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1.
Genomics ; 78(3): 124-8, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11735218

ABSTRACT

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.


Subject(s)
GTP-Binding Proteins/metabolism , Receptors, Cell Surface/genetics , Receptors, G-Protein-Coupled , Receptors, Purinergic P2 , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Cloning, Molecular , Humans , Mice , Molecular Sequence Data , Open Reading Frames , Rats , Receptors, Cell Surface/agonists , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Receptors, Purinergic P2Y , Sequence Homology, Amino Acid , Uridine Diphosphate Glucose/pharmacology
2.
J Mol Cell Cardiol ; 33(9): 1589-606, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11549339

ABSTRACT

Sphingosine 1 phosphate (S1P), an aminophospholipid, acts extracellularly as a ligand via the specific G protein-coupled receptors of the endothelial differentiation gene (EDG) 1, 3, 5, 6 and 8 receptors family and intracellularly as a second messenger in various cellular types. The aim of this work was to investigate biological activity of S1P in cardiomyocytes with respect to related sphingolipids. S1P was applied for 48 h on rat neonatal cardiomyocytes at 10 nM, 100 nM and 1 microM. S1P induced a concentration-dependent cellular hypertrophy evidenced by an increase in cell size, [3H]-phenylalanine incorporation, protein content and Brain Natriuretic Peptide (BNP) secretion. Among the lipids tested S1P exhibits the lower EC50 (67 nM) followed by dihydro-S1P (107 nM) and sphingosylphosphorylcholine (1.6 microM). The effect of S1P could be related to a stimulation of the EDG1 receptor since we showed that the EDG1 receptor is predominantly expressed at the mRNA and protein levels in rat cardiomyocytes and that specific anti-EDG1 antibodies inhibited the hypertrophic effect induced by S1P. Furthermore the expression level of most other EDG receptors for S1P appeared very low in cardiac myocytes. S1P (100 nM) increased the phosphorylation of p42/44MAPK, p38MAPK, JNK, Akt and p70(S6K), this effect being reversed by inhibitors of their respective phosphorylation which also rescue the hypertrophic phenotype. Finally, S1P stimulated actin stress fibre formation reverted by the Rho inhibitor, the C3 exoenzyme. Altogether, our results show that S1P induces cardiomyocyte hypertrophy mainly via the EDG1 receptor and subsequently via Gi through ERKs, p38 MAPK, JNK, PI3K and via Rho pathway.


Subject(s)
Cardiomegaly/pathology , Heart/drug effects , Immediate-Early Proteins/metabolism , Lysophospholipids , Receptors, G-Protein-Coupled , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Adrenergic alpha-Agonists/pharmacology , Animals , Animals, Newborn , Cardiomegaly/chemically induced , Cardiomegaly/metabolism , Cells, Cultured , Culture Media, Serum-Free , Dose-Response Relationship, Drug , Humans , Microscopy, Fluorescence , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Myocardium/cytology , Myocardium/metabolism , Peptides/immunology , Peptides/metabolism , Phenylephrine/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Structure, Tertiary , Rats , Rats, Wistar , Receptors, Cell Surface/metabolism , Receptors, Lysophospholipid , Sphingosine/chemistry , Sphingosine/metabolism , Stress Fibers/metabolism , Tissue Extracts/chemistry , Virulence Factors, Bordetella/pharmacology
3.
Yeast ; 18(8): 723-8, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11378899

ABSTRACT

Initial steps in investigating gene function often include deleting and overexpressing the gene of interest and identifying the subcellular location of the gene product. To facilitate these procedures, a number of new PCR modules, which contain selectable markers and in some cases other genetic elements (e.g promoter elements, epitope tags, and reporter genes) have been developed. These modules are used as PCR substrates to create products that can be targeted to specified locations in the yeast genome, thus modifying that genomic locus. We describe here a series of plasmids that contain a truncated version of the strong ADH1 promoter with and without amino-terminal 3HA and GST tags. Because these plasmids contain the same vector sequences as the GAL1 promoter plasmids, a constitutive and an inducible promoter can now be integrated with a minimal number of primers.


Subject(s)
Genes, Fungal , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae/genetics , Gene Expression , Plasmids , Polymerase Chain Reaction
4.
Biotechniques ; 30(3): 520-3, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11252788

ABSTRACT

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).


Subject(s)
Cloning, Molecular , Oligonucleotides/metabolism , Polymerase Chain Reaction , Recombination, Genetic , DNA, Complementary/metabolism
5.
Eur J Biochem ; 267(18): 5699-710, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10971580

ABSTRACT

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.


Subject(s)
Heat-Shock Proteins , Periplasmic Proteins , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Alanine/chemistry , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Base Sequence , Binding Sites , Blotting, Northern , Blotting, Western , COS Cells , Carrier Proteins/chemistry , Carrier Proteins/genetics , Caseins/metabolism , Cell Line , Cell Nucleus/metabolism , Chromatography, High Pressure Liquid , Cloning, Molecular , Endoplasmic Reticulum/metabolism , Escherichia coli/genetics , Fibroblasts/metabolism , High-Temperature Requirement A Serine Peptidase 1 , High-Temperature Requirement A Serine Peptidase 2 , Hot Temperature , Humans , Membrane Proteins/genetics , Mice , Microscopy, Fluorescence , Mitochondrial Proteins , Molecular Sequence Data , Mutagenesis, Site-Directed , Presenilin-1 , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Serine/chemistry , Serine Endopeptidases/biosynthesis , Subcellular Fractions/metabolism , Temperature , Time Factors , Tissue Distribution , Tunicamycin/pharmacology , Two-Hybrid System Techniques , Up-Regulation
6.
Biochem J ; 348 Pt 2: 263-72, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10816418

ABSTRACT

The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells. Both forms of recombinant Yak1p kinase were catalytically active and preferred myelin basic protein (MBP) as a substrate over several other proteins. Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164. Peptides containing each site were obtained and tested as Yak1p substrates. Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP. The maximal velocity (V(max)) values of the full-length Yak1p reaction were 110+/-21 (Ser-164) and 8.7+/-1.7 (MBP), and those of N-terminally truncated Yak1p were 560.7+/-74.8 (Ser-164) and 34. 4+/-2.2 (MBP) pmol/min per mg of protein. Although neither form of Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro. Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity. Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest that Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.


Subject(s)
Myelin Basic Protein/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Animals , Cattle , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Intracellular Signaling Peptides and Proteins , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Myelin Basic Protein/chemistry , Peptide Fragments/chemistry , Phosphorylation , Phosphoserine/metabolism , Phosphotyrosine/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Tyrosine Phosphatases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Sequence Deletion , Substrate Specificity
7.
J Biol Chem ; 275(30): 23319-25, 2000 Jul 28.
Article in English | MEDLINE | ID: mdl-10770955

ABSTRACT

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D)

Subject(s)
Membrane Glycoproteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis Regulatory Proteins , Base Sequence , CHO Cells , Calorimetry , Cricetinae , DNA Primers , Humans , Membrane Glycoproteins/genetics , Pichia/genetics , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TNF-Related Apoptosis-Inducing Ligand , Temperature , Tumor Necrosis Factor-alpha/genetics
8.
J Biol Chem ; 275(15): 10767-71, 2000 Apr 14.
Article in English | MEDLINE | ID: mdl-10753868

ABSTRACT

Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.


Subject(s)
GTP-Binding Proteins/physiology , Receptors, Cell Surface/physiology , Uridine Diphosphate Glucose/physiology , Humans , Phylogeny , Radioligand Assay , Receptors, Cell Surface/analysis
9.
J Biol Chem ; 273(25): 15605-10, 1998 Jun 19.
Article in English | MEDLINE | ID: mdl-9624152

ABSTRACT

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Mitogen-Activated Protein Kinases , Protein Kinase Inhibitors , Pyridines/pharmacology , Amino Acid Substitution , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Crystallography, X-Ray , HeLa Cells , Histidine/metabolism , Humans , JNK Mitogen-Activated Protein Kinases , Methionine/metabolism , Mitogen-Activated Protein Kinase 12 , Mitogen-Activated Protein Kinase 13 , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Protein Kinases/genetics , Structure-Activity Relationship , Threonine/metabolism , p38 Mitogen-Activated Protein Kinases
10.
Mol Pharmacol ; 51(6): 999-1006, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9187266

ABSTRACT

To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and 3.9 +/- 0.9 and 0.5 +/- 0.1 nM, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201-886. In contrast, the IC50 values for cAMP competition with either [3H]-(R)-rolipram or [3H]RP 73401 binding increased approximately 2-fold in H505N and approximately 13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in the K(m) value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4-selective inhibitors.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases , Histidine/metabolism , Isoenzymes/metabolism , Phosphodiesterase Inhibitors/metabolism , Phosphoric Diester Hydrolases/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Binding Sites , Catalysis , Cyclic Nucleotide Phosphodiesterases, Type 4 , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphodiesterase Inhibitors/pharmacology , Protein Binding , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
11.
J Biol Chem ; 272(18): 12116-21, 1997 May 02.
Article in English | MEDLINE | ID: mdl-9115281

ABSTRACT

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/metabolism , Imidazoles/metabolism , Mitogen-Activated Protein Kinases , Pyridines/metabolism , Adenosine/analogs & derivatives , Adenosine/pharmacology , Affinity Labels , Amino Acid Sequence , Binding Sites , Binding, Competitive , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/isolation & purification , Calorimetry , Cloning, Molecular , Cross-Linking Reagents , Enzyme Inhibitors/pharmacology , Escherichia coli , HeLa Cells , Humans , Imidazoles/pharmacology , Kinetics , Mutagenesis, Site-Directed , Pyridines/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae , p38 Mitogen-Activated Protein Kinases
12.
Mol Pharmacol ; 50(4): 891-9, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8863835

ABSTRACT

To identify functional domains of the 886-amino acid human recombinant cAMP-specific phosphodiesterase (PDE) subtype A (rhPDE4A), we engineered the expression of seven mutant proteins containing both NH2- and COOH-terminal truncations. The level of rhPDE4A protein expression in yeast was monitored by immunoblotting using enzyme-specific antisera. Biochemical profiles of the mutant proteins were compared with those of the full-length protein or a fully active truncated form of the enzyme (rhPDE4A Met265-886), lacking the first 264 amino acids. The smallest catalytically active fragment generated was Met332-722, which at 45 kDa is less than half the mass of the full-length enzyme (approximately 110 kDa) but spans the most highly conserved region of the PDE superfamily. Two prototypical PDE4 inhibitors, rolipram and RP 73401, inhibited cAMP hydrolyzing activity of all truncated forms of the enzyme, with IC50 values of 70-2000 nM and 0.2-0.6 nM, respectively. [3H](R)-Rolipram bound to two sites on Met265-886, a high affinity site (Kd1 = 0.7 +/- 0.3 nM) and a low affinity site (Kd2 = 34 +/- 10 nM). Interestingly, [3H](R)-rolipram failed to bind to Met332-886 with high affinity, indicating that high affinity binding is not required for inhibition of enzyme activity. Low affinity rolipram binding was still present in Met332-886 (Kd = 101 +/- 7 nM). In contrast to [3H](R)-rolipram, [3H]RP 73401 bound to a single class of high affinity sites on Met265-886 (Kd = 0.4 +/- 0.1 nM). Further truncation of the enzyme to Met332-886 had no effect on [3H]RP 73401 binding (Kd = 0.2 +/- 0.03 nM). We conclude that the catalytic center of rhPDE4A lies between amino acids 332 and 722. Furthermore, amino acids 265-332 may form a high affinity binding site for rolipram that is outside of the catalytic domain. As a more likely alternative, these amino acids may not form a distinct binding site but instead may be required for the recombinant enzyme to assume a conformation that binds rolipram at the catalytic domain with a high affinity.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases , Phosphodiesterase Inhibitors/metabolism , Phosphoric Diester Hydrolases/analysis , Phosphoric Diester Hydrolases/metabolism , Protein Structure, Tertiary , Pyrrolidinones/metabolism , Base Sequence , Benzamides/metabolism , Benzamides/pharmacology , Binding Sites , Binding, Competitive , Catalysis , Cyclic Nucleotide Phosphodiesterases, Type 4 , Kinetics , Molecular Sequence Data , Peptide Mapping , Phosphodiesterase Inhibitors/pharmacology , Pyridines/metabolism , Pyridines/pharmacology , Pyrrolidinones/pharmacology , Recombinant Proteins/metabolism , Rolipram , Structure-Activity Relationship , Tritium
13.
Blood ; 88(4): 1321-9, 1996 Aug 15.
Article in English | MEDLINE | ID: mdl-8695850

ABSTRACT

Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/genetics , Blood Platelets/enzymology , 3',5'-Cyclic-AMP Phosphodiesterases/blood , 3',5'-Cyclic-AMP Phosphodiesterases/chemistry , Amino Acid Sequence , Base Sequence , Cells, Cultured , Cloning, Molecular , Cyclic GMP/pharmacology , DNA Primers/chemistry , DNA, Complementary/genetics , Gene Expression , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphodiesterase Inhibitors/pharmacology , RNA, Messenger/genetics , Recombinant Proteins , Saccharomyces cerevisiae , Sequence Deletion , Structure-Activity Relationship
14.
Gene ; 172(1): 143-7, 1996 Jun 12.
Article in English | MEDLINE | ID: mdl-8654975

ABSTRACT

The TOR genes were first identified in Saccharomyces cerevisiae by the isolation of mutants which exhibit dominant resistance to the immunosuppressive and antifungal drug rapamycin (Rm). The originally characterized Rm-resistant (RmR) TOR1-1 and TOR2-1 alleles contain an Arg in place of a conserved Ser residue, which lies adjacent to the phosphatidylinositol (PI) kinase-related domain of TOR (Ser1972 in TOR1; Ser1975 in TOR2). Additional spontaneous RmR mutants containing Lys, Ile or Asn substitutions were subsequently isolated. As this Ser is a potential site for protein kinase C phosphorylation, we were interested in determining whether the observed RmR is due to steric hindrance of the FKBP12-Rm-TOR interaction or whether phosphorylation at this site is required to mediate the interaction. Using site-directed mutagenesis, we replaced the Ser1972 residue of TOR1 with either a conservative residue, Ala, an alternative potential phosphorylation site, Thr, or Asp to mimic phosphorylation. The TOR1 (S1972A) mutant protein retained Rm sensitivity (RmS), whereas both the Thr and Asp substitutions conferred RmR. RmS correlated with the ability to interact with FKBP12-Rm in a two-hybrid assay: both wild-type TOR1 and the S1972A mutant retained the ability to interact with FKBP12-Rm, whereas the S1972T, S1972D and S1972R mutants failed to interact. All mutant TOR1 proteins were able to complement the growth defect of tor1 null alleles, suggesting that the Ser1972 residue may not be required for TOR1 function in cycling cells. Since a TOR1(S1972A) mutant protein confers a RmS phenotype, interacts with FKBP12-Rm in a two-hybrid assay, and functions in vivo, we conclude that phosphorylation at Ser1972 is not necessary for the interaction between TOR1 and FKBP12-Rm.


Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Heat-Shock Proteins/metabolism , Mutation , Phosphatidylinositol 3-Kinases , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Polyenes/metabolism , Saccharomyces cerevisiae Proteins , Base Sequence , Binding Sites , DNA Primers , Fungal Proteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/genetics , Sirolimus , Tacrolimus Binding Proteins
15.
Curr Genet ; 29(5): 441-5, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8625423

ABSTRACT

The primary step in the aromatic amino-acid biosynthetic pathway in Saccharomyces cerevisiae is catalyzed by two redundant isozymes of 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase, either of which alone is sufficient to permit growth on synthetic complete media lacking aromatic acids (SC-Aro). The activity of one isozyme (encoded by the ARO3 gene) is feedback-inhibited by phenylalanine, whereas the activity of the other isozyme (encoded by the ARO4 gene) is feedback-inhibited by tyrosine. Transcription of both genes is controlled by GCN4. We previously cloned the ARO3 gene from the opportunistic pathogen Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine; and (2) its expression is induced in response to amino-acid deprivation, consistent with the presence of two putative GCN4-responsive promoter elements (Pereira and Livi 1993, 1995). To determine whether other DAHP synthases exist in C. albicans, we have constructed a homozygous aro3-deletion mutant strain. Such a mutant was found to be phenotypically Aro+, i. e., capable of normal growth on SC-Aro media, suggesting the presence of at least one additional isozyme. To confirm this result, a 222-bp DNA fragment was amplified by the polymerase chain reaction (PCR) from genomic DNA prepared from the homozygous aro3-deletion mutant, using a degenerate primer based on a conserved N-terminal region of Aro3p plus a degenerate comeback primer encoding a conserved region of the protein that lies within the deleted portion of the gene. The nucleotide sequence of this PCR fragment predicts a 74-amino acid DAHP synthase-related protein which shows strong homology to Aro3p from S. cerevisiae and C. albicans, but even greater homology (78% identity) to S. cerevisiae Aro4p. We conclude that cells of C. albicans contain a second Aro4p-related DAHP synthase.


Subject(s)
3-Deoxy-7-Phosphoheptulonate Synthase/genetics , Amino Acids/biosynthesis , Candida albicans/genetics , Genes, Fungal , Isoenzymes/genetics , Amino Acid Sequence , Base Sequence , Candida albicans/enzymology , Conserved Sequence , Molecular Sequence Data , Mutagenesis , Polymerase Chain Reaction , Sequence Deletion , Sequence Homology, Amino Acid
16.
J Biol Chem ; 271(14): 8488-92, 1996 Apr 05.
Article in English | MEDLINE | ID: mdl-8626550

ABSTRACT

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Chlorocebus aethiops , Cloning, Molecular , HeLa Cells , Heat-Shock Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , p38 Mitogen-Activated Protein Kinases
17.
J Pharmacol Exp Ther ; 276(2): 810-8, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8632354

ABSTRACT

Human peripheral blood monocytes were treated for 4 h with a combination of the beta-agonist salbutamol (3 microM) and the low-Km cAMP-specific phosphodiesterase (PDE4) inhibitor rolipram (30 microM) to produce a prolonged elevation of cAMP and consequent increase in PDE activity. After this treatment, isozyme-selective PDE inhibitors were used to characterize the cAMP PDE profiles of high-speed supernatants before and after DEAE-Sepharose column chromatography. These experiments, in which total soluble PDE activity was increased by 58%, showed that the increased PDE activity is due to up-regulation of PDE4 and that at least two of the four subtypes are up-regulated. Experiments in whole cells demonstrated that this relatively modest increase in PDE4 activity has significant functional consequences, reducing cAMP accumulation in response to both PGE2 and lower, though not maximal, concentrations of rolipram. Further characterization of PDE4 subtype expression in control and treated monocytes, using polymerase chain reaction and Western blotting with subtype-specific peptide antibodies, showed that resting monocytes express both mRNA and protein for PDE4A, PDE4B and PDE4D. The amount of message for PDE4A and PDE4B appeared to increase upon up-regulation, whereas mRNA for PDE4D was not detected in treated cells. Western blots showed increases in the amount of protein for both PDE4A and PDE4B after treatment. We conclude that the PDE4 subtypes are differentially regulated upon prolonged exposure to elevated cAMP, with the consequence that the PDE4 profiles of control and treated cells differ not only in total activity but also in the relative proportions of the subtypes represented.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/biosynthesis , Isoenzymes/biosynthesis , Monocytes/enzymology , RNA, Messenger/biosynthesis , Receptors, Adrenergic, beta/physiology , 3',5'-Cyclic-AMP Phosphodiesterases/genetics , Base Sequence , Cells, Cultured , Cyclic AMP/physiology , Gene Expression , Humans , Isoenzymes/genetics , Molecular Sequence Data , Up-Regulation
18.
J Biol Chem ; 270(49): 29043-6, 1995 Dec 08.
Article in English | MEDLINE | ID: mdl-7493921

ABSTRACT

CSBP1 and CSBP2 are human homologues of the Saccharomyces cerevisiae Hog1 mitogen-activated protein kinase which is required for growth in high osmolarity media. Expression of CSBP1, but not CSBP2, complemented a hog1 delta phenotype. A CSBP2 mutant (A34V) that complements hog1 delta was isolated and found to have approximately 3-fold lower kinase activity than the wild-type CSBP2. Further analysis revealed that both the kinase activity and tyrosine phosphorylation of CSBP1 and CSBP2 (A34V) is regulated by salt. In contrast, wild-type CSBP2 is constitutively active but dependent on the upstream kinase, Pbs2. Mutagenesis studies showed that reduction or elimination of CSBP2 kinase activity restores salt responsiveness as measured by tyrosine phosphorylation suggesting that too high a level of kinase activity can result in desensitization of the host cell and inability to grow in high salt.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/physiology , Mitogen-Activated Protein Kinases , Saccharomyces cerevisiae Proteins , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Enzyme Activation , Humans , Molecular Sequence Data , Mutation , Phenotype
19.
Cell Biol Int ; 19(6): 477-84, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7640661

ABSTRACT

Recombinant baculoviruses were constructed to express cDNAs encoding two distinct subtypes of human cAMP-specific phosphodiesterase (hPDE4A and hPDE4B). Infection of Spodoptera frugiperda insect cells with the appropriate recombinant baculoviruses resulted in high level production of biologically-active protein as measured by enzymatic activity and immunoblotting using subtype-specific anti-hPDE4 antisera. Both recombinant proteins showed catalytic activity with a low Km (approximately 3 microM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent Kis of 0.38 and 0.25 microM, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (Kd approximately 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/biosynthesis , Baculoviridae/genetics , Isoenzymes/biosynthesis , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 3',5'-Cyclic-AMP Phosphodiesterases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Enzyme Activation , Humans , Isoenzymes/genetics , Kinetics , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Spodoptera/enzymology , Spodoptera/virology
20.
Mol Microbiol ; 15(1): 39-54, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7752895

ABSTRACT

Transfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMI 1640 (RPMI) at 37 degrees C and 5% CO2 causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal-specific genes as well as genes expressed in response to changes in temperature, CO2 and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)- and hyphal (RPMI)-specific probes resulting in identification of a gene expressed in response to culture conditions but not regulated by the yeast-hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiae alpha-agglutinin, encoded by AG alpha 1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C. albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for agglutinin-like sequence). While the N- and C-termini of the predicted 1260-amino-acid ALS1 protein resemble those of the 650-amino-acid AG alpha 1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36-amino-acid sequence not present in AG alpha 1. These repeats are also present on the nucleotide level as a highly conserved 108 bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and, in some cases, between ALS1 alleles in the same strain, suggesting a strain-dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.


Subject(s)
Agglutinins/genetics , Candida albicans/genetics , Fungal Proteins/genetics , Genes, Fungal/genetics , Peptides/genetics , Agglutinins/chemistry , Amino Acid Sequence , Base Sequence , Blotting, Southern , Candida albicans/chemistry , Cloning, Molecular , Fungal Proteins/chemistry , Mating Factor , Molecular Sequence Data , Peptides/chemistry , Repetitive Sequences, Nucleic Acid/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Sequence Alignment
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