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 PKD1-encoded protein, "polycystin-1", has a large N-terminal extracellular portion, multiple transmembrane domains, and a short intracellular C-terminal tail with four tyrosine residues and two putative sites for serine phosphorylation. Its function in kidney development and autosomal dominant polycystic kidney disease (ADPKD) is still unknown. We have subcloned the cDNA encoding the polycystin-1 C-terminal domain (PKD1-CTD) into a prokaryotic expression vector, and site-directed mutagenesis was performed to target the four tyrosine residues and four serine residues in two putative phosphorylation sites. In vitro phosphorylation assays were conducted on both wild type and mutant PKD1-CTD fusion proteins. It was found that the wild type PKD1-CTD and all mutant fusion proteins, except S4251G/S4252G, could be phosphorylated by lysates from cultured normal human renal collecting tubule (NHCT) cells, as well as by commercially purified cAMP-dependent protein kinase (PKA). The phosphorylation of the PKD1-CTD fusion protein by NHCT lysates was greatly enhanced by cAMP and its analog 8-Br-cAMP, and inhibited by the specific PKA inhibitors PKI(6-22) and H-89. Activators and inhibitors of protein kinase C (PKC) had no effects on the phosphorylation of the PKD1-CTD fusion protein. Using commercially purified pp60(c-src) (c-src) it was also shown that the PKD1-CTD fusion protein could be phosphorylated by c-src in vitro, and that this phosphorylation could be abolished by a mutation Y4237F. By comparing the amino acid sequence at 4249-4253 (RRSSR) with the consensus sequence for PKA phosphorylation (RRXSX), we suggest that the serine residue at 4252 is the target of phosphorylation by a cAMP-dependent protein kinase in NHCT cell lysates. In addition, we suggest that Y4237 might be phosphorylated by c-src in living cells.
 ...
PMID:Identification of phosphorylation sites in the PKD1-encoded protein C-terminal domain. 1036 14

Ethanol induces translocation of the catalytic subunit (Calpha) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108-15 cells. Ethanol also induces translocation of the RIIbeta regulatory subunit of PKA to the nucleus; RI and Cbeta are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Calpha with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Calpha by the PKA inhibitor PKI. Pre-incubation of Calpha with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Calpha is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R(p))-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Calpha and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.
 ...
PMID:Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences. 1048 Sep 11

Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activity of class C L-type Ca(2+) channels which are clustered at postsynaptic sites and are important regulators of neuronal functions. We investigated a possible mechanism that could ensure rapid and efficient phosphorylation of these channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PKA and target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation contain an endogenous kinase that phosphorylates kemptide, a classic PKA substrate peptide, and also the main phosphorylation site for PKA in the pore-forming alpha(1) subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PKA inhibitory peptide PKI(5-24) and stimulated by cAMP. Physical association of the catalytic C subunit of PKA with the immunoisolated class C channel complex was confirmed by immunoblotting. A direct protein overlay binding assay performed with (32)P-labeled RIIbeta revealed a prominent AKAP with an M(r) of 280,000 in class C channel complexes. The protein was identified by immunoblotting as the microtubule-associated protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules. In vitro experiments show that MAP2B can directly bind to the alpha(1) subunit of the class C channel. Our findings indicate that PKA is an integral part of neuronal class C L-type Ca(2+) channels and suggest that the AKAP MAP2B may mediate this interaction. Neither PKA nor MAP2B were detected in immunoprecipitates of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamate receptors or class B N-type Ca(2+) channels. Accordingly, MAP2B docked at class C Ca(2+) channels may be important for recruiting PKA to postsynaptic sites.
 ...
PMID:The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons. 1051 22

The aim of this study was to determine which PGE(2) receptors and signal transduction pathways are responsible for the stimulation of oxygen uptake in liver. Hepatic parenchymal cells isolated from female Sprague-Dawley rats were incubated either with PGE(2), 17-phenyl-omega-trinor PGE(2) (an EP(1)-specific agonist), or 11-deoxy PGE(1) (an EP(2)/EP(4)-specific agonist), and oxygen consumption was measured. Both PGE(2) and 11-deoxy PGE(1) stimulated oxygen consumption. However, an EP(1) agonist was without effect. Although PGE(2) elevated intracellular calcium, this occurred at concentrations approximately 500-fold lower than that required to stimulate oxygen uptake. PGE(2)-stimulated increases in cAMP formation correlated well with the increase in oxygen consumption. Dibutyryl cAMP also increased oxygen consumption. Furthermore, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, a cell-permeable inhibitor of protein kinase A (PKA), reduced the stimulation of oxygen uptake by PGE(2). Incubation of isolated parenchymal cell mitochondria with the purified catalytic subunit of PKA and ATP increased both state 3 rates of oxygen uptake and the respiratory control ratio by approximately 50%. Activation of these events was prevented by incubation with the PKA inhibitory peptide, PKI. These findings are consistent with the hypothesis that PGE(2) stimulates oxygen consumption via an EP(2) and/or EP(4) subclass of receptors through the actions of cAMP on a cAMP-dependent protein kinase.
 ...
PMID:PGE(2) stimulates O(2) uptake in hepatic parenchymal cells: involvement of the cAMP-dependent protein kinase. 1056 11

Overexpression of cAMP-dependent protein kinase (PKA) type I isozyme is associated with cell proliferation and neoplastic transformation. The presence of PKA on the external surface of LS-174T human colon carcinoma cells has been shown. Here, we show that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular PKA (ECPKA) is present in active, free catalytic subunit (C subunit) form, and its activity is specifically inhibited by PKA inhibitory protein, PKI. Overexpression of the Calpha or RIalpha subunit gene of PKA in an expression vector, which up-regulates intracellular PKA type I, markedly up-regulates ECPKA expression. In contrast, overexpression of the RIIbeta subunit, which eliminates PKA type I, up-regulates PKA type II, and reverts the transformed phenotype, down-regulates ECPKA. A mutation in the Calpha gene that prevents myristylation allows the intracellular PKA up-regulation but blocks the ECPKA increase, suggesting that the NH(2)-terminal myristyl group of Calpha is required for the ECPKA expression. In serum of cancer patients, the ECPKA expression is up-regulated 10-fold as compared with normal serum. These results indicate that the ECPKA expression is an ordered cellular response of a living cell to actively exclude excess intracellular PKA molecules from the cell. This phenomenon is up-regulated in tumor cells and has an inverse relationship with the hormone dependency of breast cancer. Thus, the extracellular PKA may serve as a potential diagnostic and prognostic marker for cancer.
 ...
PMID:Extracellular protein kinase A as a cancer biomarker: its expression by tumor cells and reversal by a myristate-lacking Calpha and RIIbeta subunit overexpression. 1063 66

(1) In homogenates of vitellogenic follicles from Hyalophora cecropia, cyclic nucleotides promoted the transfer of label from [gamma32P]-ATP to at least four polypeptides. PKI (6-20) amide, an inhibitor of PKA (cAMP-dependent protein kinase), prevented all four reactions. Quantitative tests using kemptide as a substrate indicated that 80% of the total follicular PKA activity was localized in the follicle cells; labeling at 45, 32, and 27 kDa was particle-associated and also restricted to the follicle cells, while a 58 kDa substrate was labeled only in homogenates of the oocyte. (2) When intact follicles were incubated in [32P]-phosphate and okadaic acid, a protein phosphatase inhibitor, the 32 kDa substrate again exhibited cAMP-dependent labeling. There was thus a physiological relationship between PKA activation and 32 kDa protein phosphorylation, while exposure of at least two of the other three substrates to appropriate kinases required homogenization. The latter was illustrated by phosphorylation of the 42 kDa small subunit of vitellogenin, which occurred only when homogenization mixed the proteins of the yolk bodies with cytoplasmic kinases. (3) PKA activation is known to promote the termination of vitellogenesis, even in the absence of detectable labeling of the 32 kDa substrate. The possibility remains that phosphorylation at 32 kDa concerns later aspects of postvellogenic development that were not tested by the assay system used here.
 ...
PMID:Cyclic nucleotide-dependent protein phosphorylation in vitellogenic follicles of Hyalophora cecropia. 1064 68

1. The biophysical and pharmacological characteristics of the hyperpolarization activated non- selective cation current (If) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. 2. The cation current was detected in a large percentage (65 %) of early stage (EDS, differentiated for 7 + 3-4 days) cells at a current density of 11.4 +/- 0.6 pA pF-1 (n = 47). In late stage (LDS, differentiated for 7 + 9-12 days) cells the percentage of cells expressing If decreased (45 %), but If densities (15.5 +/- 0.9 pA pF-1, n = 20) were increased. 3. The muscarinic agonist carbachol (CCh, 1 microM) depressed basal If in EDS cells by 45.7 +/- 6.5 %, n = 5) and was without effect in LDS cardiomyocytes (n = 4). The beta-adrenoceptor agonist isoprenaline (ISO, 1 microM) stimulated If in LDS cells by 33 +/- 5.2 % (n = 6) but not in EDS cells (n = 5). 4. Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PKA, 7 microM) stimulated If in EDS cells by 37.0 +/- 2.9 %, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 microM) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 microM) completely inhibited the stimulation of the L-type Ca2+ current (ICa,L) as well as of If by ISO (1 microM). 5. Extracellular superfusion with phosphodiesterase (PDE) inhibitors - IBMX, a non-selective antagonist, Erythro-9-(2-hydoxy-3-nonyl)adenine (EHNA), a PDE2 antagonist and rolipram, a PDE4 antagonist - resulted in stimulation of ICa,L and If in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated ICa,L, but not If. 6. The present work demonstrates that If is functionally expressed during early cardiomyogenesis. Similar to ICa,L, If is regulated during embryonic development by phosphorylation via PKA. In contrast to ICa,L, If is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.
 ...
PMID:Functional expression and regulation of the hyperpolarization activated non-selective cation current in embryonic stem cell-derived cardiomyocytes. 1069 82

Long-term potentiation (LTP) has several different phases, and there is general agreement that the late phase of LTP requires the activation of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). In contrast, several studies indicate that the early LTP is not affected by interfering with the cAMP pathway. We have further tested the role of the cAMP pathway in early LTP using several types of inhibitors. Bath application of the PKA inhibitor H89 suppressed the early LTP induced by a single tetanus. Similarly, the LTP induced by a pairing protocol was decreased by postsynaptic intracellular perfusion of the peptide PKA inhibitor PKI(6-22) amide. The decrease of LTP produced by these inhibitors was evident immediately after induction. These results indicate that PKA is important in early LTP, that its locus of action is postsynaptic, and that it does not act merely by enhancing the depolarization required for LTP induction. The failure of some other inhibitors of the cAMP pathway to affect the early phase of LTP might be attributable to the saturation of some step in the cAMP pathway during a tetanus. In agreement with this hypothesis we found that application of the AC inhibitor SQ 22536 by itself did not affect the early phase of LTP, but did produce a reduction if the cAMP pathway was already attenuated by the PKA inhibitor H89. Our analysis of the results of genetic modifications of the cAMP pathway, especially the work on AC knock-outs, indicates that the genetic data are generally consistent with the pharmacological results showing the importance of this pathway in early LTP.
 ...
PMID:Inhibition of the cAMP pathway decreases early long-term potentiation at CA1 hippocampal synapses. 1084 13

The role of cAMP in cell growth and differentiation, gene expression, and neuronal function is mediated by the cAMP-dependent protein kinase (PKA). Differential expression of type I and type II PKA has been correlated with neoplastic transformation and differentiation, respectively. PKA is primarily an intracellular enzyme. However, it has been demonstrated that PKA may be associated with the plasma membrane and is exposed to the extracellular environment. Here we report the first evidence for the presence of a free extracellular kinase activity of PKA in the growth media of cultured prostate and other cancer cells, as well as in plasma samples from prostate cancer patients. This PKA activity is specific due to its phosphorylation of the PKA-specific substrate kemptide and its inhibition by the potent and specific PKA inhibitor PKI, but not by other protein kinase-inhibitory peptides. Intriguingly, this exoprotein kinase activity is cAMP independent, suggesting that only the catalytic subunit is secreted, and therefore the kinase activity is not modulated by the regulatory subunit of PKA. Western blot analysis of the culture supernatant from prostate cancer cells indicates the presence of the catalytic subunit. This increase in extracellular PKA catalytic subunit activity in prostate cancer may have profound effects on the tumorigenesis of prostate cancer and may serve as a novel marker and therapeutic target for the disease.
 ...
PMID:Extracellular catalytic subunit activity of the cAMP-dependent protein kinase in prostate cancer. 1087 81

Two novel members of the human cAMP-dependent protein kinase inhibitor (PKI) gene family, PKIB and PKIG, were cloned. The deduced proteins showed 70% and 90% identity with mouse PKIbeta and PKIgamma respectively. Both the already identified pseudosubstrate site and leucine-rich nuclear export signal motifs were defined from the 11 PKIs of different species. The PKIB and PKIG genes were mapped respectively to chromosome 6q21-22.1, using a radiation hybrid GB4 panel, and to chromosome 20q13.12-13.13, using a Stanford G3 panel. Northern-blot analysis of three PKI isoforms, including the PKIA identified previously, revealed significant differences in their expression patterns. PKIB had two transcripts of 1.9 kb and 1.4 kb. The former transcript was abundant in both placenta and brain and the latter was expressed most abundantly in placenta, highly in brain, heart, liver, pancreas, moderately in kidney, skeletal muscle and colon, and very little in the other eight tissues tested. PKIG was widely expressed as a 1.5-kb transcript with the highest level in heart, hardly detectable in thymus and peripheral blood leucocytes and was moderately expressed in the other tissues, with slightly different levels. However, PKIA was specifically expressed as two transcripts of 3.3 kb and 1.5 kb in heart and skeletal muscle. The distinct expression patterns of the three PKIs suggest that their roles in various tissues are probably different.
 ...
PMID:Cloning and mapping of human PKIB and PKIG, and comparison of tissue expression patterns of three members of the protein kinase inhibitor family, including PKIA. 1088 Mar 37


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>