse splenocytes (C57Bl/6) were pretreated with histamine (10(-4) M) for 1 h in the presence or absence of Rp-cAMPS (50 microM), an inhibitor of PKA regulatory subunit. The cells were then washed thoroughly and activated with plate-bound anti-CD3 (5 microg/ml), or PHA (1:100) or PMA + ionomycin (10 ng/ml, 1 microg/ml) for 6 h. Pretreatment with histamine inhibited IL-2 mRNA expression and secretion in cells activated with anti-CD3 or PMA, but not in cells activated with PMA + ionomycin. Rp-cAMPS prevented histamine-mediated suppression and did not itself affect IL-2 production. These results provide evidence that histamine affected IL-2 production when the cells were activated via the T cell receptor (TCR)/CD3 complex, but did not interfere with signal transduction pathways downstream of PKC leading to production of IL-2. These effects of histamine on IL-2 secretion and mRNA expression were mediated via PKA.
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PMID:Involvement of protein kinase A in histamine-mediated inhibition of IL-2 mRNA expression in mouse splenocytes. 1010 90

The mechanisms for regulating platelet HDL3 binding sites were investigated. HDL3 binding was rapid (T(1/2) association=4 minutes) and completely reversible (T(1/2) dissociation=14.5 minutes) at 4 degrees C, 22 degrees C, and 37 degrees C, and kinetic analysis yielded forward and reverse constants of 7.3x10(-4) x s(-1) and 7.13x10(3) x s(-1) x M(-1), respectively. Nevertheless, neither inhibitors of binding sites recycling or of pinocytosis, such as ammonium chloride, chloroquine, monensin, colchicine, and sodium azide, modified the binding characteristics. Moreover, when platelets were loaded with cholesterol, binding sites were not regulated (up or down). However, when exposed to high concentrations of HDL3 (1.5 g/L), apoE-free HDL (1.5 g/L), HDL2 (0.5 g/L), apoE-rich HDL (0.5 g/L), and VLDL (0.3 g/L) there was rapid downregulation of the number of binding sites in isolated permeabilized platelets, as shown by the reduction of Bmax to 66%, 58%, 45%, 53%, and 51%, respectively. Downregulation was rapid, reversible, and dose and time dependent. In contrast, LDL (up to 2.0 g/L), IDL (up to 0.1 g/L), and chylomicrons (up to 0.5 g/L) had no effect. Protein kinase C inhibitors (150 nmol/L staurosporine, 100 micromol/L H-7, and 10 nmol/L bisindolylmaleimide) inhibited downregulation up to 62% (as average value). The role of the PKC activation in regulating the activity of HDL3 binding sites also was analyzed by determining the cytosol-to-membrane translocation of enzymatic activity. Downregulation mediated by HDL3 rapidly translocated PKC activity (21% +/- 11 of total PKC activity was membrane-associated in control platelets vs. 55+/-8% in downregulated platelets, mean+/-SEM, n=3). However, agents that block sequestration (0.30 g/L, concanavalin A), and other protein kinase inhibitors, such a cAMP-dependent protein kinase inhibitors (1 micromol/L, PKI), and beta2-adrenergic receptor kinase inhibitors (100 nmol/L, heparin) had no effect. The results show that neither endocytotic response nor cholesterol-dependent mechanisms participate in the modulation of platelet HDL3 binding sites. However, a new regulatory mechanism that involves PKC-dependent downregulation of the number of binding sites may be an important pathway to regulate the thrombogenicity of lipoproteins and their effects on platelet reactivity.
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PMID:Mechanisms for regulating platelet high density lipoprotein type3 binding sites: evidence that binding sites are downregulated by a protein kinase C-dependent mechanism. 1021 79


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