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J D Corbin

Publications and source records attributed to J D Corbin.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of the isolated cAMP-binding B domain of cAMP-dependent protein kinase.

A 14.4-kDa cAMP-binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP-dependent protein kinase type I alpha regulatory subunit (RI alpha). The full-length RI alpha from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fragment was Ser-252, indicating that it encompassed the entire predicted B domain. Although the [3H]cAMP and [3H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RI alpha, the [3H]cAMP exchange rate was comparable to that of the B domain of full-length RI alpha containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high-affinity cAMP-binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2-mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP-binding domain can function independently of any other domain structures of the R subunit.

Amino Acid Sequence↗
PubMed1994

Zinc interactions and conserved motifs of the cGMP-binding cGMP-specific phosphodiesterase suggest that it is a zinc hydrolase.

cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) binds tightly to a Zn(2+)-chelate column (Francis, S. H., and Corbin, J. D. (1988) Methods Enzymol. 159, 722-729). Using three different approaches, Zn2+ is now shown to bind to cG-BPDE, and the Kd is determined to be approximately 0.5 microM, with a binding stoichiometry of approximately 3 mol of Zn2+/mol of monomer. A similar concentration range of Zn2+ (0.05-1 microM Zn2+) also supports phosphodiesterase (PDE) catalytic activity. The Zn2+ binding to cG-BPDE is not diminished by, nor is catalysis supported by, relatively high concentrations of Cu2+, Cd2+, Ca2+, or Fe2+. Neither cGMP nor 3-isobutyl-1-methylxanthine affects Zn2+ binding under the conditions used. Mn2+, Co2+, or Mg2+ supports catalysis, but only at significantly higher concentrations (4-, 15-, and 250-fold, respectively) than that required for Zn2+. Two tandem amino acid sequences, which are conserved in the catalytic domains of all characterized mammalian PDEs, resemble the single sequence motif that has been shown to coordinate Zn2+ in the catalytic sites of Zn2+ hydrolases such as thermolysin.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone.

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Identifying protein kinases in crude extracts that phosphorylate cyclic GMP-binding cyclic-GMP specific phosphodiesterase.

The main protein kinase that phosphorylates cyclic GMP-binding cyclic GMP-specific phosphodiesterase (cG-BPDE) in crude extracts of bovine lung is cyclic GMP-dependent protein kinase. This can be shown by the use of either exogenous or endogenous cG-BPDE as substrate for endogenous cyclic GMP-dependent protein kinase. The characteristics of this phosphorylation suggest a physiological significance.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1993

Autophosphorylation: a salient feature of protein kinases.

Most protein kinases catalyze autophosphorylation, a process which is generally intramolecular and is modulated by regulatory ligands. Either serine/threonine or tyrosine serves as the phosphoacceptor, and several sites on the same kinase subunit are usually autophosphorylated. Autophosphorylation affects the functional properties of most protein kinases. Members of the protein kinase family exhibit diversity in the characteristics and functions of autophosphorylation, but certain common themes are emerging.

Animals↗
PubMed1992

A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases.

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.

Amino Acid Sequence↗
PubMed1992

Phosphorylation of smg p21B/rap1B p21 by cyclic GMP-dependent protein kinase.

smg p21B/rap1B p21, a member of ras p21-like small GTP-binding protein superfamily, has been shown to be phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). We show here that this protein was also phosphorylated by cyclic GMP-dependent protein kinase (protein kinase G) in a cell-free system. The same serine residue (Ser179) in the C-terminal region was phosphorylated by both protein kinases G and A. The Km and Vmax values of smg p21B for protein kinase G were 5 x 10(-7) M and 4 x 10(-9) mol/min/mg, and those values for protein kinase A were 1 x 10(-7) M and 3 x 10(-8) mol/min/mg.

Autoradiography↗
PubMed1992

Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries.

Elevation of either cAMP or cGMP causes smooth muscle relaxation. Whether these effects are mediated through cAMP-dependent protein kinase (cAK), cGMP-dependent protein kinase (cGK), or both is unknown. Pig coronary arteries were treated with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF), relaxants which elevate cGMP, and with isoproterenol or forskolin, relaxants which elevate cAMP. Incubation of the arteries with 10 microM SNP produced a 3.3-fold increase in cGMP without altering cAMP; the cGK activity ratio (-cGMP/+cGMP) in these extracts was increased by 2.6-fold as determined by a newly developed assay, while the cAK activity ratio (-cAMP/+cAMP) was unchanged. The increase in cGK activity ratio by SNP was concentration-dependent and was nearly maximal at 30 s. Treatment of the tissue with 10 nM ANF also increased the cGK activity ratio (2.3-fold), but not that of cAK. 100 microM isoproterenol caused a 2.9-fold elevation of cAMP with no change in cGMP, but both cAK and cGK activity ratios were increased (2.3- and 1.6-fold, respectively). The increase in the cGK activity ratio could be mimicked by cAMP addition to control tissue extracts at the concentration measured in extracts of the isoproterenol-treated tissue. Forskolin (1 and 10 microM) also increased the cGK activity ratio (1.9- and 4.9-fold). The increases in cGK activity observed in extracts suggest that moderate elevation of either cGMP or cAMP causes intracellular cGK activation, thus producing relaxation of vascular smooth muscle.

Animals↗
PubMed1992

Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues.

cAMP- and cGMP-dependent protein kinases are homologous proteins and are predicted to exhibit very similar three-dimensional structures. Their cyclic nucleotide binding domains share a high degree of amino acid sequence identity. cAMP- and cGMP-dependent protein kinases are activated relatively specifically by cAMP and cGMP, respectively; and a single alanine-threonine difference between cAMP- and cGMP-binding domains partially accounts for this specificity. Thus, it would be expected that cAMP and cGMP mediate separate physiological effects. However, owing in part to the lack of absolute specificity of either enzyme and to the relatively high level of cAMP or cGMP in certain tissues, it is also possible that either cyclic nucleotide could cross-activate the other kinase. Increases in either cAMP or cGMP cause pig coronary artery relaxation. However, only cGMP-dependent protein kinase specific cyclic nucleotide analogues are very effective in causing relaxation, and cAMP elevation in arteries treated with isoproterenol or forskolin activates cGMP-dependent protein kinase, in addition to cAMP-dependent protein kinase. Conversely, increases in either cAMP or cGMP cause Cl- secretion in T-84 colon carcinoma cells, and the cGMP level in T-84 cells can be elevated sufficiently by bacterial enterotoxin to activate cAMP-dependent protein kinase. These results imply specific regulation of cAMP- and cGMP-dependent protein kinases by the respective cyclic nucleotides, but either cyclic nucleotide is able to cross-activate the other kinase in certain tissues.

Animals↗
PubMed1992

Stimulation of intestinal Cl- transport by heat-stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP.

Heat-stable enterotoxins activate guanylate cyclase, whereas heat-labile enterotoxins stimulate adenylate cyclase. Both classes of toxins cause secretory diarrhea at least in part by stimulating Cl- secretion in the intestine. The mechanism for regulation of Cl- secretion by guanosine 3',5'-cyclic monophosphate (cGMP) was investigated using cultured T84 intestinal cells as a model for intestinal crypt cells. Escherichia coli heat-stable enterotoxin (ST) markedly stimulated cGMP production in T84 cells. Cl- secretion across T84 cell monolayers cultured on permeable filters was stimulated by E. coli ST, cholera toxin, or 8-BrcAMP, but 8-BrcGMP was ineffective. cGMP analogues that are known to be potent and specific activators of cGMP-dependent protein kinase (cG-kinase) also had little effect on 36Cl- uptake by T84 cells cultured in plastic dishes. E. coli ST, forskolin, cholera toxin, or membrane-permeant cAMP analogues markedly increased 36Cl- uptake into T84 cells. The general protein kinase inhibitor, staurosporine, inhibited the stimulation of Cl- permeability elicited by E. coli ST, vasoactive intestinal peptide (VIP), or 8-BrcAMP. DEAE-Sephacel chromatography revealed a predominant type II isoform of cAMP-dependent protein kinase (cA-kinase) in T84 cells, whereas little or no cytosolic cG-kinase activity was found. Treatment of T84 cells with E. coli ST or VIP resulted in an increase in the cA-kinase activity ratio (-cAMP/+cAMP) if the cytosolic enzyme was assayed at reduced temperature (on ice).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1992

Relaxation of pig coronary arteries by new and potent cGMP analogs that selectively activate type I alpha, compared with type I beta, cGMP-dependent protein kinase.

Smooth muscle preparations of human aorta or pig coronary arteries contain nearly equal amounts of cGMP-dependent protein kinase isozymes (cGMP kinase I alpha and I beta). In order to understand the roles of these isozymes in relaxing vascular smooth muscle, several new cGMP analogs were synthesized and tested for potencies in activating each enzyme and in relaxing pig coronary arteries. Analogs modified with a derivatized phenylthio group at the 8-position were as much as 72-fold more potent in activating purified cGMP kinase I alpha than cGMP kinase I beta. Electron-donating substituents, such as hydroxy, amino, and methoxy, on the phenyl ring enhanced the potencies of these analogs in activating cGMP kinase I alpha. The most potent of these cGMP analogs [8-(4-hydroxyphenylthio)-cGMP] was 17 times more potent (EC50 = 1.1 microM) as a muscle relaxant than the most efficacious analog tested previously. Among derivatives with an 8-halo group, 8-iodo-cGMP was the most potent compound (Ka = 9 nM for I alpha and 122 nM for I beta) for both I alpha and I beta. Analogs modified at the 1,N2-position or at both the 1,N2-and 8-positions of cGMP were highly potent for activating both isozymes. Within this group, 8-I-beta-phenyl-1,N2-etheno-cGMP had Ka values of 22 nM and 17 nM for cGMP kinase I alpha and I beta, respectively, whereas the Ka values of cGMP were 110 nM and 250 nM for the two isozymes. 8-I-beta-phenyl-1,N2-etheno-cGMP was the most potent muscle relaxant tested, with EC50 of 0.4 microM. For all cGMP analogs tested, there was a positive correlation between potency for activation of cGMP kinase I alpha and that for relaxation of pig coronary arteries. Assuming that the kinase assay conditions yielded a cyclic nucleotide specificity similar to that which would exist in intact cells, it was concluded that the cGMP kinase I alpha isozyme mediates the relaxation of pig coronary artery smooth muscle caused by cGMP elevation. However, an additional role for cGMP kinase I beta in the relaxation process could not be ruled out.

Animals↗
PubMed1991

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.

Amino Acid Sequence↗
PubMed1991

Effect of epinephrine or cAMP on cAMP-bound protein kinase holoenzymes in rat heart.

Nonperfused or epinephrine-perfused rat hearts were used to examine the relative amounts of adenosine 3',5'-cyclic monophosphate (cAMP)-free and cAMP-bound holoenzymes of type II cAMP-dependent protein kinase (cAK). Crude tissue extracts of nonperfused hearts were chromatographed in the absence or presence of [3H]cAMP using DEAE-high-performance liquid chromatography. A partially resolved cAMP-free peak of cAK eluted at 0.17 M NaCl, and an asymmetric peak containing bound [3H]cAMP eluted at a slightly higher NaCl concentration. The first peak contained a tetrameric holoenzyme [2 regulatory (R) subunits and 2 catalytic (C) subunits]. From analysis of R-to-C ratios, the [3H]cAMP-bound peak contained a mixture of tetrameric and trimeric (R2C) forms. Both cAMP-free and cAMP-bound holoenzyme forms were virtually inactive without added cAMP under the conditions used. [3H]cAMP dissociation rate studies revealed that the bound cAMP in the peak fraction was equally distributed in the two different binding sites of the enzyme. Compared with the cAMP-free form, the cAMP-bound enzyme in the peak fraction exhibited enhanced binding in nonequilibrium [3H]cAMP binding assays. The cAMP-bound holoenzymes were estimated to represent at least 64% of the total type II cAK in control extracts, and the cAMP-free form was largely converted to the cAMP-bound forms by perfusing hearts with epinephrine.

Animals↗