Peptide inhibitors of cAMP-dependent protein kinase.
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Cyclic AMP↗
Biomedical subjects
H C Cheng
Publications and source records attributed to H C Cheng.
Averaged F-wave conduction velocity of peroneal nerve.
Averaged F-wave conduction velocities (a-FWCV) of peroneal nerve were measured on 38 healthy volunteers and 22 patients with clinical and electromyographic evidence of unilateral L5 or L5 + S1 radiculopathy. The peroneal nerve was stimulated supramaximally at the fibular head and the compound muscle action potential was recorded from the extensor digitorum brevis muscle. Averaged tracings from 32 consecutive stimuli were obtained. The averaged F-wave latency (Fp) and M-wave latency (Mp) were measured from the stimulating artifact to the peak of the waves. The distance (D) between the stimulating point and the T12 spinous process was measured. The value of a-FWCV was calculated as 2D/(Fp-Mp-1) m/second. In normal subjects, the average value of a-FWCV was 56 +/- 4, and there was no significant difference between males and females, nor between the right and left sides. The differences among the tests administered at different times on the same subject were also statistically insignificant. Of the 22 patients, 17 had subnormal value of a-FWCV if the lower normal limits were defined as mean minus 2.5 SD (i.e., 46 m/second) calculated from the normal control value. However, only one of them had subnormal value of FWCV calculated by the conventional method (without averaging technique). All the patients with two root (L5 + S1) involvement had subnormal a-FWCV. It is concluded that the a-FWCV is a more sensitive measure than the conventional FWCV in the assessment of L5 radiculopathy.
Adult↗
Circular dichroic investigations of secondary structure in synthetic peptide inhibitors of cAMP-dependent protein kinase: a model for inhibitory potential.
The structure of the inhibitory domain of the inhibitor protein of the cAMP-dependent protein kinase has been assessed by circular dichroism studies of synthetic inhibitory peptides. Using the inhibitory peptide PKI(5-22)amide (Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile22) [Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., & Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992] and shorter peptides of this sequence, it has been estimated that this parent peptide is composed of approximately 30% alpha-helix with the remainder being random coil with one beta-turn. The pseudosubstrate arginine cluster (Arg15-Arg19) is within the suggested region of random coil and beta-turn, representing one critical region of binding recognition by the protein kinase. The alpha-helix region proposed between Thr6 and Ile11 likewise contributes to the full biological potency and specificity of the inhibitor peptide and inhibitor protein. The removal of the two N-terminal threonines, for example, causes both a marked conformational change in the peptide and a diminishment by an order of magnitude of inhibitory activity. It is proposed that this alpha-helix region could serve one of several possibilities, including that it may provide a suitable constraint on the Tyr7 such that the hydroxyl is oriented in a position proximal to the pseudosubstrate domain, and/or may allow the optimal location of other protein kinase recognition signals. These data provide an initial description of some of the structural features of the inhibitor protein that could contribute to its high biological potency.
Amino Acid Sequence↗
Pharmacology of enoximone.
Enoximone is a new cardiotonic agent, active by both intravenous and oral routes of administration, that is being studied clinically for the treatment of patients with congestive heart failure. The animal pharmacology pertinent to the clinical development of enoximone is reviewed. Direct positive inotropic, positive chronotropic and vasodilator properties have been demonstrated for enoximone in several in vivo and in vitro preparations. However, positive inotropism and vasodilation are the principal effects of this agent with the inotropic effect being the most prominent. In anesthetized dogs, the cardiovascular effects produced by enoximone (0.1 to 1 mg/kg) were not accompanied by significant alterations in myocardial oxygen consumption. Cardiac function was improved by enoximone in anesthetized dogs given myocardial depressant amounts of propranolol. Studies in vivo and in vitro have indicated that the actions of enoximone are direct and not mediated by stimulation of adrenergic receptors, histaminic receptors, cholinergic receptors, Ca++-adenosine triphosphatase, Mg++-adenosine triphosphatase, adenyl cyclase or inhibition of Na+, K+-adenosine triphosphatase. However, enoximone reversed the depressant effects of verapamil in the dog heart-lung preparation; this suggests that its action resulted in the activation of slow calcium channels. Enoximone was found to be potent and highly selective inhibitor of a high affinity cyclic adenosine monophosphate-phosphodiesterase type IV-phosphodiesterase from dog heart, whereas standard inhibitors (e.g., 3-isobutyl-1-methylxanthine and papaverine) inhibit all 3 cardiac phosphodiesterases. Further, enoximone produced an increase in cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, in the isolated, blood perfused dog papillary muscle during the peak inotropic effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Tyrosine kinase catalyzed phosphorylation and inactivation of the inhibitor protein of the cAMP-dependent protein kinase.
The inhibitor protein of the cAMP-dependent protein kinase is a potential high affinity regulator of cAMP function. We now show that it is phosphorylated in Tyr7 by the intrinsic tyrosine kinase activity of epidermal growth factor receptor. The phosphorylated form can be readily separated from the unphosphorylated protein by high pressure liquid chromatography which has permitted the isolation of stoichiometrically phosphorylated protein. Using this method, it has been demonstrated that this phosphorylation, which occurs within the inhibitor protein's active domain, results in a 6 to 9-fold decrease in inhibitory potency. Possibly, a component of growth control could be the coupling of tyrosine kinase activity to cAMP-mediated cellular proliferation via the regulation of the efficacy of the inhibitor protein.
Animals↗
Induction of uterine leiomyomas in mice by medroxalol and prevention by propranolol.
Medroxalol hydrochloride is an antihypertensive agent with beta 1 adrenergic cardiac blocking properties, and beta 2 and some alpha 1 vasodilating activity. In previous carcinogenicity studies medroxalol was shown to induce leiomyomas of the uterus in CD-1 mice but not in Long Evans rats. In addition, there was a significant increase in endometrial stromal sarcomas in mice receiving the lowest dose of medroxalol; however, the lack of a dose response made the relationship to treatment questionable. Because of these findings, additional 18-month drug diet studies were conducted in 3 parallel segments using female CD-1 mice to determine the effects of: 1) an expanded range of doses, 2) various durations of dosing, and 3) the effect of the beta-blocker, propranolol, on leiomyoma induction. These studies confirmed the fact that chronic dietary treatment with medroxalol can lead to an increased incidence of leiomyomas in the mouse uterus, but failed to demonstrate any relationship between endometrial stromal sarcomas and medroxalol administration. A linear trend occurred in the incidence of leiomyomas and of smooth muscle hypertrophy/hyperplasia, a possible precursor to leiomyoma. Both findings were notably increased at 250 mg/kg/day or more. Doses of 50 mg/kg/day or less were considered no effect levels. At 500 mg/kg/day a treatment period of 12 months or more was required before a noticeable increase in leiomyomas occurred in mice examined after 18 months. The beta-blocker, propranolol, prevented this increase in leiomyomas, and led to the conclusion that the beta 2 agonist activity of medroxalol was involved in their induction. Propranolol did not block the spontaneous occurrence of these tumors.
Animals↗
[Radiological appearance of hemangiopericytoma].
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Adolescent↗
Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein.
Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.
Amino Acid Sequence↗
Phosphorylation of Avena phytochrome in vitro as a probe of light-induced conformational changes.
A polycation-dependent protein kinase was found to be associated with purified phytochrome preparations from etiolated Avena seedlings. This kinase and three mammalian protein kinases, the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and a Ca2+-activated phospholipid-dependent protein kinase, were used to probe light-induced conformational changes in 124-kilodalton Avena phytochrome in vitro. The red absorbing form of phytochrome (Pr) was found to be a substrate for all four protein kinases. Although the far-red absorbing form of phytochrome (Pfr) was as good a substrate as Pr with the cAMP-dependent protein kinase, the Pfr form was poorly phosphorylated by the other three protein kinases. Serine is the major amino acid residue phosphorylated on phytochrome regardless of the form of phytochrome used as substrate. Peptide mapping revealed that the sites of phosphorylation catalyzed by the cAMP-dependent protein kinase differ for Pr and Pfr forms of phytochrome. For the Pr form, the preferred site(s) of phosphorylation was near the amino terminus of the 124-kilodalton subunit. Upon photo-conversion to Pfr, this site can no longer be phosphorylated easily and a new phosphorylation site in the COOH-terminal nonchromophore domain of the molecule becomes accessible to the cAMP-dependent protein kinase. These studies of the phosphorylation of phytochrome provide a new means to study the effect of light absorption by phytochrome on the molecular conformation of the protein. The potential physiological implications of differential phosphorylation of Pr and Pfr await elucidation.
Electrophoresis, Polyacrylamide Gel↗
Cytochemical identification of the regulatory subunit of the cAMP-dependent protein kinase by use of fluorescently labeled catalytic subunit. Examination of protein kinase dissociation in hepatoma cells responding to 8-Br-cAMP stimulation.
Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization.
8-Bromo Cyclic Adenosine Monophosphate↗
A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase.
As an important new reagent for studying the cAMP-dependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein. This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr- Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by V8 protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661). Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Formula: see text), and also to a critical contribution from one or more of the 6 N-terminal residues (Formula: see text). The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of cAMP.
Amino Acid Sequence↗
Acute effect of human chorionic gonadotropin (hCG) on 17 alpha-hydroxylase and C17,20-lyase activity in neonatal or prepubertal rat testis.
In contrast to the situation in adults, desensitization of androgen production, secondary to loss of enzyme activity, was not found in testes of neonatal rats exposed to human Chorionic Gonadotropin (hCG). In the present study attention was given to the acute effects of a single injection of hCG upon the activity of testicular 17 alpha-hydroxylase, C17,20-lyase and the concentration of testosterone in the serum of 5, 10 or 28-30 day old rats was investigated. Tritiated H2O from 17 alpha-[3H]progesterone and 14CH3COOH from 21-[14C]progesterone were the products measured to evaluate hydroxylase and lyase activities respectively. Large increases in hCG in the serum were detected within 2 h of a subcutaneous injection. Testosterone, which was highest in 5 day animals, increased quickly in all animals given hCG. In 28-30-day old animals, the concentration of this steroid began to fall 24 h after injection of hCG. 17 alpha-Hydroxylase activity decreased in the testes of all animals given hCG, but only after a brief increase. Activity returned to the starting level, or above, within 24 h in 5 or 10-day old animals. In 28-30-day old rats the activity of both enzymes decreased dramatically to a nadir at 24 h, but increased thereafter. The results indicate that desensitization of testicular androgen synthesizing enzymes occurs in neonatal as well as older testes stimulated with hCG, but the desensitization was very brief in neonatal animals and no desensitization of lyase was found in 5-day old rat testes.
Aldehyde-Lyases↗
Production of leukotrienes and prostaglandins in the rat uterus during peri-implantation period.
We have measured by radioimmunoassay the production of leukotrienes (LTC4 and LTB4) and prostaglandins (PGE2 and PGF2 alpha) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC4 or LTB4 remained unaltered on days 1-3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE2 and PGF2 alpha showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: vasodilators and agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation per se.
Animals↗
An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase.
Digestion with Staphylococcus aureus V8 proteinase of the inhibitor protein of the cyclic AMP-dependent protein kinase results in the sequential formation of three active inhibitory peptides. The smallest active peptide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp . This 20-amino-acid-residue peptide has 20-40% of the activity of the native molecule and a Ki of 0.2 nM. Inhibition, as a minimum, appears to be based upon the inhibitor protein containing the recognition sequences that dictate protein-substrate-specificity. This inhibitory peptide also has sequence homology with the phosphorylation site for a protein kinase other than the cyclic AMP-dependent enzyme.
Amino Acid Sequence↗
Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase.
A limiting requirement for substrate specificity of the cAMP-dependent protein kinase is the presence of one or two basic residues located to the N-terminal side of the target substrate serine. Furthermore, circular dichroic (CD) studies have shown that binding of protein substrate involves a series of at least two independent conformational changes in the enzyme, each of which is initiated by a recognition signal on the substrate protein. The present study attempts to elucidate further the complete sequence of enzyme/ligand interactions by using the synthetic substrate peptide Kemptide and analogues differing from it at crucial points in the sequence: the Ala-peptide, where alanine is substituted for the target serine, and D-Ser-Kemptide, where the target serine is in the D rather than the L configuration. Examination of the effects of binding of these substrates on the intrinsic UV CD of the enzyme and the induced CD in the presence of Blue Dextran has revealed a third step in the substrate/enzyme binding interaction. Although sections of the conformational change at the active site are dependent on the basic subsite and the serine hydroxyl group on the peptide, respectively, the complete conformational change requires that the substrate be bound in random coil conformation. Where this does not occur, the kinetics show that the peptide will not act either as substrate or as inhibitor of the enzyme. Further, the interaction between the serine hydroxyl group and an enzyme tyrosine residue, previously observed, appears to be dependent on the correct orientation as well as the mere presence of the target -OH group.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Mouse uterine muscle as a model for studying beta 2-adrenoreceptor mediated relaxation.
Effects of isoprenaline, adrenaline and noradrenaline on contractility were studied in longitudinal strips of mouse and rat uteri in vitro. In mouse uteri, isoprenaline produced an inhibitory effect on spontaneous contractions which was antagonized by propranolol, a nonselective beta-blocker, but not by practolol, a selective beta 1-blocker. Thus, the inhibitory effect of isoprenaline is mediated by beta 2-adrenoreceptor activation. Adrenaline and noradrenaline also inhibited the spontaneous contractions of mouse uteri. In rat uteri, isoprenaline produced only an inhibitory effect whereas adrenaline initially inhibited the force and frequency of contraction but also had a secondary excitatory effect on the frequency which could be antagonized by phentolamine. Noradrenaline had an inhibitory effect on force but had only a negligible effect on frequency of rat uteri. Phenylephrine also reduced the spontaneous contractility of mouse uteri by a beta 2-mediated effect. From results generated in this study, and from the literature, it is concluded that beta 2-adrenoreceptors are predominant in nonpregnant uteri of rodents, especially the mouse, while alpha-adrenoreceptors are predominant in nonpregnant uteri of rabbit, monkey and human.
Animals↗
Prostaglandins in the uterus: modulation by steroid hormones.
Phospholipase A2 (PLA2), one of the enzymes considered to be rate-limiting in generating free arachidonic acid for prostaglandin (PG) synthesis, endogenous concentrations and in vitro production of PGs in the rat uterus were studied under various experimental conditions. Uterine PLA2 activity showed a 167-fold increase in ovariectomized rats bearing estradiol-17 beta (E2)-implants as compared to those treated with vehicle only. On the other hand, dexamethasone treatment reduced the E2-stimulable PLA2 activity by about 24-fold. The uterine PLA2 activity in the ovariectomized rat uterus was low and not altered by instillation of progesterone (P4) implants or by administration of dexamethasone. On the contrary, simultaneous placement of E2- and P4-implants prevented significantly the rise in PLA2 activity as observed under unopposed E2 exposure. Dexamethasone treatment further reduced the activity. The endogenous concentration of uterine PGF was several fold higher in the E2-implanted ovariectomized rats as compared to those without the E2-implants or carrying only P4-implants. The simultaneous treatment of the E2-implanted rats with P4 and/or dexamethasone reduced the uterine PGF concentrations considerably. The uterine PGF concentration was always lower in the ovariectomized rats under any condition if they were not treated with E2. Uterine PGE-A concentration did not change significantly between the ovariectomized rats and the ovariectomized rats carrying E2-implants. The treatment with P4 and/or dexamethasone, however, tended to decrease the PGE-A concentration. The production of PGF by the uterine homogenate increased by several fold in ovariectomized rats implanted with E2-silastic capsules as compared to those without the E2 implants. The treatments of the E2-implanted rats with P4 or dexamethasone did not alter this production. However, simultaneous exposure of E2-implanted rats to P4 and dexamethasone lowered the production rate of PGF in the uterus. The treatment of the ovariectomized rats with dexamethasone of P4 tended to elevate the uterine PGF production. The uterine PGE-A production followed more or less the same pattern. The analysis of our present data suggests that although a relationship exists between uterine PLA2 activity and PGF concentration, the role of PG synthetase could also be important in regulating PGF synthesis. Our study with dexamethasone, which showed inhibition of uterine PLA2 activity and decline in endogenous but not in vitro production of PGs, indicate that cellular integrity is essential for PLA2 to function as a rate-limiting step in PG synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
[The source, removal and inactivation of plasma 5-hydroxytryptamine].
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Animals↗