Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.     

The effect of bromodeoxyuridine and dibutyryl cyclic AMP on the induction of placental alkaline phosphate in human choriocarcinoma cells.

The effect of 5-bromo-2'-deoxyuridine (BrdUrd) and dibutyryl cyclic AMP (Bt2cAMP) on the expression of the placental isoenzyme of human alkaline phosphatase was examined in BeWo choriocarcinoma cells. By using a combination of specific immunoprecipitation and polyacrylamide-gel electrophoresis of cells labelled either metabolically with [35S]methionine or cell-surface-labelled with 125I, both BrdUrd (5 micrograms/ml) and 1 mM-Bt2cAMP were shown to result in the enhanced accumulation of a specific protein. This protein has immunochemical identity and co-electrophoreses with placental alkaline phosphatase in two-dimensional gels. These results clearly demonstrate that the induction of placental alkaline phosphatase activity in choriocarcinoma cells treated with these agents is a consequence of the accumulation of specific enzyme protein rather than of altered catalytic activity.     

Effect of the thermostable protein kinase inhibitor on intracellular localization of the catalytic subunit of cAMP-dependent protein kinase.

cAMP-dependent protein kinase mediates a variety of cellular responses in most eukaryotic cells. Many of these responses are cytoplasmic, whereas others appear to require nuclear localization of the catalytic subunit. In order to understand further the molecular basis for subcellular localization of the catalytic subunit, the effect of the heat stable protein kinase inhibitor (PKI) was investigated. The subcellular localization of the catalytic (C) subunit was determined both in the presence and absence of PKI, by microinjecting fluorescently labeled C subunit into single living cells. When injected alone, a significant fraction of the dissociated C subunit localized to the nucleus. When coin-injected with an excess of PKI, little of the C subunit localized to the nucleus, suggesting that accumulation of catalytic subunit in the nucleus requires either enzymatic activity or a nuclear localization signal. Inactivation of the catalytic subunit in vitro by treatment with N-ethylmaleimide did not prevent localization in the nucleus, indicating that enzymatic activity was not a prerequisite for nuclear localization. In an effort to search for a specific signal that might mediate nuclear localization, a complex of the catalytic subunit with a 20-residue inhibitory peptide derived from PKI (PKI(5-24)) was microinjected. In contrast to intact PKI, the peptide was not sufficient to block nuclear accumulation. In the presence of PKI(5-24), the C subunit localized to the nucleus in a fashion analogous to that of dissociated, active C subunit despite evidence of no catalytic activity in situ. Thus, nuclear localization of the C subunit appears to be independent of enzymatic activity but most likely dependent upon a signal. The signal is apparently masked by both the regulatory subunit and PKI but not by the inhibitory peptide.     

Role of unique basic residues of human pancreatic ribonuclease in its catalysis and structural stability

Human pancreatic ribonuclease (HPR) and bovine RNase A belong to the RNase A superfamily and possess similar key structural and catalytic residues. Compared to RNase A, HPR has six extra non-catalytic basic residues and high double-stranded RNA (dsRNA) cleavage activity. We mutated four of these basic residues, K6, R32, K62, and K74 to alanine and characterized the variants for function and stability. Only the variant K74A had an altered secondary structure. Whereas R32A and K62A had full catalytic activity, the mutants K6A and K74A had reduced activity on both ssRNA and dsRNA. The mutations of K62 and K74 resulted in reduction in protein stability and DNA double helix unwinding activity of HPR; while substitutions of K6 and R32 did not affect either the stability or helix unwinding activity. The reduced catalytic and DNA melting activities of K74A mutant appear to be an outcome of its altered secondary structure. The basic residues studied here, appear to contribute to the overall stability, folding, and general catalytic activity of HPR.     

Structure-function studies of two novel UDP-GlcNAc C6 dehydratases/C4 reductases. Variation from the SYK dogma

Two subfamilies of UDP-GlcNAc C6 dehydratases were recently identified. FlaA1, a short soluble protein that exhibits a typical SYK catalytic triad, characterizes one of these subfamilies, and WbpM, a large membrane protein that harbors an altered SMK triad that was not predicted to sustain activity, represents the other subfamily. This study focuses on investigating the structure and function of these C6 dehydratases and the role of the altered triad as well as additional amino acid residues involved in catalysis. The significant activity retained by the FlaA1 Y141M triad mutant and the low activity of the WbpM M438Y mutant indicated that the methionine residue was involved in catalysis. A Glu(589) residue, which is conserved only within the large homologues, was shown to be essential for activity in WbpM. Introduction of this residue in FlaA1 enhanced the activity of the corresponding V266E mutant. Hence, this glutamate residue might be responsible for the retention of catalytic efficiency in the large homologues despite alteration of their catalytic triad. Mutations of residues specific for the short homologues (Asp(70), Asp(149)-Lys(150), Cys(103)) abolished the activity of FlaA1. Among them, C103M prevented dimerization but did not significantly affect the secondary structure. The fact that we could identify subfamily-specific residues that are essential for catalysis suggested an independent evolution for each subfamily of C6 dehydratases. Finally, the loss of activity of the FlaA1 G20A mutant provided evidence that a cofactor is involved in catalysis, and kinetic study of the FlaA1 H86A mutant revealed that this conserved histidine is involved in substrate binding. None of the mutations investigated altered the substrate, product, and function specificity of these enzymes.     

The catalytic activity of muscle phosphoglucomutase in the crystalline phase

A suspension of microcrystals of phosphoglucomutase in 60% ammonium sulfate exhibits a maximal catalytic activity in substrate-velocity studies that is about 0.2 of that obtained with the soluble enzyme under the same conditions. The apparent Michaelis constants for the reaction in the crystal phase are altered to an even smaller extent, relative to that in solution, although the parameters for the monophosphate and bisphosphate are increased more than 3 and more than 5 orders of magnitude, respectively, by the sulfate present. The compatibility of larger crystals with a reaction that constitutes part of the catalytic process also is demonstrated.     

Inactivation of lignin peroxidase by phenylhydrazine and sodium azide

Lignin peroxidase (LiP) is rapidly inactivated in a concentration-dependent manner by H2O2 and either phenylhydrazine or sodium azide. Full inactivation of isozyme 2b (H8) requires approximately 50 eq of phenylhydrazine or 80 eq of sodium azide. Anaerobic incubation of isozyme 2b with [14C]phenylhydrazine and H2O2 results in 77% loss of catalytic activity and covalent binding of 0.45 mol radiolabel/mol of enzyme. Comparable but not identical results are obtained with an isozyme mixture. A lag period is observed before the peroxidative activity can be measured when an aliquot of an incubation with sodium azide is diluted into the mixture used to assay residual catalytic activity. This lag is associated with reversible accumulation of a catalytically inert species with a Compound III-like spectrum. No meso-phenyl, iron-phenyl, or N-phenyl adducts are formed with phenylhydrazine but a low yield of what appears to be delta-meso-azidoheme is obtained with sodium azide. LiP is thus less susceptible to meso heme additions and more susceptible to oxidative heme degradation than horseradish peroxidase. The data suggest that the active of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.     

Effect of High Temperature on Plant Growth and Carbohydrate Metabolism in Potato

This study was undertaken to determine the role of sucrose-metabolizing enzymes in altered carbohydrate partitioning caused by heat stress. Potato (Solanum tuberosum L.) genotypes characterized as susceptible and tolerant to heat stress were grown at 19/17[deg]C, and a subset was transferred to 31/29[deg]C. Data were obtained for plant growth and photosynthesis. Enzyme activity was determined for sucrose-6-phosphate synthase (SPS) in mature leaves and for sucrose synthase, ADP-glucose pyrophosphorylase, and UDP-glucose pyrophosphorylase in developing tubers of plants. High temperatures reduced growth of tubers more than of shoots. Photosynthetic rates were unaffected or increased slightly at the higher temperature. Heat stress increased accumulation of foliar sucrose and decreased starch accumulation in mature leaves but did not affect glucose. SPS activity increased significantly in mature leaves of plants subjected to high temperature. Changes in SPS activity were probably not due to altered enzyme kinetics. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was reduced in tubers, albeit less quickly than leaf SPS activity. There was no interaction of temperature and genotype with regard to the enzymes examined; therefore, observed differences do not account for differences between genotypes in heat susceptibility.     

Alteration of the Bacillus subtilis glutamine synthetase results in overproduction of the enzyme.

A mutational leading to glutamine auxotrophy was located near a 5-fluorouracil resistance marker in the citB-thyA region of the Bacillus subtilis chromosome. This mutation resulted in a glutamine synthetase with altered kinetic and feedback properties. The specific activity of manganese-stimulated glutamine synthetase activity in crude extracts was 18-fold higher, and the magnesium-stimulated activity was about 30% that of the wild type. Quantitation of the enzyme by precipitation with antibody prepared against pure enzyme confirmed the presence of high enzyme levels in the mutant. This mutation is very closely linked (recombination index of 0.03) to another glutamine auxotroph containing enzyme with altered electrophoretic and heat sensitivity properties. Mutations in the structural gene for glutamine synthetase may result not only in altered catalytic and regulatory properties but also in altered production of the enzyme.     

Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase

Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 μM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.     

Complementation of soluble phosphofructokinase activity in yeast mutants.

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit. The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell-free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60-100% of that of the diploid in 15-30 min at 24 degrees C. Unlike the catalytic subunit which exists in various multimeric states in cell-free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.     

The phosphotransferase activity of casein kinase II is required for its physiological function in vivo.

We have previously shown that the inviability associated with disruption of both catalytic subunits of casein kinase II in Saccharomyces cerevisiae can be rescued by plasmids expressing the catalytic subunit of the Drosophila enzyme (Padmanabha et al., 1990, Mol. Cell. Biol. 10, 4089). Here we describe the construction of mutant forms of the Drosophila catalytic subunit in which residues known to be crucial for catalytic activity in other protein kinases have been altered by site-directed mutagenesis. Mutation of either Lys66 or Asp173, which correspond to Lys72 and Asp184 of cAMP-dependent protein kinase, respectively, yields a casein kinase II catalytic subunit which fails to rescue a yeast strain lacking both endogenous catalytic subunit genes. The data indicate that the phosphotransferase activity of casein kinase II is required for its physiological function in vivo.     

Immunological and catalytic quantitation of splenic glucocerebrosidase from the three clinical forms of Gaucher disease.

The enzymatic activity of glucocerebrosidase in splenic extracts of the adult nonneurological form of Gaucher disease (type I) was 15% +/- 7% of normal, and the titer of enzyme cross-reacting material (ECRM) in these spleens was 54% +/- 9% of normal. The titer of ECRM in splenic extracts of tissues obtained from patients with the neurological forms of Gaucher disease (types II and III) was essentially the same as in type I Gaucher spleens (59% +/- 10% of normal), but the measurable catalytic activity of glucocerebrosidase in these spleens was substantially lower than that found in type I Gaucher spleens (2.3% +/- 0.6% of normal). Thus, the attentuated glucocerebrosidase activity in spleens from all three forms of Gaucher disease appears to stem from a structurally mutated enzyme that is altered in its catalytic efficiency and possibly in its antigenic expression.     

An altered adenylate cyclase in cdc35-1 cell division cycle mutant of yeast

Adenylate cyclase activity was studied in Saccharomyces cerevisiae's cell division cycle (cdc) mutant 35-1. The temperature sensitive mutant cdc35-1 was previously mapped as an allele of cyr, the adenylate cyclase gene. However, the adenylate cyclase activities of membranes prepared from cdc35-1 were not thermosensitive. The adenylate cyclase activity of cdc35-1 was found to have an altered Mn2+ dependency and did not respond to Gpp(NH)p stimulation. These results suggest that cdc35-1 mutation may not be at the catalytic site but at a site where adenylate cyclase interacts with regulatory proteins.     

Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells. Evidence for increased degradation of catalytic subunit in the presence of cAMP

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.     

The C-terminal domain of human insulin degrading enzyme is required for dimerization and substrate recognition

Insulin degrading enzyme (IDE), a zinc metalloprotease, can specifically recognize and degrade insulin, as well as several amyloidogenic peptides such as amyloid beta (Abeta) and amylin. The disruption of IDE function in rodents leads to glucose intolerance and cerebral Abeta accumulation, hallmarks of type 2 diabetes and Alzheimer's disease, respectively. Using limited proteolysis, we found that human IDE (113kDa) can be subdivided into two roughly equal sized domains, IDE-N and IDE-C. Oligomerization plays a key role in the activity of IDE. Size-exclusion chromatography and sedimentation velocity experiments indicate that IDE-N is a monomer and IDE-C serves to oligomerize IDE-N. IDE-C alone does not have catalytic activity. It is IDE-N that contains the crucial catalytic residues, however IDE-N alone has only 2% of the catalytic activity of wild type IDE. By complexing IDE-C with IDE-N, the activity of IDE-N can be restored to approximately 30% that of wild type IDE. Fluorescence polarization assays using labeled insulin reveal that IDE-N has reduced affinity to insulin relative to wild type IDE. Together, our data reveal the modular nature of IDE. IDE-N is the catalytic domain and IDE-C facilitates substrate recognition as well as plays a key role in the oligomerization of IDE.     

The role of the STAS domain in the function and biogenesis of a sulfate transporter as probed by random mutagenesis

Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the beta-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second alpha-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasma membrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.     

Activities of enzymes of sugar metabolism in cold-stored tubers of Solanum tuberosum

Storage of tubers of Solanum tuberosum at 10° or 2° for 15 days did not alter significantly the maximum catalytic activities of sucrose phosphate synthetase, sucrose synthetase, glucose-6-phosphate dehydrogenase, aldolase, and glyceraldehydephosphate dehydrogenase. The temperature coefficients of phosphofructokinase, glyceraldehydephosphate dehydrogenase, and pyruvate kinase from the tubers were shown to be higher between 2° and 10° than between 10° and 25°. The rate of sugar accumulation at 2° exceeded the activity of sucrose synthetase but was less than that of sucrose phosphate synthetase. It is suggested that sucrose accumulation at 2° is catalysed by sucrose phosphate synthetase, is not due to changes in the maximum catalytic activities of any of the above enzymes, but may be due, in part, to the susceptibility of key glycolytic enzymes to cold.     

Effects of Y361‐auto‐phosphorylation on structural plasticity of the HIPK2 kinase domain

The dual‐specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis‐auto‐phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological‐like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyzes along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP‐competitors were performed. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different conformations of the kinase domain linked to Y361 phosphorylation. In addition, our in vitro data are consistent with both the cross‐talk between the catalytic site and the activation loop of HIPK2 and the aberrant activities and accumulation previously reported for the Y361 nonphosphorylated HIPK2 in mammalian cells.