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)

Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation via the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and that this induces significant Ca(2+)/calmodulin (CaM)-independent eEF-2K activity [Redpath and Proud (1993) Biochem. J. 293, 31-34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca(2+)/CaM-independent eEF-2K activity to a similar extent, providing a mechanistic link between elevated cAMP and the inhibition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Biochem. J. 336, 525-529]. Here we describe the expression of glutathione S-transferase (GST)-eEF-2K fusion protein and the identification of two serine residues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digestion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. (32)P Radioactivity release from these peptides indicated that the sites of phosphorylation were Ser-365 and Ser-499, both of which lie C-terminal to the catalytic domain. Mutation of these sites to non-phosphorylatable residues indicated that both sites need to be phosphorylated to induce Ca(2+)/CaM-independent eEF-2K activity in vitro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenylthio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca(2+)/CaM-independent eEF-2K activity in cells. Co-expression of wild-type eEF-2K with luciferase resulted in a 2-3-fold reduction in luciferase expression. Expression of eEF-2K S499D resulted in a 10-fold reduction in luciferase expression despite the fact that this mutant was expressed at very low levels. This indicates that eEF-2K S499D is constitutively active when expressed in cells, thus leading to the suppression of its own expression. Our data demonstrate an important role for the phosphorylation of Ser-499 in the activation of eEF-2K by PKA and the inhibition of protein synthesis.
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PMID:Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity. 1117 Oct 59

Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 microM peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent Km of PKA for cAMP increased from approximately 10(-8) to 10(-6) M. The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.
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PMID:Protein nitration. 1119 83

In the Xenopus oocyte system mitogen treatment triggers the G(2)/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21(Cip1) had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.
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PMID:The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes. 1140 85

Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathione S-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified (32)P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.
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PMID:Specific phosphorylation of exogenous protein and peptide substrates by the human cytomegalovirus UL97 protein kinase. Importance of the P+5 position. 1204 83

Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) were used to study the phosphorylation and desensitization of the receptor induced by prostaglandin E1 (PGE1) or forskolin. These agents are known to increase the intracellular level of cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA). Pretreatment of cells with either agent significantly attenuated Ca2+ release induced by the agonist [1S-[1alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-indophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP). These agents also induced concentration-dependent phosphorylation of TPalpha as demonstrated by increased 32P-labeling of the receptor from cells prelabeled with 32P(i). To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for purified PKA. It was found that only the C-terminal tail fusion protein could serve as a substrate for PKA. To identify the specific serine/threonine residues in the C-terminal tail that are involved in phosphorylation, various alanine mutants of these residues were checked for their ability to serve as substrates. Ser-331 was found to be involved in PKA-mediated phosphorylation. The S331A mutant receptor overexpressed in HEK 293 cells was not phosphorylated significantly following stimulation by PGE1 or forskolin, indicating that Ser-331 was the major site of phosphorylation. Furthermore, cells overexpressing the mutant receptor became responsive to I-BOP-induced Ca2+ mobilization even after pretreatment with PGE1 or forskolin. These results indicate that Ser-331 is the primary site responsible for the phosphorylation and desensitization of the human TPalpha induced by agents that activate PKA.
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PMID:Agents that elevate cyclic AMP induce receptor phosphorylation primarily at serine 331 in HEK 293 cells overexpressing human thromboxane receptor alpha. 1214 88

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the mu-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida.
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PMID:Fibrous sheath of mammalian spermatozoa. 1267 26

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.
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PMID:Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 1487 7

A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity. The Km values for the two substrates of reaction, fructose 6-phosphate and glutamine, were determined to be 0.8 mm for fructose 6-phosphate and 1.2 mm for glutamine, which are within the ranges determined for GFAT1. The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1, whereas the serine at 235 in GFAT1 was not present in GFAT2. Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of cAMP-dependent protein kinase (PKA) inhibits its activity. Like GFAT1, GFAT2 was phosphorylated by PKA, but GFAT2 activity increased approximately 2.2-fold by this modification. When serine 202 of GFAT2 was mutated to an alanine, the enzyme not only became resistant to phosphorylation, but also the increase in activity in response to PKA also was blocked. These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA. GFAT2 was modestly inhibited (15%) by UDP-GlcNAc but not through detectable O-glycosylation. GFAT2 is, therefore, an isoenzyme of GFAT1, but its regulation by cAMP is the opposite, allowing differential regulation of the hexosamine pathway in specialized tissues.
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PMID:Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity. 1513 36

We report here that the human glutathione S-transferase P1 (GSTP1) protein, involved in phase II metabolism of many carcinogens and anticancer agents and in the regulation of c-Jun NH(2)-terminal kinase-mediated cell signaling, undergoes phosphorylation by the Ser/Thr protein kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), resulting in a significant enhancement of its metabolic activity. GSTP1 phosphorylation by PKA was glutathione (GSH)-dependent, whereas phosphorylation by PKC did not require but was significantly enhanced by GSH. In the presence of GSH, the stoichiometry of phosphorylation was 0.4 +/- 0.03 and 0.53 +/- 0.02 mol incorporated phosphate per mole of dimeric GSTP1 protein. The GSTP1 protein was phosphorylated, in the presence of GSH, by eight different PKC isoforms (alpha, betaIota, betaIotaIota, delta, epsilon, gamma, eta, and zeta), belonging to the three major PKC subclasses, albeit with various efficiencies. The catalytic efficiency, k(cat)/K(m), of the phosphorylated GSTP1 was more than double that of the unphosphorylated protein. In MGR3 human glioblastoma cells, PKA and PKC activation resulted in a significant increase in the level of phosphorylation of the GSTP1 protein and was accompanied by a 2.1- and 2.7-fold increase, respectively, in specific GSTP1 activity in the cells. Peptide phosphorylation analyses and both phosphorylation and enzyme kinetic studies with GSTP1 proteins mutated at candidate amino acid residues established Ser-42 and Ser-184 as putative phospho-acceptor residues for both kinases in the GSTP1 protein. Together, these findings show PKA- and PKC-dependent phosphorylation as a significant post-translational mechanism of regulation of GSTP1 function. The GSH-dependence of the phosphorylation suggests that under high intracellular GSH conditions, such as is present in most drug-resistant tumors, the GSTP1 protein will exist in a hyper-phosphorylated and enzymatically more active state. In normal cells, the functional activation of the GSTP1 protein by PKA- and PKC-dependent phosphorylation could represent a potentially important mechanism of cellular protection, whereas in tumors, increased phase II metabolism of anticancer drugs by the more active phosphorylated GSTP1 protein could contribute to the drug resistance and therapeutic failure frequently associated with increased activities of these Ser/Thr kinases.
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PMID:The human glutathione S-transferase P1 protein is phosphorylated and its metabolic function enhanced by the Ser/Thr protein kinases, cAMP-dependent protein kinase and protein kinase C, in glioblastoma cells. 1560 83

Claudins are integral membrane proteins essential in the formation and function of tight junctions (TJs). Disruption of TJs, which have essential roles in cell permeability and polarity, is thought to contribute to epithelial tumorigenesis. Claudin-3 and -4 are frequently overexpressed in ovarian cancer, but the molecular pathways involved in the regulation of these proteins are unclear. Interestingly, several studies have demonstrated a role for phosphorylation in the regulation of TJ complexes, although evidence for claudin phosphorylation is scarce. Here, we showed that claudin-3 and -4 can be phosphorylated in ovarian cancer cells. In vitro phosphorylation assays using glutathione S-transferase fusion constructs demonstrated that the C terminus of claudin-3 is an excellent substrate for cAMP-dependent protein kinase (PKA). Using site-directed mutagenesis, we identified a PKA phosphorylation site at amino acid 192 in the C terminus of claudin-3. Overexpression of the protein containing a T192D mutation, mimicking the phosphorylated state, resulted in a decrease in TJ strength in ovarian cancer cell line OVCA433. Our results suggest that claudin-3 phosphorylation by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. In addition, our findings may have general implications for the regulation of TJs in normal epithelial cells.
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PMID:Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells. 1590 76


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