Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of Torpedo californica and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the gamma and delta subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the gamma and delta subunits of the receptor. cAMP activation of the endogenous phosphorylation of the gamma and delta subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from T. californica and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the gamma and delta subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of (32)P per mol of receptor, respectively. The initial rates of phosphorylation of the gamma and delta subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the gamma and delta subunits had K(m) values of 4.0 and 3.3 muM and V(max) values of 2.7 and 2.1 mumol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated in vivo by a cAMP-dependent protein kinase.
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PMID:cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. 630 72

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.
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PMID:Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase. 630 13

Calmodulin level and cAMP-dependent protein kinase activity of ram germ cells at different stages of spermatogenesis have been determined. Calmodulin levels decrease during maturation. Simultaneously, calmodulin localization changes during cell differentiation. In round, elongating, and elongated spermatids, calmodulin is closely associated with the developing acrosome; in spermatozoa, it becomes present in the postacrosome, the neck region and the tail. Protein kinase activity is relatively low in testicular cells but increases dramatically during epididymal maturation of spermatozoa. A concerted regulation by cAMP and Ca2+ of biochemical events in spermatogenic cells and spermatozoa is suggested.
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PMID:Evolution of Ca2+- and cAMP-dependent regulatory mechanisms during ram spermatogenesis. 631 46

Protein kinase activity that is dependent on 3',5'-Cyclic adenosine monophosphate (cAMP-PK), [3H]cAMP binding, and cAMP-dependent protein phosphorylation were identified and partially characterized in cytosolic preparations of rat lung from day 18 of gestation to adulthood. Major cAMP-dependent phosphoproteins in lung preparations were compared to those in cytosol from purified Type II epithelial cells. Both Type I and Type II regulatory subunits of cAMP-PK were identified in fetal and adult lung. Inhibition of specific [3H]cAMP binding to lung cytosol (to the regulatory subunit of the cAMP-dependent protein kinase) followed the order of potency: cAMP greater than cGMP; adenosine, ADP, and ATP were inactive. Scatchard plots of saturation experiments with [3H]cAMP and lung cytosol were linear. Dissociation constant (KD) for cAMP binding was approximately 2-3 nM, and did not change significantly with age. In contrast, binding capacity varied significantly during development and age-related changes in binding capacity were associated with similar changes in cAMP-dependent histone kinase activity. Both [3H]cAMP binding and cAMP-dependent protein kinase activity decreased slightly before birth, reached maximal activity during the suckling period, and decreased in adulthood. cAMP enhanced histone kinase activity in rat lung cytosol at all ages studied, from day 18 of gestation to adulthood. cAMP also specifically enhanced phosphorylation of several endogenous cytosolic proteins that were identified by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major proteins whose phosphorylation was selectively enhanced by cAMP or inhibited by protein kinase inhibitor were approximately Mr = 260,000, 240,000, 97,000, 56,000, 44,000, and 28,000.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:cAMP-dependent protein kinase and protein phosphorylation in developing rat lung. 631 84

Studies were conducted to evaluate the ontogeny of cAMP-dependent protein kinases in the soluble fraction of rat ovaries obtained throughout prepubertal ovarian maturation (days 5-37). Protein kinase activity stimulated by cAMP and inhibited by the heat-stable protein kinase inhibitor, designated cAMP-dependent protein kinase activity, was already present in ovaries of 5-day-old rats. This kinase activity subsequently increased to reach maximal values between days 16 and 18, then plunged 4-fold to a nadir between days 21 and 23. cAMP-dependent protein kinase activity began to rise again by day 24, increasing 5-fold to a maximum on day 27. Experiments were conducted to evaluate the basis for the 80% reduction of cAMP-dependent protein kinase activity in the soluble extract of ovaries of 21- to 23-day-old rats. To determine whether the decrease in kinase activity was accompanied by a concomitant reduction of cAMP-binding activity, the ability of ovarian cytosol to bind [3H]cAMP was evaluated. Results showed that at the time of decreased cAMP-dependent protein kinase activity, total soluble cAMP-binding activity was not significantly reduced. Additionally, the cAMP-binding activity of the regulatory subunits RI and RII (regulatory subunits of the type I and II isoenzyme forms of cAMP-dependent protein kinase), as detected by photoaffinity labeling with 8-N3-[32P] cAMP, was not changed. To determine whether the decline in kinase activity was due to an actual disappearance of the catalytic subunit from the soluble ovarian extracts of ovaries of 21- to 23-day-old rats, the relative amounts of catalytic subunit were quantified by an enzyme-linked immunosorbent assay using an antiserum directed against bovine heart catalytic subunit. Results showed that the decrease in protein kinase catalytic activity was not due to a reduction in the amount of catalytic subunits. Experiments were conducted to determine whether the reduction of protein kinase catalytic activity in unfractionated cytosol was expressed after anion exchange chromatography. Results showed that the estimated total cAMP-stimulated protein kinase activity measured after DEAE-cellulose chromatography of the soluble ovarian extracts of 21- to 23-day-old rats was no longer depressed. Since the 80% reduction of catalytic kinase activity in ovarian extracts of 21- to 23-day-old rats was detectable in unfractionated cytosol before but not after DEAE-cellulose chromatography, we tested for the presence of an endogenous inhibitor of catalytic kinase activity in the cytosol fraction.
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PMID:Modulation of soluble ovarian adenosine 3',5'-monophosphate-dependent protein kinase activity during prepubertal development of the rat. 632 53

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanistic studies of cAMP-dependent protein kinase action. 636 50

Protein kinase N (PKN) is a serine/threonine protein kinase rapidly activated by nerve growth factor (NGF) and other agents in various cell lines. The possible involvement of PKN in the multiple pathways of the NGF mechanism of action was previously established through the use of purine analogs, some of which are apparently specific inhibitors of this kinase. Since a PKN-like activity is modulated in several cell lines by cAMP analogs and this activation requires the activity of cAMP-dependent protein kinase, the aim of the present work is to investigate possible interactions between PKN and C-PKA. Pre-incubation of the two kinases in the presence of ATP leads to potentiated phosphorylation of histone HF1, Kemptide (a substrate for C-PKA, but not for PKN), and several additional substrates. This augmented phosphorylating activity is insensitive to 6-thioguanine (an inhibitor for PKN, but not for C-PKA) and is suppressed both by the Walsh inhibitor and by the regulatory subunit of PKA. PKN-pretreated C-PKA shows a significant decrease in Km for Kemptide and a substantial increase in Vmax. C-PKA and PKN are widely expressed enzymes and the possibility of PKN-dependent modulation of PKA in intact cells would therefore have biological implications for signal transduction mechanisms.
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PMID:Nerve growth factor-activated protein kinase N modulates the cAMP-dependent protein kinase. 771 18

1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography. 2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin-dependent protein kinase and casein kinases. 3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis. 4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca2+/calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).
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PMID:Rat liver endoplasmic reticulum protein kinases. 818 36

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.
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PMID:Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway. 863 82

Protein kinases help regulate eukaryotic cell division. We investigated the regulation of cAMP-dependent protein kinase A (PKA) and casein kinase (CK) type I activity in normal cells and in cancer. To assess this activity in biopsies we suggest a new parameter--the ratio of CK activity and total PKA activity divided by cAMP concentration: CK/PKA/cAMP. In 98 samples of colon mucosa in normal, inflamed, polyp, and adenocarcinoma cells, we found this parameter to be fairly constant in normal conditions and increased 10-fold in colon cancer; the ratio does not depend on the place of biopsy or the patient's age or sex. Experiments with model systems of concanavalin A-stimulated lymphocytes and regenerating rat liver showed that in normal cell proliferation the parameter increases 2-3-fold, as compared to a 30-fold increase in cancer. Unlike normal cells, malignant cells show CK activation and decrease of cAMP; therefore, PKA activity decreases. This suggests a correlation of CK and PKA activity and significant damage to their regulation at pathological changes of tissue proliferation. To further study concerted CK and PKA regulation we used monoclonal antibodies (mAbs) against cAMP-dependent protein kinase regulatory subunit RKII beta. We produced 11 antibodies in three groups: inhibiting, which block cAMP binding with RII beta and inhibit holoenzyme formation (RS6); activating, which enhance cAMP binding and do not affect holoenzyme formation (RS28); and neutral (RS17). To investigate mAb influence on protein kinase regulation in live cells we permeabilized pheochromocytoma PC12 by digitonin. When used at 5-microM concentration for 5 min, digitonin allowed us to deliver mAb into PC12 cells at 30-34-nM concentration, leaving 68-75% viable cells. Protein kinase activity was measured within 0.5 and 4 h after incorporation of mAbs into cells. After 30 min incorporation, mAb RS6 blocked PKA activation in PC12 cells under the influence of cAMP; other mAbs showed no effect. mAb RS6 caused a 4-fold increase of free C subunit activity 4 h after incorporation. mAb RS38 decreased R2C2 activity and did not influence C subunit activity. The change of free C subunit activity caused by mAb incorporation was followed by a synchronized, well-balanced change of CK type I activity, which suggests a correlation between the two phosphorylation systems of cell proteins.
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PMID:Protein kinase A: regulation and receptor-mediated delivery of antisense oligonucleotides and cytotoxic drugs. 1211 75


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