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Sample records for adenylate kinase isoform

  1. Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

    PubMed Central

    Ruan, Qiaoqiao; Chen, Yan; Gratton, Enrico; Glaser, Michael; Mantulin, William W

    2002-01-01

    Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the

  2. Primary structure of maize chloroplast adenylate kinase.

    PubMed

    Schiltz, E; Burger, S; Grafmüller, R; Deppert, W R; Haehnel, W; Wagner, E

    1994-06-15

    This paper describes the sequence of adenylate kinase (Mg-ATP+AMP<-->Mg-ADP+ADP) from maize chloroplasts. This light-inducible enzyme is important for efficient CO2 fixation in the C4 cycle, by removing and recycling AMP produced in the reversible pyruvate phosphate dikinase reaction. The complete sequence was determined by analyzing peptides from cleavages with trypsin, AspN protease and CNBr and subcleavage of a major CNBr peptide with chymotrypsin. N-terminal Edman degradation and carboxypeptidase digestion established the terminal residues. Electrospray mass spectrometry confirmed the final sequence of 222 residues (M(r) = 24867) including one cysteine and one tryptophan. The sequence shows this enzyme to be a long-variant-type adenylate kinase, the nearest relatives being adenylate kinases from Enterobacteriaceae. Alignment of the sequence with the adenylate kinase from Escherichia coli reveals 44% identical residues. Since the E. coli structure has been published recently at 0.19-nm resolution with the inhibitor adenosine(5')pentaphospho(5')adenosine (Ap5A) [Müller, C. W. & Schulz, G. E. (1992) J. Mol. Biol. 224, 159-177], catalytically essential residues could be compared and were found to be mostly conserved. Surprisingly, in the nucleotide-binding Gly-rich loop Gly-Xaa-Pro-Gly-Xaa-Gly-Lys the middle Gly is replaced by Ala. This is, however, compensated by an Ile-->Val exchange in the nearest spatial neighborhood. A Thr-->Ala exchange explains the unusual tolerance of the enzyme for pyrimidine nucleotides in the acceptor site. PMID:8026505

  3. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  4. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    PubMed Central

    Dzeja, Petras; Terzic, Andre

    2009-01-01

    Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network. PMID:19468337

  5. Structural Studies of Archaealthermophilic Adenylate Kinase

    SciTech Connect

    Konisky, J.

    2002-07-10

    Through this DOE-sponsored program Konisky has studied the evolution and molecular biology of microbes that live in extreme environments. The emphasis of this work has been the determination of the structural features of thermophilic enzymes that allow them to function optimally at near 100 C. The laboratory has focused on a comparative study of adenylate kinase (ADK), an enzyme that functions to interconvert adenine nucleotides. Because of the close phylogenetic relatedness of members of the Methanococci, differences in the structure of their ADKs will be dominated by structural features that reflect contributions to their optimal temperature for activity, rather than differences due to phylogenetic divergence. We have cloned, sequenced and modeled the secondary structure for several methanococcal ADKs. Using molecular modeling threading approaches that are based on the solved structure for the porcine ADK, we have also proposed a general low resolution three dimensional structure for each of the methanococcal enzymes. These analyses have allowed us to propose structural features that confer hyperthermoactivity to those enzymes functioning in the hyperthermophilic members of the Methanococci. Using protein engineering methodologies, we have tested our hypotheses by examining the effects of selective structural changes on thermoactivity. Despite possessing between 68-81% sequence identity, the methanococcal AKs had significantly different stability against thermal denaturation, with melting points ranging from 69-103 C. The construction of several chimerical AKs by linking regions of the MVO and MJA AKs demonstrated the importance of cooperative interactions between amino- and carboxyl-terminal regions in influencing thermostability. Addition of MJA terminal fragments to the MVO AK increased thermal stability approximately 20 C while maintaining 88% of the mesophilic sequence. Further analysis using structural models suggested that hydrophobic interactions are

  6. Protein kinase C sensitizes olfactory adenylate cyclase

    PubMed Central

    1993-01-01

    Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory

  7. The crystal structure of human adenylate kinase 6: An adenylate kinase localized to the cell nucleus.

    PubMed

    Ren, Hui; Wang, Liya; Bennett, Matthew; Liang, Yuhe; Zheng, Xiaofeng; Lu, Fei; Li, Lanfen; Nan, Jie; Luo, Ming; Eriksson, Staffan; Zhang, Chuanmao; Su, Xiao-Dong

    2005-01-11

    Adenylate kinases (AKs) play important roles in nucleotide metabolism in all organisms and in cellular energetics by means of phosphotransfer networks in eukaryotes. The crystal structure of a human AK named AK6 was determined by in-house sulfur single-wavelength anomalous dispersion phasing methods and refined to 2.0-A resolution with a free R factor of 21.8%. Sequence analyses revealed that human AK6 belongs to a distinct subfamily of AKs present in all eukaryotic organisms sequenced so far. Enzymatic assays show that human AK6 has properties similar with other AKs, particularly with AK5. Fluorescence microscopy showed that human AK6 is localized predominantly to the nucleus of HeLa cells. The identification of a nuclear-localized AK sheds light on nucleotide metabolism in the nucleus and the energetic communication between mitochondria and nucleus by means of phosphotransfer networks.

  8. Multiforms of mammalian adenylate kinase and its monoclonal antibody against AK1.

    PubMed

    Kurokawa, Y; Takenaka, H; Sumida, M; Oka, K; Hamada, M; Kuby, S A

    1990-01-01

    An attempt has been made to determine the intracellular distribution of the multiforms of the adenylate kinase (AK) isoenzymes in mammalian tissues, to shed some light on their physiological roles, especially in energy metabolism. The adenylate kinase zymograms obtained from isoelectric focusing yielded two typical isoform patterns: (1) with a pI greater than or equal to 9 and 8.6, specific for bovine skeletal muscle, heart, aorta and brain, and (2) with a pI = 7.9 and 7.1, specific for liver and kidney. Pattern (1) was attributed to the cytosolic isoenzyme (AK1) as demonstrated by immunostaining with anti-AK1. Pattern (2) was attributed to the mitochondrial isoenzyme (AK2). These results were largely confirmed by chromatofocusing experiments. The AK1 isoenzyme was partially purified from the cytosol fraction of bovine aortic smooth muscle and had an apparent Mr of 23.5 kilodaltons. Its kinetic features are discussed from a comparative standpoint. Finally, the human serum AK1 isoform was also detected by Western blotting with a monoclonal antibody directed against crystalline porcine muscle AK1. These results are to form the basis of further studies on the 'aberrant' adenylate kinase isoenzyme from the serum of Duchenne muscular dystrophics.

  9. An aberrant adenylate kinase isoenzyme from the serum of patients with Duchenne muscular dystrophy.

    PubMed

    Hamada, M; Okuda, H; Oka, K; Watanabe, T; Ueda, K; Nojima, M; Kuby, S A; Manship, M; Tyler, F H; Ziter, F A

    1981-08-13

    The sera from patients with human Duchenne (X-linked) progressive muscular dystrophy contain elevated adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) activities, in addition to their characteristically high creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) activities. By agarose gel electrophoresis of human Duchenne dystrophic serum, the presence of an apparently normal human serum adenylate kinase together with a variant species of adenylate kinase was detected. The latter enzyme species appeared, in its mobility, to be similar to that of the normal human liver-type adenylate kinase. The presence of this aberrant liver-type adenylate kinase could also be demonstrated by characteristic (for the liver type) inhibition patterns with P1,P5-di-(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate. On the other hand, by inhibition titrations with an anti-muscle-type adenylate kinase, hemolysates from the erythrocytes of several Duchenne and Becker's dystrophics were found to contain approx. 96% muscle-type adenylate kinase and their serum approx. 97% muscle-type adenylate kinase. These same patients contained approx. 89% M-M type creatine kinase in their serum (by inhibition against anti-human muscle-type creatine kinase) indicative of the presence also of M-B plus B-B type active isoenzymes. All of these data can best be explained by the presence of a variant or mutant adenylate kinase isoenzyme in the dystrophic serum. This isoenzyme appears to resemble the liver type in its inhibition patterns with P1,P5-di(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate, and in its heat stability (compare also the agarose gel electrophoresis pattern); but structurally, it is a muscle type, or derived from a muscle type, as shown immunologically by inhibition reactions with anti-muscle-type adenylate kinase. Whether this is a fetal-type isoenzyme of adenylate kinase will require further

  10. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

    PubMed Central

    Bertrand, L; Vertommen, D; Depiereux, E; Hue, L; Rider, M H; Feytmans, E

    1997-01-01

    Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. PMID:9032445

  11. Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis.

    PubMed

    Tükenmez, Hasan; Magnussen, Helge Magnus; Kovermann, Michael; Byström, Anders; Wolf-Watz, Magnus

    2016-01-01

    Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies. PMID:27642758

  12. Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis

    PubMed Central

    Tükenmez, Hasan; Magnussen, Helge Magnus; Kovermann, Michael; Byström, Anders; Wolf-Watz, Magnus

    2016-01-01

    Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies. PMID:27642758

  13. Mechanism of adenylate kinase. Are the essential lysines essential?

    PubMed

    Tian, G C; Yan, H G; Jiang, R T; Kishi, F; Nakazawa, A; Tsai, M D

    1990-05-01

    Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2161682

  14. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  15. Zinc, a structural component of adenylate kinases from gram-positive bacteria.

    PubMed Central

    Gilles, A M; Glaser, P; Perrier, V; Meier, A; Longin, R; Sebald, M; Maignan, L; Pistotnik, E; Bârzu, O

    1994-01-01

    The recent finding that Bacillus stearothermophilus adenylate kinase contains a zinc atom coordinated to four cysteines prompted us to investigate the metal-binding properties of the enzyme from various bacteria. We conclude that zinc was present only in adenylate kinase from gram-positive species and that this property is correlated with the presence of three or four Cys residues in the sequence Cys-X2-Cys-X16-Cys-X2-Cys/Asp, in which X stands for different amino acid residues. PMID:8288548

  16. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    PubMed

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  17. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

    PubMed Central

    Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

    2015-01-01

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

  18. Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum.

    PubMed

    Cao, Wenlei; Haig-Ladewig, Lisa; Gerton, George L; Moss, Stuart B

    2006-10-01

    Proper sperm function depends on adequate ATP levels. In the mammalian flagellum, ATP is generated in the midpiece by oxidative respiration and in the principal piece by glycolysis. In locations where ATP is rapidly utilized or produced, adenylate kinases (AKs) maintain a constant adenylate energy charge by interconverting stoichiometric amounts of ATP and AMP with two ADP molecules. We previously identified adenylate kinase 1 and 2 (AK1 and AK2) by mass spectrometry as part of a mouse SDS-insoluble flagellar preparation containing the accessory structures (fibrous sheath, outer dense fibers, and mitochondrial sheath). A germ cell-specific cDNA encoding AK1 was characterized and found to contain a truncated 3' UTR and a different 5' UTR compared to the somatic Ak1 mRNA; however, it encoded an identical protein. Ak1 mRNA was upregulated during late spermiogenesis, a time when the flagellum is being assembled. AK1 was first seen in condensing spermatids and was associated with the outer microtubular doublets and outer dense fibers of sperm. This localization would allow the interconversion of ATP and ADP between the fibrous sheath where ATP is produced by glycolysis and the axonemal dynein ATPases where ATP is consumed. Ak2 mRNA was expressed at relatively low levels throughout spermatogenesis, and the protein was localized to the mitochondrial sheath in the sperm midpiece. AK1 and AK2 in the flagellar accessory structures provide a mechanism to buffer the adenylate energy charge for sperm motility.

  19. Spinach pyruvate kinase isoforms: partial purification and regulatory properties

    SciTech Connect

    Baysdorfer, C.; Bassham, J.A.

    1984-02-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K/sub m/ values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K/sub i/ for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K/sub a/ of 0.05 millimolar, and glutamate is an inhibitor with a K/sub i/ of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, the authors suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.

  20. Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity

    PubMed Central

    Thach, Trung Thanh; Luong, Truc Thanh; Lee, Sangho; Rhee, Dong-Kwon

    2014-01-01

    Streptococcus pneumoniae (pneumococcus) infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs) are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S.pneumoniae (SpAdK) serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A). Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo. PMID:25180151

  1. NMR studies of the AMP-binding site and mechanism of adenylate kinase.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y

  2. A double origin electrophoretic method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II.

    PubMed

    Murch, R S; Gambel, A M; Kearney, J J

    1986-10-01

    A rapid, reliable method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II by agarose gel electrophoresis is presented. This method uses a double origin sample application system. Unreduced sample extracts for adenylate kinase analysis are applied 13.0 cm from the anode. Reduced sample extracts for the remaining proteins of interest are applied 7.0 cm from the anode. The use of applicator foils and an increased voltage gradient result in superior resolution, linearity, and band sharpness of the allozyme patterns. Further, there is no masking of the adenylate kinase 2 band as a result of the use of a reducing agent, and carbonic anhydrase II is resolved without interference from hemoglobin as has been observed with other multisystem methods.

  3. RNA mimicry by the fap7 adenylate kinase in ribosome biogenesis.

    PubMed

    Loc'h, Jérôme; Blaud, Magali; Réty, Stéphane; Lebaron, Simon; Deschamps, Patrick; Bareille, Joseph; Jombart, Julie; Robert-Paganin, Julien; Delbos, Lila; Chardon, Florian; Zhang, Elodie; Charenton, Clément; Tollervey, David; Leulliot, Nicolas

    2014-05-01

    During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53-MDM2 pathway. This work presents the functional and structural characterization of the Fap7-Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.

  4. The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

    PubMed Central

    2012-01-01

    Background It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings. Results We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. Conclusions The data outlined serves to demonstrate the “push” mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable

  5. Human adenylate kinase 2 deficiency causes a profound haematopoietic defect associated with sensorineural deafness

    PubMed Central

    Lagresle-Peyrou, Chantal; Six, Emmanuelle M.; Picard, Capucine; Rieux-Laucat, Frédéric; Michel, Vincent; Ditadi, Andrea; Chappedelaine, Corinne Demerens-de; Morillon, Estelle; Valensi, Françoise; Simon-Stoos, Karen L.; Mullikin, James C.; Noroski, Lenora M.; Besse, Céline; Wulffraat, Nicolas M.; Ferster, Alina; Abecasis, Manuel M.; Calvo, Fabien; Petit, Christine; Candotti, Fabio; Abel, Laurent; Fischer, Alain; Cavazzana-Calvo, Marina

    2008-01-01

    Reticular dysgenesis (RD) is an autosomal recessive form of human Severe Combined Immunodeficiency, characterized by an early differentiation arrest in the myeloid lineage and impaired lymphoid maturation. In addition, affected newborns have bilateral sensorineural deafness. We have identified biallelic mutations in the adenylate kinase 2 (AK2) gene in seven patients affected with RD. These mutations resulted in the absence or a strong decrease in protein expression. We then demonstrated that restoration of AK2 expression in the bone marrow cells of RD patients overcomes the neutrophil differentiation arrest underlining its specific requirement in the development of a restricted set of haematopoietic lineages. Lastly, we established that AK2 is specifically expressed in the stria vascularis region of the inner ear, which provides an explanation to the sensorineural deafness. These results suggest a novel mechanism regulating haematopoetic cell differentiation, and involved in one of the most severe human immunodeficiency syndromes. PMID:19043416

  6. Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

    NASA Astrophysics Data System (ADS)

    Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

    2015-02-01

    Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

  7. Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

    PubMed

    Kim, H J; Nishikawa, S; Tanaka, T; Uesugi, S; Takenaka, H; Hamada, M; Kuby, S A

    1989-01-01

    An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

  8. New crystal structures of adenylate kinase from Streptococcus pneumoniae D39 in two conformations

    PubMed Central

    Thach, Trung Thanh; Lee, Sangho

    2014-01-01

    Adenylate kinases (AdKs; EC 2.7.3.4) play a critical role in intercellular homeostasis by the interconversion of ATP and AMP to two ADP molecules. Crystal structures of adenylate kinase from Streptococcus pneumoniae D39 (SpAdK) have recently been determined using ligand-free and inhibitor-bound crystals belonging to space groups P21 and P1, respectively. Here, new crystal structures of SpAdK in ligand-free and inhibitor-bound states determined at 1.96 and 1.65 Å resolution, respectively, are reported. The new ligand-free crystal belonged to space group C2, with unit-cell parameters a = 73.5, b = 54.3, c = 62.7 Å, β = 118.8°. The new ligand-free structure revealed an open conformation that differed from the previously determined conformation, with an r.m.s.d on Cα atoms of 1.4 Å. The new crystal of the complex with the two-substrate-mimicking inhibitor P 1,P 5-bis(adenosine-5′-)pentaphosphate (Ap5A) belonged to space group P1, with unit-cell parameters a = 53.9, b = 62.3, c = 63.0 Å, α = 101.9, β = 112.6, γ = 89.9°. Despite belonging to the same space group as the previously reported crystal, the new Ap5A-bound crystal contains four molecules in the asymmetric unit, compared with two in the previous crystal, and shows slightly different lattice contacts. These results demonstrate that SpAdK can crystallize promiscuously in different forms and that the open structure is flexible in conformation. PMID:25372811

  9. Three-dimensional structure of the complex between the mitochondrial matrix adenylate kinase and its substrate AMP

    SciTech Connect

    Schulz, G.E.; Diederichs, K. )

    1990-09-04

    Crystals of adenylate kinase from beef heart mitochondrial matrix (EC 2.7.4.10) complexed with its substrate AMP were analyzed by X-ray diffraction. The crystal structure was solved by multiple isomorphous replacement and solvent flattening at a resolution of 3.0 {angstrom}. There are two enzyme-substrate molecules in the asymmetric unit. The resolution was extended to 1.9 {angstrom} by model building and refinement using simulated annealing. The current R-factor is 28.4%. The model is given as a backbone tracing for residues 5-218. The enzyme can be subdivided into three domains, the relative arrangements of which differ slightly but significantly between the two crystallographically independent molecules. When compared with other adenylate kinase structures, the chain fold is similar but the observed domain arrangement differs grossly, suggesting that large parts of the enzyme move during catalysis. The observed binding site of AMP is described. Its location in conjunction with data from homologous proteins clarifies the nucleotide-binding sites of the adenylate kinases. Previous assignments of these sites derived from X-ray crystallographic and nuclear magnetic resonance analyses are discussed.

  10. Competition of Spontaneous Protein Folding and Mitochondrial Import Causes Dual Subcellular Location of Major Adenylate Kinase

    PubMed Central

    Strobel, Gertrud; Zollner, Alfred; Angermayr, Michaela; Bandlow, Wolfhard

    2002-01-01

    Sorting of cytoplasmically synthesized proteins to their target compartments usually is highly efficient so that cytoplasmic precursor pools are negligible and a particular gene product occurs at one subcellular location only. Yeast major adenylate kinase (Adk1p/Aky2p) is one prominent exception to this rule. In contrast to most mitochondrial proteins, only a minor fraction (6–8%) is taken up into the mitochondrial intermembrane space, whereas the bulk of the protein remains in the cytosol in sequence-identical form. We demonstrate that Adk1p/Aky2p uses a novel mechanism for subcellular partitioning between cytoplasm and mitochondria, which is based on competition between spontaneous protein folding and mitochondrial targeting and import. Folding is spontaneous and rapid and can dispense with molecular chaperons. After denaturation, enzymatic activity of Adk1p/Aky2p returns within a few minutes and, once folded, the protein is thermally and proteolytically very stable. In an uncoupled cell-free organellar import system, uptake of Adk1p/Aky2p is negligible, but can be improved by previous chaotropic denaturation. Import ensues independently of Hsp70 or membrane potential. Thus, nascent Adk1p/Aky2p has two options: either it is synthesized to completion and folds into an enzymatically active import-incompetent conformation that remains in the cytosol; or, during synthesis and before commencement of significant tertiary structure formation, it reaches a mitochondrial surface receptor and is internalized. PMID:12006643

  11. Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

    PubMed

    Hamada, M; Takenaka, H; Fukumoto, K; Fukamachi, S; Yamaguchi, T; Sumida, M; Shiosaka, T; Kurokawa, Y; Okuda, H; Kuby, S A

    1987-01-01

    Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

  12. An integrated approach for thermal stabilization of a mesophilic adenylate kinase.

    PubMed

    Moon, Sojin; Jung, Du-kyo; Phillips, George N; Bae, Euiyoung

    2014-09-01

    Thermally stable proteins are desirable for research and industrial purposes, but redesigning proteins for higher thermal stability can be challenging. A number of different techniques have been used to improve the thermal stability of proteins, but the extents of stability enhancement were sometimes unpredictable and not significant. Here, we systematically tested the effects of multiple stabilization techniques including a bioinformatic method and structure-guided mutagenesis on a single protein, thereby providing an integrated approach to protein thermal stabilization. Using a mesophilic adenylate kinase (AK) as a model, we identified stabilizing mutations based on various stabilization techniques, and generated a series of AK variants by introducing mutations both individually and collectively. The redesigned proteins displayed a range of increased thermal stabilities, the most stable of which was comparable to a naturally evolved thermophilic homologue with more than a 25° increase in its thermal denaturation midpoint. We also solved crystal structures of three representative variants including the most stable variant, to confirm the structural basis for their increased stabilities. These results provide a unique opportunity for systematically analyzing the effectiveness and additivity of various stabilization mechanisms, and they represent a useful approach for improving protein stability by integrating the reduction of local structural entropy and the optimization of global noncovalent interactions such as hydrophobic contact and ion pairs.

  13. The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth

    NASA Astrophysics Data System (ADS)

    Kim, Hyunjoo; Lee, Ho-June; Oh, Yumin; Choi, Seon-Guk; Hong, Se-Hoon; Kim, Hyo-Jin; Lee, Song-Yi; Choi, Ji-Woo; Su Hwang, Deog; Kim, Key-Sun; Kim, Hyo-Joon; Zhang, Jianke; Youn, Hyun-Jo; Noh, Dong-Young; Jung, Yong-Keun

    2014-02-01

    Adenylate kinase 2 (AK2), which balances adenine nucleotide pool, is a multi-functional protein. Here we show that AK2 negatively regulates tumour cell growth. AK2 forms a complex with dual-specificity phosphatase 26 (DUSP26) phosphatase and stimulates DUSP26 activity independently of its AK activity. AK2/DUSP26 phosphatase protein complex dephosphorylates fas-associated protein with death domain (FADD) and regulates cell growth. AK2 deficiency enhances cell proliferation and induces tumour formation in a xenograft assay. This anti-growth function of AK2 is associated with its DUSP26-stimulating activity. Downregulation of AK2 is frequently found in tumour cells and human cancer tissues showing high levels of phospho-FADDSer194. Moreover, reconstitution of AK2 in AK2-deficient tumour cells retards both cell proliferation and tumourigenesis. Consistent with this, AK2+/- mouse embryo fibroblasts exhibit enhanced cell proliferation with a significant alteration in phospho-FADDSer191. These results suggest that AK2 is an associated activator of DUSP26 and suppresses cell proliferation by FADD dephosphorylation, postulating AK2 as a negative regulator of tumour growth.

  14. New isoforms of Ca2+/calmodulin-dependent protein kinase II in smooth muscle.

    PubMed Central

    Zhou, Z L; Ikebe, M

    1994-01-01

    Four novel isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were found in rat aorta smooth muscle. Two of them were related to gamma-isoform of brain CaM kinase II (gamma-a). Differences in the primary structure of these isoforms were located in the variable region. One of them (gamma-b) contained 23 unique amino acid residues, whereas the other (gamma-c) did not contain this sequence. Both isoforms lacked the two segments (Val-316 to Gln-337 and Lys-353 to Leu-362) present in gamma-a. The DNA sequence of these gamma-isoforms except the variable region was exactly the same, suggesting that they are produced by alternative splicing. Another two isoforms were related to the delta-isoform of brain CaM kinase II (delta-a). delta-b contained a unique 11-residue sequence in the variable region whereas delta-c did not. As found for gamma-isoforms, the sequence analysis suggested that the three delta-isoforms are also produced by alternative splicing. Analysis of RNA by reverse transcription PCR confirmed the existence of specific messages for gamma-b, delta-a and delta-b. The variety of isoforms of CaM kinase II suggest that each isoform may play a specialized role in cell regulation. Images Figure 5 Figure 6 Figure 7 PMID:8172610

  15. Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

    PubMed Central

    Li, Dechang; Liu, Ming S.; Ji, Baohua

    2015-01-01

    Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems. PMID:26244746

  16. Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

    PubMed Central

    Beckstein, Oliver; Denning, Elizabeth J.; Perilla, Juan R.; Woolf, Thomas B.

    2009-01-01

    Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free energy calculations and our new dynamic importance sampling (DIMS) molecular dynamics (MD) method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular four ionic bonds are identified that open in a sequential, zipper-like fashion and thus dominate the free energy landscape of the transition. Transitions between the closed and open conformations only have to overcome moderate free energy barriers. Unexpectedly, the closed and open state encompass broad free energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental FRET measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS-MD computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK. PMID:19751742

  17. Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity.

    PubMed

    Ådén, Jörgen; Verma, Abhinav; Schug, Alexander; Wolf-Watz, Magnus

    2012-10-10

    Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AK(eco)) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AK(eco). Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of k(cat). Addition of the osmolyte TMAO to AK(eco) results in population shift toward the closed conformation and a significant reduction of k(cat). The Michaelis constants (K(M)) scale with the change in k(cat), which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, k(cat) and K(M) are mutually dependent, and in the case of AK(eco), any perturbation that modulates k(cat) is mirrored with a proportional response in K(M). Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AK(eco), there exists ample flexibility to obtain a specificity constant (k(cat)/K(M)) that commensurate with the exerted cellular selective pressure.

  18. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    PubMed

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  19. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    PubMed

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

  20. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation

    PubMed Central

    Pritchard, Rory A.; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S.

    2016-01-01

    Abstract Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated. PMID:26313408

  1. Identification of valine/leucine/isoleucine and threonine/alanine/glycine proton-spin systems of Escherichia coli adenylate kinase by selective deuteration and selective protonation.

    PubMed Central

    Bock-Möbius, I; Brune, M; Wittinghofer, A; Zimmermann, H; Leberman, R; Dauvergne, M T; Zimmermann, S; Brandmeier, B; Rösch, P

    1991-01-01

    Adenylate kinase from two types of Escherichia coli strains, a wild-type and a leucine-auxotrophic strain, was purified. On the one hand, growing the leucine-auxotrophic bacteria on a medium containing deuterated leucine yielded E. coli adenylate kinase with all leucine residues deuterated. On the other hand, by growing the wild-type bacteria on deuterated medium with phenylalanine, threonine and isoleucine present as protonated specimens, 80% randomly deuterated enzyme with protonated phenylalanine, threonine and isoleucine residues could be prepared. Use of these proteins enabled identification of the spin systems of these amino acid residues in the n.m.r. spectra of the protein. PMID:1991031

  2. Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    SciTech Connect

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1985-09-20

    Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 ..mu..M isoproterenol and 50 ..mu..M GTP-..gamma..-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 ..mu..M GTP-..gamma..-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of ..beta..-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes.

  3. Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells By The Traceable Kinase Method

    PubMed Central

    Chen, Xiangyu; Zhao, Xin; Abeyweera, Thushara P.; Rotenberg, Susan A.

    2012-01-01

    A previous report (Biochemistry 46: 2364–2370, 2007) described the application of The Traceable Kinase Method to identify substrates of PKCα in non-transformed human breast MCF-10A cells. Here, a non-radioactive variation of this method compared the phospho-protein profiles of three traceable PKC isoforms (α, δ and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N6-phenyl-ATP, and the resulting phospho-proteins were analyzed by Western blot with an antibody that recognizes the phosphorylated PKC consensus site. Phospho-protein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated effector protein Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, –δ, and −ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (mol phospho-CEP4/mol CEP4). Following knock-down with isoform-specific shRNA-encoding plasmids, phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC–α, –δ, or –ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and –δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms. PMID:22897107

  4. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the 'open' state

    SciTech Connect

    Buchko, G.W.; Robinson, H.; Abendroth, J.; Staker, B. L.; Myler, P. J.

    2010-04-16

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drug therapies against infectious bacterial agents. Here we report the 2.1 {angstrom} resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease melioidosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATP{sub bd}) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 {angstrom}. These two BpAdk conformations may represent 'open' Adk sub-states along the preferential pathway to the 'closed' substrate-bound state.

  5. Isoform switch of pyruvate kinase M1 indeed occurs but not to pyruvate kinase M2 in human tumorigenesis.

    PubMed

    Zhan, Cheng; Yan, Li; Wang, Lin; Ma, Jun; Jiang, Wei; Zhang, Yongxing; Shi, Yu; Wang, Qun

    2015-01-01

    Muscle type of pyruvate kinase (PKM) is one of the key mediators of the Warburg effect and tumor metabolism. Due to alternative splicing, there are at least 12 known isoforms of the PKM gene, of which PKM1 and PKM2 are two major isoforms with only a 23 amino acid sequenced difference but quite different characteristics and functions. It was previously thought the isoform switch from PKM1 to PKM2 resulted in high PKM2 expression in tumors, providing a great advantage to tumor cells. However, this traditional view was challenged by two recent studies; one study claimed that this isoform switch does not occur during the Warburg effect; the other study asserted that the isoform switch is tissue-specific. Here, we re-analyzed the RNA sequencing data of 25 types of human tumors from The Cancer Genome Atlas Data Portal, and confirmed that PKM2 was the major isoform in the tumors and was highly elevated in addition to the entire PKM gene. We further demonstrated that the expression level of PKM1 significantly declined even though there was substantially increased expression of the entire PKM gene. The proportion of PKM1 in total transcript variants also significantly declined in tumors but the proportion of PKM2 did not change accordingly. Therefore, we conclude that the isoform switch of PKM1 does indeed occur, but it switches to other isoforms rather than PKM2. Considering the change in the expression levels of PKM1, PKM2 and the entire PKM gene, we propose that the upregulation of PKM2 is primarily due to elevated transcriptional levels of the entire PKM gene, instead of the isoform switch.

  6. Interferon-. alpha. selectively activates the. beta. isoform of protein kinase C through phosphatidylcholine hydrolysis

    SciTech Connect

    Pfeffer, L.M.; Saltiel, A.R. ); Strulovici, B. )

    1990-09-01

    The early events that occur after interferon binds to discrete cell surface receptors remain largely unknown. Human leukocyte interferon (interferon-{alpha}) rapidly increases the binding of ({sup 3}H)phorbol dibutyrate to intact HeLa cells a measure of protein kinase C activation, and induces the selective translocation of the {beta} isoform of protein kinase C from the cytosol to the particulate fraction of HeLa cells. The subcellular distribution of the {alpha} and {epsilon} isoforms is unaffected by interferon-{alpha} treatment. Activation of protein kinase C by phorbol esters mimics the inhibitory action of interferon-{alpha} on HeLa cell proliferation and down-regulation of protein kinase C blocks the induction of antiviral activity by interferon-{alpha} in HeLa cells. Increased phosphatidylcholine hydrolysis and phosphorylcholine production is accompanied by diacylglycerol production in response to interferon. However, inositol phospholipid turnover and free intracellular calcium concentration are unaffected. These results suggest that the transient increase in diacylglycerol, resulting from phosphatidylcholine hydrolysis, may selectively activate the {beta} isoform of protein kinase C. Moreover, the activation of protein kinase C is a necessary element in interferon action on cells.

  7. Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases.

    PubMed

    Moon, Sojin; Bannen, Ryan M; Rutkoski, Thomas J; Phillips, George N; Bae, Euiyoung

    2014-10-01

    Local structural entropy (LSE) is a descriptor for the extent of conformational heterogeneity in short protein sequences that is computed from structural information derived from the Protein Data Bank. Reducing the LSE of a protein sequence by introducing amino acid mutations can result in fewer conformational states and thus a more stable structure, indicating that LSE optimization can be used as a protein stabilization method. Here, we describe a series of LSE optimization experiments designed to stabilize mesophilic and thermophilic adenylate kinases (AKs) and report crystal structures of LSE-optimized AK variants. In the mesophilic AK, thermal stabilization by LSE reduction was effective but limited. Structural analyses of the LSE-optimized mesophilic AK variants revealed a strong correlation between LSE and the apolar buried surface area. Additional mutations designed to introduce noncovalent interactions between distant regions of the polypeptide resulted in further stabilization. Unexpectedly, optimizing the LSE of the thermophilic AK resulted in a decrease in thermal stability. This destabilization was reduced when charged residues were excluded from the possible substitutions during LSE optimization. These observations suggest that stabilization by LSE reduction may result from the optimization of local hydrophobic contacts. The limitations of this process are likely due to ignorance of other interactions that bridge distant regions in a given amino acid sequence. Our results illustrate the effectiveness and limitations of LSE optimization as a protein stabilization strategy and highlight the importance and complementarity of local conformational stability and global interactions in protein thermal stability.

  8. Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases.

    PubMed

    Moon, Sojin; Bannen, Ryan M; Rutkoski, Thomas J; Phillips, George N; Bae, Euiyoung

    2014-10-01

    Local structural entropy (LSE) is a descriptor for the extent of conformational heterogeneity in short protein sequences that is computed from structural information derived from the Protein Data Bank. Reducing the LSE of a protein sequence by introducing amino acid mutations can result in fewer conformational states and thus a more stable structure, indicating that LSE optimization can be used as a protein stabilization method. Here, we describe a series of LSE optimization experiments designed to stabilize mesophilic and thermophilic adenylate kinases (AKs) and report crystal structures of LSE-optimized AK variants. In the mesophilic AK, thermal stabilization by LSE reduction was effective but limited. Structural analyses of the LSE-optimized mesophilic AK variants revealed a strong correlation between LSE and the apolar buried surface area. Additional mutations designed to introduce noncovalent interactions between distant regions of the polypeptide resulted in further stabilization. Unexpectedly, optimizing the LSE of the thermophilic AK resulted in a decrease in thermal stability. This destabilization was reduced when charged residues were excluded from the possible substitutions during LSE optimization. These observations suggest that stabilization by LSE reduction may result from the optimization of local hydrophobic contacts. The limitations of this process are likely due to ignorance of other interactions that bridge distant regions in a given amino acid sequence. Our results illustrate the effectiveness and limitations of LSE optimization as a protein stabilization strategy and highlight the importance and complementarity of local conformational stability and global interactions in protein thermal stability. PMID:24931334

  9. Role of water in the enzymatic catalysis: study of ATP + AMP → 2ADP conversion by adenylate kinase.

    PubMed

    Adkar, Bharat V; Jana, Biman; Bagchi, Biman

    2011-04-28

    The catalytic conversion ATP + AMP → 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate state of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

  10. Impaired activation of platelets lacking protein kinase C-theta isoform.

    PubMed

    Nagy, Bela; Bhavaraju, Kamala; Getz, Todd; Bynagari, Yamini S; Kim, Soochong; Kunapuli, Satya P

    2009-03-12

    Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-theta is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-theta-selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and alpha-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-theta, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-theta RACK peptide, which may contribute to the lower levels of granule secretion when PKC-theta function is lost. Furthermore, the level of JON/A binding to activated alpha(IIb)beta(3) receptor was also significantly decreased in PKC-theta(-/-) mice compared with wild-type littermates. PKC-theta(-/-) murine platelets showed significantly lower agonist-induced thromboxane A(2) (TXA(2)) release through reduced extracellular signal-regulated kinase phosphorylation. Finally, PKC-theta(-/-) mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl(3) in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-theta isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors. PMID:19164598

  11. A New Type of Metal-Binding Site in Cobalt- And Zinc-Containing Adenylate Kinases Isolated From Sulfate-Reducers D. Gigas And D. Desulfuricans ATCC 27774

    SciTech Connect

    Gavel, O.Y.; Bursakov, S.A.; Rocco, G.Di; Trincao, J.; Pickering, I.J.; George, G.N.; Calvete, J.J.; Brondino, C.; Pereira, A.S.; Lampreia, J.; Tavares, P.; Moura, J.J.G.; Moura, I.

    2009-05-18

    Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterized in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the 'LID' domain. The sequence {sup 129}Cys-X{sub 5}-His-X{sub 15}-Cys-X{sub 2}-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

  12. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    SciTech Connect

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  13. Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement

    PubMed Central

    Janssen, Edwin; Dzeja, Petras P.; Oerlemans, Frank; Simonetti, Arjan W.; Heerschap, Arend; Haan, Arnold de; Rush, Paula S.; Terjung, Ronald R.; Wieringa, Bé; Terzic, Andre

    2000-01-01

    Efficient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1-catalyzed β-phosphoryl transfer in mice decreases the potential of myofibers to sustain nucleotide ratios despite up-regulation of high-energy phosphoryl flux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1-deficient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1–/– muscles. Thus, AK1-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost. PMID:11101510

  14. In the Multi-domain Protein Adenylate Kinase, Domain Insertion Facilitates Cooperative Folding while Accommodating Function at Domain Interfaces

    PubMed Central

    Giri Rao, V. V. Hemanth; Gosavi, Shachi

    2014-01-01

    Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins. PMID:25393408

  15. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    SciTech Connect

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  16. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    NASA Technical Reports Server (NTRS)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  17. Protein kinase C isoforms in atherosclerosis: pro- or anti-inflammatory?

    PubMed

    Fan, Hueng-Chuen; Fernández-Hernando, Carlos; Lai, Jenn-Haung

    2014-03-15

    Atherosclerosis is a pathologic condition caused by chronic inflammation in response to lipid deposition in the arterial wall. There are many known contributing factors such as long-term abnormal glucose levels, smoking, hypertension, and hyperlipidemia. Under the influence of such factors, immune and non-immune effectors cells are activated and participate during the progression of atherosclerosis. Protein kinase C (PKC) family isoforms are key players in the signal transduction pathways of cellular activation and have been associated with several aspects of the atherosclerotic vascular disease. This review article summarizes the current knowledge of PKC isoforms functions during atherogenesis, and addresses differential roles and disputable observations of PKC isoforms. Among PKC isoforms, both PKCβ and PKCδ are the most attractive and potential therapeutic targets. This commentary discusses in detail the outcomes and current status of clinical trials on PKCβ and PKCδ inhibitors in atherosclerosis-associated disorders like diabetes and myocardial infarction. The risk and benefit of these inhibitors for clinical purposes will be also discussed. This review summarizes what is already being done and what else needs to be done in further targeting PKC isoforms, especially PKCβ and PKCδ, for therapy of atherosclerosis and atherosclerosis-associated vasculopathies in the future. PMID:24440741

  18. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes.

    PubMed

    Sundaresan, A; Risin, D; Pellis, N R

    2004-06-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  19. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  20. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  1. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  2. Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation.

    PubMed Central

    Fleming, I; MacKenzie, S J; Vernon, R G; Anderson, N G; Houslay, M D; Kilgour, E

    1998-01-01

    In this study we first established, by immunoblotting with specific antibodies, the temporal changes in cellular levels of protein kinase C (PKC) isoforms during differentiation of 3T3-F442A pre-adipocytes. Both pre-adipocyte and adipocyte 3T3-F442A cells were found to express PKC-alpha, -gamma, -delta, -epsilon, -zeta and -mu. However we were unable to detect PKC-beta, -eta or -theta. The same PKC isoform expression profile was found in rat adipocytes. The alpha, delta and gamma isoforms displayed similar temporal patterns of expression during differentiation of 3T3-F442A cells; all increased rapidly, peaking at day 2 of differentiation. Subsequently, the expression of these isoforms decreased, resulting in lower levels in fully differentiated adipocytes than in pre-adipocytes. The expression of PKC-epsilon increased steadily during differentiation, resulting in markedly elevated levels in adipocytes. Although expression of PKC-mu increased during differentiation, this was attributable to prolonged confluence rather than to the differentiation process itself. No change was observed in PKC-zeta levels during adipocyte development. Anti-sense oligodeoxynucleotides (ODNs) were used to deplete selectively the individual PKC subtypes. Each of the ODNs used effectively depleted the specific isoforms to undetectable levels and did not affect expression of the other PKC subtypes. This approach indicated that pre-adipocyte differentiation is not dependent upon PKC-zeta but that PKC-alpha,-delta and -mu each exert an inhibitory influence upon differentiation. Use of anti-sense ODNs to deplete PKC-epsilon and -gamma revealed that pre-adipocyte differentiation is dependent upon each of these isoforms. However, PKC-gamma, but not PKC-epsilon, appeared to be necessary for the clonal expansion of differentiating cells, suggesting that PKC-epsilon is required at a later phase in the differentiation process, when its expression is elevated, for the attainment and maintenance of

  3. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  4. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis.

    PubMed

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M; Hrabak, Estelle M; Harmon, Alice C; Pickard, Barbara G; Harper, Jeffrey F

    2003-08-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  5. Different Protein Kinase C Isoforms Determine Growth Factor Specificity in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Soh, Jae-Won; Yoshida, Keiko; Eves, Eva M.; Weinstein, I. Bernard; Rosner, Marsha Rich

    2000-01-01

    Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cδ (PKCδ), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCδ in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCζ-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCζ, like PKCδ, acts upstream of MEK, and PKCζ can potentiate Raf-1 activation by EGF. Inhibition of PKCζ also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCζ or PKCδ suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity. PMID:10891480

  6. Purification and stability of octameric mitochondrial creatine kinase isoform from herring (Clupea harengus) organ of vision.

    PubMed

    Niedźwiecka, Natalia; Grzyb, Katarzyna; Nona-Mołdawa, Agnieszka; Gronczewska, Jadwiga; Skorkowski, Edward F

    2015-07-01

    Creatine kinases (CKs) constitute a large family of isoenzymes that are involved in intracellular energy homeostasis. In cells with high and fluctuating energy requirements ATP level is maintained via phosphocreatine hydrolysis catalyzed by creatine kinase. In contrast to invertebrates and higher vertebrates, in poikilothermic vertebrates the adaptations for the regulation of energy metabolism by changes in the oligomeric state of CK isoforms are not well known. The present study aimed at identification of herring eye CK isoforms and focuses on factors affecting the CK-octamer stability. In addition to the CK octamer, three different dimeric isoforms of CK were detected by cellulose acetate native electrophoresis. Destabilization of octamer was studied in the presence of TSAC substrates and about 50% of octamers dissociated into dimers within 24h. Moreover, we found that the increase of temperature from 4 °C to 30 °C caused rapid inactivation of dimers in TSAC-treated samples but did not affect octameric structures. In a thermostability assay we demonstrated that octamers retain their activity even at 50 °C. Our results indicate that destabilization of the octameric structure can lead to loss of enzyme activity at higher temperatures (above 30 °C). Furthermore, our results based on N-terminal sequence analysis suggest that probably the mitochondrial s-type CK, rather than u-type, is predominantly expressed in herring eye. In conclusion the existence of four various CK isoforms in one organ may reflect complex regulation of energy metabolism in the phototransduction process in teleost fishes.

  7. Investigating the role of class-IA PI 3-kinase isoforms in adipocyte differentiation

    SciTech Connect

    Kim, Ji Eun; Shepherd, Peter R. Chaussade, Claire

    2009-02-20

    PI 3-kinases, in particular class-IA, are key signalling molecules controlling many cellular processes including growth, proliferation, migration and differentiation. In this study, we have used a collection of isoform selective PI 3-kinase inhibitors to determine whether attenuation of signalling through class-IA PI 3-kinase isoforms will impact adipocyte differentiation. First, we analysed the expression profiles and found that fibroblastic pre-adipocytes express detectable levels of p110{alpha} and p110{delta} and that after differentiation, p110{delta} levels fall while p110{alpha} levels rise, together with C/EBP{alpha} and PPAR{gamma}. When using specific inhibitors during the differentiation process, we observed that neither p110{beta} nor p110{delta} inhibition, had any significant effect. In contrast PIK-75, a selective p110{alpha} inhibitor completely abolished adipocyte differentiation as assessed by morphology, transcript and protein levels of adipocyte markers. These results indicate that long term treatment with p110{alpha} inhibitors could potentially have a severe impact on fat cell numbers in vivo.

  8. The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor.

    PubMed

    Yegutkin, Gennady G; Mikhailov, Andrey; Samburski, Sergei S; Jalkanen, Sirpa

    2006-08-01

    Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP <--> 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [alpha-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous gamma-phosphate-donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues.

  9. Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

    PubMed

    Abbot, Emily L; McCormack, James G; Reynet, Christine; Hassall, David G; Buchan, Kevin W; Yeaman, Stephen J

    2005-06-01

    The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle. PMID:15955060

  10. Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function

    PubMed Central

    Yea, Sung Su; So, Lomon; Mallya, Sharmila; Lee, Jongdae; Rajasekaran, Kamalakannan; Malarkannan, Subramaniam; Fruman, David A.

    2014-01-01

    Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110α has received considerable attention in oncology because the gene encoding p110α (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110α in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110α and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110α-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110α inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-γ and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110δinhibitor, with lesser effects of p110α inhibitors. Oral administration of mice with MLN1117, a p110α inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110α inhibitors to preserve NK cell function. PMID:24915189

  11. Alpha-isoform of Ca2+/calmodulin-dependent kinase II autophosphorylation is required for memory consolidation-specific transcription.

    PubMed

    von Hertzen, Laura S J; Giese, K Peter

    2005-08-22

    Autophosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II switches the kinase into an autonomous activity mode. This molecular switch is important for hippocampal long-term memory formation, which requires de novo gene transcription and protein synthesis. Here, we have studied whether auto-phosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II is required for gene transcription induced in the hippocampus by contextual fear conditioning. We have shown that upregulation of a nonassociative transcript, the serum and glucocorticoid-induced kinase-1 messenger RNA, is normal in alpha-isoform of Ca2+/calmodulin-dependent kinase II autophosphorylation-deficient mutant mice, whereas upregulation of an associative transcript, the nerve growth factor-inducible gene B messenger RNA, is impaired. Thus, we suggest that autophosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II is a biochemical switch that regulates association-specific consolidation processes. PMID:16056150

  12. Differential activation of NAD kinase by plant calmodulin isoforms. The critical role of domain I.

    PubMed

    Lee, S H; Seo, H Y; Kim, J C; Heo, W D; Chung, W S; Lee, K J; Kim, M C; Cheong, Y H; Choi, J Y; Lim, C O; Cho, M J

    1997-04-01

    NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.

  13. Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes*

    PubMed Central

    Ryvkin, Vladislav; Rashel, Mohammad; Gaddapara, Trivikram; Ghazizadeh, Soosan

    2015-01-01

    PKD is a family of three serine/threonine kinases (PKD-1, -2, and -3) involved in the regulation of diverse biological processes including proliferation, migration, secretion, and cell survival. We have previously shown that despite expression of all three isoforms in mouse epidermis, PKD1 plays a unique and critical role in wound healing, phorbol ester-induced hyperplasia, and tumor development. In translating our findings to the human, we discovered that PKD1 is not expressed in human keratinocytes (KCs) and there is a divergence in the expression and function of other PKD isoforms. Contrary to mouse KCs, treatment of cultured human KCs with pharmacological inhibitors of PKDs resulted in growth arrest. We found that PKD2 and PKD3 are expressed differentially in proliferating and differentiating human KCs, with the former uniformly present in both compartments whereas the latter is predominantly expressed in the proliferating compartment. Knockdown of individual PKD isoforms in human KCs revealed contrasting growth regulatory roles for PKD2 and PKD3. Loss of PKD2 enhanced KC proliferative potential while loss of PKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability. This proliferation defect was correlated with up-regulation of CDK4/6 inhibitor p15INK4B and induction of a p53-independent G1 cell cycle arrest. Simultaneous silencing of PKD isoforms resulted in a more pronounced proliferation defect consistent with a predominant role for PKD3 in proliferating KCs. These data underline the importance and complexity of PKD signaling in human epidermis and suggest a central role for PKD3 signaling in maintaining human epidermal homeostasis. PMID:25802335

  14. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

    PubMed

    Fry, D C; Byler, D M; Susi, H; Brown, E M; Kuby, S A; Mildvan, A S

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  15. Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms.

    PubMed

    Saraiva, Lucília; Silva, Rui D; Pereira, Gil; Gonçalves, Jorge; Côrte-Real, Manuela

    2006-08-01

    Mammalian protein kinase C (PKC) isoforms have been subject of particular attention because of their ability to modulate apoptotic proteins. However, the roles played by each PKC isoform in apoptosis are still unclear. Here, expression of individual mammalian PKC isoforms in Saccharomyces cerevisiae is used as a new approach to study the role of each isoform in apoptosis. The four isoforms tested, excepting PKC-delta, stimulate S. cerevisiae acetic-acid-induced apoptosis essentially through a mitochondrial ROS-dependent pathway. However, their co-expression with Bcl-xL reveals a PKC-isoform-dependent modulation of Bcl-xL anti-apoptotic activity. A yeast pathway homologue to the mammalian SAPK/JNK is responsible for acetic-acid-induced Bcl-xL phosphorylation that is differently modulated by PKC isoforms. The data obtained suggest conservation of an ancient mechanism of apoptosis regulation in yeast and mammals and offer new insights into mammalian apoptosis modulation by PKC isoforms.

  16. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model.

    PubMed

    Kim, H J; Nishikawa, S; Tokutomi, Y; Takenaka, H; Hamada, M; Kuby, S A; Uesugi, S

    1990-02-01

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.

  17. Structural and biochemical investigation of two Arabidopsis shikimate kinases: The heat-inducible isoform is thermostable

    SciTech Connect

    Fucile, Geoffrey; Garcia, Christel; Carlsson, Jonas; Sunnerhagen, Maria; Christendat, Dinesh

    2011-10-27

    The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform-specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37 C whereas the heat-induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. We determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat-induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK-mediated control of steady state concentrations of shikimate.

  18. Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3

    PubMed Central

    Hong, Bum Soo; Allali-Hassani, Abdellah; Tempel, Wolfram; Finerty, Patrick J.; MacKenzie, Farrell; Dimov, Svetoslav; Vedadi, Masoud; Park, Hee-Won

    2010-01-01

    Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKα (α1 and α2) and ChoKβ isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKα1 and ChoKβ isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKα1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKβ was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKα isoforms (α1 and α2) compared with ChoKβ. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKα2 (equivalent to Leu-419 of ChoKα1), or the corresponding residue Phe-352 of ChoKβ, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation. PMID:20299452

  19. Myophilin of Echinococcus granulosus: isoforms and phosphorylation by protein kinase C.

    PubMed

    Martin, R M; Csar, X F; Gasser, R B; Felleisen, R; Lightowlers, M W

    1997-08-01

    Myophilin is a muscle-associated antigen of the taeniid cestode Echinococcus granulosus. This protein shows a high amino acid sequence homology with calponins and calponin-like proteins, which are proposed to be associated with the regulation of smooth muscle contraction. In order to provide supportive evidence for a relationship between these proteins, we characterized myophilin using electrophoretic, biochemical and molecular biological approaches. Two-dimensional protein electrophoretic separation of E. granulosus larval proteins defined 4 isoelectric isoforms of myophilin (alpha, beta, gamma and delta), which appeared to be a consequence of post-translational modification of a single gene product. It was also demonstrated biochemically that E. granulosus myophilin undergoes specific phosphorylation in vitro by protein kinase C (PKC). Finally, myophilin homologues were identified in extracts of Taenia hydatigena and T. ovis by immunoblot. A partial cDNA of the closely related species, E. multilocularis, was isolated by cloning procedures and showed 99% homology with the E. granulosus myophilin gene. The similarities of E. granulosus myophilin with calponins in their tissue localization, protein isoforms patterns, ability to be phosphorylated in vitro by PKC, and the relatively conserved nature of the protein among related parasites suggest that myophilin may be associated with smooth muscle contraction.

  20. mRNA expression of diacylglycerol kinase isoforms in insulin-sensitive tissues: effects of obesity and insulin resistance.

    PubMed

    Mannerås-Holm, Louise; Kirchner, Henriette; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R

    2015-04-01

    Diacylglycerol kinase (DGK) isoforms regulate signal transduction and lipid metabolism. DGKδ deficiency leads to hyperglycemia, peripheral insulin resistance, and metabolic inflexibility. Thus, dysregulation of other DGK isoforms may play a role in metabolic dysfunction. We investigated DGK isoform mRNA expression in extensor digitorum longus (EDL) and soleus muscle, liver as well as subcutaneous and epididymal adipose tissue in C57BL/6J mice and obese and insulin-resistant ob/ob mice. All DGK isoforms, except for DGKκ, were detectable, although with varying mRNA expression. Liver DGK expression was generally lowest, with several isoforms undetectable. In soleus muscle, subcutaneous and epididymal adipose tissue, DGKδ was the most abundant isoform. In EDL muscle, DGKα and DGKζ were the most abundant isoforms. In liver, DGKζ was the most abundant isoform. Comparing obese insulin-resistant ob/ob mice to lean C57BL/6J mice, DGKβ, DGKι, and DGKθ were increased and DGKε expression was decreased in EDL muscle, while DGKβ, DGKη and DGKθ were decreased and DGKδ and DGKι were increased in soleus muscle. In liver, DGKδ and DGKζ expression was increased in ob/ob mice. DGKη was increased in subcutaneous fat, while DGKζ was increased and DGKβ, DGKδ, DGKη and DGKε were decreased in epididymal fat from ob/ob mice. In both adipose tissue depots, DGKα and DGKγ were decreased and DGKι was increased in ob/ob mice. In conclusion, DGK mRNA expression is altered in an isoform- and tissue-dependent manner in obese insulin-resistant ob/ob mice. DGK isoforms likely have divergent functional roles in distinct tissues, which may contribute to metabolic dysfunction. PMID:25847921

  1. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    EPA Science Inventory

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM
    IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2

    * Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue1
    1The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  2. Altered content of AMP-activated protein kinase isoforms in skeletal muscle from spinal cord injured subjects.

    PubMed

    Kostovski, Emil; Boon, Hanneke; Hjeltnes, Nils; Lundell, Leonidas S; Ahlsén, Maria; Chibalin, Alexander V; Krook, Anna; Iversen, Per Ole; Widegren, Ulrika

    2013-11-01

    AMP-activated protein kinase (AMPK) is a pivotal regulator of energy homeostasis. Although downstream targets of AMPK are widely characterized, the physiological factors governing isoform expression of this protein kinase are largely unknown. Nerve/contractile activity has a major impact on the metabolic phenotype of skeletal muscle, therefore likely to influence AMPK isoform expression. Spinal cord injury represents an extreme form of physical inactivity, with concomitant changes in skeletal muscle metabolism. We assessed the influence of longstanding and recent spinal cord injury on protein abundance of AMPK isoforms in human skeletal muscle. We also determined muscle fiber type as a marker of glycolytic or oxidative metabolism. In subjects with longstanding complete injury, protein abundance of the AMPKγ3 subunit, as well as myosin heavy chain (MHC) IIa and IIx, were increased, whereas abundance of the AMPKγ1 subunit and MHC I were decreased. Similarly, abundance of AMPKγ3 and MHC IIa proteins were increased, whereas AMPKα2, -β1, and -γ1 subunits and MHC I abundance was decreased during the first year following injury, reflecting a more glycolytic phenotype of the skeletal muscle. However, in incomplete cervical lesions, partial recovery of muscle function attenuated the changes in the isoform profile of AMPK and MHC. Furthermore, exercise training (electrically stimulated leg cycling) partly normalized mRNA expression of AMPK isoforms. Thus, physical activity affects the relative expression of AMPK isoforms. In conclusion, skeletal muscle abundance of AMPK isoforms is related to physical activity and/or muscle fiber type. Thus, physical/neuromuscular activity is an important determinant of isoform abundance of AMPK and MCH. This further underscores the need for physical activity as part of a treatment regimen after spinal cord injury to maintain skeletal muscle metabolism. PMID:24022865

  3. Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

    PubMed Central

    Strasser, R H; Benovic, J L; Caron, M G; Lefkowitz, R J

    1986-01-01

    beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors. Images PMID:3018728

  4. A Xenopus nonmuscle myosin heavy chain isoform is phosphorylated by cyclin-p34cdc2 kinase during meiosis.

    PubMed

    Kelley, C A; Oberman, F; Yisraeli, J K; Adelstein, R S

    1995-01-20

    There are two vertebrate nonmuscle myosin heavy chain (MHC) genes that encode two separate isoforms of the heavy chain, MHC-A and MHC-B. Recent work has identified additional, alternatively spliced isoforms of MHC-B cDNA with inserted sequences of 30 nucleotides (chicken and human) or 48 nucleotides (Xenopus) at a site corresponding to the ATP binding region in the MHC protein (Takahashi, M., Kawamoto, S., and Adelstein, R.S. (1992) J. Biol. Chem. 267, 17864-17871) and Bhatia-Dey, N., Adelstein, R.S., and Dawid, I.B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2856-2859). The deduced amino acid sequence of these inserts contains a consensus sequence for phosphorylation by cyclin-p34cdc2 (cdc2) kinase. In cultured Xenopus XTC cells, we have identified two inserted MHC-B isoforms and a non-inserted MHC-A isoform by immunoblotting of cell extracts. When myosin was immunoprecipitated from XTC cells and phosphorylated in vitro with cdc2 kinase, the kinase catalyzed the phosphorylation of both inserted MHC-B isoforms but not MHC-A. Isoelectric focusing of tryptic peptides generated from MHC-B phosphorylated with cdc2 kinase revealed one major phosphopeptide that was purified by reverse-phase high performance liquid chromatography and sequenced. The phosphorylated residue was Ser-214, the cdc2 kinase consensus site within the insert near the ATP binding region. The same site was phosphorylated in intact XTC cells during log phase of growth and in cell-free lysates of Xenopus eggs stabilized in second meiotic metaphase but not interphase. Moreover, Ser-214 phosphorylation was detected during maturation of Xenopus oocytes when the cdc2 kinase-containing maturation-promoting factor was activated, but not in G2 interphase-arrested oocytes. These results demonstrate that MHC-B phosphorylation is tightly regulated by cdc2 kinase during meiotic cell cycles. Furthermore, MHC-A and MHC-B isoforms are differentially phosphorylated at these stages, suggesting that they may serve

  5. The Phosphoarginine Energy-Buffering System of Trypanosoma brucei Involves Multiple Arginine Kinase Isoforms with Different Subcellular Locations

    PubMed Central

    Wadforth, Cath; Harley, Maggie; Colasante, Claudia

    2013-01-01

    Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide ‘SNL’. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed. PMID:23776565

  6. Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae.

    PubMed

    Nicastro, Raffaele; Tripodi, Farida; Gaggini, Marco; Castoldi, Andrea; Reghellin, Veronica; Nonnis, Simona; Tedeschi, Gabriella; Coccetti, Paola

    2015-10-01

    In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription.

  7. Dopamine D1 receptor and protein kinase C isoforms in spontaneously hypertensive rats.

    PubMed

    Yao, L P; Li, X X; Yu, P Y; Xu, J; Asico, L D; Jose, P A

    1998-12-01

    -Dopamine, via D1-like receptors, stimulates the activity of both protein kinase A (PKA) and protein kinase C (PKC), which results in inhibition of renal sodium transport. Since D1-like receptors differentially regulate sodium transport in normotensive and hypertensive rats, they may also differentially regulate PKC expression in these rat strains. Thus, 2 different D1-like agonists (fenoldopam or SKF 38393) were infused into the renal artery of anesthetized normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) (n=5 to 6/drug/strain). Ten or 60 minutes after starting the D1-like agonist infusion, both the infused kidney and the noninfused kidney that served as control were prepared for analysis. The D1-like agonists produced a greater diuresis and natriuresis and inhibited Na+,K+-ATPase activity in proximal tubule (PT) and medullary thick ascending limb (mTAL) to a greater extent in WKY (Delta20+/-1%) than in SHR (Delta7+/-1%, P<0.001). D1-like agonists had no effect on PKC-alpha or PKC-lambda expression in either membrane or cytosol but increased PKC-theta expression in PT in both WKY and SHR at 10 minutes but not at 60 minutes. However, membranous PKC-delta expression in PT and mTAL decreased in WKY but increased in SHR with either 10 or 60 minutes of D1-like agonist infusion. D1-like agonists also decreased membranous PKC-zeta expression in PT and mTAL in WKY but increased it in PT but not in mTAL in SHR. We conclude that there is differential regulation of PKC isoform expression by D1-like agonists that inhibits membranous PKC-delta and PKC-zeta in WKY but stimulates them in SHR; this effect in SHR is similar to the stimulatory effect of norepinephrine and angiotensin II and may be a mechanism for their differential effects on sodium transport.

  8. Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation

    PubMed Central

    Lunt, Sophia Y.; Muralidhar, Vinayak; Hosios, Aaron M.; Israelsen, William J.; Gui, Dan Y.; Newhouse, Lauren; Ogrodzinski, Martin; Hecht, Vivian; Xu, Kali; Acevedo, Paula N. Marín; Hollern, Daniel P.; Bellinger, Gary; Dayton, Talya L.; Christen, Stefan; Elia, Ilaria; Dinh, Anh T.; Stephanopoulos, Gregory; Manalis, Scott R.; Yaffe, Michael B.; Andrechek, Eran R.; Fendt, Sarah-Maria; Heiden, Matthew G. Vander

    2014-01-01

    SUMMARY Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2-deletion affects proliferation and metabolism in non-transformed, non-immortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis. PMID:25482511

  9. Facilitated interaction between the pyruvate dehydrogenase kinase isoform 2 and the dihydrolipoyl acetyltransferase.

    PubMed

    Hiromasa, Yasuaki; Roche, Thomas E

    2003-09-01

    The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd approximately 175 microM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd approximately 3 microM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in approximately 8-fold tighter binding of PDK2 to GST-L2red, which was approximately 300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound approximately 18 PDK2 dimers with a Kd similar to GST-L2. E2.E1 bound more PDK2 (approximately 27.6) than E2 with approximately 2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2.E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5'-(beta,gamma-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2.E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed "hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate. PMID:12816949

  10. Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

    SciTech Connect

    Ono, Junichi; Takada, Shoji; Saito, Shinji

    2015-06-07

    An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchical conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.

  11. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

    PubMed Central

    Kladova, A. V.; Gavel, O. Yu.; Mukhopaadhyay, A.; Boer, D. R.; Teixeira, S.; Shnyrov, V. L.; Moura, I.; Moura, J. J. G.; Romão, M. J.; Trincão, J.; Bursakov, S. A.

    2009-01-01

    Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfo­vibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 Å resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 Å resolution, respectively. Zn2+–AK and Fe2+–AK crystallized in space group I222 with similar unit-cell parameters, whereas Co2+–AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn2+–AK and Fe2+–AK forms and a dimer was present for the Co2+–AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes. PMID:19724135

  12. Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

    NASA Astrophysics Data System (ADS)

    Ono, Junichi; Takada, Shoji; Saito, Shinji

    2015-06-01

    An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchical conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.

  13. Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase

    NASA Astrophysics Data System (ADS)

    Jana, Biman; Adkar, Bharat V.; Biswas, Rajib; Bagchi, Biman

    2011-01-01

    The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

  14. Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase.

    PubMed

    Jana, Biman; Adkar, Bharat V; Biswas, Rajib; Bagchi, Biman

    2011-01-21

    The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

  15. Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase.

    PubMed

    Oliver, Sandra N; Tiessen, Axel; Fernie, Alisdair R; Geigenberger, Peter

    2008-01-01

    Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial adenylate kinase in growing tubers leads to increased rates of respiratory oxygen consumption and increased carbon fluxes into starch. Increased rates of starch synthesis were accompanied by post-translational redox-activation of ADP-glucose pyrophosphorylase (AGPase), catalysing the key regulatory step of starch synthesis in the plastid, while there were no substantial changes in metabolic intermediates or sugar levels. A similar increase in post-translational redox-activation of AGPase was found after supplying adenine to wild-type potato tuber discs to increase adenine nucleotide levels. Results provide first evidence for a link between redox-activation of AGPase and adenine nucleotide levels in plants.

  16. Bisphenol A differentially activates protein kinase C isoforms in murine placental tissue

    SciTech Connect

    Tan, Wenjuan; Huang, Hui; Wang, Yanfei; Wong, Tsz Yan; Wang, C.C.; Leung, Lai K.

    2013-06-01

    Bisphenol A is utilized to make polycarbonate plastics and is an environmental pollutant. Recent research has indicated that it is an endocrine disruptor and may interfere with reproductive processes. Our lab has previously shown that bisphenol A could regulate corticotrophin releasing hormone and aromatase in cultured placental cells. In the present study, the effect of bisphenol A on these two genes in the placenta was investigated in mice. Pregnant ICR mice were gavaged with bisphenol A at 2, 20 and 200 mg/kg body weight/day from E13 to E16 and were euthanized at E17. Compared to the control mice, increased plasma estrogen and corticotrophin releasing hormone were observed in bisphenol A-treated mice. Messenger RNA quantification indicated that placental crh but not cyp19 was induced in mice treated with bisphenol A. Tracking the related signaling pathway, we found that protein kinase C ζ/λ and δ were activated in the placentas of bisphenol A-treated mice. As the gene promoter of crh contains CRE and the half site of ERE, either phospho-PKC or estrogen could stimulate the gene transactivation. These results indicate that bisphenol A might increase plasma concentrations of estradiol, testosterone, corticotrophin releasing hormone and placental phospho-PKC ζ/λ and δ in mice. Ultimately, the incidence of premature birth in these mice could increase. - Highlights: • The pollutant bisphenol A differentially activated PKC isoforms in the placenta. • CRE-binding activity in the nuclear protein of placenta was increased. • Bisphenol A induces CRH mRNA expression in mice.

  17. Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression.

    PubMed

    Zhang, Yi; Ma, Ke; Sadana, Prabodh; Chowdhury, Farhana; Gaillard, Stephanie; Wang, Fang; McDonnell, Donald P; Unterman, Terry G; Elam, Marshall B; Park, Edwards A

    2006-12-29

    The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogen-related receptors (ERRalpha and ERRgamma). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of PDK4. Here we report that ERRalpha and ERRgamma stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1alpha to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRalpha and ERRgamma. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1alpha. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1alpha from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression. PMID:17079227

  18. Purification and characterization of an isoform of protein kinase C from bovine neutrophils

    SciTech Connect

    Dianoux, A.C.; Stasia, M.J.; Vignais, P.V. )

    1989-01-24

    Protein kinase C (PKC) from bovine neutrophils was purified 1,420-fold. Subcellular fractionation analysis of bovine neutrophil homogenate in the presence of EGTA indicated that more than 95% of the PKC activity was present in the soluble fraction. Whereas bovine brain PKC could be resolved into four isoenzymatic forms by chromatography on a hydroxylapatite column, bovine neutrophil PKC was eluted in a single peak, suggesting that it corresponded to a single isoform. The apparent molecular weight of bovine neutrophil PKC was 82,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bovine neutrophil PKC was autophosphorylated in the presence of ({gamma}-{sup 32}P)ATP, provided that the medium was supplemented with Mg{sup 2+}, Ca{sup 2+}, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of M{sub r} 48,000 and 39,000. The labeled amino acids were serine and threonine. During the course of the purification procedure of bovine neutrophil PKC, a protein of M{sub r} 23,000 was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of ({gamma}-{sup 32}P)ATP, Mg{sup 2+}, Ca{sup 2+}, phosphatidylserine, and diacylglycerol. This protein was recovered together with PKC in one of the two active peaks eluted from the Mono Q column at the second step of PKC purification. It is suggested that the M{sub r} 23,000 protein might be a natural substrate for bovine neutrophil PKC.

  19. Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

    PubMed

    Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

    2012-07-01

    Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.

  20. NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1985-08-13

    Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual

  1. Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR.

    PubMed

    Yan, H G; Tsai, M D

    1991-06-01

    Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes.

    PubMed

    Bellance, Catherine; Khan, Junaid A; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-05-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes.

  3. Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

    PubMed Central

    Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

    2012-01-01

    The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

  4. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes

    PubMed Central

    Bellance, Catherine; Khan, Junaid A.; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-01-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(–) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl­amino)­phenyl-17β-hydroxy-17-(1-propynyl)­estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes. PMID:23485561

  5. A comparative study of structural and conformational properties of casein kinase-1 isoforms: insights from molecular dynamics and principal component analysis.

    PubMed

    Singh, Surya Pratap; Gupta, Dwijendra K

    2015-04-21

    Wnt signaling pathway regulates several developmental processes in human; however recently this pathway has been associated with development of different types of cancers. Casein kinase-1 (CK1) constitutes a family of serine-threonine protein kinase; various members of this family participate in Wnt signal transduction pathway and serve as molecular switch to this pathway. Among the known six isoforms of CK1, in human, at least three isoforms (viz. alpha, delta and epsilon) have been reported as oncogenic. The development of common therapeutics against these kinases is an arduous task; unless we have the detailed information of their tertiary structures and conformational properties. In the present work, the dynamical and conformational properties for each of three isoforms of CK1 are explored through molecular dynamics (MD) simulations. The conformational space distribution of backbone atoms is evaluated using principal component analysis of MD data, which are further validated on the basis of potential energy surface. Based on these analytics, it is suggested that conformational subspace shifts upon binding to ligands and guides the kinase action of CK1 isoforms. Further, this paper as a first effort to concurrently study all the three isoforms of CK1 provides structural basis for development of common anticancer therapeutics against three isoforms of CK1.

  6. Phosphagen kinase of the giant tubeworm Riftia pachyptila. Cloning and expression of cytoplasmic and mitochondrial isoforms of taurocyamine kinase.

    PubMed

    Uda, Kouji; Tanaka, Kumiko; Bailly, Xavier; Zal, Franck; Suzuki, Tomohiko

    2005-10-30

    The giant tubeworm Riftia pachyptila lives at deep-sea hydrothermal vents along the East Pacific Rise and the Galapagos Rift. The large size and high growth rate of R. pachyptila is supported by an endosymbiotic relationship with a chemosynthetic bacterium. Elucidation of the regulation of energy metabolism of the giant tubeworm remains an interesting problem. The purpose of this study is to determine the cDNA sequence of phosphagen kinase, one of the most important enzymes in energy metabolism, and to characterize its function. Two phosphagen kinase cDNA sequences amplified from the cDNA library of R. pachyptila showed high derived amino acid sequence identity (74%) with those of cytoplasmic taurocyamine kinase (TK) and mitochondrial TK from an annelid Arenicola brasiliensis. The cytoplasmic form of the Riftia recombinant enzyme showed stronger activity for the substrates taurocyamine and also considerable activity for lombricine (21% that of taurocyamine). The mitochondrial form, which was structurally similar to mitochondrial creatine kinase, showed stronger activity for taurocyamine, and a broader activity for various guanidine compounds: glycocyamine (35% that of taurocyamine), lombricine (31%) and arginine (3%). Both forms showed no activity for creatine. The difference in substrate specificities between the cytoplasmic and mitochondrial forms might be attributable to the large difference in the amino acid sequence of the GS region and/or several key amino acid residues for establishing guanidine substrate specificity. Based on these results, we conclude that Riftia contains at least two forms of TK as phosphagen kinase. We also report the kinetic parameters, Km and kcat, of Arenicola and Riftia TKs for the first time. The Km values for taurocyamine of Arenicola and Riftia TKs ranged from 0.9 to 4.0 mM and appear to be comparable to those of other annelid-specific enzymes, lombricine kinase and glycocyamine kinase, but are significantly lower than those of

  7. A Novel Isoform of the B Cell Tyrosine Kinase BTK Protects Breast Cancer Cells from Apoptosis

    PubMed Central

    Eifert, Cheryl; Wang, Xianhui; Kokabee, Leila; Kourtidis, Antonis; Jain, Ritu; Gerdes, Michael J.; Conklin, Douglas S.

    2016-01-01

    Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large-scale loss-of-function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton’s tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer. PMID:23913792

  8. Protein kinase A catalytic subunit isoform PRKACA; History, function and physiology.

    PubMed

    Turnham, Rigney E; Scott, John D

    2016-02-15

    Our appreciation of the scope and influence of second messenger signaling has its origins in pioneering work on the cAMP-dependent protein kinase. Also called protein kinase A (PKA), this holoenzyme exists as a tetramer comprised of a regulatory (R) subunit dimer and two catalytic (C) subunits. Upon binding of two molecules of the second messenger cAMP to each R subunit, a conformational change in the PKA holoenzyme occurs to release the C subunits. These active kinases phosphorylate downstream targets to propagate cAMP responsive cell signaling events. This article focuses on the discovery, structure, cellular location and physiological effects of the catalytic subunit alpha of protein kinase A (encoded by the gene PRKACA). We also explore the potential role of this essential gene as a molecular mediator of certain disease states.

  9. Protein kinase A catalytic subunit isoform PRKACA; History, function and physiology.

    PubMed

    Turnham, Rigney E; Scott, John D

    2016-02-15

    Our appreciation of the scope and influence of second messenger signaling has its origins in pioneering work on the cAMP-dependent protein kinase. Also called protein kinase A (PKA), this holoenzyme exists as a tetramer comprised of a regulatory (R) subunit dimer and two catalytic (C) subunits. Upon binding of two molecules of the second messenger cAMP to each R subunit, a conformational change in the PKA holoenzyme occurs to release the C subunits. These active kinases phosphorylate downstream targets to propagate cAMP responsive cell signaling events. This article focuses on the discovery, structure, cellular location and physiological effects of the catalytic subunit alpha of protein kinase A (encoded by the gene PRKACA). We also explore the potential role of this essential gene as a molecular mediator of certain disease states. PMID:26687711

  10. Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor That Attenuates Disease Progression in Alzheimer’s Disease Mouse Models

    PubMed Central

    2015-01-01

    The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150’s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior. PMID:25676389

  11. Isoform-specific and Protein Kinase C-mediated Regulation of CTP:Phosphoethanolamine Cytidylyltransferase Phosphorylation*

    PubMed Central

    Pavlovic, Zvezdan; Zhu, Lin; Pereira, Leanne; Singh, Ratnesh Kumar; Cornell, Rosemary B.; Bakovic, Marica

    2014-01-01

    CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50–90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation. PMID:24519946

  12. Huntingtin-associated protein 1 (HAP1) is a cGMP-dependent kinase anchoring protein (GKAP) specific for the cGMP-dependent protein kinaseisoform.

    PubMed

    Corradini, Eleonora; Burgers, Pepijn P; Plank, Michael; Heck, Albert J R; Scholten, Arjen

    2015-03-20

    Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.

  13. Studies on the adenylate kinase isozymes from the serum and erythrocyte of normal and Duchenne dystrophic patients. Isolation, physicochemical properties, and several comparisons with the Duchenne dystrophic aberrant enzyme.

    PubMed

    Hamada, M; Sumida, M; Kurokawa, Y; Sunayashiki-Kusuzaki, K; Okuda, H; Watanabe, T; Kuby, S A

    1985-09-25

    Two species of adenylate kinase isozymes (ATP:AMP phosphotransferase, EC 2.7.4.3) from human Duchenne dystrophic serum were separated by Blue Sepharose CL-6B affinity column chromatography. One of these species was the "aberrant" adenylate kinase isozyme, found specifically in the Duchenne type of this disease (Hamada, M., Okuda, H., Oka, K., Watanabe, T., Ueda, K., Nojima, M., Kuby, S.A., Manship, M., Tyler, F., and Ziter, F. (1981) Biochim. Biophys. Acta 660, 227-237). The separated aberrant form possessed a molecular size of 98,000 (+/- 1,500), whereas the normal serum species of the enzyme was 87,000 (+/- 1,600) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by gel filtration, and by sedimentation equilibrium. The sedimentation coefficient of each species was found to be 5.8 S for the aberrant form and 5.6 S for the normal form, respectively. The subunit size (Mr = 24,700) of the aberrant enzyme in 8 M urea proved to be very similar to that of the normal human liver enzyme (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S.A. (1982) J. Biol. Chem. 257, 13120-13128), and the normal species subunit (Mr = 21,700) was found to be very similar to that of the normal human muscle enzyme (Kuby, S.A., Fleming, G., Frischat, A., Cress, M.C., and Hamada, M. (1983) J. Biol. Chem. 258, 1901-1907). Both species were tetrameric enzymes in the serum. The amino acid composition for the normal species was similar to that for the muscle-type enzyme, and that for the aberrant species was similar to the liver enzyme, but with some notable exceptions in both cases. Thus, the normal species had no tryptophan and two half-cystine residues/subunit; whereas, there was 1 tryptophan and 4 half-cystine residues/subunit of the aberrant molecule. The amino acid composition of both serum isozymes when compared to their respective muscle or liver-type enzyme differed mainly in the content of Glu, Asp, His, Leu, Ile, Gly. Kinetic properties of the two forms

  14. Na+/H+ exchanger isoform 1 induced cardiomyocyte hypertrophy involves activation of p90 ribosomal s6 kinase.

    PubMed

    Jaballah, Maiy; Mohamed, Iman A; Alemrayat, Bayan; Al-Sulaiti, Fatima; Mlih, Mohamed; Mraiche, Fatima

    2015-01-01

    Studies using pharmacological and genetic approaches have shown that increased activity/expression of the Na+/H+ exchanger isoform 1 (NHE1) play a critical role in the pathogenesis of cardiac hypertrophy. Despite the importance of NHE1 in cardiac hypertrophy, severe cerebrovascular side effects were associated with the use of NHE1 inhibitors when administered to patients with myocardial infarctions. p90 ribosomal S6 Kinase (RSK), a downstream regulator of the mitogen-activated protein kinase pathway, has also been implicated in cardiac hypertrophy. We hypothesized that RSK plays a role in the NHE1 induced cardiomyocyte hypertrophic response. Infection of H9c2 cardiomyoblasts with the active form of the NHE1 adenovirus induced hypertrophy and was associated with an increase in the phosphorylation of RSK (P<0.05). Parameters of hypertrophy such as cell area, protein content and atrial natriuretic mRNA expression were significantly reduced in H9c2 cardiomyoblasts infected with active NHE1 in the presence of dominant negative RSK (DN-RSK) (P<0.05). These results confirm that NHE1 lies upstream of RSK. Increased phosphorylation and activation of GATA4 at Ser261 was correlated with increased RSK phosphorylation. This increase was reversed upon inhibition of RSK or NHE1. These findings demonstrate for the first time that the NHE1 mediated hypertrophy is accounted for by increased activation and phosphorylation of RSK, which subsequently increased the phosphorylation of GATA4; eventually activating fetal gene transcriptional machinery. PMID:25830299

  15. Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea.

    PubMed

    Syam Prakash, S R; Jayabaskaran, Chelliah

    2006-11-01

    In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as Ca(2+)-dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of Ca(2+). The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including Ca(2+) sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8-8.6 and 35-42 degrees C, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2-9 and 35-42 degrees C, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in Ca(2+)-mediated signaling in chickpea plants.

  16. Na+/K+ATPase Regulates Sperm Capacitation Through a Mechanism Involving Kinases and Redistribution of Its Testis-Specific Isoform

    PubMed Central

    NEWTON, LARISSA D.; KRISHNAKUMAR, SULOCHANA; MENON, AJITKUMAR GOPINADHA; KASTELIC, JOHN P.; VAN DER HOORN, FRANS A.; THUNDATHIL, JACOB C.

    2016-01-01

    SUMMARY Incubation of bovine sperm with ouabain, an endogenous cardiac glycoside that inhibits both the ubiquitous (ATP1A1) and testis-specific α4 (ATP1A4) isoforms of Na+/K+ATPase, induces tyrosine phosphorylation and capacitation. The objectives of this study were to investigate: (1) fertilizing ability of bovine sperm capacitated by incubating with ouabain; (2) involvement of ATP1A4 in this process; and (3) signaling mechanisms involved in the regulation of sperm capacitation induced by inhibition of Na+/K+ATPase activity. Fresh sperm capacitated by incubating with ouabain (inhibits both ATP1A1 and ATP1A4) or with anti-ATP1A4 immunoserum fertilized bovine oocytes in vitro. Capacitation was associated with relocalization of ATP1A4 from the entire sperm head to the post-acrosomal region. To investigate signaling mechanisms involved in oubain-induced regulation of sperm capacitation, sperm preparations were pre-incubated with inhibitors of specific signaling molecules, followed by incubation with ouabain. The phosphotyrosine content of sperm preparations was determined by immunoblotting, and capacitation status of these sperm preparations were evaluated through an acrosome reaction assay. We inferred that Na+/K+ATPase was involved in the regulation of tyrosine phosphorylation in sperm proteins through receptor tyrosine kinase, nonreceptor type protein kinase, and protein kinases A and C. In conclusion, inhibition of Na+/K+ATPase induced tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. To our knowledge, this is the first report documenting both the involvement of ATP1A4 in the regulation of bovine sperm capacitation and that fresh bovine sperm capacitated by the inhibition of Na+/K+ATPase can fertilize oocytes in vitro. PMID:19834983

  17. Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform1[w

    PubMed Central

    Turner, William L.; Waller, Jeffrey C.; Vanderbeld, Barb; Snedden, Wayne A.

    2004-01-01

    NAD kinase (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD. The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge. We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively. Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs. Transcripts for both isoforms were detected in all tissues examined and throughout development. Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants. Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)-affinity chromatography, we delineated a Ca2+-dependent CaM-binding domain to a 45-residue region within the N-terminal extension of NADK2. Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein. In Arabidopsis crude extracts, CaM-dependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings. A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (KmNAD = 0.20 mM, KmMg2+−ATP = 0.17 mM). The enzyme was fully activated by conserved CaM (S0.5 = 2.2 nm) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins. Possible roles for NADKs in plants are discussed in light of our observations. PMID:15247403

  18. Non–Muscle Myosin Light Chain Kinase Isoform Is a Viable Molecular Target in Acute Inflammatory Lung Injury

    PubMed Central

    Mirzapoiazova, Tamara; Moitra, Jaideep; Moreno-Vinasco, Liliana; Sammani, Saad; Turner, Jerry R.; Chiang, Eddie T.; Evenoski, Carrie; Wang, Ting; Singleton, Patrick A.; Huang, Yong; Lussier, Yves A.; Watterson, D. Martin; Dudek, Steven M.; Garcia, Joe G. N.

    2011-01-01

    Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non–muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (∼50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody–conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6–signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation. PMID:20139351

  19. Pyruvate dehydrogenase kinase isoform 2 activity stimulated by speeding up the rate of dissociation of ADP.

    PubMed

    Bao, Haiying; Kasten, Shane A; Yan, Xiaohua; Hiromasa, Yasuaki; Roche, Thomas E

    2004-10-26

    Pyruvate dehydrogenase kinase 2 (PDK2) activity is stimulated by NADH and NADH plus acetyl-CoA via the reduction and reductive acetylation of the lipoyl groups of the dihydrolipoyl acetyltransferase (E2) component. Elevated K(+) and Cl(-) were needed for significant stimulation. Stimulation substantially increased both k(cat) and the K(m) for ATP; the fractional stimulation increased with the level of ATP. With an E2 structure lacking the pyruvate dehydrogenase (E1) binding domain, stimulation of PDK2 was retained, the K(m) for E1 decreased, and the equilibrium dissociation constant for ATP increased but remained much lower than the K(m) for ATP. Stimulation of PDK2 activity greatly reduced the fraction of bound ADP. These results fit an ordered reaction mechanism with ATP binding before E1 and stimulation increasing the rate of dissociation of ADP. Conversion of all of the lipoyl groups in the E2 60mer to the oxidized form (E2(ox)) greatly reduced k(cat) and the K(m) of PDK2 for ATP. Retention over an extended period of time of a low portion of reduced lipoyl groups maintains E2 in a state that supported much higher PDK2 activity than short-term (5 min) reduction of a large portion of lipoyl groups of E2(ox), but reduction of E2(ox) produced a larger fold stimulation. Reduction and to a greater extent reductive acetylation increased PDK2 binding to E2; conversion to E2(ox) did not significantly hinder binding. We suggest that passing even limited reducing equivalents among lipoyl groups maintains E2 lipoyl domains in a conformation that aids kinase function. PMID:15491151

  20. A single residue substitution accounts for the significant difference in thermostability between two isoforms of human cytosolic creatine kinase

    PubMed Central

    Liu, Huihui; Gao, Yan-Song; Chen, Xiang-Jun; Chen, Zhe; Zhou, Hai-Meng; Yan, Yong-Bin; Gong, Haipeng

    2016-01-01

    Creatine kinase (CK) helps maintain homeostasis of intracellular ATP level by catalyzing the reversible phosphotransfer between ATP and phosphocreatine. In humans, there are two cytosolic CK isoforms, the muscle-type (M) and the brain-type (B), which frequently function as homodimers (hMMCK and hBBCK). Interestingly, these isoenzymes exhibit significantly different thermostabilities, despite high similarity in amino acid sequences and tertiary structures. In order to investigate the mechanism of this phenomenon, in this work, we first used domain swapping and site-directed mutagenesis to search for the key residues responsible for the isoenzyme-specific thermostability. Strikingly, the difference in thermostability was found to principally arise from one single residue substitution at position 36 (Pro in hBBCK vs. Leu in hMMCK). We then engaged the molecular dynamics simulations to study the molecular mechanism. The calculations imply that the P36L substitution introduces additional local interactions around residue 36 and thus further stabilizes the dimer interface through a complex interaction network, which rationalizes the observation that hMMCK is more resistant to thermal inactivation than hBBCK. We finally confirmed this molecular explanation through thermal inactivation assays on Asp36 mutants that were proposed to devastate the local interactions and thus the dimer associations in both isoenzymes. PMID:26879258

  1. Switching of pyruvate kinase isoform L to M2 promotes metabolic reprogramming in hepatocarcinogenesis.

    PubMed

    Wong, Carmen Chak-Lui; Au, Sandy Leung-Kuen; Tse, Aki Pui-Wah; Xu, Iris Ming-Jing; Lai, Robin Kit-Ho; Chiu, David Kung-Chun; Wei, Larry Lai; Fan, Dorothy Ngo-Yin; Tsang, Felice Ho-Ching; Lo, Regina Cheuk-Lam; Wong, Chun-Ming; Ng, Irene Oi-Lin

    2014-01-01

    Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming. PMID:25541689

  2. Bisphenol A differentially activates protein kinase C isoforms in murine placental tissue.

    PubMed

    Tan, Wenjuan; Huang, Hui; Wang, Yanfei; Wong, Tsz Yan; Wang, C C; Leung, Lai K

    2013-06-01

    Bisphenol A is utilized to make polycarbonate plastics and is an environmental pollutant. Recent research has indicated that it is an endocrine disruptor and may interfere with reproductive processes. Our lab has previously shown that bisphenol A could regulate corticotrophin releasing hormone and aromatase in cultured placental cells. In the present study, the effect of bisphenol A on these two genes in the placenta was investigated in mice. Pregnant ICR mice were gavaged with bisphenol A at 2, 20 and 200mg/kg body weight/day from E13 to E16 and were euthanized at E17. Compared to the control mice, increased plasma estrogen and corticotrophin releasing hormone were observed in bisphenol A-treated mice. Messenger RNA quantification indicated that placental crh but not cyp19 was induced in mice treated with bisphenol A. Tracking the related signaling pathway, we found that protein kinase C ζ/λ and δ were activated in the placentas of bisphenol A-treated mice. As the gene promoter of crh contains CRE and the half site of ERE, either phospho-PKC or estrogen could stimulate the gene transactivation. These results indicate that bisphenol A might increase plasma concentrations of estradiol, testosterone, corticotrophin releasing hormone and placental phospho-PKC ζ/λ and δ in mice. Ultimately, the incidence of premature birth in these mice could increase.

  3. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    NASA Technical Reports Server (NTRS)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  4. Adenylate-forming enzymes.

    PubMed

    Schmelz, Stefan; Naismith, James H

    2009-12-01

    Thioesters, amides, and esters are common chemical building blocks in a wide array of natural products. The formation of these bonds can be catalyzed in a variety of ways. For chemists, the use of an activating group is a common strategy and adenylate enzymes are exemplars of this approach. Adenylating enzymes activate the otherwise unreactive carboxylic acid by transforming the normal hydroxyl leaving group into adenosine monophosphate. Recently there have been a number of studies of such enzymes and in this review we suggest a new classification scheme. The review highlights the diversity in enzyme fold, active site architecture, and metal coordination that has evolved to catalyze this particular reaction. PMID:19836944

  5. Stimulatory action of protein kinaseisoform on the slow component of delayed rectifier K+ current in guinea-pig atrial myocytes

    PubMed Central

    Toda, H; Ding, W-G; Yasuda, Y; Toyoda, F; Ito, M; Matsuura, H; Horie, M

    2007-01-01

    Background and purpose: Protein kinase C (PKC) comprises at least twelve isoforms and has an isoform-specific action on cardiac electrical activity. The slow component of delayed rectifier K+ current (I Ks) is one of the major repolarizing currents in the hearts of many species and is also potentiated by PKC activation. Little is known, however, about PKC isoform(s) functionally involved in the potentiation of I Ks in native cardiac myocytes. Experimental approach: I Ks was recorded from guinea-pig atrial myocytes, using the whole-cell configuration of patch-clamp method. Key results: Bath application of phenylephrine enhanced I Ks concentration-dependently with EC50 of 5.4 μM and the maximal response (97.1±11.9% increase, n=16) was obtained at 30 μM. Prazosin (1 μM) almost totally abolished the potentiation of I Ks by phenylephrine, supporting the involvement of α1-adrenoceptors. The stimulatory action of phenylephrine was significantly, if not entirely, inhibited by the general PKC inhibitor bisindolylmaleimide I but was little affected by Gö-6976, Gö-6983 and rottlerin. Furthermore, this stimulatory effect was significantly reduced by dialyzing atrial myocytes with PKCɛ-selective inhibitory peptide ɛV1-2 but was not significantly affected by conventional PKC isoform-selective inhibitory peptide βC2-4. Phorbol 12-myristate 13-acetate (PMA) at 100 nM substantially increased I Ks by 64.2±1.3% (n=6), which was also significantly attenuated by an internal dialysis with ɛV1-2 but not with βC2-4. Conclusions and implications: The present study provides experimental evidence to suggest that, in native guinea-pig cardiac myocytes, activation of PKC contributes to α1-adrenoceptor-mediated potentiation of I Ks and that ɛ is the isoform predominantly involved in this PKC action. PMID:17339832

  6. Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

    PubMed Central

    Hebras, Celine; McDougall, Alex

    2012-01-01

    Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and

  7. HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction.

    PubMed Central

    Gil, C; Chaib-Oukadour, I; Blasi, J; Aguilera, J

    2001-01-01

    A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn(2+)-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)gamma-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (H(C)-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-alpha, -beta, -gamma and -delta isoforms exists, whereas PKC-epsilon showed a slight decrease in its soluble fraction immunoreactivity. The PKC-zeta isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCgamma-1 and ERK-1/2. The effects shown by the H(C)-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF. The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr(490), and antibodies against Thr/Tyr phosphorylated ERK-1/2. Moreover, PLCgamma-1 phosphorylation was supported by its H(C)-TeTx-induced translocation to the membranous compartment, an event related to PLCgamma-1 activation. Since H(C)-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component. PMID:11336640

  8. Protein Kinase C (PKC)ζ Pseudosubstrate Inhibitor Peptide Promiscuously Binds PKC Family Isoforms and Disrupts Conventional PKC Targeting and Translocation

    PubMed Central

    Bogard, Amy S.

    2015-01-01

    PKMζ is generated via an alternative transcriptional start site in the atypical protein kinase C (PKC)ζ isoform, which removes N-terminal regulatory elements, including the inhibitory pseudosubstrate domain, consequently rendering the kinase constitutively active. Persistent PKMζ activity has been proposed as a molecular mechanism for the long-term maintenance of synaptic plasticity underlying some forms of memory. Many studies supporting a role for PKMζ in synaptic plasticity and memory have relied on the PKCζ pseudosubstrate-derived ζ-inhibitory peptide (ZIP). However, recent studies have demonstrated that ZIP-induced impairments to synaptic plasticity and memory occur even in the absence of PKCζ, suggesting that ZIP exerts its actions via additional cellular targets. In this study, we demonstrated that ZIP interacts with conventional and novel PKC, in addition to atypical PKC isoforms. Moreover, when brain abundance of each PKC isoform and affinity for ZIP are taken into account, the signaling capacity of ZIP-responsive pools of conventional and novel PKCs may match or exceed that for atypical PKCs. Pseudosubstrate-derived peptides, like ZIP, are thought to exert their cellular action primarily by inhibiting PKC catalytic activity; however, the ZIP-sensitive catalytic core of PKC is known to participate in the enzyme’s subcellular targeting, suggesting an additional mode of ZIP action. Indeed, we have demonstrated that ZIP potently disrupts PKCα interaction with the PKC-targeting protein A-kinase anchoring protein (AKAP) 79 and interferes with ionomycin-induced translocation of conventional PKC to the plasma membrane. Thus, ZIP exhibits broad-spectrum action toward the PKC family of enzymes, and this action may contribute to its unique ability to impair memory. PMID:26199377

  9. 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase

    PubMed Central

    Walsh, Martin J.; Brimacombe, Kyle R.; Veith, Henrike; Bougie, James M.; Daniel, Thomas; Leister, William; Cantley, Lewis C.; Israelsen, William J.; Vander Heiden, Matthew G.; Shen, Min; Auld, Douglas S.; Thomas, Craig J.; Boxer, Matthew B.

    2011-01-01

    Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described. PMID:21958545

  10. Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury

    PubMed Central

    Niyitegeka, Jean-Marie V.; Bastidas, Adam C.; Newman, Robert H.; Taylor, Susan S.

    2014-01-01

    Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αβ double knockout mice, and purified forms of recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cβ1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cβ1, and Cβ2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways. PMID:25354939

  11. Neurite outgrowth of neuroblastoma cells overexpressing alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II-effects of protein kinase inhibitors.

    PubMed

    Yamauchi, T; Yoshimura, Y; Nomura, T; Fujii, M; Sugiura, H

    1998-06-01

    Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the most abundant protein kinases in the brain and has a broad substrate specificity [M.K. Bennett, N.E. Erondu, M.B. Kennedy, Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem. 258 (1983) 12735-12744 [1]; J.R. Goldenring, B. Gonzalez, J.S. McGuire, Jr., R.J. DeLorenzo, Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins, J. Biol. Chem. 258 (1983) 12632-12640 [4]; M.B. Kennedy, P. Greengard, Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1293-1297 [10]; T. Yamauchi, H. Fujisawa, Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain, FEBS Lett. 116 (1980) 141-144 [20]; T. Yamauchi, H. Fujisawa, Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase, Eur. J. Biochem. 132 (1983) 15-21 [21

  12. Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3 Single Amino Acid near the Active Site Influences Inhibitor Sensitivity

    SciTech Connect

    Hong, Bum Soo; Allali-Hassani, Abdellah; Tempel, Wolfram; Finerty, Jr., Patrick J.; MacKenzie, Farrell; Dimov, Svetoslav; Vedadi, Masoud; Park, Hee-Won

    2010-07-06

    Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoK{alpha} ({alpha}1 and {alpha}2) and ChoK{beta} isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoK{alpha}1 and ChoK{beta} isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoK{alpha}1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoK{beta} was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoK{alpha} isoforms ({alpha}1 and {alpha}2) compared with ChoK{beta}. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoK{alpha}2 (equivalent to Leu-419 of ChoK{alpha}1), or the corresponding residue Phe-352 of ChoK{beta}, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

  13. The ζ isoform of diacylglycerol kinase plays a predominant role in regulatory T cell development and TCR-mediated ras signaling.

    PubMed

    Joshi, Rohan P; Schmidt, Amanda M; Das, Jayajit; Pytel, Dariusz; Riese, Matthew J; Lester, Melissa; Diehl, J Alan; Behrens, Edward M; Kambayashi, Taku; Koretzky, Gary A

    2013-11-26

    Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)-stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGKα and DGKζ, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGKζ, but not DGKα, suppressed the development of natural regulatory T (T(reg)) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGKα and DGKζ were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGKζ than with DGKα; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGKζ than for DGKα might lead to DGKζ exhibiting increased suppression of Ras-mediated signals compared to DGKα. Consistent with this notion, experimental studies demonstrated that DGKζ was more effective than DGKα at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGKζ is therefore one possible mechanism underlying the dominant functions of DGKζ in modulating T(reg) cell development.

  14. The ζ Isoform of Diacylglycerol Kinase Plays a Predominant Role in Regulatory T Cell Development and TCR-Mediated Ras Signaling

    PubMed Central

    Joshi, Rohan P.; Schmidt, Amanda M.; Das, Jayajit; Pytel, Dariusz; Riese, Matthew J.; Lester, Melissa; Diehl, J. Alan; Behrens, Edward M.; Kambayashi, Taku; Koretzky, Gary A.

    2014-01-01

    Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)–stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGKα and DGKζ, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGKζ, but not DGKα, suppressed the development of natural regulatory T (Treg) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGKα and DGKζ were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGKζ than with DGKα; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGKζ than for DGKα might lead to DGKζ exhibiting increased suppression of Ras-mediated signals compared to DGKα. Consistent with this notion, experimental studies demonstrated that DGKζ was more effective than DGKα at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGKζ is therefore one possible mechanism underlying the dominant functions of DGKζ in modulating Treg cell development. PMID:24280043

  15. Differential activity profile of cAMP-dependent protein kinase isoforms during long-term memory consolidation in the crab Chasmagnathus.

    PubMed

    Locatelli, Fernando; Romano, Arturo

    2005-05-01

    The isoforms of cAMP-dependent protein kinase (PKA) show distinct biochemical properties and subcellular localization, suggesting different physiological functions, and conferring the fine-tuning between the activation of cAMP-PKA cascade and the cellular response. The critical role of PKA in memory and synaptic plasticity has been extensively demonstrated both in vertebrates and invertebrates, but the role of PKA isoforms is a matter of debate. Here we present experimental data showing differential PKA activation profiles after two different experiences: an instance of associative contextual learning (context-signal learning) and a single exposure to a novel context, both in the learning and memory model of the crab Chasmagnathus. Differences were found in the temporal course of activation and in the involvement of PKA isoforms. We found increased PKA activity immediately and 6 h after context-signal training correlating with the critical periods during which pharmacological inhibition of PKA disrupts memory formation. In contrast, PKA activity increased immediately but not 6 h after single exposure to a novel context. The amounts of PKA I and PKA II holoenzymes were analyzed to determine changes in holoenzyme levels and/or differential activation induced by both experiences. Results indicate that context-induced PKA activation is at least in part due to PKA II, and that PKA activation 6 h after context-signal learning coincides with an increase in the total level of PKA I. Considering the higher sensitivity of PKA I to cAMP, its increment can account for the PKA activation found 6 h after training and is proposed as a novel mechanism providing the prolonged PKA activation during memory consolidation.

  16. Structure–Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants

    PubMed Central

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R.; Gokhale, Vijay; Brown, Mary E.; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M.; Wang, Ting; Garcia, Joe G. N.

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities. PMID:26111161

  17. Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

    PubMed

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R; Gokhale, Vijay; Brown, Mary E; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M; Wang, Ting; Garcia, Joe G N

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

  18. Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells.

    PubMed

    Wuttke, Anne; Yu, Qian; Tengholm, Anders

    2016-07-15

    PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca(2+)- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations ("spiking") at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K(+) depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca(2+) concentration ([Ca(2+)]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca(2+)]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca(2+)]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function.

  19. Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells*

    PubMed Central

    Wuttke, Anne; Yu, Qian; Tengholm, Anders

    2016-01-01

    PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca2+- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations (“spiking”) at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K+ depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca2+ concentration ([Ca2+]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca2+]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca2+]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function. PMID:27226533

  20. Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

    PubMed

    Kuby, S A; Hamada, M; Johnson, M S; Russell, G A; Manship, M; Palmieri, R H; Fleming, G; Bredt, D S; Mildvan, A S

    1989-08-01

    Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for

  1. Troponin I Assessment of Cardiac Involvement in Patients With Connective Tissue Disease and an Elevated Creatine Kinase MB Isoform Report of Four Cases and Review of the Literature.

    PubMed

    Badsha, H; Gunes, B; Grossman, J; Brahn, E

    1997-06-01

    Levels of creatine kinase MB isoform (CKMB) can be elevated in patients with myopathy, neuropathy, skeletal muscle injury, or renal failure in the absence of myocardial injury. These elevated CKMB levels make it difficult to identify cardiac involvement in conditions that can be associated with a variety of cardiac abnormalities or with symptoms that mimic them. Cardiac troponin I (cTnI), a myocardial regulatory protein, has a high specificity for cardiac muscle and can be used to clarify the etiology of CKMB elevations in such patients. In this report, four patients with diverse causes for increased CKMB levels are discussed with respect to cill.The first three patients, with tentative diagnoses of mixed connective tissue disease, amyotrophic lateral sclerosis, and polymyositis presented with increasing shortness of breath, tachycardia, nonspecific electrocardiogram changes, high creative kinase, and CKMB levels. A normal cTnI helped exclude a diagnosis of a cardiac cause of their symptoms. Patient 4 had a scleroderma variant and experienced sudden, fatal, cardiac decompensation caused by a dilated cardiomyopathy, accompanied by an increased cTnl.The cTnI is a reliable, specific, and quick wav of excluding or determining cardiac involvement in patients with connective tissue disease. As this test is inexpensive and becoming increasingly available, it could become the test of choice, especially in scenarios in which urgent management decisions are needed.

  2. Presence of enolase in the M-band of skeletal muscle and possible indirect interaction with the cytosolic muscle isoform of creatine kinase.

    PubMed Central

    Foucault, G; Vacher, M; Merkulova, T; Keller, A; Arrio-Dupont, M

    1999-01-01

    Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both alpha (ubiquitous) and beta (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or beta-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres. PMID:9931306

  3. The high-resolution crystal structure of phosphatidylinositol 4-kinase IIβ and the crystal structure of phosphatidylinositol 4-kinase IIα containing a nucleoside analogue provide a structural basis for isoform-specific inhibitor design.

    PubMed

    Klima, Martin; Baumlova, Adriana; Chalupska, Dominika; Hřebabecký, Hubert; Dejmek, Milan; Nencka, Radim; Boura, Evzen

    2015-07-01

    Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIβ and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIβ and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9 Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.

  4. An integrin-targeted, pan-isoform, phosphoinositide-3 kinase inhibitor, SF1126, has activity against multiple myeloma in vivo

    PubMed Central

    De, Pradip; Dey, Nandini; Terakedis, Breanne; Bersagel, Leif; Li, Zhi Hua; Mahadevan, Daruka; Garlich, Joseph R.; Trudel, Suzanne; Makale, Milan T.; Durden, Donald L.

    2013-01-01

    Purpose Multiple reports point to an important role for the phosphoinositide-3 kinase (PI3K) and AKT signaling pathways in tumor survival and chemoresistance in multiple myeloma (MM). The goals of our study were: (1) to generate the preclinical results necessary to justify a Phase I clinical trial of SF1126 in hematopoietic malignancies including multiple myeloma, and (2) to begin combining pan PI-3 kinase inhibitors with other agents to augment antitumor activity of this class of agent in preparation for combination therapy in Phase I/II trials. Methods We determined the in vitro activity of SF1126 with16 human MM cell lines. In vivo tumor growth suppression was determined with human myeloma (MM.1R) xenografts in athymic mice. In addition, we provide evidence that SF1126 has pharmacodynamic activity in the treatment of patients with MM. Results SF1126 was cytotoxic to all tested MM lines and potency was augmented by the addition of bortezomib. SF1126 affected MM.1R cell line signaling in vitro, inhibiting phospho-AKT, phospho-ERK, and the hypoxic stabilization of HIF1α. Tumor growth was 94% inhibited, with a marked decrease in both cellular proliferation (PCNA immunostaining) and angiogenesis (tumor microvessel density via CD31 immunostaining). Our clinical results demonstrate pharmacodynamic knockdown of p-AKT in primary patient derived MM tumor cells in vivo. Conclusions Our results establish three important points: (1) SF1126, a pan PI-3 kinase inhibitor has potent antitumor activity against multiple myeloma in vitro and in vivo, (2) SF1126 displays augmented antimyeloma activity when combined with proteasome inhibitor, bortezomib/Velcade®, and (3) SF1126 blocks the IGF-1 induced activation of AKT in primary MM tumor cells isolated from SF1126 treated patients The results support the ongoing early Phase I clinical trial in MM and suggest a future Phase I trial in combination with bortezomib in hematopoietic malignancies. PMID:23355037

  5. Distinct functionality of dishevelled isoforms on Ca2+/calmodulin-dependent protein kinase 2 (CamKII) in Xenopus gastrulation

    PubMed Central

    Gentzel, Marc; Schille, Carolin; Rauschenberger, Verena; Schambony, Alexandra

    2015-01-01

    Wnt ligands trigger the activation of a variety of β-catenin–dependent and β-catenin–independent intracellular signaling cascades. Despite the variations in intracellular signaling, Wnt pathways share the effector proteins frizzled, dishevelled, and β-arrestin. It is unclear how the specific activation of individual branches and the integration of multiple signals are achieved. We hypothesized that the composition of dishevelled–β-arrestin protein complexes contributes to signal specificity and identified CamKII as an interaction partner of the dishevelled–β-arrestin protein complex by quantitative functional proteomics. Specifically, we found that CamKII isoforms interact differentially with the three vertebrate dishevelled proteins. Dvl1 is required for the activation of CamKII and PKC in the Wnt/Ca2+ pathway. However, CamKII interacts with Dvl2 but not with Dvl1, and Dvl2 is necessary to mediate CamKII function downstream of Dvl1 in convergent extension movements in Xenopus gastrulation. Our findings indicate that the different Dvl proteins and the composition of dishevelled–β-arrestin protein complexes contribute to the specific activation of individual branches of Wnt signaling. PMID:25568338

  6. Differential regulation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) and phosphatidylinositol 3-kinase isoforms in liver and muscle of the obese diabetic (ob/ob) mouse.

    PubMed Central

    Kerouz, N J; Hörsch, D; Pons, S; Kahn, C R

    1997-01-01

    Intracellular insulin signaling involves a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (IRS) and the various isoforms of SH2 domain signaling molecules that can interact with these substrates. In this study, we have evaluated the roles of IRS-1 and IRS-2 in signaling to the phosphatidylinositol (PI) 3-kinase pathway in the ob/ob mouse, a model of the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. We find that the levels of expression of both IRS-1 and IRS-2 are decreased approximately 50% in muscle, whereas in liver the decrease is significantly greater for IRS-2 (72%) than for IRS-1 (29%). This results in differential decreases in IRS-1 and IRS-2 phosphorylation, docking of the p85alpha regulatory subunit of PI 3-kinase, and activation of this enzyme in these two insulin target tissues. In ob/ob liver there is also a change in expression of the alternatively spliced isoforms of the regulatory subunits for PI 3-kinase that was detected at the protein and mRNA level. This resulted in a 45% decrease in the p85alpha form of PI 3-kinase, a ninefold increase in the AS53/p55alpha, and a twofold increase in p50alpha isoforms. Thus, there are multiple alterations in the early steps of insulin signaling in the ob/ob mouse, with differential regulation of IRS-1 and IRS-2, various PI 3-kinase regulatory isoforms, and a lack of compensation for the decrease in insulin signaling by any of the known alternative pathways at these levels. PMID:9399964

  7. Activation of medullary dorsal horn γ isoform of protein kinase C interneurons is essential to the development of both static and dynamic facial mechanical allodynia.

    PubMed

    Pham-Dang, Nathalie; Descheemaeker, Amélie; Dallel, Radhouane; Artola, Alain

    2016-03-01

    The γ isoform of protein kinase C (PKCγ), which is concentrated in a specific class of interneurons within inner lamina II (IIi ) of the spinal dorsal horn and medullary dorsal horn (MDH), is known to be involved in the development of mechanical allodynia, a widespread and intractable symptom of inflammatory or neuropathic pain. However, although genetic and pharmacological impairment of PKCγ were shown to prevent mechanical allodynia in animal models of pain, after nerve injury or reduced inhibition, the functional consequences of PKCγ activation alone on mechanical sensitivity are still unknown. Using behavioural and anatomical approaches in the rat MDH, we tested whether PKCγ activation in naive animals is sufficient for the establishment of mechanical allodynia. Intracisternal injection of the phorbol ester, 12,13-dibutyrate concomitantly induced static as well as dynamic facial mechanical allodynia. Monitoring neuronal activity within the MDH with phospho-extracellular signal-regulated kinases 1 and 2 immunoreactivity revealed that activation of both lamina I-outer lamina II and IIi -outer lamina III neurons, including lamina IIi PKCγ-expressing interneurons, was associated with the manifestation of mechanical allodynia. Phorbol ester, 12,13-dibutyrate-induced mechanical allodynia and associated neuronal activations were all prevented by inhibiting selectively segmental PKCγ with KIG31-1. Our findings suggest that PKCγ activation, without any other experimental manipulation, is sufficient for the development of static and dynamic mechanical allodynia. Lamina IIi PKCγ interneurons have been shown to be directly activated by low-threshold mechanical inputs carried by myelinated afferents. Thus, the level of PKCγ activation within PKCγ interneurons might gate the transmission of innocuous mechanical inputs to lamina I, nociceptive output neurons, thus turning touch into pain.

  8. VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and induces context-dependent cytostasis in cancer cells.

    PubMed

    Moore, Jonathan D; Staniszewska, Anna; Shaw, Terence; D'Alessandro, Jalanie; Davis, Ben; Surgenor, Alan; Baker, Lisa; Matassova, Natalia; Murray, James; Macias, Alba; Brough, Paul; Wood, Mike; Mahon, Patrick C

    2014-12-30

    Pyruvate dehydrogenase kinase (PDK) is a pivotal enzyme in cellular energy metabolism that has previously been implicated in cancer through both RNAi based studies and clinical correlations with poor prognosis in several cancer types. Here, we report the discovery of a novel and selective ATP competitive pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, VER-246608 increased pyruvate dehydrogenase complex (PDC) activity, oxygen consumption and attenuated glycolytic activity. However, these effects were only observed under D-glucose-depleted conditions and required almost complete ablation of PDC E1α subunit phosphorylation. VER-246608 was weakly anti-proliferative to cancer cells in standard culture media; however, depletion of either serum or combined D-glucose/L-glutamine resulted in enhanced cellular potency. Furthermore, this condition-selective cytostatic effect correlated with reduced intracellular pyruvate levels and an attenuated compensatory response involving deamination of L-alanine. In addition, VER-246608 was found to potentiate the activity of doxorubicin. In contrast, the lipoamide site inhibitor, Nov3r, demonstrated sub-maximal inhibition of PDK activity and no evidence of cellular activity. These studies suggest that PDK inhibition may be effective under the nutrient-depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy.

  9. The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

    PubMed

    Kool, Martijn J; van de Bree, Jolet E; Bodde, Hanna E; Elgersma, Ype; van Woerden, Geeske M

    2016-01-01

    Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b(-/-) mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits. However, where these locomotion deficits originate is unknown. Here we made use of novel Camk2b mutants (Camk2b(f/f) and Camk2b(T287A)), to explore the molecular, temporal and brain region-specific requirements of CAMK2B for locomotion. At the molecular level we found that normal locomotion requires Calcium/Calmodulin mediated activation of CAMK2B, but CAMK2B autonomous activity is largely dispensable. At a systems level, we found that global deletion of Camk2b in the adult mouse causes only mild locomotion deficits, suggesting that the severe locomotion deficits of Camk2b(-/-) mice are largely of developmental origin. However, early onset deletion of Camk2b in cerebellum, striatum or forebrain did not recapitulate the locomotion deficits, suggesting that these deficits cannot be attributed to a single brain area. Taken together, these results provide the first insights into the molecular, temporal and region-specific role of CAMK2B in locomotion. PMID:27244486

  10. Atypical Protein Kinase C Isoform, aPKCλ, Is Essential for Maintaining Hair Follicle Stem Cell Quiescence.

    PubMed

    Osada, Shin-Ichi; Minematsu, Naoko; Oda, Fumino; Akimoto, Kazunori; Kawana, Seiji; Ohno, Shigeo

    2015-11-01

    The atypical protein kinase C (aPKC)-partition-defective (PAR) complex regulates the formation of tight junctions and apico-basal epithelial polarity. To examine the role of this complex in the epidermis, we generated mutant mice harboring epidermal-specific deletion of aPKCλ (conditional knock-out (cKO)), a major component of the aPKC-PAR complex. The mutant mice exhibited abnormal hair follicle (HF) cycling, progressive losses of pelage hairs and vibrissae, and altered differentiation into the epidermis and sebaceous gland. We found that in the aPKCλ cKO mice HF stem cell (HFSC) quiescence was lost, as revealed by the decreased expression level of quiescence-inducing factors (Fgf18 and Bmp6) produced in Keratin 6-positive bulge stem cells. The loss of quiescence dysregulated the HFSC marker expression and led to the increase in Lrig1-positive cells, inducing hyperplasia of the interfollicular epidermis and sebaceous glands, and drove an increase in Lef1-positive matrix cells, causing a prolonged anagen-like phase. Persistent bulge stem cell activation led to a gradual depletion of CD34- and α6 integrin-positive HFSC reservoirs. These results suggest that aPKCλ regulates signaling pathways implicated in HFSC quiescence. PMID:26076315

  11. Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP.

    PubMed

    Bao, Haiying; Kasten, Shane A; Yan, Xiaohua; Roche, Thomas E

    2004-10-26

    Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating. PMID:15491150

  12. Pyruvate kinase, muscle isoform 2 promotes proliferation and insulin secretion of pancreatic β-cells via activating Wnt/CTNNB1 signaling

    PubMed Central

    Wang, Suijun; Yang, Zhen; Gao, Ying; Li, Quanzhong; Su, Yong; Wang, Yanfang; Zhang, Yun; Man, Hua; Liu, Hongxia

    2015-01-01

    Failure of pancreatic β-cells is closely associated with type 2 diabetes mellitus (T2DM), an intractable disease affecting numerous patients. Pyruvate kinase, muscle isoform 2 (PKM2) is a potential modulator of insulin secretion in β-cells. This study aims at revealing roles and possible mechanisms of PKM2 in pancreatic β-cells. Mouse pancreatic β-cell line NIT-1 was used for high glucose treatment and PKM2 overexpression by its specific expression vector. Cell proliferation by Thiazolyl blue assay, cell apoptosis by annexin V-fluorescein isothiocyanate/prodium iodide staining and insulin secretion assay by ELISA were performed in each group. The mRNA and protein levels of related factors were analyzed by real-time quantitative PCR and western blot. Results showed that Pkm2 was inhibited under high glucose conditions compared to the untreated cells (P < 0.01). Its overexpression significantly suppressed NIT-1 cell apoptosis (P < 0.01), and induced cell proliferation (P < 0.05) and insulin secretion (P < 0.05). Related factors showed consistent mRNA expression changes. Protein levels of β-catenin (CTNNB1), insulin receptor substrate 1 (IRS1) and IRS2 were all promoted by PKM2 overexpression (P < 0.01), indicating the activated Wnt/CTNNB1 signaling. These results indicated the inductive roles of PKM2 in pancreatic β-cell NIT-1, including promoting cell proliferation and insulin secretion, and inhibiting cell apoptosis, which might be achieved via activating the Wnt/CTNNB1 signaling and downstream factors. This study offers basic information on the role and mechanism of PKM2 in pancreatic β-cells, and lays the foundation for using PKM2 as a potential therapeutic target in T2DM. PMID:26823761

  13. Glycogen synthase kinase 3{beta} regulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    SciTech Connect

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-10-01

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 {beta} (GSK3{beta}) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3{beta} has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3{beta} (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3{beta} delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3{beta} is required for the activation of NFAT during wound repair.

  14. Knockdown of the M2 Isoform of Pyruvate Kinase (PKM2) with shRNA Enhances the Effect of Docetaxel in Human NSCLC Cell Lines In Vitro

    PubMed Central

    Yuan, Sujuan; Zhuang, Xibing; Chen, Wei; Xing, Na; Zhang, Qi

    2016-01-01

    Purpose The aim of our study was to explore the relationships between the M2 isoform of pyruvate kinase (PKM2) and the sensitivity of human non-small cell lung cancer (NSCLC) cells to docetaxel in vitro. Materials and Methods With the method of plasmid transfection, we silenced the expression of PKM2 successfully in A549 and H460 cells. Western blotting and real-time PCR were applied to detect PKM2 expression at protein and gene levels. Cell viability was examined by CCK8 assay. Cell cycle distribution and apoptosis were examined by flow cytometry. P21 and Bax were detected. Results Expression of PKM2 mRNA and protein were significantly decreased by shRNA targeting PKM2. Silencing of PKM2 increased docetaxel sensitivity of human NSCLC A549 and H460 cells in a collaborative manner, resulting in strong suppression of cell viability. The results of flow cytometric assays suggested that knockdown of PKM2 or docetaxel treatment, whether used singly or in combination, blocked the cells in the G2/M phase, which is in consistent with the effect of the two on the expression of p21. Cells with PKM2 silencing were more likely to be induced into apoptosis by docetaxel although knockdown of PKM2 alone can't induce apoptosis significantly, which is in consistent with the effect of the two on Bax expression. Conclusion The results suggest that PKM2 knockdown could serve as a chemosensitizer to docetaxel in non-small lung cancer cells through targeting PKM2, leading to inhibition of cell viability, increase of cell arrest of G2/M phase and apoptosis. PMID:27593857

  15. Distinct Activation Mechanisms of NF-κB Regulator Inhibitor of NF-κB Kinase (IKK) by Isoforms of the Cell Death Regulator Cellular FLICE-like Inhibitory Protein (cFLIP)*

    PubMed Central

    Baratchian, Mehdi; Davis, Christopher A.; Shimizu, Akira; Escors, David; Bagnéris, Claire; Barrett, Tracey; Collins, Mary K.

    2016-01-01

    The viral FLICE-like inhibitory protein (FLIP) protein from Kaposi sarcoma-associated herpesvirus activates the NF-κB pathway by forming a stable complex with a central region (amino acids 150–272) of the inhibitor of NF-κB kinase (IKK) γ subunits, thereby activating IKK. Cellular FLIP (cFLIP) forms are also known to activate the NF-κB pathway via IKK activation. Here we demonstrate that cFLIPL, cFLIPS, and their proteolytic product p22-FLIP all require the C-terminal region of NEMO/IKKγ (amino acids 272–419) and its ubiquitin binding function for activation of the IKK kinase (or kinase complex), but none form a stable complex with IKKγ. Our results further reveal that cFLIPL requires the linear ubiquitin chain assembly complex and the kinase TAK1 for activation of the IKK kinase. Similarly, cFLIPS and p22-FLIP also require TAK1 but do not require LUBAC. In contrast, these isoforms are both components of complexes that incorporate Fas-associated death domain and RIP1, which appear essential for kinase activation. This conservation of IKK activation among the cFLIP family using different mechanisms suggests that the mechanism plays a critical role in their function. PMID:26865630

  16. Distinct Activation Mechanisms of NF-κB Regulator Inhibitor of NF-κB Kinase (IKK) by Isoforms of the Cell Death Regulator Cellular FLICE-like Inhibitory Protein (cFLIP).

    PubMed

    Baratchian, Mehdi; Davis, Christopher A; Shimizu, Akira; Escors, David; Bagnéris, Claire; Barrett, Tracey; Collins, Mary K

    2016-04-01

    The viral FLICE-like inhibitory protein (FLIP) protein from Kaposi sarcoma-associated herpesvirus activates the NF-κB pathway by forming a stable complex with a central region (amino acids 150-272) of the inhibitor of NF-κB kinase (IKK) γ subunits, thereby activating IKK. Cellular FLIP (cFLIP) forms are also known to activate the NF-κB pathway via IKK activation. Here we demonstrate that cFLIPL, cFLIPS, and their proteolytic product p22-FLIP all require the C-terminal region of NEMO/IKKγ (amino acids 272-419) and its ubiquitin binding function for activation of the IKK kinase (or kinase complex), but none form a stable complex with IKKγ. Our results further reveal that cFLIPLrequires the linear ubiquitin chain assembly complex and the kinase TAK1 for activation of the IKK kinase. Similarly, cFLIPSand p22-FLIP also require TAK1 but do not require LUBAC. In contrast, these isoforms are both components of complexes that incorporate Fas-associated death domain and RIP1, which appear essential for kinase activation. This conservation of IKK activation among the cFLIP family using different mechanisms suggests that the mechanism plays a critical role in their function.

  17. The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus

    PubMed Central

    Huante-Mendoza, Alejandro; Bravo-Patiño, Alejandro; Valdez-Alarcón, Juan J.; Finlay, B. Brett; Baizabal-Aguirre, Víctor M.

    2015-01-01

    Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus. PMID:26200352

  18. Age-related changes in brain-derived neurotrophic factor and tyrosine kinase receptor isoforms in the hippocampus and hypothalamus in male rats.

    PubMed

    Silhol, M; Bonnichon, V; Rage, F; Tapia-Arancibia, L

    2005-01-01

    A large amount of aging individuals show diminished cognitive and endocrine capabilities. The main brain areas involved in these changes are the hippocampus and hypothalamus, two regions possessing high plasticity and implicated in cognitive and endocrine functions, respectively. Among neurotrophins (considered as genuine molecular mediators of synaptic plasticity), brain-derived neurotrophic factor (BDNF) exhibits in adult rats, the highest concentrations in the hippocampus and hypothalamus. Most of neuronal effects of BDNF are mediated through high-affinity cell surface BDNF tyrosine kinase receptors (TrkB). Different TrkB isoforms are issued by alternative splicing of mRNA encoding for TrkB (trkB mRNA) generating at least three different TrkB receptors with different signaling capabilities. The goal of this study was to examine simultaneously the expression (mRNAs and proteins) of BDNF and its three specific receptors, in the hippocampus and hypothalamus throughout lifespan in rats. We observed that BDNF essentially increased during the first 2 postnatal weeks in the hippocampus and hypothalamus, with no close correlation to its mRNA levels. In these regions, mRNA encoding for BDNF full-length catalytic receptor (trkB.FL mRNA) showed no important changes throughout life but of the mRNA truncated forms of TrkB receptors (trkB.T1 mRNA and trkB.T2 mRNA) trkB.T1 mRNA strongly increased after birth, then remaining stable during aging. trkB.T2 mRNA gradually decreased from 1 postnatal week becoming undetectable in the hippocampus in old-rats. Proteins issued from these mRNAs showed substantial quantitative modifications with aging. From 2 months old, the BDNF full-length catalytic receptor (TrkB.FL) gradually and significantly decreased in the hippocampus and the hypothalamus. Of the truncated forms of TrkB receptors (TrkB.T1 and TrkB.T2) TrkB.T1, which is essentially localized in glial cells, significantly increased from the first postnatal week in the hippocampus

  19. New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response

    PubMed Central

    Taniguchi, Masaya; Okamoto, Ryuji; Ito, Masaaki; Goto, Itaru; Fujita, Satoshi; Konishi, Katsuhisa; Mizutani, Hideo; Dohi, Kaoru; Hartshorne, David J.; Itoh, Takeo

    2015-01-01

    Background & Aims Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated. Methods and Results By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%. Conclusions We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain. PMID:26512720

  20. Exploring the isoform selectivity of TGX-221 related pyrido[1,2-a]pyrimidinone-based Class IA PI 3-kinase inhibitors: synthesis, biological evaluation and molecular modelling.

    PubMed

    Marshall, Andrew J; Lill, Claire L; Chao, Mindy; Kolekar, Sharada V; Lee, Woo-Jeong; Marshall, Elaine S; Baguley, Bruce C; Shepherd, Peter R; Denny, William A; Flanagan, Jack U; Rewcastle, Gordon W

    2015-07-01

    A novel series of TGX-221 analogues was prepared and tested for their potency against the p110α, p110β, and p110δ isoforms of the PI3K enzyme, and in two cellular assays. The biological results were interpreted in terms of a p110β comparative model, in order to account for their selectivity towards this isoform. A CH2NH type linker is proposed to allow binding into the specificity pocket proposed to accommodate the high p110β-selectivity of TGX-221, although there was limited steric tolerance for substituents on the pendant ring with the 2-position most favourable for substitution.

  1. Akt isoforms in vascular disease.

    PubMed

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-08-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease.

  2. Akt isoforms in vascular disease

    PubMed Central

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-01-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease. PMID:25929188

  3. Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets.

    PubMed

    Lever, Robert A; Hussain, Azhar; Sun, Benjamin B; Sage, Stewart O; Harper, Alan G S

    2015-12-01

    Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non

  4. Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets.

    PubMed

    Lever, Robert A; Hussain, Azhar; Sun, Benjamin B; Sage, Stewart O; Harper, Alan G S

    2015-12-01

    Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non

  5. Adenylate cyclase in Arthrospira platensis responds to light through transcription.

    PubMed

    Kashith, M; Keerthana, B; Sriram, S; Ramamurthy, V

    2016-08-19

    Cyclic 3',5' adenosine monophosphate (cAMP) is a ubiquitous signaling molecule, but its role in higher plants was in doubt due to its very low concentration. In this study we wanted to look at the flux of cAMP in response to light in algae, considered to be the more primitive form of photosynthetic organisms. While it did not fluctuate very much in the tested green algae, in the cyanobacterium Arthrospira platensis its level was closely linked to exposure to light. The expression from cyaC, the major isoform of adenylate cyclase was strongly influenced by exposure of the cells to light. There was about 300 fold enhancement of cyaC transcripts in cells exposed to light compared to the transcripts in cells in the dark. Although post-translational regulation of adenylate cyclase activity has been widely known, our studies suggest that transcriptional control could also be an important aspect of its regulation in A. platensis. PMID:27311855

  6. Uridylation and adenylation of RNAs

    PubMed Central

    Song, JianBo; Song, Jun; Mo, BeiXin; Chen, XueMei

    2016-01-01

    The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3′ ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area. PMID:26563174

  7. Uridylation and adenylation of RNAs.

    PubMed

    Song, JianBo; Song, Jun; Mo, BeiXin; Chen, XueMei

    2015-11-01

    The posttranscriptional addition of nontemplated nucleotides to the 3' ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3' ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area. PMID:26563174

  8. Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

    PubMed Central

    May, Victor; Lutz, Eve; MacKenzie, Christopher; Schutz, Kristin C.; Dozark, Kate; Braas, Karen M.

    2010-01-01

    MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gαq/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways. PMID:20093365

  9. Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms

    SciTech Connect

    Kleiveland, Charlotte Ramstad Kassem, Moustapha; Lea, Tor

    2008-05-01

    The conditions used for in vitro differentiation of hMSCs contain substances that affect the activity and expression of cyclooxygenase enzymes (COX1/COX2) and thereby the synthesis of prostanoids. hMSC constitutively produce PGE2 when cultivated in vitro. In this study we have investigated effects of PGE2 on proliferation of hMSC. We here demonstrate that one of the main control molecules in the Wnt pathway, GSK-3{beta}, is phosphorylated at the negative regulatory site ser-9 after treating the cells with PGE2. This phosphorylation is mediated by elevation of cAMP and subsequent activation of PKA. Furthermore, PGE2 treatment leads to enhanced nuclear translocation of {beta}-catenin, thus influencing cell proliferation. The presence of two PKA isoforms, types I and II, prompted us to investigate their individual contribution in PGE2-mediated regulation of proliferation. Specific activation of PKA type II with synthetic cAMP analogues, resulted in enhancement of proliferation. On the other side, we found that treatment of hMSC with high concentrations of PGE2 inhibited cell proliferation by arresting the cells in G{sub 0}/G{sub 1} phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem to have opposing functions in the regulation of proliferation and differentiation in these cells.

  10. The α Isoform of Protein Kinase C Is Involved in Signaling the Response of Desmosomes to Wounding in Cultured Epithelial Cells

    PubMed Central

    Wallis, Sarah; Lloyd, Susan; Wise, Irene; Ireland, Grenham; Fleming, Tom P.; Garrod, David

    2000-01-01

    Initiation of reepithelialization upon wounding is still poorly understood. To enhance this understanding, we focus here on changes in the adhesive state of desmosomes of cultured Madin-Darby canine kidney cells in response to wounding of confluent cell sheets. Previous results show that desmosomal adhesion in Madin-Darby canine kidney cells changes from a calcium-dependent state to calcium independence in confluent cell sheets. We show that this change, which requires culture confluence to develop, is rapidly reversed upon wounding of confluent cell sheets. Moreover, the change to calcium dependence in wound edge cells is propagated to cells hundreds of micrometers away from the wound edge. Rapid transition from calcium independence to calcium dependence also occurs when cells are treated with phorbol esters that activate PKC. PKC inhibitors, including the conventional isoform inhibitor Gö6976, cause rapid transition from calcium dependence to calcium independence, even in subconfluent cells. The cellular location of the α isoform of PKC correlates with the calcium dependence of desmosomes. Upon monolayer wounding, PKCα translocates rapidly to the cell periphery, becomes Triton X-100 insoluble, and also becomes concentrated in lamellipodia. The PKCα translocation upon wounding precedes both the increase in PKC activity in the membrane fraction and the reversion of desmosomes to calcium dependence. Specific depletion of PKCα with an antisense oligonucleotide increases the number of cells with calcium-independent desmosomes. These results show that PKCα participates in a novel signaling pathway that modulates desmosomal adhesion in response to wounding. PMID:10712521

  11. Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

    SciTech Connect

    Kim, Chung Kwon; Lee, Sang Bae; Nguyen, Truong L.X.; Lee, Kyung-Hoon; Um, Sung Hee; Kim, Jihoe; Ahn, Jee-Yin

    2012-01-15

    p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

  12. Discovery of 3-(trifluoromethyl)-1H-pyrazole-5-carboxamide activators of the M2 isoform of pyruvate kinase (PKM2).

    PubMed

    Xu, Yong; Liu, Xiao-Hui; Saunders, Michael; Pearce, Scott; Foulks, Jason M; Parnell, K Mark; Clifford, Adrianne; Nix, Rebecca N; Bullough, Jeremy; Hendrickson, Thomas F; Wright, Kevin; McCullar, Michael V; Kanner, Steven B; Ho, Koc-Kan

    2014-01-15

    Activators of the pyruvate kinase M2 (PKM2) are currently attracting significant interest as potential anticancer therapies. They may achieve a novel antiproliferation response in cancer cells through modulation of the classic 'Warburg effect' characteristic of aberrant metabolism. In this Letter, we describe the optimization of a weakly active screening hit to a structurally novel series of small molecule 3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as potent PKM2 activators. PMID:24374270

  13. Upregulation of cAMP-specific PDE-4 activity following ligation of the TCR complex on thymocytes is blocked by selective inhibitors of protein kinase C and tyrosyl kinases.

    PubMed

    Michie, A M; Rena, G; Harnett, M M; Houslay, M D

    1998-01-01

    We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following ligation of the TCR (Michie, A.M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M.D. [1996] Cell. Signalling 8, 97-110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover, the PKC-activating phorbol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3 antibodies. Both the PMA- and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent induction of PDE-4 activity following crosslinking of the TCR complex. This identifies "crosstalk" occurring between the PKA and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells. PMID:9515165

  14. Comparative theoretical study of the binding of luciferyl-adenylate and dehydroluciferyl-adenylate to firefly luciferase

    NASA Astrophysics Data System (ADS)

    Pinto da Silva, Luís; Vieira, João; Esteves da Silva, Joaquim C. G.

    2012-08-01

    This is the first report of a study employing a computational approach to study the binding of (D/L)-luciferyl-adenlyates and dehydroluciferyl-adenylate to firefly luciferase. A semi-empirical/molecular mechanics methodology was used to study the interaction between these ligands and active site molecules. All adenylates are complexed with the enzyme, mostly due to electrostatic interactions with cationic residues. Dehydroluciferyl-adenylate is expected to be a competitive inhibitor of luciferyl-adenylate, as their binding mechanism and affinity to luciferase are very similar. Both luciferyl-adenylates adopt the L-orientation in the active site of luciferase.

  15. Gene expression levels of Casein kinase 1 (CK1) isoforms are correlated to adiponectin levels in adipose tissue of morbid obese patients and site-specific phosphorylation mediated by CK1 influences multimerization of adiponectin.

    PubMed

    Xu, Pengfei; Fischer-Posovszky, Pamela; Bischof, Joachim; Radermacher, Peter; Wabitsch, Martin; Henne-Bruns, Doris; Wolf, Anna-Maria; Hillenbrand, Andreas; Knippschild, Uwe

    2015-05-01

    White adipose tissue has now been recognized as an important endocrine organ secreting bioactive molecules termed adipocytokines. In obesity, anti-inflammatory adipocytokines like adiponectin are decreased while pro-inflammatory factors are over-produced. These changes contribute to the development of insulin resistance and obesity-associated diseases. Since members of the casein kinase 1 (CK1) family are involved in the regulation of various signaling pathways we ask here whether they are able to modulate the functions of adiponectin. We show that CK1δ and ε are expressed in adipose tissue and that the expression of CK1 isoforms correlates with that of adiponectin. Furthermore, adiponectin co-immunoprecipitates with CK1δ and CK1ε and is phosphorylated by CK1δ at serine 174 and threonine 235, thereby influencing the formation of adiponectin oligomeric complexes. Furthermore, inhibition of CK1δ in human adipocytes by IC261 leads to an increase in basal and insulin-stimulated glucose uptake. In summary, our data indicate that site-specific phosphorylation of adiponectin, especially at sites targeted by CK1δ in vitro, provides an additional regulatory mechanism for modulating adiponectin complex formation and function. PMID:25724478

  16. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes*

    PubMed Central

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A.

    2015-01-01

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  17. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes.

    PubMed

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A

    2015-12-18

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  18. Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression

    PubMed Central

    Nicot, Arnaud; DiCicco-Bloom, Emanuel

    2001-01-01

    Although neurogenesis in the embryo proceeds in a region- or lineage-specific fashion coincident with neuropeptide expression, a regulatory role for G protein-coupled receptors (GPCR) remains undefined. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates sympathetic neuroblast proliferation, whereas the peptide inhibits embryonic cortical precursor mitosis. Here, by using ectopic expression strategies, we show that the opposing mitogenic effects of PACAP are determined by expression of PACAP receptor splice isoforms and differential coupling to the phospholipase C (PLC) pathway, as opposed to differences in cellular context. In embryonic day 14 (E14) cortical precursors transfected with the hop receptor variant, but not cells transfected with the short variant, PACAP activates the PLC pathway, increasing intracellular calcium and eliciting translocation of protein kinase C. Ectopic expression of the hop variant in cortical neuroblasts transforms the antimitotic effect of PACAP into a promitogenic signal. Furthermore, PACAP promitogenic effects required PLC pathway function indicated by antagonist U-73122 studies in hop-transfected cortical cells and native sympathetic neuroblasts. These observations highlight the critical role of lineage-specific expression of GPCR variants in determining mitogenic signaling in neural precursors. PMID:11296303

  19. Differential distribution of phospholipase C beta isoforms and diaglycerol kinase-beta in rodents cerebella corroborates the division of unipolar brush cells into two major subtypes.

    PubMed

    Sekerková, Gabriella; Watanabe, Masahiko; Martina, Marco; Mugnaini, Enrico

    2014-03-01

    Sublineage diversification of specific neural cell classes occurs in complex as well as simply organized regions of the central and peripheral nervous systems; the significance of the phenomenon, however, remains insufficiently understood. The unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons that occur at high density in vestibulocerebellum. As they are classified into subsets that differ in chemical phenotypes, intrinsic properties, and lobular distribution, they represent a valuable neuronal model to study subclass diversification. In this study, we show that cerebellar UBCs of adult rats and mice form two subclasses-type I and type II UBCs-defined by somatodendritic expression of calretinin (CR), mGluR1α, phospholipases PLCβ1 and PLCβ4, and diacylglycerol kinase-beta (DGKβ). We demonstrate that PLCβ1 is associated only with the CR(+) type I UBCs, while PLCβ4 and DGKβ are exclusively present in mGluR1α(+) type II UBCs. Notably, all PLCβ4(+) UBCs, representing about 2/3 of entire UBC population, also express mGluR1α. Furthermore, our data show that the sum of CR(+) type I UBCs and mGluR1α(+) type II UBCs accounts for the entire UBC class identified with Tbr2 immunolabeling. The two UBC subtypes also show a very different albeit somehow overlapping topographical distribution as illustrated by detailed cerebellar maps in this study. Our data not only complement and extend the previous knowledge on the diversity and subclass specificity of the chemical phenotypes within the UBC population, but also provide a new angle to the understanding of the signaling networks in type I and type II UBCs. PMID:23503970

  20. Digitonin effects on photoreceptor adenylate cyclase.

    PubMed

    Bitensky, M W; Gorman, R E; Miller, W H

    1972-03-24

    Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.

  1. Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone.

    PubMed Central

    Holness, Mark J; Bulmer, Karen; Smith, Nicholas D; Sugden, Mary C

    2003-01-01

    Liver contains two pyruvate dehydrogenase kinases (PDKs), namely PDK2 and PDK4, which regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Starvation increases hepatic PDK2 and PDK4 protein expression, the latter occurring, in part, via a mechanism involving peroxisome proliferator-activated receptor-alpha (PPARalpha). High-fat feeding and hyperthyroidism, which increase circulating lipid supply, enhance hepatic PDK2 protein expression, but these increases are insufficient to account for observed increases in hepatic PDK activity. Enhanced expression of PDK4, but not PDK2, occurs in part via a mechanism involving PPAR-alpha. Heterodimerization partners for retinoid X receptors (RXRs) include PPARalpha and thyroid-hormone receptors (TRs). We therefore investigated the responses of hepatic PDK protein expression to high-fat feeding and hyperthyroidism in relation to hepatic lipid delivery and disposal. High-fat feeding increased hepatic PDK2, but not PDK4, protein expression whereas hyperthyroidism increased both hepatic PDK2 and PDK4 protein expression. Both manipulations decreased the sensitivity of hepatic carnitine palmitoyltransferase I (CPT I) to suppression by malonyl-CoA, but only hyperthyrodism elevated plasma fatty acid and ketone-body concentrations and CPT I maximal activity. Administration of the selective PPAR-alpha activator WY14,643 significantly increased PDK4 protein to a similar extent in both control and high-fat-fed rats, but WY14,643 treatment and hyperthyroidism did not have additive effects on hepatic PDK4 protein expression. PPARalpha activation did not influence hepatic PDK2 protein expression in euthyroid rats, suggesting that up-regulation of PDK2 by hyperthyroidism does not involve PPARalpha, but attenuated the effect of hyperthyroidism to increase hepatic PDK2 expression. The results indicate that hepatic PDK4 up-regulation can be achieved by heterodimerization of either PPARalpha or

  2. Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone.

    PubMed

    Holness, Mark J; Bulmer, Karen; Smith, Nicholas D; Sugden, Mary C

    2003-02-01

    Liver contains two pyruvate dehydrogenase kinases (PDKs), namely PDK2 and PDK4, which regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Starvation increases hepatic PDK2 and PDK4 protein expression, the latter occurring, in part, via a mechanism involving peroxisome proliferator-activated receptor-alpha (PPARalpha). High-fat feeding and hyperthyroidism, which increase circulating lipid supply, enhance hepatic PDK2 protein expression, but these increases are insufficient to account for observed increases in hepatic PDK activity. Enhanced expression of PDK4, but not PDK2, occurs in part via a mechanism involving PPAR-alpha. Heterodimerization partners for retinoid X receptors (RXRs) include PPARalpha and thyroid-hormone receptors (TRs). We therefore investigated the responses of hepatic PDK protein expression to high-fat feeding and hyperthyroidism in relation to hepatic lipid delivery and disposal. High-fat feeding increased hepatic PDK2, but not PDK4, protein expression whereas hyperthyroidism increased both hepatic PDK2 and PDK4 protein expression. Both manipulations decreased the sensitivity of hepatic carnitine palmitoyltransferase I (CPT I) to suppression by malonyl-CoA, but only hyperthyrodism elevated plasma fatty acid and ketone-body concentrations and CPT I maximal activity. Administration of the selective PPAR-alpha activator WY14,643 significantly increased PDK4 protein to a similar extent in both control and high-fat-fed rats, but WY14,643 treatment and hyperthyroidism did not have additive effects on hepatic PDK4 protein expression. PPARalpha activation did not influence hepatic PDK2 protein expression in euthyroid rats, suggesting that up-regulation of PDK2 by hyperthyroidism does not involve PPARalpha, but attenuated the effect of hyperthyroidism to increase hepatic PDK2 expression. The results indicate that hepatic PDK4 up-regulation can be achieved by heterodimerization of either PPARalpha or

  3. Zeta isoform of protein kinase C prevents oxidant-induced nuclear factor-kappaB activation and I-kappaBalpha degradation: a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity.

    PubMed

    Banan, A; Fields, J Z; Zhang, L J; Shaikh, M; Farhadi, A; Keshavarzian, A

    2003-10-01

    Oxidant damage and gut barrier disruption contribute to the pathogenesis of a variety of inflammatory gastrointestinal disorders, including inflammatory bowel disease (IBD). In our studies using a model of the gastrointestinal (GI) epithelial barrier, monolayers of intestinal (Caco-2) cells, we investigated damage to and protection of the monolayer barrier. We reported that activation of nuclear factor-kappaB (NF-kappaB) via degradation of its endogenous inhibitor I-kappaBalpha is key to oxidant-induced disruption of barrier integrity and that growth factor (epidermal growth factor, EGF) protects against this injury by stabilizing the cytoskeletal filaments. Protein kinase C (PKC) activation seems to be required for monolayer maintenance, especially activation of the atypical zeta isoform of PKC. In an attempt to investigate, at the molecular level, the fundamental events underlying EGF protection against oxidant disruption, we tested the intriguing hypothesis that EGF-induced activation of PKC-zeta prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, namely, cytoskeletal and barrier disruption. Monolayers of wild-type (WT) Caco-2 cells were incubated with oxidant (H2O2) with or without EGF or modulators. In other studies, we used the first gastrointestinal cell clones created by stable transfection of varying levels (1-5 microg) of cDNA to either overexpress PKC-zeta or to inhibit its expression. Transfected cell clones were then pretreated with EGF or a PKC activator (diacylglycerol analog 1-oleoyl-2-acetyl-glycerol, OAG) before oxidant. We monitored the following endpoints: monolayer barrier integrity, stability of the microtubule cytoskeleton, subcellular distribution and activity of the PKC-zeta isoform, intracellular levels and phosphorylation of the NF-kappaB inhibitor I-kappaBalpha, and nuclear translocation and activity of NF-kappaB subunits p65 and p50. Monolayers were also fractionated and processed to assess

  4. Adenylate cyclases involvement in pathogenicity, a minireview.

    PubMed

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

    Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed.

  5. Diuretics and the renal adenylate cyclase system

    PubMed Central

    Dawborn, J.K.; Macneil, S.; Martin, T.J.

    1977-01-01

    1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3′,5′-monophosphate (cyclic AMP)-dependent mechanism was also tested. 2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10-3M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats. 3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation. 4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin. 5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg2+, K+ and incubation at 37°C) inhibited rat cortex homogenate carbonic anhydrase activity. 6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism. PMID:202362

  6. Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

    PubMed Central

    Carricarte, V C; Bianchini, G M; Muschietti, J P; Téllez-Iñón, M T; Perticari, A; Torres, N; Flawiá, M M

    1988-01-01

    An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase. PMID:3128270

  7. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    PubMed Central

    Fewer, David P; Rouhiainen, Leo; Jokela, Jouni; Wahlsten, Matti; Laakso, Kati; Wang, Hao; Sivonen, Kaarina

    2007-01-01

    Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. PMID:17908306

  8. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

    PubMed Central

    Gueirard, P; Guiso, N

    1993-01-01

    Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different. Images PMID:8406794

  9. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  10. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  11. The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases

    PubMed Central

    Rueda, Elda M.; Johnson, Jerry E.; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J.; Sigel, Irena; Chaney, Shawnta Y.

    2016-01-01

    inner segments. The combined results indicate that glycolysis is regulated by the compartmental expression of hexokinase 2, pyruvate kinase M1, and pyruvate kinase M2 in photoreceptors, whereas the inner retinal neurons exhibit a lower capacity for glycolysis and aerobic glycolysis. Expression of nucleoside diphosphate kinase, mitochondria-associated adenylate kinase, and several mitochondria-associated creatine kinase isozymes was highest in the outer retina, whereas expression of cytosolic adenylate kinase and brain creatine kinase was higher in the cones, horizontal cells, and amacrine cells indicating the diversity of ATP-buffering strategies among retinal neurons. Based on the antibody intensities and the COX and LDH activity, Müller glial cells (MGCs) had the lowest capacity for glycolysis, aerobic glycolysis, and OXPHOS. However, they showed high expression of glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate thiokinase, GABA transaminase, and ~P transferring kinases. This suggests that MGCs utilize TCA cycle anaplerosis and cataplerosis to generate GTP and ~P transferring kinases to produce ATP that supports MGC energy requirements. Conclusions Our comprehensive and integrated results reveal that the adult mouse retina expresses numerous isoforms of ATP synthesizing, regulating, and buffering genes; expresses differential cellular and compartmental levels of glycolytic, OXPHOS, TCA cycle, and ~P transferring kinase proteins; and exhibits differential layer-by-layer LDH and COX activity. New insights into cell-specific and compartmental ATP and GTP production, as well as utilization and buffering strategies and their relationship with known retinal and cellular functions, are discussed. Developing therapeutic strategies for neuroprotection and treating retinal deficits and degeneration in a cell-specific manner will require such knowledge. This work provides a platform for future research directed at identifying the molecular targets and

  12. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  13. Parvalbumin isoforms in zebrafish.

    PubMed

    Friedberg, Felix

    2005-09-01

    By using an analysis of existing genomic information it is concluded that in zebrafish nine genes encode parvalbumin (PV). These genes possess introns that differ in size and show nucleotide variability but they contain the same number of exons, and for each corresponding exon, the number of nucleotides therein are identical in all the paralogs. This rule also applies to the multiple PV genes of other species e.g. mammals. Each of these genes displays, however, characteristic 5' and 3' UTRs which appear highly conserved between closely related species (so that orthologs among these species can be readily identified) but which show larger numbers of mutations between species that are more distant in evolution. A tree is presented which suggests that the traditional classification of PVs as alpha or beta (based mainly on charge of the protein molecule) is not sustainable. Numbers 1-9 are assigned to the various isoforms to facilitate their identification in future studies. A bifurcation of isoforms into 1 and 4; 2 and 3; 6 and 7; 8 and 9 appears to have occurred simultaneously in more recent time, i.e. perhaps approximately 60 mys ago when primates and rodents branched. PMID:16172917

  14. Interaction of Trypanosoma cruzi adenylate cyclase with liver regulatory factors.

    PubMed Central

    Eisenschlos, C; Flawiá, M M; Torruella, M; Torres, H N

    1986-01-01

    Trypanosoma cruzi adenylate cyclase catalytic subunits may interact with regulatory factors from rat liver membranes, reconstituting heterologous systems which are catalytically active in assay mixtures containing MgATP. The systems show stimulatory responses to glucagon and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or fluoride. Reconstitution was obtained by three different methods: fusion of rat liver membranes (pretreated with N-ethylmaleimide) to T. cruzi membranes; interaction of detergent extracts of rat liver membranes with T. cruzi membranes; or interaction of purified preparations of T. cruzi adenylate cyclase and of liver membrane factors in phospholipid vesicles. The liver factors responsible for the guanine nucleotide effect were characterized as the NS protein. Data also indicate that reconstitution requires the presence of a membrane substrate. PMID:2947568

  15. Aminoacyl transfer from an adenylate anhydride to polyribonucleotides

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Lacey, J. C., Jr.

    1975-01-01

    Imidazole catalysis of phenylalanyl transfer from phenylalanine adenylate to hydroxyl groups of homopolyribonucleotides is studied as a possible chemical model of biochemical aminoacylation of transfer RNA (tRNA). The effect of pH on imidazole-catalyzed transfer of phenylalanyl residues to poly(U) and poly(A) double helix strands, the number of peptide linkages and their lability to base and neutral hydroxylamine, and the nature of adenylate condensation products are investigated. The chemical model entertained exhibits a constraint by not acylating the hydroxyl groups of polyribonucleotides in a double helix. The constraint is consistent with selective biochemical aminoacylation at the tRNA terminus. Interest in imidazole as a model of histidine residue in protoenzymes participating in prebiotic aminoacyl transfer to polyribonucleotides, and in rendering the tRNA a more efficient adaptor, is indicated.

  16. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    PubMed Central

    Willner, J H; Cerri, C G; Wood, D S

    1981-01-01

    Malignant hyperthermia occurs in humans with several congenital myopathies, usually in response to general anesthesia. Commonly, individuals who develop this syndrome lack symptoms of muscle disease, and their muscle lacks specific pathological changes. A biochemical marker for this myopathy has not previously been available; we found activity of adenylate cyclase and content of cyclic AMP to be abnormally high in skeletal muscle. Secondary modification of protein phosphorylation could explain observed abnormalities of phosphorylase activation and sarcoplasmic reticulum function. PMID:6271806

  17. Engineering adenylate cyclases regulated by near-infrared window light

    PubMed Central

    Ryu, Min-Hyung; Kang, In-Hye; Nelson, Mathew D.; Jensen, Tricia M.; Lyuksyutova, Anna I.; Siltberg-Liberles, Jessica; Raizen, David M.; Gomelsky, Mark

    2014-01-01

    Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset. PMID:24982160

  18. Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

    PubMed

    Osorio-Fuentealba, Cesar; Klip, Amira

    2015-09-01

    The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase.

  19. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  20. Phorbol esters induce death in MCF-7 breast cancer cells with altered expression of protein kinase C isoforms. Role for p53-independent induction of gadd-45 in initiating death.

    PubMed Central

    de Vente, J E; Kukoly, C A; Bryant, W O; Posekany, K J; Chen, J; Fletcher, D J; Parker, P J; Pettit, G J; Lozano, G; Cook, P P

    1995-01-01

    Protein kinase C (PKC) modulates growth, differentiation and apoptosis in a cell-specific fashion. Overexpression of PKC-alpha in MCF-7 breast cancer cells (MCF-7-PKC-alpha cell) leads to expression of a more transformed phenotype. The response of MCF-7 and MCF-7-PKC-alpha cells to phorbol esters (TPA) was examined. TPA-treated MCF-7 cells demonstrated a modest cytostatic response associated with a G1 arrest that was accompanied by Cip1 expression and retinoblastoma hypophosphorylation. While p53 was detected in MCF-7 cells, evidence for TPA-induced stimulation of p53 transcriptional activity was not evident. In contrast, TPA treatment induced death of MCF-7-PKC-alpha cells. Bryostatin 1, another PKC activator, exerted modest cytostatic effects on MCF-7 cells while producing a cytotoxic response at low doses in MCF-7-PKC-alpha cells that waned at higher concentrations. TPA-treated MCF-7-PKC-alpha cells accumulated in G2/M, did not express p53, displayed decreased Cip1 expression, and demonstrated a reduction in retinoblastoma hypophosphorylation. TPA-treated MCF-7-PKC-alpha cells expressed gadd-45 which occurred before the onset of apoptosis. Thus, alterations in the PKC pathway can modulate the decision of a breast cancer cell to undergo death or differentiation. In addition, these data show that PKC activation can induce expression of gadd45 in a p53-independent fashion. Images PMID:7560079

  1. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    PubMed Central

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  2. Structural and functional investigation of the intermolecular interaction between NRPS adenylation and carrier protein domains

    PubMed Central

    Sundlov, Jesse A.; Shi, Ce; Wilson, Daniel J.; Aldrich, Courtney C.; Gulick, Andrew M.

    2012-01-01

    Summary Non-ribosomal peptide synthetases (NRPSs) are modular proteins that produce peptide antibiotics and siderophores. These enzymes act as catalytic assembly lines where substrates, covalently bound to integrated carrier domains, are delivered to adjacent catalytic domains. The carrier domains are initially loaded by adenylation domains, which use two distinct conformations to catalyze sequentially the adenylation of the substrate and the thioesterification of the pantetheine cofactor. We have used a mechanism-based inhibitor to determine the crystal structure of an engineered adenylation-carrier domain protein illustrating the intermolecular interaction between the adenylation and carrier domains. This structure enabled directed mutations to improve the interaction between non-native partner proteins. Comparison with prior NRPS adenylation domain structures provides insights into the assembly line dynamics of these modular enzymes. PMID:22365602

  3. Adenylate Energy Charge in Escherichia coli During Growth and Starvation

    PubMed Central

    Chapman, Astrid G.; Fall, Lana; Atkinson, Daniel E.

    1971-01-01

    The value of the adenylate energy charge, [(adenosine triphosphate) + ½ (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types. PMID:4333317

  4. Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis.

    PubMed

    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, Tarsis F; Pávek, Petr; Trejtnar, František; Watts, Val J; Janeba, Zlatko

    2015-08-01

    Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

  5. Isoform-specific targeting of ROCK proteins in immune cells

    PubMed Central

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform plays an exclusive role in controlling of T-cell plasticity and macrophage polarization. Specifically, selective ROCK2 inhibition shifts the balance between pro-inflammatory and regulatory T-cell subsets via concurrent regulation of STAT3 and STAT5 phosphorylation, respectively. Furthermore, the administration of an orally available selective ROCK2 inhibitor effectively ameliorates clinical manifestations in experimental models of autoimmunity and chronic graft-vs.-host disease (cGVHD). Because ROCK2 inhibition results in the suppression of M2-type macrophages while favoring polarization of M1-type macrophages, ROCK2 inhibition can correct the macrophage imbalance seen during age-related macular degeneration (AMD). In summary, the exclusive role of ROCK2 in immune system modulation argues for the development and testing of isoform-specific ROCK2 inhibitors for the treatment of inflammatory disorders. PMID:27254302

  6. Differential Roles of PML Isoforms

    PubMed Central

    Nisole, Sébastien; Maroui, Mohamed Ali; Mascle, Xavier H.; Aubry, Muriel; Chelbi-Alix, Mounira K.

    2013-01-01

    The tumor suppressor promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha in patients suffering from acute promyelocytic leukemia (APL). Treatment of APL patients with arsenic trioxide (As2O3) reverses the disease phenotype by a process involving the degradation of the fusion protein via its PML moiety. Several PML isoforms are generated from a single PML gene by alternative splicing. They share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. Here, we review the nomenclature and structural organization of the PML isoforms in order to clarify the various designations and classifications found in different databases. The functions of the PML isoforms and their differential roles in antiviral defense also are reviewed. Finally, the key players involved in the degradation of the PML isoforms in response to As2O3 or other inducers are discussed. PMID:23734343

  7. Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment.

    PubMed

    Koshimizu, Taka-aki; Tsuchiya, Hiroyoshi; Tsuda, Hidetoshi; Fujiwara, Yoko; Shibata, Katsushi; Hirasawa, Akira; Tsujimoto, Gozoh; Fujimura, Akio

    2010-02-19

    Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1-10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.

  8. The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

    PubMed Central

    Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

    1972-01-01

    Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

  9. Rat muscle 5'-adenylic acid aminohydrolase. Role of K+ and adenylate energy charge in expression of kinetic and regulatory properties.

    PubMed

    Coffee, C J; Solano, C

    1977-03-10

    The kinetic and regulatory properties of homogeneous AMP deaminase from rat skeletal muscle have ben examined with particular emphasis on (a) the role of potassium ions in the expression of these properties and (b) the role of the adenylate energy charge in the regulation of AMP deaminase activity. Although the enzyme has an absolute requirement for K+, the concentration required for maximum activation is dependent on the concentration of substrate. At saturating levels of AMP (greater than or equal 2.0 mM) maximum activation is observed with 25 mM KCl, whereas at lower substrate concentrations (0.2 mM) approximately 50 mM KCl is needed for maximum activation. Conversely, the response of enzyme activity ot increasing levels of substrate is dependent on the level of potassium ions. At substrating concentrations of K+, the saturation curve for AMP is highly sigmoidal (nh=3.2) whereas at higher KCl concentrations, the apparent cooperativity between AMP sites is almost completely abolished (nh=1.5). The inhibition by a number of phosphorylated metabolites, including ATP, GTP, creatine-P, and P1, is likewise sensitive to the concentration of K+. These results suggest that a significant amount of interaction between K+ sites and both substrate and effector sites is required for the expression of the catalytic and regulatory properties of the enzyme. The specific effects of ATP, creatine-P, and P1 on the parameters of Km and Vmax indicate that each of these profile of AMP deaminase activity generated in response to variations in the adenylate energy charge shows that within the physiological range of energy charge (0.75 to 0.95), the activity increases linearly with decreasesing energy charge and is insensitive to both the total adenylate pool size and the presence of P1 and creatine-P. These data suggest that the most important factor in the regulation of AMP deaminase activity is the state of the energy charge rather than the absolute concentrations of the individual

  10. Comparative inhibition patterns of adenylate kinases from mammals, bird, fish and microorganisms.

    PubMed

    Williams, A; Taulane, J P; Russell, P J

    1994-03-01

    The S8 inhibitions of AKs from six different sources were studied in mammals, birds, fish, and a microorganism. All AKs tested were inhibited by S8. Except for carp, all inhibited AKs from those tested were reactivated by DTT. Inhibitions of AKs by other hydrophobic inhibitors, NEM, butanol and ethanol were also studied. The inhibitions by S8 suggest that the hydrophobic pockets in the AKs cover a wide phylogenetic range. All inhibitions by S8 are reactivated by DTT. Unlike the inhibitions by S8, the characteristics of inhibitions by the other hydrophobic inhibitors differed among the AK sources tested and none was the irreversible type. The data suggest that no covalent bonds were formed with NEM. Similarly, the ability to reactivate the inhibitions by DTT differed among the AK sources. The possibility that the hydrophobic domains in the AKs may serve as part of an enzyme activity control mechanism is discussed. PMID:7749617

  11. Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary.

    PubMed Central

    Onali, P; Schwartz, J P; Costa, E

    1981-01-01

    The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by vasoactive intestinal peptide (VIP) was used as a model to investigate the molecular mechanisms triggered by the occupancy of dopamine recognition sites in rat anterior pituitary. Dopamine failed to change the basal enzyme activity, but it inhibited the stimulation of adenylate cyclase elicited by VIP. Apomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, and 2-bromo-alpha-ergocryptine mimicked the effect of dopamine, whereas (-)-sulpiride and and classical neuroleptics antagonized it. Dopamine failed to modulate the activation of pituitary adenylate cyclase by prostaglandin E1, which does not increase prolactin secretion. From these results we infer that stimulation of D-2 (dopamine) receptors may affect pituitary secretion by inhibiting the activation of anterior pituitary adenylate cyclase by VIP or other secretagogues. PMID:6171819

  12. Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies

    PubMed Central

    Jabbour, Elias; Ottmann, Oliver G.; Deininger, Michael; Hochhaus, Andreas

    2014-01-01

    The phosphoinositide 3-kinase pathway represents an important anticancer target because it has been implicated in cancer cell growth, survival, and motility. Recent studies show that PI3K may also play a role in the development of resistance to currently available therapies. In a broad range of cancers, various components of the phosphoinositide 3-kinase signaling axis are genetically modified, and the pathway can be activated through many different mechanisms. The frequency of genetic alterations in the phosphoinositide 3-kinase pathway, coupled with the impact in oncogenesis and disease progression, make this signaling axis an attractive target in anticancer therapy. A better understanding of the critical function of the phosphoinositide 3-kinase pathway in leukemias and lymphomas has led to the clinical evaluation of novel rationally designed inhibitors in this setting. Three main categories of phosphoinositide 3-kinase inhibitors have been developed so far: agents that target phosphoinositide 3-kinase and mammalian target of rapamycin (dual inhibitors), pan-phosphoinositide 3-kinase inhibitors that target all class I isoforms, and isoform-specific inhibitors that selectively target the α, -β, -γ, or -δ isoforms. Emerging data highlight the promise of phosphoinositide 3-kinase inhibitors in combination with other therapies for the treatment of patients with hematologic malignancies. Further evaluation of phosphoinositide 3-kinase inhibitors in first-line or subsequent regimens may improve clinical outcomes. This article reviews the role of phosphoinositide 3-kinase signaling in hematologic malignancies and the potential clinical utility of inhibitors that target this pathway. PMID:24425689

  13. Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain.

    PubMed

    Hungen, K V; Roberts, S; Hill, D F

    1975-08-22

    Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-brono-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation of adenylate cyclase activity of equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10 muM concentration, D-LSD not only completely eradicated the response to 10 muM dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80 muM) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1 muM D-LSD. Activation of striatal adenylate cyclase of either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the beta-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and

  14. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

    Brown, J E; Kaupp, U B; Malbon, C C

    1984-01-01

    Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor-enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1-methyl-3-isobutyl xanthine or forskolin and was increased almost 100-fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug-induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis-resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat-treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc- mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that

  15. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    PubMed

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  16. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    SciTech Connect

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  17. Molecular cloning and mRNA distribution of pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide in the lungfish.

    PubMed

    Lee, L T O; Tam, J K V; Chan, D W; Chow, B K C

    2009-04-01

    In this article, we report the isolation of a full-length cDNA clone encoding pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) from lungfish Protopterus dolloi. When comparing the deduced amino acid sequences, the lungfish PACAP was found to be highly conserved with other vertebrates; however, the PRP shares only lower levels of sequence identity with known PRP sequences. Consistently in phylogenetic analysis, the lungfish PRP, similar to sturgeon PRP, fails to cluster with other PRPs. In addition to the full-length clone, another cDNA encoding a short precursor that lacks the first 32 amino acids of the PRP was also isolated. Interestingly, similar isoforms were also identified in several nonmammalian vertebrates, and it was suggested that exon skipping of PRP/PACAP transcripts was a mechanism that regulated the expression ratio of PACAP to PRP in nonmammalian vertebrates. By real-time PCR, both long and short PRP/PACAP transcripts were found almost exclusively in the brain, and the short isoform is the more abundant transcript (3.7 times more), indicating that PACAP is the major product produced in lungfish brain. The expression patterns of lungfish and previously studied frog PRP/PACAP suggest that the PRP/PACAP gene in the tetrapod lineage may first express in the central nervous system; in the process of evolution, the functions of these peptides diversified and were later found in other tissues.

  18. Molecular cloning and mRNA distribution of pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide in the lungfish.

    PubMed

    Lee, L T O; Tam, J K V; Chan, D W; Chow, B K C

    2009-04-01

    In this article, we report the isolation of a full-length cDNA clone encoding pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) from lungfish Protopterus dolloi. When comparing the deduced amino acid sequences, the lungfish PACAP was found to be highly conserved with other vertebrates; however, the PRP shares only lower levels of sequence identity with known PRP sequences. Consistently in phylogenetic analysis, the lungfish PRP, similar to sturgeon PRP, fails to cluster with other PRPs. In addition to the full-length clone, another cDNA encoding a short precursor that lacks the first 32 amino acids of the PRP was also isolated. Interestingly, similar isoforms were also identified in several nonmammalian vertebrates, and it was suggested that exon skipping of PRP/PACAP transcripts was a mechanism that regulated the expression ratio of PACAP to PRP in nonmammalian vertebrates. By real-time PCR, both long and short PRP/PACAP transcripts were found almost exclusively in the brain, and the short isoform is the more abundant transcript (3.7 times more), indicating that PACAP is the major product produced in lungfish brain. The expression patterns of lungfish and previously studied frog PRP/PACAP suggest that the PRP/PACAP gene in the tetrapod lineage may first express in the central nervous system; in the process of evolution, the functions of these peptides diversified and were later found in other tissues. PMID:19456341

  19. Apolipoprotein E Isoforms and AMD.

    PubMed

    Toops, Kimberly A; Tan, Li Xuan; Lakkaraju, Aparna

    2016-01-01

    The cholesterol transporting protein apolipoprotein E (ApoE) occurs in three allelic variants in humans unlike in other species. The resulting protein isoforms E2, E3 and E4 exhibit differences in lipid binding, integrating into lipoprotein particles and affinity for lipoprotein receptors. ApoE isoforms confer genetic risk for several diseases of aging including atherosclerosis, Alzheimer's disease, and age-related macular degeneration (AMD). A single E4 allele increases the risk of developing Alzheimer's disease, whereas the E2 allele is protective. Intriguingly, the E4 allele is protective in AMD. Current thinking about different functions of ApoE isoforms comes largely from studies on Alzheimer's disease. These data cannot be directly extrapolated to AMD since the primary cells affected in these diseases (neurons vs. retinal pigment epithelium) are so different. Here, we propose that ApoE serves a fundamentally different purpose in regulating cholesterol homeostasis in the retinal pigment epithelium and this could explain why allelic risk factors are flipped for AMD compared to Alzheimer's disease.

  20. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

    PubMed Central

    Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C.; Bailey, Mark E. S.; Cobb, Stuart R.

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders. PMID:27315173

  1. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    PubMed

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

  2. Antagonistic Functions of Two Stardust Isoforms in Drosophila Photoreceptor Cells

    PubMed Central

    Bulgakova, Natalia A.; Rentsch, Michaela

    2010-01-01

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs–Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis. PMID:20861315

  3. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    PubMed

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  4. Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Hall, L. M.; Mullins, D. W., Jr.; Watkins, C. L.

    1985-01-01

    The interaction between amino acids and nucleotide bases is studied. The proton NMR spectrum of N-acetylphenylalanyl-AMP-anhydride is analyzed H8 and H2 signals, two upfield signals of equal size, and five phenylalanine ring proton signals are observed in the spectrum; the upfield movement of the proton and the racemization of the N-acetyl L-phenylalanine material are examined. The differences in the position of the signals due to the diastereoisomers are investigated. The separation of the D and L amino acyl adenylates using HPLC is described. H-1 NMR spectra of the isomers are examined in order to determine which isomer displays the strongest interaction between the phenyl ring and the adenine ring. The spectra reveal that the L isomer shows the highest upfield change of both H8 and H2 signals. It is noted that the phenyl ring lies over C2 of the adenine ring with the phenyl meta and para protons extended past the adenine ring and the phenyl ortho protons.

  5. Protein Kinase D family kinases: roads start to segregate.

    PubMed

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue. PMID:24847910

  6. Analysis of the Linker Region Joining the Adenylation and Carrier Protein Domains of the Modular Non-Ribosomal Peptide Synthetases

    PubMed Central

    Miller, Bradley R.; Sundlov, Jesse A.; Drake, Eric J.; Makin, Thomas A.; Gulick, Andrew M.

    2014-01-01

    Non-Ribosomal Peptide Synthetases (NRPSs) are multi-modular proteins capable of producing important peptide natural products. Using an assembly-line process the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal sub-domain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. PMID:24975514

  7. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  8. Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages.

    PubMed

    Papakonstanti, Evangelia A; Zwaenepoel, Olivier; Bilancio, Antonio; Burns, Emily; Nock, Gemma E; Houseman, Benjamin; Shokat, Kevan; Ridley, Anne J; Vanhaesebroeck, Bart

    2008-12-15

    The class IA isoforms of phosphoinositide 3-kinase (p110alpha, p110beta and p110delta) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110delta selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110delta in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110alpha, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110delta. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110alpha also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110delta.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110delta isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110delta-selective PI3K inhibitors may be more effective in inflammation than in cancer. PMID:19033389

  9. Inference of Isoforms from Short Sequence Reads

    NASA Astrophysics Data System (ADS)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  10. Diacylglycerol Kinase Inhibition and Vascular Function.

    PubMed

    Choi, Hyehun; Allahdadi, Kyan J; Tostes, Rita C A; Webb, R Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction. PMID:21547002

  11. Stimulation of hormone-responsive adenylate cyclase activity by a factor present in the cell cytosol.

    PubMed Central

    MacNeil, S; Crawford, A; Amirrasooli, H; Johnson, S; Pollock, A; Ollis, C; Tomlinson, S

    1980-01-01

    1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E. PMID:7396869

  12. Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.

    PubMed

    Striem, B J; Pace, U; Zehavi, U; Naim, M; Lancet, D

    1989-05-15

    Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.

  13. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  14. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    PubMed

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation. PMID:24646224

  15. Role of Adenylate Cyclase 1 in Retinofugal Map Development

    PubMed Central

    Dhande, Onkar S.; Bhatt, Shivani; Anishchenko, Anastacia; Elstrott, Justin; Iwasato, Takuji; Swindell, Eric C.; Xu, Hong-Ping; Jamrich, Milan; Itohara, Shigeyoshi; Feller, Marla B.; Crair, Michael C.

    2013-01-01

    The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin-dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1-dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity-dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;β2−/− double KO mice) are no worse than those in mice lacking only β2−/−, but loss of AC1 occludes map recovery in β2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye-specific segregation in the SC and dorsal LGN. PMID:22102330

  16. Evidence for a dissociable protein subunit required for calmodulin stimulation of brain adenylate cyclase.

    PubMed Central

    Toscano, W A; Westcott, K R; LaPorte, D C; Storm, D R

    1979-01-01

    An adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] preparation that is not stimulated by NaF,5'-guanylyl imidodiphosphate, or Ca2+.calmodulin has been isolated from bovine cerebral cortex by Affi-Gel Blue chromatography and calmodulin-Sepharose chromatography. Sensitivity to these effectors was restored by incubation of the adenylate cyclase preparation with detergent-solubilized protein from bovine cerebral cortex. Reconstitution of of Ca2+.calmodulin activation required the presence of 5'-guanylyl imidodiphosphate. The factor required for restoration of Ca2+.calmodulin stimulation was sensitive to heat, trypsin digestion, and N-ethylmaleimide. These observations suggest that this adenylate cyclase activity requires the presence of one or more guanyl nucleotide binding subunits for calmodulin sensitivity. PMID:293663

  17. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  18. Synthesis of amino acyl adenylates using the tert-butoxycarbonyl protecting group

    NASA Technical Reports Server (NTRS)

    Armstrong, D. W.; Seguin, R.; Saburi, M.; Fendler, J. H.

    1979-01-01

    The synthesis of amino acyl adenylates using N-tert-butoxycarbonyl-protected amino acids is reported. Anhydrous solutions containing N-tert-butoxycarbonyl alanine, phenylalanine, and methionine were combined with the anhydrous mono (tri-n-octylammonium) salt of adenosine 5'-phosphate and the resultant amino acyl adenylates were characterized by means of elemental analysis, and infrared and proton NMR spectroscopy. Amino acyl adenylate yields of up to 60% were obtained with high purity at room temperatures. The reported synthesis is considered to represent a large improvement over previous methods due to the purity of the products, normal temperature requirements, and the stability of the starting compounds, which suggests its use in investigations of prebiotic oligo- and polypeptide synthesis.

  19. Alterations in adipocyte adenylate cyclase activity in morbidly obese and formerly morbidly obese humans.

    PubMed

    Martin, L F; Klim, C M; Vannucci, S J; Dixon, L B; Landis, J R; LaNoue, K F

    1990-08-01

    Studies examining animal models of genetic obesity have identified defects in adipocyte hormone-stimulated lipolysis that involve the adenylate cyclase transmembrane signaling system, specifically those components that decrease adenylate cyclase activity. To determine whether obese people demonstrate alterations in adenylate cyclase activity that could contribute to the maintenance of obesity by inhibiting lipolysis, we examined human adipocytes from patients who were lean, obese, or formerly obese. Fat samples were obtained from the lower abdomen of 14 women who were morbidly obese (obese group), from 10 women who were formerly morbidly obese and had lost weight after gastric stapling (postobese group), and from 10 similarly aged women of normal weight (controls). Adipocyte adenylate cyclase activity was determined under ligand-free (no stimulatory or inhibitory influences present), hormone-stimulated (isoproterenol, 10(-6) mmol/L), and maximal (cells stimulated with 10 mumol/L forskolin) conditions by measuring cyclic adenosine monophosphate (cAMP) levels by radioimmunoassay. The activity of adenylate cyclase was significantly different (p less than 0.01) in the three groups. Adipocytes from obese women had lower levels of cyclase activity under both ligand-free (5% vs 16% of maximal) and hormone-stimulated conditions (76% vs 100% of maximal) than adipocytes from normal women. Postobese women had levels of hormone-stimulated cAMP identical to those of normal women but still had abnormal ligand-free levels (under 5%). These results suggest the presence of an alteration in adipocyte adenylate cyclase regulation in morbidly obese women that is not entirely corrected when weight is lost after food intake is reduced by gastric stapling. This alteration in ligand-free cAMP activity may contribute to the development and maintenance of obesity. PMID:2166354

  20. Inhibition of hormonally regulated adenylate cyclase by the beta gamma subunit of transducin.

    PubMed Central

    Bockaert, J; Deterre, P; Pfister, C; Guillon, G; Chabre, M

    1985-01-01

    Transducin (T), the GTP-binding protein of the retina activates the cGMP phosphodiesterase system, and presents analogies with the proteins GS and Gi which respectively mediate adenylate cyclase activation and inhibition by hormone receptors. These proteins are all comprised of an alpha subunit carrying the GTP-binding site and a beta gamma subunit made of two peptides. The beta peptide (35 kd) appears similar in the three proteins. We demonstrate here that purified T beta gamma inhibits adenylate cyclase from human platelet membranes. This inhibition was observed when adenylate cyclase was stimulated by GTP, prostaglandin E1 (PGE1), NaF and forskolin, but not when stimulated by GTP(gamma)S. In the presence of GTP and forskolin, the T beta gamma-induced maximal inhibition was not additive with the alpha 2-receptor-induced adenylate cyclase inhibition mediated by Gi. Both inhibitions were suppressed at high Mg2+ concentrations, which as also known to dissociate T beta gamma from T alpha-GDP. This suggests that these adenylate cyclase inhibitions are due to the formation of inactive complexes of GS alpha-GDP with T beta gamma or Gi beta gamma. T beta gamma-induced inhibition did not require detergent and could be suppressed by simple washing. T beta gamma effects are dependent on its concentration rather than on its total amount. This suggests that T beta gamma can operate in solution with no integration into the membrane. Similar inhibitory effects of T beta gamma are observed on adenylate cyclase from anterior pituitary and lymphoma S49 cell lines. PMID:3861319

  1. Changed sensitivity of adenylate cyclase signaling system to biogenic amines and peptide hormones in tissues of starving rats.

    PubMed

    Shpakov, A O; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2007-07-01

    In the myocardium and skeletal muscles of rats deprived of food for 2 days, basal activity of adenylate cyclase decreased, while the sensitivity of adenylate cyclase signaling system to the stimulating effects of non-hormonal agents (guanine nucleotides and NaF) and beta-agonist isoproterinol modulating adenylate cyclase through stimulating G proteins increased. In starving organism, the regulatory effects of hormones realizing their effects through inhibitory G proteins (somatostatin in the myocardium and bromocryptin in the brain) weakened. Their inhibitory effects on forskolin-stimulated adenylate cyclase activity and stimulating effects on binding of guanosine triphosphate decreased. In the brain of starving rats, the differences in the sensitivity of the adenylate cyclase signaling system to hormones and nonhormonal agents were less pronounced than in the muscle tissues, which attested to tissue-specific changes in the functional state of this system under conditions of 2-day starvation.

  2. Clay catalyzed polymerization of amino acid adenylates and its relationship to biochemical reactions

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The adsorption and polymerization of alanine adenylate on montmorillonite at pH 7 when either its interspacial faces or its edger are blocked by an excess of histidine or sodium hexametaphosphate was investigated. Results indicate that alanine adenylate can be adsorbed any place on the interspacial spaces of the clay; however, adsorption of its phosphate part, which is limited to the edges of the clay, is necessary for polymerization to occur. As a result, polymerization takes place only at sites on the interspacial faces bordering the edges.

  3. Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins.

    PubMed

    Zhang, Wenjun; Heemstra, John R; Walsh, Christopher T; Imker, Heidi J

    2010-11-23

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways.

  4. Activation of the Pacidamycin PacL Adenylation Domain by MbtH-Like Proteins†

    PubMed Central

    Zhang, Wenjun; Heemstra, John R.; Walsh, Christopher T.; Imker, Heidi J.

    2010-01-01

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (~70 residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways. PMID:20964365

  5. AKAP79, PKC, PKA and PDE4 participate in a Gq-linked muscarinic receptor and adenylate cyclase 2 cAMP signalling complex

    PubMed Central

    Shen, Jia X.; Cooper, Dermot M. F.

    2014-01-01

    AC2 (adenylate cyclase 2) is stimulated by activation of Gq-coupled muscarinic receptors through PKC (protein kinase C) to generate localized cAMP in HEK (human embryonic kidney)-293 cells. In the present study, we utilized a sensitive live-cell imaging technique to unravel the proteins that play essential roles in a Gq-coupled muscarinic receptor-mediated cAMP signalling complex. We reveal that, upon agonist binding to the Gq-coupled muscarinic receptor, AKAP79 (A-kinase-anchoring protein 79) recruits PKC to activate AC2 to produce cAMP. The cAMP formed is degraded by PDE4 (phosphodiesterase 4) activated by an AKAP-anchored PKA (protein kinase A). Calcineurin, a phosphatase bound to AKAP79, is not involved in this regulation. Overall, a transient cAMP increase is generated from AC2 by Gq-coupled muscarinic receptor activation, subject to sophisticated regulation through AKAP79, PKC, PDE4 and PKA, which significantly enhances acetylcholine-mediated signalling. PMID:23889134

  6. Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells.

    PubMed

    Fujiwara, Yuya; Hiraoka, Yuri; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2015-06-30

    To assess the isoform specificity of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKβ mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKβ mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKβ is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

  7. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

    PubMed Central

    Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

  8. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

    PubMed

    Kaczmarczyk, Lech; Labrie-Dion, Étienne; Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

  9. Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

    PubMed

    Lee, S H; Johnson, J D; Walsh, M P; Van Lierop, J E; Sutherland, C; Xu, A; Snedden, W A; Kosk-Kosicka, D; Fromm, H; Narayanan, N; Cho, M J

    2000-08-15

    Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.

  10. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclas...

  11. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    SciTech Connect

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. Univ. of California, Los Angeles )

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  12. Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase.

    PubMed Central

    Roelofs, J; Snippe, H; Kleineidam, R G; Van Haastert, P J

    2001-01-01

    The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase. PMID:11237875

  13. Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1.

    PubMed

    Song, Yunke; Liu, Kelvin J; Wang, Tza-Huei

    2015-01-01

    MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences.

  14. Structures of alternatively spliced isoforms of human ketohexokinase.

    PubMed

    Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

    2009-03-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

  15. Structures of alternatively spliced isoforms of human ketohexokinase

    PubMed Central

    Trinh, Chi H.; Asipu, Aruna; Bonthron, David T.; Phillips, Simon E. V.

    2009-01-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure. PMID:19237742

  16. Ozone-induced airway hyperresponsiveness: roles of ROCK isoforms.

    PubMed

    Lambert, James A; Song, Weifeng

    2015-12-15

    Acute ozone (O3) inhalation has been shown to cause airway and pulmonary epithelial injury with accompanying inflammation responses. Robust evidence exists that O3 induces airway hyperresponsiveness (AHR) in humans and in animal models. Several pathways exist that culminate in airway smooth muscle contraction, but the mechanism(s) by which O3 elicits AHR are unclear. Here, we review the recent report by Kasahara et al. (Kasahara DI, Mathews JA, Park CY, Cho Y, Hunt G, Wurmbrand AP, Liao JK, Shore SA. Am J Physiol Lung Cell Mol Physiol 309: L736-L746, 2015.) describing the role of two Rho kinase (ROCK) isoforms in O3-induced AHR utilizing a murine haploinsufficiency model. Compared with wild-type (WT) mice, the authors report that ROCK1(+/-) and ROCK2(+/-) mice exhibited significantly reduced AHR following acute exposure to O3. Additionally, WT mice treated with fasudil, an FDA-approved ROCK1/2 inhibitor, recapitulated reduction in AHR as seen in ROCK haplotypes. It was suggested that, although the two ROCK isoforms are both induced by Rho, they have different mechanisms by which they mediate O3-induced AHR: ROCK1 via hyaluronan signaling vs. ROCK2 acting downstream of inflammation at the level of airway smooth muscle contraction. These observations provide an important framework to develop novel ROCK-targeting therapies for acute O3-induced AHR.

  17. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  18. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  19. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure.

    PubMed

    Scarpace, P J; Baresi, L A; Morley, J E

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the beta-adrenergic pathway, adenylate cyclase activity and beta-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. beta-Adrenergic receptors were identified in BAT using [125I]iodocyanopindolol. Binding sites had the characteristics of mixed beta 1- and beta 2-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in beta-adrenergic receptor density due to a loss of the beta 1-adrenergic subtype. This BAT beta-adrenergic receptor downregulation was tissue specific, since myocardial beta-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. In contrast, food deprivation did not alter BAT beta-adrenergic receptor density. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. The ratio of isoproterenol-stimulated to forskolin-stimulated adenylate cyclase activity decreased in the sucrose-fed and cold-exposed rats but not in the food-deprived rats. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of beta-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability. PMID:2827501

  20. Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

    SciTech Connect

    Verkman, A.S.; Ausiello, D.A.; Jung, C.Y.; Skorecki, K.L.

    1986-08-12

    Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

  1. Properties of Adenyl Cyclase from Human Jejunal Mucosa during Naturally Acquired Cholera and Convalescence

    PubMed Central

    Chen, Lincoln C.; Rohde, Jon E.; Sharp, Geoffrey W. G.

    1972-01-01

    The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E1 (PGE1) and by fluoride was observed. The responsiveness to PGE1 was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg++ concentration was 10 mM; Mn++ at 5 mM stimulated the enzyme but could not replace Mg++ except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10-4 M. The pH optimum was 7.5 and the Michaelis constant (Km) for ATP concentration approximated 10-4 M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringer's solution at 0°C, no loss of activity was observed at 1½ and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0°C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed. Images PMID:4335441

  2. PI3K isoform dependence of PTEN-deficient tumors can be altered by the genetic context.

    PubMed

    Schmit, Fabienne; Utermark, Tamara; Zhang, Sen; Wang, Qi; Von, Thanh; Roberts, Thomas M; Zhao, Jean J

    2014-04-29

    There has been increasing interest in the use of isoform-selective inhibitors of phosphatidylinositide-3-kinase (PI3K) in cancer therapy. Using conditional deletion of the p110 catalytic isoforms of PI3K to predict sensitivity of cancer types to such inhibitors, we and others have demonstrated that tumors deficient of the phosphatase and tensin homolog (PTEN) are often dependent on the p110β isoform of PI3K. Because human cancers usually arise due to multiple genetic events, determining whether other genetic alterations might alter the p110 isoform requirements of PTEN-null tumors becomes a critical question. To investigate further the roles of p110 isoforms in PTEN-deficient tumors, we used a mouse model of ovarian endometrioid adenocarcinoma driven by concomitant activation of the rat sarcoma protein Kras, which is known to activate p110α, and loss of PTEN. In this model, ablation of p110β had no effect on tumor growth, whereas p110α ablation blocked tumor formation. Because ablation of PTEN alone is often p110β dependent, we wondered if the same held true in the ovary. Because PTEN loss alone in the ovary did not result in tumor formation, we tested PI3K isoform dependence in ovarian surface epithelium (OSE) cells deficient in both PTEN and p53. These cells were indeed p110β dependent, whereas OSEs expressing activated Kras with or without PTEN loss were p110α dependent. Furthermore, isoform-selective inhibitors showed a similar pattern of the isoform dependence in established Kras(G12D)/PTEN-deficient tumors. Taken together, our data suggest that, whereas in some tissues PTEN-null tumors appear to inherently depend on p110β, the p110 isoform reliance of PTEN-deficient tumors may be altered by concurrent mutations that activate p110α.

  3. rlk/TXK Encodes Two Forms of a Novel Cysteine String Tyrosine Kinase Activated by Src Family Kinases

    PubMed Central

    Debnath, Jayantha; Chamorro, Mario; Czar, Michael J.; Schaeffer, Edward M.; Lenardo, Michael J.; Varmus, Harold E.; Schwartzberg, Pamela L.

    1999-01-01

    Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR. PMID:9891083

  4. Characterization of two cloned human CB1 cannabinoid receptor isoforms.

    PubMed

    Rinaldi-Carmona, M; Calandra, B; Shire, D; Bouaboula, M; Oustric, D; Barth, F; Casellas, P; Ferrara, P; Le Fur, G

    1996-08-01

    We have investigated the pharmacology of two central human cannabinoid receptor isoforms, designated CB1 and CB1A, stably expressed in Chinese hamster ovary cell lines, designated as CHO-CB1 and CHO-CB1A, respectively. In direct binding assays on isolated membranes the agonist [3H]CP 55,940 bound in a saturable and highly specific manner to both cannabinoid receptor isoforms. Competition binding experiments performed with other commonly used receptor agonists showed the following rank order of potency: CP 55,940 > tetrahydrocannabinol > WIN 55212-2 > anandamide. Except for the endogenous ligand anandamide (CB1, Ki = 359.6 nM vs. CB1A, Ki = 298 nM), these agonists bound to CB1A (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 7.24,345 and 26.7 nM, respectively) with about 3-fold less affinity than to CB1 (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 2.26, 93 and 7.1 nM, respectively). The cannabinoid receptor antagonist SR 141716A also bound to CB1A (Ki = 43.3 nM) with slightly less affinity than to CB1 (Ki = 4.9 nM). Cannabinoid receptor-linked second messenger system studies performed in the CHO-CB1 and CHO-CB1A cells showed that both receptors mediated their action through the agonist-induced inhibition of forskolin-stimulated cAMP accumulation. This activity was totally blocked by pretreatment with PTX. Additionally, both isoforms activated mitogen-activated protein kinase. The selective antagonist SR 141716A was able to selectively block these responses in both cell lines, to an extent that reflected its binding characteristics. Our results show that the amino-truncated and -modified CB1 isoform CB1A exhibits all the properties of CB1 to a slightly attenuated extent.

  5. Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone*

    PubMed Central

    Higa-Nakamine, Sayomi; Maeda, Noriko; Toku, Seikichi; Yamamoto, Hideyuki

    2015-01-01

    The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer. PMID:26338704

  6. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    PubMed

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

  7. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins

    PubMed Central

    Kimberg, Daniel V.; Field, Michael; Johnson, Judith; Henderson, Antonia; Gershon, Elaine

    1971-01-01

    The effects of several prostaglandins (PG) and a highly purified preparation of cholera enterotoxin (CT) on intestinal mucosal adenyl cyclase activity and the effect of CT on intestinal mucosal cyclic 3′,5′-adenosine monophosphate concentration were determined in guinea pig and rabbit small intestine and were correlated with the effects of the same agents on ion transport. Adenyl cyclase activity, measured in a crude membrane fraction of the mucosa, was found at all levels of the small intestine with the highest activity per milligram protein in the duodenum. The prostaglandins, when added directly to the assay, increased adenyl cyclase activity; the greatest effect (2-fold increase) was obtained with PGE1 (maximal effect at 0.03 mM) and PGE2. The prostaglandins also increased short-circuit current (SCC) in isolated guinea pig ileal mucosa, with PGE1 and PGE2 again giving the greatest effects. The prior addition of theophylline (10 mM) reduced the subsequent SCC response to PGE1 and vice versa. It was concluded, therefore, that the SCC response to PGE1, like the response to theophylline, represented active Cl secretion. CT increased adenyl cyclase activity in guinea pig and rabbit ileal mucosa when preincubated with the mucosa from 1 to 2.5 hr in vitro or for 2.5 hr in vivo but not when added directly to the assay. The increments in activity caused by PGE1 and NaF were the same in CT-treated and control mucosa. Cyclic 3′,5′-AMP concentration in rabbit ileal mucosa was increased 3.5-fold after a 2 hr preincubation with CT in vitro. Phosphodiesterase activity in the crude membrane fraction of the mucosa was unaffected by either CT or PGE1. A variety of other agents including insulin, glucagon, parathormone, thyroid-stimulating hormone, L-thyroxine, thyrocalcitonin, vasopressin, and epinephrine all failed to change adenyl cyclase activity. It is concluded that CT and certain prostaglandins produce small intestinal fluid secretion by increasing mucosal adenyl

  8. Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphylococcus aureus.

    PubMed Central

    Galgiani, J N; Hewlett, E L; Friedman, R L

    1988-01-01

    Bordetella pertussis extract that contained adenylate cyclase toxin produced large increases in human neutrophil cyclic AMP levels and inhibited their oxidative burst, as reflected by luminol-enhanced chemiluminescence and superoxide release. The adenylate cyclase toxin-containing extract blocked neutrophil-mediated inhibition of N-acetylglucosamine incorporation by arthroconidia of Coccidioides immitis in a dose-dependent fashion but had no effect on neutrophil phagocytosis of Candida glabrata and only a slight inhibitory effect on arthroconidial attachment. Neither purified pertussis toxin nor extracts from Bordetella mutants lacking the adenylate cyclase toxin affected neutrophil-mediated inhibition of arthroconidial N-acetylglucosamine incorporation. These studies indicate that adenylate cyclase toxin, alone or in concert with other B. pertussis-elaborated toxins, blocks neutrophil inhibition of arthroconidia, primarily by affecting neutrophil responses other than attachment or phagocytosis. PMID:2894360

  9. Inhibition of vaccinia mRNA methylation by 2',5'-linked oligo(adenylic acid) triphosphate

    SciTech Connect

    Sharma, O.K.; Goswami, B.B.

    1981-04-01

    Extracts of interferon-treated cells synthesize unique 2',5'-linked oligo(adenylic acid) 5'-phosphates in the presence of ATP and double-stranded RNA. 2',5'-linked oligo(adenylic acid) 5'-triphosphate inhibits protein synthesis at nanomolar concentrations by activating RNase. We have observed that oligo(adenylic acid) 5'-monophosphate and 5'-triphosphate are potent inhibitors of vaccinia mRNA methylation in vitro. Both the methylation of the 5'-terminal guanine at the 7 position and the 2'-O-ribose methylation of the penultimate nucleoside are inhibited. Such inhibition of mRNA methylation is not due to degradation of the mRNA. Inhibition of the requisite modification of the 5' terminus of mRNA by 2',5'-linked oligo(adenylic acids) may be a mechanism of interferon action against both DNA and RNA viruses in which mRNAs derived from them are capped.

  10. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    PubMed

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  11. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    PubMed Central

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  12. Upregulation of adenylate cyclase 3 (ADCY3) increases the tumorigenic potential of cells by activating the CREB pathway

    PubMed Central

    Lee, Sang Jin; Hwang, Jung-Ah; Lee, Jieun; Choi, Il-Ju; Seo, Hyehyun; Park, Jong-Hoon; Suzuki, Hiromu; Yamamoto, Eiichiro; Kim, In-Hoo; Jeong, Jin Sook; Ju, Mi Ha; Lee, Dong-Hee; Lee, Yeon-Su

    2013-01-01

    Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3′,5′-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren's intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells; conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 (MMP2) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatment with a protein kinase A (PKA) inhibitor decreased MMP2 and MMP9 expression levels in ADCY3-overexpressing cells. Knockdown of ADCY3 expression by stable shRNA in human gastric cancer cells suppressed tumor growth in a tumor xenograft model. Thus, ADCY3 overexpression may exert its tumor-promoting effects via the cAMP/PKA/CREB pathway. Additionally, bisulfite sequencing of the ADCY3 promoter region revealed that gene expression was reduced by hypermethylation of CpG sites, and increased by 5-Aza-2′-deoxycytidine (5-Aza-dC)-induced demethylation. Our study is the first to report an association of ADCY3 with gastric cancer as well as its tumorigenic potentials. In addition, we demonstrate that the expression of ADCY3 is regulated through an epigenetic mechanism. Further study on the mechanism of ADCY3 in tumorigenesis will provide the basis as a new molecular target of gastric cancer. PMID:24113161

  13. Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes

    SciTech Connect

    Stadel, J.M.; Rebar, R.; Crooke, S.T.

    1987-05-01

    Desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes results in 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of ..beta..-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoproterenol- and cAMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37/sup 0/C, pH = 8.0. In both cases alkaline phosphatase treatment significantly reduced desensitization of agonist-stimulated adenylate cyclase activity by 40-60%. Similar results were obtained following alkaline phosphatase treatment of membranes from isoproterenol- and cAMP-desensitized duck erythrocytes. In addition, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with phorbol 12-mystrate 13-acetate returned adenylate cyclase activity to near control values. In all experiments inclusion of 20 mM NaPO/sub 4/ to inhibit alkaline phosphatase during treatment of membranes blocked the enzyme's effect on agonist-stimulated adenylate cyclase activity. These results demonstrate a role for phosphorylation in desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes.

  14. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-12-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.

  15. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  16. Non-co-ordinate development of beta-adrenergic receptors and adenylate cyclase in chick heart.

    PubMed Central

    Alexander, R W; Galper, J B; Neer, E J; Smith, T W

    1982-01-01

    We have studied the properties of beta-adrenergic receptors and of their interaction with adenylate cyclase in the chick myocardium during embryogenesis. Between 4.5 and 7.5 days in ovo the number of receptors determined by (-)-[3H]dihydroalprenolol ([3H]DHA) binding is constant at approx. 0.36 pmol of receptor/mg of protein. By day 9 the density decreases significantly to 0.22 pmol of receptor/mg of protein. At day 12.5--13.5 the number was 0.14--0.18 pmol of receptor/mg of protein. This number did not change further up to day 16. The same results were obtained with guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) added to the assay mixtures. There was no significant change in receptor affinity for the antagonist [3H]DHA between days 5.5 and 13. Despite the decrease in numbers of beta-adrenergic receptors, there was no change in basal, p[NH]ppG-, isoprenaline- or isoprenaline-plus-p[NH]ppG-stimulated adenylate cyclase activity between days 3 and 12 of development. We conclude that beta-adrenergic receptors and adenylate cyclase are not co-ordinately regulated during early embryonic development of the chick heart. Some of the beta-adrenergic receptors present very early in the ontogeny of cardiac tissue appear not to be coupled to adenylate cyclase since their loss is not reflected in decreased activation of the enzyme. PMID:6289805

  17. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  18. Ontogeny of fetal adenylate cyclase; mechanisms for regulation of beta-adrenergic receptors.

    PubMed

    Maier, J A; Roberts, J M; Jacobs, M M

    1989-11-01

    Transmembrane second messenger signalling systems regulate differentiation, growth and homeostatic responses during fetal development. The beta-adrenergic adenylate cyclase system is the best studied of these and has been used as a model to investigate the control of developmental processes. In tissues such as lung, heart and parotid, beta-adrenergic responsiveness of adenylate cyclase increases during development. In the developing fetal lung beta-receptor concentration increases during gestation or after glucocorticoid treatment, but cannot fully explain enhanced adrenergic responsiveness. To probe developmental and hormonal effects on beta-receptor function, we asked if advancing gestation or glucocorticoid treatment alters beta-receptor-Gs interactions in fetal rabbit lung membrane particulates. Before 25 days gestation, 1-isoproterenol competes for 3H-dihydroalprenolol (DHA), a radiolabelled beta-antagonist, with a single low affinity, later in gestation, high and low affinities of isoproterenol for the beta-receptor are present which can be shifted to the lower affinity by addition of guanyl nucleotide. High affinity binding is precociously induced in 25 days--fetal lung particulates as early as 3 h after maternal betamethasone treatment, but beta-adrenoreceptor concentration in treated fetuses was increased over controls only after 24 h of treatment. Cholera toxin catalyzed ADP ribosylation of membrane particulates showed cholera toxin substrate (Gs) was not altered by glucocorticoid treatment. Stimulation of adenylate cyclase activity with isoproterenol (100mM) and GTP (100mM) resulted in no incremental increase over that produced by GTP (100mM) alone in glucocorticoid treated or control particulates, either early or late in gestation. These data demonstrate that beta-receptor-Gs interactions are not sufficient to produce full agonist responses. Although both beta-adrenergic receptors and Gs are present in fetal rabbit lung early in gestation, interaction

  19. Circadian rhythmicity of active GSK3 isoforms modulates molecular clock gene rhythms in the suprachiasmatic nucleus.

    PubMed

    Besing, Rachel C; Paul, Jodi R; Hablitz, Lauren M; Rogers, Courtney O; Johnson, Russell L; Young, Martin E; Gamble, Karen L

    2015-04-01

    The suprachiasmatic nucleus (SCN) drives and synchronizes daily rhythms at the cellular level via transcriptional-translational feedback loops comprising clock genes such as Bmal1 and Period (Per). Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, phosphorylates at least 5 core clock proteins and shows diurnal variation in phosphorylation state (inactivation) of the GSK3β isoform. Whether phosphorylation of the other primary isoform (GSK3α) varies across the subjective day-night cycle is unknown. The purpose of this study was to determine if the endogenous rhythm of GSK3 (α and β) phosphorylation is critical for rhythmic BMAL1 expression and normal amplitude and periodicity of the molecular clock in the SCN. Significant circadian rhythmicity of phosphorylated GSK3 (α and β) was observed in the SCN from wild-type mice housed in constant darkness for 2 weeks. Importantly, chronic activation of both GSK3 isoforms impaired rhythmicity of the GSK3 target BMAL1. Furthermore, chronic pharmacological inhibition of GSK3 with 20 µM CHIR-99021 enhanced the amplitude and shortened the period of PER2::luciferase rhythms in organotypic SCN slice cultures. These results support the model that GSK3 activity status is regulated by the circadian clock and that GSK3 feeds back to regulate the molecular clock amplitude in the SCN.

  20. Defective responsiveness of adenylate cyclase to forskolin in the Drosophila memory mutant rutabaga.

    PubMed

    Dudai, Y; Sher, B; Segal, D; Yovell, Y

    1985-12-01

    The Drosophila memory mutant rutabaga (rut) has been previously shown to have a defective subpopulation (or functional state) of the enzyme adenylate cyclase. We report here that the reduced adenylate cyclase activity is also associated with a defective responsiveness of the enzyme to forskolin. Forskolin activation isotherms of the enzyme in normal membranes reveal low- and high-affinity forskolin-interacting components; the residual enzyme in the mutant shows a smaller proportion of the high-affinity response. In addition, in mutant membrane preparations, forskolin fails to shift the Km of the enzyme for free Mg2+ and for MgATP, in contrast to the situation in the normal tissue. The defect in the responsiveness to forskolin in rut is even more pronounced in a Lubrol-solubilized enzyme preparation, and is due to intrinsic properties of the cyclase system rather than to the absence (or presence) of a soluble, or detergent solubilized, factor in rut. The reduced forskolin responsiveness maps to the X chromosomal segment 12F5-6 to 13A1-5, within the region previously reported to span the locus that controls both the abortive memory and the lack of Ca2+-stimulation of adenylate cyclase in rut17. The possible relevance of the findings to postulated molecular mechanisms of short-term memory formation is discussed. PMID:3935769

  1. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  2. Persistent stimulation of adenylate cyclase and urea transport by an AVP photolabel

    SciTech Connect

    Eggena, P.; Ma, C.L.; Fahrenholz, F.; Schwartz, I.L.

    1985-07-01

    The effects of a photoaffinity label for arginine vasopressin receptors, (Phe2, Phe(p-N3)3)AVP (N3-AVP), on urea permeability and adenylate cyclase activity have been investigated in the toad urinary bladder. This compound, when activated by ultraviolet light, induced a maximal and persistent increase in the urea permeability of the intact bladder and a persistent increase in the adenylate cyclase activity of toad bladder epithelial cell homogenates. Covalent attachment of the analogue to target tissue during photolysis was equivalent at 4 and 20 degrees C. Bladders exposed to N3-AVP in the presence of AVP during photolysis were substantially less permeable to urea than controls that had been exposed to N3-AVP alone. These findings constitute further evidence in support of the previous suggestion that N3-AVP binds covalently to AVP receptors and, in addition, demonstrates that N3-AVP evokes a persistent increase in adenylate cyclase activity which, in turn, triggers a persistent increase in bladder permeability to urea.

  3. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  4. GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites.

    PubMed

    Boitz, Jan M; Jardim, Armando; Ullman, Buddy

    2016-08-01

    Purine acquisition is an essential nutritional process for Leishmania. Although purine salvage into adenylate nucleotides has been investigated in detail, little attention has been focused on the guanylate branch of the purine pathway. To characterize guanylate nucleotide metabolism in Leishmania and create a cell culture model in which the pathways for adenylate and guanylate nucleotide synthesis can be genetically uncoupled for functional studies in intact cells, we created and characterized null mutants of L. donovani that were deficient in either GMP reductase alone (Δgmpr) or in both GMP reductase and its paralog IMP dehydrogenase (Δgmpr/Δimpdh). Whereas wild type parasites were capable of utilizing virtually any purine nucleobase/nucleoside, the Δgmpr and Δgmpr/Δimpdh null lines exhibited highly restricted growth phenotypes. The Δgmpr single mutant could not grow in xanthine, guanine, or their corresponding nucleosides, while no purine on its own could support the growth of Δgmpr/Δimpdh cells. Permissive growth conditions for the Δgmpr/Δimpdh necessitated both xanthine, guanine, or the corresponding nucleosides, and additionally, a second purine that could serve as a source for adenylate nucleotide synthesis. Interestingly, GMPR, like its paralog IMPDH, is compartmentalized to the leishmanial glycosome, a process mediated by its COOH-terminal peroxisomal targeting signal. The restricted growth phenotypes displayed by the L. donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.

  5. Absolute Quantification of Endogenous Ras Isoform Abundance

    PubMed Central

    Mageean, Craig J.; Griffiths, John R.; Smith, Duncan L.; Clague, Michael J.; Prior, Ian A.

    2015-01-01

    Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data. PMID:26560143

  6. Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer

    PubMed Central

    Liu, Jinfeng; McCleland, Mark; Stawiski, Eric W.; Gnad, Florian; Mayba, Oleg; Haverty, Peter M.; Durinck, Steffen; Chen, Ying-Jiun; Klijn, Christiaan; Jhunjhunwala, Suchit; Lawrence, Michael; Liu, Hanbin; Wan, Yinan; Chopra, Vivek; Yaylaoglu, Murat B.; Yuan, Wenlin; Ha, Connie; Gilbert, Houston N.; Reeder, Jens; Pau, Gregoire; Stinson, Jeremy; Stern, Howard M.; Manning, Gerard; Wu, Thomas D.; Neve, Richard M.; de Sauvage, Frederic J.; Modrusan, Zora; Seshagiri, Somasekar; Firestein, Ron; Zhang, Zemin

    2014-01-01

    Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK. PMID:24807215

  7. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    PubMed

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions. PMID:26304132

  8. Redundancy in the World of MAP Kinases: All for One

    PubMed Central

    Saba-El-Leil, Marc K.; Frémin, Christophe; Meloche, Sylvain

    2016-01-01

    The protein kinases ERK1 and ERK2 are the effector components of the prototypical ERK1/2 mitogen-activated protein (MAP) kinase pathway. This signaling pathway regulates cell proliferation, differentiation and survival, and is essential for embryonic development and cellular homeostasis. ERK1 and ERK2 homologs share similar biochemical properties but whether they exert specific physiological functions or act redundantly has been a matter of controversy. However, recent studies now provide compelling evidence in support of functionally redundant roles of ERK1 and ERK2 in embryonic development and physiology. In this review, we present a critical assessment of the evidence for the functional specificity or redundancy of MAP kinase isoforms. We focus on the ERK1/ERK2 pathway but also discuss the case of JNK and p38 isoforms. PMID:27446918

  9. The Role of Phosphoinositide 3-Kinase Signaling in Intestinal Inflammation

    PubMed Central

    Cahill, Catherine M.; Rogers, Jack T.; Walker, W. Allan

    2012-01-01

    The phosphatidylinositol 3-kinase signaling pathway plays a central role in regulating the host inflammatory response. The net effect can either be pro- or anti-inflammatory depending on the system and cellular context studied. This paper focuses on phosphatidylinositol 3-kinase signaling in innate and adaptive immune cells of the intestinal mucosa. The role of phosphatidylinositol 3-kinase signaling in mouse models of inflammatory bowel disease is also discussed. With the development of new isoform specific inhibitors, we are beginning to understand the specific role of this complex pathway, in particular the role of the γ isoform in intestinal inflammation. Continued research on this complex pathway will enhance our understanding of its role and provide rationale for the design of new approaches to intervention in chronic inflammatory conditions such as inflammatory bowel disease. PMID:22570785

  10. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    PubMed

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

  11. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    PubMed Central

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  12. Locomotion in Lymphocytes is Altered by Differential PKC Isoform Expression

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    1999-01-01

    Lymphocyte locomotion is critical for proper elicitation of the immune response. Locomotion of immune cells via the interstitium is essential for optimal immune function during wound healing, inflammation and infection. There are conditions which alter lymphocyte locomotion and one of them is spaceflight. Lymphocyte locomotion is severely inhibited in true spaceflight (true microgravity) and in rotating wall vessel culture (modeled microgravity). When lymphocytes are activated prior to culture in modeled microgravity, locomotion is not inhibited and the levels are comparable to those of static cultured lymphocytes. When a phorbol ester (PMA) is used in modeled microgravity, lymphocyte locomotion is restored by 87%. This occurs regardless if PMA is added after culture in the rotating wall vessel or during culture. Inhibition of DNA synthesis also does not alter restoration of lymphocyte locomotion by PMA. PMA is a direct activator of (protein kinase C) PKC . When a calcium ionophore, ionomycin is used it does not possess any restorative properties towards locomotion either alone or collectively with PMA. Since PMA brings about restoration without help from calcium ionophores (ionomycin), it is infer-red that calcium independent PKC isoforms are involved. Changes were perceived in the protein levels of PKC 6 where levels of the protein were downregulated at 24,72 and 96 hours in untreated rotated cultures (modeled microgravity) compared to untreated static (1g) cultures. At 48 hours there is an increase in the levels of PKC & in the same experimental set up. Studies on transcriptional and translational patterns of calcium independent isoforms of PKC such as 8 and E are presented in this study.

  13. Pituitary adenylate cyclase-activating peptide induces long-lasting neuroprotection through the induction of activity-dependent signaling via the cyclic AMP response element-binding protein-regulated transcription co-activator 1

    PubMed Central

    Baxter, Paul S; Martel, Marc-Andre; McMahon, Aoife; Kind, Peter C; Hardingham, Giles E

    2011-01-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is a neuroprotective peptide which exerts its effects mainly through the cAMP-protein kinase A (PKA) pathway. Here, we show that in cortical neurons, PACAP-induced PKA signaling exerts a major part of its neuroprotective effects indirectly, by triggering action potential (AP) firing. Treatment of cortical neurons with PACAP induces a rapid and sustained PKA-dependent increase in AP firing and associated intracellular Ca2+ transients, which are essential for the anti-apoptotic actions of PACAP. Transient exposure to PACAP induces long-lasting neuroprotection in the face of apoptotic insults which is reliant on AP firing and the activation of cAMP response element (CRE) binding protein (CREB)-mediated gene expression. Although direct, activity-independent PKA signaling is sufficient to trigger phosphorylation on CREB’s activating serine-133 site, this is insufficient for activation of CREB-mediated gene expression. Full activation is dependent on CREB-regulated transcription co-activator 1 (CRTC1), whose PACAP-induced nuclear import is dependent on firing activity-dependent calcineurin signaling. Over-expression of CRTC1 is sufficient to rescue PACAP-induced CRE-mediated gene expression in the face of activity-blockade, while dominant negative CRTC1 interferes with PACAP-induced, CREB-mediated neuroprotection. Thus, the enhancement of AP firing may play a significant role in the neuroprotective actions of PACAP and other adenylate cyclase-coupled ligands. PMID:21623792

  14. Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

    PubMed Central

    Chen, Y; Sinha, K; Perumal, K; Reddy, R

    2000-01-01

    It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs. PMID:10999605

  15. Structural Basis of the Interaction of MbtH-like Proteins, Putative Regulators of Nonribosomal Peptide Biosynthesis, with Adenylating Enzymes*

    PubMed Central

    Herbst, Dominik A.; Boll, Björn; Zocher, Georg; Stehle, Thilo; Heide, Lutz

    2013-01-01

    The biosynthesis of nonribosomally formed peptides (NRPs), which include important antibiotics such as vancomycin, requires the activation of amino acids through adenylate formation. The biosynthetic gene clusters of NRPs frequently contain genes for small, so-called MbtH-like proteins. Recently, it was discovered that these MbtH-like proteins are required for some of the adenylation reactions in NRP biosynthesis, but the mechanism of their interaction with the adenylating enzymes has remained unknown. In this study, we determined the structure of SlgN1, a 3-methylaspartate-adenylating enzyme involved in the biosynthesis of the hybrid polyketide/NRP antibiotic streptolydigin. SlgN1 contains an MbtH-like domain at its N terminus, and our analysis defines the parameters required for an interaction between MbtH-like domains and an adenylating enzyme. Highly conserved tryptophan residues of the MbtH-like domain critically contribute to this interaction. Trp-25 and Trp-35 form a cleft on the surface of the MbtH-like domain, which accommodates the alanine side chain of Ala-433 of the adenylating domain. Mutation of Ala-433 to glutamate abolished the activity of SlgN1. Mutation of Ser-23 of the MbtH-like domain to tyrosine resulted in strongly reduced activity. However, the activity of this S23Y mutant could be completely restored by addition of the intact MbtH-like protein CloY from another organism. This suggests that the interface found in the structure of SlgN1 is the genuine interface between MbtH-like proteins and adenylating enzymes. PMID:23192349

  16. Structural basis of the interaction of MbtH-like proteins, putative regulators of nonribosomal peptide biosynthesis, with adenylating enzymes.

    PubMed

    Herbst, Dominik A; Boll, Björn; Zocher, Georg; Stehle, Thilo; Heide, Lutz

    2013-01-18

    The biosynthesis of nonribosomally formed peptides (NRPs), which include important antibiotics such as vancomycin, requires the activation of amino acids through adenylate formation. The biosynthetic gene clusters of NRPs frequently contain genes for small, so-called MbtH-like proteins. Recently, it was discovered that these MbtH-like proteins are required for some of the adenylation reactions in NRP biosynthesis, but the mechanism of their interaction with the adenylating enzymes has remained unknown. In this study, we determined the structure of SlgN1, a 3-methylaspartate-adenylating enzyme involved in the biosynthesis of the hybrid polyketide/NRP antibiotic streptolydigin. SlgN1 contains an MbtH-like domain at its N terminus, and our analysis defines the parameters required for an interaction between MbtH-like domains and an adenylating enzyme. Highly conserved tryptophan residues of the MbtH-like domain critically contribute to this interaction. Trp-25 and Trp-35 form a cleft on the surface of the MbtH-like domain, which accommodates the alanine side chain of Ala-433 of the adenylating domain. Mutation of Ala-433 to glutamate abolished the activity of SlgN1. Mutation of Ser-23 of the MbtH-like domain to tyrosine resulted in strongly reduced activity. However, the activity of this S23Y mutant could be completely restored by addition of the intact MbtH-like protein CloY from another organism. This suggests that the interface found in the structure of SlgN1 is the genuine interface between MbtH-like proteins and adenylating enzymes.

  17. A novel LKB1 isoform enhances AMPK metabolic activity and displays oncogenic properties.

    PubMed

    Dahmani, R; Just, P-A; Delay, A; Canal, F; Finzi, L; Prip-Buus, C; Lambert, M; Sujobert, P; Buchet-Poyau, K; Miller, E; Cavard, C; Marmier, S; Terris, B; Billaud, M; Perret, C

    2015-04-30

    The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism and cell polarity. We now report the identification of a novel isoform of LKB1 (named ΔN-LKB1) that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The ΔN-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although ΔN-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory autoinhibitory domain of AMPK. In contrast, ΔN-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showed that ΔN-LKB1 has an intrinsic oncogenic property. ΔN-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of ΔN-LKB1 decreased the survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both the LKB1 tumor suppressor gene and the oncogene ΔN-LKB1 are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis.

  18. Isoform-Selective Disruption of AKAP-Localized PKA Using Hydrocarbon Stapled Peptides

    PubMed Central

    2015-01-01

    A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells. PMID:24422448

  19. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  20. Crystal structure of pyridoxal kinase from the Escherichia coli pdxK gene: implications for the classification of pyridoxal kinases.

    PubMed

    Safo, Martin K; Musayev, Faik N; di Salvo, Martino L; Hunt, Sharyn; Claude, Jean-Baptiste; Schirch, Verne

    2006-06-01

    The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-A resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4' substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4' of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

  1. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster.

    PubMed

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-04-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I. PMID:24699734

  2. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster

    PubMed Central

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-01-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I. PMID:24699734

  3. The Structure of PA1221, a Non-Ribosomal Peptide Synthetase containing Adenylation and Peptidyl Carrier Protein Domains

    PubMed Central

    Mitchell, Carter A.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M.

    2012-01-01

    Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Non-Ribosomal Peptide Synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a non-fused carrier domain inter-molecularly. Finally, we have determined crystal structures of both the apo- and holo-PA1221 protein, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation-carrier domain interaction. The protein adopts a similar interface to that seen with the prior adenylation-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation. PMID:22452656

  4. Sat-Nav for T cells: Role of PI3K isoforms and lipid phosphatases in migration of T lymphocytes.

    PubMed

    Ward, Stephen G; Westwick, John; Harris, Stephanie

    2011-07-01

    Phosphoinositide 3-kinase (PI3K)-dependent signaling has been placed at the heart of conserved biochemical mechanisms that facilitate cell migration of leukocytes in response to a range of chemoattractant stimuli. This review assesses the evidence for and against PI3K-dependent mechanisms of T lymphocyte migration and whether pharmacological targeting of PI3K isoforms is likely to offer potential benefit for T cell mediated pathologies. PMID:21333676

  5. PI3K in cancer: divergent roles of isoforms, modes of activation, and therapeutic targeting

    PubMed Central

    Thorpe, Lauren M.; Yuzugullu, Haluk; Zhao, Jean J.

    2015-01-01

    Preface Phosphatidylinositol 3-Kinases (PI3Ks) are critical coordinators of intracellular signaling in response to extracellular stimuli. Hyperactivation of PI3K signaling cascades is one of the most common events in human cancers. In this Review, we discuss recent advances in our knowledge of the roles of distinct PI3K isoforms in normal and oncogenic signaling, the different ways in which PI3K can be upregulated, and the current state and future potential of targeting this pathway in the clinic. PMID:25533673

  6. The amino acid sequence of GTP:AMP phosphotransferase from beef-heart mitochondria. Extensive homology with cytosolic adenylate kinase.

    PubMed

    Wieland, B; Tomasselli, A G; Noda, L H; Frank, R; Schulz, G E

    1984-09-01

    The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three-dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.

  7. Pyruvate kinase: function, regulation and role in cancer

    PubMed Central

    Israelsen, William J.; Vander Heiden, Matthew G.

    2015-01-01

    Pyruvate kinase is an enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP in glycolysis and plays a role in regulating cell metabolism. There are four mammalian pyruvate kinase isoforms with unique tissue expression patterns and regulatory properties. The M2 isoform of pyruvate kinase (PKM2) supports anabolic metabolism and is expressed both in cancer and normal tissue. The enzymatic activity of PKM2 is allosterically regulated by both intracellular signaling pathways and metabolites; PKM2 thus integrates signaling and metabolic inputs to modulate glucose metabolism according to the needs of the cell. Recent advances have increased our understanding of metabolic regulation by pyruvate kinase, raised new questions, and suggested the possibility of non-canonical PKM2 functions to regulate gene expression and cell cycle progression via protein-protein interactions and protein kinase activity. Here we review the structure, function, and regulation of pyruvate kinase and discuss how these properties enable regulation of PKM2 for cell proliferation and tumor growth. PMID:26277545

  8. On the Dynamics of the Adenylate Energy System: Homeorhesis vs Homeostasis

    PubMed Central

    De la Fuente, Ildefonso M.; Cortés, Jesús M.; Valero, Edelmira; Desroches, Mathieu; Rodrigues, Serafim; Malaina, Iker; Martínez, Luis

    2014-01-01

    Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for

  9. Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes

    PubMed Central

    Lopez, Miguel A.; Saada, Edwin A.

    2014-01-01

    Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed “social motility,” based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies. PMID:25416239

  10. Insect stage-specific adenylate cyclases regulate social motility in African trypanosomes.

    PubMed

    Lopez, Miguel A; Saada, Edwin A; Hill, Kent L

    2015-01-01

    Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed "social motility," based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies. PMID:25416239

  11. Effects of Ca++ and Prostaglandin E1 on Vasopressin Activation of Renal Adenyl Cyclase

    PubMed Central

    Marumo, Fumiaki; Edelman, Isidore S.

    1971-01-01

    Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl2 inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca++ inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl2 reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E1 (PGE1 = 10−7m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10−4m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10−7m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE1 modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed. PMID:4329002

  12. Adenylate Energy Pool and Energy Charge in Maturing Rape Seeds 1

    PubMed Central

    Ching, Te May; Crane, Jim M.; Stamp, David L.

    1974-01-01

    A study of energy state and chemical composition of pod walls and seeds of maturing rape (Brassica napus L.) was conducted on two varieties, Victor and Gorczanski. Total adenosine phosphates, ATP, and adenylate energy charge increased with increasing cell number and cellular synthesis during the early stages, remained high at maximum dry weight accumulation and maximum substrate influx time, and decreased with ripening. A temporal control of energy supply and ATP concentration is evident in developing tissues with determined functions; whereas the association of a high energy charge and active cellular biosynthesis occurs only in tissues with a stabilized cell number. PMID:16658964

  13. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.

    PubMed Central

    Sternweis, P C; Gilman, A G

    1982-01-01

    Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-. PMID:6289322

  14. Citron rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein Citron.

    PubMed

    Di Cunto, F; Calautti, E; Hsiao, J; Ong, L; Topley, G; Turco, E; Dotto, G P

    1998-11-01

    We have identified a novel serine/threonine kinase belonging to the myotonic dystrophy kinase family. The kinase can be produced in at least two different isoforms: a approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; a approximately 54-kDa protein (CRIK-short kinase (SK)), which consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of costitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types.

  15. Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) and Vasoactive Intestinal Peptide (VIP) Regulate Murine Neural Progenitor Cell Survival, Proliferation, and Differentiation

    PubMed Central

    Scharf, Eugene; May, Victor; Braas, Karen M.; Shutz, Kristin C.

    2009-01-01

    Neural stem/progenitor cells (NPC) have gained wide interest over the last decade from their therapeutic potential, either through transplantation or endogenous replacement, after central nervous system (CNS) disease and damage. Whereas several growth factors and cytokines have been shown to promote NPC survival, proliferation, or differentiation, the identification of other regulators will provide much needed options for NPC self-renewal or lineage development. Although previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) can regulate stem/progenitor cells, the responses appeared variable. To examine the direct roles of these peptides in NPCs, postnatal mouse NPC cultures were withdrawn from epidermal growth factor (EGF) and fibroblastic growth factor (FGF) and maintained under serum-free conditions in the presence or absence of PACAP27, PACAP38, or VIP. The NPCs expressed the PAC1(short)null receptor isoform, and the activation of these receptors decreased progenitor cell apoptosis more than 80% from TUNEL assays and facilitated proliferation more than fivefold from bromodeoxyuridine (BrdU) analyses. To evaluate cellular differentiation, replicate control and peptide-treated cultures were examined for cell fate marker protein and transcript expression. In contrast with previous work, PACAP peptides downregulated NPC differentiation, which appeared consistent with the proliferation status of the treated cells. Accordingly, these results demonstrate that PACAP signaling is trophic and can maintain NPCs in a multipotent state. With these attributes, PACAP may be able to promote endogenous NPC self-renewal in the adult CNS, which may be important for endogenous self-repair in disease and ageing processes. PMID:18629655

  16. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) regulate murine neural progenitor cell survival, proliferation, and differentiation.

    PubMed

    Scharf, Eugene; May, Victor; Braas, Karen M; Shutz, Kristin C; Mao-Draayer, Yang

    2008-11-01

    Neural stem/progenitor cells (NPC) have gained wide interest over the last decade from their therapeutic potential, either through transplantation or endogenous replacement, after central nervous system (CNS) disease and damage. Whereas several growth factors and cytokines have been shown to promote NPC survival, proliferation, or differentiation, the identification of other regulators will provide much needed options for NPC self-renewal or lineage development. Although previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) can regulate stem/progenitor cells, the responses appeared variable. To examine the direct roles of these peptides in NPCs, postnatal mouse NPC cultures were withdrawn from epidermal growth factor (EGF) and fibroblastic growth factor (FGF) and maintained under serum-free conditions in the presence or absence of PACAP27, PACAP38, or VIP. The NPCs expressed the PAC1(short)null receptor isoform, and the activation of these receptors decreased progenitor cell apoptosis more than 80% from TUNEL assays and facilitated proliferation more than fivefold from bromodeoxyuridine (BrdU) analyses. To evaluate cellular differentiation, replicate control and peptide-treated cultures were examined for cell fate marker protein and transcript expression. In contrast with previous work, PACAP peptides downregulated NPC differentiation, which appeared consistent with the proliferation status of the treated cells. Accordingly, these results demonstrate that PACAP signaling is trophic and can maintain NPCs in a multipotent state. With these attributes, PACAP may be able to promote endogenous NPC self-renewal in the adult CNS, which may be important for endogenous self-repair in disease and ageing processes.

  17. Isolated neuronal growth cones from developing rat forebrain possess adenylate cyclase activity which can be augmented by various receptor agonists.

    PubMed

    Lockerbie, R O; Hervé, D; Blanc, G; Tassin, J P; Glowinski, J

    1988-01-01

    Isolated neuronal growth cones from neonatal rat forebrain were found to contain a high specific activity of adenylate cyclase (61 pmol cyclic AMP/min/mg protein) compared to the pelleted starting homogenate (5 pmol cyclic AMP/min/mg protein). Forskolin at 10(-4) M increased adenylate cyclase activity in both the pelleted homogenate and growth cone fraction by 70 and 217 pmol cyclic AMP/min/mg protein, respectively, over basal levels. The incremental effect of forskolin was 3-fold greater in the growth cone fraction than in the pelleted homogenate. However, relative to basal levels in each of the two fractions, forskolin increased adenylate cyclase activity in the growth cone fraction by only approx. 5-fold compared to 15-fold in the pelleted homogenate. Dopamine (10(-4) M), vasoactive intestinal polypeptide (10(-6) M) and isoproterenol (10(-5) M) also augmented adenylate cyclase activity in the two fractions. In the growth cone fraction, dopamine and vasoactive intestinal polypeptide produced a stimulation over basal levels by approx. 20 pmol cyclic AMP/min/mg protein while isoproterenol produced a stimulation of approx. 10 pmol cAMP/min/mg protein. The incremental effects of these receptor agonists in the growth cone fraction are approx. 5-fold greater than in the pelleted homogenate. The dopamine-sensitive adenylate cyclase activity in the growth cone fraction could be blocked by the compound SCH23390, a selective D1 receptor antagonist. At saturating concentrations, all combinations of dopamine, vasoactive intestinal polypeptide and isoproterenol were found to be completely additive on adenylate cyclase activity in the growth cone fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis*

    PubMed Central

    Boll, Björn; Taubitz, Tatjana; Heide, Lutz

    2011-01-01

    MbtH-like proteins consist of ∼70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes. PMID:21890635

  19. Interactions between neuropeptide Y and the adenylate cyclase pathway in rat mesenteric small arteries: role of membrane potential.

    PubMed Central

    Prieto, D; Buus, C; Mulvany, M J; Nilsson, H

    1997-01-01

    1. Simultaneous measurements of membrane potential and tension were performed to investigate the intracellular mechanisms of neuropeptide Y (NPY) in rat mesenteric small arteries. 2. NPY (0.1 microM) depolarized arterial smooth muscle cells from -55 to -47 mV and increased wall tension by 0.22 N m-1, representing 11% of the contraction elicited by a high-potassium solution. Isoprenaline (1 microM) and acetylcholine (1 microM) evoked hyperpolarizations of 11 and 17 mV, respectively. NPY inhibited the isoprenaline-induced effects on membrane potential without affecting those of acetylcholine. 3. Forskolin evoked sustained concentration-dependent hyperpolarizations of small mesenteric arteries. NPY (0.1 microM) inhibited the responses to 1 microM forskolin, but did not alter the stable hyperpolarization elicited by the specific activator of protein kinase A (PKA) SP-5,6-DCl-cBIMPS (0.1 mM). Forskolin increased the cyclic AMP (cAMP) content of the arteries 21-fold, and NPY inhibited the forskolin-evoked increase in cAMP levels by 91%. 4. The hyperpolarization produced by 1 microM forskolin was not affected by either charybdotoxin (0.1 microM) or 4-aminopyridine (0.5 mM), but glibenclamide (5 microM) inhibited the hyperpolarization by 70%. Glibenclamide also inhibited the hyperpolarization evoked by SP-5,6-DCl-cBIMPS by 59%. 5. Neither depolarization nor contraction caused by NPY were significantly affected by either glibenclamide (5 microM) or nifedipine (1 microM), but they were reduced by gadolinium (10 microM). However, the blocking effect of NPY on forskolin-elicited hyperpolarization was not affected by gadolinium. 6. Charybdotoxin (0.1 microM) and 4-aminopyridine (0.5 mM) strongly enhanced the depolarization and contraction caused by NPY (0.1 microM), and nifedipine (1 microM) prevented the enhanced responses to NPY in the presence of charybdotoxin. 7. These findings suggest that NPY acts through at least two different intracellular mechanisms in mesenteric small

  20. Lysyl oxidase isoforms in gastric cancer.

    PubMed

    Añazco, Carolina; Delgado-López, Fernando; Araya, Paulina; González, Ileana; Morales, Erik; Pérez-Castro, Ramón; Romero, Jacqueline; Rojas, Armando

    2016-09-01

    Gastric cancer (GC) is the fifth most frequent cancer in the world and shows the highest incidence in Latin America and Asia. An increasing amount of evidence demonstrates that lysyl oxidase isoforms, a group of extracellular matrix crosslinking enzymes, should be considered as potential biomarkers and therapeutic targets in GC. In this review, we focus on the expression levels of lysyl oxidase isoforms, its functions and the clinical implications in GC. Finding novel proteins related to the processing of these extracellular matrix enzymes might be helpful in the design of new therapies, which, in combination with classic pharmacology, could be used to delay the progress of this aggressive cancer and offer a wider temporal window for clinical intervention. PMID:27564724

  1. Structural Basis of Dscam Isoform Specificity

    SciTech Connect

    Meijers,R.; Puettmann-Holgado, R.; Skiniotis, G.; Liu, J.; Walz, T.; Wang, J.; Schmucker, D.

    2007-01-01

    The Dscam gene gives rise to thousands of diverse cell surface receptors1 thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

  2. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    PubMed

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  3. Adenylate cyclase activity in fish gills in relation to salt adaptation

    SciTech Connect

    Guibbolini, M.E.; Lahlou, B.

    1987-07-06

    The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of (..cap alpha..-/sup 32/P) - ATP into (..cap alpha..-/sup 32/P) - cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted- mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE/sub 1/. These results are interpreted in relation to the general process of osmoregulation. 27 references, 6 figures.

  4. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  5. Inhibition by methioninyl adenylate of focus formation by Rous sarcoma virus.

    PubMed

    Robert-Gero, M; Lawrence, F; Vigier, P

    1975-12-01

    Methioninyl adenylate is a specific and potent inhibitor of the enzyme methionyl-tRNA synthetase and, consequently, of protein biosynthesis. In cultures of chick embryo fibroblasts infected with Rous sarcoma virus, incubation for a 2-day period with 1 to 3 mM concentrations of this inhibitor, as late as 4 days after infection, irreversibly prevented subsequent formation of foci of transformed cells. Later addition could also elicit the irreversible disappearance of already existing foci, by phenotypic reversion and/or cell killing. Virus production in transformed cells and replication in newly infected cells were also inhibited but to a lesser degree. Under the same conditions, the same concentrations of methioninyl adenylate caused only a reversible growth arrest of normal cells. The selective toxicity of the inhibitor for transformed cells is not due to a greater affinity for the target enzyme, but it may be due to the fact that inhibition of protein biosynthesis is only partially reversible in these cells, whereas it is fully reversible in normal cells.

  6. Adenylate Cyclase Toxin (ACT) from Bordetella hinzii: Characterization and Differences from ACT of Bordetella pertussis

    PubMed Central

    Donato, Gina M.; Hsia, Hung-Lun J.; Green, Candace S.; Hewlett, Erik L.

    2005-01-01

    Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism. PMID:16267282

  7. In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

    PubMed Central

    Beushausen, S; Narindrasorasak, S; Sanwal, B D; Dales, S

    1987-01-01

    The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the

  8. 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

    SciTech Connect

    Liu, Hongbing; Herrmann, Christine H.; Chiang, Karen; Sung, Tzu-Ling; Moon, Sung-Hwan; Donehower, Lawrence A.; Rice, Andrew P.

    2010-06-25

    Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42 KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

  9. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  10. Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms

    PubMed Central

    Tatsumi, Naoya; Hojo, Nozomi; Sakamoto, Hiroyuki; Inaba, Rena; Moriguchi, Nahoko; Matsuno, Keiko; Fukuda, Mari; Matsumura, Akihide; Hayashi, Seiji; Morimoto, Soyoko; Nakata, Jun; Fujiki, Fumihiro; Nishida, Sumiyuki; Nakajima, Hiroko; Tsuboi, Akihiro; Oka, Yoshihiro; Hosen, Naoki; Sugiyama, Haruo; Oji, Yusuke

    2015-01-01

    The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms. PMID:26090994

  11. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics.

    PubMed

    Chang, Ching-Wei; Kumar, Sanjay

    2015-09-04

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces.

  12. The Arabidopsis CDPK-SnRK superfamily of protein kinases.

    PubMed

    Hrabak, Estelle M; Chan, Catherine W M; Gribskov, Michael; Harper, Jeffrey F; Choi, Jung H; Halford, Nigel; Kudla, Jorg; Luan, Sheng; Nimmo, Hugh G; Sussman, Michael R; Thomas, Martine; Walker-Simmons, Kay; Zhu, Jian-Kang; Harmon, Alice C

    2003-06-01

    The CDPK-SnRK superfamily consists of seven types of serine-threonine protein kinases: calcium-dependent protein kinase (CDPKs), CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PEP carboxylase kinase-related kinases (PEPRKs), calmodulin-dependent protein kinases (CaMKs), calcium and calmodulin-dependent protein kinases (CCaMKs), and SnRKs. Within this superfamily, individual isoforms and subfamilies contain distinct regulatory domains, subcellular targeting information, and substrate specificities. Our analysis of the Arabidopsis genome identified 34 CDPKs, eight CRKs, two PPCKs, two PEPRKs, and 38 SnRKs. No definitive examples were found for a CCaMK similar to those previously identified in lily (Lilium longiflorum) and tobacco (Nicotiana tabacum) or for a CaMK similar to those in animals or yeast. CDPKs are present in plants and a specific subgroup of protists, but CRKs, PPCKs, PEPRKs, and two of the SnRK subgroups have been found only in plants. CDPKs and at least one SnRK have been implicated in decoding calcium signals in Arabidopsis. Analysis of intron placements supports the hypothesis that CDPKs, CRKs, PPCKs and PEPRKs have a common evolutionary origin; however there are no conserved intron positions between these kinases and the SnRK subgroup. CDPKs and SnRKs are found on all five Arabidopsis chromosomes. The presence of closely related kinases in regions of the genome known to have arisen by genome duplication indicates that these kinases probably arose by divergence from common ancestors. The PlantsP database provides a resource of continuously updated information on protein kinases from Arabidopsis and other plants.

  13. Reduced basal and stimulated (isoprenaline, Gpp(NH)p, forskolin) adenylate cyclase activity in Alzheimer's disease correlated with histopathological changes.

    PubMed

    Ohm, T G; Bohl, J; Lemmer, B

    1991-02-01

    Cyclic adenosine monophosphate (cAMP) is an adenylate cyclase borne second messenger involved in basic metabolic events. The beta-adrenoceptor sensitive adenylate cyclase was studied in post-mortem hippocampi of controls and Alzheimer patients. Virtually identical subsets of each hippocampus homogenate were stimulated by 100 mumol isoprenaline, Gpp(NH)p and forskolin, respectively, in presence of an ATP-regenerating system. The determination of cAMP formed was carried out by means of a radioassay. The observed significant 50% reduction in basal as well as in stimulated adenylate cyclase activity in Alzheimer's disease is negatively correlated with semiquantitative evaluations of amyloid plaques (P less than 0.05) but not with neuritic plaques, neurofibrillary tangles or neuropil threads. This reduction in enzyme activity is obviously not due to simple cell loss alone. It is likely that the crucial point of the observed functional disturbance is at the level of the catalytic unit of the adenylate cyclase, since the same degree of reduction is maintained at all steps of the signal cascade. PMID:2054615

  14. EXPERIENCE-DEPENDENT REGULATION OF TRKB ISOFORMS IN RODENT VISUAL CORTEX

    PubMed Central

    Bracken, Bethany K.; Turrigiano, Gina G.

    2010-01-01

    Within primary visual cortex (V1), BDNF signaling through its high affinity receptor TrkB is important for normal development and experience-dependent plasticity. TrkB is expressed in several alternatively spliced isoforms, including full length TrkB (TrkB.FL), and several truncated isoforms (TrkB.T1, TrkB.T2 and TrkB.T4) that lack the intracellular tyrosine kinase domain. These isoforms are important components of BDNF signaling, yet little is known about the developmental or experience-dependent regulation of their expression. Using immunohistochemistry, we found TrkB.FL and TrkB.T1 expressed in interneurons and pyramidal neurons within V1, but not in cortical astrocytes. We used real-time PCR to quantify changes in mRNA expression of BDNF, the four TrkB isoforms, and the low affinity receptor P75NTR during normal development, and in response to visual deprivation at two different ages. BDNF expression increased between postnatal days 10 (P10) and P30, and was rapidly down-regulated by 3 days of visual deprivation during both the pre-critical period (P14–P17) and the critical period (P18–P21). Over the same developmental period expression of each TrkB isoform was regulated independently; TrkB.T1 increased, TrkB.FL and TrkB.T2 decreased, and TrkB.T4 showed transient changes. Neither brief visual deprivation nor prolonged dark-rearing induced changes in TrkB.FL or TrkB.T1 expression. However, TrkB.T4 expression was reduced by brief visual deprivation, while TrkB.T4, TrkB.T2 and P75NTR were up-regulated by prolonged dark-rearing into the critical period. Our data indicate that TrkB isoform expression can be selectively regulated by visual experience, and may contribute to experience-dependent cortical plasticity. PMID:19224567

  15. Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release

    PubMed Central

    Neeli, Indira; Radic, Marko

    2013-01-01

    In response to inflammation, neutrophils deiminate histones and externalize chromatin. Neutrophil extracellular traps (NETs) are an innate immune defense mechanism, yet NETs also may aggravate chronic inflammatory and autoimmune disorders. Activation of peptidylarginine deiminase 4 (PAD4) is associated with NET release (NETosis) but the precise mechanisms of PAD4 regulation are unknown. We observed that, in human neutrophils, calcium ionophore induced histone deimination, whereas phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), suppressed ionophore-induced deimination. Conversely, low doses of chelerythrine and sanguinarine, two inhibitors of PKC, reversed PMA inhibition and enhanced ionophore-stimulated deimination. In addition, a peptide inhibitor of PKCα superinduced ionophore activation of PAD4, thus identifying PKCα as the PMA-induced inhibitor of PAD4. At higher doses, chelerythrine, sanguinarine, and structurally unrelated PKC inhibitors blocked histone deimination, suggesting that a different PKC isoform activates histone deimination. We identify PKCζ as activator of PAD4 because a specific peptide inhibitor of this PKC isoform suppressed histone deimination. Confocal microscopy confirmed that, in the presence of PMA, NETosis proceeds without detectable histone deimination, and that ionophore cooperates with PMA to induce more extensive NET release. Broad inhibition of PKC by chelerythrine or specific inhibition of PKCζ suppressed NETosis. Our observations thus reveal an intricate antagonism between PKC isoforms in the regulation of histone deimination, identify a dominant role for PKCα in the repression of histone deimination, and assign essential functions to PKCζ in the activation of PAD4 and the execution of NETosis. The precise balance between opposing PKC isoforms in the regulation of NETosis affirms the idea that NET release underlies specific and vitally important evolutionary selection pressures. PMID:23430963

  16. Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

    PubMed Central

    Eilers, Wouter; Gevers, Wouter; van Overbeek, Daniëlle; de Haan, Arnold; Jaspers, Richard T.; Hilbers, Peter A.; van Riel, Natal; Flück, Martin

    2014-01-01

    We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII) contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE) coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis) and slow-type muscle (soleus) for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02). In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII. PMID:25054156

  17. The essential role of PIM kinases in sarcoma growth and bone invasion.

    PubMed

    Narlik-Grassow, Maja; Blanco-Aparicio, Carmen; Cecilia, Yolanda; Peregrina, Sandra; Garcia-Serelde, Beatriz; Muñoz-Galvan, Sandra; Cañamero, Marta; Carnero, Amancio

    2012-08-01

    PIM kinases are a family of serine/threonine kinases composed of three different isoforms (PIM1, PIM 2 and PIM 3) that are highly homologous. Their expression is mediated by the JAK/STAT signalling pathway, providing survival and cell cycle transition signals. PIM kinases are heavily targeted for anticancer drug discovery. However, very little is known about the relative contribution of the different isoforms to the tumourigenesis process in vivo, and how their individual inhibition might affect tumour growth. Taking advantage of genetically modified mice, we explored whether the inhibition of specific isoforms is required to prevent sarcomas induced by 3-methylcholanthrene carcinogenic treatment. We found that absence of Pim2 and Pim3 greatly reduced sarcoma growth to a similar extent to the absence of all three isoforms. This model of sarcoma generally produces bone invasion by the tumour cells. Lack of Pim2 and Pim3 reduced tumour-induced bone invasion by 70%, which is comparable with the reduction of tumour-induced bone invasion in the absence of all three isoforms. Similar results were obtained in mouse embryonic fibroblasts (MEFs) derived from these knockout (KO) mice, where double Pim2/3 KO MEFs already showed reduced proliferation and were resistant to oncogenic transformation by the RAS oncogene. Our data also suggest an important role of Gsk3β phosphorylation in the process of tumourigenesis. PMID:22623646

  18. Mode of coupling between the beta-adrenergic receptor and adenylate cyclase in turkey erythrocytes.

    PubMed

    Tolkovsky, A M; Levitzki, A

    1978-09-01

    The mode of coupling of the beta-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. A number of experimental techniques have been used: (1) measurement of the kinetics of cyclase activation to its permanetly active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentrations; (2) measurement of antagonist and agoinst binding to the beta-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the bases of these two approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor can be rejected. The binding and the kinetic data, however, can be fitted by two diametrically opposed models of receptor to enzyme coupling: (a) the precouped enzyme-receptor model where activation of the enzyme occurs, according to the following scheme: formula (see text) where H is the hormone, RE is the precoupled respetor-enzyme complex, k1 and k2 are the rate constants describing hormone binding, and k is the rate constant characterizing the formation of HRE' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. (b) The other model is the collision coupling mechanism: formula (see test) wheere KH is the horome-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE, and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. In order to establish which of the two mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed: Progressive inactivation of the beta receptor by a specific affinity label was found to cause a decrease in the maximal binding capacity of the receptor and a

  19. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  20. Functional differentiation in trematode hemoglobin isoforms.

    PubMed

    Rashid, A K; Weber, R E

    1999-03-01

    The Hbs and the major electrophoretic Hb components (isoHbs) were isolated from three species of the trematodes, Explanatum explanatum (Ee), Gastrothylax crumenifer (Gc) and Paramphistomum epiclitum (Pe), that parasitise the common Indian water buffalo Bubalus bubalis. The Hbs are monomeric and resemble the so-called nonfunctional mutant hemoglobins that have Tyr at B10 or E7 positions (replacing Leu and the His residues, respectively). However, they are capable of binding with O2 and CO. O2 equilibrium studies of trematode Hb isoforms reveal extremely high O2 affinities, with half-saturation O2 tension (P50) values up to 800 times lower than those of human hemoglobins. This correlates with Tyr residues at B10 and at the distal position (E7) that decrease the O2 dissociation rate by contributing hydrogen bonds (H-bonds) to the bound O2. These substitutions also increase the O2 association rates either due to orientation of E7-Tyr towards the solvent and/or by sterically hindering the entry of water molecules into the heme pocket. The latter may account for the low rate of autoxidation of trematode Hbs. The Hbs and their isoforms from different species exhibited pronounced variation in O2 affinity, which may relate to subtle differences in the structure of the heme pocket. The O2 affinities of the composite (unfractionated) Hbs were intermediate to those of the individual Hb isoform. The P50 values of Hbs here obtained by direct O2 equilibrium measurements differed from those calculated from kinetic data already published [Kiger, L., Rashid, A. K., Griffon, N., Haque, M., Moens, L.,Gibson, Q. H., Poyart, C., & Marden, M. C. (1998). Biophys. J. 75, 990-998.] Intermediate state(s) due to slow reorientation of E7-Tyr may account for this difference. Some Hb isoforms showed slight (either normal or reverse) Bohr effects. The hyperbolic O2 equilibrium curve, Hill coefficient (n) values near unity accord with a monomeric nature of trematode Hbs. In marked contrast to

  1. Developmental expression of two Haliotis asinina hemocyanin isoforms.

    PubMed

    Streit, Klaus; Jackson, Daniel; Degnan, Bernard M; Lieb, Bernhard

    2005-09-01

    Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

  2. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    SciTech Connect

    Ivanov, Sergey V.; Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I.

    2009-05-08

    Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

  3. The Paradoxical Signals of Two TrkC Receptor Isoforms Supports a Rationale for Novel Therapeutic Strategies in ALS

    PubMed Central

    Barcelona, Pablo F.; Galan, Alba; Aboulkassim, Tahar; Teske, Katrina; Rogers, Mary-Louise; Bertram, Lisa; Wang, Jing; Yousefi, Masoud; Rush, Robert; Fabian, Marc; Cashman, Neil

    2016-01-01

    Full length TrkC (TrkC-FL) is a receptor tyrosine kinase whose mRNA can be spliced to a truncated TrkC.T1 isoform lacking the kinase domain. Neurotrophin-3 (NT-3) activates TrkC-FL to maintain motor neuron health and function and TrkC.T1 to produce neurotoxic TNF-α; hence resulting in opposing pathways. In mouse and human ALS spinal cord, the reduction of miR-128 that destabilizes TrkC.T1 mRNA results in up-regulated TrkC.T1 and TNF-α in astrocytes. We exploited conformational differences to develop an agonistic mAb 2B7 that selectively activates TrkC-FL, to circumvent TrkC.T1 activation. In mouse ALS, 2B7 activates spinal cord TrkC-FL signals, improves spinal cord motor neuron phenotype and function, and significantly prolongs life-span. Our results elucidate biological paradoxes of receptor isoforms and their role in disease progression, validate the concept of selectively targeting conformational epitopes in naturally occurring isoforms, and may guide the development of pro-neuroprotective (TrkC-FL) and anti-neurotoxic (TrkC.T1) therapeutic strategies. PMID:27695040

  4. Characterization of the dopamine stimulated adenylate cyclase in the pedal ganglia of Mytilus edulis: interactions with etorphine, beta-endorphin, DALA, and methionine enkephalin.

    PubMed

    Stefano, G B; Catapane, E J; Kream, R M

    1981-03-01

    The dopamine-stimulated adenylate cyclase activity was studied both in vivo and in vitro in the central nervous system of the bivalve mollusc Mytilus edulis. Dopamine, epinine, and apomorphine stimulated the enzyme system. Fluphenazine, haloperidol, chlorpromaxine, and to a lesser extent BOL inhibited the dopamine-stimulated adenylate cyclase. Etorphine, beta-endorphine, DALA, and methionine enkephalin depressed cyclic AMP levels. This phenomena was naloxone reversible. In addition, the opioids inhibited the stimulation of adenylate cyclase by dopamine. This phenomena was also naloxone reversible. The study demonstrates an interaction among dopamine, the opioids, and cyclic AMP. PMID:6286125

  5. The influence of various cations on the catalytic properties of clays. [polymerization of alanine adenylate

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The polymerization of alanine adenylate in the presence of the sodium form of various clays was studied, and hectorite was found to cause more polymerization than nontronite and montmorillonite (in that order) although the differences were not great. The effect on polymerization of presaturating montmorillonite with different cations was determined. Hectorite, with increased basicity of the interspatial planes, allows polymerization of lysine, which montmorillonite does not. The general trend is that, for the same amino acid, higher degrees of polymerization are obtained when the cation in the octahedral lattice of the clay is divalent rather than trivalent. With the exchangeable cations the order is reversed, for a reason that is explained. The main role of clays in the polymerization mechanism of amino acids is concentration and neutralization of charges.

  6. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    SciTech Connect

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J. )

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.

  7. Endotoxic lipopolysaccharides stimulate steroidogenesis and adenylate cyclase in adrenal tumor cells.

    PubMed

    Wolff, J; Cook, G H

    1975-12-01

    Lipopolysaccharides (endotoxins) from Escherichia coli, Serratia marcesens and Salmonella typhosa stimulated steroid production in Y-1 adrenal tumor cells in culture with a latent period of 3-4 h. Lipid A, derived from Escherichia coli lipopolysaccharide, also stimulated steroidogenesis. Lipopolysaccharides and lipid A also stimulate adenylate cyclase activity and cause rounding of the cells. In contrast, lipopolysaccharides do not stimulate steroidogenesis in receptor-deficient adrenal tumor cells (OS-3) or Leydig tumor cells (I-10). This tends to rule out contamination by enterotoxin to which these lines respond. Although both hormone and lipopolysaccharide responses are lost in these lines, there was no interaction between these sites as judged by the failure of lipopolysaccharides to block, during their latency, the response to corticotropin in Y-1 cells. The possibility that the lipopolysaccharide effect is one on membrane conformation is discussed.

  8. Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata).

    PubMed

    Kottler, Verena A; Künstner, Axel; Koch, Iris; Flötenmeyer, Matthias; Langenecker, Tobias; Hoffmann, Margarete; Sharma, Eshita; Weigel, Detlef; Dreyer, Christine

    2015-09-01

    Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

  9. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  10. Pyruvate Kinase M2 Regulates Gene Transcription by Acting as A Protein Kinase

    PubMed Central

    Gao, Xueliang; Wang, Haizhen; Jenny, J. Yang; Liu, Xiaowei; Liu, Zhi-Ren

    2012-01-01

    Summary Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate with transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression. PMID:22306293

  11. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Astrophysics Data System (ADS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-12-01

    We studied the spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay. This led to the unexpected finding that the degrees of polymerization (DP's) of the oligo- and poly-peptides obtained depended on the amounts of polypeptides that were preadsorbed. Plotting average molecular weights obtained against c-spacings of the clay platelet aggregates which widened as a result of polypeptide addition and adsorption before the polymerization, does not permit an obvious explanation of these observations. The best correlation assigns a role to the fractional occupation of the individual intercalation layers of the polypeptides, as the adsorption increases towards a first complete mono-interlayer, then to an incipient and eventually to a complete double layer on to a third interlayer, after which the clay stacking breaks up. Spacings which correspond to an intermediate occupation of any of the three successive interlayers favor amino acids self-addition to polymers. The opposite is true for nearly empty or filled intercalation layers. We hypothesize and describe, how a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances. Moderately filled intercalation spaces may also act as sinks for the newly formed oligomers and facilitate the freeing of reaction sites for the occupation by fresh reagent. The c-spacings required for these mechanisms are the result of the intercalation of the preadsorbed polymer, but similar conditions prevail when polymers are adsorbed as they are generated during polymerization.

  12. Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the G(s) protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 micromol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 micromol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 micromol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the G(s) protein-coupled PACAP receptor to activate adenylate cyclase.

  13. Respiration in postharvest sugarbeet roots is not limited by respiratory capacity or adenylates.

    PubMed

    Klotz, Karen L; Finger, Fernando L; Anderson, Marc D

    2008-09-29

    Control of respiration has largely been studied with growing and/or photosynthetic tissues or organs, but has rarely been examined in harvested and stored plant products. As nongrowing, heterotrophic organs that are reliant on respiration to provide all of their metabolic needs, harvested plant products differ dramatically in their metabolism and respiratory needs from growing and photosynthetically active plant organs, and it cannot be assumed that the same mechanism controls respiration in both actively growing and harvested plant organs. To elucidate mechanisms of respiratory control for a harvested and stored plant product, sugarbeet (Beta vulgaris L.) root respiration was characterized with respect to respiratory capacity, adenylate levels and cellular energy status in roots whose respiration was altered by wounding or cold treatment (1 degrees C) and in response to potential effectors of respiration. Respiration rate was induced by wounding in roots stored at 10 degrees C and by cold temperature in roots stored at 1 degrees C for 11-13d. Alterations in respiration rate due to wounding or storage temperature were unrelated to changes in total respiratory capacity, the capacities of the cytochrome c oxidase (COX) or alternative oxidase (AOX) pathways, adenylate concentrations or cellular energy status, measured by the ATP:ADP ratio. In root tissue, respiration was induced by exogenous NADH indicating that respiratory capacity was capable of oxidizing additional electrons fed into the electron transport chain via an external NADH dehydrogenase. Respiration was not induced by addition of ADP or a respiratory uncoupler. These results suggest that respiration rate in stored sugarbeet roots is not limited by respiratory capacity, ADP availability or cellular energy status. Since respiration in plants can be regulated by substrate availability, respiratory capacity or energy status, it is likely that a substrate, other than ADP, limits respiration in stored sugarbeet

  14. Unexpected Discovery of Dichloroacetate Derived Adenosine Triphosphate Competitors Targeting Pyruvate Dehydrogenase Kinase To Inhibit Cancer Proliferation.

    PubMed

    Zhang, Shao-Lin; Hu, Xiaohui; Zhang, Wen; Tam, Kin Yip

    2016-04-14

    Pyruvate dehydrogenase kinases (PDKs) have recently emerged as an attractive target for cancer therapy. Herein, we prepared a series of compounds derived from dichloroacetate (DCA) which inhibited cancer cells proliferation. For the first time, we have successfully developed DCA derived inhibitors that preferentially bind to the adenosine triphosphate (ATP) pocket of PDK isoform 1 (PDK1).

  15. Brevican isoforms associate with neural membranes.

    PubMed

    Seidenbecher, Constanze I; Smalla, Karl-Heinz; Fischer, Nora; Gundelfinger, Eckart D; Kreutz, Michael R

    2002-11-01

    Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.

  16. Diacylglycerol Kinases (DGKs): Novel Targets for Improving T Cell Activity in Cancer

    PubMed Central

    Riese, Matthew J.; Moon, Edmund K.; Johnson, Bryon D.; Albelda, Steven M.

    2016-01-01

    Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the metabolism of diacylglycerol (DAG). Two isoforms of DGK, DGKα, and DGKζ, specifically regulate the pool of DAG that is generated as a second messenger after stimulation of the T cell receptor (TCR). Deletion of either isoform in mouse models results in T cells bearing a hyperresponsive phenotype and enhanced T cell activity against malignancy. Whereas, DGKζ appears to be the dominant isoform in T cells, rationale exists for targeting both isoforms individually or coordinately. Additional work is needed to rigorously identify the molecular changes that result from deletion of DGKs in order to understand how DAG contributes to T cell activation, the effect of DGK inhibition in human T cells, and to rationally develop combined immunotherapeutic strategies that target DGKs. PMID:27800476

  17. Pachastrissamine (jaspine B) and its stereoisomers inhibit sphingosine kinases and atypical protein kinase C.

    PubMed

    Yoshimitsu, Yuji; Oishi, Shinya; Miyagaki, Jun; Inuki, Shinsuke; Ohno, Hiroaki; Fujii, Nobutaka

    2011-09-15

    Sphingosine kinases (SphKs) are oncogenic enzymes that regulate the critical balance between ceramide and sphingosine-1-phosphate. Much effort has been dedicated to develop inhibitors against these enzymes. Naturally occurring pachastrissamine (jaspine B) and all its stereoisomers were prepared and evaluated for their inhibitory effects against SphKs. All eight stereoisomers exhibited moderate to potent inhibitory activity against SphK1 and SphK2. Inhibitory effects were profiled against protein kinase C (PKC) isoforms by in vitro experiments. Atypical PKCs (PKCζ and PKCι) were inhibited by several pachastrissamine stereoisomers. The improved activity over N,N-dimethylsphingosine suggests that the cyclic scaffold in pachastrissamines facilitates potential favorable interactions with SphKs and PKCs.

  18. Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin.

    PubMed

    Mas, Véronique Mansat-De; Hernandez, Hélène; Plo, Isabelle; Bezombes, Christine; Maestre, Nicolas; Quillet-Mary, Anne; Filomenko, Rodolphe; Demur, Cécile; Jaffrézou, Jean-Pierre; Laurent, Guy

    2003-02-15

    In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.

  19. The Phosphoinositide 3-Kinase Pathway in Human Cancer: Genetic Alterations and Therapeutic Implications

    PubMed Central

    Arcaro, Alexandre; Guerreiro, Ana S

    2007-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in human cancer and represents an attractive target for therapies based on small molecule inhibitors. PI3K isoforms play an essential role in the signal transduction events activated by cell surface receptors including receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs). There are eight known PI3K isoforms in humans, which have been subdivided into three classes (I-III). Therefore PI3Ks show considerable diversity and it remains unclear which kinases in this family should be targeted in cancer. The class IA of PI3K comprises the p110α, p110β and p110δ isoforms, which associate with activated RTKs. In human cancer, recent reports have described activating mutations in the PIK3CA gene encoding p110α, and inactivating mutations in the phosphatase and tensin homologue (PTEN) gene, a tumour suppressor and antagonist of the PI3K pathway. The PIK3CA mutations described in cancer constitutively activate p110α and, when expressed in cells drive oncogenic transformation. Moreover, these mutations cause the constitutive activation of downstream signaling molecules such as Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K) that is commonly observed in cancer cells. In addition to p110α, the other isoforms of the PI3K family may also play a role in human cancer, although their individual functions remain to be precisely identified. In this review we will discuss the evidence implicating individual PI3K isoforms in human cancer and their potential as drug targets in this context. PMID:19384426

  20. Expression, activation, and role of AKT isoforms in the uterus.

    PubMed

    Fabi, François; Asselin, Eric

    2014-11-01

    The three isoforms of AKT: AKT1, AKT2, and AKT3, are crucial regulators of both normal and pathological cellular processes. Each of these isoforms exhibits a high level of homology and functional redundancy with each other. However, while being highly similar and structurally homologous, a rising amount of evidence is showing that each isoform possesses specific targets as well as preferential subcellular localization. The role of AKT has been studied extensively in reproductive processes, but isoform-specific roles are yet to be fully understood. This review will focus on the role of AKT in the uterus and its function in processes related to cell death and proliferation such as embryo implantation, decidualization, endometriosis, and endometrial cancer in an isoform-centric manner. In this review, we will cover the activation of AKT in various settings, localization of isoforms in subcellular compartments, and the effect of isoform expression on cellular processes. To fully understand the dynamic molecular processes taking place in the uterus, it is crucial that we better understand the physiological role of AKT isoforms as well as their function in the emergence of diseases.

  1. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001 PMID:26735365

  2. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    PubMed Central

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  3. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

    SciTech Connect

    Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.; Guillon, G.; Homburger, V.

    1987-06-25

    We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.

  4. Pituitary Adenylate Cyclase-Activating Peptide in the Central Amygdala Causes Anorexia and Body Weight Loss via the Melanocortin and the TrkB Systems.

    PubMed

    Iemolo, Attilio; Ferragud, Antonio; Cottone, Pietro; Sabino, Valentina

    2015-07-01

    Growing evidence suggests that the pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor system represents one of the main regulators of the behavioral, endocrine, and autonomic responses to stress. Although induction of anorexia is a well-documented effect of PACAP, the central sites underlying this phenomenon are poorly understood. The present studies addressed this question by examining the neuroanatomical, behavioral, and pharmacological mechanisms mediating the anorexia produced by PACAP in the central nucleus of the amygdala (CeA), a limbic structure implicated in the emotional components of ingestive behavior. Male rats were microinfused with PACAP (0-1 μg per rat) into the CeA and home-cage food intake, body weight change, microstructural analysis of food intake, and locomotor activity were assessed. Intra-CeA (but not intra-basolateral amygdala) PACAP dose-dependently induced anorexia and body weight loss without affecting locomotor activity. PACAP-treated rats ate smaller meals of normal duration, revealing that PACAP slowed feeding within meals by decreasing the regularity and maintenance of feeding from pellet-to-pellet; postprandial satiety was unaffected. Intra-CeA PACAP-induced anorexia was blocked by coinfusion of either the melanocortin receptor 3/4 antagonist SHU 9119 or the tyrosine kinase B (TrKB) inhibitor k-252a, but not the CRF receptor antagonist D-Phe-CRF(12-41). These results indicate that the CeA is one of the brain areas through which the PACAP system promotes anorexia and that PACAP preferentially lessens the maintenance of feeding in rats, effects opposite to those of palatable food. We also demonstrate that PACAP in the CeA exerts its anorectic effects via local melanocortin and the TrKB systems, and independently from CRF.

  5. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    PubMed

    Efendiev, Riad; Dessauer, Carmen W

    2011-10-01

    3'-5'-Cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology, including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins localize the effect of cAMP in space and time by organizing receptors, AC, protein kinase A, and other components of the cAMP cascade into multiprotein complexes. In this review, we discuss how the interaction of A kinase-anchoring proteins with distinct AC isoforms affects cardiovascular physiology.

  6. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

    PubMed Central

    Wiesner, Thomas; Lee, William; Obenauf, Anna C.; Ran, Leili; Murali, Rajmohan; Zhang, Qi Fan; Wong, Elissa W. P.; Hu, Wenhuo; Scott, Sasinya N.; Shah, Ronak H.; Landa, Iñigo; Button, Julia; Lailler, Nathalie; Sboner, Andrea; Gao, Dong; Murphy, Devan A.; Cao, Zhen; Shukla, Shipra; Hollmann, Travis J.; Wang, Lu; Borsu, Laetitia; Merghoub, Taha; Schwartz, Gary K.; Postow, Michael A.; Ariyan, Charlotte E.; Fagin, James A.; Zheng, Deyou; Ladanyi, Marc; Busam, Klaus J.; Berger, Michael F.; Chen, Yu; Chi, Ping

    2016-01-01

    Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ~ 11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites1. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation. PMID:26444240

  7. Method of empirical dependences in estimation and prediction of activity of creatine kinase isoenzymes in cerebral ischemia

    NASA Astrophysics Data System (ADS)

    Sergeeva, Tatiana F.; Moshkova, Albina N.; Erlykina, Elena I.; Khvatova, Elena M.

    2016-04-01

    Creatine kinase is a key enzyme of energy metabolism in the brain. There are known cytoplasmic and mitochondrial creatine kinase isoenzymes. Mitochondrial creatine kinase exists as a mixture of two oligomeric forms - dimer and octamer. The aim of investigation was to study catalytic properties of cytoplasmic and mitochondrial creatine kinase and using of the method of empirical dependences for the possible prediction of the activity of these enzymes in cerebral ischemia. Ischemia was revealed to be accompanied with the changes of the activity of creatine kinase isoenzymes and oligomeric state of mitochondrial isoform. There were made the models of multiple regression that permit to study the activity of creatine kinase system in cerebral ischemia using a calculating method. Therefore, the mathematical method of empirical dependences can be applied for estimation and prediction of the functional state of the brain by the activity of creatine kinase isoenzymes in cerebral ischemia.

  8. Pim-1 kinase as cancer drug target: An update

    PubMed Central

    TURSYNBAY, YERNAR; ZHANG, JINFU; LI, ZHI; TOKAY, TURSONJAN; ZHUMADILOV, ZHAXYBAY; WU, DENGLONG; XIE, YINGQIU

    2016-01-01

    Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) is a serine/threonine kinase that regulates multiple cellular functions such as cell cycle, cell survival, drug resistance. Aberrant elevation of Pim-1 kinase is associated with numerous types of cancer. Two distinct isoforms of Pim-1 (Pim-1S and Pim-1L) show distinct cellular functions. Pim-1S predominately localizes to the nucleus and Pim-1L localizes to plasma membrane for drug resistance. Recent studies show that mitochondrial Pim-1 maintains mitochondrial integrity. Pim-1 is emerging as a cancer drug target, particularly in prostate cancer. Recently the potent new functions of Pim-1 in immunotherapy, senescence bypass, metastasis and epigenetic dynamics have been found. The aim of the present updated review is to provide brief information regarding networks of Pim-1 kinase and focus on its recent advances as a novel drug target. PMID:26893828

  9. The PIM family of serine/threonine kinases in cancer.

    PubMed

    Narlik-Grassow, Maja; Blanco-Aparicio, Carmen; Carnero, Amancio

    2014-01-01

    The proviral insertion site in Moloney murine leukemia virus, or PIM proteins, are a family of serine/threonine kinases composed of three different isoforms (PIM1, PIM2, and PIM3) that are highly evolutionarily conserved. These proteins are regulated primarily by transcription and stability through pathways that are controlled by Janus kinase/Signal transducer and activator of transcription, JAK/STAT, transcription factors. The PIM family proteins have been found to be overexpressed in hematological malignancies and solid tumors, and their roles in these tumors were confirmed in mouse tumor models. Furthermore, the PIM family proteins have been implicated in the regulation of apoptosis, metabolism, cell cycle, and homing and migration, which has led to the postulation of these proteins as interesting targets for anticancer drug discovery. In the present work, we review the importance of PIM kinases in tumor growth and as drug targets. PMID:23576269

  10. Magnesium regulation of the beta-receptor-adenylate cyclase complex. II. Sc3+ as a Mg2 antagonist.

    PubMed

    Maguire, M E

    1982-09-01

    Sc3+ bears the same relationship to Mg2+ as La3+ to Ca2+, a similar ionic radius but increased charge. Therefore, the possibility was investigated that Sc3+ would be a Mg2+ antagonist at Mg2+ sites on the beta-adrenergic receptor-adenylate cyclase complex of the murine S49 lymphoma cell. Sc3+ is consistently much more potent than La3+ in inhibiting adenylate cyclase regardless of the mode of activation. IC50 values for Sc3+ of 10-30 microM were observed, whereas those for La3+ were about 300 microM. However, Sc3+ does not block the ability of Mg2+ to increase beta-receptor affinity for agonist nor alter agonist affinity by itself. Furthermore, Sc3+ is a weak inhibitor of the beta-receptor-mediated inhibition of Mg2+ influx. In cyc- S49 membranes, in which the catalytic subunit of cyclase cannot interact with the nucleotide-coupling protein(s), Sc3+ is as potent as in wild-type S49 membranes and again more potent than La3+. Substrate kinetics show that Sc3+, like Mg2+, modulates adenylate cyclase activity by affecting the Vmax without altering the Km for substrate. The data suggest that Sc3+ is a specific antagonist of Mg2+ at the Mg2+ site on the catalytic subunit and support the suggestion that there are two distinct sites for Mg2+ with different functions, one site on the coupling protein(s) and one on the catalytic subunit. It was also found that an apparent complex of Sc3+ and F-, ScF4-, is a potent inhibitor of adenylate cyclase, with an IC50 of 3 microM. PMID:6292689

  11. Two Kinase Family Dramas

    PubMed Central

    Leonard, Thomas A.; Hurley, James H.

    2007-01-01

    In this issue, Lietha and colleagues (2007) report the structure of focal adhesion kinase (FAK) and reveal how FAK maintains an autoinhibited state. Together with the structure of another tyrosine kinase, ZAP-70 (Deindl et al., 2007), this work highlights the diversity of mechanisms that nature has evolved within the kinase superfamily to regulate their activity through autoinhibition. PMID:17574014

  12. Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography.

    PubMed Central

    Vauquelin, G; Geynet, P; Hanoune, J; Strosberg, A D

    1977-01-01

    The adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC 4.6.1.1] and beta-adrenergic receptor of plasma membranes of turkey erythrocytes were solubilized in an active form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer stimulated by catecholamines, NaF, or guanine nucleotides. The digitonin extract was chromatographed on an alprenolol-agarose derivative. While the bulk of protein and all the adenylate cyclase activity passed unretarded through the column, the receptor was retained. It eluted free of enzyme activity with an alprenolol solution containing 1 M NaCl; the yield was 25-30%. The protein content of the alprenolol eluates was too low to be estimated by the Lowry technique and was assessed by a more sensitive fluorometric method. Under these conditions, the beta-adrenergic receptor was purified approximately 2000-fold in a single step with retention of all its pharmacological properties. These experiments establish that the beta-adrenergic receptor and the adenylate cyclase are independent entities which may be separated on a functional basis. PMID:198798

  13. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    SciTech Connect

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.

  14. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-05-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mg protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.

  15. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

    PubMed Central

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-01-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  16. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    PubMed

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  17. Adenylate pool and energy charge in human lymphocytes and granulocytes irradiated at 632 nm (HeNe laser)

    NASA Astrophysics Data System (ADS)

    Bolognani, Lorenzo; Venturelli, T.; Volpi, N.; Zirilli, O.

    1995-05-01

    Aim of this report was to investigate the adenylate pool and the energy charge in human white blood cells exposed to increasing time (15, 30 and 60 min) of HeNe laser treatment. EDTA treated human blood diluted 1:1 with 0.88% KCl was added (1:5) with NaCl-dextran solution to allow sedimentation of red blood cells. 6 ml of the white cells floating in the supernatant were layered on 3 ml of Lymphoprep in plastic tubes and each tube was centrifuged (from 50 to 5000 X g for 5 min). Granulocytes were concentrated in the lower phase, whilst lymphocytes were in the intermediated phase. After further purification cytological homogeneity was tested by a cell counter. Granulocytes and lymphocytes were irradiated at +22°C with HeNe (Space, Valfivre equipment). On these population ATP was tested by luminometric procedure, the adenylate pool was separated by HPLC (Jasco) on neutralyzed perchloric extracts. ATP concentration increased in lymphocytes (+63.9%, p < 0.01) and in granulocytes (+25.0%, p < 0.05) after 60 min irradiation. The adenylate pool (tested by HPLC) does not change significatively in lymphocytes or granulocytes after 30 min irradiation, whilst in 60 min irradiated lymphocytes and granulocytes a significative increment was observed in nucleotide concentration. No changes were observed in energy charge according to Atkinson.

  18. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers

    PubMed Central

    Brault, Laurent; Gasser, Christelle; Bracher, Franz; Huber, Kilian; Knapp, Stefan; Schwaller, Jürg

    2010-01-01

    The identification as cooperating targets of Proviral Integrations of Moloney virus in murine lymphomas suggested early on that PIM serine/threonine kinases play an important role in cancer biology. Whereas elevated levels of PIM1 and PIM2 were mostly found in hematologic malignancies and prostate cancer, increased PIM3 expression was observed in different solid tumors. PIM kinases are constitutively active and their activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates including several cell cycle regulators and apoptosis mediators. PIM1 but not PIM2 seems also to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression. Knockdown experiments by RNA interference or dominant-negative acting mutants suggested that PIM kinases are important for maintenance of a transformed phenotype and therefore potential therapeutic targets. Determination of the protein structure facilitated identification of an increasing number of potent small molecule PIM kinase inhibitors with in vitro and in vivo anticancer activity. Ongoing efforts aim to identify isoform-specific PIM inhibitors that would not only help to dissect the kinase function but hopefully also provide targeted therapeutics. Here, we summarize the current knowledge about the role of PIM serine/threonine kinases for the pathogenesis and therapy of hematologic malignancies and solid cancers, and we highlight structural principles and recent progress on small molecule PIM kinase inhibitors that are on their way into first clinical trials. PMID:20145274

  19. Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim.

    PubMed

    Moujalled, Diane; Weston, Ross; Anderton, Holly; Ninnis, Robert; Goel, Pranay; Coley, Andrew; Huang, David C S; Wu, Li; Strasser, Andreas; Puthalakath, Hamsa

    2011-01-01

    The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy. PMID:21151042

  20. p90RSK activation contributes to cerebral ischemic damage via phosphorylation of Na+/H+ exchanger isoform 1

    PubMed Central

    Manhas, Namratta; Shi, Yejie; Taunton, Jack; Sun, Dandan

    2010-01-01

    Excessive activation of Na+/H+ exchanger isoform 1 (NHE-1) plays a role in cerebral ischemic injury. The current study investigated whether NHE-1 protein in ischemic brains is regulated by extracellular signal-regulated kinase (ERK)/90-kDa ribosomal S6 kinase (p90RSK) signaling pathways. A transient focal ischemia in mice was induced by a 60 min-occlusion of the middle cerebral artery followed by reperfusion for 3, 10, or 60 min (Rp). Expression of phosphorylated ERK 1/2 was significantly elevated in the ipsilateral hemispheres at 3 – 10 min Rp and reduced by 60 min Rp. An increase in phosphorylation of p90RSK, a known NHE-1 kinase, was also detected at 3 – 10 min Rp, which was accompanied with a transient elevation of NHE-1 phosphorylation (p-NHE-1). Stimulation of p90RSK in ischemic neurons was downstream of ERK activation because inhibition of MEK1 (MAP kinase/ERK kinase) with its inhibitor U0126 blocked phosphorylation of p90RSK. Moreover, direct inhibition of p90RSK by its selective inhibitor FMK not only reduced p-NHE-1 expression but also ischemic infarct volume. Taken together, our study revealed that reperfusion triggers a transient stimulation of the ERK/p90RSK pathway. p90RSK activation contributes to cerebral ischemic damage in part via activation of NHE-1 protein. PMID:20557427

  1. ets-2 Is a Target for an Akt (Protein Kinase B)/Jun N-Terminal Kinase Signaling Pathway in Macrophages of motheaten-viable Mutant Mice

    PubMed Central

    Smith, James L.; Schaffner, Alicia E.; Hofmeister, Joseph K.; Hartman, Matthew; Wei, Guo; Forsthoefel, David; Hume, David A.; Ostrowski, Michael C.

    2000-01-01

    The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase–stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase–Akt–JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival. PMID:11027273

  2. Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

    PubMed

    Gabreski, Nicole A; Vaghasia, Janki K; Novakova, Silvia S; McDonald, Neil Q; Pierchala, Brian A

    2016-07-29

    Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function.

  3. Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin

    PubMed Central

    Tomatis, Vanesa M.; Papadopulos, Andreas; Malintan, Nancy T.; Martin, Sally; Wallis, Tristan; Gormal, Rachel S.; Kendrick-Jones, John; Buss, Folma

    2013-01-01

    Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane. PMID:23382463

  4. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  5. Regulation of different human NFAT isoforms by neuronal activity.

    PubMed

    Vihma, Hanna; Luhakooder, Mirjam; Pruunsild, Priit; Timmusk, Tõnis

    2016-05-01

    Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear

  6. Detection of VEGF-Axxxb Isoforms in Human Tissues

    PubMed Central

    Bates, David O.; Mavrou, Athina; Qiu, Yan; Carter, James G.; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V.; Millar, Ann B.; Salmon, Andrew H. J.; Oltean, Sebastian; Harper, Steven J.

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls. PMID:23935865

  7. Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells

    PubMed Central

    Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark

    2016-01-01

    Summary Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here we report formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, which bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of ‘α4-α4’ proteasomes depends on the relative cellular levels of α4 and α3, and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of a novel mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses. PMID:26997268

  8. Content of Adenosine Phosphates and Adenylate Energy Charge in Germinating Ponderosa Pine Seeds

    PubMed Central

    Ching, Te May; Ching, Kim K.

    1972-01-01

    An average of 540 picomoles of total adenosine phosphates was found in the embryo of mature seeds of ponderosa pine (Pinus ponderosa Laws.) and 1140 picomoles in the gametophyte. Adenylate energy charges were 0.44 and 0.26, respectively. After stratification, total adenosine phosphates increased 7-fold and 6-fold in embryo and gametophyte, respectively, and energy charges rose to 0.85 and 0.75. During germination, total adenosine phosphates increased to a 20-fold peak on the 9th day in gametophytic tissue, parallel with the peak of reserve regradation and organellar synthesis, and then decreased. In embryo and seedling, total adenosine phosphates elevated 80-fold with two distinct oscillating increases of AMP and ADP. The oscillating increases occurred before the emergence of radicle and cotyledons during which the highest mitotic index prevailed in all tissues. Energy charges fluctuated between 0.65 at the rapid cell dividing stage to 0.85 at the fully differentiated stage of the seedling, while energy charges remained around 0.75 in the gametophyte. These data indicated that the content of adenosine phosphates of germinating seeds reflects growth, organogenesis, and morphogenesis, and that a compartmentalized energy metabolism must exist in dividing and growing plant cells. PMID:16658212

  9. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    PubMed Central

    Tlili, Mounira; Rouatbi, Sonia; Sriha, Badreddine; Ben Rhouma, Khémais; Sakly, Mohsen; Vaudry, David; Wurtz, Olivier; Tebourbi, Olfa

    2015-01-01

    The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP), we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h) for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM) for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC) and cytokines (IL-1α and TNF-α) in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders. PMID:26199679

  10. Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation.

    PubMed

    Leps, W T; Ensign, J C

    1979-07-01

    The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the smae levels as found during growth.

  11. Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases

    PubMed Central

    He, Weizhi; Zhang, Xuhui; Zhang, Jun; Jia, Xu; Zhang, Jing; Sun, Wenxia; Jiang, Hengyi; Chen, Dongrong; Murchie, Alastair IH

    2013-01-01

    The acquisition of antibiotic resistance by human pathogens poses a significant threat to public health. The mechanisms that control the proliferation and expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are a historically important class of antibiotics that were introduced in the 1940s. Aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug or enzymatic modification of the target rRNA through methylation or through the overexpression of efflux pumps. In our recent paper, we reported that expression of the aminoglycoside resistance genes encoding the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes was controlled by an aminoglycoside-sensing riboswitch RNA. This riboswitch is embedded in the leader RNA of the aac/aad genes and is associated with the integron cassette system. The leader RNA can sense and bind specific aminoglycosides such that the binding causes a structural transition in the leader RNA, which leads to the induction of aminoglycoside antibiotic resistance. Specific aminoglycosides induce reporter gene expression mediated by the leader RNA. Aminoglycoside RNA binding was measured directly and, aminoglycoside-induced changes in RNA structure monitored by chemical probing. UV cross-linking and mutational analysis identified potential aminoglycoside binding sites on the RNA. PMID:23880830

  12. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    PubMed Central

    Hurley, Matthew M.; Maunze, Brian; Block, Megan E.; Frenkel, Mogen M.; Reilly, Michael J.; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A.; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive.

  13. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase

    SciTech Connect

    Wysham, D.G.; Brotherton, A.F.; Heistad, D.D.

    1986-11-01

    We have studied cerebral vascular effects of forskolin, a drug which stimulates adenylate cyclase and potentiates dilator effects of adenosine in other vascular beds. Our goals were to determine whether forskolin is a cerebral vasodilator and whether it potentiates cerebral vasodilator responses to adenosine. We measured cerebral blood flow with microspheres in anesthetized rabbits. Forskolin (10 micrograms/kg per min) increased blood flow (ml/min per 100 gm) from 39 +/- 5 (mean +/- S.E.) to 56 +/- 9 (p less than 0.05) in cerebrum, and increased flow to myocardium and kidney despite a decrease in mean arterial pressure. Forskolin did not alter cerebral oxygen consumption, which indicates that the increase in cerebral blood flow is a direct vasodilator effect and is not secondary to increased metabolism. We also examined effects of forskolin on the response to infusion of adenosine. Cerebral blood flow was measured during infusion of 1-5 microM/min adenosine into one internal carotid artery, under control conditions and during infusion of forskolin at 3 micrograms/kg per min i.v. Adenosine alone increased ipsilateral cerebral blood flow from 32 +/- 3 to 45 +/- 5 (p less than 0.05). Responses to adenosine were not augmented during infusion of forskolin. We conclude that forskolin is a direct cerebral vasodilator and forskolin does not potentiate cerebral vasodilator responses to adenosine.

  14. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase

    PubMed Central

    Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J.

    2016-01-01

    Abstract Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  15. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    PubMed Central

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. DOI: http://dx.doi.org/10.7554/eLife.10766.001 PMID:26650353

  16. Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain.

    PubMed

    Wehmann, Enikő; Khayer, Bernadett; Magyar, Tibor

    2015-01-01

    Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A-D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR-RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica. PMID:25475014

  17. Adenylate cyclase 3: a new target for anti-obesity drug development.

    PubMed

    Wu, L; Shen, C; Seed Ahmed, M; Östenson, C-G; Gu, H F

    2016-09-01

    Obesity has become epidemic worldwide, and abdominal obesity has a negative impact on health. Current treatment options on obesity, however, still remain limited. It is then of importance to find a new target for anti-obesity drug development based upon recent molecular studies in obesity. Adenylate cyclase 3 (ADCY3) is the third member of adenylyl cyclase family and catalyses the synthesis of cAMP from ATP. Genetic studies with candidate gene and genome-wide association study approaches have demonstrated that ADCY3 genetic polymorphisms are associated with obesity in European and Chinese populations. Epigenetic studies have indicated that increased DNA methylation levels in the ADCY3 gene are involved in the pathogenesis of obesity. Furthermore, biological analyses with animal models have implicated that ADCY3 dysfunction resulted in increased body weight and fat mass, while reduction of body weight is partially explained by ADCY3 activation. In this review, we describe genomic and biological features of ADCY3, summarize genetic and epigenetic association studies of the ADCY3 gene with obesity and discuss dysfunction and activation of ADCY3. Based upon all data, we suggest that ADCY3 is a new target for anti-obesity drug development. Further investigation on the effectiveness of ADCY3 activator and its delivery approach to treat abdominal obesity has been taken into our consideration. PMID:27256589

  18. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3.

    PubMed

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. PMID:26650353

  19. Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate.

    PubMed

    Ribeiro, César; Esteves da Silva, Joaquim C G

    2008-09-01

    The inhibition mechanisms of the firefly luciferase (Luc) by the two major products of the reactions catalysed by Luc, oxyluciferin and dehydroluciferyl-adenylate (L-AMP), were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 microM oxyluciferin; 0.0025 to 1.25 microM L-AMP) has been measured in 50 mM Hepes buffer (pH=7.5), 10 nM Luc, 250 microM ATP and D-Luciferin (from 3.75 up to 120 microM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that oxyluciferin is a competitive inhibitor of luciferase (Ki=0.50+/-0.03 microM) while L-AMP act as a tight-binding competitive inhibitor (Ki=3.8+/-0.7 nM). The Km values obtained both for oxyluciferin and L-AMP were 14.7+/-0.7 and 14.9+/-0.2 microM, respectively. L-AMP is a stronger inhibitor of Luc than oxyluciferin and the major responsible for the characteristic flash profile of in vitro Luc bioluminescence.

  20. The adenylate energy charge as a new and useful indicator of capture stress in chondrichthyans.

    PubMed

    Guida, Leonardo; Walker, Terence I; Reina, Richard D

    2016-02-01

    Quantifying the physiological stress response of chondrichthyans to capture has assisted the development of fishing practices conducive to their survival. However, currently used indicators of stress show significant interspecific and intraspecific variation in species' physiological responses and tolerances to capture. To improve our understanding of chondrichthyan stress physiology and potentially reduce variation when quantifying the stress response, we investigated the use of the adenylate energy charge (AEC); a measure of available metabolic energy. To determine tissues sensitive to metabolic stress, we extracted samples of the brain, heart, liver, white muscle and blood from gummy sharks (Mustelus antarcticus) immediately following gillnet capture and after 3 h recovery under laboratory conditions. Capture caused significant declines in liver, white muscle and blood AEC, whereas no decline was detected in the heart and brain AEC. Following 3 h of recovery from capture, the AEC of the liver and blood returned to "unstressed" levels (control values) whereas white muscle AEC was not significantly different to that immediately after capture. Our results show that the liver is most sensitive to metabolic stress and white muscle offers a practical method to sample animals non-lethally for determination of the AEC. The AEC is a highly informative indicator of stress and unlike current indicators, it can directly measure the change in available energy and thus the metabolic stress experienced by a given tissue. Cellular metabolism is highly conserved across organisms and, therefore, we think the AEC can also provide a standardised form of measuring capture stress in many chondrichthyan species. PMID:26660290

  1. Progesterone prevents linkage of rabbit myometrial alpha 2-adrenergic receptors to inhibition of adenylate cyclase.

    PubMed

    Wu, Y Y; Riemer, R K; Goldfien, A; Roberts, J M

    1989-04-01

    The uterine response to adrenergic stimulation is determined by the hormonal milieu. This response is particularly well characterized in the rabbit. In this species, as in humans, the response of the uterus to sympathetic stimulation is alpha-adrenergically mediated contraction with elevated circulating estrogen. However, with progesterone predominance, similar stimulation inhibits uterine contractions, a response mediated by beta-adrenergic receptors acting through their second message, cyclic adenosine monophosphate. We studied the mechanisms by which sex steroids regulate myometrial adrenergic responses. In this study, we questioned whether part of the effect of sex steroids could be explained by an alteration of the coupling of the alpha 2-adrenergic receptor to the inhibition of adenylate cyclase. We found that in the progesterone-treated rabbit, although alpha 2-receptors are present, they are not linked to inhibition of cyclic adenosine monophosphate synthesis. The net synthesis of cyclic adenosine monophosphage in response to endogenous catecholamines is determined by their activation of beta-adrenergic receptors to increase and alpha 2-receptors to decrease cyclic adenosine monophosphate formation. Thus the uncoupling of alpha 2-receptors contributes to increased intracellular cyclic adenosine monophosphate in myometrium of progesterone-treated animals consistent with the reported predominance of beta-adrenergic contractile responses in this setting.

  2. Guanine-nucleotide-dependent inhibition of adenylate cyclase of rabbit heart by glucagon.

    PubMed

    Kiss, Z; Tkachuk, V A

    1984-07-16

    The present study demonstrates an inhibitory effect of glucagon on the adenylate cyclase system of rabbit heart. Inhibition was maximal (22-40%) at 0.1-0.01 microM glucagon and required the presence of 0.01-0.1 mM GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GuoPP[NH]P). Reduced or no inhibitor effect of glucagon was observed: (a) after limited proteolysis of plasma membrane proteins by trypsin, (b) in the presence of 1 mM Mn2+, (c) in the absence of Na+, and (d) during the first 10 min of incubation if GuoPP[NH]P was the activating ligand. With GTP as the activating ligand, inhibition of cyclase by glucagon occurred without delay. These data are consistent with a mediation of glucagon inhibition by a guanine-nucleotide-binding protein. In the presence of ethanol (0.2 M) or benzyl alcohol (0.05 M), agents which are known to increase the fluidity of biological membranes, glucagon increased the enzyme activity in a guanine-nucleotide-dependent manner. Activation of cyclase in the presence of alcohols was maximal (30-60%) at 0.1-1.0 microM glucagon and 0.01 mM guanine nucleotides. Data suggest that glucagon receptors can interact with both the activatory and inhibitory guanine-nucleotide-binding proteins and the physical state of membranes may play a role in determining which interaction will be preferential.

  3. Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain.

    PubMed

    Wehmann, Enikő; Khayer, Bernadett; Magyar, Tibor

    2015-01-01

    Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A-D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR-RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica.

  4. Distribution of caveolin isoforms in the lemur retina.

    PubMed

    Berta, Agnes I; Kiss, Anna L; Lukáts, Akos; Szabó, Arnold; Szél, Agoston

    2007-09-01

    The distribution of caveolin isoforms was previously evaluated in the retinas of different species, but has not yet been described in the primate retina. In this study, the distribution of caveolins was assessed via immunochemistry using isoform-specific antibodies in the retina of the black-and-white ruffed lemur. Here, we report the presence of a variety of caveolin isoforms in many layers of the lemur retina. As normal human retinas were not available for research and the retinas of primates are fairly similar to those of humans, the lemur retina can be utilized as a model for caveolin distribution in normal humans.

  5. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

    PubMed

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-11-17

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

  6. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer

    PubMed Central

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-01-01

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed. PMID:26573433

  7. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

  8. Diacylglycerol Kinase-ε: Properties and Biological Roles

    PubMed Central

    Epand, Richard M.; So, Vincent; Jennings, William; Khadka, Bijendra; Gupta, Radhey S.; Lemaire, Mathieu

    2016-01-01

    In mammals there are at least 10 isoforms of diacylglycerol kinases (DGK). All catalyze the phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PA). Among DGK isoforms, DGKε has several unique features. It is the only DGK isoform with specificity for a particular species of DAG, i.e., 1-stearoyl-2-arachidonoyl glycerol. The smallest of all known DGK isoforms, DGKε, is also the only DGK devoid of a regulatory domain. DGKε is the only DGK isoform that has a hydrophobic segment that is predicted to form a transmembrane helix. As the only membrane-bound, constitutively active DGK isoform with exquisite specificity for particular molecular species of DAG, the functional overlap between DGKε and other DGKs is predicted to be minimal. DGKε exhibits specificity for DAG containing the same acyl chains as those found in the lipid intermediates of the phosphatidylinositol-cycle. It has also been shown that DGKε affects the acyl chain composition of phosphatidylinositol in whole cells. It is thus likely that DGKε is responsible for catalyzing one step in the phosphatidylinositol-cycle. Steps of this cycle take place in both the plasma membrane and the endoplasmic reticulum membrane. DGKε is likely present in both of these membranes. DGKε is the only DGK isoform that is associated with a human disease. Indeed, recessive loss-of-function mutations in DGKε cause atypical hemolytic-uremic syndrome (aHUS). This condition is characterized by thrombosis in the small vessels of the kidney. It causes acute renal insufficiency in infancy and most patients develop end-stage renal failure before adulthood. Disease pathophysiology is poorly understood and there is no therapy. There are also data suggesting that DGKε may play a role in epilepsy and Huntington disease. Thus, DGKε has many unique molecular and biochemical properties when compared to all other DGK isoforms. DGKε homologs also contain a number of conserved sequence features that are distinctive

  9. SELECTIVE CHANGES IN BRAIN PROTEIN KINASE C ISOFORMS FOLLOWING DEVELOPMENTAL EXPOSURE TO A PCB MIXTURE.

    EPA Science Inventory

    Introduction
    Polychlorinated biphenyls (PCBs) offer a unique model to understand the major issues related to complex environmental mixtures. These environmental pollutants are ubiquitous, persistent, bioaccumulate in human body through the food chain, and exist as mixtures of ...

  10. ALTERATIONS IN BRAIN PROTEIN KINASE C ISOFORMS FOLLOWING DEVELOPMENTAL EXPOSURE TO POLYCHLORINATED BIPHENYL MIXTURE.

    EPA Science Inventory

    PCBs have been shown to alter several neurochemical end-points and are implicated in the etiology of some neurological diseases. Recent in vivo studies from our laboratory indicated that developmental exposure to a commercial PCB mixture, Aroclor 1254, caused perturbations in cal...

  11. DEVELOPMENTAL REGULATION OF PROTEIN KINASE B ACTIVATION IS ISOFORM SPECIFIC IN SKELETAL MUSCLE OF NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The postprandial activation of the insulin signaling pathway that leads to translation initiation is enhanced in skeletal muscle of the neonate and decreases with development in parallel with the developmental decline in muscle protein synthesis. Our previous study showed that the activity of protei...

  12. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity

    PubMed Central

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins. PMID:27630986

  13. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity

    PubMed Central

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins.

  14. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity.

    PubMed

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins. PMID:27630986

  15. Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.

    PubMed

    Draskovic, Petra; Saiardi, Adolfo; Bhandari, Rashna; Burton, Adam; Ilc, Gregor; Kovacevic, Miroslav; Snyder, Solomon H; Podobnik, Marjetka

    2008-03-01

    Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is able to convert the natural substrates inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) to several products with an increasing number of phospho-anhydride bonds. In this study, we structurally analyzed IPs synthesized by three mammalian isoforms of IP6K from IP 5 and IP 6. The NMR and mass analyses showed a number of products with diverse, yet specific, stereochemistry, defined by the architecture of IP6K's active site. We now report that IP6K synthesizes both pyrophosphate (diphospho) as well as triphospho groups on the inositol ring. All three IP6K isoforms share the same activities both in vitro and in vivo.

  16. Survivin isoform Delta Ex3 regulates tumor spheroid formation.

    PubMed

    Espinosa, Magali; Ceballos-Cancino, Gisela; Callaghan, Richard; Maldonado, Vilma; Patiño, Nelly; Ruíz, Víctor; Meléndez-Zajgla, Jorge

    2012-05-01

    Survivin is an important member of the Inhibitor of Apoptosis Proteins (IAPs) family and has essential roles in apoptosis and cell cycle progression. This gene is commonly upregulated in human cancer and provides an exciting diagnostic and therapeutic target. Survivin is expressed as several isoforms that are generated by alternative splicing, and some of these present antagonistic activities. Currently, information regarding the regulation of these isoforms is lacking. In this study, we sought to analyze survivin Delta Ex3 expression in a three-dimensional model of avascular tumors and its overexpression effects in processes such as proliferation, clonogenicity and apoptosis. We found a positive correlation between spheroid growth and survivin Delta Ex3 expression during the exponential phase. We demonstrated that this isoform not only decreased apoptosis but also inhibited tumor spheroid formation by decreasing proliferation and clonogenic survival. These results point toward a dual and antagonistic effect of this spliced survivin isoform in cancer development.

  17. Spatially coordinated kinase signaling regulates local axon degeneration.

    PubMed

    Chen, Mark; Maloney, Janice A; Kallop, Dara Y; Atwal, Jasvinder K; Tam, Stephen J; Baer, Kristin; Kissel, Holger; Kaminker, Joshua S; Lewcock, Joseph W; Weimer, Robby M; Watts, Ryan J

    2012-09-26

    In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3β, we show that the β isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3β in axon degeneration.

  18. Neuronal Profilin Isoforms Are Addressed by Different Signalling Pathways

    PubMed Central

    Michaelsen-Preusse, Kristin; Dresbach, Thomas; Schoenenberger, Cora-Ann; Korte, Martin; Jockusch, Brigitte M.; Rothkegel, Martin

    2012-01-01

    Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei. PMID:22470532

  19. Cell-specific expression of TLR9 isoforms in inflammation.

    PubMed

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  20. A Network of Splice Isoforms for the Mouse

    PubMed Central

    Li, Hong-Dong; Menon, Rajasree; Eksi, Ridvan; Guerler, Aysam; Zhang, Yang; Omenn, Gilbert S.; Guan, Yuanfang

    2016-01-01

    The laboratory mouse is the primary mammalian species used for studying alternative splicing events. Recent studies have generated computational models to predict functions for splice isoforms in the mouse. However, the functional relationship network, describing the probability of splice isoforms participating in the same biological process or pathway, has not yet been studied in the mouse. Here we describe a rich genome-wide resource of mouse networks at the isoform level, which was generated using a unique framework that was originally developed to infer isoform functions. This network was built through integrating heterogeneous genomic and protein data, including RNA-seq, exon array, protein docking and pseudo-amino acid composition. Through simulation and cross-validation studies, we demonstrated the accuracy of the algorithm in predicting isoform-level functional relationships. We showed that this network enables the users to reveal functional differences of the isoforms of the same gene, as illustrated by literature evidence with Anxa6 (annexin a6) as an example. We expect this work will become a useful resource for the mouse genetics community to understand gene functions. The network is publicly available at: http://guanlab.ccmb.med.umich.edu/isoformnetwork. PMID:27079421

  1. Frac-seq reveals isoform-specific recruitment to polyribosomes.

    PubMed

    Sterne-Weiler, Timothy; Martinez-Nunez, Rocio Teresa; Howard, Jonathan M; Cvitovik, Ivan; Katzman, Sol; Tariq, Muhammad A; Pourmand, Nader; Sanford, Jeremy R

    2013-10-01

    Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5'UTR as well as Alu-elements and microRNA target sites in the 3'UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms.

  2. Frac-seq reveals isoform-specific recruitment to polyribosomes

    PubMed Central

    Sterne-Weiler, Timothy; Martinez-Nunez, Rocio Teresa; Howard, Jonathan M.; Cvitovik, Ivan; Katzman, Sol; Tariq, Muhammad A.; Pourmand, Nader; Sanford, Jeremy R.

    2013-01-01

    Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5′UTR as well as Alu-elements and microRNA target sites in the 3′UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms. PMID:23783272

  3. A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

    PubMed

    Burroughs, A Maxwell; Ando, Yoshinari; de Hoon, Michiel J L; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O

    2010-10-01

    Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

  4. High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases.

    PubMed

    Demian, Douglas J; Clugston, Susan L; Foster, Meta M; Rameh, Lucia; Sarkes, Deborah; Townson, Sharon A; Yang, Lily; Zhang, Melvin; Charlton, Maura E

    2009-08-01

    Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [gamma-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIbeta and alpha) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIbeta phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets.

  5. High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases.

    PubMed

    Demian, Douglas J; Clugston, Susan L; Foster, Meta M; Rameh, Lucia; Sarkes, Deborah; Townson, Sharon A; Yang, Lily; Zhang, Melvin; Charlton, Maura E

    2009-08-01

    Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [gamma-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIbeta and alpha) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIbeta phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets. PMID:19641220

  6. Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

    PubMed Central

    Fagotti, A; Gabbiani, G; Pascolini, R; Neuville, P

    1998-01-01

    We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa. PMID:9518500

  7. Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains.

    PubMed

    Agüero-Chapin, Guillermin; Pérez-Machado, Gisselle; Sánchez-Rodríguez, Aminael; Santos, Miguel Machado; Antunes, Agostinho

    2016-01-01

    Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10-40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST searches. In this chapter we describe two workflows based on alignment-free methods intended for the identification and substrate specificity prediction of A-domains. To identify A-domains we introduce a graphical-numerical method, implemented in TI2BioP version 2.0 (topological indices to biopolymers), which in a first step uses protein four-color maps to represent A-domains. In a second step, simple topological indices (TIs), called spectral moments, are derived from the graphical representations of known A-domains (positive dataset) and of unrelated but well-characterized sequences (negative set). Spectral moments are then used as input predictors for statistical classification techniques to build alignment-free models. Finally, the resulting alignment-free models can be used to explore entire proteomes for unannotated A-domains. In addition, this graphical-numerical methodology works as a sequence-search method that can be ensemble with homology-based tools to deeply explore the A-domain signature and cope with the diversity of this class (Aguero-Chapin et al., PLoS One 8(7):e65926, 2013). The second workflow for the prediction of A-domain's substrate specificity is based on alignment-free models constructed by transductive support vector machines (TSVMs) that incorporate information of uncharacterized A-domains. The construction of the models was

  8. Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains.

    PubMed

    Agüero-Chapin, Guillermin; Pérez-Machado, Gisselle; Sánchez-Rodríguez, Aminael; Santos, Miguel Machado; Antunes, Agostinho

    2016-01-01

    Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10-40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST searches. In this chapter we describe two workflows based on alignment-free methods intended for the identification and substrate specificity prediction of A-domains. To identify A-domains we introduce a graphical-numerical method, implemented in TI2BioP version 2.0 (topological indices to biopolymers), which in a first step uses protein four-color maps to represent A-domains. In a second step, simple topological indices (TIs), called spectral moments, are derived from the graphical representations of known A-domains (positive dataset) and of unrelated but well-characterized sequences (negative set). Spectral moments are then used as input predictors for statistical classification techniques to build alignment-free models. Finally, the resulting alignment-free models can be used to explore entire proteomes for unannotated A-domains. In addition, this graphical-numerical methodology works as a sequence-search method that can be ensemble with homology-based tools to deeply explore the A-domain signature and cope with the diversity of this class (Aguero-Chapin et al., PLoS One 8(7):e65926, 2013). The second workflow for the prediction of A-domain's substrate specificity is based on alignment-free models constructed by transductive support vector machines (TSVMs) that incorporate information of uncharacterized A-domains. The construction of the models was

  9. The Secretory Pathway Kinases

    PubMed Central

    Sreelatha, Anju; Kinch, Lisa N.; Tagliabracci, Vincent S.

    2015-01-01

    Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. PMID:25862977

  10. Biochemical mechanisms of myocardial adenylate cyclase subsensitivity to isoproterenol in cardiac hypertrophy of spontaneously hypertensive rats

    SciTech Connect

    Cheon, J.W.

    1986-01-01

    The responsiveness of the myocardial adenylate cyclase (AC) system in generating cAMP was studied using isoproterenol (a beta-adrenergic receptor agonist), cholera toxin (a guanosinetriphosphatase inhibitor) and forskolin (a catalytic unit activator) in isolated myocytes of age-matched, 14-17 weeks old Wistar Kyoto normotensive rates (WKYs) and spontaneously hypertensive rats (SHRs). We found a reduction in isoproterenol-stimulated cAMP formation in myocytes of SHRs compared with WKYs. This reduction was not due to changes in isoproterenol-receptor interactions. Scatchard plot analysis of (/sup 3/H)CGP 12177 binding to beta-adrenergic receptors in isolated myocytes of WKYs and SHRs revealed to significant differences in the maximum number of binding sites or dissociation constant. There were no significant differences in Ki and IC/sub 50/ calculated from the competitive displacement of (/sup 3/H)CGP 12177 binding by (-) isoproterenol, suggesting no change in the affinity of the beta-adrenergic receptors for isoproterenol. We found no significant differences in forskolin-stimulated cAMP formation between the two groups. This suggest that the reduction in isoproterenol-stimulated cAMP formation observed in myocytes of SHRs is not due to changes in the ability of catalytic unit to convert ATP to cAMP. Interestingly, cholera toxin-stimulated cAMP formation was increased in myocytes of SHRs. One possible explanation for these observations may be increased guanosinetriphosphatase (GTPase) activation by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol was measured as the release of Pi from (..gamma..-/sup 32/P)GTP. There was an increase in isoproterenol-stimulated GTPase activity in myocytes of SHRs compared with WKYs.

  11. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

    PubMed Central

    Ohki, Mio; Sugiyama, Kanako; Kawai, Fumihiro; Tanaka, Hitomi; Nihei, Yuuki; Unzai, Satoru; Takebe, Masumi; Matsunaga, Shigeru; Adachi, Shin-ichi; Shibayama, Naoya; Zhou, Zhiwen; Koyama, Ryuta; Takahashi, Tetsuo; Tame, Jeremy R. H.; Iseki, Mineo; Park, Sam-Yong

    2016-01-01

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. PMID:27247413

  12. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating.

    PubMed

    Hurley, Matthew M; Maunze, Brian; Block, Megan E; Frenkel, Mogen M; Reilly, Michael J; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive. PMID:27597817

  13. Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.

  14. Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

    PubMed

    Dangelmaier, Carol A; Holmsen, Holm

    2014-01-01

    Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

  15. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

    PubMed

    Ohki, Mio; Sugiyama, Kanako; Kawai, Fumihiro; Tanaka, Hitomi; Nihei, Yuuki; Unzai, Satoru; Takebe, Masumi; Matsunaga, Shigeru; Adachi, Shin-Ichi; Shibayama, Naoya; Zhou, Zhiwen; Koyama, Ryuta; Ikegaya, Yuji; Takahashi, Tetsuo; Tame, Jeremy R H; Iseki, Mineo; Park, Sam-Yong

    2016-06-14

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. PMID:27247413

  16. Reconstitution of beta-adrenergic receptor with components of adenylate cyclase.

    PubMed Central

    Hekman, M; Feder, D; Keenan, A K; Gal, A; Klein, H W; Pfeuffer, T; Levitzki, A; Helmreich, E J

    1984-01-01

    Beta 1-Adrenergic receptor proteins were extracted from turkey erythrocyte membranes with lauroyl sucrose and digitonin and purified by affinity chromatography on a column of alprenolol agarose Affi-gel 10 or 15. The 5000-fold purified receptor is able to couple functionally with the stimulatory GTP-binding protein (GS) from either turkey or duck erythrocytes. Functional coupling was achieved by three different approaches. (i) Purified beta-receptor polypeptides were coupled in phospholipid (asolectin) vesicles with GS from a crude cholate or lauroyl sucrose extract of turkey erythrocyte membranes. The detergent was removed and vesicles were formed with SM-2 beads. (ii) Purified beta-receptor was reconstituted with pure, homogeneous GS in asolectin vesicles. (iii) Purified beta-receptors were either coupled in asolectin vesicles with a mixture of pure, homogeneous Gpp(NH)p-activated GS and a lauroyl sucrose extract of turkey erythrocyte membranes, or with pure, homogeneous Gpp(NH)p-activated GS alone. The decay of activity was measured on addition of GTP and hormone. In (ii) and (iii), the detergent was removed and vesicles were formed by gel filtration on Sephadex G-50 columns. In each of the three different experimental conditions, the beta-receptor was activated with l-isoproterenol and activation was blocked with d,l-propranolol. Activated GS were measured separately by means of their capacity to activate a crude Lubrol PX-solubilized adenylate cyclase preparation from rabbit myocardial membrane. The kinetics of GS activation by purified beta-receptors occupied by l-isoproterenol was first order and activation was linearly dependent on receptor concentration.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. PMID:6098472

  17. Regulation of uterine adenylate cyclase by magnesium, manganese and calcium ions

    SciTech Connect

    Rayford, W.; Sanders, R.B.

    1987-05-01

    The regulation of rat uterine adenylate cyclase (AC) by Mg/sup 2 +/, Mn/sup 2 +/ and Ca/sup 2 +/ was examined during metestrus and proestrus of the estrous cycle and Days 1 and 4 of pseudopregnancy, before and after a mild trauma to the uterus. Mg/sup 2 +/ increased cyclase activity on all days measured. Maximal enzymic activity occurred with 5-10 mM Mg/sup 2 +/ during Day 4 following a mild traumatic stimulus to the uterus. The apparent Ka for Mg/sup 2 +/ was not significantly changed during these days. AC activity as a function of Mn/sup 2 +/ concentration was biphasic. It increased with increasing concentrations of Mn/sup 2 +/ and was maximal at 1.0-2.5 mM during Day 4 following uterine trauma. Higher concentrations of Mn/sup 2 +/ were inhibitory. The apparent Ka for Mn/sup 2 +/ was 0.36 +/- 0.05 mM and was not significantly altered during the days studied. Even though the Ka for Mn/sup 2 +/ was ten-fold lower than that for Mg/sup 2 +/, the Vmaxes shown with both ions were similar. Ca/sup 2 +/ is a potent inhibitor of uterine AC activity. When measured at its I.C./sub 50/, it lowered AC activity as Mg/sup 2 +/ concentrations were increased. Ca/sup 2 +/ did not have a significant effect on AC activated by Mn/sup 2 +/. The data showed that Mg/sup 2 +/, Mn/sup 2 +/ and Ca/sup 2 +/ might have important regulatory roles in the activation and inhibition of uterine AC in the rodent.

  18. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    PubMed Central

    Hurley, Matthew M.; Maunze, Brian; Block, Megan E.; Frenkel, Mogen M.; Reilly, Michael J.; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A.; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive. PMID:27597817

  19. Pituitary adenylate cyclase-activating polypeptide prevents contrast-induced nephropathy in a novel mouse model

    PubMed Central

    Khan, Altaf-M; Maderdrut, Jerome L; Li, Min; Toliver, Herman L; Coy, David H; Simon, Eric E; Batuman, Vecihi

    2013-01-01

    We determined whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevents contrast-induced nephropathy using human renal proximal tubule epithelial (HK-2) cells and homozygous endothelial nitric oxide synthase-deficient (eNOS−/−) mice as a novel in vivo model. Cultured HK-2 cells were pretreated with 10−9–10−6 mol/L PACAP or vasoactive intestinal peptide (VIP) for 1 h, and then exposed to ionic (Urografin) or nonionic (iohexol) contrast media at 50 mg iodine/mL for 24 h. Male eNOS−/− mice received Urografin (1.85 g iodine/kg) intravenously after water deprivation for 24 h, and PACAP38 (10 μg) intraperitoneally 1 h before and 12 h after Urografin injection. Urografin and iohexol increased lactate dehydrogenase and kidney injury molecule 1 in the culture medium, induced apoptosis, and inhibited cell proliferation in HK-2 cell cultures. PACAP38 and VIP reduced these changes in a dose-dependent manner. PACAP38 was more potent than VIP. In eNOS−/− mice, Urografin raised serum creatinine and cystatin C levels, caused renal tubule damage, induced apoptosis, and promoted neutrophil influx. Urografin also increased kidney protein levels of proinflammatory cytokines, and kidney mRNA levels of proinflammatory cytokines, kidney injury biomarkers, and enzymes responsible for reactive oxygen and nitrogen species. PACAP38 significantly reduced these Urografin-induced changes in eNOS−/− mice. This study shows that both Urografin and iohexol are toxic to HK-2 cells, but Urografin is more toxic than iohexol. Urografin causes acute kidney injury in eNOS−/− mice. PACAP38 protects HK-2 cells and mouse kidneys from contrast media and is a potential therapeutic agent for contrast-induced nephropathy. PMID:24400164

  20. Quantification of the Adenylate Cyclase Toxin of Bordetella pertussis In Vitro and during Respiratory Infection

    PubMed Central

    Eby, Joshua C.; Gray, Mary C.; Warfel, Jason M.; Paddock, Christopher D.; Jones, Tara F.; Day, Shandra R.; Bowden, James; Poulter, Melinda D.; Donato, Gina M.; Merkel, Tod J.

    2013-01-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis. PMID:23429530

  1. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice

    PubMed Central

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1). Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory. PMID:23060763

  2. Pituitary Adenylate Cyclase-Activating Polypeptide induces a depressive-like phenotype in rats

    PubMed Central

    Seiglie, Mariel P.; Smith, Karen L.; Blasio, Angelo; Cottone, Pietro; Sabino, Valentina

    2015-01-01

    Major Depressive Disorder (MDD) is a chronic, life-threatening psychiatric condition characterized by depressed mood, psychomotor alterations, and a markedly diminished interest or pleasure in most activities, known as anhedonia. Available pharmacotherapies have limited success and the need for new strategies is clear. Recent studies attribute a major role to the pituitary adenylate cyclase-activating polypeptide (PACAP) system in mediating the response to stress. PACAP knockout mice display profound alterations in depressive-like behaviors and genetic association studies have demonstrated that genetic variants of the PACAP gene are associated with MDD. However, the effects of PACAP on depressive-like behaviors in rodents have not yet been systematically examined. The present study investigated the effects of central administration of PACAP in rats on depressive-like behaviors, using well-established animal models that represent some of the endophenotypes of depression. We used intracranial self-stimulation (ICSS) to assess the brain reward function, saccharin preference test to assess anhedonia, social interaction to assess social withdrawal, and forced swim test (FST) to assess behavioral despair. PACAP raised the current threshold for ICSS, elevation blocked by the PACAP antagonist PACAP(6-38). PACAP reduced the preference for a sweet saccharin solution, and reduced the time the rats spent interacting with a novel animal. Interestingly, PACAP administration did not affect immobility in the FST. Our results demonstrate a role for the central PACAP/PAC1R system in the regulation of depressive-like behaviors, and suggest that hyperactivity of the PACAP/PAC1R system may contribute to the pathophysiology of depression, particularly the associated anhedonic symptomatology and social dysfunction. PMID:26264905

  3. With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl- cotransporters.

    PubMed

    Mercado, Adriana; de Los Heros, Paola; Melo, Zesergio; Chávez-Canales, María; Murillo-de-Ozores, Adrián R; Moreno, Erika; Bazúa-Valenti, Silvana; Vázquez, Norma; Hadchouel, Juliette; Gamba, Gerardo

    2016-07-01

    The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.

  4. Structurally homologous binding of plant calmodulin isoforms to the calmodulin-binding domain of vacuolar calcium-ATPase.

    PubMed

    Yamniuk, Aaron P; Vogel, Hans J

    2004-02-27

    The discovery that plants contain multiple calmodulin (CaM) isoforms having variable sequence identity to mammalian CaM has sparked a flurry of new questions regarding the intracellular role of Ca(2+) regulation in plants. To date, the majority of research in this field has focused on the differential enzymatic regulation of various mammalian CaM-dependent enzymes by the different plant CaM isoforms. However, there is comparatively little information on the structural recognition of target enzymes found exclusively in plant cells. Here we have used a variety of spectroscopic techniques, including nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, to study the interactions of the most conserved and most divergent CaM isoforms from soybean, SCaM-1, and SCaM-4, respectively, with a synthetic peptide derived from the CaM-binding domain of cauliflower vacuolar calcium-ATPase. Despite their sequence divergence, both SCaM-1 and SCaM-4 interact with the calcium-ATPase peptide in a similar calcium-dependent, stoichiometric manner, adopting an antiparallel binding orientation with an alpha-helical peptide. The single Trp residue is bound in a solvent-inaccessible hydrophobic pocket on the C-terminal domain of either protein. Thermodynamic analysis of these interactions using isothermal titration calorimetry demonstrates that the formation of each calcium-SCaM-calcium-ATPase peptide complex is driven by favorable binding enthalpy and is very similar to the binding of mammalian CaM to the CaM-binding domains of myosin light chain kinases and calmodulin-dependent protein kinase I.

  5. Phosphoinositide-dependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes.

    PubMed

    Hresko, Richard C; Murata, Haruhiko; Mueckler, Mike

    2003-06-13

    By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607-21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1-3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt. PMID:12682057

  6. Concanavalin A amplifies both beta-adrenergic and muscarinic cholinergic receptor-adenylate cyclase-linked pathways in cardiac myocytes.

    PubMed Central

    Rocha-Singh, K J; Hines, D K; Honbo, N Y; Karliner, J S

    1991-01-01

    Concanavalin A (Con A) is a tetrameric plant lectin that disrupts plasma membrane-cytoskeletal interactions and alters plasma membrane fluidity. We used Con A as a probe to explore beta-adrenergic and muscarinic cholinergic receptor-mediated regulation of cAMP in intact neonatal rat ventricular myocytes. Preincubation with Con A, 0.5 micrograms/ml, attenuated 1 microM (-)-norepinephrine (NE)-induced downregulation of beta-adrenergic receptors and resulted in a 50% augmentation of cAMP accumulation stimulated by 1 microM NE. Con A also augmented forskolin (1-10 microM)-stimulated cAMP accumulation by an average of 37% (P less than 0.05); however, Con A preincubation had no effect on basal or cholera toxin-stimulated cAMP content. The muscarinic cholinergic agonist carbachol (1-100 microM) decreased 1 microM NE-stimulated cAMP generation by an average of 32% (n = 7, P less than 0.05); preincubation with Con A further enhanced the inhibitory effect of carbachol by 18% (n = 7, P less than 0.05). Carbachol (1 microM) for 2 h decreased muscarinic cholinergic receptor density in whole cells by 33%; preincubation with Con A prevented this receptor downregulation. Con A pretreatment did not affect (-)-isoproterenol- or forskolin-stimulated adenylate cyclase activity in cell homogenates, suggesting that an intact cytoarchitecture is necessary for Con A to augment cAMP formation. We conclude that Con A, through its modulation of beta-adrenergic and muscarinic cholinergic receptor signaling, amplifies both stimulatory and inhibitory adenylate cyclase-linked pathways in intact neonatal ventricular myocytes. These data suggest the possibility that plasma membrane-cytoskeletal interaction is an important regulator of transmembrane signaling because interference with this interaction results in alterations in cAMP accumulation mediated by both beta-adrenergic- and muscarinic cholinergic-adenylate cyclase pathways. PMID:1653274

  7. Oxymetazoline inhibits adenylate cyclase by activation of serotonin-1 receptors in the OK cell, an established renal epithelial cell line.

    PubMed

    Murphy, T J; Bylund, D B

    1988-07-01

    The nonselective alpha-adrenergic agonist oxymetazoline inhibits parathyroid hormone (PTH)-stimulated cAMP production in intact OK cells, an epithelial cell line derived from an American opossum kidney. This inhibition, however, is not blocked by alpha 2-adrenergic receptor antagonists. After excluding several alternate hypotheses to explain this anomalous activity of oxymetazoline, we hypothesized that oxymetazoline activates a receptor in OK cells that is negatively coupled to adenylate cyclase but distinct from the alpha 2-adrenergic receptor. Prior exposure of OK cells to pertussis toxin blocks the inhibitory response to oxymetazoline, suggesting involvement of a guanine nucleotide-binding regulatory protein. Screening various compounds for attenuation of PTH-stimulated adenylate cyclase showed that serotonin (5HT) is a potent and fully efficacious agonist. Desensitization of alpha 2-receptor-mediated inhibition of cAMP production by epinephrine did not alter the response to either 5HT or oxymetazoline, indicating that these compounds do not produce their effect by activating alpha 2-adrenergic receptors. The 5HT1 receptor-selective antagonist methiothepin, but not ketanserin (5HT2-selective) or ICS-205,930 (5HT3-selective), blocked the response to both 5HT and oxymetazoline. The potency of methiothepin for antagonizing oxymetazoline-induced inhibition of PTH-stimulated cAMP production was not significantly different from its potency for the 5HT-induced effect. These data indicate that OK cells express a 5HT1 receptor that is negatively coupled to adenylate cyclase and that oxymetazoline is an agonist at these receptors.

  8. Inhibition of class IA PI3K enzymes in non-small cell lung cancer cells uncovers functional compensation among isoforms.

    PubMed

    Stamatkin, Christopher; Ratermann, Kelley L; Overley, Colleen W; Black, Esther P

    2015-01-01

    Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies while normal cell proliferation requires pathway functionality. Although inhibitors of the PI3K pathway are in clinical trials or approved for therapy, an understanding of the functional activities of pathway members in specific malignancies is needed. In lung cancers, the PI3K pathway is often aberrantly activated by mutation of genes encoding EGFR, KRAS, and PIK3CA proteins. We sought to understand whether class IA PI3K enzymes represent rational therapeutic targets in cells of non-squamous lung cancers by exploring pharmacological and genetic inhibitors of PI3K enzymes in a non-small cell lung cancer (NSCLC) cell line system. We found that class IA PI3K enzymes were expressed in all cell lines tested, but treatment of NSCLC lines with isoform-selective inhibitors (A66, TGX-221, CAL-101 and IC488743) had little effect on cell proliferation or prolonged inhibition of AKT activity. Inhibitory pharmacokinetic and pharmacodynamic responses were observed using these agents at non-isoform selective concentrations and with the pan-class I (ZSTK474) agent. Response to pharmacological inhibition suggested that PI3K isoforms may functionally compensate for one another thus limiting efficacy of single agent treatment. However, combination of ZSTK474 and an EGFR inhibitor (erlotinib) in NSCLC resistant to each single agent reduced cellular proliferation. These studies uncovered unanticipated cellular responses to PI3K isoform inhibition in NSCLC that does not correlate with PI3K mutations, suggesting that patients bearing tumors with wildtype EGFR and KRAS are unlikely to benefit from inhibitors of single isoforms but may respond to pan-isoform inhibition.

  9. Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease*

    PubMed Central

    Liu, Chang; Song, Xiaomin; Nisbet, Rebecca

    2016-01-01

    Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue. PMID:26861879

  10. Apoptosis induced by a HIPK2 full-length-specific siRNA is due to off-target effects rather than prevalence of HIPK2-Δe8 isoform.

    PubMed

    Di Rocco, Giuliana; Verdina, Alessandra; Gatti, Veronica; Virdia, Ilaria; Toietta, Gabriele; Todaro, Matilde; Stassi, Giorgio; Soddu, Silvia

    2016-01-12

    Small interfering RNAs (siRNAs) are widely used to study gene function and extensively exploited for their potential therapeutic applications. HIPK2 is an evolutionary conserved kinase that binds and phosphorylates several proteins directly or indirectly related to apoptosis. Recently, an alternatively spliced isoform skipping 81 nucleotides of exon 8 (Hipk2-Δe8) has been described. Selective depletion of Hipk2 full-length (Hipk2-FL) with a specific siRNA that spares the Hipk2-Δe8 isoform has been shown to strongly induce apoptosis, suggesting an unpredicted dominant-negative effect of Hipk2-FL over the Δe8 isoform. From this observation, we sought to take advantage and assessed the therapeutic potential of generating Hipk2 isoform unbalance in tumor-initiating cells derived from colorectal cancer patients. Strong reduction of cell viability was induced in vitro and in vivo by the originally described exon 8-specific siRNA, supporting a potential therapeutic application. However, validation analyses performed with additional exon8-specific siRNAs with different stabilities showed that all exon8-targeting siRNAs can induce comparable Hipk2 isoform unbalance but only the originally reported e8-siRNA promotes cell death. These data show that loss of viability does not depend on the prevalence of Hipk2-Δe8 isoform but it is rather due to microRNA-like off-target effects. PMID:26625198

  11. Apoptosis induced by a HIPK2 full-length-specific siRNA is due to off-target effects rather than prevalence of HIPK2-Δe8 isoform

    PubMed Central

    Di Rocco, Giuliana; Verdina, Alessandra; Gatti, Veronica; Virdia, Ilaria; Toietta, Gabriele; Todaro, Matilde; Stassi, Giorgio; Soddu, Silvia

    2016-01-01

    Small interfering RNAs (siRNAs) are widely used to study gene function and extensively exploited for their potential therapeutic applications. HIPK2 is an evolutionary conserved kinase that binds and phosphorylates several proteins directly or indirectly related to apoptosis. Recently, an alternatively spliced isoform skipping 81 nucleotides of exon 8 (Hipk2-Δe8) has been described. Selective depletion of Hipk2 full-length (Hipk2-FL) with a specific siRNA that spares the Hipk2-Δe8 isoform has been shown to strongly induce apoptosis, suggesting an unpredicted dominant-negative effect of Hipk2-FL over the Δe8 isoform. From this observation, we sought to take advantage and assessed the therapeutic potential of generating Hipk2 isoform unbalance in tumor-initiating cells derived from colorectal cancer patients. Strong reduction of cell viability was induced in vitro and in vivo by the originally described exon 8-specific siRNA, supporting a potential therapeutic application. However, validation analyses performed with additional exon8-specific siRNAs with different stabilities showed that all exon8-targeting siRNAs can induce comparable Hipk2 isoform unbalance but only the originally reported e8-siRNA promotes cell death. These data show that loss of viability does not depend on the prevalence of Hipk2-Δe8 isoform but it is rather due to microRNA-like off-target effects. PMID:26625198

  12. Isoform- and tissue-specific regulation of the Ca(2+)-sensitive transcription factor NFAT in cardiac myocytes and heart failure.

    PubMed

    Rinne, Andreas; Kapur, Nidhi; Molkentin, Jeffery D; Pogwizd, Steven M; Bers, Donald M; Banach, Kathrin; Blatter, Lothar A

    2010-06-01

    Nuclear factors of activated T cells (NFATs) are Ca(2+)-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca(2+) concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca(2+) overload, leading to spontaneous Ca(2+) waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be

  13. Protein kinase C controls activation of the DNA integrity checkpoint

    PubMed Central

    Soriano-Carot, María; Quilis, Inma; Bañó, M. Carmen; Igual, J. Carlos

    2014-01-01

    The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans. PMID:24792164

  14. Allosteric equilibrium model explains steady-state coupling of beta-adrenergic receptors to adenylate cyclase in turkey erythrocyte membranes.

    PubMed

    Ugur, O; Onaran, H O

    1997-05-01

    We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we

  15. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    SciTech Connect

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L.

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  16. Allosteric equilibrium model explains steady-state coupling of beta-adrenergic receptors to adenylate cyclase in turkey erythrocyte membranes.

    PubMed Central

    Ugur, O; Onaran, H O

    1997-01-01

    We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we

  17. Activation of phosphatidylinositol 3-kinase is required for transcriptional activity of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: assessment of the role of protein kinase B and p70 S6 kinase.

    PubMed Central

    Fernández de Mattos , S; de los Pinos E, E; Joaquin, M; Tauler, A

    2000-01-01

    Previous studies have demonstrated that the F isoform of6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase(6PF2K/Fru-2,6-BPase) is transcriptionally regulated by growth factors. The aim of this study was to investigate the importance of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway in the regulation of 6PF2K/Fru-2,6-BPase gene expression. We have completed studies using chemical inhibitors and expression vectors for the proteins involved in this signalling cascade. Treatment of cells with LY 294002, an inhibitor of PI 3-kinase, blocked the epidermal growth factor (EGF)-dependent stimulation of 6PF2K/Fru-2,6-BPase gene transcription. Transient transfection of a constitutively active PI 3-kinase was sufficient to activate transcription from the F-type 6PF2K/Fru-2,6-BPase promoter. In contrast, co-transfection with a dominant-negative form of PI 3-kinase completely abrogated the stimulation by EGF, and down-regulated the basal promoter activity. In an attempt to determine downstream proteins that lie between PI 3-kinase and 6PF2K/Fru-2,6-BPase gene expression, the overexpression of a constitutively active form of protein kinase B (PKB) was sufficient to activate 6PF2K/Fru-2,6-BPase gene expression, even in the presence of either a dominant-negative form of PI 3-kinase or LY 294002. The over-expression of p70/p85 ribosomal S6 kinase or the treatment with its inhibitor rapamycin did not affect 6PF2K/Fru-2,6-BPase transcription. We conclude that PI 3-kinase is necessary for the transcriptional activity of F-type 6PF2K/Fru-2,6-BPase, and that PKB is a downstream effector of PI 3-kinase directly involved in the regulation of 6PF2K/Fru-2,6-BPase gene expression. PMID:10861211

  18. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stres